Design, synthesis, and in vitro activity of novel 2′- O -substituted 15-membered azalides

Article abstract of DOI:10.1021/jm201676t

Malaria remains one of the most widespread human infectious diseases, and its eradication will largely depend on antimalarial drug discovery. Here, we present a novel approach to the development of the azalide class of antimalarials by describing the design, synthesis, and characterization of novel 2′-O-substituted-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A derivatives consisting of different quinoline moieties covalently liked to a 15-membered azalide scaffold at position 2′. By multistep straightforward synthesis, 19 new, stable, and soluble compounds were created and biologically profiled. Most active compounds from the 4-amino-7-chloroquinoline series showed high selectivity for P. falciparum parasites, and in vitro antimalarial activity improved 1000-fold over azithromycin. Antimalarial potency was equivalent to chloroquine against the sensitive strain (3D7A) and up to 48-fold enhanced over chloroquine against the chloroquine-resistant strain (W2). Concurrently, the antibacterial activity of the compounds was eliminated, thus facilitating the development of malaria-specific macrolide agents.

Full text of DOI:10.1021/jm201676t

Article
pubs.acs.org/jmc
Design, Synthesis, and in Vitro Activity of Novel 2′-O-Substituted 15-
Membered Azalides
Dijana Pesic,†,§ Kristina Starcevic,†,∥ Ana Toplak,†,⊥ Esperanza Herreros,‡ Jaume Vidal,‡
̌
́
̌
́
Maria Jesus Almela,‡ Dubravko Jelic,†,§ Sulejman Alihodzic,†,§ Radan Spaventi,†,§ and Mihaela Peric*,†,#
́
̌
́
́
†GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz baruna Filipovica
‡Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres
Cantos (Madrid), Spain
́
29, 10000 Zagreb, Croatia
S
* Supporting Information
ABSTRACT: Malaria remains one of the most widespread
human infectious diseases, and its eradication will largely
depend on antimalarial drug discovery. Here, we present a
novel approach to the development of the azalide class of
antimalarials by describing the design, synthesis, and character-
ization of novel 2′-O-substituted-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A derivatives consisting of different
quinoline moieties covalently liked to a 15-membered azalide
scaffold at position 2′. By multistep straightforward synthesis,
19 new, stable, and soluble compounds were created and
biologically profiled. Most active compounds from the 4-amino-7-chloroquinoline series showed high selectivity for P. falciparum
parasites, and in vitro antimalarial activity improved 1000-fold over azithromycin. Antimalarial potency was equivalent to
chloroquine against the sensitive strain (3D7A) and up to 48-fold enhanced over chloroquine against the chloroquine-resistant
strain (W2). Concurrently, the antibacterial activity of the compounds was eliminated, thus facilitating the development of
malaria-specific macrolide agents.
applying a hybrid molecule strategy could ensure an approach
that is both expedient and affordable.
INTRODUCTION
■
Malaria is by far the most serious and widespread parasitic
disease in humans. Despite enormous global control efforts that
have resulted in a reduction of the estimated mortality, malaria
still caused 781000 deaths in 2009.1 The protozoal parasites
that cause the infection belong to the genus Plasmodium with
species Plasmodium vivax, Plasmodium ovale, Plasmodium
malariae, Plasmodium knowlesi, and Plasmodium falciparum
known to infect humans. P. falciparum, the species responsible
for the most serious form of the disease, has acquired resistance
to the widest range of antimalarial drug monotherapies;
therefore, the World Health Organisation is arguing against
monotherapeutic approaches to malaria treatment.2 Conse-
quently, combination therapies including a fast-eliminating drug
with a slow-eliminating partner agent have been developed over
the past decades, which are now part of standard clinical
practice and have contributed to a reduction in the emergence
and spread of drug-resistant parasites.3
An additional strategy to overcome drug-resistant malaria
parasites would be the design and introduction of hybrid
molecules. Such chemical entities are defined as chemicals
having two or more structural domains that bring distinct
biological properties into one molecule.4,5 This would
eventually enable more efficient parasite eradication and
consequently restrain the emergence of resistance. As new
malaria drug candidates are expected to be more active than
their parent compounds while remaining a low-cost treatment,6
Macrolide antibiotics, pioneered by azithromycin (AZI), have
demonstrated in vitro7 and in vivo activity in malaria
prophylaxis8,9 and treatment, both as monotherapy10 and in
combinations.11−13 AZI is of particular interest due to its
proven safety record and favorable pharmacokinetic parameters
such as its long half-life enabling once-daily dosing.14 However,
AZI as a monotherapy was proven inadequate in treating
malaria,10 while its therapeutic value in different combination
therapies is still under evaluation in human clinical stud-
ies.11,12,15−19
In the design of novel hybrid molecules, we were guided by
the idea of creating macrolide compounds that would have
enhanced antimalarial efficacy while retaining AZI pharmaco-
kinetic properties. Our previous work has shown that modifying
a 15-membered azalide scaffold at position 9a-N with various
aromate substituents could yield these desired properties.20,21
We envisaged our new molecules comprising a (chloro)-
quinoline moiety covalently linked to a macrolide scaffold at
position 2′ (Figure 1). Novel design of the 15-membered
azalide would bring favorable pharmacokinetic properties of
AZI together with superior quinoline activity against Plasmo-
dium while enabling the elimination of antibacterial activity due
Received: December 13, 2011
Published: March 1, 2012
© 2012 American Chemical Society
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Figure 1. Schematic representation of novel azalide-quinoline concept.
a
Scheme 1. Synthesis of 2′-O-(3-Aminopropyl)-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (7)
a
Reagents and conditions: (a) DMA/DMA, chloroform, reflux. (b) Ac2O, DMAP, TEA, DCM, rt. (c) MeOH, rt, 45 °C. (d) Acrylonitrile, NaH, t-
BuOH. (e) Hydrogen, PtO2, AcOH; LiOH, MeOH/water.
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a
Scheme 2. Synthesis of 2′-O-(3-Carboxyethyl)-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (10)
a
Reagents and conditions: (a) Methyl acrylate, NaH, t-BuOH. (b) HCOOH, MeOH, rt. (c) LiOH, THF/water.
to the position on the macrolide scaffold used for derivatization.
The exclusion of antibacterial action is a desirable property for a
new antimalarial because it would allow the eradication of the
parasites without affecting the commensal bacteria or the
Taking into account the azalide's delicate and complex
structure and several functional groups with similar reactivity, it
was very important to elaborate a protecting group strategy that
would allow regioselective alkylation at position 2′. The 2′-OH
group is the most reactive group of both erythronolides and
azalides; thus, it should be initially protected and later
selectively deprotected. Vicinal diols at C-11 and C-12 are
often protected as cyclic carbonates, which allowed further
transformations on carbohydrate hydroxyl groups.24,27 How-
ever, this strategy was not useful in this case because cyclic
carbonates are unstable in basic conditions used in the key step
of 2′-OH etherification. Cyclic acetals, on the other hand, are
stable in neutral and basic media and seemed as an attractive
alternative for 11-OH, 12-OH protection. N,N-Dimethylaceta-
mide dimethyl acetal (DMA/DMA) is a commercially available
reagent used in mild transacetalysation without the need for
acid catalysis.28,29 Cyclic amide acetal 2 was readily prepared by
treatment of 1 with DMA/DMA in chloroform under reflux.
Reaction of 2 with acetic anhydride in the presence of
dimethylaminopyridine (DMAP) resulted in the protection of
both C-4″ and C-2′ hydroxyl groups and the formation of 3.
Treatment of 3 with methanol allowed selective deacetylation
of the 2′-OH group due to autocatalytic participation of
neighboring 3′-dimethylamino group.24,25 Furthermore, under
these reaction conditions, acetal protection was almost
quantitatively converted into 11,12-O-methoxyethylidne deriv-
ative 4. The hydrolytic instability of this acetal was actually
expected in view of its orthoester structure.29 Michael
condensation of 4 with acrylonitrile in the presence of sodium
hydride in t-BuOH and subsequent reduction of the resulting
cyanoethyl aduct 5 afforded primary amine 6. It is worth noting
here that cyclic acetal protection was cleaved in this step to
afford diacetate 6. Final hydrolysis of diacetate 6 afforded 2′-O-
(3-aminopropyl)-9-deoxo-9a-methyl-9a-aza-9a-homoerythro-
mycin A (7) in an overall 20% yield. Using an analogous
synthetic procedure, free acid azalide derivative 10 was
obtained in a 15% overall yield from 4 (Scheme 2).
spread of antibacterial macrolide resistance.
AZI's antibacterial activity arises from the inhibition of
protein synthesis on bacterial ribosomes. The 2′-OH position
on desosamine sugar contributes greatly to macrolides'
antibacterial activity and to their ribosome binding by forming
important interactions with the ribosome exit tunnel in the 50S
subunit.22 Consequently, there is limited evidence for the
pursuit of chemical transformations at the 2′-position of the
macrolide scaffold and, if available, report mainly the
introduction of protecting groups.23−25 Chemical modification
of this position is challenging, and we have developed novel
synthetic approaches for obtaining 2′-alkylated 15-membered
azalide derivatives. Furthermore, by this alteration and by the
introduction of quinoline moieties at this position, 19 novel
compounds with unique biological profiles were created, thus
making an important step forward in the discovery of novel
antimalarial agents. Herein, we report the original synthesis and
biological characterization of novel 2′-O-substituted-9-deoxo-
9a-methyl-9a-aza-9a-homoerythromycin A derivatives where a
quinoline moiety was linked via an aminoalkyl linker at the 2′
position (Figure 1).
RESULTS
■
Chemistry. As a part of the synthetic program focused on
new hybrid macrolide/chloroquinoline analogues, we inves-
tigated the construction of compounds with the goal of
identifying novel azalide class of antimalarials. The complexity
of the azalide scaffold in combination with our goal to explore
structural variations posed a formidable synthetic challenge.
Our basic synthetic approach envisioned construction of
appropriately functionalized and protected azalide compounds
that would permit the exploration of C-2′ position modifica-
tions and limited variation in the basic structure of the
desosamine amino sugar (Scheme 1). The crucial intermediate
2′-O-(3-aminopropyl) derivative 7 allowed a smooth mod-
ification of the primary amino group by amidations and
aminations at the C-2′ position.26
Having the macrolide intermediates needed for the
preparation of hybride molecules, we turned our attention to
quinoline partners. Therefore, the commercially available 4,7-
dichloro-quinoline (11) was regioselectively turned into 7-
chloro-4-(substituted)aminoquinolines 12−14 (Scheme 3).
