Novel hybrid molecules based on 15-membered azalide as potential antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2012.01.039

Malaria remains the most prevalent tropical disease, and due to the spread of resistant parasites novel therapeutics are urgently needed. Azithromycin has shown potential in malaria treatment so we designed hybrid azalide molecules with the aim to improve activity against and selectivity for the malaria parasite. Novel hybrid molecules comprising 4-aminoquinoline moiety covalently liked to 15-membered azalide scaffold at position C-3′ were synthesized and biologically evaluated. Antimalarial testing against Plasmodium falciparum sensitive and resistant strains confirmed the improved in vitro activity over azithromycin and chloroquine. Selectivity of the compounds (HepG2 IC ₅₀/P. falciparum IC₅₀ ratio) for the parasite was high (100-2700) and their antibacterial activity diminished. Even though oral bioavailability determined for compound 12 was low, novel quinoline C-3′-substituted 15-membered azalides represent an interesting subclass of antimalarial macrolides that need further research and evaluation.

Full text of DOI:10.1016/j.ejmech.2012.01.039

European Journal of Medicinal Chemistry 49 (2012) 365e378
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel hybrid molecules based on 15-membered azalide as potential
antimalarial agents
,
1
,
,
,
3
,3
ꢀ ꢁ a
Kristina Starcevic , Dijana Pesic 3, Ana Toplak a 2, Goran Landek a , Sulejman Alihodzic
,
ꢀ
ꢁ a
ꢀ ꢁ a
Esperanza Herreros b, Santiago Ferrer b, Radan Spaventi a , Mihaela Peric
,
3
,
ꢁ a
*
a GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz baruna Filipovica 29, 10000 Zagreb, Croatia
b Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Malaria remains the most prevalent tropical disease, and due to the spread of resistant parasites novel
therapeutics are urgently needed. Azithromycin has shown potential in malaria treatment so we designed
hybrid azalide molecules with the aim to improve activity against and selectivity for the malaria parasite.
Novel hybrid molecules comprising 4-aminoquinoline moiety covalently liked to 15-membered azalide
scaffold at position C-30 were synthesized and biologically evaluated. Antimalarial testing against
Plasmodium falciparum sensitive and resistant strains confirmed the improved in vitro activity over
azithromycin and chloroquine. Selectivity of the compounds (HepG2 IC50/P. falciparum IC50 ratio) for the
parasite was high (100e2700) and their antibacterial activity diminished. Even though oral bioavailability
determined for compound 12 was low, novel quinoline C-30-substituted 15-membered azalides represent
an interesting subclass of antimalarial macrolides that need further research and evaluation.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 10 November 2011
Received in revised form
16 January 2012
Accepted 17 January 2012
Available online 26 January 2012
Keywords:
Malaria
Antimalarial
Azalide
Quinoline
Hybrid molecules
Synthesis
Plasmodium falciparum
1. Introduction
existing drugs, have urged the need for discovery of new anti-
malarial agents [2].
Malaria is the most important parasitic disease worldwide,
affecting approximately 225 million people and killing 781 thou-
sand, the majority of whom are young children in sub-Saharan
Africa [1]. The disease is caused by five different species of
malaria parasite from the genus Plasmodium with Plasmodium
falciparum being the most virulent and deadly. The increasing
resistance of P. falciparum to currently available drugs, and espe-
cially to chloroquine (CQ), high costs and severe side effects of
Up to the late 1990’s parasitic diseases were treated with single-
drug therapy. Nowadays, World Health Organisation recommends
the use of two or more agents as the best way to minimize treat-
ment failures and the spread of drug resistance [1,3]. In the last
decade the concept of hybrid molecules has emerged as a new
approach in the design of novel chemotherapeutics including
antimalarial agents [4e7]. Hybrid molecules are defined as chem-
ical entities combined from two active components linked with
either a resistant or a labile linker unit that bring distinct biological
properties into one molecule. By implementing this strategy
several advantages over drug combination in addressing the issues
associated with the rapid emergence of drug resistance, potency,
solubility, route of administration, pharmacokinetics, metabolism
and safety could be achieved [8e11]. By adopting this approach,
a courageous step forward for scientific community involved in
malaria control and eradication could be accomplished.
Abbreviations: AZ, azithromycin; CQ, chloroquine; P. falciparum, Plasmodium
falciparum.
* Corresponding author. Present address: School of Medicine, Center for Trans-
ꢀ
lational and Clinical research, Department for Intercellular Communication, Salata
2, 10000 Zagreb, Croatia. Tel.: þ385 1 4590070; fax: þ385 1 4566724.
ꢁ
E-mail address: mihaela.peric@mef.hr (M. Peric).
Present address: Faculty of Veterinary Medicine, Department of Animal
1
Azithromycin (AZ) is a potent chemotherapeutic agent with
extended spectrum of antibacterial activity and favourable phar-
macokinetic properties [12,13]. It is an azalide antibiotic derived
semi-synthetically from macrolide erythromycin [14]. Macrolide
antibiotics, particularly AZ, have demonstrated in vitro and in vivo
Husbandry, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia.
2
Present address: Biotechnology, GBB, University of Groningen, Nijenborgh 4, NL
9747 AG Groningen, Netherlands.
3
ꢀ
ꢀ
ꢁ
Present address: Galapagos istrazivacki centar d.o.o, Prilaz baruna Filipovica 29,
10000 Zagreb, Croatia.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.039

ꢀ ꢁ
K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
366
activity in malaria prophylaxis and treatment experimental models
[15e17]. It also showed the potential to be an effective, well-
tolerated clinical agent for the prophylaxis of malaria [18e20]. AZ
effectiveness was not sufficient to be used as a monotherapy for
malaria but in combinations with CQ [21,22], quinine [23], artesu-
nate [24] and sulfadoxine-pyrimethamine [25] it proved useful as
an antimalarial agent. AZ has predominant activity against eryth-
rocytic stages of the parasite and also shows moderate activity
against hepatic stages in experimental models and in humans
[17,26]. Notably, AZ displays good safety profile for young children
and pregnant women [27]. Extensive clinical trials provided so far
little evidence for the superiority or equivalence of AZ therapy to
currently used antimalarials [28], so novel, more efficient macro-
lide agents would provide valuable follow-up candidates to
potentially replace AZ or even expand its therapeutic spectrum.
In our previous work we reported antimalarial activity of novel
15-membered azalides with improved in vitro [29,30] and in vivo
[31] activity over AZ. In the present article, we report the synthesis
of hybrid entities containing a 4-aminoquinoline moiety linked to
properly C-30 modified 15-membered azalide. These two pharma-
cophores were linked by linkers (L) of different length and structure
(Fig. 1) with the aim to increase their antimalarial activity (espe-
cially against resistant parasites), selectivity for the parasite and
diminish antibacterial activity. Such properties would allow effi-
cient eradication of the parasites without affecting the gut bacterial
flora and potentially contributing to the spread of macrolide
resistance among commensal and consequently pathogenic
bacteria.
2. Results and discussion
2.1. Chemistry
Novel C-30-substituted 15-membered azalide compounds were
designed to explore the chemical space around the desosamine
sugar, as so far functionally rather unexplored constituent of aza-
lide scaffold. The other goal was to use inexpensive starting
materials and short synthesis procedures to comply with a strict
requirement for obtaining a low cost antimalarial agent. Novel
compounds were envisaged to contain different functionalities in
the C-30 position linker at the desosamine sugar, as it was outlined
on Fig. 1. Also, as it is well known that azalides undergo decom-
position in aqueous acidic media in the form of cladinose sugar
glycosidic bond hydrolysis, the selective cleavage of cladinose was
exploited to prepare the decladinosyl derivatives.
For the preparation of hybrid macrolide-4-aminoquinoline
molecules, the synthesis of the key intermediates, 30-aminopropyl
derivatives 2a, 2b, 4a, 4b and 5 was performed and optimized
(Scheme 1).
AZ
CQ
L1 =
N
H
N
9
O
9a
H
10
8
HO
N
11
AZ
N
Cl
OH
O
L2
L3 = AZ
L =
=
N
O7H
CQ
HO
O
O
6
5
12
13
O
3'
H
N
2' 4'
CQ
4
+
+
N
H
1'O5'
O
1
HN
3
2
N
O
O
CQ
AZ
N
H
4
1''
2''
5''
4''
3''
H
N
OH
CQ
L5
=
AZ
N
O
N
N
AZ
L
CQ
R2
N
N
O
HO
OH
N
L
HO
OH
O
O
R1
O
OH
O
O
R1 = H, -L-cladinosyl =
2 = H, -Cl
O
R
AZ
Fig. 1. Structures of azithromycin (AZ), chloroquine (CQ) and the design of the new class of antimalarial agents based on 15-membered azalide-4-aminoquinoline hybrid approach.

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
367
N
NH2
N
N
OH
O
OH
O
N
O
N
O
HO
HO
HO
O
HO
O
OH
OH
iii
ii
O
O
R1
R1
O
O
N
N
OH
O
N
O
HO
OH
O
4a R1=Cladinosyl
4b R1=H
NH
O
HO
O
HO
OH
3a R1=Cladinosyl
3b R1=H
i
HO
O
OH
O
O
R1
O
R1
O
1a R1=Cladinosyl
1b R1=H
2a R1=Cladinosyl
2b R1=H
N
iv
OH
O
N
O
HO
HO
O
OH
O
R1
O
5
R1=Cladinosyl
Scheme 1. Reagents and conditions: i, I2, MeOH, 500 W lamp for 2.5 h then r.t., 24 h or DEAD, acetone, r.t., 24 h; ii, acrylonitrile, 60 ꢁC, w24 h; iii, H2/PtO2/C, glacial acetic acid, 5 Barr,
r.t., 24 h; iv propargyl bromide, THF, r.t.
Functionalization of the desosamine sugar was performed by
demethylation of C-30dimethylamino group following two
protocols. The first protocol, an iodine-promoted demethylation
in MeOH by 500 W lamp irradiation was used for demethylation
of decladinosyl derivative 1b, gave very high yield of compound
2b [32]. The second one, a diethyl azodicarboxylate (DEAD)
mediated reaction of 1a gave almost quantitative yield of the
desmetyl compound 2a [33,34]. Michael addition of 2a and 2b
with excess of acrylonitrile and subsequent reduction of the
resulting cyanoethyl adducts 3a and 3b afforded primary amines
4a and 4b. Subjecting intermediate 2a to propargyl bromide in
tetrahydrofuran resulted in the formation of 30-N-(2-propyn-1-yl)
derivative 5.
in Scheme 2 and is performed as previously described [35]. The
azide compound 11 was synthesized using the standard chemical
approach for such compounds via methansulfonyl intermediate.
The final hybrid compounds (12e21) were prepared by coupling
4-amino-7-chloroquinoline derivatives 7e11 with macrolide
subunits 2a, 4a and 4b (Scheme 3 and 4).
The amidation of primary amines 4a and 4b with acid 8 pro-
ceeded in the presence of EDC ꢀ HCl, HOBt and triethylamine in
dichloromethane at room temperature (Scheme 3). Nucleophilic
substitution of the chlorine atom in commercially available 4,7-
dichloroquinoline 6 led to the formation of the compound 16.
Azalides undergo decomposition in aqueous acidic media
comprising hydrolysis of cladinose sugar glycosidic bond and this
property was used for the preparation of decladinosyl derivatives
15 and 17.
Parallel to the synthesis of macrolide intermediates, the prep-
aration of quinoline intermediates was initiated. Thus the
commercially available 4,7-dichloroquinoline
lectively transformed to 7-chloro-4-(substituted)aminoquinolines
7e11. The synthesis procedures for compounds 7 and 8 is presented
6
was regiose-
Dehalogenated compounds 14, 18 and 19 were successfully
prepared by hydrogenation of parent compounds in ethanol in the
presence of palladium on charcoal.
Cl
Cl
N
HN
i
COOH
7
Cl
N
N
ii
Cl
HN
6
iii
COOH
8
Cl
N
Cl
N
Cl
N
v
iv
HN
NH
NH
+
OH
OSO2CH3
10
-N
N
N
9
11
Scheme 2. Synthesis of 4-amino-7-chloroquinoline intermediates. Reagents and conditions: i, glycine; ii, 4-aminobutanoic acid; iii, 3-aminopropanol, 130e140 ꢁC, 24 h;
iv, methansulfonyl chloride TEA, THF, 0 ꢁC, 45 min, 0 ꢁC; v, sodium azide, DMF (anhydrous), 55 ꢁC, over night.

ꢀ
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368
K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
N
N
NH
NH
Cl
O
O
NH
NH
N
N
OH
O
OH
O
N
O
N
O
HO
HO
ii
HO
O
HO
O
OH
OH
O
i
O
R1
R1
O
O
NH2
N
12 R1=Cladinosyl
13R1=H
14 R1=Cladinosyl
15 R1=H
iii
OH
O
N
O
HO
HO
O
OH
Cl
O
R1
iv
O
N
N
4a R1=Cladinosyl
4b R1=H
NH
NH
N
N
OH
N
ii
OH
O
HO
N
HO
HO
HO
O
OH
OH
O
O
O
O
O
R1
O
R1
O
O
18 R1=Cladinosyl
19 R1=H
16 R1=Cladinosyl
17 R1=H
iii
Scheme 3. Synthesis of novel 4-aminoquinoline derivatives of 15-membered azalides. Reagents and conditions: i, 8, EDC ꢀ HCl, HOBt, triethylamine, DCM, r.t., over night; ii, H2, Pd/
C, Ethanol, 5 Barr, 4 h; iii, 3 M HCl, r.t., 1 h; iv, 4,7-dichloroquinoline, DIPEA, NaHCO3, DMSO, 110 ꢁC, 20 h; iii, 3 M HCl, r.t., 1 h; ii, H2, Pd/C, Ethanol, 5 Barr, 4 h.
Cl
N
NH
O
N
OH
O
N
O
HO
HO
O
OH
O
i
R1
O
N
OH
O
NH
O
HO
20 R1=Cladinosyl
HO
O
OH
O
R1
O
ii
2a R1=Cladinosyl
Cl
N
O
H
N
OH
O
N
O
HO
N
HO
O
OH
O
R1
O
21 R1=Cladinosyl
Scheme 4. Synthesis of 30-N-acyl derivatives of 15-membered azalides. Reagents and conditions: i, 8, EDC ꢀ HCl, HOBt, triethylamine, DCM, r.t., over night; ii, 7, EDC ꢀ HCl, HOBt,
triethylamine, DCM, microwave irradiation, 100 ꢁC, 5 min.

