Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin

Article abstract of DOI:10.1021/jm201615t

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation w

Full text of DOI:10.1021/jm201615t

Article
pubs.acs.org/jmc
Antimalarial Activity of 9a-N Substituted 15-Membered Azalides with
Improved in Vitro and in Vivo Activity over Azithromycin
†,○
Mihaela Peric,*,†,∥ Andrea Fajdetic,†,○ Renata Rupcic,†,○ Sulejman Alihodzic,†,○ Dinko Ziher,
̌
́
́
̌
́
̌
́
Mirjana Bukvic Krajacic,†,○ Kirsten S. Smith,‡,# Zrinka Ivezic-Schonfeld,†,● Jasna Padovan,†,○
́
̌
́
́
̈
Goran Landek,†,○ Dubravko Jelic,†,○ Antun Hutinec,†,○ Milan Mesic,†,○ Arba Ager,§ William Y. Ellis,‡
́
́
Wilbur K. Milhous,‡,⊥ Colin Ohrt,‡ and Radan Spaventi†,○
†GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz baruna Filipovica
́
29, 10000 Zagreb, Croatia
‡Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring,
Washington, DC 20910, United States
§Department of Microbiology and Immunology, University of Miami, Miami, Florida 33177, United States
#U.S. Army Medical Materiel Development Activity, 1430 Veterans Drive, Fort Detrick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide
investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next
generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine
functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited
marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high
selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative
showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for
compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than
azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.
multidrug resistant parasites. This further fosters the need for
the discovery of new antimalarial drugs.2
INTRODUCTION
■
Malaria continues to be one of the most widespread infectious
diseases of our time. This disease is caused by infection with
one or more protozoan parasites of the genus Plasmodium in
humans. Plasmodium falciparum is responsible for the most
serious form of the disease that could be complicated by coma
and death if left untreated. The malaria infection is estimated to
affect 250 million people worldwide leading to almost 1 million
fatal outcomes each year.1 Sub-Saharan Africa is the most
distressed region where the vast majority of deaths occur in
children under five years of age and in pregnant women. Even
though during the past century a large number of drugs for the
treatment of malaria have been developed, their utility in curing
malaria is declining due to the selection of single-drug or
Azithromycin is a semisynthetic macrolide antibiotic that has
become a blockbuster drug due to its wide spectrum of activity
and superior pharmacokinetic and safety properties. Antima-
larial potential of azithromycin was discovered by Walter Reed
Army Institute of Research.3−6 It was demonstrated that
azithromycin is a slow acting antimalarial7,8 that exerts its
activity by inhibiting protein synthesis on the prokaryote-like
ribosome in Plasmodium organelle, the apicoplast.9 Currently,
azithromycin is being extensively evaluated in clinical trials to
assess its potential use and combination partner in different
Received: November 29, 2011
Published: December 12, 2011
© 2011 American Chemical Society
1389
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Novel 15-Membered Azalides
a
Reagents and conditions: i, 3 or 4, arylhalide, DMSO, 100 °C, 14 h; ii, 3−5, aldehyde, TEA, NaBH4, MeOH, rt, 24 h; iii, 3−5, carboxylic acid,
HOBT, EDCxHCl, TEA, DCM, rt, 24 h; iv, 3 (to compound 14), CH2CHCOR4, CHCl3, reflux, 48 h; v, LiOH, H2O, THF, rt, 3 h; vi, 15,
(alkyl)aryl amine, HOBT, EDCxHCl, TEA, DCM, rt, 24 h; vii, H2, 10% Pd/C, EtOH, 4 bar, rt.
malaria-related indications.10−19 As no evidence for the
superiority or equivalence to other antimalarials has been
confirmed so far, the future of azithromycin for the treatment
action,21 oral bioavailability, long half-life, and good safety
record proven in children and pregnant women, we propose
more potent analogues from this class could offer properties
needed for malaria treatment and prophylaxis.
of malaria is uncertain.20
We recently reported extensive synthesis of new 9a-
substituted 15-membered azalide compound libraries compris-
ing of (thio)urea derivatives, investigated their in vitro and in
vivo antimalarial activity,22 and analyzed the structure−activity
relationship.22,23 This research led to the discovery of novel
urea and thiourea derivatives of 15-membered azalides with
promising in vitro antimalarial activity. Here we report
additional work undertaken in this area to avoid initial
Antimalarial drug discovery today is focusing efforts on
creating new molecules that are more potent and safer than
currently available drugs and at the same time affordable to the
target underprivileged population.2 These drugs should be
dosed orally and infrequently to ensure better patient
compliance. A very important feature of any potential new
class is the activity against strains resistant to other antimalarial
drug classes.7 Because of azithromycin-specific mode of
1390
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
Chart 1. Structures of the Substituents (R) Used in This Study
difficulties with in vivo efficacy. A set of 42 novel 9a-N
substituted amide and amine azalide compounds were
synthesized, their in vitro antimalarial activity was determined,
and structure−activity relationship (SAR) was established.
Furthermore, five compounds were selected for further
profiling by investigating their pharmacokinetic properties
and in vivo antimalarial efficacy in an adapted Plasmodium
berghei mouse model.
8−10 were obtained by reductive alkylation of amines 3−5
with appropriate aldehydes in the presence of triethylamine
(TEA) and sodium borohydride. Amides 11−13 were prepared
by acylation of corresponding amines 3−5 with various acids in
the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide (EDC) and 1-hydroxybenzotriazole (HOBT) in dichloro-
methane at room temperature. On the other hand, amides 16
were obtained by esterification of carboxylic acid 15 with
various amines using the above-described conditions for the
preparation of amides 11−13. Starting carboxylic acid 15 was
prepared by the Michael-type addition of amine 2 with methyl
or ethyl acrylate followed by ester hydrolysis of 14 under basic
conditions.29
CHEMISTRY
■
Identification of promising lead molecules as starting points for
new antimalarial programs represents a persistent challenge for
scientists within the field.24 Because a preliminary library of 9a-
N substituted urea and thiourea azalide derivatives has set forth
compounds with improved in vitro antimalarial activity over
azithromycin,22,23 we expanded research in this field. Here we
describe the discovery of novel classes of 9a-N substituted
azalides that were designed to additionally explore the chemical
space around 9a-N substituted 15-membered azalide scaffolds.
In the view of the stringent requirement for novel antimalarials
to enable low cost therapy for uncomplicated malaria and
intermittent preventive treatment (less than one US dollar per
treatment),2 we designed novel 9a-N substituted derivatives as
low cost compounds by using inexpensive starting materials and
short synthetic procedures. Structural features that guided the
design of novel macrolides included (1) replacement of the
ureido/thioureido group with an amido and amino function-
ality and (2) retaining the aryl or heteroaryl group for
improving antimalarial activity.25
RESULTS AND DISCUSSION
■
In Vitro Activity. The in vitro activity of 42 amides and
amines of 15-membered azalides was evaluated against P.
falciparum strain TM91C235, a multidrug resistant clone from
Southeast Asia and HepG2 hepatocellular carcinoma cell line.
The results presented as IC50 values are shown in Table 1 for
compounds from two amine classes and in Table 2 for two
different amide scaffolds.
Results from antimalarial in vitro screening indicated a high
percentage of compounds (>90%) with substantially improved
activity over azithromycin (up to >100-fold for the most potent
compounds) and a majority of compounds (>60%) with IC50 at
least 10-fold enhanced. As already observed with (thio)urea
derivatives,25 classes of compounds described here also exhibit
higher in vitro potencies against resistant strains than against
the sensitive strain (data not shown).
The presented library discloses compounds from an early
lead-optimization phase where the influence of major structural
features on the antimalarial activity was investigated, primarily
the removal of cladinose and desosamine sugars, type of linker
at the 9a-N position and the nature of the aromatic substituent
R (Scheme 1). As sugar substiutents, L-cladinose and D-
desosomine, present in azithromycin have been shown to
contribute substantially to its antibacterial activity,30 we
investigated the significance of sugar unit(s) on antimalarial
Here we report the synthesis of 42 novel compounds
belonging to four new series of 15-membered azalides (Scheme
1). Three series were completed starting from corresponding
primary amine intermediates 3−5, the synthesis and character-
ization of which was described previously.26−28 Amine
compounds 6 and 7 were prepared by the substitution of
primary amines 3 and 4 with aryl and heteroaryl halides at
elevated temperature. The chlorine atom from the quinoline
moiety was removed by catalytic hydrogenation of compounds
6u and 7u yielding compounds 6t and 7t. Amine compounds
1391
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
Table 1. In Vitro Activity of 9a-N Substituted Amine Derivatives 6−10 Determined against P. falciparum TM91C235 Strain and
HepG2 Hepatocellular Carcinoma Cell Line
a
b
des = D-desosamine. clad = L-cladinose, ND = not determined.
activity by comparing activities of paired analogues with the
same 9a-N substituent and different sugar content (11p, 13p;
8o, 9o; 8n, 9n; 8t, 9t; 11h, 12h; 8r, 9r, 10r; 10l, 11l, 12l; 8s,
9s, 10s; 8f, 9f; 6t, 7t; 6u, 7u). All aglycones (10r, 10s, and 13p)
were 3−4-fold less active in comparison to their decladinosyl
analogues (9r and 9s) and 24−37-fold less active than
analogues with both sugars (8r, 8s, and 11p). Furthermore,
the activity of decladinosyl derivatives was lower than the
activity of corresponding analogues with both sugars (i.e., IC50
of 8o < 9o; 11h < 12h; 6t < 7t) with one exception (IC50 of 6u
> 7u).
The influence of the linker at the 9a-N position and the
nature of the aromatic moiety on antimalarial activity were
analyzed in more detail by examining the amine (6 and 8) and
amide (11 and 16) compounds containing both sugars.