An appropriate combination of the 4-amino-7-chloroquino-
line derivative and a macrolide subunit allowed synthesis of the
final hybrid compounds (Schemes 4 and 5). Amidation of
amino acids 12 and 13 with primary amine 7 proceeded in the
Starting from available precursor, 9-deoxo-9a-methyl-9a-aza-
9a homoerythromycin A (1), the method provided an easy
access to a variety of novel analogues having various groups
tethered to the C-2′ position of the azalide skeleton.
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Scheme 3. Synthetic Approach to 4-Amino-7-
chloroquinoline Intermediates
Scheme 5. Synthesis of Novel 15-Membered Azalides: 4-
Aminoquinoline Derivative 25 Prepared by Amidation of 10
investigate the influence of cladinose sugar removal on
biological activity (Scheme 4).
We also investigated whether the presence of a chlorine atom
in the quinoline ring was necessary for the activity of the hybrid
molecules. Catalytic hydrogenation afforded a set of com-
pounds 16, 18, 20, and 23 in good yields.
Furthermore, the aminoquinoline moiety was substituted
with a quinolinyl methanamine moiety (26−29), as well as the
linking position of the quinoline ring, to investigate its influence
on antiparasitic activity. Compounds 26a, 26b, and 26c were
prepared by reductive amination of 7 with quinoline
carbaldehydes (Scheme 6). Additionally, N-methyl derivatives
(27 and 28) were prepared to verify the influence of these
structural features on antimalarial activity.
presence of 1-hydroxybenzotriazole (HOBt) and PS-carbodii-
mide (PS-CDI) or HOBt and N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDCxHCl) (Scheme 4). By
analogy, amide 25 was obtained under similar conditions by
coupling azalide acid 10 and amine 14. Nucleophilic
substitution of the chlorine atom at position 4 in 11 with
amine 7 led to the formation of compound 22.
A characteristic common to both erythronolides and azalides
is decomposition in aqueous acidic media comprising
hydrolysis of a cladinose sugar glycosidic bond.30−32 Several
decladinosyl derivatives were prepared (17, 18, 21, and 24) to
For the purpose of investigating the chemical stability of the
2′-ether bond, a set of solubility and solution stability studies
a
Scheme 4. Synthesis of Novel 15-Membered Azalides: 4-Aminoquinoline Derivatives Prepared by Amidation of Amine 7
a
Reagents and conditions: (a) 12, HOBt, PS-CDI, DCM, rt. (b) 13, HOBt, EDCxHCl, TEA, DCM, rt. (c) H2/Pd/C. (d) H+/H2O. (e) 11, DIPEA/
DMSO.
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a
Scheme 6. Synthesis of Quinolinyl Methanamine Derivatives 26−29
a
Reagents and conditions: (a) TEA, NaBH4, rt. (b) HCHO, HCOOH, CHCl3, rt. (c) 3 M HCl, rt.
were performed using compound 22. Results revealed a high
stability at pH 5−6 and degradation at pH 1.5−3,33 attributable
to a very well-known decomposition of 15-membered azalides:
hydrolysis of the cladinose sugar glycosidic bond.30−32 The
newly introduced 2′-ether linkage was found to be stable in
variable media, pH, and temperatures. Water solubility of the
2′-ether derivatives was determined to be >10 mg/mL (pH 4),
which increased up to 100-fold for some derivatives when
prepared in acetic salt form.
Biology. The in vitro antimalarial activity of the compounds
was compared with that of AZI and chloroquine (CHL). Using
the methodology described in the in vitro screening protocol,
compounds listed in Tables 1 and 2 were profiled for their
antimalarial activity against two different P. falciparum strains
(the CHL-resistant W2 and CHL-sensitive 3D7A strains) and
their inhibition of human hepatocellular carcinoma cell line
HepG2 (used for determining potential cytotoxicity). The
results of the biological evaluation were expressed as the drug
concentration resulting in 50% inhibition (IC50) of parasite or
cell growth. Also, the antibacterial activity was verified by
determining the minimal inhibitory concentration (MIC) of the
compounds against phenotypically macrolide-sensitive strains
of three Gram-positive bacterial species: Streptococcus pneumo-
niae, Streptococcus pyogenes, and Staphylococcus aureus.
the CHL-sensitive strain (3D7A). A remarkable characteristic
of these novel molecules was its activity against the W2 strain,
which was improved, as compared to CHL, for almost all of the
tested compounds.
Among the 19 compounds assayed against the 3D7A and W2
strains, six compounds (15, 19, 22, 23, 24, and 25) exhibited
activity with an IC50 in the low nanomolar range, which is
equivalent to CHL and several orders of magnitude superior to
AZI. Compounds 15, 19, and 22−25 showed greatly enhanced
activity against the W2 (chloroquinoline-resistant) strain as
compared to CHL and consistently low IC50 values against
both strains tested.
Overall, the best antimalarial activity was observed for the
compounds that retained both sugars and were substituted with
4-amino-7-chloroquinoline moiety (15, 19, and 22). Activity
decreased when the chlorine atom on the quinoline core was
substituted with hydrogen (15 vs 16, 17 vs 18, 19 vs 20, and 22
vs 23) and when cladinose was removed from the azalide
backbone (15 vs 17, 19 vs 21, and 22 vs 24). Modification of
the linker by insertion of the amide moiety also resulted in
decreased antiparasitic activity (22 vs 15 and 22 vs 19). When
the 4-aminoquinoline moiety was replaced with quinolinyl-
methanamine substituents (Table 2, compounds 26−29),
activity against the 3D7A strain weakened substantially.
However, for compounds 26a−c and 27a,b (derivatives with
both sugars present), activity against the CHL-resistant W2
strain remained in the low nanomolar range.
As shown in Tables 1 and 2, all tested compounds exhibited
in vitro antimalarial activity with a substantial improvement
over AZI (3−843-fold increase). Interestingly, the tested
compounds followed the trend of AZI in exhibiting lower
IC50 values against the CHL-resistant strain (W2) than against
The inhibition of the human hepatocellular carcinoma
HepG2 cell line was determined to assess the cytotoxicity of
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Table 1. In Vitro Activity of 4-Aminoquinoline Derivatives of 2′-O-Substituted 9-Deoxo-9a-methyl-9a-aza-9a-homoerythromycin
A: Antimalarial Activity against Two P. falciparum Strains (W2 and 3D7A), Inhibition of Human Hepatocellular Carcinoma
HepG2 Cell Line (Cytotoxicity), and Antibacterial Activity against Three Macrolide Sensitive Bacterial Strains (S. pneumoniae,
S. pyogenes, and S. aureus)
*
ND, not determined.
the compounds as well as their selectivity for the P. falciparum
parasites. Selectivity is defined as the ratio between HepG2 and
P. falciparum IC50 values whereby a compound with a value
above 100 is considered to be selective for the parasite. The
results demonstrated that the factor of selectivity for all of the
compounds was greatly above 100 for the W2 strain (from 231
up to 4356), as well as for the 3D7A strain for 4-
aminoquinoline derivatives and (2- and 4-quinolinyl)-
methamine (from 132 to 2240), while only the (3-quinolinyl)-
methamine derivatives had selectivity lower than 100 (49 for
27b, 88 for 26b, and for 28b and 29b selectivity could not be
precisely calculated).
Because of the fact that the 2′ position of macrolide
antibiotics is known for its importance in enabling ribosome
binding and antibacterial effect, the potency of the novel
compounds on bacterial pathogens was verified. The bacteria
tested represent the species most affected by the action of
macrolide antibiotics, and the strains used were all fully
sensitive to AZI. The data demonstrated a complete loss of
antibacterial activity for the compounds tested, with some
negligible activity of compounds 22 and 23 remaining against S.
pneumoniae and S. aureus.
DISCUSSION
■
Novel antimalarial agents are urgently needed since the
implementation of an effective antimalarial drug policy is
often hindered by the emergence of resistant strains resulting in
treatment failures. Macrolides, and AZI in particular, have
shown the potential to be used in malaria treatment and
prophylaxis. The current bottleneck in AZI implementation as
an effective antimalarial agent is the lack of strong clinical
evidence for overall equivalence or superiority over existing
therapies, probably due to its low efficacy when used as a
monotherapy.19 Our work on novel and improved azalides for
the treatment of malaria has already lead to the identification of
several classes of 15-membered macrolides with improved in
vitro and in vivo activity over AZI.20 Encouraged by these
findings, we extended our investigations with a focus on the
most active examples. Key chemical features of the most
promising compound included an AZI-based scaffold with a
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Table 2. In Vitro Activity of Quinolinyl-Methanamine Derivatives of 2′-O-Substituted 9-Deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A Derivatives: Antimalarial Activity against Two P. falciparum Strains (W2 and 3D7A), Inhibition of Human
Hepatocellular Carcinoma HepG2 Cell Line (Cytotoxicity), and Antibacterial Activity against Three Macrolide Sensitive
Bacterial Strains (S. pneumoniae, S. pyogenes, and S. aureus)
chloroquinoline substituent attached at the 9a-N position,
which resulted in the increased in vitro and in vivo potency and
favorable pharmacokinetic and druglike properties.20,21 A step
forward was achieved with the class of azalides presented here
in that different quinoline substituents were added to the 15-
membered azalide scaffold at the 2′-O position. This combined
the favorable synergistic properties of these two pharmaco-
phores observed in our previous compounds by utilizing a
chemically novel and alternative method, which allowed even
further improvements in their properties. Within the presented
set of molecules, the most active antimalarial azalides so far
were obtained (15 and 22) with very favorable in vitro profiles
(i.e., cytotoxicity and selectivity).
In our drug design, we aimed for the elimination of
antibacterial activity. An antibacterially inactive drug in the
contemporary treatment of malaria would definitively have a
therapeutic and ethical advantage in that such a drug would not
affect the emergence and spread of bacterial resistance nor
would it further narrow the spectrum of accessible antibiotics to
already vulnerable and underprivileged target populations in
malaria endemic countries.
action. The elimination of antibacterial potency indicated that
2′-OH position is highly important for target binding and that
the addition of quinoline substituents at this position leads to
the disruption of the compound-ribosome steric complemen-
tarity.