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
369
Reactions of 2a with chloroquinoline acids 7 and 8 were per-
formed under the similar reaction conditions (EDC ꢀ HCl, HOBt,
triethylamine, DCM) yielding azalide amides 20 and 21 (Scheme 4).
Nuclear magnetic resonance (NMR) spectra of amides 20 and 21
in DMSO-d6 solution at room temperature revealed that some
resonance lines were doubled. Using calculated proton peak
intensities of aromatic protons, the ratio of two sets was established
to be approximately 1.5:1. The largest difference in the chemical
shifts between two sets of resonance lines was observed for atoms
in the vicinity of sugar rings, while all other atoms showed no or
very little difference in the chemical shifts. These findings suggest
the presence of two isomers in the solution, most probably arising
from slow rotation around the amide bond. It is known from the
literature that a single CeN bond in amides has some double bond
character, due to the resonance effect, so cis and trans isomers exist
in the equilibrium in a solution [36].
To investigate the chemical properties of the linker, the amide
bond was substituted by the triazole ring as its bioisostere [37,38].
The copper-(I)-catalyzed coupling of azides and terminal acety-
lenes creates 1,4-disubstituted 1,2,3-triazole linkages, which share
useful topological and electronic features with nature’s ubiquitous
amide connectors. However, unlike amides, triazoles are not
susceptible to cleavage hydrolytically or otherwise [39].
the cladinose sugar from the 15-membered azalide scaffold. All
decladinosyl derivatives (13, 15, 17, 19) had their antibacterial
activity almost completely eliminated, as already observed for
some other classes of 15-membered azalides [41]. In case of the
linker with amide moiety the presence of chlorine on the quinoline
substituent contributed to the increase in the antibacterial activity
against streptococci (12 vs. 18) while the same was not observed for
the alkyl linked pair (14 and 16). For the directly linked amide
derivatives 20 and 21 antibacterial activity was slightly enhanced in
the compound with the ethyl spacer. 30-linker [42].
The triazole ring in C-30 linker as bioisoster of amide moiety
increased antibacterial activity of compound 22 even more then the
amide derivative itself [42].
2.2.2. In vitro antimalarial activity and cytotoxicity
The in vitro antimalarial activity of the prepared compounds has
been compared with that of AZ and CQ. The set of 6 compound
representing 30 analogue pairs listed in Table 1 were profiled for
their antimalarial activity against two different P. falciparum
strains: CQ resistant W2 strain and CQ sensitive 3D7A strain. As
already observed with antibacterial activity, antimalarial activity of
the decladinosyl derivatives was lower in comparison to their cla-
dinosyl analogues (13 vs. 12 and 17 vs. 16). The directly linked
compounds, 16 and 17, were the most active pair and activities
exceeded both AZ (IC50s were 300e3838 fold lower) and CQ
(1,5e216 fold lower) against two tested P. falciparum strains. The
selectivity for the parasite (defined as the ratio between HepG2 IC50
and P. falciparum IC50s, if >100 compound is considered to be
selective for the parasite) for this pair was very good (over 1000
fold) but the IC50s measured were considered to be insufficient for
the progression of these compounds to the next stage of evaluation.
The 30-N-derivative 22 was prepared via regioselective [1 þ 3]
dipolar cycloaddition reaction of 15-membered azalide derivative 5
and 3-(7-chloroquinolin-4-ylamino)propyl azide 11 (Scheme 5).
The control of regioselectivity was achieved by using copper (I) e
catalyst in DMF/water mixture. The reaction proceeded very fast
under microwave irradiation with high yield.
2.2. Biological evaluation of prepared compounds
For the directly linked amide derivative 21 the determined IC50
s
were 206 and 1151 fold lower than AZ while IC50s compared to CQ
were 1,6 and 33 fold lower. Its propyl analogue, 20, was less active
but still retaining improvement over AZ (111 and 29 fold against
3D7A and W2, respectively) and CQ against W2 strain (5 fold). The
compound 13 had the lower activity determined with only 9e18
2.2.1. In vitro antibacterial activity
Antibacterial activity was determined for all new compounds on
a panel of 5 pathogenic bacterial species and compared to AZ and
CQ (Tables 1 and 2). The bacteria tested represent the species most
affected by the action of macrolide antibiotics and the strains used
were all fully sensitive to AZ. Antibacterial activity of novel C-30 15-
membered azalide derivatives was substantially inferior to that of
AZ. This output was anticipated since C-30dimethylamino group
strongly contributes to the macrolide binding to the bacterial
ribosomes [40]. Other structural feature that most evidently
affected weakening of the antibacterial activity was the removal of
fold increase in the IC50
s over AZ. Its cladinosyl analogue,
compound 12 had the activity improved over AZ 56 and 150 fold
whereas compared to CQ this increase was only 9 fold against W2
strain. The cytotoxicity and selectivity of compound 12 was in the
acceptable range and when combined with overall properties
(activity, straightforwardness of synthesis and estimated cost of
Cl
N
HN
Cl
N
N
NH
N
N
N
N
N
O
OH
HO
HO
OH
O
N
O
HO
OH
-N
N
+
N
HO
OH
11
O
O
O
O
O
O
O
O
O
OH
OH
O
O
22
5
Scheme 5. Synthesis of 30-N-derivative of 15-membered azalide via “Click” reaction. Reagents and conditions: 11, sodium L-ascorbate, CuSO4$5H2O, 1/1 DMF/water, microwave
irradiation, 60 ꢁC, 5 min.

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370
K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
Table 1
Antibacterial activity (MIC/
m
gmlꢂ1) of novel 4-aminoquinoline derivatives of 30-N-substituted-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin against three macrolide
sensitive bacterial strains (S. pneumoniae, S. pyogenes and S. aureus) compared to azithromycin (AZ).
N
R2
OH
HO
N
O
HO
OH
O
O
O
O
R1
MIC (
AZ
m
g/ml)
12
13
14
15
16
17
18
19
20
21
22
R1
R2
clada clad
H
clad
H
clad
H
clad
H
clad
clad
clad
CH3
strains
S. aureus ATCC13709
S. pneumoniae SP030
S. pyogenes 3565
M. catarrhalis ATCC23246 0.06
H. influenzae ATCC49247
1
>64
4
16
4
>64
>64
>64
>64
>64
>64
4
16
8
>64
>64
>64
>64
>64
>64
8
16
1
>64
>64
>64
32
>64
32
32
4
>64
>64
>64
>64
>64
>64
>64
8
16
4
16
2
4
8
>64
16
0.5
0.5
2
0.06
0.125
2
>64
>64
>64
>64
>64
>64
a
Cladinosyl.
goods) this compound presented the best candidate for further
macrolide molecules so the parameters established for compound
profiling in the pharmacokinetic study.
12 are well in line with the previous data [13].
2.2.3. Pharmacokinetics
3. Discussion
The metabolic stability of compound 12 was evaluated in mouse,
rat and human liver microsomes and intrinsic clearance (CLi) of
CLi<0.6 mL/min*g liver indicated that the compound is stable in all
three species. The pharmacokinetic study was performed in CD-1
mice and PK parameters were determined in both the blood and
plasma (Table 3). Following intravenous administration compound
12 was characterized by a moderate systemic clearance, a large
volume of distribution, a very long half-life and low oral bioavail-
ability. The moderate clearance, long half-life and large volume of
distribution are all characteristics of most 14- to 16-membered
The need to design a new generation of antimalarial drugs that
can combat the most drug-resistant forms of the malarial parasite is
well recognized. Efforts of medicinal chemistry to bring novel,
effective and affordable agents to the market are obviously needed
but at the same time seem to be more and more difficult [43]. Our
work on antimalarial macrolides has lead us to identify the
compounds that could be classified as hybrid molecules since two
distinct chemical elements were present (azalide and chlor-
oquinoline moieties) [31]. The chemical “synergy” was very well
translated both to antimalarial activity and to pharmacokinetic
properties. In the continuation of these efforts we synthesized pilot
series of new compounds with the goal to further explore the
opportunities of this promising class. With the current set of
compounds we investigated whether the ease of synthesis could be
combined with the desired biological properties. As it is well
Table 2
Antimalarial activity of 4-aminoquinoline derivatives of 30-N-substituted-9-deoxo-
9a-methyl-9a-aza-9a-homoerythromycin determined against two P. falciparum
strains: W2 (strain resistant to chloroquine and pyrimethamine) and 3D7A (stan-
dard sensitive strain); and cytotoxicity evaluated in HepG2 cell line compared to
azithromycin (AZ) and chloroquine (CQ).
P. falciparum
IC50 (nM)
Cytotoxicity
IC50 M)
Table 3
(m
Pharmacokinetic parameters of compound 12 estimated in blood after intravenous
and oral gavage administration (12.5 mg/kg) to CD-1 mice.
3D7A
W2
HepG2
AZ
CQ
12
13
16
17
20
21
11513
16
77
636
3
11
2680
431
48
295
2
9
93
13
203
116
>50
>50
3
14
>50
27
Compound
CL
Vss
(L/kg)
116a
t1/2
(hr)
63.9b
DNAUCPO
(ng*hr/mL)
F
(%)
B/Pc
(mL/min/kg)
47.7a
12
13.28
5
1
a
% Extrapolated AUC > 20%.
r Value < 0.9.
B/P (blood/plasma) values were calculated as the ratio of AUC0ꢂt measured in
b
c
104
10
parallel.

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
371
known that azalides are potent antibacterial agents with desos-
amine C-30-dimethylamino group significantly contributing to their
binding for the 50S subunit of the 70S prokaryotic ribosome [40],
our compounds were envisaged as hybrid molecules comprising
azalide scaffold with bulky 4-aminoquinoline substituent linked at
the C-30 position. By such modification, we were aiming at elimi-
nation of antibacterial activity and retaining the antimalarial
potency. In the development of novel macrolides for malaria we
considered a separation of antibacterial and antiparasitic action to
be an essential task. While optimizing the efficacy against the
parasite any residual activity against bacteria could present an
obstacle since the consequences of sub-optimal dosing of the
antibiotic would contribute significantly to the spread of macrolide
resistance making this class of safe and effective antibacterial
agents useless to vulnerable paediatric population of malaria
endemic countries. Also, the results from the antibacterial activity
were used as an indirect marker of the macrolide mode of action
since their target are bacterial 70S ribosomes also present within
the apicoplast organelle of the parasite [44]. With the strategy
employed a set of promising compounds was obtained where
antibacterial activity was substantially diminished while their
antimalarial activity significantly improved over AZ. The substitu-
tion on C-30-dimethylamino group with 4-aminoquinoline-linker
substituents decreased the antibacterial potency of novel deriva-
tives by 4 to >100 fold but the additional removal of the cladinose
most efficiently eliminated the antibacterial activity. The level of
correlation between antibacterial activity and mode of action in the
parasite can not be precisely estimated due to the potential
differences in compound uptake and accumulation between
organisms but as previously mentioned tested pathogens share the
same target so it is likely that the inhibition of that target retards
the growth of both the bacteria and the parasite. The current set of
data can simply be used as an indicator for possible weakening of
ribosome target binding. On the other hand, the antimalarial
activity of analogues 13, 16, 21 was substantially increased over AZ
and even over CQ, with higher potencies recorded against CQ
resistant parasite. The obtained results indicate the existence of
a mechanism that enables the delivery of the chloroquinoline
moiety to the target site (the food vacuole) probably by increasing
the uptake into the parasite and/or overcoming the accumulation
obstacles that CQ alone faces [45]. The mechanism could be of
purely physicochemical nature since the tested compounds have
higher lipophilicity (according to the calculated o/w partition and
distribution coefficients clogP/clogD values, data not shown) than
their parent compounds and could penetrate membranes more
efficiently thus increasing their accumulation within the cells, as
already shown for some other macrolide compounds [46]. In
addition, the mechanism could have the biochemical background
like changing the substrate specificity for the transport proteins
involved in CQ efflux, or a combination of both.
(12e22), comprising 4-aminoquinoline moiety covalently liked to
macrolide scaffold, were synthesized by a straightforward synthetic
procedure. The novel C-30-substituted 4-aminoquinoline 15-
membered azalides represent a promising subclass of antima-
larial macrolides that exhibit high in vitro activity and selectivity
against sensitive and resistant P. falciparum parasites com-
plemented by the reduced antibacterial action. More compounds
would need to be synthesized and their efficacy determined in
order to verify the full potential of this class as novel antimalarial
agents.
5. Experimental
5.1. Chemistry
All solvents and reagents (SigmaeAldrich, Merck) were used as
supplied and all solvents are of the highest purity unless otherwise
noted. Mass spectra were obtained on a Waters Micromass ZQmass
spectrometer for ESþ-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 100e2000 Da, with a scan time of 1.5 s and
an interscan delay of 0.1 s in a continuum mode. Reserpine was
used as the external mass calibrant lock mass ([M þ H]þ
¼
609.2812 Da). The elemental composition was calculated using
a MassLynx v4.1 for the [M þ H]þ and the mass error quoted
within ꢃ5 ppm range. NMR spectra were recorded on a Bruker
Avance DRX500 or Bruker Avance DPX300 spectrometer in CDCl3
or DMSO and chemical shifts are reported in ppm using TMS as an
internal standard. In synthetic procedures, column chromatog-
raphy was carried out over Merck Kieselgel 60 (230e400 mesh) or
on SPE cartrige with average size silica 50 mm. Thin layer chro-
matography was performed on 0.24 mm silica gel plates Merck TLC
60F254. The eluent used was indicated and the solvent ratios refer
to the volume. In general, organic solutions were dried with
anhydrous Na2SO4 or K2CO3, evaporation and concentrations were
carried out under reduced pressure below 40 ꢁC, unless otherwise
noted.
The purity of each compound was determined on an Agilent
1100 Series LC/MSD trap. The analysis was carried out using MS
and UV detection at 240 nm on a YMC Pack Pro C18 column,
150 mm ꢀ 4.6 mm i.d., 3
mm particle size. Gradient elution at
flow rate 1.0 ml/min started with 10% of acetonitrile/90% of
40 mM ammonium acetate buffer solution and ended after
15 min with 90% of acetonitrile/10% of 40 mM ammonium acetate
buffer solution. The ESI source was operated in the positive ion
mode. Nitrogen was used as nebulizing 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.
Using the described method the determined purity for each
compound was ꢄ95%.
The in vivo evaluation of compound 12 showed typical macro-
lide pharmacokinetic profile but with low oral exposure probably
due to the low absorption from the intestine [13]. So, despite the
fact that compounds active against P. falciparum in vitro were
identified, the fact remains that the determined oral exposure and
oral bioavailability for compound 12 were low, further develop-
ment for this compound is unlikely. In order to fully appreciate this
class of compounds as novel antimalarial agents more analogues
would need to be synthesized and their biological profile deter-
mined in the efficacy experiments.
5.1.1. 3-O-Decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A (1b)
9-Deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 1a (10 g,
0.013 mol) was dissolved in methanol (100 ml) and water (50 ml)
and pH was adjusted to 1.79 with 2 N HCl. The reaction mixture was
stirred at r.t. 24 h. After 24 h the reaction mixture was stirred at
50 ꢁC for 4 h and 30 min. Then the reaction mixture was stirred at
r.t. for next 24 h. The methanol was evaporated. DCM (50 ml) was
added, pH adjusted to 1.79 and layers separated (3 times). Then
DCM (80 ml) was added to the aqueous layer, pH adjusted to 9.02,
and the layers were separated (3 times). The organic layers (sepa-
rated at pH 9.02) were combined and washed with sat. NaCl
4. Conclusions
Following multistep synthetic route novel C-30-substituted-9-
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A, eleven derivatives

