Discovery of novel benzo[ a ]phenoxazine SSJ-183 as a drug candidate for malaria

Article abstract of DOI:10.1021/ml100120a

Malaria is a serious infectious disease caused by protozoan parasites in tropical and subtropical regions. Even inhabitants of temperate zones are exposed to the danger of malaria infection because of travel and global warming. Novel, effective, safe, and inexpensive drugs are required to treat malaria and contribute to the global goal of eradication. A search for new antimalarial agents has been performed by the synthesis of new benzo[a]phenoxazines, followed by biological evaluations. The derivative SSJ-183 (5), having a 4-aminopyridine group, showed an IC₅₀ value against Plasmodium falciparum of 7.6 nM and a selectivity index of >7300. Cure was achieved by three oral doses of 5 at 100 mg/kg to mice infected with the Plasmodium berghei ANKA strain. The safety of 5 was supported by acute toxicity testing in mice with single doses up to 2000 mg/kg po, chromosome aberration test, in vitro as well as in vivo micronucleus tests, and phototoxicity studies in mice. Thus, 5 is a promising candidate as a new antimalarial agent.

Full text of DOI:10.1021/ml100120a

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Discovery of Novel Benzo[a]phenoxazine SSJ-183 as a Drug
Candidate for Malaria
Jian-Feng Ge,†,‡ Chika Arai,† Mei Yang,† Abu Bakar Md.,† Jun Lu,† Nasser S. M. Ismail,†,§
Sergio Wittlin, Marcel Kaiser, Reto Brun, Susan A. Charman,^ Tien Nguyen,^ Julia Morizzi,^
Isamu Itoh,# and Masataka Ihara*,†
†Drug Discovery Science Research Center, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, ‡College of
Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215006, China, §Pharmaceutical
Chemistry Department, Faculty of Pharmacy Ain Shams University, Elkhalifa AlMaamoon Street, 11566 Abbasseya, Cairo,
Egypt, Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, and University of Basel, Basel, Switzerland,
^Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus,
381 Royal Parade, Parkville, Victoria 3052, Australia, and #Synstar Japan Co., Ltd., Tokyo Tech Yokohama Venture Plaza W401,
4259-3 Nagatsuta-cho, Midori-ku, Yokohama, 226-8510, Japan
ABSTRACT Malaria is a serious infectious disease caused by protozoan parasites
in tropical and subtropical regions. Even inhabitants of temperate zones are
exposed to the danger of malaria infection because of travel and global warming.
Novel, effective, safe, and inexpensive drugs are required to treat malaria and
contribute to the global goal of eradication. A search for new antimalarial agents
has been performed by the synthesis of new benzo[a]phenoxazines, followed by
biological evaluations. The derivative SSJ-183 (5), having a 4-aminopyridine group,
showed an IC50 value against Plasmodium falciparum of 7.6 nM and a selec-
tivity index of >7300. Cure was achieved by three oral doses of 5 at 100 mg/kg
to mice infected with the Plasmodium berghei ANKA strain. The safety of 5 was
supported by acute toxicity testing in mice with single doses up to 2000 mg/kg po,
chromosome aberration test, in vitro as well as in vivo micronucleus tests,
and phototoxicity studies in mice. Thus, 5 is a promising candidate as a new
antimalarial agent.
KEYWORDS Antimalarial activity, benzo[a]phenoxazine, Plasmodium falciparum,
Plasmodium berghei, oral administration
n tropical and subtropical regions, malaria is one of the
most perilous infectious diseases caused by protozoan
parasites. Each year, 500 million cases of malaria occur,
Further searches based on the combinatorial synthetic
approach8 provided various in vivo active rhodacyanine
derivatives.9,10 Among them, SSJ-127 (2) showed cure of
P. berghei-infected mice with subcutaneous administration,11
but the rhodacyanine derivatives did not exhibit any oral
activity. Phenoxazinium derivatives, a type of DLC exempli-
fied by 3 and 4, displayed potent in vitro activity and oral
efficacy.12-15 However, cytotoxicity was observed at low
micromolar concentrations in several phenoxazinium
derivatives.12,14 Because the electrophilicity of the carbon
atom at the 1 position in the phenoxazinium skeleton would
be troublesome, we examined the addition of a benzene ring
to the phenoxazinium framework. Thus, we have prepared
and evaluated various benzo[a]phenoxazine derivatives. The
corresponding salts of benzo[a]phenoxazines are a variant of
DLC candidates. Herein, we disclose the details of the
discovery and testing of SSJ-183 (5), having high in vitro
I
and nearly 1 million people die, the majority of whom are
young children and pregnant women.1-4 Because of global
warming, even inhabitants of temperate zones are in danger
of exposure to malaria infection. No vaccine is currently
available for malaria, and the resistance of the protozoa to
clinically used chemotherapeutic agents is increasingly com-
mon. Thus, novel, effective, safe, and inexpensive drugs are
highly desired to control malaria.
