Synthesis, structure-activity relationship, and antimalarial activity of ureas and thioureas of 15-membered azalides

Article abstract of DOI:10.1021/jm2001585

Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from

Full text of DOI:10.1021/jm2001585

ARTICLE
pubs.acs.org/jmc
Synthesis, StructureꢀActivity Relationship, and Antimalarial Activity
of Ureas and Thioureas of 15-Membered Azalides
||
†,ꢁ
Mirjana Bukviꢀc Krajaꢁciꢀc,†,ꢁ Mihaela Periꢀc,*,†, Kirsten S. Smith,‡,f Zrinka Iveziꢀc Sch€onfeld,†,^ Dinko Ziher,
ꢁ
Andrea Fajdetiꢀc,†,ꢁ Nedjeljko Kujundꢁziꢀc,† Wolfgang Sch€onfeld,†,# Goran Landek,†,ꢁ Jasna Padovan,†,ꢁ
Dubravko Jeliꢀc,†,ꢁ Arba Ager,§ Wilbur K. Milhous,‡,¥ William Ellis,‡ Radan Spaventi,†,ꢁ and Colin Ohrt‡
†GlaxoSmithKline Research Centre Zagreb Ltd., Prilaz Baruna Filipoviꢀca 29, 10000 Zagreb, Croatia
‡Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring,
Washington, D.C. 20910, United States
§Department of Microbiology and Immunology, University of Miami, Miami, Florida 33177, United States
S Supporting Information
b
ABSTRACT:
Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent
for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class.
Ureas and thioureas of 15-membered azalides, N00-substituted 9a-(N0-carbamoyl-β-aminoethyl), 9a-(N0-thiocarbamoyl-
β-aminoethyl), 9a-[N0-(β-cyanoethyl)-N0-(carbamoyl-β-aminoethyl)], 9a-[N0-(β-cyanoethyl)-N0-(thiocarbamoyl-β-aminoethyl)],
9a-{N0-[β-(ethoxycarbonyl)ethyl]-N0(carbamoyl-β-aminoethyl)}, and 9a-[N0-(β-amidoethyl)-N0-(carbamoyl-β-aminoethyl)] of
9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results
obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin),
particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a-position. The improved in
vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher
volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of
molecules represents a good starting platform for the design of novel antimalarial azalides.
’ INTRODUCTION
appear to have developed multiple drug resistance, and this
remains an issue in antimalarial treatment and prevention.2 This
fact further underscores the urgent necessity to find new
efficacious and safe compounds to maintain and improve the
management and prevention of malaria in a new era of intensive
malaria control, elimination, and eradication.2
Macrolides belong to the polyketide class of natural products.
Macrolide antibiotics are an old and well-established class of
antibacterial agents that have long played a significant role in the
chemotherapy of infectious diseases.3 The azalide azithromycin,
a 15-membered macrolide, is characterized by an enhanced
antibacterial profile and improved pharmacokinetic properties
Malaria is a serious disease with half of the world’s population
at risk of malaria, particularly those in sub-Saharan Africa and
regions of Asia. An estimated 250 million cases led to nearly 1
million deaths in 2006, of which 91% were in Africa and 85%
were of children under 5 years of age.1 The disease is caused by a
parasite, protozoa of the Plasmodia genus, which is transmitted
by the female Anopheles mosquito. There are five species that
infect humans, with P. falciparum causing the vast majority of the
fatalities.
Drugs of diverse chemical classes such as chloroquine, me-
floquine, halofantrine, artemisinin, atovaquone/proguanil, dox-
ycycline, and primaquine have been developed for the treatment
and/or prophylaxis of malaria. However, while successful
against some strains of malaria, many strains of Plasmodium
Received: October 29, 2010
Published: April 08, 2011
r
2011 American Chemical Society
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Figure 1. Ureas and thioureas of 15-membered azalides.14
in comparison to other 14-membered ring macrolides. Azithro-
mycin is of particular interest because of its apparent safety in
pregnancy.4 Furthermore, since it is now available in generic
forms, its cost has substantially decreased.
Since these compounds were obtained by a straightforward
and affordable chemical procedure, a set of compounds was
selected for antimalarial profiling. Herein we report the in vitro
and in vivo efficacy of these compounds against three Plasmo-
dium falciparum strains. Their excellent antimalarial activity
encouraged us to continue and extend our investigation to novel
classes of ureas and thioureas of 15-membered azalides. In this
paper we also report the synthesis and antimalarial activity of
novel N00-substituted 9a-(N0-carbamoyl/thiocarbamoyl-β-aminoethyl)
derivatives.
It has been known for almost 2 decades that azithromycin has
the potential for therapeutic use in malaria.5ꢀ7 The in vitro
activity of azithromycin against P. falciparum is in the low
micromolar range (IC50 values are between 0.5 and 10 μg/mL)
in a 68 h assay.8 Azithromycin is a slow acting antimalarial that
targets the parasite apicoplast, and its prokariotic ribosomes and
effects of its action increase with prolonged incubation time
(from one to two generations), giving IC50 in the nanomolar
range.9 Azithromycin does not appear to be cross-resistant with
standard antimalarial drugs. Although resistance can be induced
in vitro,9 the rapidity of loss to resistance in the field when used in
combination with other antimalarial drugs is unknown (as is its
ability to protect other antimalarial drugs from the emergence of
resistance).
In the 1990s, azithromycin was shown to have prophylactic
activity on malaria when administered daily and weekly by the
Walter Reed Army Institute of Research.6,10ꢀ12 Development
was discontinued when the maximum tolerated daily dose only
demonstrated an approximate efficacy of 65% in nonimmune
adults (although efficacy was very high against P. vivax).11,12
Despite the insufficient prophylactic activity, based on the
activity observed with azithromycin, a three-pronged approach
was encouraged for the treatment and/or prophylaxis of malaria
including a better tolerated formulation,13 drug combinations,8
and the development of new analogues with increased potency
and longer half-lives.
In this manuscript, we present the initial efforts made toward
the discovery of novel antimalarial azalides with improved
activity, efficacy, and safety while being affordable to the target
patient population.2
As macrolide antibiotics exert their activity through the 50S
ribosomal subunit, present both in bacteria and in Plasmodium
apicoplast, the goal for the project was to identify new azalide
derivatives with antibacterial activity comparable to that of
azithromycin. Furthermore, the next generation antimalarial
azalides needed to be produced by a low-step, high-yield
synthetic route resulting in a low-cost final drug product. In
our previous paper we have reported the synthesis of ureas and
thioureas of 15-membered azalides and their comparable or improved
antibacterial activity over that of azithromycin (Figure 1).14
’ CHEMISTRY
We have previously developed an efficient synthetic metho-
dology to access a wide range of N00-substituted 9a-(N0-carba-
moyl/thiocarbamoyl-γ-aminopropyl) derivatives 1ꢀ5 (Figure 1).14
This report will highlight the synthesis and in vitro antimalarial
activity of a series of novel 9a-(N0-carbamoyl/thiocarbamoyl-
β-aminoethyl) analogues 12ꢀ15 and 19ꢀ22.
In this work we have used 9-deoxo-9a-aza-9a-homoerythro-
mycin A (7) as a convenient source of starting material for further
chemical modification leading to novel classes of N00-substituted
9a-(N0-carbamoyl-β-aminoethyl) (12), 9a-(N0-thiocarbamoyl-
β-aminoethyl) (14), 9a-[N0-(β-cyanoethyl)-N0-(carbamoyl-β-amino-
ethyl)] (19), 9a-[N0-(β-cyanoethyl)-N0-(thiocarbamoyl-β-ami-
noethyl)] (20), 9a-{N0-[β-(ethoxycarbonyl)ethyl]-N0-(carbamoyl-
β-aminoethyl)} (21), and 9a-[N0-(β-amidoethyl)-N0-(carbamoyl-
β-aminoethyl)] (22) derivatives (Scheme 1). The 9a-N-β-ami-
noethyl precursor 10 was synthesized in three steps starting from
compound 7. It is well-known that the simple 9a-N-alkyl
derivatives of 7 can be prepared by a straightforward reductive
amination of 7 with aldehydes, while other 9a-N-alkyl analogues
require alkyl halide reaction with 30-N-oxide 8.15 Thus, in order
to prevent quaternization at the 30-nitrogen, the basic desosa-
mine sugar 30-N-oxide 8 was prepared by oxidation with H2O2.