ꢀ ꢁ
K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
372
(6 ꢀ 60 ml). The organic layer was dried over Na2SO4 and evapo-
(d, J ¼ 10.38 Hz, 1H), 4.47e4.53 (m, 1H), 3.85 (d, J ¼ 10.07 Hz, 1H),
3.64 (s, 1H), 3.60e3.63 (m, 1H), 3.59e3.62 (m, 1H), 3.17e3.23 (m,
1H), 3.14 (d, J ¼ 8.85 Hz, 1H), 2.72e2.79 (m, 1H), 2.65e2.72 (m, 1H),
2.62 (d, J ¼ 6.41 Hz, 1H), 2.41e2.45 (m, 6H), 2.24e2.27 (m, 1H), 2.00
(dd, J ¼ 3.81, 11.75 Hz, 1H), 1.87e1.96 (m, 1H), 1.82 (d, 2H), 1.59e1.62
(m, 1H), 1.53e1.56 (m, 1H), 1.42e1.51 (m, 1H), 1.28e1.33 (m, 6H),
1.25 (d, J ¼ 6.10 Hz, 3H), 1.19e1.20 (m, 1H), 1.13 (d, J ¼ 6.71 Hz, 3H),
1.09 (s, 3H), 1.04 (d, J ¼ 7.32 Hz, 3H), 0.96 (d, J ¼ 6.41 Hz, 3H), 0.89
rated yielding 74% (7.166 g, 0.012 mol) of title compound as a white
powder. 1H NMR (400 MHz, DMSO-d6)
d 6.20 (br. s., 1H), 5.07
(d, J ¼ 6.59 Hz, 1H), 4.95 (d, J ¼ 11.00 Hz, 1H), 4.55 (d, J ¼ 7.40 Hz,
1H), 4.31 (s, 1H), 4.19 (s, 1H), 4.04 (d, J ¼ 8.09 Hz, 1H), 3.38e3.45 (m,
3H), 3.28e3.33 (m, 1H), 2.99e3.11 (m, 1H), 2.67 (q, J ¼ 7.00 Hz, 1H),
2.45e2.48 (m,1H), 2.40 (ddd, J ¼ 4.50,10.00,12.00 Hz,1H), 2.30 (dd,
J ¼ 3.00, 12.00 Hz, 1H), 2.21 (s, 9H), 2.09 (q, J ¼ 7.30 Hz, 1H), 2.04
(t, J ¼ 12.00 Hz, 1H), 1.68e1.82 (m, 2H), 1.59 (d, J ¼ 12.37 Hz, 1H),
1.36e1.45 (m, 2H), 1.27e1.39 (m, 1H), 1.05e1.14 (m, 10H), 0.97
(s, 3H), 0.94 (d, J ¼ 6.59 Hz, 3H), 0.87 (d, J ¼ 7.17 Hz, 3H), 0.83
(d, J ¼ 6.82 Hz, 3H), 0.75 (t, J ¼ 7.22 Hz, 3H). 13C NMR (101 MHz,
(t, J ¼ 7.20 Hz, 3H). 13C NMR (126 MHz, CDCl3-d)
d 177.6, 105.8, 93.9,
78.0, 77.8, 74.1, 73.9, 73.7, 69.9, 62.0, 61.0, 57.3, 44.5, 41.4, 36.8, 36.3,
32.7, 29.6, 26.2, 21.3, 21.0, 16.3, 11.1, 8.3, 7.9.
DMSO-d6):
d
175.27, 102.42, 87.59, 76.75, 76.21, 75.96, 73.46, 72.27,
5.1.4. 30-(N-Demethyl)-30-N-(
b-cyanoethyl)-9-deoxo-9a-methyl-
70.44, 68.75, 67.90, 64.62, 61.28, 43.24, 40.75, 40.34, 40.34, 36.09,
35.32, 30.63, 26.02, 25.76, 21.16, 21.06, 20.52, 17.60, 16.13, 10.34,
8.27, 6.22.
9a-aza-9a-homoerythromycin A (3a)
30-N-(Demethyl)-9-deoxo-9a-methyl-9a-aza-9a-homoery-
thromycin A 2a (6.14 g, 8.4 mmol) in acrylonitrile (70 ml) was
refluxed for 18 h. The acrylonitrile was evaporated under reduced
pressure to afford 96% of the title product (6.37 g, 0.0081 mol) as
a white powder. MS m/z: (ES): MHþ ¼ 788; 1H NMR (500 MHz,
5.1.2. 30-N-(Demethyl)-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A (2a)
9-Deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 1a (20 g;
26.7 mmol) was dissolved in 250 ml of acetone, and diluted diethyl
azodicarboxylate (DEAD, 40% in toluene) (14.6 g; 0.085 mol) in
acetone (32 ml) was added slowly over 1 h. The reaction was stirred
at room temperature for 24 h and evaporated to dryness. The white
residue was diluted in MeOH (80 ml) and saturated NHþ4 Clꢂ (80 ml)
and refluxed for 1 h. After evaporation to dryness, the residue was
taken up with water (110 ml) and the pH adjusted to 9.4 with
aqueous ammonium hydroxide and finally extracted with DCM
(4 ꢀ 100 ml). The organic layer was dried over Na2SO4 and evap-
orated to dryness. The residue was washed with small amount of
DCM (50e80 ml) to afford 77.9% of the title product (15.282 g,
0.021 mol). MS m/z: (ES): MHþ ¼ 735. 1H NMR (500 MHz, CDCl3-d)
DMSO-d6)
d
4.84 (d, J ¼ 4.88 Hz, 1H), 4.74 (dd, J ¼ 2.75, 10.07 Hz,
1H), 4.40 (d, J ¼ 7.32 Hz, 1H), 4.29 (br. s., 2H), 4.20 (d, J ¼ 7.32 Hz,
1H), 4.16 (d, 2.44 Hz, 1H), 4.04e4.12 (m, 1H), 4.02
J
¼
(d, J ¼ 3.05 Hz, 1H), 3.62e3.72 (m, 1H), 3.51 (d, J ¼ 7.02 Hz, 1H),
3.44 (d, J ¼ 8.54 Hz, 1H), 3.38 (dd, J ¼ 2.29, 11.75 Hz, 2H), 3.24 (s,
3H), 3.04e3.10 (m, 2H), 2.92 (dd, J ¼ 7.32, 9.46 Hz, 1H), 2.61e2.70
(m, 3H), 2.36 (d, J ¼ 10.99 Hz, 1H), 2.26 (d, J ¼ 13.00 Hz, 1H), 2.25
(s, 3H), 2.20 (s, 3H), 2.11 (dd, J ¼ 11.44 Hz, 1H), 1.84e1.92 (m, 2H),
1.72e1.82 (m, J ¼ 2.59, 7.37, 7.37, 7.51, 14.51 Hz, 1H), 1.65 (ddd,
J ¼ 1.83, 10.99 Hz, 1H), 1.53 (d, J ¼ 13.50 Hz, 1H), 1.51 (dd, J ¼ 4.50,
13.00 Hz, 1H), 1.32e1.43 (m, 1H), 1.27 (dd, J ¼ 8.24, 14.04 Hz, 1H),
1.17 (s, 3H), 1.15 (d,
J
¼
6.10 Hz, 4H), 1.14 (s, 3H), 1.08
(d, J ¼ 7.32 Hz, 3H), 1.06 (d, J ¼ 5.80 Hz, 3H), 1.01 (s, 3H), 0.94
d
5.34 (br. s., 1H), 5.15 (d, J ¼ 4.58 Hz, 1H), 4.71 (dd, J ¼ 2.29, 9.92 Hz,
(d, J ¼ 7.32 Hz, 6H), 0.85 (d, J ¼ 7.02 Hz, 3H), 0.79 (t, J ¼ 7.48 Hz,
1H), 4.42 (d, J ¼ 7.32 Hz, 1H), 4.25 (d, J ¼ 1.83 Hz, 1H), 4.01e4.14
(m, 1H), 3.66 (s, 1H), 3.62 (d, J ¼ 7.02 Hz, 1H), 3.51e3.60 (m, 1H),
3.34 (s, 3H), 3.27 (dd, J ¼ 7.63, 9.46 Hz, 1H), 3.04 (d, J ¼ 9.46 Hz, 1H),
2.70e2.76 (m, 1H), 2.65e2.70 (m, 1H), 2.57e2.64 (m, 1H), 2.56
(d, J ¼ 9.46 Hz, 1H), 2.48 (s, 3H), 2.36 (d, J ¼ 15.26 Hz, 1H), 2.32 (s,
3H), 2.03e2.08 (m, 1H), 1.99e2.02 (m, 1H), 1.96e2.02 (m, 2H),
1.87e1.95 (m, 1H), 1.72 (d, J ¼ 14.34 Hz, 1H), 1.59 (dd, J ¼ 4.88,
15.26 Hz, 1H), 1.46 (ddd, J ¼ 7.32, 9.77, 14.34 Hz, 1H), 1.33
(d, J ¼ 6.41 Hz, 3H), 1.32 (s, 3H), 1.26 (s, 3H), 1.23e1.24 (m, 2H), 1.22
(d, J ¼ 6.10 Hz, 3H), 1.19 (d, J ¼ 7.32 Hz, 3H), 1.07e1.12 (m, 6H), 0.98
(d, J ¼ 7.63 Hz, 3H), 0.92 (d, J ¼ 5.80 Hz, 3H), 0.89 (t, J ¼ 7.48 Hz, 3H).
3H); 13C NMR (75 MHz, DMSO-d6)
d/ppm: 175.02, 156.45, 119.98,
102.11, 94.43, 82.92, 77.33, 76.27, 74.84, 73.53, 72.69, 72.46, 70.55,
68.62, 66.85, 64.72, 64.48, 61.37, 60.84, 60.36, 77.33, 76.27, 74.84,
73.53, 72.69, 72.46, 70.55, 68.62, 66.85, 64.72, 64.48, 61.37, 60.84,
60.36, 50.01, 48.72, 44.65, 41.57, 35.87, 35.67, 34.57, 32.79, 29.24,
27.35, 26.01, 22.03, 21.29, 20.93, 18.35, 17.63, 16.79, 14.73, 14.44,
10.87, 9.03, 6.69.
5.1.5. 30-N-(Demethyl)-30-N-(
b-cyanoethyl)-3-O-decladinosyl-9-
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (3b)
A solution of 30-N-(demethyl)-3-O-decladinosyl-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A 2b (1.8192 g, 3.15 mmol)
in acrylonitrile (20 ml) was heated to 80 ꢁC over night (24 h).
The acrylonitrile was evaporated to afford crude product
(1.2043 g), which was purified by column chromatography
(DCM:MeOH:NH4OH ¼ 90:9:0.5) yielding 37% of the title product
(727 mg, 0.0012 mol). MS m/z: (ES): MHþ ¼ 630. 1H NMR (500 MHz,
13C NMR (75 MHz, CDCl3-d)
d 102.3, 94.7, 83.7, 78.1, 77.9, 77.5, 74.2,
74.3, 73.7, 73.6, 73.0, 70.0, 68.4, 65.7, 62.6, 60.5, 49.5, 45.5, 42.3,
42.3, 36.8, 36.2, 34.7, 32.8, 27.5, 26.7, 22.0, 21.6, 21.3, 21.1, 18.2, 16.3,
14.7, 11.2, 9.4, 7.3.
5.1.3. 30-N-(Demethyl)-3-O-decladinosyl-9-deoxo-9a-methyl-9a-
aza-9a-homoerythromycin A (2b)
DMSO-d6)
d
6.22 (br. s., 1H), 5.13 (d, J ¼ 6.41 Hz, 1H), 4.95
A solution of 3-O-decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A 1b (7.16 g, 0.0121 mol), NaOAc anhydrous
(4.97 g, 0.061 mol) and I2 (4.61 g, 0.018 mol) in MeOH (150 ml) was
irradiated with a 500 W lamp for 2 h and 30 min. The reaction
mixture become colourless and an additional amount of I2 (1.15 g)
was added. The reaction mixture was stirred at r.t. for 24 h. The 1 M
Na2S2O3 added and the solvent evaporated. To the crude residue
water (200 ml) and DCM (100 ml) were added and the pH was
adjusted to pH 4 and layers separated (2 ꢀ 100 ml). To the aqueous
layer DCM (100 ml) was added and the pH adjusted to 9.5
(2 ꢀ 100 ml). Layers were separated and the organic layer was
washed with water. The organic layer was dried over Na2SO4 and
evaporated yielding 97% of the title product (6.809 g, 0.012 mol).
(dd, J ¼ 1.98, 11.14 Hz, 1H), 4.56 (d, J ¼ 7.32 Hz, 1H), 4.31 (s, 1H), 4.22
(d, J ¼ 2.75 Hz, 1H), 4.06 (d, J ¼ 7.93 Hz, 1H), 3.43e3.47 (m, 1H),
3.41e3.43 (m, 2H), 3.31e3.34 (m, 1H), 3.11 (ddd, J ¼ 2.90, 7.32,
9.92 Hz, 1H), 2.77e2.87 (m, 1H), 2.69e2.74 (m, 1H), 2.65e2.69
(m, 1H), 2.56e2.63 (m, 2H), 2.49e2.56 (m, 1H), 2.46e2.51
(m, 1H), 2.30 (d, J ¼ 10.07 Hz, 1H), 2.25 (s, 3H), 2.22 (s, 3H), 2.10
(d, J ¼ 7.32 Hz, 1H), 2.05 (t, J ¼ 12.05 Hz, 1H), 1.75e1.81 (m, 1H),
1.71e1.74 (m, 1H), 1.54e1.61 (m, 1H), 1.41e1.46 (m, 1H), 1.35e1.40
(m, 1H), 1.32 (dd, J ¼ 5.19, 14.34 Hz, 1H), 1.19 (d, J ¼ 11.29 Hz, 1H),
1.07e1.15 (m, 9H), 0.98 (s, 4H), 0.95 (d, J ¼ 6.71 Hz, 3H), 0.88
(d, J ¼ 7.32 Hz, 3H), 0.84 (d, J ¼ 7.02 Hz, 3H), 0.76 (t, J ¼ 7.48 Hz, 3H).
13C NMR (126 MHz, DMSO-d6)
d 175.5, 120.1, 102.5, 87.8, 77.0, 76.4,
76.1, 73.6, 72.5, 70.8, 69.0, 68.0, 64.5, 61.5, 50.4, 43.5, 40.9, 36.1, 35.5,
33.7, 26.2, 25.9, 21.4, 21.2, 20.7, 17.8, 16.9, 16.3, 10.5, 8.5, 6.3.
MS m/z: (ES): MHþ ¼ 577.2. 1H NMR (500 MHz, CDCl3-d)
d 4.83