We have searched new hit compounds against malaria
parasites from our compound library, taking into account the
π-delocalized lipophilic cation (DLC) hypothesis5 in which
hydrophobic cations containing delocalized π-electrons are
accumulated into mitochondria and inhibit metabolic acti-
vity. According to the working hypothesis, the rhodacyanine
derivative MKT-077 (1)6 (Figure 1) was first identified as a
potential lead compound possessing potent in vitro activity
against Plasmodium falciparum.7 However, very poor effi-
cacy was observed in vivo against Plasmodium berghei.
Received Date: May 30, 2010
Accepted Date: July 6, 2010
Published on Web Date: July 12, 2010
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new compounds 5 and 11-26, prepared by the similar
method, are recorded in the Supporting Information.
We evaluated compounds 5-26 (Figures 1 and 2) for
their in vitro activity (IC50) against P. fa lci par um K1, a
strain resistant to chloroquine and pyrimethamine, and
determined the cytotoxicity to mammalian cells. They
were further screened by in vivo efficacy against P. be rghe i
strain NK-65 via the oral route (po) according to estab-
lished protocols (Table 1).10 For the in vivo screening, test
compounds were formulated in 5% glucose and 10%
ethanol solution or in 7% Tween 80 and 3% ethanol
solution to provide a dose of 100 mg/kg. The resulting
suspensions were administered to female ICR mice as a
single oral dose.
Benzo[a]phenoxazinium 6,16 having no substituent at the
6-position, showed weak activity against P. falciparum K1.
The activity of Nile red (7) possessing an oxygen substi-
tuent was also weak, while the introduction of a nitrogen
functionality increased the potency and reduced the IC50
value of Nile blue A (8) to 0.0156 μM. Although a low
cytotoxicity was observed for 8, the in vivo activity was
poor. The phenyl and tolyl derivatives 916 and 1016
showed low activities in both in vitro (IC50: 0.191 and
0.232 μM) and in vivo tests. Higher activities were
achieved in the in vitro test of compounds possessing
hetero aromatic rings on the nitrogen at the 6-position.
Therefore, several derivatives 11-14 were prepared
and evaluated, but their in vivo efficacies were unsatis-
factory. Improved in vivo efficacies were observed for the
compounds 15, 16, and 5 carrying a pyridine ring.
Among them, 5 exhibited the best activity: an IC50 value
of 0.0076 μM against P. falci paur m K1, an IC50 value of
55.7 μM in the cytotoxicity test, a selectivity of 7334, and
>99.9% inhibition of P. berghei NK-65. In addition to the
inhibition of parasitemia, the mean survival days (MSD)
after a single dose were extended to 14.6 days as com-
pared to approximately 6 days for an untreated control.
Very similar results were obtained, when single oral
doses of 100 mg/kg were administered to NMRI female
mice infected with P. bergh ei ANKA strain21 in three
independent experiments (n = 3 mice per experiment,
average inhibition = 99%, and average MSD = 16; data
not shown).
Figure 1. Structures of previous examples of antimalarial com-
pounds and the candidate 5.
Scheme 1. Synthesis of Benzo[a]phenoxazine 5 and Correspond-
ing Quaternary Salts
and in vivo antimalarial activity when administered to
P. berghei-infected mice via the oral route.
Although numerous analogues were prepared and as-
sessed, only the 4-aminopyridine derivatives are presented
here. Thus, the dimethyl derivative 17 showed potent
activity but a shorter survival as compared to 5. With
longer substituents at the 9-position, 18 and 19 were less
active. Morpholine compound 20 displayed good activity.
