Synthesis and biological activity of 4″-O-acyl derivatives of 14- and 15-membered macrolides linked to ω-quinolone-carboxylic unit

Article abstract of DOI:10.1016/j.bmc.2010.06.050

The synthesis and antimicrobial activity of a new class of macrolide antibiotics which consist of a macrolide scaffold and a quinolone unit covalently connected by an appropriate linker are described. Optimization of several synthetic steps and structural

Full text of DOI:10.1016/j.bmc.2010.06.050

Bioorganic & Medicinal Chemistry 18 (2010) 6547–6558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of 400-O-acyl derivatives of 14- and
15-membered macrolides linked to
x-quinolone-carboxylic unit
a,
a
a
a
a
*
ˇ
´
´
ˇ ´
Maja Matanovic Škugor , Vlado Štimac , Ivana Palej , Ðurdjica Lugaric , Hana Cipcic Paljetak ,
a
a
b
a
d,
´
´
´
Darko Filic , Marina Modric , Ivica Ðilovic , Dubravka Gembarovski , Stjepan Mutak
Vesna Erakovic Haber , David J. Holmes , Zrinka Ivezic-Schoenfeld , Sulejman Alihodzic
,
a
c
d,à
´
´
ˇ ´ a
a
´
GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovica 29, HR-10000 Zagreb, Croatia
b Laboratory of General and Inorganic Chemistry, Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102-a, 10000 Zagreb, Croatia
c GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
d PLIVA Research Institute Ltd, Prilaz baruna Filipovi´ca 29, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 22 June 2010
The synthesis and antimicrobial activity of a new class of macrolide antibiotics which consist of a
macrolide scaffold and a quinolone unit covalently connected by an appropriate linker are described.
Optimization of several synthetic steps and structural properties of lead compound 26 are discussed.
Promising antibacterial properties of this compound and some of its analogues are reported.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Macrolide
Quinolone
Antibacterial activity
Resistance
1. Introduction
Recent papers report on the synthesis of new types of macrolide
derivatives, in particular those derived from erythromycin and
Macrolide antibiotics represent an established class of antibacte-
rial compounds, in particular effective in a treatment of respiratory
infections.1–3 Among them, 9-deoxo-9a-methyl-9a-aza-9a-homo-
erythromycin A (Azithromycin) is characterized by its enhanced
chemical stability and exhibited characteristic pharmacokinetic
properties in a comparison to erythromycin and clarithromycin,
two closely related antibiotics.4–6
clarithromycin, and their in vitro antibacterial acitivty.11–13
Our intensive work on azithromycin1,14,15 and its derivatives,16–18
have revealed new chemical spacesforfurther derivatizations. Inthis
paper we disclose the synthesis of a new class of azythromycin deriv-
atives, exemplified to some detail on the presentlead compound, and
their in vitro antimicrobial activity.
However, continuous increase in number of infections caused
by bacteria resistant to one or multiple antibiotic classes is of great
concern.7,8 In fact, spread of resistance among common respiratory
pathogens including Streptococcus pneumoniae is recognized by
Infectious Diseases Society of America as one of the three major
areas of concern that creates a need for new antibiotics.8 There
are several drug discovery strategies aiming at overcoming this is-
sue by intensive search for alternative approach, targeting bacterial
virulence factors, cell division mechanisms, or targets specific for
narrow range of bacterial species.9 Nevertheless, the clinical phase
2 or 3 current development pipelines consist almost entirely of
new compounds derived from or belonging to old, well established
antibiotic classes, such as b-lactams, quinolones, or macrolides.10
2. Results and discussion
2.1. Retrosynthetic considerations
For the last few years our team has been investigating a new
class of antimicrobial compounds which consist of a macrolide
scaffold and a quinolone unit, covalently connected by a suitable
linker.19,20 For this class of molecules, we introduced a term ‘mac-
rolones’. Among recently designed compounds, compound 26 be-
came a lead within its series characterized by an adequate length
and number of heteroatoms in the linker, as well as selected posi-
tions of its attachment to azithromycin and the quinolone units.
Novel macrolones from this series are shown in Figure 1.
General procedure for target macrolones 26–34 is presented in
Scheme 1.
* Corresponding author. Tel.: +385 16051028; fax: +385 16051019.
E-mail address: maja.m.matanovic-skugor@gsk.com (M.M. Škugor).
Macrolone 26, as well as its congeners 27–28, and analogues
with macrocyclic scaffolds 29–34 are characterized by the struc-
ture comprising of a linker connecting the quinolone 3-carboxylic
ˇ
Present address: Hercegovacka 99, HR-10000 Zagreb, Croatia.
à
Present address: Nabriva Therapeutics AG, Leberstrasse 20, Vienna, Austria.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.050

6548
M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
O
O
O
O
O
O
N
O
O
H
N
N
N
O
O
N
O
N
N
O
O
O
N
N
O
OH
HO
O
N
O
N
O
N
O
OH
HO
HO
OH
HO
OH
HO
N
O
HO
HO
HO
HO
HO
HO
OH
HO
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OR
OR
OR
OR
OR
OR
O
OR
O
O
O
O
O
O
29
33
34
31
30
32
-R
-R
O
N
O
O
N
O
O
OH
O
O
O
OH
O
O
26
O
O
O
O
OH
O
N
27
O
O
O
O
OH
O
N
28
Figure 1. Macrolone derivatives 26–34.
X
X
X
R2
R
R
1
1
R2
N
R
R2
1
O
N
R3
O
O
N
O
O
R3
O
O
HO
R3
O
HO
O
O
O
O
(ii)
(i)
O
O
O
O
O
O
O
O
O
(iii)
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
O
O
OH
O
O
O
N
R
26-34
X = -C(=O)-NH-, -C(=O), -N(-CH3)-CH2-,
-C(=N-OCH2CH3)-, -C(=N-OCH2OCH2CH2OCH3)-
X = -C(=O)-NH-, -C(=O), -N(-CH3)-CH2-,
-C(=N-OCH2CH3)-, -C(=N-OCH2OCH2CH2OCH3)-
R1 = H, CH3
R1 = H, CH3
R2 = R3 = H or R2R3 = -CON(H or CH3)-
R2 = R3 = H or R2R3 = -CON(H or CH3)-
R = alkyl, cycloalkyl
Scheme 1. General procedure for target macrolones 26–34. Reagents and conditions: (i) Ac2O, NaHCO3, DCM, rt, 4 h; (ii) EDACxHCl, DMAP, DCM, 0 °C to rt, 4–24 h; (iii) MeOH,
55 °C, 24 h.
acid unit and the macrolide scaffold at the C400-O position of the
cladinose sugar. All linkers contain the terminal C–C6 bond to
quinolone unit, two ether C–O–C bonds in the linker, and a weaker
C400–O–CO terminal ester bond to the cladinose.
This structural aspect of the macrolone 26, along with the high-
er complexity of macrolide scaffold versus quinolone carboxylic
acid unit, suggested convergent synthetic approach to target com-
pounds, as outlined in Scheme 1.
2.2. Synthesis
Azithromycin contains three secondary hydroxyl groups whose
reactivity, in esterification reaction, is approximately in the order:
20-OH ꢀ 400-OH > 11-OH.24,25 Nevertheless, under standard condi-
tions, a mixture of various mono-, di- and tri-ester derivatives is
generally obtained. In a view of this order of reactivity, protection
of 20-OH is essential, and can be affected under properly selected
mild acylation condition. Based on previous experience of our
team,15 and of the others,26 the 20-O-acetyl was selected as the pro-
tecting group on all scaffolds.
Different synthetic approaches are known for a preparation of
the quinolone 3-carboxylic acid derivatives.21,22 Using the com-
mercially available 2-fluoro-5-iodobenzoic acid as a starting mate-
rial, iodoquinolinic acids are prepared as outlined in Scheme 2.
N-Allyl derivative 12 was prepared by an alternative approach23
presented in Scheme 3.
The syntheses of 20-O-acetil-protected macrolides15 and deriva-
tives of quinolone carboxylic acid21–23 are repeatedly reported in
the literature, although the later needed notable modifications to
improve workability and the overall yield. Due to the sensitivity
of macrolide unit, minimal transformations were envisaged after
linking of quinolone unit to macrolide precursor. This concept sug-
gested formation of the C400-O ester bond in the last step, after one
C–C bond and the two ether C–O–C bonds were formed on the lin-
ker-quinolone unit.
A
more detailed synthesis of macrolones 26–34 will be
Synthesis of intermediary dicarboxylic acids 23–25 were next
target for the final linkage of N1-substituted quinolonic acid deriv-
atives 10–12 to the selected macrolide scaffolds.
Construction of the linker in these compounds started from the
commercially available O-benzyl-protected 1,2-ethanediol, which
described hereafter. Many improvements and modifications of
the separate steps on the route to these target compounds cumu-
lated with synthetic protocol for the preparation of the lead
compound 26.