Moreover, the antimalarial activity of analogues 15, 19, 22,
and 25 was substantially increased over AZI and CHL, with
stronger inhibition of the CHL-resistant parasite. The obtained
results point to the existence of a mechanism delivering the
chloroquinoline moiety to the target site (the food vacuole)
possibly by contributing to its uptake and accumulation into
this organelle. The mechanism could have a physicochemical
rationale since these compounds have higher lipophilicity
(according to the calculated log P values, data not shown) than
their parent compound and could thus penetrate membranes
more efficiently. In addition, the mechanism could involve
biochemical reasons like changing the substrate specificity for
the transport proteins involved in CHL efflux.
The results reported here demonstrate a proof of concept for
the linkage of AZI and a 4-amino-7-chloroquinoline moiety in a
single molecule that retains, and evidently enhances, the
antimalarial activity of the parent compounds. This novel
generation of 15-membered azalides deserves additional
investigation, and some of the later stage assays are already
under way to assess the developability and mode of action of
the most promising leads from this series.
The addition of quinoline substituents on the 2′-O position
of 15-membered azalide scaffold completely abolished anti-
bacterial activity of the new derivatives. This effect was
irrespective of the removal of the cladinose.
Furthermore, because the target of macrolide antibiotics is
70S prokaryotic ribosome, which is also present in the parasite
apicoplast organelle, we considered the testing of antibacterial
activity as an indirect measurement of the macrolide mode of
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were expressed in μg/mL. The panel consisted of two macrolide-
sensitive streptococci clinical isolates (S. pneumoniae SP030 and S.
pyogenes 3565) and one reference strain obtained from American Type
Culture Collection (S. aureus ATCC13709). These organisms are the
major bacterial pathogens targeted by macrolide antibiotics. The
method for automated preparation of microtiter plates with compound
double dilutions was programmed for TECAN robotic system in
GEMINI pipetting software.35
CONCLUSION
■
With malaria still devastating endemic countries, novel
antimalarials are urgently needed. Here, we describe a novel
concept in antimalarial drug design: a set of hybrid molecules
obtained by covalently linking quinoline moieties to an azalide
macrolactone scaffold. Following an original multistep synthetic
route, 19 novel 2′-O-substituted-9-deoxo-9a-methyl-9a-aza-9a-
homoerythromycin A derivatives were obtained and analyzed.
Screening of the in vitro antimalarial and cytotoxic activity of
the aforementioned compounds enabled the identification of
compounds with high selectivity for the P. falciparum parasite,
antimalarial activity comparable to CHL against a sensitive
strain (3D7A), and greatly enhanced activity against a CHL-
resistant strain (W2). At the same time, the antibacterial activity
of the compounds was eliminated, facilitating the development
of malaria-specific macrolide agents. Taken all together, 2′-O-
substituted-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A
derivatives with a covalently linked 4-amino-7-chloroquinoline
moiety are recognized as promising lead molecules for
preclinical development. The exact mode of action of the
presented compounds is not yet fully elucidated, and detailed
target analyses, as well as in vivo profiling, are currently in
progress.
Chemistry. General Methods. All commercial reagents
(Merck, Sigma-Aldrich) were used as provided unless otherwise
indicated, and all solvents were of the highest purity unless
otherwise noted. NMR spectra were recorded on a Bruker
Avance DRX500 or Bruker Avance DPX300 spectrometer in
CDCl3 or DMSO, and chemical shifts were reported in ppm
using TMS as an internal standard. 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
interscan 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 elemental composition was calculated using
a MassLynx v4.1 for the [M + H]+ and the mass error quoted
within 5 ppm range. In synthetic procedures, column
chromatography was carried out over Merck Kieselgel 60
(230−400 mesh) or on an solid-phase extraction (SPE)
cartridge with average size silica of 50 μm. Thin-layer
chromatography was performed on 0.24 mm silica gel plates
Merck TLC 60F254. The indicated eluent was used, and the
solvent ratios refer to the volume. In general, organic solutions
were dried with anhydrous Na2SO4 or K2CO3, and 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 with MS and UV
detection at 240 nm on a YMC Pack Pro C18 column, 150 mm × 4.6
mm i.d., 3 μm particle size. The gradient elution at a flow rate of 1.0
mL/min started with 10% acetonitrile/90% 40 mM ammonium
acetate buffer solution and ended after 15 min with 90% acetonitrile/
10% 40 mM ammonium acetate buffer solution. The ESI source was
operated in the positive ion mode. Nitrogen was used as a nebulizing
and drying gas at 350 °C. The m/z range scanned in the MS
measurement was 200−1200. Complete system control, data
acquisition, and processing were done using the ChemStation for
LC/MSD version 4.2 from Agilent. The acceptable range of purity for
each compound was ≥95%.
EXPERIMENTAL SECTION
■
Biology. P. falciparum Inhibition in Vitro. The activity of the
test compounds against P. falciparum in vitro was determined
using a modification of the semiautomated microdilution
technique of Desjardins.34 Strains used were the standard
sensitive 3D7A and CHL/pyrimethamine-resistant W2 strains.
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 yield a hematocrit of 3% and a parasitemia of
0.25−0.55%. [3H]Hypoxanthine was added to give a final
concentration of 12.5−16 μCi/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
fiberglass strips, and hypoxanthine incorporation was deter-
mined by scintillation spectrophotometry. From the concen-
tration−response curves analyzed by nonlinear regression, the
50% inhibitory concentrations (IC50) for each test compound
were calculated.
Cytotoxicity Assay. The detailed protocol for cytotoxicity
measurement is described elsewhere.35 In brief, Hep G2 cells were
maintained in complete RPMI 1640 medium supplemented with 10%
fetal bovine serum at 37 °C in a 5% CO2 atmosphere. The cytotoxicity
assay was performed using the MTS CellTiter 96 AQueous One
Solution Cell Proliferation Assay (Promega, United States). Each
culture in the 96-well plates contained 50000 cells, which were
exposed to serial dilutions (1:2) of tested compounds (initially
dissolved in DMSO and subsequently diluted in supplemented RPMI
1640 medium). 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 IC50 was
determined based on the obtained response curves.
2′-O-(3-Aminopropyl)-9-deoxo-9a-methyl-9a-aza-9a-homoery-
thromycin A (7). Compound 6 (486 mg, 0.54 mmol) was dissolved in
THF (4,5 mL), and LiOH (6 mL of 0.5 M) was added, heated at 40
°C for 2 h, and stirred at rt for an additional 72 h. H2O (10 mL) was
added to the reaction mixture, followed by extraction with EtOAc.
Organic layers were collected and dried over Na2SO4. Solvent was
evaporated yielding 380 mg (87%) of the white solid. MS (ES+): 806
1
[MH]+. H NMR (600 MHz, DMSO-d6) δ/ppm: 0.79 (t, J = 7.4 Hz,
3H), 0.84 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 7.6 Hz, 3H), 0.94 (d, J = 6.7
Hz, 3H), 1.01 (s, 3H), 1.07 (d, J = 6.1 Hz, 3H), 1.09 (d, J = 7.5 Hz,
3H), 1.10−1.11 (m, 1H), 1.13 (s, 3H), 1.15 (d, J = 6.3 Hz, 3H), 1.17
(s, 3H), 1.31 (dd, J = 14.8, 7.9 Hz, 1H), 1.34−1.40 (m, 1H), 1.51 (d, J
= 14.8 Hz, 1H), 1.51 (d, J = 15.0 Hz, 1H), 1.54−1.61 (m, 2H), 1.61−
1.66 (m, 1H), 1.73−1.81 (m, 1 H), 1.85−1.89 (m, 1H), 1.90−1.94 (m,
1H), 2.13 (dd, J = 11.9, 10.5 Hz, 1 H), 2.20 (s, 3H), 2.22 (s, 6H), 2.25
(d, J = 15.0 Hz, 1H), 2.35 (dd, J = 11.0, 3.5 Hz, 1H), 2.46 (ddd, J =
12.2, 10.4, 4.1 Hz, 1H), 2.64−2.70 (m, 3H), 2.72−2.77 (m, 1H), 2.85
(dd, J = 10.1, 7.5 Hz, 1H), 2.91 (d, J = 9.6 Hz, 1H), 3.22 (s, 3H),
3.42−3.44 (m, 1H), 3.47−3.50 (m, 1H), 3.54 (d, J = 7.2 Hz, 1H),
3.62−3.68 (m, 1H), 3.87−3.92 (m, 1H), 4.06 (dq, J = 9.5, 6.2 Hz,
1H), 4.20 (dd, J = 4.0, 1.9 Hz, 1H), 4.31 (s, 1H), 4.32−4.35 (m, 1H),
4.40 (d, J = 7.3 Hz, 1H), 4.75 (dd, J = 10.2, 2.7 Hz, 1H), 4.85 (d, J =
5.1 Hz, 1H). 13C NMR (600 MHz, DMSO-d6) δ/ppm: 177.02, 101.84,
Antibacterial Screen. The compounds were tested against
bacteria by a standard broth microdilution method36 with the
exception that for the testing of Streptococcus strains, lysed horse
blood in the broth was substituted with 5% horse serum. Bacteria were
grown on appropriate agar plates (Becton Dickinson, United States):
Columbia agar with 5% sheep blood for streptococci and Mueller−
Hinton agar for S. aureus. AZI and CHL were used as controls. MICs
3223
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Journal of Medicinal Chemistry
Article
94.26, 82.18, 79.72, 77.22, 76.98, 76.22, 74.78. 73.50, 72.75, 72.42,
70.55, 68.55, 66.77, 64.58, 63.78, 61.34, 48.75, 44.69, 41.66, 41.43,
40.16, 38.62, 35.64, 34.52, 30.57, 30.49, 27.35, 25.91, 22.04, 21.34,
20.91, 20.84, 18.45, 17.58, 14.72, 10.86, 8.18, 6.68.
2′-O-[3-({2-[(4-Quinolinyl)amino]ethanoyl}amino)propyl]-9-
deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (16). To an
ethanol solution of 15 (200 mg, 0.195 mmol in 30 mL), Pd/C
(10%) was added (60 mg, 0.056 mmol), and the reaction mixture was
stirred under hydrogen atmosphere (4 barr). After the 4 h catalyst was
filtered off, EtOAc (20 mL) and water (20 mL) were added to the
filtrate, and the pH was adjusted to 4 (1 M HCl). The water layer was
then extracted with DCM (2 × 30 mL), and the layers were separated.