Furthermore, substitution of a methyl group at the 11-
position improved the safety, and two derivatives 21 and
22 provided good in vivo efficacy. Because the hydrochlor-
ides 21 and 22 gave similar in vivo activities as that of 5,
benzo[a]phenoxazine is apparently absorbed through the
gut as the hydrochloride when administered po. The
absence of cytotoxicity of compounds 23 and 24, having
a bromine atom on the A ring, was encouraging; however,
both showed low in vivo activity, possibly due to their poor
solubility and poor oral bioavailability. On the other hand,
A number of benzo[a]phenoxazine derivatives have been
prepared by known methods (Scheme 1).16-20 As a typical
example, the synthesis of 5 is shown in Scheme 1. Namely,
the nitrate salt of 6 (X = NO3)16 was reacted with 4-amino-
pyridine for 24 h in refluxing ethanol. Chromatographic
purification of the product on silica gel revealed 5, mp
247-248 ꢀC, in 38% yield. Notably, 5 is very stable as a free
amine for a long period under ambient conditions. All tested
compounds were prepared by the same method as des-
cribed above. When 5 was converted into a salt by treatment
with hydrochloric acid as usual, the elemental analysis of the
salt, mp >300 ꢀC, indicated that the molecular ratio of 5 and
hydrogen chloride must be 1:2.
Furthermore, the quaternary salts 25 and 26 were pre-
pared by treatment of 5 with alkyl halides in acetonitrile
under refluxing conditions. The physical and spectral data of
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Figure 2. Structures of tested benzo[a]phenoxazine derivatives.
high cytotoxicity was observed for the quaternary salts 25
and 26.
Trypanosoma cruzi, and 6500 nM for Leishmania donovani)
as compared to P. fa lciparum strain K1. Interestingly, the
deep purple/blue color of the compound formulation was
not detected in the urine, eyes, and organs of mice treated
with a single oral dose of 100 mg/kg, although the proto-
type molecule, methylene blue, in this class stains tissues
and urine. In vitro and in vivo activities of 5 are much
better than those of methylene blue. Furthermore, no
hemolysis was proved in blood taken from G6PD-deficient
patient at Jichi Medical University, and the details will be
discussed in the future.
In vivo pharmacokinetic studies in rats were carried out
according to the reported procedure (Figure 3).21 After oral
dosing, 5 had a bioavailability of approximately 30%. After
iv administration, the terminal half-life was approximately
5.5 h, and 5 demonstrated a high volume of distribution and
high clearance.
To gain additional information on the in vivo efficacy
of 5, we carried out a dose-response experiment in NMRI
female mice infected with P. berghei GFP ANKA strain21
(Table 2). High efficacy was observed by the po route with
cures achieved by oral administration of three daily doses of
100 mg/kg.
In other evaluations with compound 5, we detected no
lethality at doses up to 2000 mg/kg po using 20 mice.
Furthermore, no effects were found at 1000 μM (- and
þ S9) in a chromosome aberration test, at 2.0 μM (- and
þ S9) in an in vitro micronucleus test and at 1000 mg/kg ꢀ 2
in an in vivo rat micronucleus test. No phototoxicity was
detected in mice dosed at 300 mg/kg po. In binding assays
against 80 receptors, only two human recombinant re-
ceptors, A3 and D3, were inhibited ∼80% with 1 μM, and
no inhibitions were noted with other receptors. The selec-
tivity was further supported by g1000-fold higher IC50
values of 5 against three other protozoal parasites (36000
nM for Trypanosoma brucei rhodesiense, 11300 nM for
In summary, in a search for novel antimalarial agents,
new benzo[a]phenoxazines were synthesized and sub-
jected to biological evaluation. The derivative 5 possessing
the 4-aminopyridine moiety showed an IC50 value against
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Table 1. Evaluation of Benzo[a]phenoxazines: In Vitro Activity against P. falciparum K1, Cytotoxicity toward L-6 Myoblasts, and in Vivo
Activity against P. berghei NK-65 (n = 3 Mice)
IC50 (μM)a
compound
P. falciparum K1
cytotoxicity L-6
selectivityb
inhibition (%)c
MSD
6; X = Cl
0.606
0.868
0.0156
0.191
0.232
0.063
0.027
0.1125
0.005
0.015
0.023
0.0076
0.0081
0.05
3.82
65.9
6.3
76.0
ND
15
ND
7
6.3
8
43.14
164.9
123.2
179.8
67.9
2765
863
15
6.6
9
0
6.3
10
531
0
6.3
11
2855
2515
1493
2734
1052
5088
7334
20390
1342
327
14.7
81
6.3
12
10.3
6.3
13
167.9
13.6
11.4
3.3
14
6.3
15
15.8
>99.9
62.7
>99.9
>99.9
15.3
5.6
8.3
16
117.0
55.7
10.0
14.6
10.0
6.3
5
17
165.1
67.1
18
19
0.195
0.029
0.18
63.8
6.3
20
96.9
3342
481
97.9
99.7
>99.9
12.7
14.2
22.0
39
7.3
21
86.6
13.0
15.7
6.3
22
0.017
0.038
0.011
14.8
887
23
>190.2
>190.2
1.56
>5007
>17290
174
24
6.6
25
0.009
0.024
0.019-0.066
6.6
26
0.75
NDd
31
7.3
chloroquine
ND
>99.9
12.6
a Mean of two independent assays. b Calculated as IC50 for L-6 cells/IC50 for P. falciparum. c Parasitemia was determined on day 4 after infection. The
difference between the mean value of the control groupand those of treatedgroups is calculated and expressed as a percent relative tothe control group.