Generally, phenyl derivatives (8a, 11b, and 16b, IC50 of 104,
233, and 261, respectively) were significantly more active in
comparison to other tested monocyclic aromatic derivatives,
that is, five-membered heteroaromatic oxazole derivative 11p
(IC50 of 878) and six-membered heteroaromatic pyridine
derivatives (8m, 8n, and 8o, IC50 of 1478, 391, and 568,
respectively). On the other hand, phenyl derivatives (8f, 11i,
11h, and 16h) were less active than their naphthyl analoques
1392
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
Table 2. In Vitro Activity of 9a-N Substituted Amide Derivatives 11−13 and 16 Determined against P. falciparum TM91C235
Strain and HepG2 Hepatocellular Carcinoma Cell Line
a
b
des = D-desosamine. clad = L-cladinose, ND = not determined.
(8a, 11b, and 16b). Quinolyl derivatives (i.e., 6t, 6u, 8r, and 8s)
showed excellent antimalarial activity, suggesting that steric
bulk and heteroatom in bicyclic aromatic system might have a
positive impact on the antimalarial potency. Introduction of a
chlorine atom on the quinoline moiety retained superior
antimalarial activity irrespective of the presence of cladinose
(6u and 7u). However, antimalarial activity significantly
decreased when the methoxy group was positioned on C-6 of
the quinoline ring (compound 6v, IC50 of 854) in contrast to
the positive influence of electron donating groups on naphthyl
ring derivatives (8j and 11l).
Table 3. Structure−Activity Relationship of Amide (16b,
16h, 11b, 11h) and Amine (8a, 8f) Linked Derivatives
Compared with Urea Derivatives,31 All Containing
Analogous Aromate Substitutents (IC50 Determined against
P. falciparum TM91C235 Strain)
SAR observations regarding linker influence were expanded
comparing our earlier published 9a-N substituted 15-membered
azalides having urea and thiourea moieties in the linker22,23
with amide and amine analogues reported here. The most
populated analogue sets with naphthyl and phenyl aromatic
moieties (Table 3) reveal a significant effect of the linker used
on antimalarial activity. The activity increased among classes as
follows: urea > amide (16b, 16h, 11b, 11h) > amine (8a, 8f).
Occasionally, compounds with increased antimalarial potency
also showed a slight increase in cytotoxicity (Tables 1 and 2).
However, IC50 values determined in HepG2 cell line were
relatively high (85% of compounds had IC50 ≥ 20 μM)
generating favorable selectivity index (ratio between HepG2
IC50 and P. falciparum IC50 above 100) in almost all cases where
calculations were possible. The overall selectivity index for
novel 9a-N substituted amides (11−13 and 16) and amines
(6−10) was superior to the selectivity index of previous classes
1393
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
Table 4. Pharmacokinetic Parameters Estimated in Blood after Intravenous (IV) and Oral Gavage (PO) Administration to CD-
a
1 Mice (5 mg/kg IV and 25 mg/kg PO)
CLs (mL/min/kg)
Vss (L/kg)
T1/2 (h)
oral F (%)
fu
CLu (mL/min/kg)
azithromycin
16 5.3
4.0 0.2
7.7 1.5
10.2 0.3
23.3 3.3
10.5 3.3
8.8 3.6
2.9 1.3
4.9 1.5
4.9 1.1
5.0 1.3
7.8 2.1
8.3 1.1
17.8 9.4
14.4 1.1
8.7 1.6
6.9 0.7
16.5 3.5
72
0.69
0.42
0.39
0.57
0.43
0.13
23.2
9.5
20
6u
6t
20.4
1.7
8t
19.5
12.7
23.6
18
11c
16k
54
81
a
CL = blood clearance, Vss = steady-state volume of distribution, T1/2 = half life, F = bioavailability, fu = fraction unbound in mouse plasma, CLu =
a
b
CLs/fu, assuming Cbl/Cpl = 1, = n = 1, = n = 2.
Chart 2. Structures of the Azalide Compounds Used for in Vivo Study in the Mouse Model
consisting of urea and thiourea derivatives22,23 due to an overall
marked improvement in antimalarial activity accompanied by
decrease in cytotoxicity.
Preliminary Pharmacokinetics and in Vivo Antima-
larial Activity. The in vivo pharmacokinetic profiles (Table 4)
were investigated in the mouse for five compounds (Chart 2).
Two amides 16k and 11c and three amines 6u, 6t, 8t were
selected based on their favorable in vitro profile (IC50 against P.
falciparum < 200 nM and HepG2 IC50 > 30 μM) and high
solubility when prepared as acetic salts (≥10 mg/kg).
In vitro metabolic stability studies in liver microsomes
(mouse, rat, and human) indicated good stability across species,
resulting in low intrinsic clearance (CLi < 0.6 mL/min·g liver),
with the exception of compound 16k which showed a low to
moderate intrinsic CLi (1.8 mL/min·g liver) in human liver
microsomes. Plasma protein binding (PPB) in the mouse
suggested these compounds have low PPB ranging from 43 to
61%, with the exception of compound 16k which was 87%
protein bound.
In line with the in vitro mouse microsomal stability results,
all compounds had a very low blood CL (<15% LBF), with the
exception of compound 11c which had a slightly higher CL (ca.
26% LBF). When correcting the systemic clearance for protein
binding, compound 11c had a moderate clearance, and
compound 16k had a high clearance. Overall, the tested
compounds were characterized by a moderate (compounds 6t,
8t, and 6u) to a large (compounds 11c and 16k) volume of
distribution and a very long half-life. Compounds with an
ureido linker functionality connecting the aromatic moiety with
the macrolide 15-membered ring scaffold had a very low oral
bioavailability (FPO < 1%) in the mouse, and no improvement
was observed with the insertion of a thiourea motif into the
linker (data not shown). Replacement of the (thio)urea
functionality with an amide in the linker resulted in a significant
increase in oral bioavailability (16k, 11c). A substantially
improved oral exposure and oral bioavailability were obtained
when an aminoquinoline moiety was present as aromatic
substituent on the azalide unit (6u, 8t).
In Vivo Efficacy Studies in Mice Models. The in vivo
efficacy was evaluated for five selected azalide derivatives using
the P. berghei mouse model, a model that is routinely used to
screen larger numbers of compounds at this stage of discovery.
As the efficacy in the P. berghei murine model with our previous
set of compounds with low to moderate oral bioavailability was
modest upon oral administration, the intravenous route was
chosen for further screening in order to enable direct delivery
of compound to the site of the infection (the blood). In
addition, it has been shown that azithromycin can have a lower
bioavailability when administered orally after food intake,32 and
as infected animals in this model need to have food ad libidum
at all times, the IV route was chosen to avoid potential
absorption issues. The dosing regimen included administration
of 5 mg/kg of compound twice daily on days 3, 4, and 5
postinfection, while azithromycin, used as a control, was applied
at doses 5 mg/kg and 15 mg/kg in the same dosing regimen.
None of the compounds tested, including azithromycin,
cured mice (i.e., cleared them of parasitemia until day 31) at the
doses administered. In order to evaluate and compare the
compound efficacy, survival time of the last animal was used to
assess the time needed to delay malaria-related deaths among
treated animals (Table 5).
Azithromycin treated mice had a survival time ranging from
17 to 27 days at 5 mg/kg and 19 to 29 days at a dose of 15 mg/
kg, depending on the experiment. Animals treated with amine
derivatives 6u, 6t, and 8t had longer survival times of +9, +3,
and +3 days, respectively, in comparison to the azithromycin-
treated group at an equivalent dose. The amide derivatives 11c
and 16k exhibited shorter survival times than azithromycin
group, −12 and −9 days, respectively.
On the basis of this initial efficacy screening result,
compound 6u was selected to be further profiled in a dose
ranging study using the same animal model (Table 6) at doses
1394
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
their chemical modifications seem a valid stream for
investigation. The amide and amine series of 15-membered
azalides presented in this publication demonstrate a marked
improvement in in vitro antimalarial activity in comparison to
azithromycin, as well as over our previous set of compounds.22
Nevertheless, improved in vivo activity in comparison to
azithromycin for compounds screened in the P. berghei mouse
model was only observed with three (6u, 6t, and 8t) of five
amine derivatives following intravenous dosing. Although the
overall disposition kinetics for all five amine derivatives were
similar (low systemic clearance, moderate to large volume of
distribution, very long half-lives), unbound clearance values for
these compounds differ. The unbound clearance correlates well
with the in vivo efficacy results in the P. berghei mouse model
such that the most efficacious compounds, 6u, 6t, and 8t, have
a low clearance, suggesting that the relative efficacy of these
compounds may be influenced by the AUC/IC50 ratio.
Furthermore, it has been reported that macrolides’ lipophilicity
and cationic nature influence their accumulation and retention
in different cell types;33 thus unequal distribution into different
blood cell types could also be the reason for their different
efficacy effects in this murine model.
The fact, however, remains that the level of improvement in
in vitro activity did not fully translate in vivo. Even though
aforementioned differences in unbound clearance in vivo could
contribute to this effect, the most likely cause of the lack of in
vitro−in vivo correlation is the difference in parasite species
used in these two assays: P. falciparum vs P. berghei. This effect
was already presumed with our previous set of compounds and
has also been observed with some other compound classes.22
The P. berghei murine model is more convenient for screening
of numerous compounds and is thus used frequently in early
stages of drug discovery; however it is not necessarily
representative of P. falciparum human malaria. Thus, future
evaluation of the most active compounds should be performed
in some of the available P. falciparum animal models such as the
Aotus monkey or its nonprimate replacement the humanized
mouse model.36,37
Table 5. In Vivo Efficacy of Five Selected Compounds in the
P. berghei Mice Model (at 5 mg/kg, IV, BID, for 3 days)
Expressed As Survival Relative to Azithromycin
a
survival relative to azithromycin
(
days)
azithromycin
0
+9
6u
6t
+3
8t
+3
11c
16k
−12
−9
a
Survival of the last animal alive in the compound dosing group
compared to the survival of the last animal in the azithromycin group
expressed in days (more (+) or less (−) than azithromycin).