By the addition of 1 M NaOH, the pH of the water layer was adjusted
to pH 6.5 and extracted with DCM (2 × 30 mL), and the layers were
separated. The organic layers at pH 6.5 were combined, and water was
added. By addition of NH4OH, the pH was adjusted at pH 9.5, and the
layers were separated. The crude product obtained after evaporation of
the solvent was recrystallized from diisopropyl-ether yielding 120 mg
2′-O-(3-Carboxyethyl)-9-deoxo-9a-methyl-9a-aza-9a-homoery-
thromycin A (10). A 0.5 M concentration of LiOH was added (25
mL) to a solution of 9 (3.2 g) in THF (50 mL).26 The reaction was
stirred at room temperature for 48 h, and the solvent was evaporated
to yield 2.78 g of crude product subsequently purified on an SPE
column (50 g). A total of 130.7 mg of the title product was isolated.
1
MS (ES+): 821.4 [MH]+. H NMR (500 MHz, DMSO-d6) δ/ppm:
0.79 (t, J = 7.5 Hz, 3H), 0.85 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 7.5 Hz,
3H), 0.95 (d, J = 6.5 Hz, 3H), 1.02 (s, 3H), 1.09 (m, 3H), 1.11 (m,
3H), 1.14 (s, 3H), 1.15 (d, J = 6.0 Hz, 3H), 1.18 (s, 3H), 1.19 (m,
1H), 1.32 (m, 1H), 1.39 (m, 1H), 1.50 (m, 1H), 1.51 (d, J = 15 Hz,
1H), 1.76 (m, 1H), 1.78 (m, 1H), 1.88 (m, 1H), 1.93 (m, 1H), 2.15
(m, 1H), 2.18 (m, 1H), 2.22 (bs, 3H), 2.26 (d, J = 15 Hz, 1H), 2.32
(m, 1H), 2.35 (m, 1H), 2.40 (s, 6H), 2.66 (m, 1H), 2.67 (m, 1H), 2.68
(m, 1H), 2.91 (d, J = 9 Hz, 1H), 3.02 (dd, J1 = 10.5, 7.5 Hz, 1H), 3.21
(s, 3H), 3.43 (bs, 1H), 3.53 (d, J = 7.0 Hz, 1H), 3.68 (m, 1H), 3.71
(m, 1H), 3.81 (m, 1H), 4.04 (m, 1H), 4.16 (m, 1H), 4.23 (bs, 1H),
4.35 (bs, 1H), 4.44 (d, J = 7.5 Hz, 1H), 4.75 (dd, J1 = 10.5, 2.5 Hz,
1H), 4.85 (d, J = 4.5 Hz, 1H), 7.60 (bs, 1H). 13C NMR (500 MHz,
DMSO-d6) δ/ppm: 7.13, 8.55, 11.32, 15.15, 18.05, 18.91, 21.30, 21.38,
21.67, 22.48, 26.34, 27.71, 31.21, 34.97, 36.12, 36.89, 40.56, 41.87,
41.98, 45.13, 49.24, 61.88, 64.68, 65.12, 67.02, 68.48, 68.92, 72.90,
73.21, 73.97, 75.25, 76.68, 77.48, 77.68, 78.51, 83.05, 94.75, 102.08,
173.68, 177.47.
2′-O-[3-({2-[(7-Chloro-4-quinolinyl)amino]ethanoyl}amino)-
propyl]-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (15).
PS-Carbodiimide resin (PS-CDI; loading, 1.2 mmol/g) (325 mg,
0.403 mmol) was added to a dry reaction vessel. Compound 12 (77
mg, 0.326 mmol) and 1-hydroxybenzotriazole hydrate (29.3 mg, 0.217
mmol), dissolved in a mixture of DCM (5 mL) and DMF (2.5 mL),
were added to the dry resin. The mixture was stirred at room
temperature for 5 min upon which 7 (250 mg, 0.310 mmol), dissolved
in DCM (5 mL), was added. The reaction mixture was heated by
microwave irradiation at 70 °C for 6 min. HOBt was scavenged using
PS-trisamine (loading, 4.11 mmol/g) (420 mg, 1.73 mmol) for 3 h at
room temperature. The product was filtered off, and the resin was
washed with DCM (2 × 10 mL). After evaporation of filtrate, 278 mg
of white foam was obtained. Crude material was dissolved in 3 mL of
EtOAc and precipitated with the addition of n-hexane. Isolated
precipitate was further recrystallized from acetone/petroleum ether
(bp 35−60 °C), and 68 mg of the title product was isolated. Further
crystallization from filtrate resulted in isolation of additional amounts
(85 mg) of the title product (46%). MS (ES+): 1024 [MH]+. 1H NMR
(500 MHz, DMSO-d6) δ/ppm: 8.39 (d, J = 5.5 Hz, 1H), 8.23 (d, J =
9.2 Hz, 1H), 8.11 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.68 (t,
J = 6.0 Hz, 1H), 7.49 (dd, J = 8.9, 2.1 Hz, 1H), 6.25 (d, J = 5.2 H, 1H),
4.84 (d, J = 4.9 Hz, 1H), 4.75 (dd, J = 10 Hz, 1H), 4.37 (d, J = 7.3 Hz,
1H), 4.29 (m, 2H), 4.24 (d, J = 7.6 Hz, 1H), 4.21 (dd, J = 4.0, 2.0 Hz,
1H), 4.06 (m, 1H), 3.86 (dd, J = 6.9, 5.5 Hz, 2H), 3.79 (m, 1H), 3.60
(m, 1H), 3.52 (d, J = 7.3 Hz, 1H), 3.46 (m, 2H), 3.23 (m, 1H), 3.20
(s, 3H), 3.12 (ddd, J = 12.8, 6.4 Hz, 1H), 2.91 (dd, J = 9.0, 7.8 Hz,
1H), 2.81 (dd, J = 9.9, 7.5 Hz, 1H), 2.66 (m, 2H), 2.45 (m, 1H), 2.33
(dd, J = 11.9, 1.3 Hz, 1H), 2.25 (d, J = 11.6 Hz, 1H), 2.22 (s, 6H), 2.20
(s, 3H), 2.12 (dd, J = 11.9 Hz, 1H), 1.91 (m, 1H), 1.88 (br. s., 1H),
1.77 (m, 1H), 1.57 (m, 3H), 1.49 (m, 2H), 1.38 (m, 1H), 1.31 (dd, J =
13.6, 7.8 Hz, 1H), 1.16 (s, 3H), 1.15 (d, J = 6.1 Hz, 3H), 1.12 (s, 3H),
1.09 (d, J = 7.3 Hz, 4H), 1.06 (d, J = 5.8 Hz, 3H), 1.01 (s, 3H), 0.93
(d, J = 6.7 Hz, 3H), 0.91 (d, J = 7.6 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H),
0.80 (t, J = 7.3 Hz, 3H). 13C NMR (DMSO-d6) δ/ppm: 177.45,
168.63, 152.13, 150.56, 149.31, 133.84, 127.86, 124.67, 124.51, 117.89,
102.41 (C-1′), 99.43, 94.78, 82.66, 80.22, 77.75, 77.52, 76.76, 75.23,
74.02, 73.2, 72.95, 70.36, 68.96, 67.18, 65.06, 64.24, 61.77, 49.21,
46.21, 45.18, 42.21, 41.90, 41.16, 37.00, 36.09, 35.03, 31.81, 30.23,
27.85, 26.39, 22.47, 21.77, 21.38, 21.38, 18.90, 18.06, 15.17, 11.34,
8.80, 7.20. HRMS (ESI) m/z calcd for C52H87N5O13Cl (M + H+),
1024.5989; found, 1024.5996.
1
(62%) of the title product. MS (ES+): 990.6 [MH]+. H NMR (500
MHz, DMSO-d6) δ/ppm: 8.38 (d, J = 5.5 Hz, 1H), 8.18 (d, J = 7.6 Hz,
1H), 8.08 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.63 (ddd, J = 7.6, 1.2 Hz,
1H), 7.49 (t, J = 6.0 Hz, 1H), 7.45 (ddd, J = 7.5, 2.0 Hz, 1H), 6.23 (d,
J = 5.2 Hz, 1H), 4.84 (d, J = 4.9 Hz, 1), 4.75 (dd, J = 10.2, 2.6 Hz,
1H), 4.37 (d, J = 7.3 Hz, 1H), 4.29 (s, 1H), 4.28 (d, J = 4.3 Hz, 1H),
4.25 (d, J = 7.3 Hz, 1H), 4.21 (dd, J = 4.0, 1.8 Hz, 1H), 4.07 (m, 1H),
3.85 (dd, J = 6.0 Hz, 1H), 3.80 (dt, J = 9.2, 5.6 Hz, 1H), 3.59 (m, 1H),
3.53 (d, J = 7.3 Hz, 1H), 3.46 (m, 2H), 3.21 (s, 3H), 3.24 (m, 1H),
3.13 (m, 2H), 2.91 (dd, J = 9.5, 7.6 Hz, 1H), 2.81 (dd, J = 10.1, 7.3 Hz,
1H), 2.67 (m, 2H), 2.45 (m, 1H), 2.33 (dd, J = 12.0, 2.0 Hz, 1H), 2.25
(d, J = 15.0 Hz, 2H), 2.22 (s, 6H), 2.20 (s, 3H), 2.12 (dd, J = 11.6 Hz,
1H), 1.91 (m, 1H), 1.88 (m, 1H), 1.77 (m, 1H), 1.58 (m, 3H), 1.50
(d, J = 14.6 Hz, 1H), 1.49 (d, J = 15.0 Hz, 1H), 1.38 (m, 1H), 1.32
(dd, J = 14.8, 7.8 Hz, 1H), 1.16 (s, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.13
(m, 1H), 1.12 (s, 3H), 1.10 (d, J = 7.6 Hz, 3H), 1.06 (d, J = 6.1 Hz,
3H), 1.01 (s, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 7.2 Hz, 3H),
0.84 (d, J = 6.7 Hz, 3H), 0.79 (t, J = 7.3 Hz, 3H). 13C NMR (DMSO-
d6) δ/ppm: 176.86, 168.26, 150.31, 149.73, 147.98, 128.80, 128.58,
123.81, 121.47, 118.68, 101.83, 98.39, 94.24, 82.10, 79.66, 77.21,
76.98, 76.21, 74.66, 73.45, 72.62, 72.41, 69.82, 68.37, 66.61, 64.50,
63.66, 61.20, 48.63, 45.74, 44.63, 41.65, 41.32, 40.57, 36.43, 35.52,
34.47, 31.23, 29.68, 27.27, 25.83, 21.89, 21.19, 20.82, 20.78, 18.33,
17.49, 14.59, 10.76, 8.25, 6.64. HRMS (ESI) m/z calcd for
C52H88N5O13 (M + H+), 990.6379; found, 990.6364.