M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
6549
O
O
O
I
I
COOH
F
I
COOCl
(ii)
(i)
O
(iii)
O
+
F
N
O
N
F
2
1
O
F
O
O
O
O
I
I
I
(iv)
O
(v)
OH
O
NH
R
N
R
N
R
10 R=ethyl
11 R=cyclopropyl
5
6
R=ethyl
R=cyclopropyl
3
4
R=ethyl
R=cyclopropyl
Scheme 2. Synthesis of 1-N-ethyl (10) and 1-N-cyclopropyl (11) 6-iodoquinolonic acid. Reagents and conditions: (i) (COCl)2, THF/DCM (1:50), rt, 4 h; (ii) TEA, toluene, 90 °C,
6 h; (iii) R-NH2, THF/EtOH (1:1.5), rt, 3 h; (iv) K2CO3, DMF, 140 °C, 1 h; (v) NaOH, THF/H2O (1:1), 80 °C, 3 h.
(iii)
(iv)
(i)
(ii)
7
8
12
9
Scheme 3. Synthesis of 1-N-allyl-6-iodoquinolonic acid (12). Reagents and conditions: (i) 2-ethoxymethylene-malonic acid diethyl ester, 105 °C, 10 min; (ii) polyphosphoric
acid, 110 °C, 2 h; (iii) allyl bromide, K2CO3, DMF, 65 °C, 1 h; (iv) NaOH, THF/H2O (1:1), 80 °C, 3 h.
was quantitatively propargylated to give 13, and submitted to
Sonogashira-type27 C–C coupling with 6-iodoquinolone-3-carbox-
ylic acids 10–12, Scheme 4. This reaction proved more effective
with acids 10–12 than with the esters analogue 5, 6 and 9, and
was completed on 20 g scale with 78–82% yield. Initially the isola-
tion required column chromatography on silica-gel with an eluent
system CH2Cl2/MeOH/concd NH3 (90:15:1.5), with yield of about
50%. The isolation was substantially improved by addition of water
to the reaction mixture and controlled acid–base extraction.
Extraction of the product by di-iso-propyl ether at pH 12 elimi-
nates PPh3O, while excess of carboxylic acids 14–16 remains in
the basic aqueous solution. Treatment of this solution with char-
coal, followed by filtering through Celite eliminates traces of the
metal. Upon acidification to pH 6 pure 14–16 were isolated.
Deprotection of terminal hydroxyl group by hydrogenation
with concomitant reduction of the triple bond in 14–16 was com-
pleted by Pd/C/H2 in MeOH/DCM affording 17–19 in 85–91% yield.
Michael addition of alcohols 17–19 to acrylonitrile was sub-
stantially improved when DBU in acrylonitrile as the solvent was
replaced by 10% aq NaOH. Specifically, polymerization of acryloni-
trile was suppressed by lowering the reaction temperature from
80 °C with DBU to 5–10 °C with 10% aq NaOH, and diminishing mo-
lar ratio of acrylonitrile from ca. 60:1 to ca. 5:1. Under these con-
ditions the retro-Michael reaction decreased from approx. 20% to
less than 4%. The intermediates 20–22 were selectively precipi-
tated from aqueous solution at pH 6.3 and gave products with high
purity. Hydrolysis of nitriles generally requires harsh conditions
and often a mixture of amide and carboxylic acid is formed.28,29
Since compounds 20–22 comprise b-keto carboxylic acid unit, un-
der such conditions 3-carboxylic group can decarboxylate. In the
first experiments intensive decarboxylation was actually observed,
accompanied by the retro-Michael reaction of b-cyanoethoxy unit
back to the alcohols 17–19 and acrylonitrile. Proper modification of
hydrolytic conditions avoided both difficulties. In the first step
hydrolysis of cyano to amido group was completed in concd sulfu-
ric acid at 20–22 °C over 24 h. HPLC control revealed complete and
selective transformation of compounds 20–22 into amide, which
was not isolated. This two-step one-pot procedure was success-
fully completed by water addition of concd H2SO4/H2O to the ratio
1.5:1. Hydrolysis was completed at 70 °C over 24 h. Diacids 23–25
was achieved in high over all yield, while retro-Michael reaction
was completely prevented.
The most challenging aspect was the requirement for site-selec-
tive acylation at 400-OH of 20-O-protected macrolide by dicarboxylic
acid 23–25 in the last step of macrolone synthesis. The three hy-
droxyl groups in 20-O-protected macrolide and two carboxylic
groups in 23 imply the risk of the formation of six structurally iso-
meric monoesters, beside possible formation of some diesters.
Extensive experimentation with 23 has shown that less acidic
3-carboxylic group is not activated with EDAC ꢁ HCl coupling re-
agent as effectively as the terminal one. The pKa of b-keto-carbox-
ylic group in 23 is estimated to 5.5–6.5,30,31 respectively, while pKa
of b-alkoxy-carboxylic group is found between 3 and 3.5.28 EDAC is
known to activate the carboxylic function by a mechanism that in-
cludes formation of an acyl-guanidinium intermediate.32,33 In our
case selective activation of the terminal, more acidic carboxylic
group in 23 resulted in the formation of guanidinium-like species
which enter ulterior activation by DMAP before acyl transfer to
C400-OH group, Scheme 5.
The elaborated synthetic route to compound 26, was applied
with minor modifications to prepare target macrolones 27 and
28 with azithromycin as the scaffold and macrolones 29–34 with
other macrolide scaffolds, Table 1.
The mass spectra for all new compounds were in agreement
with the proposed structure. These results were further confirmed
by the NMR spectra of all compounds, which revealed two sets of
signals in the aliphatic and aromatic regions arising from the pro-
tons and carbons of macrolide and quinolone moieties, respectively,
as well as signals reflecting the number of methylene groups in the
linker.
The newly formed ester carbonyl resonates in the 13C NMR
spectrum around 170 ppm in 26–34. The significant deshielding

6550
M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
O
O
(i)
O
OH
I
O
O
OH
Br
+
+
13
N
R
10 R=ethyl
11 R=cyclopropyl
12 R=allyl
O
O
O
O
O
(ii)
HO
O
OH
O
OH
(iii)
N
R
N
R
17 R=ethyl
14 R=ethyl
18 R=cyclopropyl
19 R=propyl
15 R=cyclopropyl
16 R=allyl
O
O
O
O
O
HO
O
O
OH
OH
O
N
(iv)
O
N
R
(v) (vi)
N
R
23 R=ethyl
24 R=cyclopropyl
25 R=propyl
20 R=ethyl
21 R=cyclopropyl
22 R=propyl
Scheme 4. Synthesis of dicarboxylic acid derivatives 23–25. Reagents and conditions: (i) THF, NaH, 0–35 °C, 24 h; (ii) CH3CN/TEA (1:1), CuI, Pd(PPh3)2Cl2, 50 °C, 24 h; (iii)
MeOH/DCM (3:1), Pd/C/H2, rt, 5 bar, 24 h; (iv) CH3CN, 10%NaOH, 15–20 °C, 2 h; (v) H2SO4 concd, 20–22 °C, 24 h; (vi) H2O, 75 °C, 24 h.
+
N
N
H
N
H
H
bifuracated
H-bonding
O
O
O
O
N
N
O
O
O
H+
N +
N
+
N
N
N
N
C
OH-
activated acyl species I
N
N
N
N
H
H
+
N
H
O
O
N
O
O
O
O
N
activated acyl species II
Scheme 5. Mechanism of site-selective activation of dicarboxylic acid derivatives by EDAC.
of the 400-H signal in the 1H NMR, together with its long-range
coupling to the new ester carbonyl signal, provided evidence that
the esterification occurred at the 400-OH group of the macrolide
scaffold.
All target macrolones proved chemically stable and the lead
compound 26 tended to crystallize from some organic solvents
as solvates. High-quality crystals were obtained from di-iso-propyl
ether. Single-crystal X-ray analysis was completed and packing of

M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
6551
Table 1
Antibacterial activity of macrolones 26–34, given as minimum inhibitory concentration (MIC) in units of
l
g/mL
Phenotype
Organism & strain
S. aureus S. pneumoniae S. pyogenes S. aureus S. pneumoniae S. pyogenes S. pneumoniae S. pyogenes
S. aureus
ATCC13709 PK1
eryS
H. influenzae M. catarrhalis
Ci137
M
2 Finland
M
90256
iMLS
134 GR M
iMcLS
Finland 11 58 Spain
166 GR-Micro ATCC 49247 ATCC 23246
cMLS
M
iMLS
cMLS
AZM
TEL
26
27
28
29
30
31
32
0.5
>64
60.125
0.25
8
0.25
60.125
8
0.5
>64
60.125
0.25
60.125
60.125
60.125
4
4
0.25
8
>64
0.25
0.25
0.25
60.125
60.125
60.125
0.25
60.125
0.5
>64
>64
0.25
>64
16
60.125
0.5
0.25
0.5
60.125
1
60.125
1
1
2
1
2
1
1
2
2
1
4
1
60.125
60.125
60.125
0.5
0.5
0.25
-
60.125
60.125
1
60.125
60.125
60.125
60.125
60.125
1
60.125
60.125
0.5
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
60.125
1
60.125
1
60.125
60.125 60.125
60.125 60.125
60.125 60.125
1
60.125
60.125
60.125
60.125
0.25
0.5
1
33
34
60.125
60.125 60.125
60.125
60.125
60.125
1
AZM = azithromycin; TEL = telithromycin; iMLS = inducible resistance to macrolide, licosamide and streptogramin (MLS) antibiotics; iMcL = inducible resistance to macrolide
and constitutive resistance to lincosamide antibiotics; cMLS = constitutive MLS resistance; M = efflux mediated macrolide resistance.
Figure 2. PyMOL39 stereo drawing of 26. Hydrogen atoms are omitted for clarity.
Figure 3. The crystal packing of 26 projected down a axis. Hydrogen atoms have been omitted for clarity. Molecules are colored according to symmetry equivalence.
the molecules in the unit cell is shown in Figures 2 and 3. The
asymmetric unit (space group P1) contains two symmetrically
independent molecules of compound 26 and six molecules of di-
iso-propyl ether. There are only very week hydrogen bonds of the
type C–Hꢂ ꢂ ꢂO between molecules. These very week intermolecular
bonds are the reason for quick loss of the solvent molecules and
fast decomposition of the crystals.
2.3. Biological results
Antibacterial activity of compounds 26–34 was determined by a
standard broth microdilution method.34 Azithromycin and telithro-
mycin were used as controls and results are shown in Table 1 and are
expressed as minimum inhibitory concentrations (MICs) in units of
lg/mL. The organisms tested represented relevant Gram-positive (S.

