Azithromycin-sulfonamide conjugates as inhibitors of resistant Streptococcus pyogenes strains

Article abstract of DOI:10.1016/j.ejmech.2006.08.008

Novel hybrid compounds 6a-6d, conjugates of 15-membered azalides and sulfonamides, i.e. unsubstituted, 4-aryl- and 4-heteroaryl-aminosulfonyl derivatives of 9a-[N′-(phenylcarbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A were synthesized and characterized by IR, one- and two-dimensional NMR spectroscopies and MALDI-TOF and MS/MS mass spectrometry. The new compounds were evaluated in vitro against a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. 9a-{N′-[4-(Aminosulfonyl)phenyl]carbamoyl} - (6a) and 9a-{N′-[4-(phenylaminosulfonyl)phenyl]carbamoyl} - (6b) derivatives showed improvements in activity against inducible resistant Streptococcus pyogenes in comparison with macrolide antibiotic azithromycin and starting material 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2). In addition, the synthesized azithromycin-sulfonamide conjugates 6a-6d showed good antibacterial activity against sensitive S. pyogenes and Streptococcus pneumoniae strains. The kinetics of degradation in the artificial gastric juice showed that the most active compounds, 6a and 6b, exhibited azithromycin like stability. The cleavage of the cladinose sugar was found to be the main decomposition pathway leading to inactive 7a and 7b, prepared also as analytical standards by the alternative synthetic route together with 7c and 7d.

Full text of DOI:10.1016/j.ejmech.2006.08.008

European Journal of Medicinal Chemistry 42 (2007) 138e145
http://www.elsevier.com/locate/ejmech
Original article
Azithromycinesulfonamide conjugates as inhibitors of resistant
Streptococcus pyogenes strains
a,
Mirjana Bukvic Krajacic , Predrag Novak , Mario Cindric , Karmen Brajsa ,
b,c
a
b
*
Miljenko Dumic , Nedjeljko Kujundzic
´
´
´
ˇ
ꢀ
a
b
´
´
ꢀ
a
´
PLIVA e Research and Development Ltd., Prilaz baruna Filipovica 29, HR-10000 Zagreb, Croatia
b
´
PLIVA e Research Institute Ltd., Prilaz baruna Filipovica 29, HR-10000 Zagreb, Croatia
c Department of Analytical Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
Received 13 April 2006; received in revised form 13 July 2006; accepted 13 August 2006
Available online 28 September 2006
Abstract
Novel hybrid compounds 6ae6d, conjugates of 15-membered azalides and sulfonamides, i.e. unsubstituted, 4-aryl- and 4-heteroaryl-amino-
sulfonyl derivatives of 9a-[N0-(phenylcarbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A were synthesized and characterized by
IR, one- and two-dimensional NMR spectroscopies and MALDI-TOF and MS/MS mass spectrometry.
The new compounds were evaluated in vitro against a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. 9a-
{N0-[4-(Aminosulfonyl)phenyl]carbamoyl} e (6a) and 9a-{N0-[4-(phenylaminosulfonyl)phenyl]carbamoyl} e (6b) derivatives showed improve-
ments in activity against inducible resistant Streptococcus pyogenes in comparison with macrolide antibiotic azithromycin and starting material
9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2). In addition, the synthesized azithromycinesulfonamide conjugates 6ae6d showed good
antibacterial activity against sensitive S. pyogenes and Streptococcus pneumoniae strains.
The kinetics of degradation in the artificial gastric juice showed that the most active compounds, 6a and 6b, exhibited azithromycin like
stability. The cleavage of the cladinose sugar was found to be the main decomposition pathway leading to inactive 7a and 7b, prepared also
as analytical standards by the alternative synthetic route together with 7c and 7d.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 15-Membered azalides; Azithromycinesulfonamide conjugates; Antimicrobial activity; Structural elucidation; Acid stability
1. Introduction
clinical isolates [3,4] both in hospital settings and in the com-
munity, significant efforts have been devoted to chemical mod-
ifications of the existing macrolides. One of the main types of
bacterial resistance is connected to methylases which are re-
sponsible for developing macrolide, lincosamide and streptog-
ramin (MLS) resistance mechanisms of the inducible (iMLS)
or constitutive (cMLS) types.
As part of our continuous synthetic efforts focused on a prep-
aration of 9a-substituted azalide derivatives [5,6] we have
recently reported the synthesis of novel sulfonylureas of 15-
membered azalides which showed significant activity against
inducible resistant S. pyogenes strains [7]. Those results promp-
ted us to further explore modifications at position 9a of an aza-
lide lactone ring, especially by the carbamoyl group linked
sulfonamides, such as sulfanilamide, sulfabenz, sulfapyridine
Macrolide antibiotic azithromycin 1 (Fig. 1), the first repre-
sentative of the azalide class, is well-established antimicrobial
agent that has been widely prescribed for the treatment of re-
spiratory tract infections owing to its high efficacy and safety
[1,2]. Over the past decade, the increasing incidence of bacte-
rial resistance to a large number of antibacterial agents is be-
coming a major threat to successful treatment of infectious
diseases. Due to a rapid emergence of macrolide-resistant
Streptococcus pneumoniae and Streptococcus pyogenes among
* Corresponding author. Tel.: þ385 1 372 2566; fax: þ385 1 372 1954.
´
E-mail address: mirjana.bukvic.krajacic@pliva.hr (M. Bukvic Krajacic).
ꢀ ´
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.08.008

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
139
and sulfamethoxazole (Fig. 1, Scheme 1). Macrolide antibiotics
exert their antibacterial activity by binding to ribosomal 23S
RNA and thus blocking the bacterial protein synthesis [8,9].