2′-O-[3-({2-[(7-Chloro-4-quinolinyl)amino]ethanoyl}amino)-
propyl]-3-O-decladinosyl-9-deoxo-9a-methyl-9a-aza-9a-homoery-
thromycin A (17). Compound 15 (0.08 g, 0.078 mmol) in HCl (10
mL, 3 M) was stirred at room temperature for 30 min. The reaction
mixture was diluted with EtOAc (20 mL), the pH was adjusted to pH
9.5 (by addition of 6 M NaOH), and the layers were separated.
Organic extracts were washed with water (2 × 20 mL). The combined
organic layers were evaporated and purified by column chromatog-
raphy (eluent DCM:MeOH:NH4OH = 90:9:1.5) yielding 50 mg
1
(74%) of the title product. MS (ES+): 866.59 [MH]+. H NMR (500
MHz, DMSO-d6) δ/ppm: 8.40 (d, J = 5.2 Hz, 1H), 8.24 (d, J = 8.9 Hz,
1H), 8.11 (t, J = 5.5 Hz, 1H), 7.81 (d, J = 2.1 Hz, 1H), 7.68 (t, J = 5.8
Hz, 1H), 7.49 (dd, J = 8.9, 2.1 Hz, 1H), 6.27 (d, J = 5.2 Hz, 1H), 5.14
(d, J = 6.7 Hz, 1H), 4.95 (dd, J = 11.1, 1.4 Hz, 1H), 4.74 (d, J = 7.3
Hz, 1H), 4.05 (d, J = 7.6 Hz, 1H), 3.86 (dd, J = 5.2, 3.4 Hz, 2H), 3.83
(m, 1H), 3.53 (s, 1H), 3.43 (m, 2H), 3.35 (m, 1H), 3.29 (m, 1H), 3.22
(m, 1H), 3.11 (m, 1H), 2.84 (dd, J = 9.6, 7.8 Hz, 1H), 2.69 (dd, J =
13.0, 6.6 Hz, 1H), 2.54 (m, 1H), 2.43 (ddd, J = 14.0, 9.6, 5.0 Hz, 1H),
2.27 (m, 1H), 2.22 (s, 3H), 2.21 (s, 6H), 2.06 (m, 2H), 1.78 (m, 2H),
1.57 (m, 3H), 1.39 (m, 2H), 1.31 (dd, J = 13.5, 5.0 Hz, 1H), 1.17 (m,
1H), 1.12 (m, 6H), 1.07 (s, 3H), 0.97 (s, 3H), 0.94 (d, J = 6.4 Hz,
3H), 0.86 (d, J = 7.3 Hz, 3H), 0.82 (d, J = 7.0 Hz, 3H), 0.77 (t, J = 7.3
Hz, 3H). 13C NMR (500 MHz, DMSO-d6) δ/ppm: 175.97, 168.63,
152.2, 150.52, 149.32, 133.82, 127.8, 124.68, 124.50, 117.89, 100.71,
99.47, 84.29, 79.83, 76.84, 76.55, 76.53, 74.01, 72.82, 70.12, 69.18,
67.89, 64.13, 61.81, 46.19, 43.67, 41.25, 41.02, 37.18, 21.81, 21.56,
21.01, 18.21, 16.89, 10.91, 8.58, 6.69. HRMS (ESI) m/z calcd for
C44H73N5O10Cl (M + H+), 866.5046; found, 866.5039.
2′-O-[3-({4-[(7-Chloro-4-quinolinyl)amino]butanoyl}amino)-
propyl]-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (19).
To a solution of 7 (0.18 g, 0.22 mmol) in DCM (10 mL), 4-[(7-
3224
dx.doi.org/10.1021/jm201676t | J. Med. Chem. 2012, 55, 3216−3227

Journal of Medicinal Chemistry
Article
chloro-4-quinolinyl)amino]butanoic acid (0.066 g, 0.25 mmol), HOBt
(0.037 g, 0.286 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide (0.071 g, 0.37 mmol), and triethylamine (TEA) (0.25 mL) were
added, and the reaction mixture was stirred overnight at rt. Water was
added, the organic layer separated, and the pH of the water layer was
adjusted to pH 6.5 and extracted with DCM. To the water layer, DCM
was added, and the pH was adjusted to pH 9.0 and extracted with
DCM. DCM layers at pH 6.5 and 9.0 were combined and dried over
Na2SO4. DCM was evaporated affording 0.25 g of the title product as a
light yellow powder. MS (ES+): 1052.7 [MH]+. 1H NMR (500 MHz,
CDCl3) δ/ppm: 8.42 (m, 1H), 8.23 (m, 1H), 8.05 (m, 1H), 8.01 (m,
1H), 7.81 (t, J = 5.2 Hz, 1H), 7.40 (m, 2H), 6.31 (m, 2H), 5.23 (m,
1H), 5.18 (d, J = 4.9 Hz, 1H), 4.73 (m, 1H), 4.38 (d, J = 7.3 Hz, 1H),
4.32 (d, J = 2.4 Hz, 1H), 4.10, 3.42 (m, 2H), 4.06 (m, 1H), 3.67 (m,
1H), 3.65 (d, J = 7.3 Hz, 1H), 3.48 (m, 1H), 3.61 (m, 1H), 3.30 (m,
1H), 3.34 (m, 2H), 3.30 (s, 3H), 3.05 (dd, J = 9.0, 7.5 Hz, 1H), 2.95
(dd, J = 10.1, 7.6 Hz, 1H), 2.70 (m, 1H), 2.69 (m, 1H), 2.59 (m, 1H),
2.56 (m, 1H), 2.07 (m, 1H), 2.44 (m, 2H), 2.34 (s, 6H), 2.33 (s, 3H),
2.33 (m, 1H), 1.61 (m, 1H), 2.16 (m, 2H), 2.04 (m, 1H), 1.95 (m,
1H), 1.89 (m, 1H), 1.67 (m, 1H), 1.46 (m, 1H), 1.75 (m, 1H), 1.24
(m, 1H), 1.69 (m, 1H), 1.63 (m, 1H), 1.29 (m, 1H), 1.32 (d, J = 6.1
Hz, 3H), 1.30 (s, 3H), 1.26 (s, 3H), 1.20 (d, J = 5.9 Hz, 3H), 1.19 (d, J
= 7.5 Hz, 3H), 1.10 (d, J = 7.0 Hz, 3H), 1.09 (s, 3H), 0.94 (d, J = 7.6
Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR
(500 MHz, CDCl3) δ/ppm: 179.0, 173.5, 151.7, 149.8, 147.2, 136.1,
126.8, 125.8, 123.2, 117.5, 103.1, 98.0, 94.5, 82.7, 79.8, 78.3, 77.6, 77.3,
74.6, 73.9, 73.4, 73.2, 72.2, 70.2, 68.3, 65.6, 65.1, 62.9, 49,4, 45.5, 44.5,
44.2, 42.5, 40.6, 38.4, 36.3, 34.7, 34.6, 29.0, 28.9, 27.6, 26.8, 22.3, 23.0,
21.8, 21.5, 21.5, 18.3, 16.4, 14.8, 11.4, 8.5, 7.5. HRMS (ESI) m/z calcd
for C54H91N5O13Cl (M + H+), 1052.6302; found, 1052.6294.
2′-O-{3-[(7-Chloro-4-quinolinyl)amino]propyl}-9-deoxo-9a-meth-
yl-9a-aza-9a-homoerythromycin A (22). To a solution of 7 (25.0 g,
0.031 mol) in DMSO (250 mL), 4,7-dichloroquinoline (30.7 g, 155
mmol) and Trisma Base (18.77 g, 155 mmol) were added. The
reaction mixture was heated at 105 °C for 18 h. The reaction mixture
was cooled to room temperature and evaporated to yield a slurry
product. The slurry product was resolved in 500 mL of dichloro-
methane and 1500 mL of water. The pH of the mixture was adjusted
to pH 5.0 by the addition of 1 M HCl, and layers were separated. The
water layer was extracted at pH 5 with DCM (5 × 500 mL). The layers
were separated. Further DCM (500 mL) was added, the pH was
adjusted to pH 6.0 by the addition of 1 M NaOH, and the layers were
separated. The water layer was extracted at pH 6.0−6.5 with DCM (22
× 500 mL). The organic layers extracted at pH 6.0−6.5 were dried
over Na2SO4, and the organic solvent evaporated yielding 17.91 g of
the crude product as light yellow crystals. The crude product of 17.91
g was recrystallized from acetonitrile to afford 12.89 g (43%) of the
title product. MS (ES+): 967.6 [MH]+. 1H NMR (500 MHz, DMSO-
d6) δ ppm: 8.37 (d, J = 5.2 Hz, 1H), 8.21 (d, J = 9.2 Hz, 1H), 7.75 (d, J
= 2.1 Hz, 1H), 7.41 (dd, J = 9.2, 2.1 Hz, 1H), 7.25 (t, J = 5.2 Hz, 1H),
6.43 (d, J = 5.5 Hz, 1H), 4.82 (d, J = 4.9 Hz, 1H), 4.72 (dd, J = 10.1,
2.4 Hz, 1H), 4.41 (d, J = 7.3 Hz, 1H), 4.31 (m, 2H), 4.19 (d, J = 7.6
Hz, 1H), 4.14 (dd, J = 4.0, 1.8 Hz, 1H), 4.05 (m, 1H), 3.88 (m, 1H),
3.63 (m, 2H), 3.49 (d, J = 7.0 Hz, 1H), 3.37 (d, J = 8.2 Hz, 1H), 3.30
(m, 2H), 3.22 (s, 3H), 2.90 (dd, J = 9.2, 7.6 Hz, 1H), 2.86 (dd, J =
10.1, 7.6 Hz, 1H), 2.55 (m, 3H), 2.29 (d, J = 11.3 Hz, 1H), 2.24 (s,
6H), 2.23 (d, J = 4.5, 1H), 2.18 (br. s., 3H), 1.91 (m, 2H), 1.81 (m,
3H), 1.75 (m, 1H), 1.59 (dd, J = 10.5, 3.2 Hz, 1H), 1.49 (dd, J = 15.0,
4.9 Hz, 1H), 1.43 (d, J = 14.6 Hz, 1H), 1.37 (m, 1H), 1.14 (d, J = 6.2
Hz, 3H), 1.13 (s, 6 Me, 6H), 1.10 (m, 1H), 1.10 (m, 1H), 1.06 (d, J =
5.8 Hz, 3H), 1.06 (d, J = 7.0 Hz, 3H), 1.03 (s, 3H), 0.94 (d, J = 6.7 Hz,
3H), 0.83 (d, J = 7.3 Hz, 3H), 0.79 (t, J = 7.0 Hz, 3H), 0.78 (d, J = 6.8
Hz, 3H). 13C NMR (DMSO-d6) δ/ppm: 177.39, 152.09, 150.45,
149.42, 133.60, 127.87, 124.37, 124.28, 117.86, 102.33, 98.98, 94.67,
82.65, 80.10, 77.73, 77.40, 76.69, 75.23, 73.95, 73.19, 72.84, 69.95,
68.96, 67.12, 65.07, 64.31, 61.85, 49.18, 45.14, 42.01, 41.85, 41.31,
39.88, 36.04, 35.00, 32.43, 28.87, 27.77, 26.20, 22.38, 21.77, 21.38,
21.31, 18.90, 18.04, 15.10, 11.32, 8.70, 7.07. HRMS (ESI) m/z calcd
for C46H77N5O10Cl (M + H+), 967.5774; found, 967.5759.