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
373
5.1.6. 30-(N-Demethyl)-30-N-(
9a-aza-9a-homoerythromycin A (4a)
30-(N-Demethyl)-30-N-(
-cyanoethyl)-9-deoxo-9a-methyl-9a-
g-aminopropyl)-9-deoxo-9a-methyl-
(10 ml). According to TLC, the reaction was finished, so water
(20 ml) was added and the layers separated. The organic layer was
dried over Na2SO4. Solvent was evaporated affording crude product
(0.205 g) which was further purified by column chromatography
(eluent DCM/MeOH/NH4OH ¼ 90/9/1.5) yielding 5.7% of the title
product (30 mg, 0.04 mmol) as white powder. MS m/z: (ES):
b
aza-9a-homoerythromycin A 3a (6.37 g, 8.08 mmol) was dissolved
in glacial acetic acid (150 ml) and hydrogenated in Parr reactor over
PtO2 (1.3 g) for 24 h at 5.0 Barr. The catalyst was filtered off. Filtrate
(acetic acid) was evaporated. Water (50 ml) and DCM (100 ml)
were added to reaction mixture and pH adjusted to 9.5. Layers
were separated and the aqueous layer was extracted by DCM
(3 ꢀ 100 ml). The organic layers were combined and dried over
Na2SO4 then evaporated yielding 5.9 g crude product. The crude
product was combined with another batch (prepared in a similar
manner) and purified by column chromatography on silica-gel
eluting with CH2Cl2:MeOH:NH4OH ¼ 90:9:1.5, resulting 38% of
the title product (2.44 g, 0.003 mol) as a white powder. MS m/z:
MHþ ¼ 773. 1H NMR (500 MHz, DMSO-d6)
d
4.84 (d, J ¼ 4.88 Hz,
1H), 4.74 (dd, J ¼ 2.75, 10.07 Hz, 1H), 4.40 (d, J ¼ 7.32 Hz, 1H), 4.29
(br. s., 2H), 4.20 (d, J ¼ 7.32 Hz, 1H), 4.16 (d, J ¼ 2.44 Hz, 1H),
4.04e4.12 (m, 1H), 4.02 (d, J ¼ 3.05 Hz, 1H), 3.62e3.72 (m, 1H), 3.51
(d, J ¼ 7.02 Hz, 1H), 3.44 (d, J ¼ 8.54 Hz, 1H), 3.38 (dd, J ¼ 2.29,
11.75 Hz, 2H), 3.24 (s, 3H), 3.04e3.10 (m, 2H), 2.92 (dd, J ¼ 7.32,
9.46 Hz, 1H), 2.61e2.70 (m, 3H), 2.36 (d, J ¼ 10.99 Hz, 1H), 2.26 (d,
J ¼ 13.00 Hz,1H), 2.25 (s, 3H), 2.20 (s, 3H), 2.11 (dd, J ¼ 11.44 Hz,1H),
1.84e1.92 (m, 2H), 1.72e1.82 (m, J ¼ 2.59, 7.37, 7.37, 7.51, 14.51 Hz,
1H), 1.65 (ddd, J ¼ 1.83, 10.99 Hz, 1H), 1.53 (d, J ¼ 13.50 Hz, 1H), 1.51
(dd, J ¼ 4.50, 13.00 Hz, 1H), 1.32e1.43 (m, 1H), 1.27 (dd, J ¼ 8.24,
14.04 Hz, 1H), 1.17 (s, 3H), 1.15 (d, J ¼ 6.10 Hz, 4H), 1.14 (s, 3H), 1.08
(d, J ¼ 7.32 Hz, 3H), 1.06 (d, J ¼ 5.80 Hz, 3H), 1.01 (s, 3H), 0.94
(d, J ¼ 7.32 Hz, 6H), 0.85 (d, J ¼ 7.02 Hz, 3H), 0.79 (t, J ¼ 7.48 Hz, 3H).
(ES): MHþ ¼ 792. 1H NMR (500 MHz, DMSO-d6)
d 7.52 (br. s., 1H),
4.84 (d, J ¼ 4.88 Hz, 1H), 4.74 (dd, J ¼ 2.44, 10.07 Hz, 1H), 4.41
(d, J ¼ 7.02 Hz, 1H), 4.16 (d, J ¼ 2.44 Hz, 1H), 4.04e4.12 (m, 1H),
3.60e3.74 (m, 1H), 3.51 (d, J ¼ 7.02 Hz, 1H), 3.44 (s, 1H), 3.23 (s, 3H),
3.05 (dd, J ¼ 7.78, 9.61 Hz, 1H), 2.91 (d, J ¼ 9.46 Hz, 1H), 2.62e2.72
(m, 2H), 2.54e2.61 (m, 3H), 2.42e2.49 (m, 1H), 2.35e2.38 (m, 1H),
2.31e2.35 (m, 1H), 2.26 (d, J ¼ 14.95 Hz, 1H), 2.20 (s, 3H), 2.17
(s, 3H), 2.11 (t, J ¼ 11.75 Hz, 1H), 1.83e1.92 (m, 2H), 1.70e1.82
(m, 1H), 1.58 (d, J ¼ 12.82 Hz, 1H), 1.51e1.54 (m, 1H), 1.45e1.51
(m, 2H), 1.37 (ddd, J ¼ 7.48, 9.99, 14.27 Hz, 1H), 1.26 (dd, J ¼ 8.09,
13.58 Hz, 1H), 1.17 (s, 3H), 1.15 (d, J ¼ 6.10 Hz, 3H), 1.13 (br. s., 3H),
1.03e1.11 (m, 7H), 1.01 (s, 3H), 0.90e0.97 (m, 6H), 0.85
(d, J ¼ 6.71 Hz, 3H), 0.79 (t, J ¼ 7.48 Hz, 3H). 13C NMR (75 MHz,
13C NMR (75 MHz, DMSO-d6)
d/ppm: 176.91, 101.94, 94.32, 82.50,
81.73, 77.23, 77.16, 76.18, 74.76, 74.35, 73.46, 72.59, 72.42, 70.65,
68.49, 66.73, 64.56, 62.81, 61.26, 48.67, 44.51, 43.54, 41.71, 41.43,
35.59, 35.52, 34.53, 32.33, 27.18, 25.91, 21.91, 21.26, 20.86, 20.79,
18.30, 17.55, 14.58, 10.76, 8.91, 6.63. HRMS (ES) calcd for C40 H73 N2
O12 (M þ Hþ) 773.5164, found 773.5149.
5.1.9. N-(7-Chloro-4-quinolinyl)glycine (7)
DMSO-d6)
d
/ppm: 177.04, 102.18, 94.42, 82.69, 77.29, 76.26, 74.79,
4,7-Dichloroquinoline (4.4 g, 22.22 mmol), glycine (3.34 g,
44.4 mmol) and phenol (12.02 g, 128 mmol) were placed into
a reaction flask and stirred at 120 ꢁC for 18 h. An additional 1 eq. of
glycine (1.668 g, 22.22 mmol) was added. The reaction mixture was
stirred for 3 h and then cooled to r.t. and diluted with EtOAc (50 ml).
The precipitate was filtered off, washed with EtOAc and then dis-
solved in 10% Na2CO3 (supported by heating). Into this solution
toluene (5 mL) was added, the solution cooled to 5 ꢁC and the pH
adjusted to 5.7 (by 6 M HCl). The precipitate was filtered off,
washed with water and toluene, and dried under reduced pressure
at 60 ꢁC over night to yield 71.3% of the title product (3.75 g,
0.016 mol). MS m/z: (ES): MHþ ¼ 237. 1H NMR (300 MHz, DMSO-d6)
73.53, 72.72, 72.47, 70.33, 68.64, 67.06, 64.66, 64.42, 61.37, 77.29,
76.26, 74.79, 73.53, 72.72, 72.47, 70.33, 68.64, 67.06, 64.66, 64.42,
61.37, 50.41, 48.71, 44.63, 41.82, 41.55, 36.84, 35.67, 34.58, 30.46,
27.33, 26.01, 22.02, 21.40, 20.94, 18.39, 17.62, 14.71, 10.87, 8.97, 6.70.
5.1.7. 30-N-(Demethyl)-30-N-(
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (4b)
30-N-(Demethyl)-30-N-(
-cyanoethyl)-3-O-decladinosyl-9-
g-aminopropyl)-3-O-decladinosyl-9-
b
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 3b (0.6086 g,
0.97 mmol) was dissolved in glacial acetic acid (40 ml) and
hydrogenated in a Parr reactor over PtO2 (0.218 g) for 24 h at 5.0
Barr. The catalyst was filtered off. The filtrate (acetic acid) was
cooled, DCM (100 ml) was added and the pH was adjusted with 40%
NaOH to pH 9.5. Then the reaction mixture was extracted by DCM
(3 ꢀ 50 ml). Layers were separated; the organic layer was dried over
Na2SO4 and evaporated yielding 85% of the title product (0.5243 g,
0.83 mmol) as a white powder. MS m/z: (ES): MHþ ¼ 633. 1H NMR
d
8.40 (1 H, d, J ¼ 5.5 Hz), 8.18 (1 H, d, J ¼ 8.9 Hz), 7.81 (1 H,
d, J ¼ 2.1 Hz), 7.43 (1 H, dd, J ¼ 8.9, 2.2 Hz), 6.38 (1H, d, J ¼ 5.5 Hz),
3.99 (2 H, CH2, s). 13C NMR (75 MHz, DMSO-d6)
171.35 (C]O),
d
151.98, 150.72, 149.29, 134.12, 127.84, 124.86, 124.15, 117.98, 99.80,
45.27 (CH2).
(500 MHz, DMSO-d6)
d
6.21 (br. s., 1H), 4.95 (dd, J ¼ 1.68, 11.14 Hz,
5.1.10. 3-(7-Chloroquinolin-4-ylamino)propyl alcohol (9)
1H), 4.57 (d, J ¼ 7.63 Hz, 1H), 3.42 (s, 3H), 3.33 (d, J ¼ 9.16 Hz, 1H),
3.09 (dd, J ¼ 7.63, 9.77 Hz, 1H), 2.68 (q, J ¼ 6.61 Hz, 1H), 2.53e2.62
(m, 3H), 2.44e2.48 (m, 2H), 2.36 (dt, J ¼ 6.56, 12.51 Hz, 1H), 2.30
(d, J ¼ 10.07 Hz, 1H), 2.22 (s, 3H), 2.18 (s, 3H), 2.06e2.12 (m, 1H),
2.00e2.06 (m, 1H), 1.75e1.81 (m, 1H), 1.71e1.75 (m, 1H), 1.56e1.63
(m, 1H), 1.47 (t, J ¼ 6.71 Hz, 2H), 1.40e1.44 (m, 1H), 1.36e1.40
(m, 1H), 1.30e1.37 (m, 1H), 1.05e1.14 (m, 10H), 0.98 (s, 3H), 0.95
(d, J ¼ 6.71 Hz, 3H), 0.88 (d, J ¼ 7.32 Hz, 3H), 0.84 (d, J ¼ 7.02 Hz, 3H),
4,7-Dichloroquinoline (5.0 g, 25.2 mmol) and 3-aminopropanol
(23.17 ml, 303 mmol) were heated with stirring at 130e140 ꢁC for
24 h. After cooling, the reaction was poured into water (150 ml) and
filtered off, and the solid was dried then boiled in ethyl acetate
(50 ml) to give 86% of the title compound (5.024 g, 0.021 mol). MS
m/z: (ES): MHþ ¼ 237. 1H NMR (500 MHz, DMSO-d6)
d 8.39
(d, J ¼ 5.50 Hz, 1H), 8.25 (d, J ¼ 9.00 Hz,1H), 7.77 (d, J ¼ 2.50 Hz,1H),
7.43 (dd, J ¼ 2.00, 9.00 Hz, 1H), 7.29 (t, J ¼ 5.50 Hz, 1H), 6.46
(d, J ¼ 5.50 Hz, 1H), 4.59 (t, J ¼ 5.00 Hz, 1H) 3.55 (q, J ¼ 6.00 Hz, 2H),
3.31e3.34 (m, 2H), 1.81 (quin, J ¼ 6.50 Hz, 2H); 13C NMR (125 MHz,
0.76 (t, J ¼ 7.32 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
d 175.4,102.7,
87.9, 76.9, 76.4, 76.1, 73.6, 72.5, 70.5, 69.0, 68.2, 64.7, 61.5, 50.7, 43.4,
41.0, 39.0, 37.0, 36.3, 35.5, 31.1, 30.5, 26.2, 26.0, 21.3, 21.3, 20.6, 17.8,
16.3, 10.5, 8.5, 6.4.
DMSO-d6) d/ppm: 152.30, 150.49, 149.45, 133.69, 127.86, 124.41,
124.36, 117.83, 98.97, 58.94, 39.88, 31.44.
5.1.8. 30-N-(Demethyl)-30-N-(1-propynyl)-9-deoxo-9a-methyl-9a-
aza-9a-homoerythromycin A (5)
5.1.11. 3-(7-Chloroquinolin-4-ylamino)propyl
methanesulfonate (10)
Methansulfonyl chloride (0.181 ml) was added to a solution of 3-
(7-chloroquinolin-4-ylamino)propyl alcohol (0.5 g, 2.112 mmol),
triethylamine (0.66 ml) and THF (10 ml) at 0 ꢁC. The mixture was
A mixture of 30-N-(demethyl)-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A 2a (0.5 g, 0.680 mmol) and propargyl
bromide (0.121 g, 0.816 mmol) were stirred over night at r.t. in THF