Strategies which involved macrolide conjugates incorporating
heteroaromatic rings [10], as well as macrolideenucleoside
and macrolideenucleobase conjugates [11] have already been
introduced and these compounds showed an increased affinity
for the ribosome. Similarly, we expect that by introducing
novel interactive groups into the azalide backbone further im-
provements in activity might be achieved. Therefore, the pres-
ent paper describes the synthesis, structure elucidation and
antibacterial in vitro evaluation of novel hybrid compounds,
conjugates of 15-membered azalides and sulfonamides, e.g.
the 4-aminosulfonyl e (6a), 4-phenylaminosulfonyl e (6b),
4-[(pyridyl-2-yl)aminosulfonyl] e (6c), and 4-[(5-methylisoxa-
zol-3-yl)aminosulfonyl] e (6d), derivatives of 9a-[N0-(phenyl-
carbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin
A (Scheme 1).
temperature, furnished the decladinosyl derivatives 7a and
7b, alternatively prepared as the analytical standards, by the
same type of reactions as mentioned above, i.e. via 2 /
3 / 5 / 7aed pathway (Scheme 1).
2.2. Structural characterization
Structures of all synthesized compounds were determined by
IR and NMR spectroscopies and mass spectrometry (MALDI-
TOF MS and MS/MS). The assignments of proton and carbon
chemical shifts were made by the combined use of one- (1H
and APT) and two-dimensional (gCOSY, gHSQC and gHMBC)
NMR spectra. With respect to 2 and 3 several new resonances
were observed in NMR spectra of compounds 6ae6d and
7ae7d. The additional singlet at approximately 155 ppm in
the 13C NMR spectra was observed reflecting the presence of
a carbamoyl carbon atom. Strong bands found at approximately
1660e1634 cmꢁ1 in IR spectra were assigned to carbamoyl
C]O stretching vibrations. The proton and carbon chemical
shifts in the regions 6.8e8.0 ppm (DMSO-d6), 7.9e8.1 ppm
(pyridine-d5) and 6.1e8.3 (CDCl3) for protons and 116e
170 ppm, for carbons were diagnostic for substituted benzene,
pyridine and isoxazole rings. Carbon and proton chemical shifts
of all compounds are given in Section 4. The mass spectra ex-
hibited precursor ions that are consistent with masses of the re-
spective compounds 6ae6d and 7ae7d as shown in Scheme 1.
Elemental composition was determined by accurate mass mea-
surements in the mass range m/z 749.5613e1046.5423 with the
observed mass accuracy better than 4 ppm. Accurately mea-
sured masses together with calculated molecular formulas and
IR bands of compounds 6ae6d and 7ae7d are displayed in
Table 1. A typical fragmentation pattern of 6b with theoretically
calculated masses is shown in Fig. 2. Accurately measured
masses of 6b product ions revealed predominantly ions pro-
duced after a loss of benzensulfonamido moiety at the position
9a, e.g. m/z 735.4954. Other product ions were formed by a loss
of desosamine sugar and one water molecule (m/z 851.4437 and
m/z 833.4339), or by a loss of both sugars and one or two water
molecules (m/z 694.3341, m/z 676.3221 and m/z 658.3160).
Also, the mass spectra exhibited product ions formed by a si-
multaneous carbamoyl and two glycosidic bond cleavages (des-
osamine and cladinose) giving m/z 420.2959, m/z 402.2843 and
m/z 384.2741, respectively. Similar fragmentation was ob-
served for other compounds too.
In order to study the stability of the most active compounds,
an acid hydrolysis was performed and degradation products
were analyzed and biologically evaluated.
2. Results and discussion
2.1. Chemistry
The compounds 6ae6d were prepared by the reaction of 9-
deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2), the last
intermediate in the azithromycin (1) synthesis [12,13] with
4-(chlorosulfonyl)phenylisocyanate in toluene at 0e5 ꢀC
(Scheme 1). The smoothly formed 9a-{[4-(chlorosulfonyl)-
phenyl]carbamoyl} e derivative 4 was transformed without
the isolation into the compounds 6ae6d, by the reaction
with 2 equiv of ammonia, aniline, 2-aminopyridine and 5-
methyl-3-aminoisoxasole, respectively. The regioselective hy-
drolysis of 6a and 6b in 1 M hydrochloric acid at room
O
S
H2N
H2N
H2N
NH
2
O
Sulfanilamide
N
9a
O
S N
O
N
O
OH
HO
H
HO
OH
2.3. Acid stability
Sulfabenz
O
O
O
O
H
N
N
It is known that in aqueous acidic media 14-membered
erythromycin derivatives underwent intramolecular cyclization
reactions [14,15] to form inactive by-products with unfavoura-
ble side effects. On the other hand 15-membered azithromycin
derivatives were found to be more stable with no cyclization
products. The main decomposition pathway for azithromycin
included hydrolysis of the cladinose sugar glycosidic bond
leading to an inactive decladinosyl derivative [16].
O
O
S
N
OH
O
Sulfapyridine
O
Azithromycin
O
H
1
H2N
S
N
O
O
Sulfamethoxazole
The acidic stability studies of the most active compounds
6a and 6b were carried out in 0.01 M HCl aqueous
Fig. 1. Structures of azithromycin, sulfanilamide, sulfabenz, sulfapyridine and
sulfamethoxazole.

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
140
O
S
O
O
S
c
b
a
d
Cl
HN
NH R'
HN
N
b c
O
O
O
HN
N
9a
10
9
N
O
N
N
O
8
OH
HO
OH
HO
OH
HO
HO
HO
7
HO
3'
11
OH
OH
OH
i
12
6
5
ii
i
2' 4'
1'
13O
O5'
14
15
O
O
O
O
O
4
1
3
2
O
OR
O
OR
O
OR
6a-6d R = Cladinosyl 6a,6b
4R = Cladinosyl
5R = H
2 R = Cladinosyl
3 R = H
ii
ii
7a-7d R = H
7a,7b
O
a
b
c
d
R = Cladinosyl =
1''
5''
c'
b'
a'
e'
a' N
2'' 4''
a' N
O
3''
OH
d'
R'
d'
H
c'
O
b'
CH3
c'
c'
b'
b'
Scheme 1. Synthesis of azithromycinesulfonamide conjugates 6ae6d and 7ae7d. Reagents and conditions: i, 4-(chlorosulfonyl)phenylisocyanate, toluene,
0e5 ꢀC, 1 h; ii, 1 M hydrochloric acid, r.t., 12 h; iii, 2 equiv of different amines: ammonia, aniline, 2-aminopyridine and 5-methyl-3-aminoisoxasole.
hydrochloric acid at 37 ꢀC, as a model of artificial gastric
juice. We were particularly interested to see whether the hy-
drolysis of the cladinose glycosidic bond was the main degra-
dation pathway for 6a and 6b or would decomposition include
also cleavages of 9a-carbamoyl and sulfonamide bonds.