2′-O-{3-[(4-Quinolinyl)amino]propyl}-9-deoxo-9a-methyl-9a-
aza-9a-homoerythromycin A (23). To an ethanol solution of 22 (150
mg, 0.15 mmol in 25 mL), Pd/C (10%) was added (75 mg, 0.07
mmol), and the reaction mixture was stirred under hydrogen
atmosphere (5 barr). After 4 h, the catalyst was filtered off, and the
solvent was evaporated to yield 130 mg of crude product, which was
further purified by column chromatography (eluent DCM:MeOH:N-
H4OH = 90:9:1.5) yielding 90 mg (64%) of the white powder. MS
1
(ES+): 933.6 [MH]+. H NMR (500 MHz, DMSO-d6) δ/ppm: 8.36
(d, J = 5.2 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H),
7.59 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.08 (t, J = 5.3 Hz,
1H), 6.40 (d, J = 5.5 Hz, 1H), 4.83 (d, J = 4.9 Hz, 1H), 4.73 (dd, J =
10.2, 2.9 Hz, 1H), 4.43 (d, J = 7.3 Hz, 1H), 4.30 (m, 2H), 4.20 (d, J =
7.3 Hz, 1H), 4.17 (m, 1H), 4.07 (m, 1H), 3.90 (dt, J = 8.9, 5.8 Hz,
1H), 3.65 (m, 2H), 3.52 (d, J = 7.3 Hz, 1H), 3.39 (m, 3H), 3.23 (s,
3H), 2.91 (m, 1H), 2.87 (dd, J = 9.5, 7.0 Hz, 1H), 2.58 (m, 3H), 2.29
(d, J = 11.0 Hz, 1H), 2.24 (d, J = 15.0 Hz, 1H), 2.23 (s, 6H), 2.19 (s,
3H), 1.99 (dd, J = 11.6, 11.0 Hz, 1H), 1.86 (m, 4H), 1.76 (m, 1H),
1.60 (m, 1H), 1.50 (dd, J = 15.0, 5.0 Hz, 1H), 1.47 (dd, J = 11.8, 1.0
Hz, 1H), 1.37 (m, 1H), 1.19 (m, 1H), 1.15 (s, 3H), 1.15 (d, J = 7.0
Hz, 3H), 1.14 (s, 3H), 1.11 (d, J = 11.9 Hz, 1H), 1.06 (d, J = 6.3 Hz,
3H), 1.06 (d, J = 7.0 Hz, 3H), 1.02 (s, 3H), 0.93 (d, J = 6.7 Hz, 3H),
0.90 (d, J = 7.3 Hz, 3H), 0.80 (d, J = 7.5 Hz, 3H), 0.79 (t, J = 6.5 Hz,
3H). 13C NMR (DMSO-d6) δ/ppm: 177.42, 150.83, 150.33, 148.59,
129.33, 128.95, 124.01, 121.94, 119.25, 102.36, 98.45, 94.69, 82.65,
80.13, 77.74, 77.4, 76.71, 75.23, 73.97, 73.21, 72.89, 70.09, 68.93,
67.16, 65.06, 64.38, 61.79, 49.18, 45.14, 42.09, 41.90, 41.2, 40.38,
36.07, 35.01, 32.23, 29.04, 27.81, 26.28, 22.40, 21.79, 21.3, 21.33,
18.90, 18.09, 15.11, 11.33, 8.78, 7.11. HRMS (ESI) m/z calcd. for
C50H85N4O12 (M + H+), 933.6164; found, 933.6171.
2′-O-[3-({2-[(7-Chloro-4-quinolinyl)amino]ethyl}amino)-3-
oxopropyl]9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A
(25). To a solution of 10 (95 mg, 0.12 mmol) in DCM (10 mL),
TEA (0.11 mL, 0.81 mmol), HOBt (18 mg, 0.13 mmol), compound
14 (27 mg, 0.12 mmol), and EDCxHCl (33 mg, 0.1725 mmol) were
added. The reaction mixture was stirred at room temperature for 42 h.
To the reaction mixture, 30 mL of water was added (pH 8.0) and
extracted with DCM (3 × 30 mL). The organic layers were collected
and dried on Na2SO4. The solvent was evaporated to afford 130.7 mg
of yellowish solid, which was purified on Isolute SPE 10 g column
eluting with DCM/[MeOH/NH4OH] resulting in 62 mg (50%) of
1
the title product. MS (ES+) m/z: 1024.59 [MH]+. H NMR (600
MHz, DMSO-d6) δ/ppm: 8.65 (t, J = 5.5 Hz, 1H), 8.39 (d, J = 5.4 Hz,
1H), 8.13 (d, J = 9.1 Hz, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.45 (dd, J =
9.0, 2.0 Hz, 1H), 7.44 (m, 1H), 6.54 (d, J = 5.4 Hz, 1H), 4.83 (d, J =
4.7 Hz, 1H), 4.74 (dd, J = 10.1, 2.6 Hz, 1H), 4.39 (d, J = 7.3 Hz, 1H),
4.29 (br. s., 2H), 4.24 (br. s., 1H), 4.19 (d, J = 2.3 Hz, 1H), 4.04 (m,
1H), 3.88 (m, 1H), 3.66 (m, 1H), 3.63 (m, 1H), 3.52 (d, J = 7.0 Hz,
1H), 3.43 (d, J = 7.7 Hz, 1H), 3.35 (br. s., 4H), 3.14 (s, 3H), 2.89 (br.
s., 1H), 2.85 (dd, J = 10.1, 7.5 Hz, 1H), 2.66 (m, 2H), 2.43 (m, 1H),
2.33 (br. s., 1H), 2.30 (m, 2H), 2.22 (d, J = 15.2 Hz, 1H), 2.20 (s, 3H),
2.18 (s, 6H), 2.11 (dd, J = 12.0 Hz, 1H), 1.90 (m, 2H), 1.76 (m, 1H),
1.60 (m, 1H), 1.49 (m, 2H), 1.38 (m, 1H), 1.30 (dd, J = 14.0, 7.5 Hz,
1H), 1.16 (s, 3H), 1.14 (d, J = 6.1 Hz, 3H), 1.11 (s, 3H), 1.08 (d, J =
7.5 Hz, 4H), 1.06 (d, J = 5.9 Hz, 3H), 1.01 (s, 3H), 0.93 (d, J = 6.6 Hz,
3H), 0.88 (d, J = 7.5 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.79 (t, J = 7.4
Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ/ppm: 176.8, 171.5,
151.6, 149.8, 148.8, 133.1, 127.3, 123.9, 123.4, 117., 101.7 (C-1′), 98.3,
94.1, 82.3, 78.9, 77.1, 76.9, 76.1, 74.7, 73.4, 72.6, 72.3, 68.4, 67.4, 66.6,
64.5, 63.5, 61.2, 48.5, 44.5, 42.3, 41.6, 41.3, 40.3, 37.7, 36.4, 35.5, 34.4,
30.0, 27.2, 25.8, 21.8, 21.1, 20.8, 19.9, 18.3, 17.4, 14.5, 10.7, 8.1, 6.6.
HRMS (ESI) m/z calcd for C52H87N5O13Cl (M + H+), 1024.5989;
found, 1024.5978.