Table 6. Efficacy of Compound 6u in Plasmodium berghei
Infected Mice Following Three Days of Intravenous (IV)
and Intraperitoneal (IP) Dosing
a
survival of cured animals
compound
6u
dose (mg/kg)
IV
IP
20
15
10
20
15
10
10
5
5/5
5/5
1/5
2/5
1/5
0/5
2/5
0/5
0/5
0/5
0/5
5/5
3/5
0/5
4/5
3/5
2/5
2/5
0/5
0/5
0/5
0/5
azithromycin
chloroquine
2.5
1.25
control
a
(no. of mice without parasitemia on day 31)/(total no. of mice at
doses).
of 20, 15, and 10 mg/kg BID for 3 days by IV (intravenous)
and IP (intraperitoneal) route. Azithromycin and chloroquine
were included as comparator drugs and administered in the
same dosing. Azithromycin was administered at equivalent
doses as 6u, whereas chloroquine was dosed at lower doses 10,
5, 2.5, and 1.25 mg/kg due to safety issues.
Overall, compound 6u has several interesting properties that
justify its progression into further screens as a promising
antimalarial drug candidate: it is in vitro superior to
azithromycin and has macrolide-like pharmacokinetics and in
vivo efficacy in doses lower than azithromycin and comparable
to chloroquine (when molar concentrations were considered).
Key chemical features of compound 6u include its two
distinctive pharmacologically active elements, the azalide and
chloroquinoline moieties, which could both contribute to the
overall potency. Some medicinal chemistry approaches
advocate hybrid chemical entities, comprising two pharmaco-
phores, that offer a dual mode of action by affecting two
different targets. In order to validate if compound 6u could be
considered a hybrid molecule, a set of assays verifying whether
it possesses macrolide mode of action (like inhibition of
apicoplast protein synthesis and/or delayed death phenotype)
as well as aminoquinoline mode of action (inhibition of heme
polymerization) will be performed. Further evaluation of
compound 6u, such as extensive evaluation of its ADME
properties and safety are also warranted in order to assess its
full potential for the clinical use in malaria treatment and
prophylaxis.
Upon IV administration, compound 6u cured 5/5 mice at
doses of 20 and 15 mg/kg and also IP at a dose of 20 mg/kg. At
equivalent doses and administration routes, azithromycin cured
only 2/5, 1/5, and 4/5 mice, respectively. Chloroquine only
cured mice at the highest dose 10 mg/kg (2 out of 5) both
following IV and IP administration. Following IV admin-
istration of 6u and azithromycin at 10 mg/kg, 1/5 mice and 0/5
mice were cured respectively, whereas upon IP administration
at this dose 0/5 mice were cured with compound 6u and 2/5
mice were cured with azithromycin, respectively. These in vivo
results demonstrate superior efficacy of compound 6u to
azithromycin upon IV administration. The in vivo efficacy
observed in the P. berghei murine model confirmed an
improvement in potency of this new derivative over
azithromycin, as already observed in the in vitro P. falciparum
test.
DISCUSSION
■
Novel antimalarial agents are urgently needed, particularly the
ones that would enable infrequent dosing, long lasting effect,
and parasite clearance efficacy. Macrolides are a class of anti-
infective agents that could provide these desired properties, and
1395
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
8.27 (d, J = 9.16 Hz, 1H), 7.77 (d, J = 2.14 Hz, 1H), 7.43 (dd, J = 2.14,
9.16 Hz, 1H), 7.28 (t, J = 5.34 Hz, 1H), 6.47 (d, J = 5.80 Hz, 1H), 4.90
(dd, J = 2.80, 9.80 Hz, 1H), 4.79 (d, J = 4.88 Hz, 1H), 4.42 (d, J = 7.02
Hz, 1H), 4.29 (s, 1H), 4.23 (d, J = 7.63 Hz, 1H), 4.07 (dq, J = 6.30,
9.60 Hz, 1H), 4.02 (d, J = 6.60 Hz, 1H), 3.95−3.97 (m, 1H), 3.61 -
3.70 (m, 1H), 3.53 (d, J = 6.90 Hz, 1H), 3.52 (d, J = 6.71 Hz, 1H),
3.23−3.27 (m, 2H), 3.22 (s, 3H), 3.03 (dd, J = 7.63, 10.07 Hz, 1H),
2.93−2.99 (m, 1H), 2.90 (dd, J = 6.71, 9.46 Hz, 1H), 2.76 (q, J = 6.71
Hz, 1H), 2.72 (dq, J = 6.00, 7.50 Hz, 2H), 2.62−2.65 (m, 1H), 2.57−
2.62 (m, 1H), 2.42 (ddd, J = 4.12, 10.30, 12.13 Hz, 1H), 2.27 (d, J =
14.65 Hz, 1H), 2.21 (s, 6H), 2.09 (dd, J = 9.31, 13.28 Hz, 1H), 1.87−
1.98 (m, 3H), 1.73−1.83 (m, 2H), 1.55−1.61 (m, 1H), 1.45−1.52 (m,
2H), 1.33−1.43 (m, 2H), 1.17 (s, 3H), 1.15 (d, J = 6.41 Hz, 3H), 1.13
(s, 3H), 1.10 (d, J = 7.32 Hz, 3H), 1.07 (d, J = 6.10 Hz, 3H), 1.04−
1.06 (m, 1H), 1.01 (d, J = 6.70 Hz, 3H), 1.00 (s, 3H), 0.99 (d, J = 7.63
Hz, 3H), 0.84 (d, J = 7.02 Hz, 3H), 0.80 (t, J = 7.32 Hz, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 176.3, 152.0, 150.2, 149.3, 133.4, 127.7,
124.3, 124.1, 117.4, 102.0, 98.8, 95.4, 77.5, 76.6, 75.1, 74.4, 72.6, 70.6,
67.1, 64.9, 64.8, 48.9, 48.7, 41.0, 40.5, 34.9, 30.0, 25.7, 21.4, 21.4, 21.0,
18.6, 18.3, 11.0, 9.3; MS (ESI) m/z calcd for C49H82N4O12Cl (M +
H+) 953.5618; found 953.5638.
9a-[3-(7-Chloro-quinolin-4-ylamino)propyl]-3-O-decladino-
syl-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (7u).
Compound 7u (0.32 g, Y = 21%) was obtained according to the
described procedure for compound 6u, starting from intermediate 4
(1.5 g, 1.89 mmol) and 4,7-dichloroquinoline (0.75 g, 3.39 mmol); 1H
NMR (500 MHz, DMSO-d6) δ 8.39 (d, J = 5.19 Hz, 1H), 8.29 (d, J =
9.16 Hz, 1H), 7.78 (d, J = 2.14 Hz, 1H), 7.44 (dd, J = 2.14, 9.16 Hz,
1H), 7.20 (t, J = 4.88 Hz, 1H), 6.45 (d, J = 5.49 Hz, 1H), 5.03 (d, J =
9.80 Hz, 1H), 5.00 (d, J = 5.80 Hz, 1H), 4.51−4.67 (m, 1H), 4.40 (d, J
= 7.32 Hz, 1H), 4.29 (s, 1H), 4.22−4.29 (m, 1H), 3.52 (br. s., 1H),
3.46−3.50 (m, 1H), 3.44 (br. s., 1H), 3.34−3.40 (m, 1H), 3.18−3.30
(m, 2H), 3.09 (dd, J = 8.00, 10.00 Hz, 1H), 2.83−2.99 (m, 1H), 2.75
(br. s., 1H), 2.63−2.72 (m, 1H), 2.54−2.61 (m, 1H), 2.47−2.50 (m,
1H), 2.45 (ddd, J = 3.80, 10.00, 12.50 Hz, 1H), 2.22 (s, 6H), 1.98−
2.09 (m, 1H), 1.85−1.97 (m, 3H), 1.74−1.85 (m, 3H), 1.54−1.64 (m,
1H), 1.41 (ddd, J = 7.17, 10.68, 14.19 Hz, 1H), 1.14 (d, J = 7.02 Hz,
3H), 1.10 - 1.14 (m, 2H), 1.08 (s, 3H), 1.09 (d, J = 6.30 Hz, 3H), 1.01
(s, 3H), 0.98 (d, J = 6.71 Hz, 3H), 0.84−0.89 (m, 6H), 0.78 (t, J =
7.32 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) δ 7.96, 8.24, 10.60,
15.92, 17.73, 20.94, 21.26, 21.45, 26.50, 26.96, 29.12, 30.12, 36.43,
39.11, 40.30, 40.30, 40.99, 43.72, 50.91, 64.42, 68.29, 70.16, 73.31,
74.35, 75.97, 76.15, 76.15, 90.17, 98.57, 117.39, 123.80, 124.14,
127.35, 133.22, 148.98, 150.08, 151.79, 175.34; MS (ESI) m/z calcd
for C41H66N4O9Cl (M+H+) 795.4675; found 795.4683.
CONCLUSIONS
■
We report here the synthesis and antimalarial activity of the
novel class of potent macrolides substituted with amide and
amine function at the 9a-N position of the 15-membered
azalide scaffold. The compounds were synthesized via a simple
and inexpensive chemical procedure using easily available
building blocks to respond to the demand for low-cost novel
antimalarial agents. When compared to our previous class of
compounds, 15-membered substituted (thio)ureas,22 these
classes exhibited marked improvements with regard to the in
vitro antimalarial activity, selectivity, PK properties, and in vivo
efficacy. The best compound from this novel set of molecules,
compound 6u, showed improvement over azithromycin >100
fold in the P. falciparum in vitro assay that translated in the in
vivo efficacy after IV dosing by curing mice more efficiently
than azithromycin and comparable to chloroquine. Detailed
pharmacology, safety, and mode of action studies are
additionally needed to reveal the true potential of this class,
and compound 6u in particular, for its clinical use in the
treatment and prophylaxis of malaria.