A combined use of LC/UV and LC/MS methods to study
kinetics of hydrolysis for parent compounds and their degrada-
tion products was employed. In the LC/UV chromatogram,
peaks at retention times 9.41 min, 8.05 min, 11.41 min and
10.45 min were attributed to 6a, 7a, 6b and 7b, respectively.
The mass spectra of the reaction mixtures after 10 h showed
only precursor ions at m/z 775.42 and m/z 851.49, which
stemmed from the cleavage of the cladinose sugars giving de-
cladinosyl compounds 7a and 7b, respectively. No further deg-
radation products were observed under the studied conditions.
The hydrolytic degradation of 6a and 6b was shown to fol-
low pseudo first-order kinetics as was also observed for azi-
thromycin [16]. Reaction rate constants (k) were determined
from the exponential fit analysis of the plot of the reactant con-
centration (6a and 6b) versus time. The calculated rate con-
stants for hydrolytic degradation at different temperatures
and times for 50% decay (t1/2) are listed in Table 2.
concentrations (MICs). MIC values for all compounds (Table
3) were determined in comparison with azithromycin (1) and
9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2) on
a panel of sensitive and resistant Gram-positive bacterial
strains (Staphylococcus aureus, S. pneumoniae and S. pyogenes)
and on Gram-negative strains (Escherichia coli, Haemophilus
influenzae and Moraxella catarrhalis).
In Table 3 it is clearly seen that 6a and 6b possess two to
three times better activity against iMLS resistant S. pyogenes
strain (MIC 2 mg/ml) when compared to both azithromycin 1
and starting cyclic amine 2. These activities are comparable
to those observed for azalide sulfonylureas reported previously
[7]. The introduction of 4-aryl- and 4-heteroaryl-aminosulfonyl
groups in 6be6d does not improve the activity against iMLS re-
sistant S. pyogenes in comparison with 6a and azalide sulfonyl-
ureas [7]. Compounds 6ae6d are found to be inactive against
pathogens with an efflux gene (mef ) and against pathogens
with a constitutively resistant S. pyogenes. New azithromy-
cinesulfonamide conjugates 6a and 6b exhibit somewhat lower
activity than azithromycin (1) and starting amine 2 against sen-
sitive S. pneumonia (MIC 0.5 and 1 mg/ml) and S. pyogenes
(MIC 2 mg/ml) strains. Furthermore, 6c and 6d showed in gen-
eral lower activity against most of the tested bacterial strains ex-
cept for sensitive S. aureus and M. catarrhalis where better
activity was observed in comparison with 6a and 6b analogs.
All synthesized azithromycinesulfonamide conjugates 6ae
6d are inactive against Gram-negative H. influenzae and E. coli
strains.
The activation energies of hydrolysis of glycosidic bonds
for 6a and 6b were determined next, and were found to be
90.5 kJ/mol (21.8 kcal/mol) and 89.9 kJ/mol (21.6 kcal/mol),
respectively (Fig. 3). These values were similar to that ob-
tained for azithromycin which amounted to 105.9 kJ/mol
(25.3 kcal/mol) [16].
To confirm the structures of degradation products 7a and 7b
and to support the proposed hydrolytic pathway their isolation
and characterization were subsequently performed.
The decladinosyl derivatives 7ae7d didn’t show any anti-
bacterial activity at all against tested bacterial strains, as al-
ready observed for the related macrolides [7].
2.4. In vitro antimicrobial activity
3. Conclusion
The antibacterial screening of azithromycinesulfonamide
conjugates 6ae6d and 7ae7d was performed by a standard di-
lution assay for the determination of minimal inhibitory
The coupling of a benzenesulfonamido moiety to the 9a po-
sition of 15-membered azalide scaffold via carbamoyl linker
has proven to be a promising method to tackle the resistance

Table 1
Spectroscopic data for the synthesized compounds 6ae6d and 7ae7d
Compound number
R
R0
Molecular formula
IR (cmꢁ1
)
Calculated
Measured
monoisotopic
m/z [MþH]þ
933.5106
monoisotopic
m/z [MþH]þ
933.5129
6a
6b
Cladinosyl
Cladinosyl
H
C44H76N4O15
S
S
3439, 2974, 2937, 1722, 1644, 1591, 1520, 1462, 1333, 1160, 834
3418, 2973, 2937, 1725, 1650, 1591, 1523, 1498, 1158, 1464, 836, 756
C50H80N4O15
1009.5419
1009.5433
6c
Cladinosyl
Cladinosyl
C49H79N5O15
S
3444, 2972, 2935, 1728, 1634, 1592, 1525, 1464, 1383, 1165, 836, 775
3434, 2961, 2932, 1728, 1642, 1591, 1523, 1466, 1272, 1165, 947, 836, 751
1010.5371
1014.5321
1010.5410
1014.5355
N
N
O
CH3
6d
C48H79N5O16
S
7a
7b
H
H
H
C
36H62N4O12
S
3444, 2975, 2937, 1723, 1651, 1592, 1538, 1456, 1328, 1160, 835, 756
3444, 2975, 2937, 1723, 1660, 1591, 1525, 1497, 1159, 836, 756
775.4163
851.4476
775.4168
851.4490
C42H66N4O12
S
7c
H
H
C41H65N5O12
S
3444, 2969, 2935, 1724, 1634, 1592, 1526, 1462, 1385, 1285, 1164, 836, 775
3444, 2972, 2934, 1725, 1651, 1592, 1520, 1464, 1269, 1165, 944, 836, 754
852.4428
856.4378
852.4433
856.4391
N
N
O
7d
C40H65N5O13
S
CH3

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
142
-4
-4,5
-5
O
S
H
N
H
N
O
6b
6a
O
735.5007
N
-5,5
-6
N
HO
HO
HO
OH
-6,5
3,15
3,2
3,25
3,3
3,35
3,4
O
O
O
O
1/T (K)x103
O
Fig. 3. Arrhenius plot of temperature versus pseudo first-order reaction rates of
6a and 6b.
