Synthesis and antimalarial property of orally active phenoxazinium salts

Article abstract of DOI:10.1021/jm070201e

Phenoxazinium salts were found to display good antimalarial efficacy in vivo against Plasmodium berghei. Several compounds provided 100% parasitemia clearance at a dose of 20-30 mg kg^-1 x 4 days (ip) and good survival effects without obvious acute toxicity. They also showed excellent potency by oral administration. A preliminary pharmacokinetic study revealed that the oral availability of 1a was excellent.

Full text of DOI:10.1021/jm070201e

©
Copyright 2007 by the American Chemical Society
Volume 50, Number 10
May 17, 2007
Letters
Synthesis and Antimalarial Property of Orally
Active Phenoxazinium Salts
synthesis of new antimalarial candidates possessing the DLC
10-12
structure.
Recently, we have found several compounds that
show excellent in vivo potency against P. bergei in the mouse
1
0
,
†
†
†
model when administered intraperitoneally (ip). However,
further development is still required, because these compounds
showed poor to moderate efficacy as oral treatments. We
describe here the biological properties and partial structure-
activity relationship study of a class of phenoxazinium salts 1
as orally active antimalarial candidates.
Kiyosei Takasu,* Tsubasa Shimogama, Chie Satoh,
‡
‡
†,§
Marcel Kaiser, Reto Brun, and Masataka Ihara
Department of Organic Chemistry, Graduate School of
Pharmaceutical Sciences, Tohoku UniVersity, Aobayama,
Sendai 980-8578, Japan, and Swiss Tropical Institute,
Socinstrasse 57, CH-4002 Basel, Switzerland
Several research groups have sporadically reported on cationic
dyes with a heteroaromatic cationic skeleton that demonstrate
ReceiVed February 21, 2007
13-15
antimalarial properties
the antimalarial efficacy of methylene blue (2).
since Guttmann and Ehrich reported
Abstract: Phenoxazinium salts were found to display good antimalarial
efficacy in vivo against Plasmodium berghei. Several compounds
16,17
Vennerstrom
-
1
and co-workers re-examined the in vitro antimalarial potencies
of xanthene, azine, oxazine, and thiazine dyes and reported that
several of them displayed strong in vitro activity against drug-
provided 100% parasitemia clearance at a dose of 20-30 mg kg
×
4
days (ip) and good survival effects without obvious acute toxicity.
They also showed excellent potency by oral administration. A
preliminary pharmacokinetic study revealed that the oral availability
of 1a was excellent.
13
resistant P. falciparum. In connection with our working
hypothesis, the above dyes intrigued us as DLC compounds.
At the outset of our program, the in vivo evaluation of the
antimalarial potency of commercially available dyes (Figure 1)18
was carried out using rhodent malaria P. berghei NK-65 (drug-
sensitive strain) in mice. The assay was performed according
Malaria is one of the most important infectious diseases in
the world, as it remains a major health problem. It infects more
than 300 million people per year and causes more than one
million deaths annually, mostly among young children in sub-
Saharan Africa. Malaria is re-emerging as the biggest infectious
killer and is currently a first priority tropical disease of the World
Health Organization.1,2 A major contributor to malarial morbid-
ity and mortality is almost certainly the increasing resistance
of malaria parasites to available chemotherapeutics, such as
1
9
to Peters’ 4-day suppressive test protocol. The suppression
(%) of malaria protozoa on day 4 is determined by comparing
the parasitemia of infected mice, which are injected with the
test compounds for 4 days with that of untreated controls. Table
1 summarizes parasitemia suppression data at a dose of 5.0 mg
a
3,4
-1
chloroquine (CQ ) and pyrimethamine. Therefore, strong
interest has been directed at the search for new antimalarial
agents that can be dosed orally.5 As part of our studies to
develop synthetic antimalarial compounds, we have earlier
reported that rhodacyanines, having a π-delocalized lipophilic
kg × 4 days (ip), along with reported EC values in vitro
5
0
13
against P. falciparum from the literature. Phenoxazinium salt
-7
1
a (basic blue 3), whose purity was ∼60% in the commercial
source, showed 50% parasitemia suppression without several
apparent symptoms of acute toxicity, such as diarrhea and body
weight loss (entry 1). Phenothiazinium 2 (methylene blue) and
xanthenium 3 (pyronin Y) displayed less of a suppression effect
than 1a, although they showed no apparent acute toxicity during
the medication period (entries 2 and 3). However, phenazinium
8
cationic (DLC) structure, exhibit strong antimalarial efficacies
9
in vitro against the Plasmodium falciparum parasite. In the
course of our investigation, we were striving for the design and
*
To whom correspondence should be addressed. Phone: +81-22-795-
878. Fax: +81-22-795-6877. E-mail: kay-t@mail.pharm.tohoku.ac.jp.
4
(Janus green B) resulted in animal death before day 4 (entry
6
†
‡
§
4). Further in vivo screening of commercial phenoxazinium salts
5-7 was carried out, but all the compounds showed weak
suppression effects (entries 5-7). The results indicated that
phenoxazinium salt would be more promising as a lead
compound compared to the other heteroaromatic cations, and
Tohoku University.
Swiss Tropical Institute.
Current address: Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo
1
42-8501, Japan.
a
Abbreviations: CQ, chloroquine; DLC, π-delocalized lipophilic cations;
Piv, pivaloyl; MSD, mean survival days.
1
0.1021/jm070201e CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/19/2007