The following reaction of 8 with bromacetonitrile and subse-
quent reduction of the resulting cyanomethyl compound 9
involved the simultaneous deoxygenation at the 30-dimethylami-
no group and afforded the precursor 10 to ureas 12, 19, 21, 22
and thioureas 14, 20. Intermediates 16 and 17 were obtained by
Michael addition of ethyl acrylate followed by amidation.
9a-[N0-(β-Cyanoethyl)-β-aminoethyl] derivative 18 was pre-
pared by Michael addition of acrylonitrile according to a proce-
dure described previously.14
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Scheme 1. Synthesis of Novel Ureas and Thioureas of 15-Membered Azalidesa
a Reagents and conditions: (i) H2O2, MeOH, room temp; (ii) bromoacetonitrile, K2CO3, DCM, room temp; (iii) LiB(Et)3H, THF, room temp;
(iv) alkyl or aryl isocyanate/isothiocyanate, CH2Cl2, room temp, 1 h; (v) 0.25 M hydrochloric acid, room temp; (vi) ethyl acrylate, ethanol, room temp;
(vii) acrylonitrile, methanol, reflux, 22 h; (viii) 25% ammonia solution, ethanol, room temp.
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Table 1. Antimalarial Activity of Substituted 9a-(N0-Carbamoyl-β-aminoethyl) 12, 13, 19, 21, 22 and 9a-(N0-Thiocarbamoyl-
β-aminoethyl) 14, 15, 20 Derivatives against Three Plasmodium falciparum Strains and Cytotoxicity against Two Immortal Human
Cell Lines THP-1 (Monocytes) and Hep G2 (Hepatocytes)a
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Table 1. Continued
a (/) ND: not determined.
The addition of isocyanates or thioisocyanates to primary and
secondary amines is an efficient method for the preparation of
substituted ureas and thioureas, respectively.16 N00-Aryl and alkyl
substituted derivatives 12ꢀ15 and 19ꢀ22 were obtained by the
reaction of the corresponding isocyanates and thioisocyanates
with amines 10, 16, 17, and 18 in high yield. The reaction
proceeded smoothly in an aprotic solvent and at room tempera-
ture to form less polar products (Scheme 1). It is remarkable that
no carbamate was formed in these reactions, since hydroxy
groups are quite reactive with isocyanates and isothiocyanates.
Also, site selective addition to secondary amino groups in the
presence of primary amide in compound 17 was a noteworthy
outcome.
The structures of all synthesized compounds were determined
by NMR spectroscopy and mass spectrometry. The assignments
of proton and carbon chemical shifts were made by a combined
use of one- (1H and APT) and two-dimensional (COSY, HSQC,
and HMBC) NMR spectra. Carbon and proton chemical shifts of
all compounds are given in the experimental part.
’ IN VITRO ANTIMALARIAL AND CYTOTOXIC ACTIVITY
The preliminary library contained a total of 36 urea and
thiourea derivatives of 15-membered azalides, more specifically,
17 N00-substituted 9a-(N0-carbamoyl/thiocarbamoyl-γ-amino-
propyl)-derivatives (1-5) and 19 N00-substituted 9a-(N0-carba-
moyl/thiocarbamoyl-β-aminoethyl)-derivatives (12ꢀ15 and
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19-22). The in vitro antimalarial activity was evaluated against
three Plasmodium falciparum strains: TM91C235 (multidrug
resistant), D6 (chloroquine sensitive), and W2 (chloroquine/
pyrimethamine resistant) (Table 1) and compared to azithro-
mycin 6 as standard. Among these, TM91C235 was considered the
most important strain because of its multidrug resistance profile.
Excellent in vitro activity of the tested library was confirmed by the
total number of compounds more active than azithromycin against
all three strains (30 compounds out of 36). An overall analysis of the
data revealed an interesting property of these novel macrolides,
mainly that higher in vitro potency was seen against one or both
resistant strains than against the sensitive D6 strain.
By structureꢀactivity relationship (SAR) analysis, the influ-
ence of the cladinose sugar, nature of the substituent R0, and type
of the linker (variations of alkyl chains on 9a-N position on
macrolide ring) on the in vitro activity was investigated for this
set of compounds. Each feature was analyzed separately; how-
ever, it was also noted that the combination of key structural
features contributed significantly to the enhancement of the
antimalarial activity.
Initially, the significance of the cladinose sugar was explored.
An evident trend was observed for a tested set of compounds
where the absence of the cladinosyl moiety decreased antimalar-
ial activity up to 10-fold (decladinosyl derivative 3d in compar-
ison to cladinosyl derivative 2d with IC50 of 1543 and 156 μM,
respectively). The removal of the cladinose sugar was shown to
be the most influential structural feature that resulted in the
reduction of in vitro antimalarial activity.
Table 2. Pharmacokinetic Parameters Estimated in Blood
after Intravenous (iv) and Oral Gavage (po) Administration
to SpragueꢀDawley Rats (10 mg/kg iv and 30 mg/kg po)a
CL ((mL/min)/kg) Vd (L/kg) T1/2 (h) oral F (%)
azithromycin
11.0
4.0
20.0
10.4
2.6
24.0
30.0
13.4
41.5
15.2
33.0
3.4
2d
2fb
4g
5f
2.3
1.3
7.7
23.3
31.7
18.4
21
24.5
a CL, blood clearance; Vd, apparent volume of distribution at the terminal
phase based on drug concentration in blood, T1/2, half-life; oral F, oral
bioavailability, b The iv parameters were determined in one rat.
In vitro cytotoxicity measured for the selected set of com-
pounds revealed that all compounds showed relatively low
cytotoxicity in vitro (IC50 g 4 μM). SAR analysis indicted that
(1) thiourea derivatives showed slightly higher cytotoxicity than
corresponding urea analogues (i.e., 14d > 12d, 14e > 12e, 14h >
12h) and (2) an ethyl chain between 9a-N and urea/thiourea
functionality increases cytotoxicity in comparison to a propyl
chain derivatives (i.e., 12i > 1i, 12h > 1h, 14d > 2d, 14f > 2f).
In general, the antimalarial activity of compounds tested was
accompanied by a decrease in IC50 determined for Hep G2 and
THP-1 cell lines. Despite this fact, most active compounds
showed P. falciparum IC50 more than 2 log units above cellular
inhibition IC50, and this marked them as potent and selective
compounds for further profiling.17
Further exploration of the SAR was focused on the nature of
the substituents R0, R2, and the length of the linkers (Table 1):
linker A, chain between 9a-N position on macrolide ring and N0
position on carbamoyl/thiocarbamoyl group; linker B, chain
between N00 position on carbamoyl/thiocarbamoyl group
and alkyl/aromatic moiety R0. The observations on these SAR
features include the following: (1) Introduction of aromatic
groups significantly improved in vitro activity over azithromycin
6 (up to 88-fold) and over alkyl groups in the same position
(1b, 4b, 12a, 12b). (2) Reduction of activity was approximately
10-fold by the introduction of additional substituents R2 on urea
and thiourea moieties: β-cyanoethyl, β-(ethoxycarbonyl)ethyl,
and β-amidoethyl chains. For example, activities of 19i, 21i, and
22i were significantly reduced in comparison to 12i. (3) There
was no clear-cut effect on in vitro activity for all derivatives with
respect to the linker A length. The more active urea derivatives in
the in vitro assay had short linker A (1h, 1i, 1j vs 12h, 12i, 12j),
while in thiourea series derivatives with longer linker A (propyl
chain) showed similar or equal activity compared with the com-
pounds with shorter and/or more rigid ethyl chain (2d, 2e, 2f, 2h
and 14d, 14e, 14f, 14h). (4) There was slightly improved
antimalarial activity for compounds with a longer linker B
(compound 14f with propyl linker more active than compounds
with shorter linkers 14c, 14d, and 14e). (5) A combination of the
structural features, urea/thiourea and naphthyl/phenyl R0 aro-
matic moieties, enhances antimalarial activity. The most active
urea and thiourea analogues demonstrated that ureas with
naphthyl moiety exhibit better activity than their thiourea
analogues (e.g., 12h vs 14h with IC50 of 42 and 130 μM,
respectively). However, thioureas with a phenyl substituent
demonstrated slightly better activity than urea derivatives. For
example, compounds 14e and 14d with thiourea moiety showed
better activity than urea analogues 12e and 12d (IC50 of 128,
106, 243, 170 μM, respectively).