O
O
[M+H]+
851.4476
833.4370
was better than 90% according to LC/MS measurements.
MALDI-TOF MS spectra in reflectron mode were obtained
on an Applied Biosystems Voyager DE STR instrument (Foster
City, CA). The laser wavelength was 337 nm (N2 laser) and la-
ser frequency amounted to 20 Hz. Desorbed ions were acceler-
ated to 2.0 kV. Delayed extraction was 150 ns. Calibrant and
analyte spectra were obtained in positive ion mode. Calibration
type was internal with calibrants azithromycin and angiotensin
II dissolved in a-cyano-4-hydroxycinnamic acid matrix in the
mass range m/z 749.5613e1046.5423. Accurately measured
MS/MS analyses were performed with a quadrupole-time of
flight mass spectrometer Q-TOF Micro equipped with the
lock-spray (Micromass, Manchester, UK). Azithromycin was
used as an internal lock-mass standard. NMR experiments
were carried out on a Bruker Avance DRX500 spectrometer
operating at 500.13 MHz for 1H and 125.77 MHz for 13C,
respectively, with a 5 mm diameter inverse detection probe
and a z-axis gradient coil. Standard spectral conditions were
used. The solvents were pyridine-d5, DMSO-d6 and CDCl3
and all experiments were performed at ambient temperature.
TMS was used as the internal standard. IR spectra were
recorded in KBr pellets on a Nicolet Magna 760 FT-IR spec-
trometer. The number of scans was 16 and the resolution was
OH
m/z 1009.5419
420.2961
402.2855
384.2750
694.3373
676.3268
658.3162
Fig. 2. Proposed fragmentation pattern of the compound 6b based on accurate
mass MS/MS experiments with theoretically calculated masses.
problems. Hence, newly synthesized azalides 6a and 6b dis-
played a strong antibacterial activity against inducible resis-
tant S. pyogenes.
In acidic conditions 6a and 6b showed azithromycin like sta-
bility giving only inactive decladinosyl degradation products.
The results presented here together with those previously
reported for azalide sulfonylureas [7] can serve as a good plat-
form for further derivatization and development of antimicro-
bial agents with improved activity against resistant S. pyogenes
strains. Next steps in this direction should also include charac-
terization of binding modes and interactions with ribosome by
using biochemical and structural methods.
4 cmꢁ1
.
4. Experimental
4.1.1. General procedure for the preparation of 6ae6d and
7ae7d
4.1. Chemistry
The solution of 2 or 3 (1.84 mmol) and 4-(chlorosulfonyl)-
phenyl isocyanate (1.84 mmol) in dry toluene (30 ml) was
stirred for about 2 h at temperature 0e5 ꢀC. In the reaction
mixture amine (23% water solution of ammonia (30.0 mmol),
aniline (4.6 mmol), 2-aminopyridine (5.2 mmol) or 5-methyl-
3-aminoisoxasole (5.8 mmol) was added and the reaction mix-
ture was stirred for about 2 h at room temperature. The crude
product was filtered, wherefrom by column chromatography
on silica gel using solvent system CH2Cl2:CH3OH ¼ 9:1 pure
compound was obtained.
Analytical TLC was performed on Merck 60 F254 plates us-
ing CH2Cl2:CH3OH:NH3(25%) ¼ 90:9:1.5 as eluents. Column
chromatography was performed on a Merck Silica gel 60
(0.043e0.060 mm). The purity of the isolated compounds
Table 2
Pseudo first-order reaction rate constants and t1/2 values for 6a and 6b
Comp.
6a
6b
t/ꢀC
kb/minꢁ1
ta1/2/min
kb/minꢁ1
ta1/2/min
25
30
37
40
a
2.29 ꢂ 10ꢁ3
3.77 ꢂ 10ꢁ3
8.31 ꢂ 10ꢁ3
1.39 ꢂ 10ꢁ2
303
184
83
1.88 ꢂ 10ꢁ3
3.21 ꢂ 10ꢁ3
7.10 ꢂ 10ꢁ3
1.12 ꢂ 10ꢁ2
369
216
98
4.1.1.1. 9-Deoxo-9-dihydro-9a-{N0-[4-(aminosulfonyl)phenyl]-
50
62
carbamoyl}-9a-aza-9a-homoerythromycin
A
86.2%. H NMR (pyridine) d 3.12 (1H, H-2), 4.67 (1H, H-
(6a). Yield
1
Calculated according to t1/2 ¼ ln 2/k.
b
Standard deviation was better than 0.6 ꢂ 10ꢁ3 minꢁ1 in all experiments.