Single oral administration (100 mg/kg) was given on day 1. d ND, not determined.
Table 2. In Vivo Results for 5 Orally Administrated to n = 3 Mice/
Dose Once Daily for Three Consecutive Days to P. berghei GFP
ANKA Strain (Data from 2 to 3 Independent Experiments)
mg/kg
inhibition (%)a
MSD (% of cured animals)
3 ꢀ 100
3 ꢀ 30
3 ꢀ 10
>99.9
99
>30.0 (100%)
27.2 (78%)
4.0b
26
a Determined on day 4 postinfection. b Mice were euthanized on day
4 based on a lack of sufficient efficacy.
P. falciparum of 7.6 nM and a selectivity index of >7300.
Cure was achieved with three daily oral doses to mice
infected with P. berghei ANKA strain at a dosage of 100
mg/kg. The safety of 5 was demonstrated by a single dose
toxicity test, the highest dose being 2000 mg/kg, a chromo-
some aberration test, in vitro and in vivo micronucleus
tests, and a phototoxicity test. Binding assays against 80
receptors supported a high selectivity. Urine and eyes
were not colored during treatment, and no color was
detected in organs after 7 days of administration. On the
basis of the above findings, compound 5 appears to be a
promising candidate for further testing. We have also
developed a more efficient synthesis of 5, and a report
Figure 3. Plasma concentration vs time profile of 5 in male
Sprague-Dawley rats following intravenous (2.8 mg/kg, filled
symbols) and oral (21.5 mg/kg, open symbols) administration.
of this new synthesis as well as the investigation of the
mechanism of action of 5 will be published in due course.
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SUPPORTING INFORMATION AVAILABLE General syn-
thetic procedure, melting point, and spectral data for all new tested
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Dyes Pigm. 2008, 79, 22–39.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should
be addressed. Tel and Fax: þ81-3-5498-6391. E-mail: m-ihara@
hoshi.ac.jp.
(14) Ge, J.-F.; Arai, C.; Kaiser, M.; Wittlin, S.; Brun, R.; Ihara, M.
Synthesis and in vitro antiprotozoal activities of water-solu-
ble, inexpensive 3,7-bis(dialkylamino)phenoxazin-5-ium de-
rivatives. J. Med. Chem. 2008, 51, 3654–3658.
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codynamics and pharmacokinetics study of phenoxazinium
derivatives for antimalarial agent. Bioorg. Med. Chem. 2009,
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Funding Sources: This study was supported by the Creation and
Support Program for Start-ups from Universities, Japan Science
Technology Agency (JST), and the Program for Promotion of Funda-
mental Studies in Health Sciences of the National Institute of
Biomedical Innovation (NIBIO).
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ACKNOWLEDGMENT We thank Professor Terumi Nakajima
and Professor Toshio Honda, Hoshi University, and Dr. Hiroyuki
Togashi and Seiki Sakanoue, Synstar Japan Co., Ltd., for their
encouragement. We also thank Professor Hiroyuki Matsuoka, Jichi
Medical University, for in vitro work using blood from a G6PD
patient.
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