’ PRELIMINARY PHARMACOKINETICS AND IN VIVO
ANTIMALARIAL ACTIVITY
On the basis of in vitro activity (IC50 against P. falciparum
strain TM91C235 of <200 nM and HepG2 IC50 of >9 μM) and
their structural similarity, several compounds were screened for
metabolic stability in vitro (1h, 1i, 1j, 2d, 2e, 2f, 2h, 4g, 4j, 5d, 5e, 5f)
using human and mouse liver microsomes, and only a few were
selected for in vivo rat pharmacokinetic studies in order to
determine their pharmacokinetic profiles (Table 2). In vitro, all
compounds demonstrated good metabolic stability with t1/2
greater than 120 min (t1/2 = 103 min for compound 5e in
human liver microsomes). As was observed with azithromycin
and in line with the in vitro data, these analogues had a low
systemic clearance, moderate to high volume of distribution, and
a very long half-life; however, the oral bioavailability was low
(2d, 2f) to moderate (4g). Nevertheless, all 12 compounds (1h, 1i, 1j,
2d, 2e, 2f, 2h, 4g, 4j, 5d, 5e, 5f) were progressed to be tested
in vivo in the mouse P. berghei model, as the dosing regimen was
subcutaneous. Compounds were administered subcutaneously at
160/40/10 mg/kg twice daily for 3 days (total doses of 960/240/
60 mg/kg, high/medium/low dose), including azithromycin,
which was used as a comparator. The results from the efficacy
model were expressed as a curative dose 50 (CD50), defined as
the daily dose of compound that cures 50% of mice. Animals that
have a negative blood smear on the last day of the experiment are
considered cured. The results of the efficacy experiments are
presented in Table 3.
Azithromycin cured all treated mice at the high and medium
doses with a CD50 of 50 mg/kg, while in the low dose only three
of five mice were cured. The most active of the compounds tested
belonged to the 9a-(N0-carbamoyl-γ-aminopropyl) (1) and
9a-(N0-thiocarbamoyl-γ-aminopropyl) (2) groups. Compound
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Table 3. Efficacy of Novel Macrolides in Plasmodium berghei
as multiresistant malaria parasites emerge.2,18 This paper reports
the in vitro and in vivo results for a novel class of antimalarial
compounds: the 9a-N-substituted azalides. Azithromycin, an
azalide antibiotic currently tested in clinical settings for the
treatment and prophylaxis of malaria, has low in vitro activity
against P. falciparum strains8 as well as low clinical efficacy when
used as monotherapy.11,12,19 Therefore, a novel macrolide deri-
vative with an equivalent safety profile and more potent anti-
malarial activity is of interest to all involved in malaria eradication
and control. The most prominent feature in antimalarial activity
of novel N00-substituted urea and thiourea derivatives was a
substantial improvement of their in vitro activity against
P. falciparum strains when compared to azithromycin. The most
outstanding increase in activity was observed against resistant
P. falciparum strains TM91C235 and W2, where the IC50 values
of the most active compound 12i were 88 and 76 times lower
when compared to azithromycin, respectively. The increases in in
vitro potency were not that pronounced for the drug sensitive
P. falciparum strain D6, where the improvement over azithro-
mycin was no more than 14 times.
Infected Mice after 3 Days of Subcutaneous Dosinga
(no. of mice without parasitemia
P. berghei in vivo
on day 31)/(total no. of mice) at
doses 960, 240, 60 mg/kg
cmpd
CD50 (mg/kg)
1h
1i
150
>960
397.5
285
3/5, 3/5, 2/5
2/5, 0/5, 0/5
4/4, 0/5, 1/5
4/5, 2/5, 2/5
3/5, 1/5, 0/5
3/5, 1/5, 0/5
4/5, 3/5, 1/5
5/5, 2/5, 1/5
4/5, 0/5, 1/5
1/5, 1/5, 2/5
0/4, 1/5, 0/5
3/5, 0/5, 0/5
5/5, 5/5, 3/5
0/5
1j
2d
2e
2f
780
780
2h
4g
4j
195
420
510
5d
5e
5f
>960
>960
800
azithromycin
controlb
50
On the basis of such encouraging in vitro antimalarial activity
over azithromycin, structural pairing, and good in vitro metabolic
stability, a total of 12 compounds were tested for their in vivo
efficacy in the P. berghei mouse model. The curative effects in vivo
were in line with previously observed in vitro SAR trends, with
naphthyl substituted urea and thiourea derivatives (1h, 1j, 2h)
and the thiourea phenyl derivative (2d) being the most active,
while compounds with a β-cyanoethyl substituent on urea and
thiourea moieties had lower curative outcomes than their analo-
gues lacking this side chain. Nevertheless, the substantially
improved in vitro activity in comparison to azithromycin was
not fully translated in vivo, leading to at least 3-fold higher CD50
in the mouse model. Preliminary in vitro microsomal stability
data indicated that these compounds had good metabolic
stability, as was confirmed by low clearances in vivo for the
compounds tested. In comparison to azithromycin, known for its
extensive tissue distribution,3 these derivatives had a tendency
toward higher volumes of distribution, in line with their increased
lipophilic character (approximately 2ꢀ3 log units higher than
azithromycin, according to calculated log P values, data not
shown) because of the presence of strong lipophilic aromatic
phenyl and naphthyl rings in the 9a-N substituent. More
extensive tissue penetration could lead to lower concentrations
at the site of action (blood), resulting in lower efficacy in vivo.
Finally, the in vitro activity against human malaria parasite P.
falciparum is considered to be the guiding tool for the progres-
sion of compounds to one of the standard in vivo mouse models
in malaria drug discovery; however, the data gathered in this
research suggest that the P. berghei mouse model used may not be
appropriate for the evaluation of this class of compounds, as
previously observed for some other compound classes.20 Overall,
with increased in vitro activity, promising PK properties, and
modest in vivo efficacy, this series of molecules represents a good
starting platform for the design of novel antimalarial azalides. On
the basis of the data reported here, other new series were
proposed and synthesized.
a Groups of four to five P. berghei infected mice were treated subcutaneously
on days þ3, þ4, þ5 postinfection with macrolides suspended in palm oil at
doses 160, 40, and 10 mg/kg twice daily for 3 days. Antimalarial activity was
measured by curative dose 50, the daily dose that cures 50% of the mice.
Animals that have a negative blood smear on the last day of the experiment
are considered cured. b Mice that received only palm oil vehicle.
1h cured three mice at the high and medium doses and two mice
at the low dose, while its thiourea derivative 2h cured all four
mice at the high dose (this was the only group that contained
only four mice), three mice at the medium dose, and one mouse
at the low dose. The CD50 values were determined to be 150 and
195 mg/kg, respectively. Compound 2d had a CD50 of 285 mg/kg
(four mice at the high dose and two mice at both the medium
and low doses were cured), while its 9a-N0-(β-cyanoethyl)
analogue 5d was found to be much less active, curing only one
mouse at the high and medium doses and two mice at the low
dose with CD50 being larger than 960 mg/kg. In contrast to this
analogue example, compound 1j and its 9a-N0-(β-cyanoethyl)
analogue 4j did not exhibit such difference in their in vivo efficacy,
as both had a similar pattern of efficacy (four mice cured at the high
dose, zero mice in the middle dose, and one mouse at low dose) and
their CD50 values were 398 and 510 mg/kg, respectively. Analogues
2f and 5f had no curative effect at the low dose, while at the high
dose three mice were cured. At the medium dose one mouse was
cured by 2f and no mice by 5f, so their respective calculated CD50
values were 780 and 800 mg/kg. The least active pair of analogues in
this set of compounds was 2e and 5e, where 2e exhibited a CD50 of
780 mg/kg whereas 5e had CD50 of >960 mg/kg. Compound 2e
cured three mice in high dose and one mouse at the medium dose,
while 5e cured only one mouse at the medium dose. Compound 4g
without an analogue in this efficacy experiment exhibited a CD50 of
420 mg/kg and cured five mice at the high dose, two mice at the
medium dose, and one mouse at the lower dose. Interestingly, the
only 2-naphthyl analogue tested, 1i, cured only two mice at the high
dose and as such had a CD50 of >960 mg/kg.