3), 2.45 (1H, H-4), 4.05 (1H, H-5), 2.23, 1.91 (2H, H-7),

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
143
Table 3
In vitro antibacterial activity of novel azithromycinesulfonamide conjugates 6ae6d and their decladinosyl analogs 7ae7d
Test
compound
Strain/MIC (mg/ml)
S. aureus
Ery-S
S. pneumoniae
Ery-S
S. pyogenes
Ery-S
S. pyogenes
iMLS ermTR
S. pyogenes
cMLS
S. pyogenes
M mefA
M. catarrhalis
ATCC-0324
H. influenzae
ATCC-0529
E. coli
ATCC-0001
1
0.5
2
ꢃ0.125
ꢃ0.125
8
16
64
>64
>64
>64
>64
>64
>64
2
16
ꢃ0.125
NT
0.25
1
1
4
2
6a
0.25
1
0.25
64
16
2
2
4
>64
32
>64
64
32
32
>64
>64
>64
>64
>64
6b
6c
0.5
4
2
8
32
2
>64
>64
>64
>64
>64
>64
4
64
6d
7ae7d
16
>64
8
>64
8
>64
>64
>64
>64
iMLS: inducible resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics; cMLS: constitutive MLS resistance; M: efflux mediated macrolide
resistance; NT: not tested.
2.85 (1H, H-8), 4.34 (1H, H-11), 5.75 (1H, H-13), 2.23, 1.7
(2H, H-14) 0.99 (3H, H-15), 4.92 (1H, H-10), 3.44 (1H, H-
20), 2.68 (1H, H-30), 2.23 (6H, 30 NMe2), 1.42, 1.2 (2H, H-
40), 3.98 (1H, H-50), 5.12 (1H, H-100), 2.39, 1.5 (2H, H-200),
3.44 (3H, 300 OMe), 3.23 (1H, H-400), 4.51 (1H, H-500), 1.35
(3H, 2 Me), 1.64 (3H, 4 Me), 2.01 (3H, 6 Me), 1.08 (1H,
H-8 Me), 1.29 (3H, 12 Me), 1.29 (3H, 50 Me), 1.29 (3H,
300 Me), 1.49 (3H, 500 Me), 8.63 (2H, phenyl, b), 8.19 (2H,
phenyl, c), 8.01 (1H, phenyl, d); 13C NMR (pyridine)
d 176.9 (C-1), 45.7 (C-2), 79.9 (C-3), 40.9 (C-4), 85.6 (C-
5), 73.8 (C-6), 40 (C-7), 28.5 (C-8), 56.2 (C-9), 75.0 (C-
11), 73.8 (C-12), 77.5 (C-13), 22.9 (C-14), 11.8 (C-15),
103.1 (C-10), 71.3 (C-20), 65.3 (C-30), 40.2 (30 NMe2), 29.6
(C-40), 68.2 (C-50), 96.5 (C-100), 35.6 (C-200), 74.8 (C-300),
49.6 (300 OMe), 78.6 (C-400), 66.1 (C-500), 13.8 (2 Me), 10.0
(4 Me), 30.2 (6 Me), 20.7 (8 Me), 10 (10 Me), 16.2 (12
Me), 21.9 (50 Me), 21.6 (300 Me), 19.2 (500 Me), 156.7
(NCONH), 145.0 (phenyl, a), 128.4 (phenyl, b), 118.5 (phe-
nyl, c), 138.1 (phenyl, d).
128.8 (phenyl, c0), 127.5 (phenyl, b), 123.9 (phenyl, d0),
120.1 (phenyl, b0).
4.1.1.3. 9-Deoxo-9-dihydro-9a-{N0-[4-(2-pyridylaminosulfonyl)-
phenyl]carbamoyl}-9a-aza-9a-homoerythromycin A (6c). Yield
1
42.1%. H NMR (DMSO) d 2.61 (1H, H-2), 4.00 (1H, H-3),
2.50 (1H, H-4), 3.39 (1H, H-5), 2.08 (1H, H-8), 3.65 (1H, H-
10), 3.34 (1H, H-11), 5.03 (1H, H-13), 1.76, 1.34 (2H, H-14),
0.79 (3H, H-15), 4.41 (1H, H-10), 3.00 (1H, H-20), 2.5 (1H,
H-30), 2.24 (6H, 30 NMe2), 1.57, 1.15 (2H, H-40), 3.64 (1H,
H-50), 4.75 (1H, H-100), 2.27, 1.48 (2H, H-200), 3.21 (3H, 300
OMe), 2.89 (1H, H-400), 4.02 (1H, H-500), 1.07 (3H, 2 Me),
0.88 (3H, 4 Me), 1.28 (3H, 6 Me), 0.90 (1H, H-8 Me), 0.88
(3H, 12 Me), 1.11 (3H, 50 Me), 1.13 (3H, 300 Me), 1.06 (3H,
500 Me), 8.03 (1H, pyridyl, e0), 7.70 (2H, phenyl, b), 7.66 (1H,
pyridyl, c0), 7.43 (2H, phenyl, c), 7.10 (1H, pyridyl, b0), 6.86
(1H, pyridyl, d0); 13C NMR (DMSO) d 175.6 (C-1), 44.6 (C-
2), 78.2 (C-3), 84.0 (C-5), 73.6 (C-6), 27.6 (C-8), 75 (C-12),
76 (C-13), 21.6 (C-14), 11.1 (C-15), 101.9 (C-10), 70.5 (C-20),
64.5 (C-30), 40.1 (30 NMe2), 29.4 (C-40), 67.0 (C-50), 95.1 (C-
100), 34.7 (C-200), 72.6 (C-300), 48.8 (300 OMe), 77.3 (C-400),
64.8 (C-500), 15.4 (2 Me), 9.11 (4 Me), 28.3 (6 Me), 19.9 (8
Me), 9.1 (10 Me), 14.2 (12 Me), 21.3 (50 Me), 20.8 (300 Me),
18.4 (500 Me), 155.4 (NCONH), 152.7 (pyridyl, a0), 144.5 (pyr-
idyl, e0), 144.4 (phenyl, a), 139.4 (pyridyl, c0), 133.4 (phenyl, d),
127.2 (phenyl, b), 116.0 (pyridyl, d0), 113.0 (pyridyl, b0).
4.1.1.2. 9-Deoxo-9-dihydro-9a-{N0-[4-(phenylaminosulfonyl)-
phenyl]carbamoyl}-9a-aza-9a-homoerythromycin
A
(6b).