EXPERIMENTAL SECTION
■
Chemistry. All commercial reagents (Merck, Sigma-Aldrich) were
used as provided unless otherwise indicated, and all solvents are of the
highest purity unless otherwise noted. The purity of final compounds
was assessed by the analytical LC-MS method and found to be ≥95%
unless otherwise stated. The LC-MS analyses were performed using
Waters Acquity UPLC instrument equipped with diode-array detector
and MS detector, Waters SQD, using the following method: column,
Waters Acquity UPLC BEH C18, 2.1 × 50 mm, 1.7 μm particles;
mobile phase A, 0.1% HCOOH in water, mobile phase B, 0.1%
HCOOH in CH3CN, isocratic 5% B in 1.5 min, then gradient 5−80%
B in 7.25 min followed by 1.25 min at 90% B.
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. 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 chromatography was carried out over Merck Kieselgel 60
(230−400 mesh) or on SPE cartrige with average size silica 50 μm.
Thin layer chromatography was performed on 0.24 mm silica gel plates
Merck TLC 60F254. The eluent used was indicated and solvent ratios
refer to volume. In general, organic solutions were dried with
anhydrous Na2SO4 or K2CO3, evaporation and concentration were
carried out under reduced pressure below 40 °C, unless otherwise
noted.
9a-[3-(9H-Purin-6-ylamino)propyl]-9-deoxo-9-dihydro-9a-
aza-9a-homoerythromycin A (6q). 6-Chloro-9H-purine (0.56 g,
3.61 mmol) and diisopropylethyl amine (1 mL) were added to a
solution of intermediate 3 (0.5 g, 0.63 mmol) in n-butanole (2 mL).
The reaction mixture was stirred at 80 °C for 18 h, than H2O (100
mL) and CH2Cl2 (100 mL) were added, and the pH adjusted to 10,
the layers were separated and the CH2Cl2 layer was washed with brine
(50 mL), dried over K2CO3, and evaporated in a vacuum. The residue
was precipitated from EtOAc-hexane and purified by column
chromatography on silica gel using solvent system DCM/MeOH/
1
NH4OH = 90:9:0.5 yielding compound 6q (0.30 g, Y = 52%); H
NMR (500 MHz, DMSO-d6) δ 8.17 (br. s., 1H), 8.05 (br. s., 1H), 7.64
(br. s., 1H), 4.90 (dd, J = 2.44, 10.07 Hz, 1H), 4.79 (d, J = 4.88 Hz,
1H), 4.41 (d, J = 7.32 Hz, 1H), 4.28 (s, 1H), 4.21 (d, J = 7.63 Hz, 1H),
4.06 (dq, J = 6.20, 9.60 Hz, 1H), 4.02 (dd, J = 1.68, 5.95 Hz, 1H), 3.94
(d, J = 7.00 Hz, 1H), 3.62−3.69 (m, 1H), 3.51 (d, J = 7.00 Hz, 1H),
3.51 (d, J = 6.41 Hz, 1H), 3.38−3.50 (m, 2H), 3.22 (s, 3H), 3.05 (dd, J
= 7.63, 9.77 Hz, 1H), 2.91 (dd, J = 7.63, 9.46 Hz, 1H), 2.84−2.89 (m,
1H), 2.71 (dq, J = 4.00, 7.30 Hz, 1H), 2.66−2.77 (m, 1H), 2.54−2.59
(m, 1H), 2.56 (dd, J = 3.00, 11.50 Hz, 1H), 2.42 (ddd, J = 4.00, 10.00,
12.00 Hz, 1H), 2.27 (d, J = 14.95 Hz, 1H), 2.22 (s, 6H), 2.04 (t, J =
11.60 Hz, 1H), 1.92−1.98 (m, 1H), 1.82 - 1.91 (m, 2H), 1.73−1.81
(m, 1H), 1.66−1.74 (m, 1H), 1.54−1.63 (m, 1H), 1.50−1.56 (m, 1H),
1.50 (dd, J = 4.88, 14.65 Hz, 1H), 1.34−1.43 (m, 1H), 1.28−1.35 (m,
1H), 1.18 (s, 3H), 1.15 (d, J = 6.10 Hz, 3H), 1.13 (s, 3H), 1.10 (d, J =
All final compounds were isolated as amorphous solid.
9a-[3-(7-Chloro-quinolin-4-ylamino)propyl]-9-deoxo-9-dihy-
dro-9a-aza-9a-homoerythromycin A (6u). 4,7-Dichloroquinoline
(3.0 g, 15.5 mmol) was added to a solution of intermediate 3 (4.09 g,
5.05 mmol) in DMSO (12 mL). The reaction mixture was stirred at
100 °C for 14 h and then the mixture of H2O (100 mL) and EtOAc
(100 mL) was added. The solvents were separated and the EtOAc
layer was washed with brine (50 mL), dried over K2CO3, and
evaporated in a vacuum. The residue was precipitated from EtOAc-
hexane. The precipitated solid was filtered and purified by column
chromatography on silica gel using solvent system DCM/MeOH/
NH4OH = 90:9:0.5. Work up of the chromatography fraction and
precipitation from EtOAc/hexane yielded compound 6u (1.2 g, Y =
1
25%); H NMR (500 MHz, DMSO-d6) δ 8.39 (d, J = 5.19 Hz, 1H),
1396
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
7.32 Hz, 3H), 1.07 (d, J = 5.80 Hz, 3H), 1.05 - 1.07 (m, 1H), 1.00 (s,
3H), 0.97 - 1.00 (m, 6H), 0.83 (d, J = 6.71 Hz, 3H), 0.79 (t, J = 7.32
Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 9.31, 9.31, 10.90, 14.92,
18.13, 18.39, 20.89, 21.28, 21.36, 22.09, 26.96, 27.24, 27.72, 29.77,
34.74, 39.43, 40.23, 40.23, 40.23, 40.27, 44.16, 48.74, 49.75, 60.49,
62.86, 64.58, 64.76, 67.03, 70.60, 72.65, 73.38, 74.30, 75.23, 76.32,
77.29, 77.81, 82.53, 94.90, 101.91, 139.10, 152.28, 176.23; MS (ESI)
m/z calcd for C45H80N7O12 (M + H+) 910.5865; found 910.5859.
9a-[3-(Quinolin-4-ylamino)propyl]-9-deoxo-9-dihydro-9a-
aza-9a-homoerythromycin A (6t). To a solution of compound 6u
(50 mg, 0.05 mmol) in EtOH (15 mL), 10% Pd/C (30 mg) was added
and the reaction mixture was hydrogenated in Parr apparatus at 4 bar
of hydrogen pressure for 24 h. The catalyst was filtrated off and solvent
evaporated under reduced pressure. Product was purified by column
chromatography (SPE column 5 g, eluent: DCM/MeOH/NH4OH =
90:9:0.5) and then precipitated from EtOAc:hexane yielding
equiv) was than added and the reaction mixture was stirred overnight
at room temperature. The MeOH was evaporated and the residue
extracted between DCM and water yielding crude products which
were purified on SPE column eluating gradiently starting from 100%
DCM and ending with mixture of DCM/MeOH/NH4OH = 90:9:0.5
yielding compounds 8−10.
9a-{3-[(Quinolin-4-ylmethyl)amino]propyl}-9-deoxo-9-dihy-
dro-9a-aza-9a-homoerythromycin A (8t). White powder (Y =
1
40%); H NMR (500 MHz, DMSO-d6) δ 8.83 (d, J = 4.27 Hz, 1H),
8.21 (d, J = 8.24 Hz, 1H), 8.02 (d, J = 8.54 Hz, 1H), 7.74 (t, J = 7.48
Hz, 1H), 7.60 (t, J = 7.48 Hz, 1H), 7.55 (d, J = 4.27 Hz, 1H), 4.93 (d, J
= 10.38 Hz, 1H), 4.80 (d, J = 4.88 Hz, 1H), 4.43 (d, J = 7.32 Hz, 1H),
4.23 (d, J = 7.32 Hz, 1H), 4.21 (s, 2H), 4.12 (s, 1H), 4.04 (dq, J =
6.30, 9.60 Hz, 1H), 3.96 - 3.99 (m, 1H), 3.99 (d, J = 5.80 Hz, 1H),
3.66 (dq, J = 5.65, 10.53 Hz, 1H), 3.53 (s, 1H), 3.50 (d, J = 6.41 Hz,
1H), 3.21 (s, 3H), 3.03 (ddd, J = 1.00, 7.32, 10.00 Hz, 1H), 2.95−3.02
(m, 1H), 2.82 (t, J = 7.78 Hz, 1H), 2.63−2.76 (m, 3H), 2.53−2.59 (m,
2H), 2.37−2.46 (m, 2H), 2.24 (d, J = 14.00 Hz, 1H), 2.20 (s, 6H),
2.13 (dd, J = 9.16, 12.82 Hz, 1H), 1.86−1.97 (m, 2H), 1.73−1.82 (m,
1H), 1.62−1.72 (m, 1H), 1.54−1.60 (m, 2H), 1.45−1.53 (m, 1H),
1.30−1.44 (m, 3H), 1.17 (s, 3H), 1.08−1.13 (m, 9H), 1.06 (d, J = 6.10
Hz, 3H), 0.96−1.01 (m, 9H), 0.95−0.98 (m, 1H), 0.88 (d, J = 6.71
Hz, 3H), 0.78 (t, J = 7.32 Hz, 3H); 13C NMR (126 MHz, DMSO-d6)
δ 176.59, 150.49, 147.93, 146.47, 129.77, 129.25, 127.05, 126.51,
124.25, 120.07, 102.19, 95.32, 82.97, 78.34, 77.62, 76.89, 75.53, 74.53,
73.86, 72.99, 70.96, 67.39, 65.14, 64.87, 63.54, 59.48, 49.23, 49.09,
48.90, 47.46, 44.55, 40.64, 40.64, 40.64, 40.52, 35.05, 30.29, 28.58,
27.68, 26.96, 22.87, 21.73, 21.49, 21.27, 18.76, 18.22, 15.37, 11.23,
9.74, 8.93; MS (ESI) m/z calcd for C50H85N4O12 (M+H+) 933.6164;
found 933.6162.