’ CONCLUSIONS
’ DISCUSSION
Given the enormous burden malaria is causing to the affec-
ted population and the limited resources for their healthcare,
the usual requirements for efficacy, safety, compliance, and
The development of new antimalarial drugs is an urgent
clinical need, as many drugs on the market are losing efficacy
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affordability are particularly stringent for novel antimalarials. Our
research on the antimalarial macrolides has contributed substan-
tially to the area of antimalarial drug discovery. A novel class of
antimalarial agents, 15-membered azalide urea and thiourea
derivatives, with substantially improved in vitro antimalarial
activity over azithromycin and comparable pharmacokinetic
properties was invented. Straightforward and affordable synthetic
procedures for the preparation of novel ureas and thioureas of
15-membered azalides (N00-substituted 9a-(N0-carbamoyl/thiocar-
bamoyl-γ-aminopropyl) derivatives and N00-substituted 9a-(N0-
carbamoyl/thiocarbamoyl-β-aminoethyl) derivatives) were developed.
In our research some compounds showed promising efficacy
in the mouse model, although the model used may not be fully
appropriate for the evaluation of this class of compounds.
This research established a good starting platform for the
exploration of a wider chemical space around this novel series
that will direct medicinal chemistry toward further optimization
of the 9a-N substituent.
(2 ꢁ 200 mL). The combined organic layers were evaporated
under reduced pressure, and the residue was precipitated from DCM
and diisopropyl ether, yielding 8 (21.5 g, 94.3% yield). MS (ESþ)
m/z: 751.6.
9-Deoxo-9-dihydro-30-N-oxide-9a-cyanomethyl-9a-aza-
9a-homoerythromycin A (9). To a DCM (200 mL) solution of 8
(20 g, 26.63 mmol) K2CO3 (7.35 g, 53.26 mmol) was added, and the
reaction mixture was stirred for 10 min at room temperature. Then
bromoacetonitrile (3.71 mL, 53.26 mmol) was added, and the reaction
mixture was stirred overnight at room temperature. The reaction
mixture was washed with brine, yielding after evaporation 20 g of the
crude product. Precipitation from water yielded intermediate 9 (7.1 g,
31.3% yield). MS (ESþ) m/z: 790.6.
9-Deoxo-9-dihydro-9a-(β-aminoethyl)-9a-aza-9a-homo-
erythromycin A (10). To the solution of 9 (3 g, 3.80 mmol) in THF
(25 mL), LiB(Et)3H (10 mL, 1 M THF solution) was added dropwise
over 20 min at ꢀ20 ꢀC. The mixture was stirred for 10 min at ꢀ20 ꢀC to
complete conversion. To the reaction mixture water (50 mL) and DCM
(50 mL) were added, and gradient extraction was performed at pH 4.5
and 10. Evaporation of the combined organic extracts at pH 10 yielded
1.6 g of the crude product. Column chromatography using elution
system DCM/MeOH/NH4OH = 90:9:0.5 yielded 10 (0.87 g, 29.5%
yield). MS (ESþ) m/z: 778.5.
3-O-Decladinosyl-9-deoxo-9-dihydro-9a-(β-aminoethyl)-
9a-aza-9a-homoerythromycin A (11). A solution of intermediate
10 (1.5 g, 1.93 mol) in 0.25 N HC1 (50 mL) was stirred for 20 h at room
temperature. To the reaction mixture DCM (50 mL) was added, and
gradient extraction was performed at pH 1.1 and 9.5. Evaporation of the
combined organic extracts at pH 9.5 yielded 0.98 g of crude product.
Column chromatography using elution system DCM/MeOH/NH4OH =
90:9:1.5 yielded compound 11 (0.76 g, 62.7% yield). 1H NMR
(500 MHz, DMSO-d6) δ: 5.08 (d, 1H, H-13), 4.98 (d, 1H, 3 OH), 4.53
(d, 1H, H-10), 3.51 (s, 1H, H-11), 3.45 (d, 1H, H-5), 3.43 (br s, 1H,
H-50), 3.32ꢀ3.35 (m, 1H, H-3), 3.08 (dd, 1H, H-20), 2.70ꢀ2.80 (m, 1H,
β-CH2), 2.65 (d, 1H, H-10), 2.52ꢀ2.53 (m, 2H, H-9, R-CH2),
2.45ꢀ2.48 (m, 2H, H-30, H-2), 2.40ꢀ2.45 (m, 1H, β-CH2),
2.24ꢀ2.27 (m, 2H, H-9, R-CH2), 2.22 (s, 6H, 30NMe2), 2.07ꢀ2.12
(m, 1H, H-4), 1.76ꢀ1.85 (m, 1H, H-14), 1.65ꢀ1.72 (m, 1H, H-8),
1.56ꢀ1.64 (m, 2H, H-7, H-40), 1.37 (ddd, 1H, H-14), 1.15 (s, 3H, 6
Me), 1.13 (d, 3H, 50 Me), 1.12 (d, 3H, 2 Me), 1.10ꢀ1.11 (m, 1H, H-40),
1.08ꢀ1.10 (m, 1H, H-7), 0.97 (s, 3H, 12 Me), 0.97 (d, 3H, 10 Me), 0.89
(d, 3H, 4 Me), 0.83 (d, 3H, 8 Me), 0.75 (t, 3H, 15 Me). 13C NMR
(75 MHz, DMSO-d6) δ: 174.8, 102.5, 86.9, 76.4, 76.1, 75.3, 73.9, 73.5,
70.5, 68.1, 64.6, 61.1, 43.8, 40.6, 40.2, 37.6, 36.1, 30.5, 29.6, 25.9, 21.3,
21.1, 20.9, 16.7, 16.1, 10.5, 8.5, 8.4. MS (ESþ) m/z: 620.6.
’ EXPERIMENTAL SECTION
Chemistry. TLC was performed on Merck 60 F254 plates using
DCM/MeOH/NH4OH = 90:9:0.5 as eluents. Column chromatography
was performed on Merck silica gel 60 (0.043ꢀ0.060 mm).
NMR spectra were recorded on a Bruker Avance DRX500 and Bruker
Avance DPX 300 spectrometer operating at 500 and 300 MHz for 1H.
The chemical shifts were reported as δ ppm relative to TMS, using the
residual solvent peak as the reference unless otherwise noted. The
following abbreviations were used to express the multiplicities:
s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet. The
solvents used were pyridine-d5 and DMSO-d6. All spectra were recorded
at ambient temperature.
Mass spectra were obtained on a Waters Micromass ZQmass spectro-
meter for ESþ MS. Electrospray positive ion mass spectra were acquired
using a Micromass Q-Tof2 hybrid quadrupole time-of-flight mass
spectrometer, equipped with a Z-spray interface, over a mass range of
100ꢀ2000 Da, with a scan time of 1.5 s and an interscan delay of 0.1 s in
continuum mode. Reserpine was used as the external mass calibrant lock
mass ([M þ H]þ = 609.2812 Da). The elemental composition was
calculated using a MassLynx, version 4.1, for the [M þ H]þ, and the
mass error quoted was within the (5 ppm range.
The purity of each compound was determined on an Agilent 1100
series LC/MSD trap. The analysis was carried out using MS and UV
detection at 240 nm on a YMC Pack Pro C18 column, 150 mm ꢁ 4.6 mm
i.d., 3 μm particle size. Gradient elution at a flow rate of 1.0 mL/min
started with 10% of acetonitrile/90% of 40 mM ammonium acetate
buffer solution and ended after 15 min with 90% of acetonitrile/10% of
40 mM ammonium acetate buffer solution. The ESI source was operated
in the positive ion mode. Nitrogen was used as nebulizing and drying gas
at 350 ꢀC. The m/z range scanned in the MS measurement was
200ꢀ1200. Complete system control, data acquisition, and processing
were done using the ChemStation for LC/MSD, version 4.2, from
Agilent. By use of the described method, the determined purity for each
compound was g95%.