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M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
pneumoniae, Streptococcus pyogenes and Staphylococcus aureus) and
Gram-negative (Haemophilus influenzae and Moraxella catarrhalis)
respiratory tract pathogens, and were either sensitive or resistant
to macrolide antibiotics. Macrolide resistance was due to two major
mechanisms—production of efflux pumps (M), or ribosome modifi-
cation by methylation. Methylase expression was inducible (iMLS)
or constitutive (cMLS).
required to reach complete site-selective acylation at 400-OH of
cladinose unit. Besides, only one of the two carboxylic groups of
quinolone-linker precursors was activated by EDAC ꢁ HCl.
All novel compounds exhibited improved antibacterial activity
against the tested macrolide resistant bacteria (superior to teli-
thromycin), and selected compounds had good potency against
the wide panel of recent clinical isolates of H. influenzae (compara-
ble to azithromycin). This new class of macrolide derivatives repre-
sents a promising approach for the delivery of novel antimicrobials
with improved spectrum of activity for the treatment of infections
caused by respiratory pathogens.
All analogues showed MIC 6 0.125
lg/mL against erythromycin
sensitive S. pneumoniae and S. pyogenes.
Azithromycin derivatives 26–28 had very high antibacterial
potency against all tested organisms. It is noteworthy that S. pyog-
enes strains are fully covered, regardless the resistance mechanism,
and activity against cMLS S. pyogenes clearly exceeds that of mar-
keted macrolide or ketolide antibiotics, as telithromycin has MIC
Within the investigates series, compound 26 exhibited the most
promising biological profile. Further experiments in animal models
would indicate the potential use of these compounds as new anti-
bacterial agents.
16 lg/mL and 26–28 of 6 0.125 lg/mL. As 26 was the most potent
compound, in order to investigate influence of a macrolide scaffold,
dicarboxylic acid 23 was attached to six different macrolides. Com-
pounds 29, 32 and 34 retained high overall potency. However,
activity of compounds 30, 31 and 33 against iMLS S. aureus strain
was impaired, and potency against cMLS S. pneumoniae and
S. pyogenes strains was reduced for 31 and 33. All derivatives had
good activity against H. influenzae, comparable to that of azithro-
mycin. Therefore, 26–28 and 34 were additionally tested against
the wide range (n = 43) of recent clinical isolates of H. influenzae
and summary of the obtained MIC data is shown in Table 2, given
as MIC50 (concentration needed to inhibit growth 50% of tested iso-
lates), MIC90 (concentration needed to inhibit growth 90% of tested
isolates) and range of MIC values.
Pharmacokinetic properties of two selected compounds (26 and
27) where determined in rats and are summarized in Table 3. Both
compounds are characterized by moderate blood clearance (35%
and 43% of liver blood flow, respectively), large volume of distribu-
tion (10.4 and 10.5 L/kg, respectively) and long half-life (9.4 and
6.7 h, respectively) supporting once daily dosing. The mean oral
bioavailability from a solution formulation was 22% for compound
26 and 16% for compound 27.
4. Experimental
4.1. General
All commercial reagents (Merck, Sigma–Aldrich) were used as
provided unless otherwise indicated, and all solvents are of high
purity unless otherwise noted.
1D and 2D NMR spectra (1H, APT, COSY, HSQC, HMBC) were re-
corded at 25 °C in DMSO-d6 with TMS as the internal standard on
Bruker Avance DRX500 spectrometer using QNI probe and Bruker
Avance DPX300 spectrometer using dual 1H/13C probe.
Mass spectra were obtained on a Waters Micromass ZQmass
spectrometer for ES+-MS. Electrospray positive ion mass spectra
were acquired using a Micromass Q-Tof2 hybrid quadrupole time-
of-flight mass spectrometer, equipped with a Z-spray interface, over
a mass range of 100–2000 Da, with a scan time of 1.5 s and an inter-
scan delay of 0.1 s in a continuum mode. Reserpine was used as the
external mass calibrant lock mass ([M+H]+ = 609.2812 Da). The ele-
mental composition was calculated using a MassLynx v4.1 for the
[M+H]+ and the mass error quoted within 5 ppm range.
All 20-O-acetyl-macrolides were prepared according to pub-
lished literature.15
Considering these results, we can conclude that the current lead
molecule 26 has the most favorable antibacterial profile, other new
macrolones showing comparable or lower activity. Further in vivo
experiments with these compounds could confirm their improved
in vitro antibacterial activity. Additional studies of correlation of
their physico-chemical and biological properties are envisaged.
All final compounds were isolated as amorphous solid except by
compound 26.
Reaction flow and purity of products were monitored by thin
layer chromatography (TLC) on Merck Kieselgel 60 (230–400
mesh) using specifically solvent systems indicated in the protocol.
I2, UV-light (254 nm) and H2SO4, followed by heating to >120 °C
were used for detection.
3. Conclusion
We have presented the synthesis of macrolones 26–34 and have
shown that only protection of the 20-OH group of macrolide unit is
4.2. Biological methods and materials
4.2.1. In vitro antibacterial activity assays
Table 2
Antimicrobial profiling of selected compounds against H. influenzae (n = 43)
Minimum inhibitory concentrations (MICs) were determined by
the broth microdilution method as described by CLSI guidelines,34
except that for Streptococcus medium, lysed blood was substituted
with 5% horse serum. Dilutions of tested compounds were pre-
pared using TECAN Genesis 150. Bacteria were grown on appropri-
ate agar plates (by Becton Dickinson, USA)—Columbia agar with 5%
sheep blood for Streptococci, and M. catarrhalis, chocolate agar for
H. influenzae and Mueller-Hinton agar for Staphylococci.
26
27
28
34
AZM
MIC 50
MIC 90
Range
1
2
1
2
1
2
2
4
1–8
1
2
0.5–2
0.5–2
0.5–2
0.25–2
MIC in units of lg/mL.
AZM = azithromycin.
Table 3
4.2.2. In vivo rat pharmacokinetic studies
Summary of the pharmacokinetics of 26 and 27 in rats
PK po/iv crossover studies were performed in male Sprague-
Dawley rats. Animals were dosed at target doses of 30 mg/kg orally
(po) and 5 mg/kg intravenously (iv). Compounds were adminis-
tered as a solution in 1.0% DMSO, 20% Encapsin (w/v) and saline
(iv) or water (po), pH 3.5–4.0. Animals where fed for iv and fasted
for po administration. Blood was collected at several timepoints up
to 1800 min after administration, hemolyzed with water in a 1:1
CLa % LBF
Vdss L/kg
T1/2 hours
Oral Fc
%
b
Compound
26
27
35
43
10.4
10.5
9.4
6.7
22
16
a
b
c
CL: moderate blood clearance.
Vdss: volume of distribution.
Oral F: oral bioavailability.

M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
6553
ratio and stored at ꢃ20 °C until analysis. Samples were extracted
by deproteinization and the supernatants were analyzed for parent
compound by LC/MS.
1H NMR (500 MHz, DMSO) d: 7.77–7.71 (m, 1H), 7.60 (br s, 1H),
7.55 (dd, 1H), 7.02 (dd, 1H), 3.89 (q, 2H), 3.50 (q, 2H), 1.21 (t, 3H),
0.94 (t, 3H).
13C NMR (125 MHz, DMSO) d: 186.9, 166.0, 159.8, 159.5, 156.3,
138.7, 138.6, 136.76, 136.3, 136.2, 134.0, 133.8, 117.8, 117.6, 99.4,
87.5, 58.8, 44.5, 15.7, 13.7.
4.3. Crystal structure elucidation
Single crystal structure determination was done in Department
of Chemistry, Faculty of Science, University of Zagreb, Croatia.
Data were collected on the Oxford Diffraction Xcalibur diffractom-
eter with Saphire 3 CCD detector. Data reduction was done with
the CRYSALIS program package.35 Program package WINGX36was used
for structure solution refinement. The structure was solved by di-
rect methods (SIR92)37 and refined by least squares method based
on F2 by using all reflexes. It was refined in overlapping blocks
(SHELXL-97).38 All non-hydrogen atoms, except for the solvate
molecule atoms were refined anisotropically. Hydrogen atoms
were generated on geometrical basis (C–H = 0.95 Å, Uiso(H) = 1.2
Ueq of the corresponding C atom; Uiso(H) = 1.5 Ueq(C) for the
methyl group. The reading model was used for refinement of H
atoms.
4.4.4. Ethyl 3-(cyclopropylamino)-2-[(2-fluoro-5-iodophenyl)-
carbonyl]-2-propenoate (4)
The title compound 4 as a yellow solid was prepared in 72%
yield starting from compound 2 (23.8 g, 0.06 mol) and cyclopropyl-
amine (6.7 mL, 0.096 mol) according to the procedure used to pre-
pare compound 3.
MS (ES) m/z 403.86 [M+H]+.
This compound was used in the next step without further
purification.
4.4.5. Ethyl 1-ethyl-6-iodo-4-oxo-1,4-dihydro-3-quinolinecarb-
oxylate (5)
Compound 3 (130.79 g, 0.33 mol) was dissolved in dry DMF
(787 mL) at rt K2CO3 (94.42 g, 0.67 mol) was added and the mix-
ture was stirred at 140 °C for 1 h. The reaction was monitored by
TLC in a solvent system EtOAc/cyclohexane = 1:1. The reaction
mixture was filtered and the filtrate evaporated under reduced
pressure. To the oily residue THF (300 mL) was added under stir-
ring until a precipitate was formed. The precipitate was filtered
off yielding 93.35 g (76%) of the title compound 5 as a yellow solid.
4.4. Synthetic procedures
4.4.1. 2-Fluoro-5-iodo-benzyl chloride (1)
Into a solution of 2-fluoro-5-iodo-benzoic acid (50 g, 0.188 mol)
in DCM (250 mL) and DMF (3 mL) at rt oxalyl chloride (19.9 mL,
0.223 mol) was added dropwise. Reaction mixture was stirred at
rt for 4 h. The reaction was monitored by TLC in a solvent system
EtOAc/n-hexane = 1:1. The excess of oxalyl chloride was removed
by evaporation under reduced pressure to give the title compound
1 as oily residue (51.93 g, 97%). The residue was used in the next
step as is.
HRMS (ES) calcd for
372.0096.
C
14H14INO3 [M+H]+ 372.0097 found
1H NMR (500 MHz, DMSO) d: 8.71 (s, 1H), 8.50 (d, 1H), 8.05 (dd,
1H), 7.64 (d, 1H), 4.39 (q, 2H), 4.23 (q, 2H), 1.36 (t, 3H), 1.29 (t, 3H).
13C NMR (125 MHz, DMSO) d: 171.3, 164.3, 149.2, 140.6, 138.0,
134.8, 129.9, 119.6, 110.5, 90.0, 59.7, 47.9, 14.2, 14.1.
4.4.2. Ethyl 3-(dimethylamino)-2-[(2-fluoro-5-iodophenyl)car-
bonyl]-2-propenoate (2)
4.4.6. Ethyl 1-cyclopropyl-6-iodo-4-oxo-1,4-dihydro-3-quino-
linecarboxylate (6)
The title compound 6 as a yellow solid was prepared in 60%
yield starting from compound 4 according to the procedure used
to prepare compound 5.
A solution of compound 1 (51.93 g, 0.18 mol) in dry toluene
(430 mL) was added to the stirring solution of ethyl 3-(dimethyl-
amino)acrylate (26.9 g, 0.19 mol) in dry TEA (33.9 mL, 0.24 mol)
at rt The solution was stirred at 90 °C for 6 h. The reaction was
monitored by TLC in a solvent system EtOAc/n-hexane = 1:1. After
cooling to rt insoluble material was filtered off and the solvent was
evaporated to dryness. The residue was triturated in n-hexane
(200 mL), the precipitate was collected on filter, dried in vacuum
and then precipitated from EtOH/Et2O = 1:2, affording 47.17 g
(67%) of the title compound 2 as yellow solid.
HRMS (ES) calcd for
384.0105.
C
15H14INO3 [M+H]+ 384.0097 found
1H NMR (500 MHz, DMSO) d: 8.48 (s, 1H), 8.44 (d, 1H), 8.11 (dd,
1H), 7.87 (d, 1H), 4.22 (q, 2H), 3.64 (m, 1H), 1.28 (t, 3H), 1.23 (m,
2H), 1.11 (m, 2H).
13C NMR (125 MHz, DMSO) d: 171.4, 164.1, 148.6, 140.6, 139.9,
134.4, 129.2, 120.0, 110.3, 90.3, 59.8, 34.63, 14.2, 7.5.
HRMS (ES) calcd for C14H15FINO3 [M+H]+ 392.0159 found
392.0137.
1H NMR (500 MHz, DMSO) d: 7.75–7.78 (ov, 2H), 7.66 (dd, 1H),
7.03 (dd, 1H), 3.87 (q, 2H), 2.78 (s, 6H), 0.88 (t, 3H).
13C NMR (125 MHz, DMSO) d: 184.3, 166.6, 159.8, 158.0, 157.8,
139.6, 132.9, 132.8, 137.3, 118.1, 117.9, 101.1, 87.7, 59.0, 42.1,
13.6.
4.4.7. Diethyl {[(4-iodophenyl)amino]methylidene}propane-
dioate (7)
Iodoaniline (10.00 g, 0.046 mol) and 2-ethoxymethylene-
malonic acid diethyl ester (9.22 mL, 0.046 mol) were stirred at
105 °C for 10 min and raw product precipitated from the reaction
mixture. The mixture was cooled to rt and n-hexane (100 mL)
was added. The suspension was filtered yielding 16.37 g (92%) of
the title compound 7 as a yellow solid.
4.4.3. Ethyl 3-(ethylamino)-2-[(2-fluoro-5-iodophenyl)car-
bonyl]-2-propenoate (3)
A stirred solution of compound 2 (95.43 g, 0.24 mol) in a mix-
ture EtOH/THF = 1.5:1 (298 mL) was cooled in an ice-bath to
5–10 °C. Into it cold ethylamine (2 M solution in THF, 242.85 mL,
0.49 mol) was added dropwise. The reaction was stirred at rt for
3 h and monitored by TLC in a solvent system EtOAc/cyclohex-
ane = 1:1. Into the mixture n-hexane was added. The resulting pre-
cipitate was filtered off, washed with n-hexane and dried affording
75.26 g (79%) of the title compound 3 as a yellow solid. The product
was used in the next step without further purification.
HRMS (ES) calcd for C14H15FINO3 [M+H]+ 392.0159 found
392.0165.
HRMS (ES) calcd for
390.0215.
C
14H16INO4 [M+H]+ 390.0202 found
1H NMR (500 MHz, DMSO) d: 8.94 (s, 1H), 8.56 (d, 1H), 8.11 (dd,
1H), 7.29 (d, 1H), 4.23 (q, 2H), 4.16 (q, 2H), 1.27 (t, 3H), 1.24 (t, 3H).
13C NMR (125 MHz, DMSO) d: 166.6, 164.7, 153.6, 150.0, 147.0,
146.6, 115.1, 95.0, 86.84, 59.9, 59.7, 14.3, 14.1.
4.4.8. Ethyl 6-iodo-4-oxo-1,4-dihydro-3-quinolinecarboxylate (8)
A mixture of compound 7 (16.00 g, 0.04 mol) and polyphospho-
ric acid (45 g) was stirred at 110 °C for 2 h. The reaction was mon-