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
374
stirred for 45 min at 0 ꢁC and diluted with NaHCO3 solution (20 ml).
The reaction was extracted with diethyl ether (20 ml then
2 ꢀ 10 ml). The organic extracts were dried over Na2SO4, filtered,
and evaporated to yield of the title compound (0.524 g,
1.588 mmol). The crude product was used in next step without
further purification. MS m/z: (ES): MHþ ¼ 315.19.
18.2, 16.3, 14.9, 11.2, 9.5, 7.4. HRMS (ES) calcd for C53 H89 N5 O13 Cl
(M þ Hþ) 1038.6145, found 1038.6143.
5.1.14. 30-N-(Demethyl)-30-N-[3-({4-[(7-chloro-4-quinolinyl)
amino]butanoyl}amino)propyl]-3-O-decladinosyl-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A (13)
To a solution of 30-N-(demethyl)-30-N-(
g-aminopropyl)-3-O-
5.1.12. 3-(7-Chloroquinolin-4-ylamino)propyl azide (11)
decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A
4b (0.2 g, 0.316 mmol) in DCM (8 ml), 4-[(7-chloro-4-quinolinyl)
amino]butanoic acid (0.093 g, 0.354 mmol), HOBt (0.0564 g,
0.4172 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(0.104 g, 0.544 mmol) and Et3N (0.25 ml) were added and the
reaction mixture was stirred over night at r.t. NaHCO3 (10 ml) was
added, the organic layer separated, and the pH of the aqueous layer
was adjusted to 6.5 and extracted with DCM (30 ml). DCM (30 ml)
was added to the aqueous layer, the pH adjusted to 9.0 and
extracted with DCM (3 ꢀ 30 ml). The DCM layers at pH 6.5 and 9.0
were combined and dried over Na2SO4. DCM was evaporated
affording crude product (0.22 g) which was further purified by
column chromatography (eluent DCM:MeOH:NH4OH ¼ 90:9:0.5)
yielding 28% of the title product (0.078 g, 0.089 mmol) as a light
yellow powder. MS m/z: (ES): MHþ ¼ 881. 1H NMR (500 MHz,
To
a suspension of 3-(7-chloroquinolin-4-ylamino)propyl
methanesulfonate 10 (0.524 g, 1.588 mmol) in anhydrous DMF
(15 ml), sodium azide (0.207 g, 3.18 mmol) was added, and the
mixture heated over night at 55 ꢁC. According to TLC, the reaction
was complete, so water (20 ml) was added. The product was
extracted with toluene (3 ꢀ 50 ml). The organic layer was washed
with saturated brine and dried over Na2SO4. The solvent was
evaporated to afford 96% of the title product (0.4 g, 1.52 mmol). MS
m/z: (ES): MHþ ¼ 262.19. 1H NMR (500 MHz, DMSO-d6)
d 8.40
(d, J ¼ 5.19 Hz, 1H), 8.27 (d, J ¼ 9.16 Hz, 1H), 7.79 (d, J ¼ 2.14 Hz, 1H),
7.45 (dd, J ¼ 2.29, 9.00 Hz, 1H), 7.32 (t, J ¼ 5.04 Hz, 1H), 6.48
(d, J ¼ 5.49 Hz, 1H), 3.49 (t, J ¼ 6.87 Hz, 2H), 3.20e3.38 (m, 2H), 1.91
(t, J ¼ 6.87 Hz, 2H); 13C NMR (126 MHz, DMSO-d6)
d 151.8, 150.0,
149.0, 133.4, 127.5, 124.1, 117.5, 98.6, 48.5, 39.5, 27.1; HRMS (ES)
calcd for C12 H12 N4 O Cl (M þ Hþ) 263.0700, found 263.0706.
CDCl3)
d
8.42 (d, J ¼ 5.19 Hz, 1H), 7.90 (d, J ¼ 1.83 Hz, 1H), 7.88
(d, J ¼ 8.85 Hz, 1H), 7.34 (dd, J ¼ 1.83, 8.85 Hz, 1H), 6.87 (br. s., 1H),
6.81 (br. s., 1H), 6.25 (d, J ¼ 5.49 Hz, 1H), 4.75 (d, J ¼ 10.68 Hz, 1H),
4.43 (d, J ¼ 7.32 Hz, 1H), 3.79 (d, J ¼ 10.07 Hz, 1H), 3.64 (s, 1H), 3.57
(s, 1H), 3.41e3.45 (m, 1H), 3.35e3.39 (m, 1H), 3.28e3.32 (m, 1H),
3.26e3.29 (m, 2H), 3.24e3.27 (m, 1H), 2.69e2.77 (m, 1H),
2.61e2.65 (m, 1H), 2.48e2.54 (m, 3H), 2.41e2.46 (m, 2H), 2.37
(s, 3H), 2.34e2.36 (m, 1H), 2.28e2.33 (m,1H), 2.14 (s, 3H), 2.10e2.13
(m, 2H), 2.04e2.07 (m, 1H), 1.92e1.94 (m, 1H), 1.86e1.93 (m, 1H),
1.62e1.65 (m, 1H), 1.57e1.64 (m, 2H), 1.56e1.59 (m, 1H), 1.49e1.54
(m, 1H), 1.45 (dd, J ¼ 5.80, 14.34 Hz, 1H), 1.31 (s, 3H), 1.26e1.29
(m, 1H), 1.25e1.27 (m, 3H), 1.21 (d, J ¼ 6.71 Hz, 3H), 1.12
(d, J ¼ 6.71 Hz, 3H), 1.07 (s, 3H), 1.05 (d, J ¼ 7.32 Hz, 3H), 0.91
(d, J ¼ 7.02 Hz, 3H), 0.87 (t, J ¼ 7.20 Hz, 3H). 13C NMR (126 MHz,
5.1.13. 30-N-(Demethyl)-30-N-[3-({4-[(7-chloro-4-quinolinyl)
amino]butanoyl}amino)propyl]-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A (12)
To
a
solution of 30-N-(demethyl)-30-N-(
g-aminopropyl)-9-
deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A
4a (0.1 g,
0.126 mmol) in DCM (8 ml), 4-[(7-chloro-4-quinolinyl)amino]
butanoic acid (0.039 g, 0.165 mmol), HOBt (0.0216 g,
0.169 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(0.042 g, 0.22 mmol) and Et3N (0.1 ml) were added and the
reaction mixture was stirred over night at r.t. NaHCO3 (10 ml) was
added, the organic layer separated, pH of the aqueous layer was
adjusted to 6.5 and extracted with DCM. DCM (10 ml) was added
to the aqueous layer, pH adjusted to 9.0 and extracted with DCM
(10 ml). The DCM layers at pH 6.5 and 9.0 were combined and
dried over Na2SO4. The solvent was evaporated affording crude
product (102 mg). The crude product was combined with another
CDCl3)
d 177.5, 173.6, 151.5, 150.5, 148.6, 134.9, 127.9, 125.2, 122.4,
117.4, 106.6, 98.2, 94.5, 79.5, 77.5, 75.7, 74.3, 73.1, 71.0, 70.8, 69.8,
65.4, 62.6, 51.4, 44.4, 43.9, 42.1, 38.2, 37.2, 36.2, 35.9, 34.5, 29.1, 26.7,
26.6, 25.9, 23.1, 21.2, 21.1, 20.9, 16.0, 10.9, 8.0, 7.7.
batch (prepared in a similar manner, starting from 30-N-(
g-ami-
nopropyl)-9-deoxo-9a-aza-9a-homoerythromycin A (0.2 g)) and
purified by column chromatography on silica gel (eluent
CH2Cl2:MeOH:NH4OH ¼ 90:9:1.5) resulting in 64.6% of the title
product (0.084 g, 0.081 mmol) as white powder. MS m/z: (ES):
5.1.15. 30-N-(Demethyl)-30-N-[3-({4-[(4-quinolinyl)amino]
butanoyl}amino)propyl]-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A (14)
To
a
solution of 30-N-(demethyl)-30-N-[3-({4-[(7-chloro-4-
MHþ ¼ 1039. 1H NMR (500 MHz, CDCl3)
d
8.41 (d, J ¼ 5.80 Hz, 1H),
quinolinyl)amino]butanoyl}amino)propyl]-9-deoxo-9a-methyl-9a-
aza-9a-homoerythromycin A 12 (0.3553 g, 0.342 mmol) in ethanol
(50 mL), Pd/C (10%) (0.180 g, 1.691 mmol) was added and the
reaction mixture was stirred under a hydrogen atmosphere (5 barr).
After 4 h the catalyst was filtered off and the solvent evaporated
yielding crude product (0.325 g) which was further purified by
column chromatography (eluent DCM:MeOH:NH4OH ¼ 90:9:1.5)
yielding 79% of the title product (0.271 g, 0.27 mmol) as a white
powder. MS m/z: (ES): MHþ ¼ 1004. 1H NMR (500 MHz, DMSO-d6)
7.98 (d, J ¼ 8.85 Hz, 1H), 7.95 (d, J ¼ 1.83 Hz, 1H), 7.66 (br. s., 1H),
7.39 (br. s., 1H), 7.37 (dd, J ¼ 1.98, 9.00 Hz, 1H), 6.27 (d, J ¼ 5.80 Hz,
1H), 5.11 (d, J ¼ 4.58 Hz, 1H), 4.71 (d, J ¼ 8.85 Hz, 1H), 4.40
(d, J ¼ 7.32 Hz, 1H), 4.25 (d, J ¼ 2.44 Hz, 1H), 4.00e4.10 (m, 1H),
3.66 (s, 1H), 3.64 (d, J ¼ 7.02 Hz, 1H), 3.48e3.53 (m, 1H), 3.38
(q, J ¼ 5.70 Hz, 2H), 3.30e3.34 (m, 1H), 3.28 (d, J ¼ 5.19 Hz, 2H),
3.24 (s, 3H), 3.03 (d, J ¼ 9.46 Hz, 1H), 2.72e2.75 (m, 1H), 2.68e2.71
(m, 1H), 2.58e2.64 (m, 1H), 2.55 (d, J ¼ 10.68 Hz, 1H), 2.41e2.48
(m, 3H), 2.33 (s, 3H), 2.29 (d, J ¼ 15.26 Hz, 1H), 2.21 (s, 3H),
2.07e2.15 (m, 2H), 2.03e2.06 (m, 1H), 2.01e2.04 (m, 1H),
1.97e2.01 (m, 1H), 1.85e1.95 (m, 1H), 1.74 (d, J ¼ 14.34 Hz, 1H),
1.62e1.67 (m, 2H), 1.60e1.63 (m, 1H), 1.56 (dd, J ¼ 4.88, 15.26 Hz,
1H), 1.41e1.53 (m, 1H), 1.30e1.34 (m, 6H), 1.23e1.29 (m, 2H),
1.17e1.23 (m, 9H), 1.09 (s, 3H), 1.08e1.12 (m, 3H), 0.99
(d, J ¼ 7.63 Hz, 3H), 0.81e0.94 (m, 6H). 13C NMR (75 MHz, CDCl3)
d
8.37 (d, J ¼ 5.19 Hz,1H), 8.18 (d, J ¼ 8.24 Hz,1H), 7.90 (t, J ¼ 5.50 Hz,
1H), 7.76 (d, J ¼ 8.24 Hz, 1H), 7.59 (dd, J ¼ 7.63, 8.24 Hz, 1H), 7.40
(dd, J ¼ 7.63 Hz, 1H), 7.21 (t, J ¼ 5.19 Hz, 1H), 6.43 (d, J ¼ 5.19 Hz, 1H),
4.82 (d, J ¼ 4.88 Hz, 1H), 4.74 (dd, J ¼ 2.44, 10.07 Hz, 1H), 4.40
(d, J ¼ 7.32 Hz, 1H), 4.30 (d, J ¼ 8.00 Hz, 1H), 4.29 (s, 1H), 4.20
(d, J ¼ 7.02 Hz, 1H), 4.14 (d, J ¼ 2.75 Hz, 1H), 4.03e4.10 (m, 1H), 4.01
(d, J ¼ 1.83 Hz, 1H), 3.62e3.69 (m, 1H), 3.50 (d, J ¼ 7.02 Hz, 1H), 3.43
(d, J ¼ 7.93 Hz, 1H), 3.25 (q, J ¼ 6.61 Hz, 2H), 3.19 (s, 3H), 3.08e3.14
(m, 2H), 3.03e3.07 (m, 1H), 2.87e2.92 (m, 1H), 2.61e2.69 (m, 2H),
2.47e2.51 (m, 2H), 2.42e2.48 (m, 2H), 2.36e2.40 (m,1H), 2.32e2.36
(m, 1H), 2.20e2.24 (m, 3H), 2.20 (s, 3H), 2.14 (s, 3H), 2.10
d
178.6, 173.5, 151.1, 150.5, 147.7, 135.4, 127.1, 125.5, 122.8, 117.3,
103.2, 98.0, 94.9, 84.4, 78.1, 78.0, 77.6, 74.3, 73.9, 73.6, 73.1, 71.5,
70.0, 68.8, 65.7, 64.6, 62.6, 52.5, 49.3, 45.3, 44.1, 42.3, 41.8, 39.4,
36.4, 34.8, 34.5, 29.8, 27.4, 26.8, 26.1, 23.0, 22.0, 21.6, 21.3, 21.2,