9-Deoxo-9-dihydro-9a-{N0-[β-(ethoxycarbonyl)ethyl]-
β-aminoethyl}-9a-aza-9a-homoerythromycin A (16). To the
EtOH (10 mL) solution of 10 (1.22 g, 1.57 mmol), ethyl acrylate
(0.08 mL, 1.22 mmol) was added. The reaction mixture was stirred for 8
days at room temperature. After completing the reaction, the solvent was
evaporated and the residue was extracted with DCM (3 ꢁ 30 mL). The
crude product was purified by the solid phase extraction technique (SPE
1
10 g), yielding the title compound (0.69 g, 62.9% yield). H NMR
(500 MHz, DMSO-d6) δ: 5.52 (br s, 1H, 6 OH), 4.95 (dd, 1H, H-13),
4.83 (d, 1H, H-100), 4.43 (d, 1H, H-10), 4.26 (d, 1H, 400OH), 4.04ꢀ4.11
(m, 1H, H-500), 4.05 (q, 2H, CH2/Et), 3.97ꢀ4.00 (m, 1H, H-3), 3.97
(br s, 1H, 20 OH), 3.94 (s, 1H, 12 OH), 3.63ꢀ3.71 (m, 1H, H-50), 3.52
(d, 1H, H-5), 3.46 (s, 1H, H-11), 3.22 (s, 3H, 300OMe), 3.06ꢀ3.11
(m, 1H, R-CH2), 3.00ꢀ3.06 (m, 1H, H-20), 2.87ꢀ2.93 (m, 1H, H-400),
2.76ꢀ2.80 (m, 1H, R0-CH2), 2.72ꢀ2.76 (m, 3H, β-CH2), 2.67ꢀ2.71
(m, 1H, H-10), 2.51ꢀ2.54 (m, 1H, H-9), 2.41ꢀ2.48 (m, 2H, β0-CH2),
2.39ꢀ2.41 (m, 1H, H-30), 2.31ꢀ2.37 (m, 1H, R-CH2), 2.29 (d, 1H,
H-200), 2.22 (s, 6H, 30NMe2), 2.15ꢀ2.20 (m, 1H, H-9), 1.92ꢀ1.99 (m,
1H, H-4), 1.80ꢀ1.83 (m, 1H, H-8), 1.73ꢀ1.79 (m, 1H, H-14), 1.59 (dd,
9-Deoxo-9-dihydro-30-N-oxide-9a-aza-9a-homoerythro-
mycin A (8). To a solution of 9-deoxo-9-dihydro-9a-aza-9a-homoer-
ythromycin A (7) (20 g, 27.21 mmol) in MeOH (80 mL) at 0 ꢀC, a 30%
water solution of H2O2 (30 mL) was added dropwise over 30 min. The
reaction mixture was stirred for an additional 1.5 h at room temperature.
After detection of complete transformation the reaction mixture was
poured into iceꢀwater (400 mL) and DCM (200 mL). A saturated
water solution of Na2SO3 (150 mL) was added to remove excess H2O2.
The layers were separated, and the water layer was extracted with DCM
3602
dx.doi.org/10.1021/jm2001585 |J. Med. Chem. 2011, 54, 3595–3605

Journal of Medicinal Chemistry
ARTICLE
1H, H-40), 1.50ꢀ1.55 (m, 1H, H-7), 1.51 (dd, 1H, H-200), 1.37 (ddd,
1H, H-14), 1.28 (dd, 1H, H-7), 1.20 (s, 3H, 6 Me), 1.18 (t, 3H, CH3/
Et), 1.16 (d, 3H, 500 Me), 1.14 (s, 3H, 300 Me), 1.11 (d, 3H, 2 Me), 1.08
(d, 3H, 50 Me), 1.06ꢀ1.06 (m, 1H, H-40), 1.00 (d, 3H, 4 Me), 0.96ꢀ0.98
(m, 6H, 10 me, 12 Me), 0.85 (d, 3H, 8 Me), 0.78 (t, 3H15 Me). 13C
NMR (75 MHz, DMSO-d6) δ: 175.7, 172.0, 101.9, 95.2, 82.6, 78.4, 77.3,
76.4, 74.9, 74.0, 73.6, 72.6, 70.6, 67.0, 66.0, 64.8, 64.5, 60.3, 59.6, 51.4,
48.7, 48.0, 44.8, 44.2, 40.3, 40.3, 40.2, 34.8, 34.2, 29.9, 28.7, 26.5, 22.0,
21.4, 20.9, 20.9, 18.4, 17.3, 15.3, 14.0, 10.8, 9.5, 8.8. MS (ESþ)
m/z: 878.5.
General Procedure for the Preparation of Ureas 12, 13, 19,
21, 22 and Thioureas 14, 15, 20. To the solution of 10, 11, 16, or
18 in dichloromethane, 20% excess isocyanate or isothiocyanate was
added, and the reaction mixture was stirred about 1 h at 0ꢀ5 ꢀC. The
crude product was evaporated, wherefrom by column chromatography
on silica gel using solvent system CH2Cl2/CH3OH/NH3(25%) =
90:9:1.5 the pure compound was obtained.
9-Deoxo-9-dihydro-9a-N-(N0-ethylcarbamoyl-β-aminoethyl)-9a-aza-
1
9a-homoerythromycin A (12a). Yield: 0.30 g, 61.7%. H NMR (500
MHz, pyridine-d5) δ: 6.57 (t, 1H, NH), 5.43 (dd, 1H, H-13), 5.25
(d, 1H, H-100), 5.00 (d, 1H, H-10), 4.69 (d, 1H, H-3), 4.64 (m, 1H,
H-500), 4.25 (d, 1H, H-11), 4.17 (d, 1H, H-5), 4.02 (m, 1H, H-50), 3.86
(m, 1H, β-CH2), 3.71 (dd, 1H, H-20), 3.63 (m, 1H, R-CH2), 3.54 (s, 3H,
300OMe), 3.50 (m, 1H, β-CH2), 3.36 (m, 2H, CH2/Et), 3.31 (m, 1H,
H-400), 3.19 (m, 1H, H-2), 3.15 (m, 1H, H-10), 2.84 (m, 1H, H-30), 2.76
(m, 1H, R-CH2), 2.76 (d, 1H, H-9), 2.54 (m, 1H, H-4), 2.48 (d, 1H,
H-200), 2.39 (m, 1H, H-9), 2.32 (s, 6H, 30NMe2), 2.30 (m, 1H, H-8),
2.19 (m, 1H, H-14), 1.99, (m, 1H, H-7), 1.86 (m, 1H, H-7), 1.82 (s, 3H,
6 Me), 1.73 (m, 1H, H-14), 1.65 (s, 3H, 300 Me), 1.64 (d, 3H, 4 Me), 1.60
(dd, 1H, H-200), 1.56 (d, 1H, H-40), 1.42 (d, 3H, 10 Me), 1.37 (d, 3H, 2
Me), 1.35 (s, 3H, 12 Me), 1.34 (d, 3H, 500 Me), 1.33 (d, 3H, 50 Me), 1.25
(m, 1H, H-40), 1.12 (t, 3H, CH3/Et), 0.97 (d, 3H, 8 Me), 0.94 (t, 3H, 15
Me). 13C NMR (125 MHz, pyridine-d5) δ: 178.0, 159.9, 103.8, 97.2,
84.4, 80.8, 79.9, 79.2, 76.9, 75.7, 74.1, 72.0, 68.5, 66.7, 66.4, 66.0, 62.1,
53.4, 50.2, 46.2, 41.4, 40.9, 40.1, 36.2, 35.9, 31.2, 30.4, 28.2, 22.8, 22.6,
22.3, 22.0, 19.9, 18.7, 16.6, 16.4, 11.9, 11.0, 9.7. MS (ESþ) m/z: 849.1.