2
282 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Letters
Scheme 1. Synthesis of Phenoxazinium Salts
Figure 1. Heteroaromatic cations tested for in vivo antimalarial activity.
Table 1. Antimalarial Activity of 1a and 2-7
in vitroa
in vivob
entry
cmpd
EC50 (nM)
suppression (%)
c
NTi
4.0
450
4.9
1
2
3
4
5
6
7
1a
50.6
15.9
20.6
toxic
11.0
15.6
21.8
d
2
e
3
f
4
f
5
5.5
g
6
670
h
NTi
7
a
Reported data in ref 13 (against P. falciparum W2 strain). b Reported
-
1
data in ref 13 (against P. berghei NK-65 at a dose of 5.0 mg kg × 4
c
d
days). Approximately 60% dye content. Approximately 80% dye content.
e
f
Approximately 50% dye content. Approximately 65% dye content.
Approximately 90% dye content. Greater than 95% dye content. Not
g
h
i
Table 2. In Vitro Antimalarial Activity against CQ-Resistant P.
falciparum K1
tested.
entry
cmpd
1aa
EC50 (nM)
selectivity
that fully substituted nitrogen atoms on both ends of the
phenoxazinium framework would be crucial for good in vivo
antimalarial efficacy. Additionally, we observed that there was
no good correlation between in vitro and in vivo efficacies
1
2
3
4
5
6
7
8
4.7
2.8
12.6
2.4
28
200
270
1500
830
2400
700
350
2300
440
160
32
b
1a
1
1b‚ /2ZnCl2
1g
1h‚HCl
1j‚HCl
1k‚HCl
CQ
(entries 2 and 5 vs entries 3 and 6).
Next we synthesized several phenoxazinium salts, including
1
a, with high purity.20 Compounds 1a-g were prepared by the
reaction of m-aminophenol 8 with p-nitrosoaniline 9 in the
a
b
Purchased from MP biochemicals (∼60% purity). Synthetic compound
>95% purity).
2
1
presence of acid, followed by auto-oxidation by air. Each
compound was purified by column chromatography on silica
gel and, if necessary, through an ion-exchange process. As an
alternative route, 1 can be prepared by a two step sequence from
(
of piperazine-containing phenoxaziniums, 1h, 1j, and 1l, was
not so strong (entries 5-7). Namely, it was made clear that the
introduction of additional hydrophilic groups into 1 resulted in
a decrease in antimalarial efficacy.
22
m-anisidine 10. Namely, 1a was synthesized from 3-methoxy-
-nitrosoaniline 11, which was readily obtained from 10
Scheme 1). The overall chemical yield of 1a was higher in the
4
(
The in vivo potency of synthetic 1a-l was also evaluated
-
1
latter method.
using the above-mentioned procedure (5.0 mg kg × 4 days,
ip), and observation of malaria-infected mice after the end of
treatment was continued in order to record their mean survival
days. The results are summarized in Table 3. Synthetic basic
blue 3 (1a), as the pure form, provided 81% suppression of
parasitemia on day 4 (Table 3, entry 1) without obvious signs
of acute toxicity. Namely, the in vivo activity of 1a is also
increased in proportion to its purity (see Table 1, entry 1). The
tetramethyl analog 1b showed a 40% suppressive effect (entry
2), whereas the activity of its semi-ZnCl2 salt markedly
decreased (entry 3). The phenoxazinium salts 1c-g and 1h‚
With several synthetic phenoxazinium salts in hand, selected
compounds were assessed for their in vitro antimalarial activity
against P. falciparum K1 (CQ-resistant strain) according to the
2
3
procedures described by Desjardins and co-workers. Their
cytotoxicities were determined using a rat skeletal myoblast cell
line, L-6. Selectivity, defined by the ratio EC50 (L-6)/EC50 (P.
falciparum), was also determined. Both the commercially
available (∼60% purity) and synthetic (>95% purity) 1a
exhibited very strong antimalarial activity in proportion to the
purity (Table 2, entries 1 and 2). The selective toxicity of 1a
was excellent as well. The semi-zinc salt of 1b, which can be
obtained as a stable crystalline form of 1b, also showed good
activity (entry 3). Furthermore, the dibutyl analog 1g also shows
strong activity (entry 4). On the contrary, the activity of a series
5
HCl, whose R substituent is a hydrogen atom, provided good
to excellent parasitemia suppression (60-94%) at the same dose
(entries 4-9). Notably, the mean survival days of the mice
treated with 1d-g are significantly prolonged as compared with