6554
M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
itored by TLC in a solvent system EtOAc/cyclohexane = 1:1. Water
(1 L) was added, the precipitate filtered off and washed with water.
The cake was suspended in DMF (15 mL), stirred for 10 min, fil-
tered off and dried at 60 °C in vacuum yielding 8.5 g (62%) of the
title compound 8.
1H NMR (300 MHz, DMSO) d: 9.08 (s, 1H), 8.62 (d, 1H), 8.21 (dd,
1H), 7.73 (d, 1H), 6.06 (m, 1H), 5.28 (dd, 1H), 5.22 (d, 2H), 5.16 (dd,
1H).
13C NMR (75 MHz, DMSO) d: 176.9, 166.0, 150.4, 142.4, 139.2,
134.5, 132.6, 127.5, 121.2, 118.9, 108.8, 92.4, 55.8.
HRMS (ES) calcd for
343.9807.
C
12H10INO3 [M+H]+ 343.9784 found
4.4.13. ({[2-(2-Propyn-1-yloxy)ethyl]oxy}methyl)benzene (13)
2-Benzyloxyethanol (107 g, 5 mL, 0.7 mol) was dissolved in THF
(500 mL), stirred under N2 atmosphere for 20 min and then cooled
to 0–5 °C. Into this solution NaH (33.8 g, 60% in min. oil, 0.84 mol)
was added in small portions. The reaction mixture was stirred at
35 °C for 30 min and then cooled to 0–5 °C. Propargyl bromide
(80% solution in toluene, 100.4 g, 94 mL, 0.84 mol) was added
dropwise and the stirring continued at rt under N2 atmosphere
overnight. The reaction was monitored by TLC in a system EtOAc/
n-hexane = 1:4. THF was evaporated under reduced pressure. To
the residue water (150 mL) was added and the mixture extracted
with EtOAc (3 ꢁ 100 mL). Organic phase was dried over Na2SO4
and evaporated under reduced pressure yielding 131.12 g (98%)
of the title compound 13 as yellow oil. The product was used in
the next step without further purification.
1H NMR (500 MHz, DMSO) d: 8.57 (s, 1H), 8.40 (d, 1H), 7.97 (dd,
1H), 7.43 (d, 1H), 4.22 (q, 2H), 1.27 (t, 3H).
13C NMR (75 MHz, DMSO) d: 171.5, 164.3, 149.8, 140.6, 134.7,
129.7, 120.3, 109.3, 90.4, 60.0, 14.2.
4.4.9. Ethyl 6-iodo-4-oxo-1-(2-propen-1-yl)-1,4-dihydro-3-
quinolinecarboxylate (9)
To the solution of compound 8 (4 g, 0.012 mol) in DMF (40 mL)
K2CO3 (3.22 g, 0.023 mol) and allyl bromide (1.48 mL, 0.018 mol)
were added. The mixture was stirred at 65 °C for 1 h. The reaction
was monitored by TLC in a solvent system EtOAc/cyclohex-
ane = 1:1. Reaction mixture was diluted with water (50 mL) and
the resulting suspension filtered. The cake was dried at 50 °C in
vacuum for 4 h yielding 3.30 g (74%) of the title compound 9 as a
yellowish solid.
MS (ES) m/z 190.92 [M+H]+.
HRMS (ES) calcd for
384.0099.
C
15H14INO3 [M+H]+ 384.0097 found
1H NMR (300 MHz, DMSO) d: 7.33–7.35 (m, 5H), 4.57 (s, 2H),
4.21 (d, 2H), 3.72 (m, 2H), 3.65 (m, 2H), 2.43 (t, 1H).
1H NMR (300 MHz, DMSO) d: 8.71 (s, 1H), 8.50 (d, 1H), 8.04 (dd,
1H), 7.52 (d, 1H), 6.04 (m, 1H), 5.25 (dd, 1H), 5.11 (dd, 1H), 5.03 (d,
2H), 4.23 (q, 2H), 1.29 (t, 3H).
13C NMR (75 MHz, DMSO) d: 138.4, 128.5, 128.4, 127.8, 127.7,
73.3, 69.2, 61.2, 58.5, 19.8.
13C NMR (75 MHz, DMSO) d: 171.7, 164.4, 149.9, 140.7, 138.7,
134.9, 132.6, 129.9, 120.3, 118.1, 110.8, 90.4, 60.0, 54.7, 14.4.
4.4.14. 1-Ethyl-4-oxo-6-[3-({2-[(phenylmethyl)oxy]ethyl}oxy)-
1-propyn-1-yl]-1,4-dihydro-3-quinolinecarboxylic acid (14)
Into a solution of compound 10 (21 g, 0.06 mol) in a mixture
MeCN/TEA = 1:1 (568 mL) at rt CuI (1.17 mg, 0.0061 mol) was
added. The mixture was stirred for 20 min at rt and then warmed
to 50 °C. Into that solution (2-prop-2-ynyloxy-ethoxymethyl)-ben-
zene (13) (29 g, 0.15 mol) and Pd(OPh3)2Cl2 (2.15 g, 0.003 mol) were
added and the mixture was stirred at 50 °C overnight. The reaction
4.4.10. 1-Ethyl-6-iodo-4-oxo-1,4-dihydro-3-quinolinecarboxylic
acid (10)
Compound
5 (93.35 g, 0.25 mol) and NaOH (46.26 mg,
1.16 mmol) were dissolved in a mixture THF/H2O =1:1 (900 mL).
Reaction mixture was stirred for 3 h at 80 °C. The reaction was mon-
itored by TLC in a solvent system DCM/MeOH/NH4OH = 90:15:
1.5. THF was evaporated under reduced pressure. The residual solu-
tion was diluted with water and pH adjusted to 4.5 using 6 M aq HCl.
The resulting suspension was filtered, the cake washed with water
anddriedat50 °Cfor4 h yielding84.19 g (98%)ofthetitlecompound
10 as a white solid.
was monitored by TLC in
a solvent system DCM/MeOH/
NH4OH = 90:15:1.5. After 24 h starting compound was consumed.
To the reaction mixture water (800 mL) and i-Pr2O (600 mL) were
added, pH was adjusted to 12 using 40% aq NaOH and the layers were
separated. Aqueous layer was extracted with i-Pr2O (2 ꢁ 400 mL).
Aqueous layer was treated with charcoal at 80 °C over 10 min and
filtered over Celite. After cooling to 25–35 °C pH was adjusted to 6
using 5% aq HCl. The resulting suspension was filtered and the cake
dried at 50 °C in vacuum yielding 20.26 g (82%) of the title com-
pound 14 as yellow solid.
HRMS (ES) calcd for
343.9774.
C
12H10INO3 [M+H]+ 343.9784 found
1H NMR (300 MHz, DMSO) d: 9.06 (s, 1H), 8.57 (d, 1H), 8.21 (dd,
1H), 7.84 (d, 1H), 4.58 (q, 2H), 1.42 (t, 3H).
13C NMR (75 MHz, DMSO) d: 176.4, 165.8, 149.6, 142.3, 138.5,
134.3, 127.2, 120.4, 108.4, 92.1, 49.2, 14.6.
HRMS (ES) calcd for
406.1639.
C
24H23NO5 [M+H]+ 406.1654 found
1H NMR (300 MHz, CDCl3) d: 8.78 (s, 1H), 8.57 (d, 1H), 7.83 (dd,
1H), 7.57 (d, 1H), 7.38–7.27 (m, 5H), 4.60 (s, 2H), 4.48 (s, 2H), 4.39
(q, 2H), 3.81 (m, 2H), 3.71 (m, 2H), 1.59 (t, 3H).
4.4.11. 1-Cyclopropyl-6-iodo-4-oxo-1,4-dihydro-3-quinoline-
carboxylic acid (11)
The title compound 11 as a white solid was prepared in 96%
yield starting from compound 6, according to the procedure used
to prepare compound 10.
13C NMR (75 MHz, CDCl3) d: 177.8, 166.8, 148.1, 138.5, 138.0,
136.7, 130.7, 128.4–127.7, 126.5, 121.0, 116.5, 109.4, 87.9, 84.3,
73.4, 69.5, 69.3, 59.1, 49.8, 14.9.
HRMS (ES) calcd for
355.9777.
C
13H10INO3 [M+H]+ 355.9784 found
1H NMR (300 MHz, DMSO) d: 8.75 (s, 1H), 8.58 (d, 1H), 8.28 (dd,
1H), 8.07 (d, 1H), 3.84 (m, 1H), 1.30 (m, 2H), 1.19 (m, 2H).
13C NMR (75 MHz, DMSO) d: 176.7, 165.6, 149.1, 142.3, 140.6,
133.9, 126.8, 120.8, 108.0, 92.4, 36.0, 7.7.
4.4.15. 1-Cyclopropyl-4-oxo-6-[3-({2-[(phenylmethyl)oxy]ethyl}
oxy)-1-propyn-1-yl]-1,4-dihydro-3-quinolinecarboxylic acid
(15)
The title compound 15 as a yellowish solid was prepared in 78%
yield starting from compound 11 according to the procedure used
to prepare compound 14.
4.4.12. 6-Iodo-4-oxo-1-(2-propen-1-yl)-1,4-dihydro-3-quino-
linecarboxylic acid (12)
HRMS (ES) calcd for
418.1666.
C
25H23NO5 [M+H]+ 418.1654 found
The title compound 12 was prepared in 93% yield starting
from compound 9 according to the procedure used to prepare
compound 10.
1H NMR (300 MHz, DMSO) d: 8.42 (s, 1H), 8.27 (d, 1H), 8.02 (d,
1H), 7.63 (dd, 1H), 7.27 (m, 5H), 4.54–4.52 (ov, 4H), 3.85 (m, 1H),
3.72 (t, 2H), 3.63 (t, 2H), 1.31 (m, 2H), 1.19 (m, 2H).
HRMS(ES)calcdforC13H10INO3 [M+H]+ 355.9784found355.9773.