9-Deoxo-9-dihydro-9a-[N0-(β-cyanoethyl)-N0-(2-naphthyl)carbamo-
yl-β-aminoethyl]-9a-aza-9a-homoerythromycin A (19i). Yield: 0.22 g,
81.4%. 1H NMR (500 MHz, DMSO-d6) δ: 8.62 (s, 1H, NH), 8.04 (d,
1H, naphthyl, a), 7.77ꢀ7.81 (m, 2H, naphthyl, d, e), 7.74 (d, 1H,
naphthyl, h), 7.66 (dd, 1H, naphthyl, c), 7.40ꢀ7.44 (m, 1H, naphthyl, f),
7.32ꢀ7.36 (m, 1H, naphthyl, g), 5.33 (br s, 1H, 6OH), 4.98 (dd, 1H,
H-13), 4.79 (d, 1H, H-100), 4.46 (br s, 1H, 12OH), 4.44 (d, 1H, H-10),
4.41 (d, 1H, 11OH), 4.25 (d, 1H, 400OH), 4.03ꢀ4.11 (m, 1H, H-500),
4.02 (dd, 1H, H-3), 3.96 (br s, 1H, 20OH), 3.66ꢀ3.70 (m, 1H, H-50),
3.62ꢀ3.66 (m, 2H, H-50), 3.51ꢀ3.56 (m, 2H, H-11, H-5), 3.44ꢀ3.51
(m, 2H, β-CH2), 3.22 (s, 3H, 300OMe), 3.02ꢀ3.08 (m, 1H, H-20),
2.97ꢀ3.02 (m, 1H, R-CH2), 2.89 (dd, 1H, H-400), 2.83 (d, 1H, H-10),
2.79 (t, 2H, β0-CH2), 2.75 (d, 1H, H-2), 2.67 (dd, 1H, H-9), 2.59ꢀ2.65
(m, 1H, R-CH2), 2.39ꢀ2.46 (m, 1H, H-30), 2.26 (d, 1H, H-200), 2.21 (s,
6H, 30NMe2), 2.16ꢀ2.19 (m, 1H, H-9), 1.92ꢀ1.97 (m, 1H, H-4),
1.88ꢀ1.92 (m, 1H, H-8), 1.76ꢀ1.85 (m, 1H, H-14), 1.55ꢀ1.62 (m, 1H,
H-40), 1.53ꢀ1.56 (m, 1H, H-7), 1.48 (dd, 1H, H-200), 1.41 (ddd, 1H,
H-14), 1.32 (d, 1H, H-7), 1.24 (s, 3H, 6Me), 1.15 (d, 3H, 500Me),
1.10ꢀ1.13 (m, 6H, 2Me, 300Me), 1.08ꢀ1.09 (m, 1H, H-40), 1.07 (d, 3H,
50Me), 1.04 (s, 3H, 12Me), 1.01ꢀ1.04 (m, 3H, 10Me), 1.01 (s, 3H,
4Me), 0.89 (d, 3H, 8Me), 0.82 (t, 3H, H-15). 13C NMR (126 MHz,
DMSO-d6) δ: 175.9, 154.6, 137.9, 133.4, 129.1, 127.5, 127.2, 126.8,
125.9, 123.8, 121.3, 119.2, 115.3, 101.9, 95.3, 82.9, 78.4, 77.2, 76.6, 76.2,
74.1, 73.6, 72.6, 70.6, 67.0, 64.8, 64.5, 63.9, 59.4, 50.1, 48.7, 46.5, 44.2,
43.1, 40.2, 39.9, 39.6, 34.8, 29.8, 28.1, 26.8, 22.1, 21.3, 21.2, 20.8, 18.4,
18.1, 16.7, 15.2, 10.9, 9.4, 8.8. MS (ESI) m/z calcd for C53H86N5O13
[M þ H]þ 1000.6222; found 1000.6255.
9-Deoxo-9-dihydro-9a-[N0-(β-amidoethyl)-β-aminoethyl]-
9a-aza-9a-homoerythromycin A (17). To the EtOH (10 mL)
solution of 16 (0.44 g, 0.50 mol), 25% ammonia solution (8 mL) was
added. The reaction mixture was stirred at room temperature for 3 days.
After evaporation of the solvent the crude residue was dissolved in DCM
(30 mL). Water (30 mL) was added, and the pH was adjusted to 9.6.
After extraction with DCM (2 ꢁ 30 mL) the combined organic layers
were dried over K2CO3 and the solvent was evaporated, yielding the title
compound 17 (0.30 g, 72.2% yield). 1H NMR (500 MHz, DMSO-d6) δ:
4.91 (dd, 1H, H-13), 4.82 (d, 1H, H-100), 4.43 (d, 1H, H-10), 4.03ꢀ4.11
(m, 1H, H-500), 3.95 (d, 1H, H-3), 3.62ꢀ3.70 (m, 1H, H-50), 3.53
(d, 1H, H-5), 3.48 (s, 1H, H-11), 3.24ꢀ3.27 (m, 1H, R-CH2), 3.22
(s, 3H, 300OMe), 3.03 (dd, 1H, H-10), 2.88ꢀ2.95 (m, 2H, R0-CH2),
2.83ꢀ2.88 (m, 1H, H-400), 2.70ꢀ2.79 (m, 4H, H-2, H-10, β-CH2),
2.44ꢀ2.49 (m, 2H, R-CH2, H-9), 2.38ꢀ2.44 (m, 1H, H-30), 2.28 (d, 1H,
H-200), 2.23ꢀ2.26 (m, 2H, β0-CH2), 2.22 (s, 6H, 30NMe2), 2.16ꢀ2.21
(m, 1H, H-9), 1.90ꢀ1.99 (m, 1H, H-4), 1.74ꢀ1.82 (m, 1H, H-14),
1.68ꢀ1.73 (m, 1H, H-8), 1.59 (dd, 1H, H-40), 1.49ꢀ1.55 (m, 2H, H-7,
H-200), 1.35ꢀ1.44 (m, 1H, H-14), 1.26 (br s, 1H, H-7), 1.23 (s, 3H, 6
Me), 1.18 (d, 3H, 500 Me), 1.13 (s, 3H, 300 Me), 1.12 (d, 3H, 2 Me), 1.08
(s, 1H, H-40), 1.07 (s, 3H, 50Me), 1.02 (d, 3H, 4 Me), 0.97ꢀ1.00 (m, 6H,
10 Me, 12 Me), 0.85 (d, 3H, 8 Me), 0.79 (t, 3H, 15 Me). 13C NMR
(75 MHz, DMSO-d6) δ: 175.5, 173.7, 101.9, 95.6, 82.4, 79.1, 77.3, 76.8,
76.0, 74.0, 72.6, 70.6, 67.0, 65.2, 64.8, 64.5, 60.1, 48.9, 48.7, 45.6, 44.4,
44.0, 40.3, 39.9, 38.6, 35.0, 32.3, 29.8, 28.9, 26.4, 21.6, 21.4, 21.0, 20.8,
18.6, 17.7, 15.6, 10.7, 9.6, 8.7. MS (ESþ) m/z: 850.6.
9-Deoxo-9-dihydro-9a-[N0-(β-cyanoethyl)-β-aminoethyl]-
9a-aza-9a-homoerythromycin A (18). To the MeOH (15 mL)
solution of intermediate 10 (0.92 g, 1.18 mol), a solution of acrylonitrile
(0.08 mL, 1.221 mmol) in MeOH (2 mL) was added dropwise. The
reaction mixture was refluxed for 22 h. After the reaction was completed,
the solvent was evaporated and the residue extracted with DCM (3 ꢁ
30 mL). The crude product was purified by the solid phase extraction
technique (SPE 10 g), yielding the title compound (0.63 g, 89.0% yield).
1H NMR (500 MHz, DMSO-d6) δ: 4.98 (dd, 1H, H-13), 4.81 (d, 1H,
H-100), 4.43 (d, 1H, H-10), 4.26 (d, 1H, 400 OH), 4.08 (dq, 1H, H-500),
3.97 (s, 1H, 12 OH), 3.92ꢀ3.96 (m, 2H, 20OH), 3.63ꢀ3.71 (m, 1H,
H-50), 3.52 (d, 1H, H-5), 3.46 (br s, 1H, H-11), 3.22 (s, 3H, 300OMe),
3.10ꢀ3.16 (m, 1H, R-CH2), 3.03 (ddd, 1H, H-20), 2.91 (dd, 1H, H-400),
2.78ꢀ2.83 (m, 1H, R0-CH2), 2.74ꢀ2.77 (m, 1H, H-2), 2.71ꢀ2.74 (m,
1H, H-10), 2.69ꢀ2.71 (m, 1H, R0-CH2), 2.67ꢀ2.68 (m, 1H, β-CH2),
2.57 (t, 2H, β0-CH2), 2.52ꢀ2.53 (m, 1H, H-9), 2.46ꢀ2.48 (m, 1H, β-
CH2), 2.39ꢀ2.45 (m, 1H, H-30), 2.34ꢀ2.38 (m, 1H, R-CH2), 2.28
(d, 1H, H-200), 2.21 (s, 6H, 30NMe2), 2.16ꢀ2.20 (m, 1H, H-9),
1.92ꢀ2.01 (m, 1H, H-4), 1.75ꢀ1.83 (m, 1H, H-14), 1.72ꢀ1.75 (m,
1H, H-8), 1.59 (dt, 1H, H-40), 1.49ꢀ1.55 (m, 1H, H-7), 1.52 (dd, 1H,
H-200), 1.32ꢀ1.42 (m, 1H, H-14), 1.24ꢀ1.29 (m, 1H, H-7), 1.22 (s, 3H,
6 Me), 1.18 (d, 3H, 500Me), 1.14 (s, 3H, 300Me), 1.12 (d, 3H, 2 Me), 1.08
(d, 3H, 50Me), 1.06 (br s, 1H, H-40), 1.01 (d, 3H, 4 Me), 0.96ꢀ0.99 (m,
6H, 10 Me, 12 Me), 0.84 (d, 3H, 8 Me), 0.78 (t, 3H, 15 Me). 13C NMR
(75 MHz, DMSO-d6) δ: 175.6, 120.0, 101.8, 95.4, 82.5, 78.8, 77.3, 76.5,
75.2, 74.0, 73.7, 72.6, 70.6, 67.0, 65.9, 64.8, 64.5, 60.3, 51.5, 48.7, 47.7,
44.8, 44.3, 40.2, 38.9, 34.9, 29.8, 29.0, 26.6, 21.8, 21.4, 20.9, 20.8, 18.5,
17.6, 17.4, 15.5, 10.7, 9.6, 9.0. MS (ESþ) m/z: 831.4.