Yield 41.0%. 1H NMHR (DMSO) d 2.54 (1H, H-2), 4.01
(1H, H-3), 2.05 (1H, H-4), 3.38 (1H, H-5), 2.05 (1H, H-8),
4.01 (1H, H-11), 5.03 (1H, H-13), 1.76, 1.28 (2H, H-14),
0.79 (3H, H-15), 4.45 (1H, H-10), 3.45 (1H, H-20), 2.5 (1H,
H-30), 2.50 (6H, 30 NMe2), 1.46, 1.11 (2H, H-40), 3.67 (1H,
H-50), 4.76 (1H, H-100), 2.26, 1.48 (2H, H-200), 3.22 (3H, 300
OMe), 2.90 (1H, H-400), 4.01 (1H, H-500), 1.06 (3H, 2 Me),
0.89 (3H, 4 Me), 1.27 (3H, 6 Me), 0.88 (1H, H-8 Me),
0.88 (3H, 12 Me), 1.11 (3H, 50 Me), 1.14 (3H, 300 Me),
1.06 (3H, 500 Me), 10.1 (SO2NH ), 7.57 (2H, phenyl, b),
7.44 (2H, phenyl, c), 7.20 (2H, phenyl, c0), 7.07 (2H, phenyl,
b0), 6.99 (1H, phenyl, d0); 13C NMR (DMSO) d 175.9 (C-1),
78.2 (C-3), 85 (C-5), 73.7 (C-6), 27.9 (C-8), 75 (C-11), 75.1
(C-12), 76.1 (C-13), 21.8 (C-14), 11.3 (C-15), 101.6 (C-10),
71 (C-20), 64.8 (C-30), 40.2 (30 NMe2), 29.9 (C-40), 66.7
(C-50), 96.2 (C-100), 34.8 (C-200), 72.8 (C-300), 49.0 (300
OMe), 77.4 (C-400), 65.0 (C-500), 15.4 (2 Me), 10.9 (4 Me),
27.9 (6 Me), 20.4 (8 Me), 9.2 (10 Me), 14.8 (12 Me), 21.3
(50 Me), 21.1 (300 Me), 18.5 (500 Me), 155.5 (NCONH),
145.1 (phenyl, a), 138.1 (phenyl, a0), 131.2 (phenyl, d),
4.1.1.4. 9-Deoxo-9-dihydro-9a-{N0-[4-(5-methyl-3-isoxazolyla-
minosulfonyl)phenyl]carbamoyl}-9a-aza-9a-homoerythromycin
A (6d). Yield 61.7%. 1H NMR (DMSO) d 2.62 (1H, H-2), 4.01
(1H, H-3), 1.74 (1H, H-4), 3.44 (1H, H-5), 2.08 (1H, H-8),
3.44 (1H, H-11), 5.04 (1H, H-13), 1.66, 1.34 (2H, H-14),
0.79 (3H, H-15), 4.42 (1H, H-10), 3.09 (1H, H-20), 2.77 (1H,
H-30), 2.43 (6H, 30 NMe2), 1.77, 1.52 (2H, H-40), 3.66 (1H,
H-50), 4.75 (1H, H-100), 2.24, 1.49 (2H, H-200), 3.20 (3H, 300
OMe), 2.89 (1H, H-400), 3.99 (1H, H-500), 1.08 (3H, 2 Me),
0.87 (3H, 4 Me), 1.28 (3H, 6 Me), 0.88 (1H, 8 Me), 0.87
(3H, 12 Me), 1.09 (3H, 50 Me), 1.12 (3H, 300 Me), 1.09 (3H,
500 Me), 7.62 (2H, phenyl, b), 7.46 (2H, phenyl, c), 6.05
(1H, isoxazolyl, b0), 2.24 (3H, isoxazolyl, Me); 13C NMR
(DMSO) d 175.8 (C-1), 44.6 (C-2), 78.2 (C-3), 39.9 (C-4),

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
144
84 (C-5), 73.7 (C-6), 27.8 (C-8), 78.1 (C-11), 75 (C-12), 78.1
(C-13), 21.8 (C-14), 11.3 (C-15), 101.7 (C-10), 70.0 (C-20),
64.8 (C-30), 40.1 (30 NMe2), 30.1 (C-40), 66.8 (C-50), 95.8
(C-100), 34.9 (C-200), 72.8 (C-300), 48.9 (300 OMe), 77.4 (C-
400), 65.0 (C-500), 14.6 (2 Me), 8.2 (4 Me), 28 (6 Me), 20.2
(8 Me), 13.6 (12 Me), 21.3 (50 Me), 21.1 (300 Me), 18.5 (500
Me), 155.6 (NCONH), 170 (isoxazolyl, c0), 160 (isoxazolyl,
a0), 14.0 (isoxazolyl, Me), 144.7 (phenyl, a), 132.0 (phenyl,
d), 127.2 (phenyl, b), 119 (phenyl, c).
e0), 7.74 (2H, phenyl, b), 7.64 (1H, phenyl, c0), 7.42 (2H, phe-
nyl, c), 7.38 (1H, pyridyl, b0), 6.80 (1H, pyridyl, d0); 13C NMR
(CDCl3) d 176 (C-1), 44.7 (C-2), 75.3 (C-3), 38.1 (C-4), 90
(C-5), 74.1 (C-6), 29.1 (C-8), 74.1 (C-12), 78.4 (C-13), 20.9
(C-14), 10.4 (C-15), 105.4 (C-10), 68.9 (C-20), 64.6 (C-30),
39.6 (30 NMe2), 27.4 (C-40), 69.9 (C-50), 15.5 (2 Me), 7.0 (4
Me), 27 (6 Me), 19 (8 Me), 16.3 (12 Me), 20.2 (50 Me),
155.8 (NCONH), 142.8 (phenyl, a), 141.5 (pyridyl, e0),
140.8 (pyridyl, c0), 133.3 (phenyl, d), 127.4 (phenyl, b),
117.9 (phenyl, c), 114.5 (pyridyl, d0), 114.1 (pyridyl, b0).