9a-{3-[(3-Phenylpropanoyl)amino]propyl}-9a-aza-9-deoxo-
9-dihydro-9a-homoerythromycin A (11c). To a solution of 3-
phenylpropanoic acid (0.28 g, 1.89 mmol) in dry DCM (100.0 mL),
TEA (2.63 mL, 18.9 mmol), HOBT (0.512 g, 3.79 mmol),
intermediate 3 (1.5 g, 1.89 mmol) and EDC × HCl (1.45 g, 7.58
mmol) were added. The reaction mixture was stirred at room
temperature overnight, and then H2O (50 mL) was added and
extracted with DCM (3 × 50 mL). Combined organic layers were
washed with brine (50 mL), dried over Na2SO4, and evaporated in
vacuum. The residue was purified by column chromatography on
silicagel using solvent system DCM/MeOH/NH4OH = 90:7:0.5 to
yield compound 11c (1.29 g, 74%).
1
compound 6t (41 mg, Y = 89%); H NMR (500 MHz, DMSO-d6)
δ 8.38 (d, J = 5.19 Hz, 1H), 8.21 (d, J = 7.63 Hz, 1H), 7.76 (d, J = 8.24
Hz, 1H), 7.59 (td, J = 1.22, 7.63 Hz, 1H), 7.32−7.45 (m, 1H), 7.13 (t,
J = 5.19 Hz, 1H), 6.44 (d, J = 5.49 Hz, 1H), 4.91 (dd, J = 2.75, 10.07
Hz, 1H), 4.79 (d, J = 4.88 Hz, 1H), 4.43 (d, J = 7.02 Hz, 1H), 4.29 (s,
1H), 4.23 (d, J = 6.71 Hz, 1H), 4.04−4.10 (m, 1H), 4.04 (dd, J = 2.00,
6.00 Hz, 1H), 4.00 (d, J = 7.30 Hz, 1H), 3.62−3.70 (m, J = 2.00, 6.20,
6.20, 6.20, 11.00 Hz, 1H), 3.54 (d, J = 7.30 Hz, 1H), 3.52 (d, J = 7.50
Hz, 1H), 3.23−3.27 (m, 2H), 3.22 (s, 3H), 3.04 (dd, J = 7.48, 9.92 Hz,
1H), 2.94−3.01 (m, 1H), 2.91 (dd, J = 5.65, 8.70 Hz, 1H), 2.76 (q, J =
7.02 Hz, 1H), 2.71 (dq, J = 1.00, 7.50 Hz, 1H), 2.62−2.66 (m, 1H),
2.60 (dd, J = 3.50, 13.20 Hz, 1H), 2.43 (ddd, J = 3.80, 10.30, 12.00 Hz,
1H), 2.27 (d, J = 14.65 Hz, 1H), 2.22 (s, 6H), 2.10 (dd, J = 9.92, 12.66
Hz, 1H), 1.87 - 2.00 (m, 3H), 1.72 - 1.84 (m, 2H), 1.59 (ddd, J = 1.50,
3.00, 11.00 Hz, 1H), 1.50 (dd, J = 4.88, 14.65 Hz, 1H), 1.45−1.51 (m,
1H), 1.33−1.42 (m, 2H), 1.19 (s, 3H), 1.15 (d, J = 6.10 Hz, 3H), 1.13
(s, 3H), 1.11 (d, J = 7.02 Hz, 3H), 1.08−1.11 (m, 1H), 1.07 (d, J =
5.80 Hz, 3H), 1.01 (d, J = 6.50 Hz, 3H), 1.01 (s, 3H), 0.99 (d, J = 7.63
Hz, 3H), 0.84 (d, J = 6.71 Hz, 3H), 0.80 (t, J = 7.32 Hz, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 9.25, 9.33, 10.90, 14.84, 18.09, 18.38,
20.89, 21.25, 21.34, 22.30, 25.65, 27.10, 27.84, 29.86, 34.72, 40.23,
40.23, 40.45, 40.90, 44.16, 48.57, 48.72, 59.58, 62.62, 64.56, 64.79,
66.99, 70.52, 72.66, 73.43, 74.27, 75.03, 76.37, 77.28, 77.82, 82.57,
94.88, 98.04, 101.93, 118.76, 121.65, 123.58, 128.55, 128.79, 148.11,
149.88, 150.47, 176.30; MS (ESI) m/z calcd for C49H83N4O12 (M +
H+) 919.6008; found 919.5990.
1H NMR (500 MHz, DMSO-d6) δ 0.79 (t, J = 7.3 Hz, 3 H), 0.86 (d,
J = 6.7 Hz, 3 H), 0.98 (m, 6 H), 1.00 (s, 3 H), 1.07 (d, J = 5.8 Hz, 3
H), 1.10 (d, J = 7.3 Hz, 4 H), 1.13 (s, 3 H), 1.15 (d, J = 6.1 Hz, 3 H),
1.20 (s, 3 H), 1.36 (m, 2 H), 1.43 (m, 1 H), 1.45 (m, 1 H), 1.50 (dd, J
= 14.8, 4.4 Hz, 1H), 1.60 (m, 2 H), 1.77 (m, J = 13.0, 7.3, 7.3, 7.3, 2.0
Hz, 1 H), 1.88 (br. s.,1 H), 1.94 (dq, J = 7.6, 6.0 Hz, 1 H), 2.06 (t, J =
11.4 Hz, 1 H), 2.22 (s, 6 H), 2.27 (d, J = 15.0 Hz, 1 H), 2.34 (t, J = 7.8
Hz, 2 H), 2.44 (m, 2 H), 2.54 (m, 1 H), 2.71 (m, 2 H), 2.80 (t, J = 7.8
Hz, 3 H), 2.91 (t, J = 8.4 Hz, 1 H), 2.98 (m, 2 H), 3.04 (m, 1 H), 3.22
(s, 3 H), 3.50 (m, 2 H), 3.66 (dq, J = 10.0, 5.9 Hz, 1 H), 3.96 (d, J =
6.1 Hz, 1 H), 4.01 (d, J = 4.9 Hz, 1 H), 4.06 (dq, J = 9.6, 6.3 Hz, 1 H),
4.23 (d, J = 7.3 Hz, 1 H), 4.28 (s, 1 H), 4.42 (d, J = 7.0 Hz, 1 H), 4.79
(d, J = 4.3 Hz, 1 H), 4.88 (d, J = 10.1, Hz, 1 H), 7.17 (m, 3 H), 7.25
(m, 2 H), 7.80 (t, J = 5.2 Hz, 1 H); 13C NMR (101 MHz, DMSO-d6)
δ 9.60, 9.63, 11.25, 15.23, 18.42, 18.75, 21.24, 21.59, 21.70, 22.62,
27.35, 27.58, 28.17, 30.33, 31.39, 35.05, 37.32, 40.61, 40.81, 44.49,
48.81, 49.08, 60.13, 63.10, 64.87, 65.11, 67.30, 70.90, 72.99, 73.72,
74.58, 75.32, 76.64, 77.61, 78.13, 82.86, 95.20, 102.27, 126.06, 128.39,
128.47, 141.65, 171.30, 176.61; MS (ESI) m/z calcd for C49H86N3O13
(M + H+) 924.6161; found 924.6139.
9a-[3-(Quinolin-4-ylamino)propyl]-3-O-decladinosyl-9-
deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (7t). Accord-
ing to the procedure described for compound 6t starting from the 7t
1
(0.31 g, 0.39 mmol) product 7u (0.22 g, Y = 75%) was obtained; H
NMR (500 MHz, DMSO-d6) δ 8.38 (d, J = 5.19 Hz, 1H), 8.23 (d, J =
8.54 Hz, 1H), 7.76 (d, J = 8.24 Hz, 1H), 7.51−7.65 (m, 1H), 7.35−
7.44 (m, 1H), 7.03 (t, J = 4.88 Hz, 1H), 6.42 (d, J = 5.49 Hz, 1H), 5.03
(dd, J = 1.00, 12.00 Hz, 1H), 5.00 (d, J = 5.80 Hz, 1H), 4.42 (d, J =
7.32 Hz, 1H), 4.29 (s, 1H), 4.25−4.29 (m, 1H), 3.53 (d, J = 5.80 Hz,
1H), 3.46−3.50 (m, 1H), 3.45 (s, 1H), 3.35−3.43 (m, 1H), 3.17−3.30
(m, 2H), 3.09 (t, J = 8.70 Hz, 1H), 2.85−2.99 (m, 1H), 2.76 (br. s.,
1H), 2.65−2.72 (m, 1H), 2.53−2.62 (m, 1H), 2.47−2.50 (m, 1H),
2.45 (ddd, J = 4.00, 10.00, 12.00 Hz, 1H), 2.21 (s, 6H), 2.00−2.08 (m,
1H), 1.93−1.98 (m, 1H), 1.87−1.93 (m, 1H), 1.75−1.86 (m, 3H),
1.67−1.76 (m, 1H), 1.53−1.62 (m, 1H), 1.35−1.47 (m, 1H), 1.14 (d, J
= 6.71 Hz, 3H), 1.09 (s, 3H), 1.08−1.13 (m, 2H), 1.09 (d, J = 6.10 Hz,
3H), 1.01 (s, 3H), 0.98 (d, J = 6.71 Hz, 3H), 0.88 (d, J = 7.50 Hz, 3H),
0.87 (d, J = 6.30 Hz, 3H), 0.78 (t, J = 7.48 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ 7.96, 8.26, 10.60, 15.91, 17.74, 20.94, 21.25, 21.49,
26.52, 27.02, 29.11, 30.16, 36.37, 39.27, 40.30, 40.30, 40.98, 50.79,
58.19, 60.39, 64.41, 68.27, 70.17, 73.32, 74.34, 75.99, 76.17, 76.42,
89.86, 98.03, 103.37, 118.76, 121.70, 123.52, 128.50, 128.86, 148.18,
149.89, 150.55, 175.31; MS (ESI) m/z calcd for C41H69N4O9 (M +
H+) 761.5065; found 761.5052.