9-Deoxo-9-dihydro-9a-{N0-[β-(ethoxycarbonyl)ethyl]-N0-(2-napht-
hyl)carbamoyl-β-aminoethyl}-9a-aza-9a-homoerythromycin A (21i).
1
Yield: 0.18 g, 82.3%. H NMR (500 MHz, DMSO-d6) δ: 8.52 (s, 1H,
NH), 8.02 (d, 1H, naphthyl, a), 7.79 (d, 1H, naphthyl, e), 7.77 (d, 1H,
naphthyl, d), 7.73 (d, 1H, naphthyl, h), 7.63 (dd, 1H, naphthyl, c),
7.39ꢀ7.44 (m, 1H, naphthyl, g), 7.31ꢀ7.36 (m, 1H, naphthyl, f), 5.45 (br
s, 1H, 6OH), 4.97 (dd, 1H, H-13), 4.79(d, 1H, H-100), 4.43ꢀ4.46(m, 2H,
H-10), 4.34 (br s, 1H, 11OH), 4.25 (d, 1H, 400OH), 4.09 (q, 2H, ethyl,
CH2), 4.04ꢀ4.07 (m, 1H, H-500), 4.01ꢀ4.04 (m, 1H, H-3), 3.98 (brs, 1H,
20OH), 3.63ꢀ3.71 (m, 1H, H-50), 3.56ꢀ3.62 (m, 2H, R00-CH2), 3.54
(d, 1H, H-5), 3.53 (br s, 1H, H-11), 3.43 (t, 2H, β-CH2), 3.22 (s, 3H,
3603
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ARTICLE
300OMe), 3.02ꢀ3.07 (m, 1H, H-20), 2.96ꢀ3.02 (m, 1H, R-CH2), 2.89
(dd, 1H, H-400), 2.79ꢀ2.85 (m, 1H, H-10), 2.75 (quin, 1H, H-2), 2.68
(dd, 1H, H-9), 2.62 (t, 2H, β00-CH2), 2.58 (br s, 1H, R-CH2), 2.39ꢀ2.46
(m, 1H, H-30), 2.26 (d, 1H, H-200), 2.21 (s, 6H, 30NMe2), 2.18ꢀ2.20 (m,
1H, H-9), 1.92ꢀ1.97 (m, 1H, H-4), 1.87ꢀ1.92 (m, 1H, H-8), 1.75ꢀ1.84
(m, 1H, H-14), 1.57ꢀ1.61 (m, 1H, H-40), 1.51ꢀ1.55 (m, 1H, H-7),
1.43ꢀ1.50 (m, 1H, H-200), 1.37ꢀ1.43 (m, 1H, H-14), 1.31ꢀ1.37 (m, 1H,
H-7), 1.23 (s, 3H, 6Me), 1.20 (t, 3H, ethyl, CH3), 1.15 (d, 3H, 500Me),
1.10ꢀ1.13 (m, 6H, 2Me, 300Me), 1.08ꢀ1.10 (m, 1H, H-40), 1.07 (d, 3H,
50Me), 1.04 (s, 3H, 12Me), 1.03 (d, 3H, 10Me), 1.00 (d, 3H, 4Me), 0.90
(d, 3H, 8Me), 0.82 (t, 3H, H-15). 13C NMR (126 MHz, DMSO-d6) δ:
176.1, 171.6, 154.8, 138.2, 133.5, 129.0, 127.6, 127.3, 126.8, 126.0, 123.8,
121.2, 114.9, 101.9, 95.2, 82.8, 78.2, 77.3, 76.7, 76.0, 74.1, 73.6, 72.6, 70.6,
67.1, 64.9, 64.5, 64.0, 60.0, 59.4, 49.9, 48.8, 46.3, 44.2, 43.2, 40.3, 39.9, 39.8,
34.8, 33.3, 29.8, 28.2, 26.5, 22.1, 21.4, 21.2, 20.9, 18.4, 18.0, 15.2, 14.0, 10.9,
9.5, 8.8. MS (ESI) m/z calcd for C55H91N4O15 [M þ H]þ 1047.6481;
found 1047.6488.
were inoculated into the intraperitoneal cavity of male mice that weighed
24ꢀ30 g. By day 3 postinfection, parasitemia ranged from 1.0% to 3.7%.
Each drug suspended in peanut oil was administered subcutaneously
(sc) twice daily from day 3 to day 5 postinfection. The volume of drug
suspension given depends on the weight of the mouse and the drug
concentration of the suspension. In general, the volume is given at
0.01 mL/g of body weight. Five mice were used in each dosage group.
Blood films were taken on day 6 and biweekly for 31 days. Mice that were
blood film 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., 12ꢀ14 days).
Mice losing g20% of their body weights were sacrificed.
Cytotoxicity Assay. Two immortal cell lines, human monocytic
leukemia (THP-1, monocyte) and human Caucasian hepatocyte carci-
noma (Hep G2, epithelial), were obtained from the European Collec-
tion of Cell Cultures. Cells were maintained in completed RPMI 1640
(Cambrex) medium supplemented with 10% fetal bovine serum
(Gibco) at 37 ꢀC in 5% CO2 atmosphere. Cells were passaged twice a
week to keep them in rapid growth phase.
The compounds were examined for cytotoxic effects by continuous
drug exposure by the CellTiter 96 Aqueous One solution cell prolifera-
tion assay (MTS Assay, Promega). A 15 μL amount of the MTS reagent
was added directly to cell lines cultured in microtiter plates. After 1ꢀ4 h
of incubation, the absorbance at 490 nm was recorded with a spectro-
photometric plate reader (Ultra, TECAN). MTS assay measures cell
viability through quantification of reducing equivalents such as NADH,
using mitochondrial succinate dehydrogenase activity of living cells. The
method was programmed for TECAN robotic system in GEMINI
pipetting software.25 The results were expressed as IC50 values.
Preliminary Pharmacokinetics. Metabolic Stability Assay. Selected
compounds were evaluated in several secondary assays including meta-
bolic stability assessments using in vitro liver microsomes. Sample stocks
at 10 or 20 mM (depending on solubility) in DMSO were diluted to a
final concentration of 1 μM into a mixture containing, 0.5 mg/mL of
prewarmed pooled human or mouse liver microsomes (BD Gentest),
1.3 mM NADP (Sigma), 3.3 mM MgCl2 (Sigma), and 0.1 M PBS, pH
7.4, using a TECAN Genesis robotic liquid handler. The reaction was
started with the addition of 1 U/mL glucose 6-phosphate dehydrogen-
ase G6PD. The mixture was incubated on a shaking platform at 37 ꢀC,
and aliquots were taken and quenched with the addition of an equal
volume of cold acetonitrile at 0, 10, 20, 30, and 60 min. Samples were
centrifuged at 3700 rpm for 10 min at 20 ꢀC to remove debris. Sample
quantification was carried out by LCMS, and metabolic half-life (t1/2) was
calculated by log plots of the total ion chromatograph area remaining.