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
375
(t, J ¼ 11.75 Hz, 1H), 1.83e1.92 (m, 4H), 1.73e1.81 (m, 1H), 1.50e1.57
(m, 4H), 1.47 (dd, J ¼ 5.00, 15.00 Hz, 1H), 1.32e1.42 (m, 1H), 1.26
(dd, J ¼ 8.09, 14.50 Hz, 1H), 1.16 (s, 3H), 1.14 (d, J ¼ 6.10 Hz, 3H), 1.09
(s, 3H), 1.03e1.06 (m, 7H), 1.01 (s, 3H), 0.92e0.96 (m, 6H), 0.84
(d, J ¼ 6.71 Hz, 3H), 0.79 (t, J ¼ 7.32 Hz, 3H). 13C NMR (126 MHz,
(d, J ¼ 4.88 Hz, 1H), 4.75 (dd, J ¼ 2.79, 10.12 Hz, 1H), 4.40
(d, J ¼ 7.33 Hz, 1H), 4.26e4.32 (m, 2H), 4.18 (d, J ¼ 7.33 Hz, 1H), 4.16
(dd, J ¼ 1.74, 4.54 Hz, 1H), 4.12 (d, J ¼ 2.44 Hz, 1H), 4.07 (dq, J ¼ 6.00,
9.00 Hz, 1H), 3.61e3.68 (m, 1H), 3.51 (d, J ¼ 6.98 Hz, 1H), 3.44
(d, J ¼ 8.72 Hz, 1H), 3.34e3.37 (m, 2H), 3.15 (s, 3H), 3.08e3.13
(m, 1H), 2.90 (dd, J ¼ 6.89, 9.16 Hz, 1H), 2.65e2.71 (m, 2H),
2.62e2.65 (m,1H), 2.51e2.54 (m,1H), 2.48e2.50 (m,1H), 2.33e2.38
(m, 1H), 2.22e2.27 (m, 1H), 2.22 (s, 3H), 2.20 (s, 3H), 2.10e2.15
(m, 1H), 1.88e1.92 (m, 1H), 1.85e1.88 (m, 1H), 1.73e1.81 (m, 3H),
1.56e1.60 (m, 1H), 1.51e1.55 (m, 1H), 1.47e1.50 (m, 1H), 1.37
(ddd, J ¼ 7.24, 10.03, 14.31 Hz, 1H), 1.23e1.31 (m, 1H), 1.17 (s, 3H),
1.14 (d, J ¼ 6.28 Hz, 3H), 1.10e1.11 (m, 1H), 1.09 (s, 3H), 1.09
(d, J ¼ 7.00 Hz, 3H), 1.04 (d, J ¼ 6.11 Hz, 3H), 1.01 (s, 3H), 0.97
(d, J ¼ 7.50 Hz, 3H), 0.94 (d, J ¼ 6.80 Hz, 3H), 0.84e0.86 (m, 3H), 0.79
DMSO-d6)
d 177.34, 172.04, 150.88, 150.09, 148.54, 129.24, 128.84,
123.93, 121.83, 119.06, 102.52, 98.25, 94.72, 83.04, 77.60, 77.60,
76.55, 75.17, 73.84, 73.02, 72.76, 70.62, 68.94, 67.29, 64.96, 64.23,
61.66, 51.52, 48.98, 44.92, 42.45, 42.09, 41.85, 37.08, 36.63, 35.99,
34.84, 33.12, 30.85, 27.71, 27.65, 26.30, 24.12, 22.34, 21.71, 21.20,
21.20, 18.71, 17.96, 15.03, 11.20, 9.34, 7.00. HRMS (ES) calcd for C53
H90 N5 O13 (M þ Hþ) 1004.6535, found 1004.6530.
5.1.16. 30-N-(Demethyl)-30-N-[3-({4-[(4-quinolinyl)amino]
butanoyl}amino)propyl]-3-O-decladinosyl-9-deoxo-9a-methyl-9a-
aza-9a-homoerythromycin A (15)
(t, J ¼ 7.41 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d 176.98, 151.81,
150.06, 149.01, 133.15, 127.34, 123.94, 123.80, 117.40, 102.25, 98.54,
94.48, 82.76, 77.36, 77.31, 76.30, 74.88, 73.57, 72.69, 72.52, 70.51,
68.58, 66.98, 64.69, 64.22, 61.36, 51.01, 48.67, 44.63, 41.83, 41.49,
40.32, 36.52, 35.67, 34.62, 31.09, 27.28, 26.10, 25.99, 22.00, 21.33,
20.89, 20.85, 18.37, 17.64, 14.71, 10.85, 9.05, 6.72. HRMS (ES) calcd
for C49 H82 N4 O12 Cl (M þ Hþ) 953.5618, found 953.5601.
30-N-(Demethyl)-30-N-[3-({4-[(4-quinolinyl)amino]butanoyl}
amino)propyl]-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A 17 (0.1 g, 0.1 mmol) was dissolved in 3 M HCl (5 mL) and stirred at
r.t. for 1 h. The reaction mixture was diluted with water and the pH
of the aqueous layer was adjusted to pH 9.5 and extracted with
DCM (3 ꢀ 20 ml). The organic extracts (pH 9.5) were washed with
water and dried over Na2SO4. The solvent was evaporated yielding
86.2% of the title product (0.073 g, 0.086 mmol) as pale yellow
crystals. MS m/z: (ES): MHþ ¼ 846. 1H NMR (500 MHz, DMSO-d6)
5.1.18. 30-N-(Demethyl)-30-N-{3-[(7-chloro-4-quinolinyl)amino]
propyl}-3-O-decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A (17)
d
8.37 (d, J ¼ 5.49 Hz, 1H), 8.19 (d, J ¼ 8.24 Hz, 1H), 7.92
30-N-(Demethyl)-30-N-{3-[(7-Chloro-4-quinolinyl)amino]
(t, J ¼ 5.49 Hz, 1H), 7.76 (d, J ¼ 8.54 Hz, 1H), 7.59 (t, J ¼ 7.63 Hz, 1H),
7.40 (t, J ¼ 7.63 Hz, 1H), 7.21 (t, J ¼ 5.19 Hz, 1H), 6.43 (d, J ¼ 5.49 Hz,
1H), 6.21 (br. s., 1H), 5.16 (d, J ¼ 6.41 Hz, 1H), 4.95 (dd, J ¼ 1.83,
11.29 Hz, 1H), 4.56 (d, J ¼ 7.32 Hz, 1H), 4.32 (s, 1H), 4.30
(d, J ¼ 2.14 Hz, 1H), 4.06 (d, J ¼ 8.24 Hz, 1H), 3.38e3.44 (m, 3H), 3.35
(br. s., 1H), 3.21e3.28 (m, 2H), 3.06e3.14 (m, 3H), 2.67
(q, J ¼ 6.71 Hz, 1H), 2.51e2.55 (m, 1H), 2.44e2.49 (m, 2H), 2.38
(ddd, J ¼ 6.41, 6.56, 12.66 Hz, 1H), 2.29 (d, J ¼ 10.07 Hz, 1H),
2.23e2.25 (m, 2H), 2.22 (s, 3H), 2.16 (s, 3H), 2.11 (q, J ¼ 7.22 Hz, 1H),
2.04 (dd, J ¼ 11.75 Hz, 1H), 1.90 (quin, J ¼ 7.10 Hz, 2H), 1.70e1.81
(m, 2H), 1.54e1.59 (m, 1H), 1.47e1.54 (m, 3H), 1.36e1.45 (m, 1H),
1.29e1.35 (m, 1H), 1.13e1.18 (m, 1H), 1.08e1.12 (m, 9H), 0.97 (s, 3H),
0.95 (d, J ¼ 6.71 Hz, 3H), 0.89 (d, J ¼ 7.02 Hz, 3H), 0.83 (d, J ¼ 7.02 Hz,
propyl}-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 16
(0.1 g, 0.1 mmol) was dissolved in 3 M HCl (5 mL) and stirred at r.t.
for 1 h. The reaction mixture was diluted with water (5 ml) and the
pH of the water layer was adjusted to pH 9.5 and extracted with
DCM (3 ꢀ 20 ml). The organic extracts (pH 9.5) were washed with
water (3 ꢀ 10 ml) and dried over Na2SO4. The solvent evaporated
yielding 26.8% of the title product (0.0213 g, 0.027 mmol) as pale
yellow crystals. MS m/z: (ES): MHþ ¼ 795. 1H NMR (600 MHz,
DMSO-d6)
d
8.38 (d, J ¼ 5.41 Hz, 1H), 8.25 (d, J ¼ 9.07 Hz, 1H), 7.76
(d, J ¼ 2.27 Hz, 1H), 7.43 (dd, J ¼ 2.27, 9.07 Hz, 1H), 7.35
(t, J ¼ 5.32 Hz, 1H), 6.50 (d, J ¼ 5.58 Hz, 1H), 5.15 (d, J ¼ 6.45 Hz, 1H),
4.97 (dd, J ¼ 2.18, 11.08 Hz, 1H), 4.59 (d, J ¼ 7.50 Hz, 1H), 4.40
(d, J ¼ 2.62 Hz, 1H), 4.33 (s, 1H), 4.06 (d, J ¼ 8.20 Hz, 1H), 3.40e3.46
(m, 3H), 3.35e3.37 (m, 1H), 3.32 (s, 2H), 3.12e3.18 (m, 1H),
2.67e2.72 (m,1H), 2.62e2.66 (m,1H), 2.54e2.58 (m,1H), 2.51e2.54
(m, 1H), 2.51e2.51 (m, 1H), 2.28e2.34 (m, 1H), 2.23 (s, 6H), 2.12
(d, J ¼ 7.15 Hz, 1H), 2.04e2.10 (m, 1H), 1.71e1.82 (m, 4H), 1.57e1.63
(m, 1H), 1.42e1.45 (m, 1H), 1.37e1.41 (m, 1H), 1.34 (dd, J ¼ 6.00,
14.00 Hz, 1H), 1.17 (d, J ¼ 11.50 Hz, 1H), 1.09e1.13 (m, 9H), 0.99
(s, 3H), 0.96 (d, J ¼ 6.63 Hz, 3H), 0.91 (d, J ¼ 7.33 Hz, 3H), 0.85
(d, J ¼ 6.98 Hz, 3H), 0.77 (t, J ¼ 7.33 Hz, 3H). 13C NMR (75 MHz,
3H), 0.75 (t, J ¼ 7.32 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
d 175.78,
172.17, 151.03, 150.25, 148.66, 129.36, 128.99, 124.08, 121.99, 119.21,
103.20, 98.42, 88.42, 77.33, 76.76, 76.48, 73.98, 72.80, 70.83, 69.31,
68.52, 64.80, 61.85, 51.65, 43.77, 42.60, 41.29, 37.30, 36.94, 36.62,
35.82, 33.28, 31.59, 27.69, 26.54, 26.27, 24.27, 21.70, 21.57, 21.06,
18.15, 16.66, 10.89, 8.81, 6.74. HRMS (ES) calcd for C45 H76 N5 O10
(M þ Hþ) 846.5592, found 846.5580.
5.1.17. 30-N-(Demethyl)-30-N-{3-[(7-chloro-4-quinolinyl)amino]
DMSO-d6) d 175.22, 151.71, 149.94, 148.90, 133.05, 127.23, 123.83,
propyl}-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (16)
123.75, 117.29, 102.61, 98.42, 87.82, 76.74, 76.20, 75.95, 73.44, 72.27,
70.41, 68.64, 67.92, 64.33, 61.26, 51.04, 43.23, 40.43, 40.21, 36.50,
36.50, 35.31, 31.39, 25.95, 25.73, 25.60, 21.12, 20.96, 20.52, 17.56,
16.05, 10.30, 8.21, 6.17. HRMS (ES) calcd for C41 H68 N4 O9 Cl
(M þ Hþ) 795.4675, found 795.4667.
To
a g-aminopropyl)-9-
solution of 30-N-(demethyl)-30-N-(
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 4a (0.5 g, 0.63
mmol) in DMSO (20 mL), 4,7-dichloroquinoline (0.624 g, 3.15
mmol), NaHCO3 (111 mg), and DIPEA (0.33 ml, 1.9 mmol) were
added. The reaction mixture was heated at 110 ꢁC for 20 h. The
reaction mixture was evaporated to dryness. Crude residue was
diluted with EtOAc (50 ml) and washed with water (50 ml) at pH 5.
Then pH was adjusted at pH 9 and non organic layer was extracted
with EtOAc (3 ꢀ 50 ml). The combined organic layers were dried
over Na2SO4 and the organic solvent evaporated. Crude product
was purified by column chromatography (eluent DCM: MeOH:
NH4OH ¼ 90:9:1.5) yielding 11.5% of the title product (0.069 g,
0.072 mmol) as a light yellow crystals. MS m/z: (ES): MHþ ¼ 953.5.
5.1.19. 30-N-(Demethyl)-30-N-{3-[(4-quinolinyl)amino]propyl}-9-
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (18)
To a solution of 30-N-(demethyl)-30-N-{3-[(7-chloro-4-quinolinyl)
amino]propyl}-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A 16 (0.120 g, 0.126 mmol) in ethanol (20 mL), Pd/C (10%) (0.060 g,
0.564 mmol) was added and the reaction mixture was stirred under
a hydrogen atmosphere (5 barr). After 4 h the catalyst was filtered
off and the solvent was evaporated yielding 60% of the title
compound (0.070 g, 0.076 mmol) as a white powder. MS m/z: (ES):
1H NMR (600 MHz, DMSO-d6)
d
8.37 (d, J ¼ 5.41 Hz, 1H), 8.24
(d, J ¼ 9.07 Hz, 1H), 7.76 (d, J ¼ 2.27 Hz, 1H), 7.40 (dd, J ¼ 2.27,
MHþ ¼ 919. 1H NMR (500 MHz, DMSO-d6)
d
8.38 (d, J ¼ 5.49 Hz,
9.07 Hz, 1H), 7.30e7.36 (m, 1H), 6.50 (d, J ¼ 5.58 Hz, 1H), 4.83
1H), 8.23 (d, J ¼ 8.24 Hz, 1H), 7.78 (d, J ¼ 8.24 Hz, 1H), 7.64

