Novel diaryl ureas with efficacy in a mouse model of malaria

Article abstract of DOI:10.1016/j.bmcl.2012.12.022

Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cultures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum. Compound 18 demonstrated EC₅₀ values of 37 and 55 nM versus in vitro cultured parasite strains and promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at day 31 post-treatment when administered subcutaneously at 256 mg/kg. This series of compounds provides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue antimalarial drug discovery efforts.

Full text of DOI:10.1016/j.bmcl.2012.12.022

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1022–1025
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel diaryl ureas with efficacy in a mouse model of malaria
John W. Anderson a, Dimitri Sarantakis a, Jacek Terpinski a, T. R. Santha Kumar b, Han-Chun Tsai c,
Mack Kuo c, Arba L. Ager d, William R. Jacobs Jr. e,f, Guy A. Schiehser a, Sean Ekins g, James C. Sacchettini c,
David P. Jacobus a, David A. Fidock b, Joel S. Freundlich h,i,
⇑
a Department of Medicinal Chemistry, Jacobus Pharmaceutical Company, 37 Cleveland Lane, Princeton, NJ 08540, USA
b Department of Microbiology & Immunology, Columbia University Medical Center, New York, NY 10032, USA
c Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843-2128, USA
d Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136, USA
e Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
f The Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA
g Collaborations in Chemistry, 5616 Hilltop Needmore Road, Fuquay Varina, NC 27526, USA
h Department of Pharmacology & Physiology, UMDNJ—New Jersey Medical School, Newark, NJ 07103, USA
i Department of Medicine, Ruy V. Lourenço Center for Emerging and Reemerging Pathogens, UMDNJ—New Jersey Medical School, Newark, NJ 07103, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cul-
tures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum.
Compound 18 demonstrated EC50 values of 37 and 55 nM versus in vitro cultured parasite strains and
promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at
day 31 post-treatment when administered subcutaneously at 256 mg/kg. This series of compounds pro-
vides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue anti-
malarial drug discovery efforts.
Received 22 October 2012
Revised 28 November 2012
Accepted 10 December 2012
Available online 20 December 2012
Keywords:
Plasmodium falciparum
Malaria
Ó 2012 Elsevier Ltd. All rights reserved.
Drug discovery
Diaryl urea
2Traditional treatments for Plasmodium falciparum malaria such
as chloroquine and pyrimethamine-sulfadoxine have in many
areas succumbed to drug resistance, and evidence now suggests
emerging resistance to the new first-line artemisinins in Western
Cambodia.1 Infection is rampant, resulting in ꢀ650,000 deaths
per year.2 Clearly, a need exists for novel therapies that are orally
bioavailable, economical, and safe and whose activities are not
compromised by existing resistance.3–5
Our laboratories have been interested in fatty acid biosynthesis
in both Plasmodium spp. and Mycobacterium tuberculosis. Initial
investigations into the mycobacterial target of the front-line drug
isoniazid (INH) led to the discovery that the INH–NAD adduct
binds the enoyl acyl carrier protein reductase InhA (also known
as FabI or ENR).6 This sparked efforts by us and others to study tri-
closan-inspired small molecule inhibitors of InhA that do not re-
quire KatG activation of INH for M. tuberculosis,7 and that inhibit
the P. falciparum homolog (PfFabI, previously termed PfENR) in
biochemical assays with purified enzyme.8–10 Interestingly, the
plasmodial FabI is not essential for the intra-erythrocytic stage of
the parasite and instead is required for normal progression of the
liver-stage of infection.11,12
Efforts to optimize the antimalarial efficacy of small molecule
triclosan analogs began with the hypothesis that the phenol could
be replaced by organic functionality capable of maintaining some
of the hydrogen-bonding interactions that have been shown crys-
tallographically to be important for enzyme inhibition.13 The key
interactions in this case involve the PfFabI Tyr-277 phenol and
the 20-OH of the ribose unit of the bound NAD+ co-factor. The com-
pounds (Table 1) were prepared via adaptation of standard syn-
thetic methods common to diaryl ether assembly14 and feature a
variety of 1-substituents, ranging from an ether and a nitrile to car-
boxylic acid derivatives, amines, sulfonamides, and ureas. Triclosan
methyl ether was prepared by methylation of the parent with
methyl iodide in the presence of potassium carbonate to afford 1.
The synthesis of triclosan 1-substituted analogs relied significantly
on the preparation of anilines 5a and 5b (Scheme 1). The anilines
were synthesized beginning with the coupling of the respective
o-chloronitrobenzene with 2,4-dichlorophenol in DMSO. The
resulting diaryl ethers underwent reduction to afford anilines 5a
and b. Aniline 5a was carried in three straightforward steps,
involving diazotization, the Sandmeyer reaction, and the
Rosenmund-von Braun reaction,15 to benzonitrile 2. The 1-cyano
⇑
Corresponding author. Tel.: +1 973 972 7165; fax: +1 973 972 1141.
E-mail address: freundjs@umdnj.edu (J.S. Freundlich).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.022