9a-{3-[(Naphtalen-1-yl-acetyl)amino]propyl}-3-O-decladino-
syl-9a-aza-9-deoxo-9-dihydro-9a-homoerythromycin A (12h).
PS-Carbodiimide resin (PS-CDI, loading: 1.2 mmol/g) (105.2 mg,
0.126 mmol) was added to a dry reaction vessel. The naphtalen-1-yl-
acetic acid (186.2 mg, 0.095 mmol) dissolved in dry DCM (1.5 mL),
was added to the dry resin. The mixture was stirred at room
temperature for 1 h upon which intermediate 4 (50 mg, 0.063 mmol)
dissolved in dry DCM (0.8 mL) was added. The reaction mixture was
General Procedure for Reductive Alkylation. Appropriate
aldehyde (1 equiv), TEA (0.3 equiv), and macrolide intermediate 3−5
(1.2 equiv) were added to degasses MeOH solution and the reaction
mixture was stirred under N2 at room temperature for 1 h. NaBH4 (2
1397
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
stirred for 2 h at room temperature, filtered and the resin was washed
13C NMR (101 MHz, DMSO-d6) δ ppm: 9.41, 10.98, 14.93, 18.19,
18.45, 20.96, 21.35, 21.40, 22.08, 26.53, 27.77, 30.07, 32.37, 34.79,
40.24, 40.38, 44.18, 47.09, 48.79, 59.96, 64.59, 64.88, 67.02, 70.56,
72.72, 73.78, 74.31, 76.42, 77.33, 77.89, 82.60, 94.97, 101.90, 174.20,
176.30.
9a-{1-[(1S)-1-(1-Naphthalenyl)ethyl]amino)propanoyl}-9-
deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (16k). TEA
(62.4 μL, 0.45 mmol), HOBT (12.2 mg, 0.09 mmol), (1S)-1-(1-
naphthalenyl)ethanamine (8.5 mg, 0.05 mmol), and EDC × HCl (34.5
mg, 0.18 mmol) were added to a solution of intermediate 3 (36.3 mg,
0.045 mmol) in dry DCM (3.0 mL). The reaction mixture was stirred
at room temperature overnight. Solvent was evaporated under reduced
pressure giving 98.2 mg of yellowish crude product which was purified
on preparative LC-MS (XTerra Prep RP18 column, 5 μm, 19 × 100
mm) using gradient system for elution: (0.1% HCOOH in H2O/
CH3CN) in which HCOOH: CH3CN was changed from 95:5 to
60:40 to give the title compound (14.9 mg, yield 35%).
with DCM (4 × 1.5 mL). The filtrate was evaporated to dryness
1
affording the compound 12h (57.6 mg, 95%); H NMR (400 MHz,
DMSO-d6) δ 0.76 (t, J = 7.34 Hz, 3 H), 0.83 (d, J = 6.70 Hz, 3 H),
0.87 (d, J = 7.17 Hz, 3 H), 0.93 (d, J = 6.59 Hz, 3 H), 0.99 (s, 3 H),
1.08−1.23 (m, 11 H), 1.30−1.43 (m, 1 H), 1.45 - 1.56 (m, 1 H),
1.56−1.69 (m, 2 H), 1.69−1.84 (m, 3 H), 1.91−2.04 (m, 2 H), 2.28
(s, 6 H), 2.31−2.45 (m, 2 H), 2.52−2.64 (m, 2 H), 2.67−2.76 (m, 2
H), 2.92−3.08 (m, 2 H), 3.08−3.16 (m, 1 H), 3.35−3.60, 3.64 (s, 2
H), (m, 4 H), 4.23 (br. s., 1 H), 4.52 (d, J = 7.17 Hz, 1 H), 5.00 (d, J =
10.98 Hz, 1 H), 7.38−7.50 (m, 3 H), 7.73 (s, 1 H), 7.78−7.88 (m, 3
H), 8.05 (t, J = 5.26 Hz, 1 H); 13C NMR (101 MHz, DMSO-d6) δ
8.34, 10.67, 15.98, 17.83, 21.07, 21.29, 21.71, 26.61, 28.22, 30.42,
37.24, 40.22, 42.58, 43.65, 64.62, 68.16, 70.11, 73.47, 74.39, 75.89,
76.44, 125.44, 126.01, 127.13, 127.35, 127.45, 127.55, 131.74, 132.98,
134.28, 163.75, 169.78, 175.27. MS (ESI) m/z calcd for C44H71N3O10
(M + H+) 801.5139; found 801.5146.
1H NMR (400 MHz, DMSO-d6) δ 0.78 (t, J = 7.34 Hz, 3 H), 0.85
(d, J = 6.70 Hz, 3 H), 0.93−1.02 (m, 9 H), 1.04−1.16 (m, 13 H), 1.18
(s, 3 H), 1.30−1.43 (m, 3 H), 1.47 (d, J = 6.82 Hz, 3 H), 1.51 (d, J =
4.39 Hz, 1 H), 1.59 (m, 1 H), 1.69−1.81 (m, 1 H), 1.83−1.98 (m, 2
H), 2.00−2.12 (m, 1 H), 2.23 (s, 6 H), 2.28 (m, 1 H), 2.36−2.46 (m, 2
H), 2.55 (m, 1 H), 2.64−2.78 (m, 3 H), 2.86−2.94 (m, 1 H), 3.01−
3.07 (m, 2 H), 3.08−3.17 (m, 1 H), 3.21 (s, 3 H), 3.45−3.53 (m, 2 H),
3.63−3.72 (m, 1 H), 3.95−4.03 (m, 1 H), 4.03−4.10 (m, 2 H), 4.23
(dd, J = 8.09, 3.01 Hz, 1 H), 4.27 (br. s., 1 H), 4.42 (d, J = 7.17 Hz, 1
H), 4.78 (d, J = 4.05 Hz, 1 H), 4.85 - 4.93 (m, 1 H), 5.62−5.72 (m, 1
H), 7.43−7.58 (m, 4 H), 7.81 (d, J = 8.09 Hz, 1 H), 7.92 (d, J = 8.44
Hz, 1 H), 8.08 (d, J = 8.21 Hz, 1 H), 8.44 (d, J = 7.98 Hz, 1 H); 13C
NMR (101 MHz, DMSO-d6) δ 9.44, 9.78, 10.99, 14.87, 18.16, 18.43,
20.96, 21.33, 21.40, 21.49, 22.28, 27.72, 29.99, 33.40, 34.77, 40.29,
43.83, 44.11, 48.79, 64.61, 64.87, 67.04, 70.54, 72.73, 73.45, 74.28,
74.96, 76.44, 77.32, 77.82, 82.60, 94.91, 101.93, 122.27, 123.13,
125.40, 125.53, 126.06, 127.17, 128.58, 130.38, 133.35, 140.23, 170.38,
176.28; MS (ESI) m/z calcd for C52H86N3O13 (M + H+) 960.6161;
found 960.6176.
Biology. Compound Susceptibility Testing. The in vitro
antimalarial activity of novel 15-membered azalide analogues was
determined using the tritiated hypoxanthine incorporation assay of
Desjardins et al.,38 as modified by Milhous et al.39 with the exception
of exposing the parasite to the drug for 48 h.7 The P. falciparum clone
used was TM91C235,40 a strain from Southeast Asia that shows high
level resistance to mefloquine and a number of other antimalarials.
Compounds and control antimalarials (chloroquine, mefloquine, and
azithromycin) were diluted 2-fold over 11 different concentration and
IC50's were determined using a nonlinear logistic dose response
program. The presented IC50's represent averaged values in cases
where multiple results were generated and relative errors were usually
<30% and did not exceed 50%, which is standard for this type of assay.
Cytotoxicity Assay. The assay used to measure the influence of
compounds on the proliferation of different human cell lines was
described in detail elswhere.41 In brief, human epithelial cell line
HepG2 was purchased from ECACC. Cells were maintained and
tested in complete RPMI 1640 medium (Institute of Immunology,
Zagreb, Croatia) supplemented with 10% Fetal Bovine Serum
(Gibco), penicillin (100 U/mL), streptomycin (100 mg/mL), and
amphotericin B (0.25 mg/mL) at 37 °C in a 5% CO2 atmosphere.
Cytotoxicity assay was performed by using the MTS CellTiter 96
AQueous One Solution Cell Proliferation Assay (Promega, USA).
Culture wells in 96-well plates contained 50 000 HepG2 cells and were
incubated with 10 concentrations of serially diluted compounds (1:2)
for 24 h at 37 °C in 5% CO2. MTS reagent was added directly to the
culture wells. Mixture was incubated for the additional 2 h at 37 °C in
5% CO2, and absorbance recorded at 490 nm using a spectrophoto-
metric plate reader (Ultra, TECAN, USA). The results were expressed
as IC50 values. In cases where multiple results were generated averaged
values are presented.
3-(9-Deoxo-9-dihydro-9a-aza-9a-homoerythromycin A) pro-
pionic acid methyl ester (14). Methyl acrylate (24.5 mL, 272.1
mmol) was added to a solution of intermediate 2 (4.0 g, 5.44 mmol) in
CHCl3 (80.0 mL). Reaction mixture was stirred under reflux (60 °C)
for 2 days. After evaporation of organic solvent, crude product (4.58 g)
was obtained. Crude product was purified using a solid phase
extraction (SPE) technique on a LC-Si (50 g) cartridge with the
FlashMaster II instrument and gradient system for elution: DCM/
DCM: MeOH: NH4OH = 90: 5: 0.5) in which (DCM/MeOH/
NH4OH = 90:5:0.5) was increased from 0 to 100% giving after
evaporation of solvent the title product as white powder (2.95 g, 65%);
MS (ES+) m/z: [MH]+ = 821.5.