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2283
Table 5. In Vivo Antimalarial Activity by Oral Route (po)
Table 3. In Vivo Antimalarial Activity against P. berghei at a Dose of
-
1
a
5
.0 mg kg (ip)
medication
program
% supp. MSDb mice alive
%
supp.
MSDb
(d)
entry
1
cmpd
1aa
on day 4
(d)
on day 30
entry
cmpd
on day 4
25 mg kg-1 day-1
4 days
×
×
×
82.3
7.6
0/5
1
2
3
4
5
6
7
8
9
1ac
1b
1b‚ /2ZnCl2
80.6
45.9
17.1
71.7
77.0
85.3
90.3
83.1
60.4
4.2
NDe
6.7
5.6
6.7
9.4
10.0
11.4
16.0
8.6
5.3
5.3
2
3
4
5
6
7
8
50 mg kg day-1
-
1
98.6
100
100
100
100
100
100
14
0/5
3/5
3/5
2/5
0/5
0/5
0/5
1
4 days
-
1
1c
1d
1e
1f
90 mg kg day-1
>24
>28
>13
14
4 days
100 mg kg day-1
-
1
×
4 days
-
1
1g
30 mg kg /8 h ×
1h‚HCl
12 times
-
1
10
11
12
13
1i
15 mg kg / 8 h ×
1j‚HCl
1k‚HCl
17.4
19.9
90.6
12 times
100 mg kg day-1
-
1
×
×
13
5.4
22.7
d
CQ
1 day
1h‚HCl 100 mg kg day-1
-
1
17
a
In vivo evaluation was carried out according to Peters’ four-day
4
days
suppressive protocol using five ICR-mice. b MSD ) mean survival days.
MSD for untreated mice (control) is 5.0-5.3 days. Synthetic 1a (>95%
c
a
b
Puchased 1a (∼60% purity). MSD ) mean survival days. MSD for
d
-1
e
purity). 10 mg kg × 4 days were dosed. Not determined.
Table 4. Dose-Efficacy Response of 1a, 1e, and 1h‚HCl
untreated mice (control) is 5.6 days.
Table 6. Pharmacokinetic Property of 1aa
MSDb
(d)
dose
% supp.
on day 4
entry
cmpd
(mg kg-1)
AUC0
t1/2
(miin)
Cmax
(min)
BA
(%)
-480 min
(mg mL min-1)
1aa
NDc
route
1
2
3
4
5
6
7
8
9
2.5
5.0
10
30.5
46.3
80.4
95.8
58.7
85.3
99.9
100
54.8
78.5
98.1
100
NDc
>11
>14
7.8
b
iv
43.0
41.4
5.8
59.2
pob
10
96.3
20
a
b
Puchased 1a (∼60% purity). Average of three animals administered
1e
2.5
5.0
10
20
5.0
10
-
1
at 10 mg kg as a saline solution.
10
13
>17
6
d
days (entries 1, 3, and 8). Beyond our expectation, phenoxazi-
mium salts showed good oral bioavailability. Moreover, 60%
of mice (3/5) treated with 1a at a dose of more than 90 mg
1h‚HCl
1
1
1
0
1
2
7.4
17.6
16.4
20
30
-
1
-1
kg day survive beyond day 30; however, for 1h‚HCl, none
of the treated mice was alive on day 30. The antimalarial effect
of 1a given by the oral route was also dose-dependent between
a
b
Purchased 1a (∼60% purity). MSD ) mean survival days. MSD for
c
d
untreated mice (control) is 5.0-5.2 days. Not determined. Two of five
mice treated survived on day 30.
-
1
20 and 100 mg kg × 4 days (entries 1-4). When the
medication program was changed to short-span dosing (three
times a day), the total treatment dose could be reduced to 45
untreated mice. The in vivo antimalarial potency of 1f (5.0 mg
-
1
-1
kg day ) is comparable to that of CQ at a dose of 10 mg
-
1
-1
mg kg day for complete parasitemia clearance. However,
the survival effect was not prolonged (entries 5 and 6). A single
-
1
-1
kg day (entry 13). On the contrary, the introduction of a
5
hydroxy or acyloxy group as an R substituent lead to significant
loss of suppression and survival effects (entries 10-12). The
results indicated that the hydrogen atom as the R substituent
-
1
dosing on day 1 at 100 mg kg provided 100% parasitemia
suppression, but none of the treated mice survived beyond day
5
3
0 (entry 7).