J. W. Anderson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1022–1025
1023
Table 1
In vitro activities of triclosan derivatives with a 1-substituent against cultured P.
falciparum strains
Compound
R
EC50
(
lM) 3D7
EC50(lM) Dd2
Triclosan
1
2
OH
OCH3
CN
2.9
100
20
3.8
76
43
3
4
5a
6
7
8
9
10
11
12
13
COOH
CONH2
NH2
CH2NH2
C(CH3)2NH2
NHSO2Me
NHSO2Ph
NHAc
>150
73
52
2.3
7.2
38
4.7
33
>150
60
55
4.3
1.8
60
5.1
48
NHBz
NHC(O)NH2
NHC(O)NHPh
9.7
18
0.73
9.4
18
1.2
Scheme 2. Synthesis of various 1-substituted triclosan amides, sulfonamides, and
ureas. Reagents and conditions: (a) RSO2Cl, NEt3, DCM; (b) Ac2O or PhC(O)Cl and
NEt3, DCM; (c) (i) (Cl3CO)2CO, NEt3, DCM, ꢁ78 °C to rt; (ii) R2NH2; (d) R2NCO, tol.
triclosan 2 was then hydrolyzed to afford amide 4 and carboxylic
acid 3. Reduction of 2 with lithium aluminum hydride afforded
aminomethyl 6. Utilization of Ciganek’s methylorganocerium
reagent,16 generated in situ from cerium(III) chloride and 1 equiv
of methyllithium reduced 2 to
a
,
a
-dimethylamine 7. Aniline 5a
A set of follow-up diaryl ureas (Scheme 2, Table 2) was prepared
to probe the structure–activity relationship pertinent to growth
inhibition of in vitro-cultured P. falciparum, regardless of molecular
target. It is clear from a select subset of analogs that the
substitution pattern on the non-diaryl ether aryl moiety affected
antimalarial activity against both strains of P. falciparum. In
particular, the 3-position favored electron-withdrawing groups
such as trifluoromethyl or cyano. The 4-position also preferred
electron-withdrawing groups such as cyano, nitro, fluoro, and
chloro. This was exemplified in the most potent compounds in
this series: 18 (3-CF3-4-ClC6H3), 21 (3-CF3-4-NO2C6H3), and 24
(3,4-diCNC6H3).
The in vivo activity of a subset of the urea derivatives was
assessed utilizing the Plasmodium berghei rodent malaria model
(Table 2).18 Briefly, ꢀ5 weeks old CD-1 mice (Charles River
Laboratories) were infected intraperitoneally with 106 P. berghei
(KBG-173 line) parasitized red blood cells on day 0. Drug dosing
(bid, divided dose) was initiated on day 3 and continued on days
4 and 5. Subcutaneous administration was achieved using a peanut
oil suspension of the compound. Activity was defined by the frac-
tional survival at day 31. The infected control mice survived an
provided a starting point for the synthesis of a limited set of sulfon-
amide and amide analogs 8–11 via reaction with a sulfonyl chlo-
ride, anhydride or acid chloride under traditional conditions
(Scheme 2). Two routes to ureas were devised, involving either di-
rect reaction of aniline 5a or 5b with a commercial isocyanate or
activation with triphosgene followed by mixing with a commercial
amine. Two simple urea analogs 12 and 13 were initially made.
While none of the phenol replacements displayed significant
activity (IC50 >10 l
M) in a PfFabI inhibition assay,13 both amino-
methyl 6 and diaryl urea 13 demonstrated efficacy in growth inhi-
bition assays17 with cultured 3D7 (drug-sensitive) and Dd2
(resistant to chloroquine and pyrimethamine–sulfadoxine) P. falci-
parum strains. Molecular modeling studies rationalized how the
loss of hydrogen-bonding (perhaps through both donating and
accepting) upon replacement of the 1-OH may lead to abrogation
of potent binding to, and hence reduced inhibition of PfFabI. Chem-
ical inspection of 6 and 13 led to the prioritization of the diaryl
urea for optimization based on its superior whole-cell efficacy
and the potential to readily explore a range of aryl substituents,
not belonging to the diaryl ether subunit.
Scheme 1. Synthesis of various 1-substituted triclosan derivatives. Reagents and conditions: (a) KOH, DMSO, 100 °C; (b) H2, Ra-Ni, EtOH; (c) t-BuONO, BF3ꢂEt2O, THF, 0 °C; (d)
NaI, acetone; (e) CuCN, DMF, 150 °C; (f) NaOH, H2O2, EtOH, 50 °C; (g) 6 N HCl(aq), 2-methoxyethanol, 80 °C.