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K. Starcevic et al. / European Journal of Medicinal Chemistry 49 (2012) 365e378
376
(t, J ¼ 7.63 Hz, 1H), 7.54 (br. s., 1H), 7.43 (t, J ¼ 7.63 Hz, 1H), 6.54 (d,
J ¼ 5.49 Hz, 1H), 4.85 (d, J ¼ 4.58 Hz, 1H), 4.76 (dd, J ¼ 2.14, 10.07 Hz,
1H), 4.42 (d, J ¼ 7.32 Hz, 1H), 4.34 (br. s., 1H), 4.14 (br. s., 1H),
4.02e4.11 (m, 1H), 3.63e3.71 (m, 1H), 3.51 (d, J ¼ 6.71 Hz, 1H), 3.45
(s, 1H), 3.35e3.41 (m, 2H), 3.18 (s, 3H), 3.13 (dd, J ¼ 7.78, 9.61 Hz,
1H), 2.91 (d, J ¼ 9.46 Hz, 1H), 2.73e2.81 (m, 1H), 2.68e2.72 (m, 1H),
2.65e2.68 (m, 1H), 2.53e2.59 (m, 1H), 2.52e2.54 (m, 1H),
2.40e2.47 (m, 2H), 2.21e2.28 (m, 7H), 1.88e1.91 (m, 2H), 1.78e1.84
(m, 2H), 1.75e1.80 (m, 1H), 1.61 (d, J ¼ 11.90 Hz, 1H), 1.51e1.56
(m, 1H), 1.47e1.53 (m, 1H), 1.34e1.43 (m, 1H), 1.30e1.34 (m, 1H),
1.19 (s, 3H), 1.16 (d, J ¼ 6.10 Hz, 3H), 1.12e1.14 (m, 1H), 1.10 (s, 3H),
1.09 (d, J ¼ 7.63 Hz, 3H), 1.05 (d, J ¼ 6.41 Hz, 3H), 1.03 (s, 3H),
0.95e1.00 (m, 6H), 0.86 (d, J ¼ 6.71 Hz, 3H), 0.80 (t, J ¼ 7.32 Hz, 3H).
6.5 and 9.0 were combined and dried over Na2SO4. The DCM was
evaporated affording crude product (0.58 g) which was further
purified by column chromatography (eluent DCM/MeOH/
NH4OH ¼ 90/9/1.5) yielding 9.2% the title product (0.054 g,
0.055 mmol) as a light yellow powder. MS m/z: (ES): MHþ ¼ 981. 1H
NMR (500 MHz, DMSO-d6)
d
8.38 (t, J ¼ 4.73 Hz, 1H), 8.23 (dd,
J ¼ 6.56, 9.00 Hz, 1H), 7.77 (t, J ¼ 1.98 Hz, 1H), 7.43 (t, J ¼ 2.44 Hz,
1H), 7.41 (t, J ¼ 2.44 Hz, 1H), 7.32 (s, 1H), 6.52 (d, J ¼ 5.49 Hz, 1H),
6.49 (d, J ¼ 5.49 Hz, 1H), 5.01 (d, J ¼ 5.19 Hz, 1H), 4.85 (d, J ¼ 5.19 Hz,
1H), 4.81 (d, J ¼ 4.88 Hz, 1H), 4.74e4.76 (m, 1H), 4.72e4.74 (m, 1H),
4.64 (d, J ¼ 4.58 Hz, 1H), 4.48e4.51 (m, 1H), 4.46 (t, J ¼ 7.48 Hz, 1H),
4.27e4.32 (m, 2H), 4.19e4.22 (m, 1H), 4.16e4.18 (m, 1H), 4.13 (br. s.,
1H), 4.09e4.12 (m, 1H), 4.04e4.08 (m, 1H), 3.80e3.84 (m, 1H),
3.76e3.79 (m, 1H), 3.64e3.70 (m, 1H), 3.55 (d, J ¼ 7.02 Hz, 1H), 3.51
(d, J ¼ 7.32 Hz, 1H), 3.44 (d, J ¼ 7.32 Hz, 1H), 3.30 (s, 3H), 3.24e3.28
(m, 2H), 3.15e3.20 (m, 1H), 3.10 (s, 1H), 2.94 (dd, J ¼ 6.87, 9.31 Hz,
1H), 2.88e2.92 (m, 1H), 2.81 (s, 3H), 2.71 (s, 3H), 2.63e2.69 (m, 2H),
2.58e2.62 (m, 2H), 2.44e2.47 (m, 2H), 2.36 (d, J ¼ 9.77 Hz, 1H), 2.28
(d, J ¼ 15.56 Hz, 1H), 2.20 (s, 3H), 2.16 (d, J ¼ 15.50 Hz, 1H),
2.08e2.12 (m, 1H), 1.85e1.94 (m, 4H), 1.73e1.81 (m, 1H), 1.54e1.58
(m, 1H), 1.53e1.57 (m, 1H), 1.50e1.57 (m, 1H), 1.47e1.50 (m, 1H),
1.40e1.44 (m, 1H), 1.37e1.40 (m, 1H), 1.32e1.40 (m, 1H), 1.22e1.31
(m, 1H), 1.12e1.20 (m, 9H), 1.09 (d, J ¼ 7.32 Hz, 3H), 1.04e1.08
(m, 3H), 1.00 (s, 6H), 0.91e0.97 (m, 6H), 0.85 (d, J ¼ 7.02 Hz, 3H),
13C NMR (126 MHz, DMSO-d6)
d 172.4, 151.2, 149.7, 147.0, 129.7,
127.9, 124.5, 122.2, 118.9, 102.7, 98.5, 94.9, 83.2, 77.7, 76.6, 75.1, 74.0,
73.2, 70.8, 68.6, 67.4, 65.1, 64.6, 62.1, 51.4, 49.1, 45.1, 42.0, 40.9, 37.1,
36.2, 35.0, 31.5, 27.6, 26.3, 22.5, 21.8, 21.4, 21.3, 18.8, 18.1, 15.2, 11.3,
9.5, 7.3. HRMS (ES) calcd for C49 H83 N4 O12 (M þ Hþ) 919.6008,
found 919.6016.
5.1.20. 30-N-(Demethyl)-30-N-{3-[(4-quinolinyl)amino]propyl}-3-
O-decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A (19)
To a solution of 30-N-(demethyl)-30-N-{3-[(7-chloro-4-quinolinyl)
amino]propyl}-3-O-decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A 17 (0.120 g, 0.151 mmol) in ethanol (20 mL),
Pd/C (10%) (0.060 g, 0.262 mmol) was added and the reaction
mixture was stirred under a hydrogen atmosphere (5 barr). After 4 h
the catalyst was filtered off and the solvent was evaporated yielding
crude product (88 mg) which was further purified by column
chromatography (eluent DCM:MeOH:NH4OH ¼ 90:9:1.5) yielding
50% of the title comopund (0.058 g, 0.076 mmol) as a white powder.
0.79 (t, J ¼ 7.48 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d 172.8, 152.4,
150.6, 149.7, 134, 127.9, 124.7, 124.6, 118.1, 102.9, 99.2. 95.0, 83.5,
77.8, 77.8, 76.9, 75.6, 74.2, 73.5, 73.2, 70.3, 69.2 67.1, 65.3, 62.2, 58.2,
54.6, 49.4, 49.2, 45.4, 42.8, 42.2, 42.1, 36.5, 36.2, 35.1, 31.1, 29.7, 27.9,
26.9, 26.4, 24.1, 22.6, 21.7, 21.5, 21.4, 19.2, 18.3, 15.3, 11.4, 9.5, 7.3.
HRMS (ES) calcd for C50 H82 N4 O13 Cl (M þ Hþ) 981.5567, found
981.5559.
MS m/z: (ES): MHþ ¼ 761.71. 1H NMR (500 MHz, DMSO-d6)
d
8.37 (d,
5.1.22. 30-N-(Demethyl)-30-N-[4-(7-chloro-quinolin-4-ylamino)
acetyl]-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (21)
To a solution of 30-N-(demethyl)-9-deoxo-9a-methyl 9a-aza-9a-
homoerythromycin A 2a (0.1 g, 0.136 mmol) in DCM (4 ml), N-(7-
chloro-4-quinolinyl)glycine (0.039 g, 0.163 mmol), HOBt (0.035 g,
0.231 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(0.034 g, 0.177 mmol) and Et3N (0.152 ml) were added and the
mixture heated by microwave (BIOTAGE) at 100 ꢁC for 5 min
NaHCO3 (10 ml) was added and the organic layer separated. The
DCM layer was dried over Na2SO4. DCM was evaporated affording
crude product (0.121 g) which was further purified by column
chromatography (eluent DCM/MeOH/NH4OH ¼ 90/9/1.5) yielding
23% of the title product (0.030 g, 0.031 mmol) as a white powder.
J ¼ 5.49 Hz,1H), 8.18 (d, J ¼ 8.24 Hz,1H), 7.76 (d, J ¼ 8.24 Hz,1H), 7.58
(t, J ¼ 7.63 Hz,1H), 7.40 (t, J ¼ 7.63 Hz,1H), 7.19 (t, J ¼ 5.34 Hz,1H), 6.46
(d, J ¼ 5.49 Hz,1H), 5.15 (d, J ¼ 6.41 Hz,1H), 4.97 (dd, J ¼ 1.68, 11.14 Hz,
1H), 4.59 (d, J ¼ 7.32 Hz,1H), 4.36 (d, J ¼ 2.14 Hz,1H), 4.33 (s,1H), 4.07
(d, J ¼ 8.24 Hz,1H), 3.39e3.47 (m, 3H), 3.35e3.37 (m,1H), 3.28e3.33
(m, 2H), 3.16 (ddd, J ¼ 2.14, 7.32, 10.00 Hz, 1H), 2.66e2.71 (m, 1H),
2.64 (t, J ¼ 6.41 Hz, 1H), 2.52e2.59 (m, 2H), 2.46e2.49 (m, 1H), 2.31
(d, J ¼ 9.77 Hz, 1H), 2.24 (s, 3H), 2.23 (s, 3H), 2.11e2.16 (m, 1H), 2.07
(t, J ¼ 11.90 Hz,1H),1.71e1.82 (m, 4H),1.61 (dd, J ¼ 3.36,11.29 Hz,1H),
1.42e1.47 (m, 1H), 1.37e1.42 (m, 1H), 1.30e1.39 (m, 1H), 1.18
(q, J ¼ 12.21 Hz, 1H), 1.08e1.13 (m, 9H), 0.99 (s, 3H), 0.96
(d, J ¼ 6.71 Hz, 3H), 0.91 (d, J ¼ 7.32 Hz, 3H), 0.85 (d, J ¼ 7.02 Hz, 3H),
0.77 (t, J ¼ 7.32 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
d
175.80,
MS m/z: (ES): MHþ ¼ 953. 1H NMR (600 MHz, DMSO-d6)
d 8.39
151.08, 150.32, 148.69, 129.36, 128.95, 124.07, 121.99, 119.24, 103.13,
98.47, 88.28, 77.30, 76.77, 76.50, 74.00, 72.82, 70.97, 69.28, 68.49,
64.82, 61.84, 51.72, 43.77, 41.29, 40.98, 37.20, 36.64, 35.87, 32.12,
26.56, 26.46, 26.30, 21.73, 21.57, 21.08, 18.18, 16.68, 10.90, 8.85, 6.75.
HRMS (ES) calcd for C41 H68 N4 O9 (M þ Hþ) 761.5065, found
761.5059.
(d, J ¼ 5.41 Hz, 1H), 8.36 (d, J ¼ 5.23 Hz, 1H), 8.16e8.20 (m, 1H),
7.79e7.82 (m, 1H), 7.44e7.51 (m, 1H), 7.36 (s, 1H), 7.31 (s, 1H), 6.41
(d, J ¼ 5.58 Hz,1H), 6.34 (d, J ¼ 5.58 Hz,1H), 5.42 (d, J ¼ 5.58 Hz,1H),
4.85 (d, J ¼ 4.88 Hz, 1H), 4.84 (s, 1H), 4.79 (d, J ¼ 4.19 Hz, 1H), 4.74
(dd, J ¼ 2.79, 10.12 Hz, 1H), 4.54 (d, J ¼ 7.50 Hz, 1H), 4.47
(d, J ¼ 7.33 Hz, 1H), 4.43e4.45 (m, 1H), 4.33e4.36 (m, 1H),
4.32e4.37 (m, 1H), 4.32 (s, 1H), 4.26e4.29 (m, 1H), 4.22
(d, J ¼ 5.23 Hz, 1H), 4.15e4.17 (m, 1H), 4.10 (dq, J ¼ 5.93, 9.42 Hz,
1H), 4.02e4.07 (m, 1H), 3.80e3.85 (m, 1H), 3.66e3.74 (m, 1H), 3.54
(d, J ¼ 6.98 Hz,1H), 3.43e3.47 (m,1H), 3.26 (s, 3H), 3.24 (s, 3H), 2.94
(s, 3H), 2.90e2.94 (m, 1H), 2.77 (s, 3H), 2.64e2.70 (m, 2H),
2.35e2.40 (m, 1H), 2.27 (d, J ¼ 14.83 Hz, 1H), 2.21 (d, J ¼ 2.79 Hz,
3H), 2.09e2.17 (m, 1H), 1.91e1.94 (m, 1H), 1.87e1.90 (m, 1H),
1.71e1.81 (m, 2H), 1.57e1.61 (m, 1H), 1.53e1.56 (m, 1H), 1.46e1.50
(m, 1H), 1.34e1.40 (m, 1H), 1.25e1.32 (m, 1H), 1.21 (s, 3H),
1.17e1.19 (m, 3H), 1.16 (s, 3H), 1.11e1.14 (m, 3H), 1.02e1.09 (m, 3H),
1.02 (s, 3H), 1.01 (s, 3H), 0.98 (d, J ¼ 7.50 Hz, 3H), 0.93e0.96 (m, 6H),
0.86 (d, J ¼ 6.98 Hz, 3H), 0.76e0.81 (m, 3H). 13C NMR (75 MHz,
5.1.21. 30-N-(Demethyl)-30-N-[4-(7-chloroquinolin-4-ylamino)
butanoyl]-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin (20)
To a solution of 30-N-(demethyl)-9-deoxo-9a-methyl 9a-aza-9a-
homoerythromycin A 2a (0.5 g, 0.6 mmol) in DCM (30 ml), 4-[(7-
chloro-4-quinolinyl)amino]butanoic acid (0.181 g, 0.68 mmol),
HOBt (0.108 g, 0.8 mmol), 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide (0.196 g, 1.02 mmol) and Et3N (0.8 ml) were added
and the reaction mixture was stirred over night at r.t. Water was
added, the organic layer separated, pH of the aqueous layer was
adjusted to 6.5 and extracted with DCM (30 ml). To the aqueous
layer DCM (30 ml) was added, pH adjusted to 9.0, layers separated
and extracted again with DCM (3 ꢀ 30 ml). The DCM layers at pH
DMSO-d6) d 176.9,168.1, 151.5, 149.8,148.7,133.2, 127.3, 124.2,123.5,