2′-O-{3-[(4-Quinolinylmethyl)amino]propyl}-9-deoxo-9a-methyl-
9a-aza-9a-homoerythromycin A (26a). To a solution of 7 (1 g, 1.24
mmol) in MeOH (35 mL), TEA (0.585 mL, 4.2 mmol), 4-
quinolinecarbaldehyde (164 mg, 1.04 mmol), and the reaction mixture
were stirred at room temperature for 18 h, after which NaBH4 (94 mg,
2.48 mmol) was added. The reaction mixture was stirred for an
additional 3 h, and after that time, the solvent was evaporated. The
3225
dx.doi.org/10.1021/jm201676t | J. Med. Chem. 2012, 55, 3216−3227

Journal of Medicinal Chemistry
Article
residue was dissolved in water, and the pH was adjusted to pH 9.5 and
extracted with DCM. The combined organic layers were dried over
anhydrous Na2SO4. Evaporation of the solvent yielded 1.3 g of yellow
powder product, which was further purified by column chromatog-
raphy, (eluentDCM:MeOH:NH4OH = 90:9:1.5) yielded 0.35 g (33%)
ASSOCIATED CONTENT
* Supporting Information
Additional experimental procedures and spectroscopic informa-
tion for selected compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
1
of the title product. MS (ES+): 947.66 [MH]+. H NMR (600 MHz,
DMSO-d6) δ/ppm: 0.78 - 0.82 (m, 6H), 0.93 (t, J = 7.2 Hz, 6H), 1.02
(m, 3H), 1.05 (d, J = 5.9 Hz, 3H), 1.06−1.07 (m, 1H), 1.08 (d, J = 7.5
Hz, 3H), 1.11 (s, 3H), 1.14 (d, J = 6.1 Hz, 3H), 1.15 (s, 3H), 1.24−
1.30 (m, 1H), 1.33−1.42 (m, 1H), 1.48 (d, J = 14.8 Hz, 1H), 1.49 (d, J
= 15.0 Hz, 1H), 1.54−1.58 (m, 1H), 1.63−1.73 (m, 2H), 1.74−1.81
(m, 1H), 1.83−1.86 (m, 1H), 1.87−1.92 (m, 1H), 2.05 (d, J = 11.2
Hz, 1H), 2.16 (s, 6H), 2.19 (s, 3H), 2.22 (d, J = 15.0 Hz, 1H), 2.32 (d,
J = 12.0 Hz, 1H), 2.44 (ddd, J = 12.2, 10.3, 4.2 Hz, 1H), 2.63−2.68
(m, 2H), 2.68−2.74 (m, 2H), 2.81 (dd, J = 10.1, 7.5 Hz, 1H), 2.89
(dd, J = 9.4, 7.5 Hz, 1H), 3.14 (s, 3H), 3.44 (d, J = 8.2 Hz, 1H), 3.51
(d, J = 7.2 Hz, 1H), 3.53−3.58 (m, 1H), 3.58−3.64 (m, 1H), 3.81−
3.86 (m, 1H), 4.02−4.07 (m, 1H), 4.14−4.16 (m, 1H), 4.17 (br. S.,
1H), 4.17 (s, 2H), 4.29 (br. s., 2H), 4.39 (d, J = 7.5 Hz, 1H), 4.75 (dd,
J = 10.1, 2.8 Hz, 1H), 4.83 (d, J = 4.9 Hz, 1H), 7.54 (d, J = 4.4 Hz,
1H), 7.60 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.74 (ddd, J = 8.3, 6.9, 1.3
Hz, 1H), 8.02 (dd, J = 8.5, 1.0 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.82
(d, J = 4.4 Hz, 1H). 13C NMR (600 MHz, DMSO-d6) δ/ppm: 176.91,
149.97, 147.46, 146.26, 129.33, 128.76, 126.52, 126.01, 123.69, 119.46,
101.79, 94.18, 82.13, 79.49, 77.20, 76.95, 76.20, 74.70, 73.46, 72.63,
72.41, 70.26, 68.40, 66.6, 64.52, 63.79, 61.26, 48.98, 48.53, 46.76,
44.61, 41.63, 41.36, 40.59, 35.57, 34.48, 31.31, 29.55, 27.25, 25.81,
21.88, 21.23), 20.83, 20.76, 18.34, 17.53, 14.61, 10.80, 8.23, 6.67.
HRMS (ESI) m/z calcd for C51H87N4O12 (M + H+), 947.6321; found,
947.6321.
AUTHOR INFORMATION
Corresponding Author
*Tel: 385-1-4590070. Fax: 385-1-4566724. E-mail: mihaela.
peric@mef.hr.
■
Present Addresses
§Galapagos istrazivack
̌ ́
i centar d.o.o, Prilaz baruna Filipovica 29,
̌
10000 Zagreb, Croatia.
∥Faculty of Veterinary Medicine, Department of Animal
Husbandry, V. Heinzela 55, 10000 Zagreb, Croatia.
⊥Biotechnology, GBB, University of Groningen, Nijenborgh 4,
NL 9747 AG Groningen, Netherlands.
#University of Zagreb School of Medicine, Center for
Translational and Clinical Research, Department for Inter-
̌
cellular Communication, Salata 2, 10000 Zagreb, Croatia.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We express gratitude to Dragica Muhvic
for incalculable assistance with the synthesis and for their
technical support. Also, we are indebted to Biserka Metelko and
Goran Landek for performing NMR experiments, Zeljko
■
́
and Đurđica Lugaric
́
̌
2′-O-{3-[Methyl(4-quinolinylmethyl)amino]propyl}-9-deoxo-9a-
methyl-9a-aza-9a-homoerythromycin A (27a). To a solution of 26a
(0.15 g, 0.158 mmol) in chloroform (5 mL), formaldehyde (0.028
mL) and formic acid (0.149 mL, 4.05 mmol) were added. The reaction
mixture was stirred at 60 °C. After 18 h, the reaction mixture was
diluted with DCM and water. Layers were separated, and the organic
layer was washed with brine and dried over Na2SO4. Evaporation of
the solvent yielded 0.16 g of crude product, which was further purified
by column chromatography (eluent DCM:MeOH:NH4OH =
90:9:1.5) yielding 0.1 g (65%) of the title product. MS (ES+):
Osman for the mass spectroscopy support, and Bill Leavens
for performing of HRMS spectra. The work was partially
supported by Medicines for Malaria Venture, Zurich, Switzer-
land.
ABBREVIATIONS USED
■
AZI, azithromycin; CHL, chloroquine; PS-CDI, polymer-
supported carbodiimide; SPE column, solid-phase extraction
column; TEA, triethylamine; DIPEA, diisopropylethylamine
1
961.67 [MH]+. H NMR (600 MHz, DMSO-d6) δ/ppm: 0.73 (d, J =
6.8 Hz, 3H), 0.80 (t, J = 7.5 Hz, 3H), 0.87 (d, J = 7.5 Hz, 3H), 0.94 (d,
J = 6.8 Hz, 3H), 1.03 (s, 3H), 1.05 (d, J = 6.1 Hz, 3H), 1.08 (d, J = 7.3
Hz, 3H), 1.10 (m, 1H), 1.12 (s, 3H), 1.12 (d, J = 4.5 Hz, 3H), 1.14 (s,
3H), 1.17−1.24 (m, 1H), 1.38 (ddd, J = 14.3, 10.1, 7.3 Hz, 1H), 1.45
(d, J = 14.0 Hz, 1H), 1.49 (dd, J = 14.9, 5.0 Hz, 1H), 1.53−1.58 (m,
1H), 1.69 (quin, J = 7.0 Hz, 2H), 1.74−1.80 (m, 1H), 1.81−1.84 (m,
1H), 1.85−1.88 (m, 1H), 2.03 (t, J = 11.7 Hz, 1H), 2.18 (s, 3H), 2.18
(br. s., 3H), 2.19 (s, 6H), 2.23 (d, J = 14.8 Hz, 1H), 2.30 (d, J = 10.6
Hz, 1H), 2.42−2.45 (m, 1H), 2.45−2.49 (m, 2H), 2.61−2.64 (m, 1H),
2.64−2.68 (m, 1H), 2.80 (dd, J = 10.0, 7.4 Hz, 1H), 2.89 (dd, J = 9.4,
7.5 Hz, 1H), 3.16 (s, 3H), 3.44 (d, J = 8.9 Hz, 1H), 3.50 (d, J = 7.2 Hz,
1H), 3.51−3.56 (m, 1H), 3.58−3.65 (m, 1H), 3.70 (dt, J = 9.0, 6.7 Hz,
1H), 3.85−3.94 (m, 2H), 4.00−4.07 (m, 1H), 4.14 (d, J = 7.5 Hz,
1H), 4.17 (dd, J = 4.2, 1.9 Hz, 2H), 4.28 (d, J = 8.2 Hz, 1H), 4.29 (s,
1H), 4.35 (d, J = 7.3 Hz, 1H), 4.75 (dd, J = 10.1, 2.8 Hz, 1H), 4.83 (d,
J = 4.9 Hz, 1H), 7.47 (d, J = 4.4 Hz, 1H), 7.58 (ddd, J = 8.3, 6.9, 1.3
Hz, 1H), 7.74 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 8.01 (dd, J = 8.5, 0.8 Hz,
1H), 8.28 (dd, J = 8.5, 0.8 Hz, 1H), 8.81 (d, J = 4.4 Hz, 1H). 13C
NMR (DMSO-d6) δ/ppm: 177.06, 150.04, 147.92, 144.71, 129.44,
128.97, 127.14, 126.04, 124.51, 121.24, 101.96, 94.36, 82.17, 79.85,
77.44, 77.11, 76.45, 74.89, 73.68, 72.81, 72.62, 69.92, 68.55, 66.80,
64.71, 63.92, 61.45, 58.60, 54.39, 48.78, 44.81, 42.14, 41.89, 41.54,
40.96, 35.75, 34.71, 32.60, 27.80, 27.43, 25.99, 22.00, 21.81, 21.40,
21.04, 18.52, 17.75, 14.78, 10.98, 8.49, 6.90. HRMS (ESI) m/z calcd
for C52H89N4O12 (M + H+), 961.6477; found, 961.6491.
REFERENCES
■
(1) WHO. World Malaria Report 2010; World Health Organisation:
Geneva, 2010.
(2) WHO. Guidelines for the Treatment of Malaria, 2nd ed.; WHO
Press, World Health Organisation: Geneva, 2010.
(3) Guerin, P. J.; Bates, S. J.; Sibley, C. H. Global resistance
surveillance: Ensuring antimalarial efficacy in the future. Curr. Opin.
Infect. Dis. 2009, 22, 593−600.
(4) Biot, C.; Chibale, K. Novel approaches to antimalarial drug
discovery. Infect. Disord.: Drug Targets 2006, 6, 173−204.
(5) Meunier, B. Hybrid molecules with a dual mode of action: Dream
or reality? Acc. Chem. Res. 2008, 41, 69−77.
(6) Wells, T. N.; Alonso, P. L.; Gutteridge, W. E. New medicines to
improve control and contribute to the eradication of malaria. Nat. Rev.
Drug Discovery 2009, 8, 879−891.
(7) Gingras, B. A.; Jensen, J. B. Activity of azithromycin (CP-62,993)
and erythromycin against chloroquine-sensitive and chloroquine-
resistant strains of Plasmodium falciparum in vitro. Am. J. Trop. Med.