M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
6555
13C NMR (75 MHz, DMSO) d: 177.1, 165.4, 149.0, 140.6, 138.3,
4.4.20. 6-[3-({2-[(2-Cyanoethyl)oxy]ethyl}oxy)propyl]-1-ethyl-
136.2, 134.4, 127.9–126.6, 125.0, 119.8, 119.2, 107.8, 88.2, 83.9,
71.9, 68.7, 68.9, 58.1, 35.9, 7.5.
4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (20)
Compound 17 (27.32 g, 0.085 mol) was dissolved in 10% aq
NaOH (328 mL, 0.85 mol) and the solution cooled to 5–10 °C.
Acrylonitrile (28.18 mL, 0.43 mol) was added dropwise and the
reaction mixture was stirred at 15–20 °C for 2 h. The reaction
was monitored by TLC in a solvent system DCM/MeOH/concd
NH3 = 90:15:1.5. To the mixture water (250 mL) was added and
pH was adjusted to 6.3 using 6 M aq HCl. The precipitate was fil-
tered off and the cake washed with water yielding 44.51 g of wet
title compound 20 as a yellowish solid. The product was used in
next step without drying.
4.4.16. 4-Oxo-6-[3-({2-[(phenylmethyl)oxy]ethyl}oxy)-1-pro-
pyn-1-yl]-1-(2-propen-1-yl)-1,4-dihydro-3-quinolinecarboxylic
acid (16)
The title compound 16 as a yellowish solid was prepared in 80%
yield starting from compound 12 according to the procedure used
to prepare compound 14.
HRMS (ES) calcd for
418.1651.
C
25H24NO5 [M+H]+ 418.1654 found
1H NMR (300 MHz, CDCl3) d: 8.90 (s, 1H), 8.32 (d, 1H), 7.87 (dd,
1H), 7.50 (d, 1H), 7.36–7.19 (m, 5H), 6.07 (m, 1H), 5.27 (d, 1H), 5.23
(d, 2H), 5.20 (d, 1H), 4.53 (s, 2H), 4.47 (s, 2H), 3.74 (m, 2H), 3.63 (m,
2H).
HRMS (ES) calcd for
373.1757.
C
20H24N2O5 [M+H]+ 373.1763 found
1H NMR (300 MHz, DMSO) d: 8.57 (s, 1H), 8.09 (d, 1H), 7.67 (d,
1H), 7.59 (dd, 1H), 4.34 (q, 2H), 3.62 (t, 2H), 3.58 (m, 2H), 3.52 (m,
2H), 3.42 (t, 2H), 2.76–2.74 (ov, 4H), 1.34 (t, 3H).
13C NMR (75 MHz, CDCl3) d: 177.8, 166.6, 150.4, 138.7, 136.5,
136.3, 132.6, 129.6, 128.9–127.8, 125.9, 121.1, 119.7, 119.0,
108.7, 88.1, 84.8, 72.4, 69.3, 69.2, 58.5, 55.9.
13C NMR (75 MHz, DMSO) d: 177.4, 167.2, 146.7, 137.2, 137.0,
132.6, 127.5, 125.1, 119.2, 116.5, 107.0, 69.6, 69.3, 69.2, 65.2,
47.3, 31.0, 30.7, 18.0, 14.4.
4.4.17. 1-Ethyl-6-{3-[(2-hydroxyethyl)oxy]propyl}-4-oxo-1,4-
dihydro-3-quinolinecarboxylic acid (17)
4.4.21. 6-[3-({2-[(2-Cyanoethyl)oxy]ethyl}oxy)propyl]-1-cyclo-
propyl-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (21)
The title compound 21 (1.83 g, wet) as a yellowish solid was
prepared from starting compound 18 (1.34 g, 0.004 mol) according
to the procedure used to prepare compound 20.
Hydrogenation of compound 14 (10 g, 0.025 mol) was per-
formed in a mixture MeOH/DCM = 3:1 (120 mL) in the presence
of 10% Pd/C (2 g, 20 wt %) at 5 bar pressure for 24 h. The reaction
was monitored by TLC in a solvent system DCM/MeOH/NH4OH =
90:15:1.5. After filtration of the catalyst, the solution was concen-
trated to dryness under reduced pressure yielding 6.9 g (87%) of
the title compound 17 as a yellow solid. The product was used in
the next step without further purification.
HRMS (ES) calcd for
385.1744.
C
21H24N2O5 [M+H]+ 385.1763 found
1H NMR (300 MHz, DMSO) d: 8.74 (s, 1H), 8.23 (d, 1H), 8.17 (d,
1H), 7.89 (dd, 1H), 3.85 (m, 1H), 3.64 (t, 2H), 3.61–3.52 (ov, 4H),
3.45 (t, 2H), 2.86 (t, 2H), 2.77 (t, 2H), 1.87 (m, 2H), 1.33 (m, 2H),
1.21 (m, 2H).
HRMS (ES) calcd for
320.1486.
C
17H21NO5 [M+H]+ 320.1498 found
1H NMR (300 MHz, DMSO) d: 9.02 (s, 1H), 8.19 (s, 1H), 7.98 (d,
1H), 7.84 (dd, 1H), 4.62 (q, 2H), 3.52 (m, 2H), 3.43–3.39 (ov, 4H),
2.86 (t, 2H), 1.88 (m, 2H), 1.43 (t, 3H).
13C NMR (75 MHz, DMSO) d: 178.6, 166.8, 148.8, 141.3, 140.2,
135.7, 125.7, 125.1, 120.5, 119.3, 107.9, 70.4, 70.2, 70.0, 66.1,
36.6, 31.9, 31.5, 18.9, 8.3.
13C NMR (75 MHz, DMSO) d: 177.6, 166.3, 148.7, 140.5, 137.5,
135.1, 125.7, 124.7, 118.2, 107.5, 72.2, 69.4, 60.4, 49.1, 31.2, 30.8,
14.8.
4.4.22. 6-[3-({2-[(2-Cyanoethyl)oxy]ethyl}oxy)propyl]-4-oxo-1-
propyl-1,4-dihydro-3-quinolinecarboxylic acid (22)
The title compound 22 (0.90 g, wet) as a yellowish solid was
prepared from starting compound 19 (0.73 g, 0.0023 mol) accord-
ing to the procedure used to prepare compound 20.
4.4.18. 1-Cyclopropyl-6-{3-[(2-hydroxyethyl)oxy]propyl}-4-oxo-
1,4-dihydro-3-quinolinecarboxylic acid (18)
The title compound 18 as a yellowish solid was prepared in 91%
yield starting from compound 15 according to the procedure used
to prepare compound 17.
HRMS (ES) calcd for
387.1916.
C
21H27N2O5 [M+H]+ 387.1920 found
1H NMR (300 MHz, DMSO) d: 8.84 (s, 1H), 8.16 (d, 1H), 7.89 (d,
1H), 7.75 (dd, 1H), 4.44 (t, 2H), 3.63 (t, 2H), 3.56 (m, 2H), 3.51 (m,
2H), 3.42 (t, 2H), 2.81 (t, 2H), 2.78 (t, 2H), 1.89 (m, 2H), 1.80 (m,
2H), 0.91 (t, 3H).
HRMS (ES) calcd for
332.1482.
C
18H21NO5 [M+H]+ 332.1498 found
1H NMR (300 MHz, DMSO) d: 8.72 (s, 1H), 8.22 (dd, 1H), 8.16 (d,
1H), 7.88 (dd, 1H), 3.83 (m, 1H), 3.52 (t, 2H), 3.43–3.39 (ov, 4H),
2.85 (t, 2H), 1.87 (m, 2H), 1.32 (m, 2H), 1.19 (m, 2H).
13C NMR (75 MHz, DMSO) d: 178.2, 166.4, 148.4, 141.0, 139.8,
135.3, 125.3, 124.7, 118.9, 107.5, 72.5, 69.6, 60.6, 36.3, 31.5, 31.2,
7.9.
13C NMR (75 MHz, DMSO) d: 177.1, 166.8, 148.6, 139.0, 137.9,
134.5, 126.6, 125.2, 119.7, 118.1, 107.0, 70.0, 69.8, 69.7, 65.7,
54.8, 31.4, 31.1, 22.4, 18.4, 11.0.
4.4.23. 6-[3-({2-[(2-Carboxyethyl)oxy]ethyl}oxy)propyl]-1-
ethyl-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (23)
Into an aqueous solution of H2SO4 (20 wt %, 208 mL) compound
20 (44.51 g, wet precipitate from the previous step) was added in
portions while keeping the temperature at 5–10 °C. The reaction
mixture was stirred at 10–20 °C for 1 h and then at rt for additional
18 h. The mixture was added dropwise into a stirring water
(310 mL) while cooled to 10–20 °C. The resulting mixture was stir-
red at 70–80 °C for 24 h. The reaction was monitored by TLC in a
solvent systems DCM/MeOH/NH4OH = 90:15:1.5 and DCM/
MeOH/MeCN/concd NH3 = 4:4:1:2. The mixture was cooled in an
ice-bath, diluted with water and pH adjusted to 1.3 using 40% aq
NaOH. The resulting suspension was filtered, the cake washed with
water and dried in vacuum at 60 °C yielding 39.06 g of the title
compound 23 as a white solid.
4.4.19. 6-{3-[(2-Hydroxyethyl)oxy]propyl}-4-oxo-1-propyl-1,4-
dihydro-3-quinolinecarboxylic acid (19)
The title compound 19 as a yellowish solid was prepared in 85%
yield starting from compound 16 according to the procedure used
to prepare compound 17.
HRMS (ES) calcd for
334.1667.
C
18H24NO5 [M+H]+ 334.1654 found
1H NMR (500 MHz, DMSO) d: 9.02 (s, 1H), 8.19 (d, 1H), 8.00 (d,
1H), 7.84 (dd, 1H), 4.52 (t, 2H), 3.51 (m, 2H), 3.40 (ov, 4H), 2.84 (t,
2H), 1.88 (m, 2H), 1.83 (m, 2H), 0.91 (t, 3H).
13C NMR (125 MHz, DMSO) d: 177.9, 166.6, 149.3, 140.8, 138.0,
135.4, 125.8, 124.9, 118.7, 107.6, 72.4, 69.6, 60.6, 55.3, 31.4, 31.1,
22.4, 10.9.