1024
J. W. Anderson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1022–1025
Table 2
Profile of diaryl ureas tested for in vitro activity against cultured P. falciparum strains and in vivo efficacy in a P. berghei mouse model
Compound
Ar
R
EC50
(
lM) 3D7
EC50
(l
M) Dd2
SC dose (mg/kg)
FSurvival @ 31da,b,c
14
15
16
17
18
18
19
19
20
1-Naphthyl
2-Naphthyl
4-ClPh
3,4-Cl2Ph
3-CF3-4-ClPh
CF3
CF3
Cl
Cl
CF3
1.2
0.61
2.0
0.81
0.41
5.0
256
256
256
256
256
128
256
128
ndd
nd
NES
NES
NES
NES
4/7
EXT
1/7
2/7
nd
1.9
2.5
0.037
0.13
0.18
0.055
0.15
0.18
3-CF3-4-FPh
CF3
CF3
3-CF3-4-OMePh
nd
21
22
23
24
25
3-CF3-4-NO2Ph
3-CF3-4-NH2Ph
3-CF3-4-CNPh
3,4-CN2Ph
CF3
CF3
CF3
CF3
CF3
0.072
1.5
0.13
0.081
0.20
0.072
3.6
0.075
0.14
0.20
nd
nd
nd
nd
nd
nd
nd
nd
3,4-Cl2Ph
nd
nd
a
b
c
FSurvival = proportion of animals living at day 31.
NES = no extension of survival beyond infected control animals (8 days).
EXT = extension of survival beyond infected control animals (8 days), but no survival at day 31.
nd = not determined.
d
average of 8 days whereas non-infected control mice survived the
entire 31 days of the study. Compounds with comparatively lower
in vitro activity, such as 14–17, failed to exhibit an extension of
survival beyond the control animals. 19, exhibiting better whole-
cell efficacy, allowed survival of 2 of 7 and 1 of 7 animals at 31 days
post-infection, at the 128 and 256 mg/kg doses, respectively. Diaryl
urea 18, displaying the most potent whole-cell efficacy to date in
this family, when dosed at 128 mg/kg, enabled extension of sur-
vival of the infected mice beyond the control. Promisingly, dosing
at 256 mg/kg of 18 demonstrated 4 of 7 animals surviving 31 days
post-infection. While many factors contribute to the in vivo effi-
cacy of a small molecule antimalarial, it is clear that the diaryl ur-
eas’ ability to inhibit the growth of P. falciparum in vitro was
correlated with their efficacy in an in vivo P. berghei mouse model
of infection.
It is interesting to note that phenoxy-substituted ureas have
been previously reported as potent inhibitors of Plasmodium spp.,
based on solely in vitro data. These include the compound
WR268961 (Supplementary data Fig. S1),19 where the urea linkage
is para- with respect to the oxygen of the diaryl ether unit instead
of ortho- as in 13–25. WR268961 abrogated parasite growth with
an EC50 = 87 nM (W2 strain) and 460 nM (D6 strain), and modestly
inhibited the P. falciparum cysteine protease plasmepsin 2 (PfPM2;
identification. This chemotype was also found amongst hits against
P. falciparum dihydroorotate dehydrogenase26 that lacked whole-
cell activity.
In order to more quantitatively compare the diaryl ureas re-
ported herein with those disclosed in the literature as antimalarials,
we leveraged a total of thirty-four diaryl ureas generated in our lab-
oratories (Supplementary data Table 1) to generate a common fea-
tures pharmacophore (Accelrys Discovery Studio 2.5.5) with five
hydrophobic features and two hydrogen bond acceptors. Figure 1
depicts the top 3 active compounds that mapped to it. This pharma-