Hyg. 1992, 47, 378−382.
(8) Anderson, S. L.; Berman, J.; Kuschner, R.; Wesche, D.; Magill, A.;
Wellde, B.; Schneider, I.; Dunne, M.; Schuster, B. G. Prophylaxis of
Plasmodium falciparum malaria with azithromycin administered to
volunteers. Ann. Intern. Med. 1995, 123, 771−773.
(9) Taylor, W. R.; Richie, T. L.; Fryauff, D. J.; Picarima, H.; Ohrt, C.;
Tang, D.; Braitman, D.; Murphy, G. S.; Widjaja, H.; Tjitra, E.; Ganjar,
A.; Jones, T. R.; Basri, H.; Berman, J. Malaria prophylaxis using
3226
dx.doi.org/10.1021/jm201676t | J. Med. Chem. 2012, 55, 3216−3227

Journal of Medicinal Chemistry
Article
azithromycin: A double-blind, placebo-controlled trial in Irian Jaya,
Indonesia. Clin. Infect. Dis. 1999, 28, 74−81.
erythromycin antibiotics. 4. Structure-activity relationships of eryth-
romycin esters. J. Med. Chem. 1972, 15, 635−638.
(24) Baker, W. R.; Clark, J. D.; Stephens, R. L.; Kim, K. H.
Modification of macrolide antibiotics. Synthesis of 11-deoxy-11-
(carboxyamino)-6-O-methylerythromycin A 11,12-(cyclic esters) via
an intramolecular Michael reaction of O-carbamates with an
.alpha.,.beta.-unsaturated ketone. J. Org. Chem. 1988, 53, 2340−2345.
(25) Jones, P. H.; Perun, T. J.; Rowley, E. K.; Baker, E. J. Chemical
modifications of erythromycin antibiotics. 3. Synthesis of 4″ and 11
esters of erythromycin A and B. J. Med. Chem. 1972, 15, 631−634.
(26) Alihodzic, S.; Pesic, D.; Peric, M. Preparation of 9-deoxy-9a-
methyl-9a-aza-9a-homoerythromycin A derivatives and their use for
the treatment of malaria. PCT/EP2008/059848.
(10) Dunne, M. W.; Singh, N.; Shukla, M.; Valecha, N.;
Bhattacharyya, P. C.; Dev, V.; Patel, K.; Mohapatra, M. K.; Lakhani,
J.; Benner, R.; Lele, C.; Patki, K. A multicenter study of azithromycin,
alone and in combination with chloroquine, for the treatment of acute
uncomplicated Plasmodium falciparum malaria in India. J. Infect. Dis.
2005, 191, 1582−1588.
(11) Noedl, H.; Krudsood, S.; Chalermratana, K.; Silachamroon, U.;
Leowattana, W.; Tangpukdee, N.; Looareesuwan, S.; Miller, R. S.;
Fukuda, M.; Jongsakul, K.; Sriwichai, S.; Rowan, J.; Bhattacharyya, H.;
Ohrt, C.; Knirsch, C. Azithromycin combination therapy with
artesunate or quinine for the treatment of uncomplicated Plasmodium
falciparum malaria in adults: A randomized, phase 2 clinical trial in
Thailand. Clin. Infect. Dis. 2006, 43, 1264−1271.
(12) Miller, R. S.; Wongsrichanalai, C.; Buathong, N.; McDaniel, P.;
Walsh, D. S.; Knirsch, C.; Ohrt, C. Effective treatment of
uncomplicated Plasmodium falciparum malaria with azithromycin-
quinine combinations: A randomized, dose-ranging study. Am. J. Trop.
Med. Hyg. 2006, 74, 401−406.
(27) Djokic, S.; Kobrehel, G.; Lopotar, N.; Kamenar, B.; Nagl, B.;
Drvos,
̌
D. Erythromycin series. Part 13.1 Synthesis and structure
elucidation of 10-Dihydro-10-deoxo-11-methyl-11-azaerythromycin A.
J. Chem. Res. (S) 1988, 152−153.
(28) Hanessian, S.; Moralioglu, E. Chemistry of 1-(dimethylamino)-
ethylidene and [alpha]-(dimethylamino)benzylidene acetals: Utility as
blocking group for diols and for selective esterification. Tetrahedron
Lett. 1971, 12, 813−816.
(13) Krudsood, S.; Silachamroon, U.; Wilairatana, P.; Singhasivanon,
P.; Phumratanaprapin, W.; Chalermrut, K.; Phophak, N.; Popa, C. A
randomized clinical trial of combinations of artesunate and
azithromycin for treatment of uncomplicated Plasmodium falciparum
malaria in Thailand. Southeast Asian J. Trop. Med. Public Health 2000,
31, 801−807.
(29) Hanessian, S.; Moralioglu, E. Chemistry and synthetic utility of
α-(diamino)benzylidene and 1-(dimethylamino)ethylidene acetals.
Can. J. Chem. 1972, 50, 233−245.
(30) Istuk, Z. M.; Mutak, S.; Kujundzic, N.; Kragol, G. Novel 9a-
carbamoyl- and 9a-thiocarbamoyl-3-decladinosyl-6-hydroxy and 6-
methoxy derivatives of 15-membered macrolides. Bioorg. Med. Chem.
2007, 15, 4498−4510.
(31) Pestka, S.; LeMahieu, R. A. Inhibition of [14C]chloramphenicol
binding to Escherichia coli ribosomes by erythromycin derivatives.
Antimicrob. Agents Chemother. 1974, 6, 39−45.
(32) Pestka, S.; LeMahieu, R. A. Effect of erythromycin analogues on
binding of [14C]erythromycin to Escherichia coli ribosomes.
Antimicrob. Agents Chemother. 1974, 6, 479−488.
(33) Filic, D.; Starcevic, K.; Pesic, D.; Jurmanovic, S.; Meenan, E.;
Spezzaferri, A.; Dilovic, I.; Prugovecki, B. Crystal structure and
physicochemical properties of crystalline form of 2′-O-{3-[(7-chloro-4-
quinolinyl)amino]propyl}-9-deoxo-9a-methyl-9a-aza-9a- homoery-
thromycin A. J. Pharm. Sci. 2011, 100, 2586−2598.
(34) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Quantitative assessment of antimalarial activity in vitro by a
semiautomated microdilution technique. Antimicrob. Agents Chemother.
1979, 16, 710−718.
(14) Schonfeld, W.; Kirst, H. A. Macrolide Antibiotics; Birkhauser
̈
Verlag: Basel, 2002.
(15) Dunne, M. W.; Singh, N.; Shukla, M.; Valecha, N.;
Bhattacharyya, P. C.; Patel, K.; Mohapatra, M. K.; Lakhani, J.; Devi,
C. U.; Adak, T.; Dev, V.; Yadav, R. S.; Lele, C.; Patki, K. A double-
blind, randomized study of azithromycin compared to chloroquine for
the treatment of Plasmodium vivax malaria in India. Am. J. Trop. Med.
Hyg. 2005, 73, 1108−1111.
(16) Chico, R. M.; Pittrof, R.; Greenwood, B.; Chandramohan, D.
Azithromycin-chloroquine and the intermittent preventive treatment
of malaria in pregnancy. Malar. J. 2008, 7, 255.
(17) Kalilani, L.; Mofolo, I.; Chaponda, M.; Rogerson, S. J.; Alker, A.
P.; Kwiek, J. J.; Meshnick, S. R. A randomized controlled pilot trial of
azithromycin or artesunate added to sulfadoxine-pyrimethamine as
treatment for malaria in pregnant women. PLoS One 2007, 2, e1166.
(18) Sykes, A.; Hendriksen, I.; Mtove, G.; Mandea, V.; Mrema, H.;
Rutta, B.; Mapunda, E.; Manjurano, A.; Amos, B.; Reyburn, H.;
Whitty, C. J. Azithromycin plus artesunate versus artemether-
lumefantrine for treatment of uncomplicated malaria in Tanzanian
children: a randomized, controlled trial. Clin. Infect. Dis. 2009, 49,
1195−1201.
(35) Verbanac, D.; Jelic, D.; Stepanic, V.; Tatic, I.; Ziher, D.; Kostrun,
S. Combined in silico and in vitro approach to drug screening. Croat.
Chem. Acta 2005, 78, 133−139.
(36) NCCLS Approved Standard. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. M7-A7 26(2),
2003.
(19) van Eijk, A. M.; Terlouw, D. J. Azithromycin for treating
uncomplicated malaria. Cochrane Database Syst. Rev. 2011, 2,
CD006688.
(20) Bukvic, K. M.; Peric, M.; Smith, K. S.; Schonfeld, Z. I.; Ziher, D.;
Fajdetic, A.; Kujundzic, N.; Schonfeld, W.; Landek, G.; Padovan, J.;
Jelic, D.; Ager, A.; Milhous, W. K.; Ellis, W.; Spaventi, R.; Ohrt, C.
Synthesis, structure-activity relationship, and antimalarial activity of
ureas and thioureas of 15-membered azalides. J. Med. Chem. 2011, 54,
3595−3605.
(21) Peric, M.; Fajdetic, A.; Rupcic, R.; Alihodzic, S.; Ziher, D.;
Bukvic, K. M.; Smith, K. S.; Ivezic-Schonfeld, Z.; Padovan, J.; Landek,
G.; Jelic, D.; Hutinec, A.; Mesic, M.; Ager, A.; Ellis, W. Y.; Milhous, W.
K.; Ohrt, C.; Spaventi, R. Antimalarial Activity of 9a-N Substituted 15-
Membered Azalides with Improved in Vitro and in Vivo Activity over
Azithromycin. J. Med. Chem. 2012, 55, 1389−1401.
(22) Schlunzen, F.; Zarivach, R.; Harms, J.; Bashan, A.; Tocilj, A.;
Albrecht, R.; Yonath, A.; Franceschi, F. Structural basis for the
interaction of antibiotics with the peptidyl transferase centre in
eubacteria. Nature 2001, 413, 814−821.
(23) Martin, Y. C.; Jones, P. H.; Perun, T. J.; Grundy, W. E.; Bell, S.;
Bower, R. R.; Shipkowitz, N. L. Chemical modification of
3227
dx.doi.org/10.1021/jm201676t | J. Med. Chem. 2012, 55, 3216−3227