6556
M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
HRMS (ES) calcd for C20H25NO7 [M+H]+ 392.1709 found
392.1690.
1H NMR (300 MHz, DMSO) d: 9.02 (s, 1H), 8.19 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 4.60 (q, 2H), 3.62 (t, 2H), 3.52 (m, 2H), 3.49 (m,
2H), 3.41 (t, 2H), 2.83 (t, 2H), 2.45 (t, 2H), 1.87 (m, 2H), 1.43 (t, 3H).
13C NMR (75 MHz, DMSO) d: 177.4, 172.5, 166.1, 148.4, 140.3,
137.3, 134.9, 125.4, 124.5, 118.0, 107.4, 69.5, 69.3, 69.2, 66.2,
48.9, 34.7, 31.0, 30.5, 14.5.
and pH adjusted to 4.3 using solution NH3/H2O 1:1. Aqueous layer
was extracted with fresh DCM (2 ꢁ 200 mL). Combined DCM ex-
tracts at pH 4.3 were washed with water at pH 8.5, dried over
K2CO3 and evaporated under reduced pressure, precipitated from
i-PrOAc/di-i-Pr2O yielding 38.12 g (50%) of the title compound 26
as a white solid.
HRMS (ES) calcd for C58H95N3O18 [M+H]+ 1122.6689 found
1122.6647.
1H NMR (300 MHz, DMSO) d: 9.03 (s, 1H), 8.18 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 4.91 (d, 1H), 4.73 (dd, 1H), 4.59 (q, 2H), 4.55 (d,
1H), 4.43 (d, 1H), 4.33 (m, 1H), 4.17 (dd, 1H), 3.66 (m, 1H), 3.64 (m,
2H), 3.50 (ov, 2H), 3.47 (ov, 1H), 3.45 (ov, 1H), 3.43 (ov, 2H), 3.38 (t,
2H), 3.22 (s, 3H), 3.05 (dd, 1H), 2.81 (t, 2H), 2.67 (ov, 1H), 2.67 (ov,
1H), 2.59 (m, 2H), 2.40 (m, 1H), 2.35 (dd, 1H), 2.31 (d, 1H), 2.21 (s,
3H), 2.18 (s, 3H), 2.11 (t, 1H), 1.88 (ov, 1H), 1.85 (ov, 2H), 1.85 (ov,
1H), 1.78 (m, 1H), 1.66 (dd, 1H), 1.59 (m, 1H), 1.51 (d, 1H), 1.42 (t,
3H), 1.37 (m, 1H), 1.27 (dd, 1H), 1.12 (s, 3H), 1.1 (s, 3H), 1.09 (ov,
1H), 1.08 (d, 3H), 1.07 (dd, 3H), 1.03 (d, 3H), 1.01 (s, 3H), 0.96 (d,
3H), 0.94 (d, 3H), 0.84 (d, 3H), 0.79 (t, 3H).
4.4.24. 6-[3-({2-[(2-Carboxyethyl)oxy]ethyl}oxy)propyl]-1-
cyclopropyl-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (24)
The title compound 24 (1.34 g) as a white solid was prepared
from starting compound 21 (1.83 g, wet) according to the proce-
dure used to prepare compound 23.
HRMS (ES) calcd for
404.1694.
C
21H25NO7 [M+H]+ 404.1709 found
1H NMR (300 MHz, DMSO) d: 8.72 (s, 1H), 8.24 (d, 1H), 8.16 (d,
1H), 7.88 (dd, 1H), 3.85 (m, 1H), 3.61 (t, 2H), 3.54–3.47 (ov, 4H),
3.42 (t, 2H), 2.84 (t, 2H), 2.45 (t, 2H), 1.87 (m, 2H), 1.31 (m, 2H),
1.19 (m, 2H).
13C NMR (75 MHz, DMSO) d: 177.4, 177.0, 170.9, 166.1, 148.5,
140.2, 137.4, 134.9, 125.5, 124.6, 118.0, 107.5, 102.0, 94.3, 82.7,
78.0, 77.3, 76.3, 74.9, 73.5, 72.4, 72.0, 70.5, 69.4, 69.7, 69.2, 68.6,
66.8, 66.1, 64.8, 62.2, 61.4, 48.8, 44.5, 41.6, 40.2, 35.7, 34.9, 34.2,
31.1, 31.0, 30.6, 27.3, 26.0, 22.0, 21.6, 20.9, 20.5, 17.6, 14.5, 10.9,
8.9, 6.7, 4.9.
13C NMR (75 MHz, DMSO) d: 177.7, 172.5, 165.9, 147.9, 140.5,
139.3, 134.9, 124.9, 124.3, 118.4, 107.1, 69.6, 69.4, 69.2, 66.2,
35.8, 34.7, 31.0, 30.6, 7.5.
4.4.25. 6-[3-({2-[(2-Carboxyethyl)oxy]ethyl}oxy)propyl]-4-oxo-
1-propyl-1,4-dihydro-3-quinolinecarboxylic acid (25)
The title compound 25 (0.74 g) as a white solid was prepared
from starting compound 22 (0.90 g, wet) according to the proce-
dure used to prepare compound 23.
4.4.27. 400-O-(3-{2-[3-(3-Carboxy-1-cyclopropyl-4-oxo-1,4-
dihydro-quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-9-deoxo-
9a-methyl-9a-aza-9a-homoerythromycin A (27)
HRMS (ES) calcd for
406.1851.
C
21H28NO7 [M+H]+ 406.1866 found
The title compound 27 as a white solid was prepared in 53%
yield starting from compound 24 according to the procedure used
to prepare compound 26.
1H NMR (300 MHz, DMSO) d: 8.67 (s, 1H), 8.19 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 4.53 (t, 2H), 3.61 (t, 2H), 3.51 (m, 2H), 3.48 (m,
2H), 3.41 (t, 2H), 2.83 (t, 2H), 2.42 (t, 2H), 1.88 (m, 2H), 1.81 (m,
2H), 0.91 (t, 3H).
HRMS (ES) calcd for C59H95N3O18 [M+H]+ 1134.6689 found
1134.6666.
1H NMR (300 MHz, DMSO) d: 8.73 (s, 1H), 8.22 (d, 1H), 8.16 (d,
1H), 7.87 (dd, 1H), 4.91 (d, 1H), 4.74 (dd, 1H), 4.56 (d, 1H), 4.43 (d,
1H), 4.34 (m, 1H), 4.17 (dd, 1H), 3.84 (m, 1H), 3.69 (m, 1H), 3.66 (m,
2H), 3.47 (d, 1H), 3.43 (m, 1H), 3.38 (t, 2H), 3.33–3.37 (ov, 4H), 3.23
(s, 3H), 3.04 (t, 1H), 2.83 (t, 2H), 2.67 (m, 1H), 2.58 (t, 2H), 2.40 (m,
1H), 2.38 (d, 1H), 2.33 (d, 1H), 2.21 (s, 6H), 2.19 (s, 3H), 2.11 (t, 1H),
1.89 (m, 1H), 1.83–1.86 (ov, 3H), 1.78 (m, 1H), 1.67 (dd, 1H), 1.59
(m, 1H), 1.51 (s, 1H), 1.36 (m, 1H), 1.32 (m, 2H), 1.25 (m, 1H),
1.18 (m, 2H), 1.13 (s, 3H), 1.09 (d, 3H), 1.08 (d, 3H), 1.07–1.09
(ov, 1H), 1.03 (d, 3H), 1.01 (s, 6H), 0.95 (d, 3H), 0.94 (d, 3H), 0.85
(dd, 3H), 0.79 (t, 3H).
13C NMR (75 MHz, DMSO) d: 177.9, 173.1, 166.6, 149.3, 140.3,
138.0, 135.4, 125.9, 124.9, 118.6, 107.6, 70.0, 69.8, 69.6, 66.8,
55.3, 35.4, 31.4, 31.0, 22.4, 10.9.
4.4.26. 400-O-(3-{2-[3-(3-Carboxy-1-ethyl-4-oxo-1,4-dihydro-
quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A (26)
Compound 23 (34.17 g, 0.09 mol) was dissolved in DCM
(155 mL) and the solution cooled to 0 °C in an ice-bath under N2
atmosphere. Into the solution EDACxHCl (33.35 g, 0.17 mol),
20-O-acetyl-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A
13C NMR (75 MHz, DMSO) d: 177.7, 177.0, 170.9, 165.9, 148.0,
140.4, 139.4, 134.8, 124.9, 124.3, 118.4, 107.1, 102.0, 94.3, 82.7,
78.0, 77.4, 76.3, 74.9, 73.5, 72.4, 70.5, 69.7, 69.4, 69.2, 68.6, 66.8,
66.1, 64.8, 62.2, 61.4, 48.8, 44.6, 41.7, 41.6, 40.3, 35.8, 35.7, 35.0,
34.3, 31.1, 30.7, 30.2, 27.4, 26.0, 22.0, 21.6, 20.5, 20.2, 17.7, 17.6,
14.6, 10.9, 8.9, 7.5, 6.7.
(53.12 g, 0.07 mol) and DMAP (24.60 g, 0.20 mol) were added.
The reaction mixture was stirred at 0–5 °C for 2 h and then at rt
for additional 4 h. The reaction was monitored by TLC in a solvent
system DCM/MeOH/concd NH3 = 90:15:1.5. The mixture was con-
centrated under reduced pressure to dryness. The residue was dis-
solved in i-PrOAc (400 mL) and the solution washed with satd aq
NaHCO3 (2 ꢁ 200 mL). Onto the organic layer water was added
and pH was adjusted to 6.3 using a mixture AcOH/H2O = 1:1. Or-
ganic layer was separated and washed once more with fresh water
at pH 6.3. Combined aqueous layers were extracted with i-PrOAc
(4 ꢁ 50 mL). Combined organic layers were dried over K2CO3 and
concentrated to dryness under reduced pressure. The residue was
dissolved in MeOH (800 mL) and the solution stirred at 55 °C for
18 h. The reaction was monitored by TLC in a solvent system
DCM/MeOH/concd NH3 = 90:15:1.5. MeOH was evaporated under
reduced pressure. The residue was dissolved in EtOAc (250 mL),
water (250 mL) was added and pH adjusted to 3.3–3.5 using AcOH.
Organic layer was separated and extracted once more with water
(200 mL). Combined aqueous layers were washed with DCM
(2 ꢁ 100 mL). Onto aqueous layer fresh DCM (300 mL) was added
4.4.28. 400-O-(3-{2-[3-(3-Carboxy-4-oxo-1-propyl-1,4-dihydro-
quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A (28)
The title compound 28 as a white solid was prepared in 51%
yield starting from compound 25 according to the procedure used
to prepare compound 26.
HRMS (ES) calcd for C59H97N3O18 [M+H]+ 1136.6845 found
1136.6842.
1H NMR (300 MHz, DMSO) d: 9.02 (s, 1H), 8.18 (d, 1H), 7.98 (d,
1H), 7.82 (dd, 1H), 4.95 (d, 1H), 4.75 (dd, 1H), 4.56 (q, 2H), 4.53 (d,
1H), 4.43 (d, 1H), 4.34 (m, 1H), 4.17 (d, 1H), 3.69 (m, 1H), 3.66 (t,
2H), 3.53–3.45 (ov, 4H), 3.47 (ov, 1H), 3.45 (ov, 1H), 3.38 (ov, 2H),
3.22 (s, 3H), 3.05 (dd, 1H), 2.81 (t, 2H), 2.67 (m, 2H), 2.58 (t, 2H),
2.41 (m, 1H), 2.34 (dbr, 1H), 2.28 (d, 1H), 2.22 (s, 6H), 2.19 (s, 3H),