cophore is also able to select 23 compounds out of the 451 with the
diaryl urea substructure present in the GSK library of antimalarial
hits4 (up to 100 conformers per molecule generated using the
FAST algorithm in CAESAR). Slight variants on the diaryl urea scaf-
fold, such as TCMDC-140251 (Supplementary data Fig. S1;
XC50 = 190 nM against 3D7 strain) containing aryl and 1-indolinyl
moieties, mapped well to the model. Interestingly, TCMDC-
139010 exhibited a poor fit because it lacked three of the hydropho-
bic features. The pharmacophore differs from that constructed by
Zhang et al. which contained 2–3 hydrophobic features and 2–3
hydrogen bond acceptors.25 Not surprisingly, the 3 most active
diaryl ureas from the paper by Zhang et al. failed to map to our phar-
macophore; most likely, the 4-aminoquinaldine-derived diaryl
ureas present different features than the triclosan-derived diaryl ur-
eas. Distinctions in the arrangement of hydrophobic and hydrogen
bonding features in the diaryl ureas from Zhang et al. and in this
study may enable these molecules to target different proteins in
Plasmodium falciparum. The pharmacophore developed in this study
may be leveraged to search other databases (e.g. compound vendor
libraries and approved drugs) to identify novel compounds with
antimalarial activity.
IC50 = 17 lM). The diaryl ureas presented herein, however, do not
appear to significantly target the plasmepsins as they equally inhi-
bit the growth of both wild type and knockouts of PfPM1 through 4
(see Supplementary data Table S1), attained via a genetic disrup-
tion methodology in the Dd2 background.20 GlaxoSmithKline dis-
closed the whole-cell efficacy of screening hit TCMDC-139010
(Supplementary data Fig. S1; XC50 = 930 nM vs 3D7 strain), but
without information concerning the biochemical target.4 We also
reported in 2005 the preparation of triclosan-based 40-ureas (Sup-
plementary data Fig. S1) that were less potent against cultured par-
A novel class of diaryl ureas derived initially from triclosan has
been disclosed with regard to their potent in vitro efficacy against
cultured drug-sensitive and drug-resistant strains of P. falciparum.
Importantly, family members such as 18 demonstrate promising
in vivo activity in a P. berghei mouse model of infection. Further
investigation of the structure–activity relationship of these triclo-
san derivatives is necessary to further improve their antimalarial
asite (EC50 values of ꢀ100
lM) than 12–25, but exhibited IC50
values of ꢀ100 nM against purified PfFabI assayed in vitro.14 More
generally, the diaryl urea class of small molecules has been previ-
ously reported in the literature to exhibit potent efficacy against
cultured parasites21–25 but without definitive biological target

J. W. Anderson et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1022–1025
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Acknowledgments
This work has been supported by funding from the Medicines
for Malaria Venture. S.E. kindly acknowledges Accelrys Inc. for pro-
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