Rat Pharmacokinetis. Male SpragueꢀDawley rats weighing 250ꢀ300 g
(IFFA CREADO, Lyon, France) were dosed in a crossover design. Each
compound was administered intravenously at 10 mg/kg, followed by a 2 day
washout period, and orally at 30 mg/kg free base equivalent.
9-Deoxo-9-dihydro-9a-[N0-(β-amidoethyl)-N0-(2-naphthyl)carbamoyl-
β-aminoethyl]-9a-aza-9a-homoerythromycin A (22i). Yield: 0.23 g,
73.2%. 1H NMR (500 MHz, DMSO-d6) δ: 8.52 (s, 1H, NH), 8.04 (d,
1H, naphthyl, a), 7.77 (d, 1H, naphthyl, d), 7.75 (d, 1H, naphthyl, e),
7.73 (d, 1H, naphthyl, h), 7.61 (dd, 1H, naphthyl, c), 7.38ꢀ7.42 (m, 1H,
naphthyl, f), 7.32ꢀ7.36 (m, 1H, naphthyl, g), 5.42 (br s, 1H, 6OH), 4.98
(dd, 1H, H-13), 4.80 (d, 1H, H-100), 4.44ꢀ4.48 (m, 2H, H-10, 11OH),
4.32 (br s, 1H, 12OH), 4.24 (d, 1H, 400OH), 4.04ꢀ4.07 (m, 1H, H-500),
4.01ꢀ4.04 (m, 1H, H-3), 3.97 (br s, 1H, 20OH), 3.64ꢀ3.72 (m, 1H,
H-50), 3.56ꢀ3.62 (m, 2H, H-11, H-5), 3.56 (d, 1H, β-CH2), 3.20 (s, 3H,
300OMe), 3.00ꢀ3.06 (m, 1H, R-CH2), 2.96ꢀ3.02 (m, 1H, H-20), 2.90
(dd, 1H, H-400), 2.79ꢀ2.83 (m, 1H, β-CH2), 2.75 (quin, 1H, H-2), 2.66
(dd, 1H, H-9), 2.60 (t, 2H, CH2), 2.54 (br s, 1H, R-CH2), 2.39ꢀ2.46
(m, 1H, H-30), 2.23 (d, 1H, H-200), 2.19 (s, 6H, 30NMe2), 2.15ꢀ2.18 (m,
1H, H-9), 1.92ꢀ1.97 (m, 1H, H-4), 1.85ꢀ1.90 (m, 1H, H-8),
1.75ꢀ1.84 (m, 1H, H-14), 1.58ꢀ1.62 (m, 1H, H-40), 1.50ꢀ1.54 (m,
1H, H-7), 1.44ꢀ1.50 (m, 1H, H-200), 1.38ꢀ1.43 (m, 1H, H-14),
1.31ꢀ1.36 (m, 1H, H-7), 1.20 (s, 3H, 6Me), 1.13 (d, 3H, 500Me),
1.10ꢀ1.12 (m, 6H, 2Me, 300Me), 1.06ꢀ1.08 (m, 1H, 50Me), 1.07 (d, 3H,
12Me), 1.03 (s, 3H), 1.02 (d, 3H, 10Me), 1.00 (d, 3H, 4Me), 0.94 (d,
3H, 8Me), 0.85 (t, 3H, H-15). 13C NMR (126 MHz, DMSO-d6) δ:
175.9, 173.6, 154.6, 138.0, 132.8, 129.4, 128.2, 127.4, 126.8, 126.1, 123.6,
121.4, 115.3, 101.4, 95.7, 82.1, 78.4, 77.3, 76.4, 74.0, 73.6, 72.6, 70.6,
67.0, 65.9, 64.5, 60.0, 59.4, 51.9, 48.8, 48.3, 44.6, 44.2, 40.3, 40.0, 39.8,
34.8, 34.3, 29.9, 28.6, 26.5, 22.1, 21.4, 21.0, 20.9, 18.4, 17.7, 15.2, 14.0,
10.9, 9.5, 8.8. MS (ESI) m/z calcd. for C55H91N4O15 [M þ H]þ
1047.6481; found 1047.6488.
Biology. Compound Susceptibility Testing. The susceptibility of
different malaria strains to azithromycin analogues was determined using
the tritiated hypoxanthine incorporation assay of Desjardins et al.,21 as
modified by Milhous et al.,22 except that the drug exposure period was 48 h.
The P. falciparum clones used were W2, D6, and TM91C235.23 W2 from
Vietnam is a mefloquine-sensitive strain resistant to chloroquine and
pyrimethamine. D6 clone from West Africa is sensitive to all antimalarials,
with a decreased in vitro sensitivity to mefloquine. TM91C235 is a strain
from Thailand that is highly resistant to mefloquine and a number of other
antimalarials. Compounds and control antimalarials (chloroquine, meflo-
quine, and azithromycin) were diluted 2-fold over 11 different concentra-
tion, and IC50 values were determined using a nonlinear logistic dose
response program. The presented IC50 values 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.
In Vivo Antimalarial Studies of P. berghei in Mice. The in vivo
efficacies of the new compounds were determined by a modified
Thompson test.24 This test measures the survival of mice and parasite
clearance following administration of the drug on days 3ꢀ5 postinfec-
tion. In brief, 5 ꢁ 106 P. berghei infected erythrocytes (KBG-173 strain)
Compounds were formulated in 1%/20% Encapsin (w/v) and saline.
Blood samples were collected up to 24 h postdosing and analyzed by
LCMS/MS. Pharmacokinetic parameters were calculated using Win-
Nonlin (Pharsight, U.S.).
’ ASSOCIATED CONTENT
S
Supporting Information. Additional spectroscopic in-
b
formation for selected compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 385-1-4590070. Fax: 385-1-4566724. E-mail: mihaela.
peric@mef.hr.
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Journal of Medicinal Chemistry
ARTICLE
Present Addresses
University of Zagreb School of Medicine, Center for Transla-
azithromycin and doxycycline as prophylaxis for malaria in Western
Kenya. Clin. Infect. Dis. 1998, 26, 146–150.
ꢁ
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D. B.; Tulyayon, S.; Permpanich, B.; Wimonwattrawatee, T.; Chuanak,
N.; Laoboonchai, A.; Sookto, P.; Brewer, T. G.; McDaniel, P.; Eamsila,
C.; Yongvanitchit, K.; Uhl, K.; Kyle, D. E.; Keep, L. W.; Miller, R. E.;
Wongsrichanalai, C. Randomized, controlled, double-blind trial of daily
oral azithromycin in adults for the prophylaxis of Plasmodium vivax
malaria in Western Thailand. Am. J. Trop. Med. Hyg. 2005, 73, 842–849.
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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.
tional and Clinical Research, Salata 2, 10000 Zagreb.
^Nabriva Therapeutics AG, Leberstrasse 20, 1110 Vienna,
Austria.
#Green River Pharma AG, Kreuzstrasse 26, 8008 Z€urich,
Switzerland.
¥University of South Florida, 13201 Bruce B. Downs Boulevard,
MDC 56, Tampa, FL 33612.
ꢁGalapagos Istraꢁzivaꢁcki Centar d.o.o., Prilaz Baruna Filipoviꢀca
29, 10000 Zagreb, Croatia.
fUS Army Medical Materiel Development Activity, 1430
Veterans Dr., Fort Detrick, MD 21702.
(13) Chandra, R.; Liu, P.; Breen, J. D.; Fisher, J.; Xie, C.; LaBadie, R.;
Benner, R. J.; Benincosa, L. J.; Sharma, A. Clinical pharmacokinetics and
gastrointestinal tolerability of a novel extended-release microsphere
formulation of azithromycin. Clin. Pharmacokinet. 2007, 46, 247–259.
(14) Bukviꢀc Krajaꢁciꢀc, 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.
Notes
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.
(15) 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
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antimicrobial activity of novel sulfonylureas of 15-membered azalides.
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’ ACKNOWLEDGMENT
The authors thank Dr. Lucia Gerena for the hard work with
the in vitro susceptibility testing of P. falciparum strains. The
authors also acknowledge the support provided by Renata
Rupcic with the scale-up synthesis of certain compounds. This
study was supported by the Military Infectious Diseases Research
Program and U.S. Army Medical Research and Material
Command.
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