4.1.1.5.
nosulfonyl)phenyl]carbamoyl}-9a-aza-9a-homoerythronolide
5-O-Desosaminyl-9-deoxo-9-dihydro-9a-{N0-[4-(ami-
4.1.1.8. 5-O-Desosaminyl-9-deoxo-9-dihydro-9a-N-{N0-[4-(5-
methyl-3-isoxazolylaminosulfonyl)phenyl]carbamoyl}-9a-aza-
9a-homoerythronolide A (7d). Yield 54.4%. 1H NMR (CDCl3)
d 2.66 (1H, H-2), 3.87 (1H, H-3), 3.47 (1H, H-5), 1.8 (1H, H-
8), 4.60 (1H, H-13), 1.95, 1.59 (2H, H-14), 0.92 (3H, H-15),
4.36 (1H, H-10), 3.32 (1H, H-20), 2.47 (1H, H-30), 2.33 (6H,
30 NMe2), 1.71, 1.33 (2H, H-40), 3.56 (1H, H-50), 1.30 (3H,
2 Me), 0.93 (3H, 4 Me), 1.31 (3H, 6 Me), 1.06 (1H, H-8
Me), 1.13 (3H, 12 Me), 1.20 (3H, 50 Me), 7.64 (2H, phenyl,
b), 7.40 (2H, phenyl, c), 6.16 (1H, isoxazolyl, c0), 2.32 (3H,
isoxazolyl, c0-Me); 13C NMR (CDCl3) d 176 (C-1), 44 (C-
2), 74.6 (C-3), 38.6 (C-4), 90 (C-5), 74.1 (C-6), 29 (C-8), 74
(C-12), 77.1 (C-13), 21.4 (C-14), 10.8 (C-15), 105.6 (C-10),
69.3 (C-20), 64 (C-30), 40.1 (30 NMe2), 28.8 (C-40), 69 (C-
50), 16.0 (2 Me), 7.4 (4 Me), 26 (6 Me), 19 (8 Me), 16.7 (12
Me), 20.8 (50 Me), 156.1 (NCONH), 170.6 (isoxazolyl, c0),
157.6 (isoxazolyl, a0), 142 (phenyl, a), 133 (phenyl, d),
128.1 (phenyl, b), 117.9 (phenyl, c), 95.5 (isoxazolyl, b’),
12.6 (isoxazolyl, c’-Me).
A
1
(7a). Yield 28.8%. H NMR (pyridine) d 3.02 (1H, H-2), 4.30
(1H, H-3), 1.69 (1H, H-4), 4.41 (1H, H-5), 2.2 (1H, H-8), 4.3
(1H, H-11), 5.7 (1H, H-13), 2.36, 1.77 (2H, H-14), 1.01 (3H,
H-15), 5.1 (1H, H-10), 3.61 (1H, H-20), 2.62 (1H, H-30), 2.21
(6H, 30 NMe2), 1.53, 1.19 (2H, H-40), 3.49 (1H, H-50), 1.58
(3H, 2 Me), 1.26 (3H, 4 Me), 1.76 (3H, 6 Me), 1.15 (1H, H-8
Me), 1.40 (3H, 12 Me), 1.16 (3H, 50 Me), 8.16 (2H, phenyl,
b), 7.92 (2H, phenyl, c); 13C NMR (pyridine) d 176 (C-1),
45.6 (C-2), 77 (C-3), 39.1 (C-4), 92.8 (C-5), 74 (C-6), 40.3 (C-
7), 28 (C-8), 75 (C-12), 78 (C-13), 23.1 (C-14), 11.4 (C-15),
103.3 (C-10), 69.9 (C-20), 65.4 (C-30), 40.3 (30 NMe2), 29.2 (C-
40), 69.2 (C-50), 16.7 (2 Me), 8.9 (4 Me), 27 (6 Me), 21.1 (8
Me), 19.9 (12 Me), 21.5 (50 Me), 156.7 (NCONH), 138 (phenyl,
a), 127.6 (phenyl, b), 118 (phenyl, c), 144.8 (phenyl, d).
4.1.1.6.
5-O-Desosaminyl-9-deoxo-9-dihydro-9a-N-{N0-[4-
(phenylaminosulfonyl)phenyl]-carbamoyl}-9a-aza-9a-homoer-
1
ythronolide A (7b). Yield 20.0%. H NMR (CDCl3) d 2.67
(1H, H-2), 3.86 (1H, H-3), 3.57 (1H, H-5), 1.9 (1H, H-8),
4.59 (1H, H-13), 1.96, 1.56 (2H, H-14), 0.93 (3H, H-15),
4.35 (1H, H-10), 3.31 (1H, H-20), 2.55 (1H, H-30), 2.30 (6H,
30 NMe2), 1.70, 1.30 (2H, H-40), 3.57 (1H, H-50), 1.30 (3H,
2 Me), 0.94 (3H, 4 Me), 1.31 (3H, 6 Me), 1.07 (1H, 8 Me),
1.13 (3H, 12 Me), 1.20 (3H, 50 Me), 7.57 (2H, phenyl, b),
7.37 (2H, phenyl, c), 7.21 (2H, phenyl, c0), 7.09 (2H, phenyl,
b0), 7.06 (1H, phenyl, d0); 13C NMR (CDCl3) d 178.2 (C-1),
44.7 (C-2), 75.4 (C-3), 38.1 (C-4), 90 (C-5), 74.1 (C-6), 58
(C-9), 27.6 (C-8), 74.1 (C-12), 78.4 (C-13), 20.9 (C-14),
10.4 (C-15), 105.3 (C-10), 68.9 (C-20), 64.6 (C-30), 39.6 (30
NMe2), 27.6 (C-40), 69.8 (C-50), 15.5 (2 Me), 7.0 (4 Me), 27
(6 Me), 19.6 (8 Me), 16.3 (12 Me), 20.4 (50 Me), 155.8
(NCONH), 152.8 (phenyl, a0), 143.3 (phenyl, a), 136.3 (phe-
nyl, d), 128.7 (phenyl, c0), 127.8 (phenyl, b), 124.5 (phenyl,
d0), 120.9 (phenyl, b0), 117.8 (phenyl, c).