1H NMR (400 MHz, CDCl3) δ ppm: 0.85 (t, J = 7.40 Hz, 3 H),
0.91 (d, J = 6.90 Hz, 3 H), 1.03 (s, 3 H), 1.04 (d, J = 8.36 Hz, 3 H),
1.09 (d, J = 6.90 Hz, 3 H), 1.17−1.21 (m, 10 H), 1.27 (s, 3 H), 1.28
(d, J = 6.18 Hz, 3 H), 1.31−1.39 (m, 1 H), 1.41−1.49 (m, 1 H), 1.52−
1.56 (dd, J = 4.85 Hz, J = 15.20 Hz, 1 H), 1.60−1.70 (m, 2 H), 1.80−
1.88 (m, 1 H), 1.90−1.96 (m, 1 H), 1.96−2.00 (m, 1 H), 2.12−2.20
(m, 1 H), 2.25 (s, 6 H), 2.31 (d, J = 15.20 Hz, 1 H), 2.36−2.45 (m, 2
H), 2.58−2.68 (m, 3 H), 2.72−2.75 (m, 1 H), 2.77−2.81 (m, 1 H),
2.99 (t, J = 9.79 Hz, 1 H) 3.20 (dd, J = 7.27 Hz, J = 10.18 Hz, 1 H)
3.29 (s, 3 H), 3.35−3.43 (m, 1 H), 3.44−3.50 (m, 1 H), 3.59 (d, J =
6.91 Hz, 1 H), 3.66 (s, 3H), 3.69 (d, J = 5.09 Hz, 1 H), 4.01−4.06 (m,
1 H), 4.16 (dd, J = 2.42 Hz, J = 6.55 Hz, 1 H), 4.34 (d, J = 5.09 Hz, 1
H), 4.40 (d, J = 7.27 Hz, 1 H), 4.76 (dd, J = 2.55 Hz, J = 10.06 Hz, 1
H), 4.95 (dd, J = 4.61, 1 H),
13C NMR (101 MHz, CDCl3) δ ppm: 8.34, 9.61, 11.11, 15.29,
16.36, 18.26, 21.30, 21.31, 21.52, 22.57, 27.11, 26.78, 28.78, 28.94,
32.97, 34.97, 40.32, 40.57, 41.07, 44.85, 46.24, 49.39, 51.90, 60.56,
64.38, 65.51, 65.55, 68.81, 70.88, 72.77, 74.13, 74.26, 74.81, 77.66,
78.02, 79.04, 82.96, 95.57, 103.11, 173.54, 177.39.
3-(9-Deoxo-9-dihydro-9a-aza-9a-homoerythromycin A) pro-
pionic acid (15). A solution of LiOH (0.28 g, 6.72 mmol) in water
(25.0 mL) was added to a solution of intermediate 14 (2.4 g, 2.92
mmol) in THF (25.0 mL). Reaction mixture was stirred at room
temperature for 3 h. Brine was added to the reaction mixture (30 mL)
and extracted with CH2Cl2 (3 × 30 mL). The combined organic
extracts were dried over anhydrous Na2SO4. After evaporation the title
product was obtained (2.30 g, 98%); MS (ES+) m/z: [MH]+ = 807.5.
1H NMR (400 MHz, DMSO-d6) δ ppm: 0.79 (t, J = 7.45 Hz, 3 H),
0.87 (d, J = 6.75 Hz, 3 H), 0.98 (d, J = 7.45 Hz, 3 H), 1.01 (s, 3 H),
1.02 (d, J = 7.74 Hz, 3 H), 1.07 (d, J = 5.91 Hz, 3 H), 1.10 (d, J = 7.17
Hz, 3 H), 1.13 (s, 3 H), 1.16 (d, J = 6.05 Hz, 3 H), 1.20 (s, 3 H),
1.33−1.42 (m, 2 H), 1.43−1.48 (m, 1 H), 1.51 (dd, J = 4.91 Hz, J =
14.91 Hz, 1 H), 1.58−1.63 (m, 1 H), 1.73−1.81 (m, 1 H), 1.87−1.95
(m, 2 H), 2.13 (dd, J = 10.13 Hz, J = 12.94 Hz, 1 H), 2.24 (s, 6 H),
2.25 − 2.34 (m, 2 H), 2.36−2.45 (m, 2 H), 2.40−2.49 (m, 2 H), 2.57−
2.62 (m, 1H), 2.68−2.82 (m, 3 H), 2.91 (d, J = 9.42 Hz, 1 H) 3.05
(dd, J = 7.45 Hz, J = 9.98 Hz, 1 H) 3.08−3.14 (m, 1H), 3.22 (s, 3 H),
3.48 (s, 1 H), 3.51 (d, J = 6.47 Hz, 1 H), 3.64 - 3.70 (m, 1H), 3.99−
4.02 (m, 1 H), 4.03−4.09 (m, 1 H), 4.43 (d, J = 7.17 Hz, 1 H), 4.79
(d, J = 4.78 Hz, 1 H), 4.90 (dd, J = 2.39, J = 10.13, 1 H).
In Vivo Efficacy Studies in P. berghei Murine Model. A modified
Thompson test was used to test the in vivo efficacies of the new
compounds.42 The test measures the survival of the mice and parasite
1398
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
clearance following administration of the drug on days 3 to 5
postinfection. Male mice that weighed 24−30 g were inoculated with 5
× 106 P. berghei-infected erythrocytes (KBG-173 strain) into the
intraperitoneal cavity. By day 3 postinfection, parasitemia usually
ranged from 1.0 to 3.7%. Each drug was administered in an aqueous
solution (saline, or saline with addition of 0.2% ethanol and 0.02%
acetic acid for less soluble compounds, azithromycin and 8t) IV twice
daily from days 3 to 5 postinfection. In general, the volume of drug
suspension is given at 0.01 mL/gram of body weight. Five mice were
used in each dosage group and blood films were taken from each
mouse on day 6 and biweekly until day 31. Mice that had their blood
smears negative on day 31 postinfection were considered cured.
Compounds were considered active when the survival time of the
treated mice was greater than twice that of the control mice (i.e., 12 to
14 days). Mice losing ≥20% of their body weight were sacrificed.
Pharmacokinetic Studies. Intrinsic Clearance (CLi) Assay.
Intrinsic clearance (CLi) values were determined in mouse, rat and
human liver microsomes. Test compounds (1.0 μM) were incubated at
37 °C for 60 min in 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).
The final concentration of solvent was 1% of the final volume. Samples
were analyzed by LC-MS/MS. The intrinsic clearance (CLi) was
determined from the first order elimination constant by nonlinear
regression using Excel (Microsoft Exel 2003), corrected for the volume
of the incubation and assuming 52.5 mg of microsomal protein/g of
liver for all species. Values for CLi were expressed as mL/min/g of
liver.
Present Addresses
∥University of Zagreb School of Medicine, Center for
̌
Translational and Clinical research, Salata 2, 10000 Zagreb.
⊥University of South Florida, 13201 Bruce B. Downs
Boulevard, MDC 56, Tampa, FL 33612.
○Galapagos Research Centre, Prilaz baruna Filipovica
́
29,
10000 Zagreb, Croatia.
●Nabriva Therapeutics AG, Leberstrasse 20, 1110 Vienna,
Austria.
ACKNOWLEDGMENTS
■
The authors thank Dr. Lucia Gerena for the hard work with the
in vitro susceptibility testing of P. falciparum strains. This study
was supported by the Military Infectious Diseases Research
Program and US Army Medical Research and Materiel
Command. The U.S. government authors declare no competing
financial interests. The opinions or assertions contained herein
are the private views of the authors, and are not to be construed
as official, or as reflecting the views of the Department of the
Army, the Department of Defense, or the United States
government.
ABBREVIATIONS USED
■
IV, intravenuously; IP, intraperitoneally; PO, per os (oral
gavage); BID, bis in die (twice daily)
Plasma Protein Binding (PPB). PPB was determined using the
ultrafiltration method at concentrations of 1 mg/mL. Test compounds
were spiked to mouse plasma (preincubated at 37C for 15 min) to a
final concentration of 1 μg/mL. Aliquots (25 ul) were taken at t = 0
and t = 15 min and spiked with 25 μL of PBS. The remaining samples
were filtered filtered through a 10 kDa filter (Milipore Multiscreen
Ultracel 10 MAU01010 filger, Milipore, USA) while centrifuging for
30 min at 37C at 2850 g. Aliquots of the filtrate were deproteinized
and analyzed by LC-MS/MS.
In Vivo Pharmacokinetic Studies. Male CD-1 mice (N = 3 per
route) were dosed intravenously (IV) at 5 mg/kg (2 mL/kg dosing
volume) and orally (PO), fasted, at a dose of 25 mg/kg (10 mL/kg).
Dosing solutions were prepared in 100% saline (2.5 mg/mL) both for
IV and PO administration for all compounds with the exception of
compounds 9, 12, and 14. Dosing solutions for compounds 9, 12, and
14 were prepared by dissolving Ethanol, acetic acid and saline. After
both routes of administration, blood samples were collected from the
tail vain up to 24 h and by cardiac puncture at the last time point (30
h), hemolyzed and frozen until analysis. Samples were prepared for
analysis by protein precipitation with two volumes of acetonitrile
containing internal standard and analyzed by LC-MS/MS in positive
ion mode with electrospray. Quantitation was performed using
multiple reaction monitoring (MRM) at the specific transitions for
each compound. Noncompartmental analysis for all compounds was
performed using WinNonlin, version 4.1.1 (Pharsight, Mountain View,
CA).
REFERENCES
■
(1) World Malaria Report; Geneva: World Health Organisation, 2008.
(2) 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.
(3) 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.
(4) Gingras, B. A.; Jensen, J. B. Antimalarial activity of azithromycin
and erythromycin against Plasmodium berghei. Am. J. Trop. Med. Hyg.
1993, 49, 101−105.
(5) Kuschner, R. A.; Heppner, D. G.; Andersen, S. L.; Wellde, B. T.;
Hall, T.; Schneider, I.; Ballou, W. R.; Foulds, G.; Sadoff, J. C.; Schuster,
B. Azithromycin prophylaxis against a chloroquine-resistant strain of
Plasmodium falciparum. Lancet 1994, 343, 1396−1397.