M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
6557
2.12 (tbr, 1H), 1.88 (ov, 1H), 1.86–1.80 (ov, 2H), 1.83 (ov, 1H), 1.77
(m, 1H), 1.67 (dd, 1H), 1.61 (m, 1H), 1.52 (d, 1H), 1.38 (m, 1H), 1.27
(ddbr, 1H), 1.12 (s, 3H), 1.09 (d, 6H), 1.05 (ov, 1H), 1.02 (d, 3H),
1.01 (s, 6H), 0.95 (ov, 6H), 0.92 (t, 3H), 0.85 (d, 3H), 0.80 (t, 3H).
13C NMR (75 MHz, DMSO) d: 177.6, 177.2, 171.1, 166.3, 149.0,
140.5, 137.8, 135.1, 125.6, 124.7, 118.4, 107.4, 102.2, 94.5, 82.9,
78.2, 77.6, 76.5, 75.1, 73.7, 72.6, 70.62, 70.6, 69.9, 69.6, 69.5,
68.8, 66.3, 65.0, 32.4, 61.7, 55.0, 49.0, 44.7, 41.8, 41.5, 40.4, 35.9,
35.2, 34.4, 31.2, 30.8, 30.3, 27.5, 26.2, 22.2, 21.8, 21.4, 21.1, 20.7,
17.9, 17.8, 14.8, 11.1, 10.7, 9.1, 6.9.
13C NMR (75 MHz, DMSO) d: 177.2, 174.8, 170.8, 169.6, 166.2,
148.3, 140.0, 137.3, 134.6, 125.8, 124.6, 118.8, 107.0, 101.7, 95.0,
82.6, 78.0, 77.9, 76.3, 75.8, 73.7, 72.2, 70.5, 69.8, 69.7, 69.6, 69.2,
68.1, 66.7, 66.1, 64.6, 62.2, 48.8, 48.7, 44.2, 44.1, 40.2, 38.8, 38.7,
37.0, 34.9, 34.3, 31.0, 30.6, 30.0, 21.5, 21.0, 20.9, 20.3, 18.5, 17.7,
16.9, 15.7, 14.5, 14.3, 10.5, 8.9.
4.4.31. 400-O-(3-{2-[3-(3-Carboxy-1-ethyl-4-oxo-1,4-dihydro-
quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-6-O-methyl-8a-
aza-8a-homoerythromycin A (31)
The title compound 31 as a white solid was prepared in 41%
yield starting from compounds 23 and 20-O-acetyl-6-O-methyl-
8a-aza-8a-homoerythromycin A according to the procedure used
to prepare compound 29.
4.4.29. 400-O-(3-{2-[3-(3-Carboxy-1-ethyl-4-oxo-1,4-dihydro-quin-
oline-6-yl)-propoxy]-ethoxy}-propionyl)-clarithromycin (29)
Compound 23 (300 mg, 0.77 mmol) was dissolved in dry DMF
(4 mL) and the solution cooled to 0 °C under N2 atmosphere. To this
solution EDACxHCl (293 mg, 1.53 mmol) was added and the mixture
was stirred for 5 min. Then, 20-O-acetylclarithromycin (468 mg,
0.59 mmol) solution in dry DCM (4 mL) was added followed by addi-
tion of DMAP (281 mg, 2.3 mmol). The mixture was stirred at rt for
24 h and monitored on TLC in a solvent system CH2Cl2/MeOH/concd
NH3 = 90:15 1.5. Saturated NaHCO3 (20 mL) and EtOAc (20 mL) were
added and the layers separated. Aqueous layer was extracted with
EtOAc (2 ꢁ 20 mL). Combined organic layers were dried over
K2CO3 and concentrated to dryness. The residue was dissolved in
MeOH (100 mL) and the solution stirred at 55 °C for 24 h. MeOH
was evaporatedandthe residuepurifiedby columnchromatography
on silica-gel in a solvent system DCM/MeOH/concd NH3 = 90:9:1.5.
The residue was precipitated from EtOAc/n-hexane yielding 205
mg (Y = 31%) of the title compound 29 as a white solid.
HRMS (ES) calcd for C58H94N3O19 [M+H]+ 1136.6482 found
1136.6501.
1H NMR (300 MHz, DMSO) d: 9.03 (s, 1H), 8.18 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 7.54 (d, 1H), 4.94 (d, 1H), 4.88 (dd, 1H), 4.60 (q,
2H), 4.56 (d, 1H), 4.39 (d, 1H), 4.30 (m, 1H), 4.06 (s, 1H), 3.95 (m,
1H), 3.86 (d, 1H), 3.69–3.62 (ov, 3H), 3.54 (d, 1H), 3.52–3.46 (ov,
4H), 3.40–3.37 (ov, 3H), 3.22 (s, 3H), 3.03 (dd, 1H), 2.97 (s, 3H),
2.83 (t, 2H), 2.64 (dq, 1H), 2.61–2.52 (ov, 2H), 2.40–2.35 (ov, 2H),
2.29 (d, 1H), 2.21 (s, 6H), 1.94 (m, 1H), 1.87–1.84 (ov, 3H), 1.68
(dd, 1H), 1.63 (dd, 1H), 1.59 (br d, 1H), 1.42 (t, 3H), 1.37 (m, 1H),
1.20 (s, 3H), 1.11–1.07 (ov, 6H), 1.05 (ov, 1H), 1.03–1.01 (ov, 6H),
1.01 (s, 3H), 0.98 (d, 3H), 0.97–0.94 (ov, 6H), 0.80 (t, 3H).
13C NMR (75 MHz, DMSO) d: 177.9, 177.8, 173.5, 171.3, 166.6,
148.9, 140.7, 137.8, 135.3, 125.9, 125.0, 118.5, 107.9, 102.7, 94.6,
79.0, 78.5, 78.4, 76.3, 76.0, 74.5, 72.8, 71.2, 70.9, 70.1, 69.8, 69.7,
67.3, 66.5, 65.1, 62.7, 51.7, 49.3, 49.2, 45.3, 42.4, 41.9, 40.7, 40.6,
35.4, 34.4, 31.4, 31.3, 31.0, 30.7, 24.6, 21.9, 21.6, 20.9, 20.4, 18.3,
17.3, 15.0, 14.9, 14.3, 11.6, 9.8, 9.3.
HRMS (ES) calcd for C58H92N2O19 [M+H]+ 1121.6373 found
1121.6404.
1H NMR (300 MHz, DMSO) d: 9.04 (s, 1H), 8.19 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 5.06 (dd, 1H), 4.86 (d, 1H), 4.60 (q, 2H), 4.56 (d,
1H), 4.43 (d, 1H), 4.27 (dq, 1H), 3.68 (m, 2H), 3.66 (t, 2H), 3.62 (br s,
1H), 3.60 (d, 1H), 3.55 (d, 1H), 3.50 (t, 2H), 3.46 (t, 2H), 3.39 (t, 2H),
3.21 (s, 3H), 3.04 (dd, 1H), 2.95 (dq, 1H), 2.91 (s, 3H), 2.85 (dq, 1H),
2.82 (t, 2H), 2.64–2.59 (m, 2H), 2.54 (m, 1H), 2.41 (m, 1H), 2.36 (d,
1H), 2.21 (s, 6H), 1.87–1.84 (m, 2H), 1.86 (ov, 1H), 1.83 (m, 1H),
1.76 (dd, 1H), 1.67 (dd, 1H), 1.59 (br d, 1H), 1.45 (dd, 1H), 1.42
(t, 3H), 1.39 (m, 1H), 1.23 (ov, 2H), 1.17 (ov, 1H), 1.13 (s, 3H),
1.09 (d, 3H), 1.06 (d, 3H), 1.04 (ov, 12H), 0.76 (t, 3H).
4.4.32. 11-N,12-O-Carbonyl-400-O-(3-{2-[3-(3-carboxy-1-ethyl-4-
oxo-1,4-dihydro-quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-
11-desoxy-6-O-methyl-11-methylamino-erythromycin A (32)
The title compound 32 as a white solid was prepared in 27%
yield starting from compounds 23 and 20-O-acetyl-11-N,12-O-
carbonyl-11-desoxy-6-O-methyl-11-methylamino-erythromycin
according to the procedure used to prepare compound 29.
A
HRMS (ES) calcd for C60H93N3O19 [M+H]+ 1160.6482 found
13C NMR (75 MHz, DMSO) d: 219.7, 177.4, 175.0, 170.9, 166.1,
148.5, 140.2, 137.4, 134.9, 125.5, 124.6, 118.1, 107.5, 101.9, 95.4,
79.9, 79.2, 77.8, 77.7, 75.9, 74.1, 72.1, 70.6, 69.6, 69.4, 69.2, 68.8,
66.8, 66.1, 64.5, 62.4, 50.1, 48.9, 48.8, 44.2, 43.7, 40.2, 38.6, 38.2,
37.9, 34.9, 34.2, 31.0, 30.6, 30.1, 21.5, 20.6, 20.3, 19.7, 18.1, 17.6,
16.9, 15.6, 14.6, 11.9, 10.4, 8.9.
1160.6508.
1H NMR (300 MHz, DMSO) d: 9.04 (s, 1H), 8.19 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 4.85 (d, 1H), 4.78 (dd, 1H), 4.59 (q, 2H), 4.57
(d, 1H), 4.44 (d, 1H), 4.26 (dq, 1H), 3.66 (ov, 3H), 3.63 (ov, 1H),
3.53–3.46 (ov, 6H), 3.39 (t, 2H), 3.21 (s, 3H), 3.07–3.01 (ov, 2H),
2.87 (ov, 1H), 2.85 (s, 3H), 2.82 (t, 2H), 2.64–2.59 (ov, 2H), 2.45
(m, 1H), 2.42 (m, 1H), 2.35 (d, 1H), 2.23 (s, 6H), 2.09 (s, 3H),
1.85 (m, 2H), 1.81 (ov, 1H), 1.71–1.68 (ov, 3H), 1.62 (br d, 1H),
1.54 (ov, 2H), 1.42 (t, 3H), 1.38 (s, 3H), 1.22 (s, 3H), 1.16 (d,
3H), 1.10 (ov, 7H), 1.05 (ov, 3H), 1.04 (ov, 3H), 1.02 (s, 3H),
0.88 (d, 3H), 0.78 (t, 3H).
4.4.30. 9-Ethyloxymino-400-O-(3-{2-[3-(3-carboxy-1-ethyl-4-
oxo-1,4-dihydro-quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-
erythromycin A (30)
The title compound 30 as a white solid was prepared in 27%
yield starting from compounds 23 and 20-O-acetyl-9-ethyloxyimi-
no-erythromycin A according to the procedure used to prepare
compound 29.
13C NMR (75 MHz, DMSO) d: 215.2, 177.4, 176.2, 170.9, 166.1,
156.5, 148.5, 140.2, 137.4, 134.9, 125.5, 124.5, 118.1, 107.5,
101.9, 95.2, 82.4, 78.7, 78.1, 77.7, 76.3, 75.1, 72.1, 70.5, 69.7,
69.4, 69.3, 66.9, 66.1, 64.5, 62.4, 61.2, 49.4, 48.9, 48.8, 44.9, 44.5,
40.2, 38.6, 38.5, 38.0, 34.9, 34.1, 31.9, 31.0, 30.6, 30.1, 21.5, 21.4,
20.3, 19.6, 18.0, 17.8, 15.4, 14.6, 13.4, 13.3, 10.1, 8.8.
HRMS (ES) calcd for C59H95N3O19 [M+H]+ 1150.6638 found
1150.6667.
1H NMR (300 MHz, DMSO) d: 8.98 (s, 1H), 8.18 (d, 1H), 7.95 (d,
1H), 7.79 (dd, 1H), 5.12 (dd, 1H), 4.96 (q, 2H), 4.83 (d, 1H), 4.57 (q,
2H), 4.55 (d, 1H), 4.45 (d, 1H), 4.28 (m, 1H), 3.88 (d, 1H), 3.69–3.63
(ov, 3H), 3.50–3.44 (ov, 6H), 3.38 (t, 2H), 3.21 (s, 3H), 3.05 (ov, 1H),
3.02 (dd, 1H), 3.00 (m, 1H), 2.81 (ov, 3H), 2.67–2.57 (ov, 2H), 2.39
(m, 1H), 2.33 (d, 1H), 2.19 (s, 6H), 1.92 (m, 1H), 1.89–1.83 (ov, 3H),
1.66 (dd, 1H), 1.58 (br d, 1H), 1.47–1.36 (ov, 5H), 1.28 (s, 3H), 1.16
(t, 3H), 1.11 (d, 3H), 1.09 (ov, 1H), 1.08–1.06 (ov, 9H), 1.03 (d, 3H),
1.00–0.97 (ov, 9H), 0.75 (t, 3H).
4.4.33. 400-O-(3-{2-[3-(3-Carboxy-1-ethyl-4-oxo-1,4-dihydro-quin-
oline-6-yl)-propoxy]-ethoxy}-propionyl)-roxythromycin (33)
The title compound 33 as a white solid was prepared in 30%
yield starting from compounds 23 and 20-O-acetyl-roxythromycin
according to the procedure used to prepare compound 29.
HRMS (ES) calcd for C61H99N3O21 [M+H]+ 1210.6849 found
1210.6946.