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377
117.2, 102.1, 101.2, 99.4, 94.3, 83.2, 82.6, 77.2, 76.2, 74.7, 73.4, 72.6,
72.3, 69.4, 68.5, 66.5, 64.8, 61.3, 57.1, 48.8, 48.6, 44.6, 43.7, 42.7, 41.8,
41.4, 36.0, 35.5, 35.2, 34.4, 28.0, 27.2, 26.5, 25.9, 21.9, 20.9, 20.8, 18.2,
17.5, 14.6, 10.7, 9.1, 8.9, 6.6. HRMS (ES) calcd for C48 H78 N4 O13 Cl
(M þ Hþ) 953.5254, found 953.5236.
staphylococci. Antibacterial activity of compounds was determined
by a standard broth microdilution method [47] except that in the
medium for testing Streptococcus, lysed horse blood was
substituted with 5% horse serum. Minimum inhibitory concentra-
tions (MICs) were expressed in units of mg/mL. AZ and CQ were used
as controls. Double dilutions of tested compounds were prepared
using TECAN Genesis 150 [48].
5.1.23. 30-N-(Demethyl)-30-N-[(1-{3-[(7-chloro-4-quinolinyl)
amino]propyl}-1H-1,2,3-triazol-4-yl)methyl]-9-deoxo-9a-methyl-
9a-aza-9a-homoerythromycin A (22)
5.2.2. Antimalarial screen
3-(7-Chloroquinolin-4-ylamino)propyl azide 11 (33.4 mg,
0.127 mmol) and 30-N-(1-propynyl)-9-deoxo-9a-aza-9a-homoery-
thromycin A 5 (50 mg, 0.084 mmol) were suspended in a 1:1
The activity of the test compounds against P. falciparum in vitro
was determined using a modification of the semi-automated
microdilution technique of Desjardins [49,50]. Strains used were
standard sensitive 3D7A and CQ/pyrimethamine resistant W2
strain. Compounds were dissolved in DMSO and subsequently
diluted in culture medium (RPMI 1640) supplemented with 10% v/v
human plasma. Serial 1:2 drug dilutions were prepared in triplicate
in microtiter plates. Culture medium supplemented with 10% v/v
human plasma and type A Rhesus positive human erythrocytes
infected with P. falciparum strains (3D7A or W2) was added to these
in order to yield a hemocrit of 3% and a parasitaemia of 0.25e0.55%.
[3H]Hypoxanthine was added to give a final concentration of
mixture of DMF and water (4 ml). Sodium L-ascorbate (5.13 mg) was
added followed by copper(II) sulphate pentahydrate (0.807 mg)
and the heterogeneous mixture was heated by microwave at 60 ꢁC
for
5 min, at which point TLC analysis indicated complete
consumption of the reactants. The reaction mixture was dilluted
with water (5 ml), cooled in ice and a precipitate was formed. It was
impossible to filter the mixture so the product was isolated by
extraction with DCM (3 ꢀ 5 ml). The organic layer was dried over
Na2SO4 and evaporated to afford crude product (0.067 g). Crude
product was purified on Biotage SP1 flash purifiation system
chromatography (5 g) in eluent (DCM:(NH4OH:MeOH) ¼ 9:10.5)
yielding 25.3% of the title product (0.022 g, 0.021 mmol). MS m/z:
12.5e16 m
Ci/mL of culture. The plates were incubated at 37 ꢁC in
a modular incubator flushed with a mixture of 5% oxygen, 3%
carbon dioxide, and 92% nitrogen. After incubation for 48 h
particulate matter was harvested on fibre-glass strips and hypo-
xanthine incorporation determined by scintillation spectropho-
tometry. From the concentrationeresponse curves, analyzed by
non-linear regression, the 50% inhibitory concentrations (IC50) for
each test compound was calculated.
(ES): MHþ
¼
1034. 1H NMR (600 MHz, DMSO-d6)
d 8.39
(d, J ¼ 5.41 Hz, 1H), 8.26 (d, J ¼ 9.07 Hz, 1H), 8.06 (s, 1H), 7.79
(d, J ¼ 2.27 Hz, 1H), 7.46 (dd, J ¼ 2.27, 9.07 Hz, 1H), 7.34
(t, J ¼ 5.00 Hz, 1H), 6.44 (d, J ¼ 5.58 Hz, 1H), 4.81 (d, J ¼ 5.06 Hz, 1H),
4.74 (dd, J ¼ 2.88, 10.03 Hz, 1H), 4.46 (t, J ¼ 6.98 Hz, 2H), 4.38
(d, J ¼ 7.33 Hz, 1H), 4.29 (d, J ¼ 7.60 Hz, 1H), 4.28 (s, 1H), 4.15e4.17
(m, 1H), 4.14 (dd, J ¼ 1.74, 4.54 Hz, 1H), 4.03e4.08 (m, 1H), 4.00
(br. s., 1H), 3.76 (d, J ¼ 13.96 Hz, 1H), 3.64e3.69 (m, 1H), 3.63
(d, J ¼ 13.96 Hz, 1H), 3.50 (d, J ¼ 6.98 Hz, 1H), 3.43 (d, J ¼ 8.20 Hz,
1H), 3.27e3.31 (m, 2H), 3.10e3.14 (m, 1H), 3.07 (s, 3H), 2.89
(dd, J ¼ 8.00, 10.00 Hz, 1H), 2.61e2.70 (m, 2H), 2.51e2.56 (m, 1H),
2.32e2.37 (m, 1H), 2.20 (s, 3H), 2.19e2.22 (m, 1H), 2.18 (s, 3H),
2.17e2.20 (m, 2H), 2.07e2.14 (m, 1H), 1.84e1.90 (m, 2H), 1.73e1.81
(m, 1H), 1.62 (dd, J ¼ 3.75, 10.73 Hz, 1H), 1.52 (d, J ¼ 14.50 Hz, 1H),
1.47 (dd, J ¼ 4.97, 14.92 Hz, 1H), 1.32e1.42 (m, 1H), 1.23e1.29
(m, 1H), 1.17 (s, 4H), 1.14 (d, J ¼ 6.11 Hz, 3H), 1.08 (s, 3H), 1.07
(d, J ¼ 5.93 Hz, 3H), 1.05 (d, J ¼ 7.50 Hz, 3H), 1.01 (s, 3H), 0.91e0.95
(m, 6H), 0.85 (d, J ¼ 6.80 Hz, 3H), 0.79 (t, J ¼ 7.41 Hz, 3H). 13C NMR
5.2.3. Cytotoxicity assay
The detailed protocol for cytotoxicity measurement was
described elsewhere [48]. In brief, HepG2 cells were maintained in
complete RPMI 1640 medium supplemented with 10% Foetal
Bovine Serum at 37 ꢁC in a 5% CO2 atmosphere. Cytotoxicity assay
was performed by using the MTS CellTiter 96 AQueous One Solu-
tion Cell Proliferation Assay (Promega, USA). Each culture in 96-
well plate contained 50 000 cells which were exposed to serial
dilutions (1:2) of tested compounds (initially dissolved in DMSO
and subsequently diluted in supplemented RPMI 1640 medium).
The method was programmed for the TECAN robotic system in
GEMINI pipetting software [48]. Plates were incubated for 24 h at
37 ꢁC in 5% CO2. After the addition of MTS reagent and 2 h of
incubation at 37 ꢁC in 5% CO2, the absorbance at 490 nm was
recorded and IC50s calculated based on the 10 points dose response
curves.
(75 MHz, DMSO-d6): d 177.06, 151.87, 149.87, 148.99, 145.77, 133.35,
127.46, 124.03, 124.03, 123.41, 117.42, 102.11, 98.64, 94.39, 82.73,
77.26, 77.26, 76.24, 74.89, 73.54, 72.70, 72.44, 70.45, 68.68, 66.92,
64.65, 63.22, 61.40, 49.37, 48.50, 47.19, 44.62, 41.78, 41.56, 39.71,
36.40, 35.73, 34.52, 31.24, 28.42, 27.38, 26.02, 22.07, 21.44, 20.90,
20.90, 18.44, 18.44, 17.67, 14.74, 10.92, 10.92, 9.03, 9.03, 6.72, 6.72.
HRMS (ES) calcd for C52 H85 N7 O12 Cl (M þ Hþ) 1034.5945, found
1034.5956.
5.2.4. Pharmacokinetic studies
Metabolic stability was assessed by measuring intrinsic clear-
ance (CLi) values in the mouse, rat and human liver microsomes.
Test compounds (1.0 m
M) were incubated at 37 ꢁC for 60 min in
5.2. Biology
50 mM potassium phosphate buffer (pH 7.4) containing 0.5 mg
microsomal protein/mL. The reaction was started by addition of
cofactor (2.0 mM NADP, 20 mM Glucose-6-phosphate, 2.0 mM
MgCl2 and 4.0 U/ml Glucose-6-phosphate dehydrogenase).
Samples were analyzed by LC-MS/MS and the intrinsic clearance
(CLi) determined from the first order elimination constant by non-
linear regression using Excel (Microsoft Exel 2003), corrected for
the volume of the incubation and assuming 52.5 mg microsomal
protein/g liver for all species. Values for CLi were expressed as mL/
min/g liver.
For the in vivo pharmacokinetic study, compound 12 was dosed
intravenously (IV) at 12.5 mg/kg (10 mL/kg dosing volume) and
orally (PO) at a dose of 12.5 mg/kg (20 mL/kg) to fasted female CD-1
mice (N ¼ 3 per route). Dosing solutions were prepared in 100%
5.2.1. Antibacterial screen
The organisms used in testing represented macrolide sensitive
Gram-positive and Gram-negative pathogens that are primarily
targeted by macrolide antibiotics. The panel consisted of macrolide
sensitive bacterial isolates: two clinical strains Streptococcus
pneumoniae SP030 and Streptococcus pyogenes 3565, and strains
obtained from American Type Culture Collection Staphylococcus
aureus ATCC13709, Haemophilus influenzae ATCC49247 and Mor-
axella catarrhalis ATCC23246. Bacteria were grown on appropriate
agar plates (by Becton Dickinson, USA) e Columbia agar with 5%
sheep blood for streptococci and M. catarrhalis, Mueller-Hinton
chocolate agar for H. influenzae and Mueller-Hinton agar for

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samples were collected from the tail vain up to 24 h and by cardiac
puncture at the last time point (48 h), hemolyzed and frozen until
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experiments, Zeljko Osman for the mass spectroscopy support, Bill
ꢀ
ꢀ
ꢁ
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