would be essential for the antimalarial property of phenox-
azinium salts.
Compounds 1a (purchased), 1e, and 1h‚HCl, which showed
no apparent body weight loss of the tested mice during the
medication period (5.0 mg kg day ), were selected to
investigate their dose-efficacy response via ip administration.
As shown in Table 4, significant improvements in the suppres-
sion of parasitemia were observed in accordance with the
amounts of drugs in all cases. Interestingly, 40% of mice (among
A preliminary pharmacokinetic study using Wister rats was
performed (Table 6). The half-life values of 1a (purchased) by
intravenous (iv) and po administration were determined as 5.8
and 59 min, respectively. The Cmax by the oral route was reached
-
1
-1
-
1
within 10 min. The oral bioavailability of 1a (10 mg kg ) was
determined to be excellent (96% BA). These results clearly show
that phenoxazinium salts, such as 1a, would be a new and
promising class of candidate orally available antimalarials, but
it will be necessary to improve the plasma clearance.
-
1
the five tested mice) treated with 1e at a dose of 20 mg kg
days survive beyond day 30. Nevertheless, 1e and 1h‚HCl
showed apparent acute toxicity, including body weight loss and
×
In conclusion, we have chosen phenoxazinium salts 1 as a
class of antimalarial active compounds among the several types
of heteroaromatic cations and have evaluated the in vitro and
in vivo antimalarial potency against P. falciparum (CQ-resistant
K1 strain) and P. berghei (NK-65 strain), respectively. Some
compounds were found to possess promising in vitro and in
vivo efficacy. Compounds 1a and 1h‚HCl are also orally
bioavailable. Currently, further pharmacodynamic and toxico-
logical studies of 1 are under consideration, and the synthesis
of phenoxazinium salts possessing improved pharmacokinetic
properties are now progressing.
4
-
1
diarrhea, at doses of more than 20 or 30 mg kg × 4 days,
although the growth of malaria parasites was almost perfectly
suppressed at these doses. It was evident that ip administration
of the tested compounds at a dose of less than 20 mg kg day
would afford the best effects.
As a next stage, the antimalarial efficacy of 1a (purchased)
and 1h‚HCl by oral administration (po) was assessed. Prelimi-
nary toxicity tests indicated that the 50% lethal dose (LD50) of
both compounds was more than 200 mg kg (po). As can be
seen from Table 5, both compounds showed 100% clearance
-1
-1
-
1
Acknowledgment. We thank Dr. Masayuki Kawakami, Dr.
-
1
of parasitemia on day 4 at a dose of 90 or 100 mg kg × 4
Hiroshi Kitaguchi, and Dr. Kozo Sato for their helpful discus-

2
284 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Letters
sions. This work was financially supported by Grants-in-Aid
from Japan Science and Technology Agency (JST), the Takeda
Science Foundation.
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Their Inhibition of P. Vinckei and P. Yoelii nigeriensis In Vivo.
Biochem. Pharmacol. 1996, 51, 693-700.
Supporting Information Available: Experimental procedures
and characterization data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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JM070201E