6558
M. M. Škugor et al. / Bioorg. Med. Chem. 18 (2010) 6547–6558
1H NMR (500 MHz, DMSO) d: 9.04 (s, 1H), 8.19 (d, 1H), 7.99 (d,
data. We would also like to thank NMR-team: Ana Cikoš and Biser-
ka Metelko.
ˇ
1H), 7.83 (dd, 1H), 5.13 (dd, 1H), 5.05 (m, 2H), 4.82 (d, 1H), 4.60 (q,
2H), 4.56 (d, 1H), 4.44 (d, 1H), 4.29 (dq, 1H), 3.92 (nrdd, 1H), 3.66
(ov, 6H), 3.50 (ov, 3H), 3.41 (ov, 6H), 3.38 (d, 1H), 3.24 (s, 3H), 3.21
(s, 3H), 3.03 (d, 1H), 2.86 (ov, 4H), 2.81 (ov, 3H), 2.70 (dq, 1H), 2.55
(ov, 2H), 2.41 (m, 1H), 2.33 (d, 1H), 2.21 (s, 6H), 1.86 (ov, 4H), 1.68
(dd, 1H), 1.60 (m, 1H), 1.50 (ov, 2H), 1.42 (t, 3H), 1.38 (m, 1H), 1.28
(s, 3H), 1.17 (ov, 4H), 1.09 (ov, 9H), 1.02 (d, 3H), 1.00 (0v, 9H), 0.76
(t, 3H).
References and notes
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13C NMR (125 MHz, DMSO) d: 177.9, 176.0, 171.5, 171.4, 171.3,
166.6, 149.0, 140.7, 137.8, 135.4, 125.9, 125.0, 119.5, 107.9, 102.3,
97.1, 95.5, 83.0, 79.1, 78.4, 76.3, 75.4, 74.2, 72.6, 71.6, 70.9, 70.8,
70.1, 69.8, 69.6, 67.3, 67.2, 66.5, 65.2, 62.7, 58.4, 49.4, 49.3, 44.6,
40.6, 39.8, 39.4, 37.6, 35.4, 34.8, 31.4, 31.0, 30.3, 26.8, 26.7, 21.4,
21.3, 20.9, 19.1, 18.2, 17.5, 16.3, 15.1, 15.0, 11.0, 9.4.
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4.4.34. 11-Amino-11-N,12-O-carbonyl-400-O-(3-{2-[3-(3-Car-
boxy-1-ethyl-4-oxo-1,4-dihydro-quinoline-6-yl)-propoxy]-
ethoxy}-propiony)-11-desoxy-6-O-methyl-erythromycin A (34)
The title compound 34 as a white solid was prepared in 70%
yield starting from compounds 23 and 20-O-acetyl-11-amino-11-
N,12-O-carbonyl-11-desoxy-6-O-methyl-erythromycin A accord-
ing to the procedure used to prepare compound 26.
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14. Ðokic, S.; Kobrehel, G.; Lazarevski, G.; Lopotar, N.; Tamburašev, Z.; Kamenar, B.;
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HRMS (ES) calcd for C59H91N3O19 [M+H]+ 1146.6325 found
1146.6326.
1H NMR (300 MHz, DMSO) d: 9.03 (s, 1H), 8.19 (d, 1H), 7.98 (d,
1H), 7.83 (dd, 1H), 7.71 (s, 1H), 4.87 (dd, 1H), 4.84 (d, 1H), 4.60 (q,
2H), 4.55 (d, 1H), 4.43 (d, 1H), 4.27 (m, 1H), 3.62–3.67 (ov, 4H),
3.47–3.52 (ov, 5H), 3.46 (d, 1H), 3.39 (t, 2H), 3.21 (s, 3H), 3.04
(dd, 1H), 2.95 (dq, 1H), 2.83 (ov, 1H), 2.81 (t, 2H), 2.8 (s, 3H),
2.61 (ov, 1H), 2.53 (ov, 1H), 2.35–2.41 (ov, 2H), 2.33 (d, 1H), 2.20
(s, 6H), 1.85 (m, 2H), 1.82 (ov, 1H), 1.76 (ov, 1H), 1.69 (dd, 1H),
1.68 (m, 1H), 1.60 (m, 1H), 1.54 (m, 1H), 1.50 (ov, 1H), 1.42 (t,
3H), 1.37 (s, 3H), 1.22 (s, 3H), 1.16 (d, 3H), 1.07–1.09 (ov, 6H),
1.06 (ov, 1H), 1.01–1.04 (ov, 9H), 0.97 (d, 3H), 0.79 (t, 3H).
13C NMR (75 MHz, DMSO) d: 216.2, 177.4, 176.4, 170.8, 166.1,
157.2, 148.5, 140.2, 137.3, 134.9, 125.5, 124.6, 118, 107.6, 101.9,
95.1, 83.1, 79.1, 77.9, 77.8, 76.4, 74.9, 72.1, 70.5, 69.6, 69.4, 69.2,
66.9, 66.1, 64.6, 62.4, 56.7, 49.5, 48.9, 48.8, 44.6, 44.4, 40.2, 38.4,
39.1, 36.9, 34.9, 34.1, 30.9, 30.6, 30.1, 19.5, 21.5, 21.4, 20.3, 17.9,
17.6, 15.2, 14.5, 13.4, 13.1, 10.3, 8.7.
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ˇ ´
4.4.35. 400-O-(3-{2-[3-(3-Carboxy-1-ethyl-4-oxo-1,4-dihydro-
quinoline-6-yl)-propoxy]-ethoxy}-propionyl)-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A (26) diisopropyl ether
solvate single crystals
Compound 26 (24.4 g) was dissolved by heating in isopropylac-
etate (41.5 mL). The resulting solution was cooled to room temper-
ature and added into diisopropylether (487 mL) during 45 min
under vigorous stirring. The obtained precipitate was filtered off
and the mother liquor was maintained in closed vessel at room
temperature for 48 h. Generated single crystals were sent for sin-
gle-crystal X-ray diffraction studies without isolation.
35. Oxford Diffraction, Oxford Diffraction Ltd, Xcalibur CCD System, CrysAlis
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´
We wish to thank Professor Vitomir Šunjic for constructive and
helpful suggestions during preparation of manuscript. We grate-
fully acknowledge Erick Garver for conducting the rat pharmacoki-
netic studies and Jasna Padovan for useful comments regarding PK