(6) Andersen, S. L.; Oloo, A. J.; Gordon, D. M.; Ragama, O. B.;
Aleman, G. M.; Berman, J. D.; Tang, D. B.; Dunne, M. W.; Shanks, G.
D. Successful double-blinded, randomized, placebo-controlled field
trial of azithromycin and doxycycline as prophylaxis for malaria in
western Kenya. Clin. Infect. Dis. 1998, 26, 146−150.
(7) Ohrt, C.; Willingmyre, G. D.; Lee, P.; Knirsch, C.; Milhous, W.
Assessment of azithromycin in combination with other antimalarial
drugs against Plasmodium falciparum in vitro. Antimicrob. Agents
Chemother. 2002, 46, 2518−2524.
(8) Yeo, A. E.; Rieckmann, K. H. Increased antimalarial activity of
azithromycin during prolonged exposure of Plasmodium falciparum in
vitro. Int. J. Parasitol. 1995, 25, 531−532.
(9) Sidhu, A. B.; Sun, Q.; Nkrumah, L. J.; Dunne, M. W.; Sacchettini,
J. C.; Fidock, D. A. In vitro efficacy, resistance selection, and structural
modeling studies implicate the malarial parasite apicoplast as the target
of azithromycin. J. Biol. Chem. 2007, 282, 2494−2504.
(10) 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.
(11) 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
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional spectroscopic information for selected compounds is
available. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Address: University of Zagreb School of Medicine Center for
Translational and Clinical Research Salata 2 10000 Zagreb,
̌
Croatia. E-mail: mihaela.peric@mef.hr. Tel: 385-1-4590070.
Fax: 385-1-4566724.
1399
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
the treatment of Plasmodium vivax malaria in India. Am. J. Trop. Med.
Hyg. 2005, 73, 1108−1111.
(25) Bukvic, K. M.; Peric, M.; Smith, K. S.; Ivezic-Schonfeld, Z.;
̈
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.
(26) Bright, G. M.; Nagel, A. A.; Bordner, J.; Desai, K. A.; Dibrino, J.
N.; Nowakowska, J.; Vincent, L.; Watrous, R. M.; Sciavolino, F. C.;
English, A. R. Synthesis, in vitro and in vivo activity of novel 9-deoxo-
9a-AZA-9a-homoerythromycin A derivatives; a new class of macrolide
antibiotics, the azalides. J. Antibiot. (Tokyo) 1988, 41, 1029−1047.
(27) Krajacic, M. B.; Kujundzic, N.; Dumic, M.; Cindric, M.; Brajsa,
K.; Metelko, B.; Novak, P. Synthesis, characterization and in vitro
antimicrobial activity of novel sulfonylureas of 15-membered azalides.
J. Antibiot. (Tokyo) 2005, 58, 380−389.
(28) Mercep, M.; Mesic, M.; Tomaskovic, L. Compounds with anti-
inflamatory activity. 10/830858 [US 7579334 B2]. 2009.
(29) Alihodzic, S.; Fajdetic, A.; Hutinec, A.; Ivezic, Z.; Kujundzic, N.;
Rupcic, R. 9a-N substitutes azalides for the treatment of malaria. WO
2007/125414 A3. 2007.
(30) 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.
(31) Bukvic, K. M.; Novak, P.; Dumic, M.; Cindric, M.; Paljetak, H.
C.; Kujundzic, N. Novel ureas and thioureas of 15-membered azalides
with antibacterial activity against key respiratory pathogens. Eur. J.
Med. Chem. 2009, 44, 3459−3470.
(32) Curatolo, W.; Foulds, G.; LaBadie, R. Mechanistic study of the
azithromycin dosage-form-dependent food effect. Pharm. Res. 2010,
27, 1361−1366.
(33) Stepanic, V.; Kostrun, S.; Malnar, I.; Hlevnjak, M.; Butkovic, K.;
Caleta, I.; Duksi, M.; Kragol, G.; Makaruha-Stegic, O.; Mikac, L.; Ralic,
J.; Tatic, I.; Tavcar, B.; Valko, K.; Zulfikari, S.; Munic, V. Modeling
cellular pharmacokinetics of 14- and 15-membered macrolides with
physicochemical properties. J. Med. Chem. 2011.
(12) 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.
(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.
(14) Krudsood, S.; Buchachart, K.; Chalermrut, K.; Charusabha, C.;
Treeprasertsuk, S.; Haoharn, O.; Duangdee, C.; Looareesuwan, S. A
comparative clinical trial of combinations of dihydroartemisinin plus
azithromycin and dihydroartemisinin plus mefloquine for treatment of
multidrug resistant falciparum malaria. Southeast Asian J. Trop. Med.
Public Health 2002, 33, 525−531.
(15) 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.
(16) 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.
(17) 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
azithromycin: a double-blind, placebo-controlled trial in Irian Jaya,
Indonesia. Clin. Infect. Dis. 1999, 28, 74−81.
(18) Luntamo, M.; Kulmala, T.; Mbewe, B.; Cheung, Y. B.; Maleta,
K.; Ashorn, P. Effect of repeated treatment of pregnant women with
sulfadoxine-pyrimethamine and azithromycin on preterm delivery in
Malawi: a randomized controlled trial. Am. J Trop. Med. Hyg. 2010, 83,
1212−1220.
(19) Thriemer, K.; Starzengruber, P.; Khan, W. A.; Haque, R.;
Marma, A. S.; Ley, B.; Vossen, M. G.; Swoboda, P.; Akter, J.; Noedl, H.
Azithromycin combination therapy for the treatment of uncomplicated
falciparum malaria in Bangladesh: an open-label randomized,
controlled clinical trial. J Infect. Dis. 2010, 202, 392−398.
(20) van Eijk, A. M.; Terlouw, D. J. Azithromycin for treating
uncomplicated malaria. Cochrane. Database. Syst. Rev. 2011, 2,
CD006688.
(34) Schonfeld, W.; Kirst, H. A. Macrolide Antibiotics; Birkhauser
̈
Verlag: Basel, 2002.
(35) Dong, Y.; Wittlin, S.; Sriraghavan, K.; Chollet, J.; Charman, S.
A.; Charman, W. N.; Scheurer, C.; Urwyler, H.; Santo, T. J.; Snyder,
C.; Creek, D. J.; Morizzi, J.; Koltun, M.; Matile, H.; Wang, X.;
Padmanilayam, M.; Tang, Y.; Dorn, A.; Brun, R.; Vennerstrom, J. L.
The structure-activity relationship of the antimalarial ozonide
arterolane (OZ277). J. Med. Chem. 2010, 53, 481−491.
(36) Angulo-Barturen, I.; Jimenez-Diaz, M. B.; Mulet, T.; Rullas, J.;
Herreros, E.; Ferrer, S.; Jimenez, E.; Mendoza, A.; Regadera, J.;
Rosenthal, P. J.; Bathurst, I.; Pompliano, D. L.; Gomez de las, H. F.;
Gargallo-Viola, D. A murine model of falciparum-malaria by in vivo
selection of competent strains in non-myelodepleted mice engrafted
with human erythrocytes. PLoS One 2008, 3, e2252.
(21) Fichera, M. E.; Roos, D. S. A plastid organelle as a drug target in
(37) Jimenez-Diaz, M. B.; Mulet, T.; Viera, S.; Gomez, V.; Garuti, H.;
Ibanez, J.; Varez-Doval, A.; Shultz, L. D.; Martinez, A.; Gargallo-Viola,
D.; Angulo-Barturen, I. Improved murine model of malaria using
Plasmodium falciparum competent strains and non-myelodepleted
NOD-scid IL2Rgammanull mice engrafted with human erythrocytes.
Antimicrob. Agents Chemother. 2009, 53, 4533−4536.
(38) 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.
(39) Milhous, W. K.; Weatherly, N. F.; Bowdre, J. H.; Desjardins, R.
E. In vitro activities of and mechanisms of resistance to antifol
antimalarial drugs. Antimicrob. Agents Chemother. 1985, 27, 525−530.
(40) Oduola, A. M.; Milhous, W. K.; Weatherly, N. F.; Bowdre, J. H.;
Desjardins, R. E. Plasmodium falciparum: induction of resistance to
mefloquine in cloned strains by continuous drug exposure in vitro.
Exp. Parasitol. 1988, 67, 354−360.
apicomplexan parasites. Nature 1997, 390, 407−409.
(22) Bukvic, K. M.; Peric, M.; Smith, K. S.; Ivezic-Schonfeld, Z.;
̈
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.
(23) Hutinec, A.; Rupcic, R.; Ziher, D.; Smith, K. S.; Milhous, W.;
Ellis, W.; Ohrt, C.; Ivezic-Schonfeld, Z. An automated, polymer-
̈
assisted strategy for the preparation of urea and thiourea derivatives of
15-membered azalides as potential antimalarial chemotherapeutics.
Bioorg. Med. Chem. 2011, 19, 1692−1701.
(24) Gamo, F. J.; Sanz, L. M.; Vidal, J.; de, C. C.; Alvarez, E.;
Lavandera, J. L.; Vanderwall, D. E.; Green, D. V.; Kumar, V.; Hasan, S.;
Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos, J. F.
Thousands of chemical starting points for antimalarial lead
identification. Nature 2010, 465, 305−310.
1400
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401

Journal of Medicinal Chemistry
Article
(41) Verbanac, D.; Jelic, D.; Stepanic, V.; Tatic, I.; Ziher, D.; Kostrun,
S. Combined in silico and in vitro approach to drug screening. Croatica
Chem. Acta 2005, 78, 133−139.
(42) Ager, A. L. Experimental models: rodent malaria models (in
vivo). In Handbook of Experimental Pharmacology: Antimalarial Drugs;
Peters, W.; Richards, W. H. G., Eds.; Springer-Verlag: New York,
1984; pp 225−254.
1401
dx.doi.org/10.1021/jm201615t | J. Med. Chem. 2012, 55, 1389−1401