4.1.2. General procedure for the hydrolysis of 6a and 6b
The suspension of 6a or 6b (0.55 mmol) in 1 M hydro-
chloric acid (5 ml) was stirred for 12 h at room temperature;
the pH was adjusted to 9.5e10 by adding 1 M NaOH and
was extracted with CH2Cl2 (3 ꢂ 4 ml). The combined organic
layers were washed with water, dried over anhydrous Na2SO4,
evaporated to dryness under reduced pressure to give crude
product wherefrom by chromatography on silica gel column
using CH2Cl2:CH3OH:NH3(25%) ¼ 90:9:1.5 as eluents pure
7a or 7b was obtained. The spectroscopic data of so obtained
compound were identical to the spectroscopic data of 7a or 7b
obtained by the general procedure for preparation of 6ae6d
and 7ae7d.
4.2. Acid stability of 6a and 6b
4.2.1. Preparation of 6a hydrolyzate in 0.01 M HCl
4.1.1.7. 5-O-Desosaminyl-9-deoxo-9-dihydro-9a-N-{N0-[4-(2-
pyridylaminosulfonyl)phenyl]-carbamoyl}-9a-aza-9a-homoery-
thronolide A (7c). Yield 47.0%. 1H NMR (CDCl3) d 2.63 (1H,
H-2), 3.84 (1H, H-3), 3.65 (1H, H-5), 1.8 (1H, H-8), 3.5 (1H,
H-11), 4.61 (1H, H-13), 1.94, 1.55 (2H, H-14), 0.92 (3H, H-
15), 4.34 (1H, H-10), 3.31 (1H, H-20), 2.6 (1H, H-30), 2.29
(6H, 30 NMe2), 1.71, 1.26 (2H, H-40), 3.59 (1H, H-50), 1.29
(3H, 2 Me), 0.93 (3H, 4 Me), 1.29 (3H, 6 Me), 1.07 (1H, 8
Me), 1.12 (3H, 12 Me), 1.22 (3H, 50 Me), 8.30 (1H, pyridyl,
Solution of 6a in 0.01 M HCl of concentration 1 mg/ml
(100 ml) was stirred for 10 h at room temperature. After isola-
tion and purification procedures, described in Section 4.1.2,
1
the pure 7a was obtained. Its H and 13C NMR spectra were
identical to the spectra of the 7a authentic sample.
4.2.2. HPLC assay and decay data
The solution of 6a or 6b in 0.01 M HCl aqueous hydro-
chloric acid (concentration of 1 mg/ml) was held at 25 ꢀC,

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M. Bukvic Krajacic et al. / European Journal of Medicinal Chemistry 42 (2007) 138e145
ꢀ ´
145
30 ꢀC, 37 ꢀC and 40 ꢀC for 10 h. The sample of the solution
(20 ml) was taken each 20 min, and the course of reaction
was monitored by the reverse phase HPLC. Each experiment
was repeated three times, and displayed data represent average
values.
ranging from 64 to 0.125 mg/ml. After 24 h incubation, optical
density was detected by measuring absorbance at 600 nm.
MIC is defined as the concentration that shows 90% growth
inhibition, and was determined by broth dilution methods,
National Committee for Clinical Laboratory Standards (NCCLS,
M7-A2 protocols). All screening procedures were performed
on a Tecan Genesis 150 robot unit.
An Agilent 1100 Series LC/MSD trap, SL model with an
electrospray interface (ESI), a binary pump, degasser, auto-
sampler, thermostatted column compartment and diode array
detector were used. The analysis was carried out with UV de-
tection at 240 nm on a YMC Pack Pro C18 column,
150 mm ꢂ 4.6 mm i.d., 3 mm particle size with a mobile phase
consisted of channel A 40 mM ammonium acetate buffer solu-
tion (pH ¼ 5) and channel B acetonitrile. Gradient elution at
flow rate 1.0 ml/min started with 10% of channel B and ended
after 15 min with 90% of channel B. The ESI source was op-
erated in the positive ion mode. Nitrogen was used as nebuliz-
ing and drying gas at 350 ꢀC. The m/z range scanned in the MS
measurement was 200e1200. Complete system control, data
acquisition and processing were done using the ChemStation
for LC/MSD version 4.2 from Agilent.
References
[1] V.R. Iacoviello, S.H. Zinner, in: W. Schonfeld, H.A. Kirst (Eds.), Macro-
¨
lide Antibiotics, Birkhauser Verlag, Basel, 2002, pp. 15e24.
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[2] W. Schonfeld, S. Mutak, in: W. Schonfeld, H.A. Kirst (Eds.), Macrolide
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[3] A. Tomasz, Am. J. Med. 107 (1999) 55Se59S.
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Reaction rate constants were determined by using exponen-
tial fit method available within Excel program. The activation
energies for hydrolysis of the glycosidic bond in 6a and 6b
were calculated according to the Arrhenius equation (1).
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ln k ¼ ln A ꢁ Ea=RT
ð1Þ
where A is the temperature factor, Ea is the activation energy, R
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thermodynamic temperature.
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4.3. In vitro antimicrobial activity
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J. Chem. Res., Synop. (1988) 152e153.
Antibacterial activity data given in Table 3 were obtained
by microdilution test in MuellereHinton media. Test sub-
stances and standards were dissolved in DMF (Merck) at con-
centration 5 mg/ml. Solutions of the substances were prepared
in MuellereHinton broth media with final concentration
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