Synthesis and in vitro antiprotozoal activities of water-soluble, inexpensive phenothiazinium chlorides

Article abstract of DOI:10.1016/j.dyepig.2010.09.001

Tropical diseases are serious infectious diseases caused by protozoan parasites in tropical and subtropical regions. Novel, effective, safe, and inexpensive drugs are required to treat the parasites and contribute to the global goal of eradication. A series of phenothiazinium chlorides were synthesized and evaluated for in vitro activities against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani. Notably, 3,7-bis(piperidinyl)phenothiazinium chloride showed IC₅₀ of 0.097 μmol L^-1 against T. cruzi and 3,7-bis(benzyl(methyl)amino) phenothiazinium chloride exhibited IC₅₀ of 0.081 μmol L ^-1 against L. donovani, although the cytotoxicities of these compounds against L-6 cells were observed at low concentration.

Full text of DOI:10.1016/j.dyepig.2010.09.001

Dyes and Pigments 89 (2011) 44e48
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis and in vitro antiprotozoal activities of water-soluble,
inexpensive phenothiazinium chlorides
,
a
c
c
Yue-Ting Lu a, Chika Arai b, Jian-Feng Ge a , Wu-Sheng Ren , Marcel Kaiser , Sergio Wittlin ,
*
Reto Brun c, Jian-Mei Lu a, Masataka Ihara b
,
**
a Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, 199 Ren’Ai Road, Suzhou 215123, China
b Institute of Medicinal Chemistry, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
c Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Received in revised form
20 August 2010
Accepted 5 September 2010
Available online 15 September 2010
Tropical diseases are serious infectious diseases caused by protozoan parasites in tropical and subtropical
regions. Novel, effective, safe, and inexpensive drugs are required to treat the parasites and contribute to
the global goal of eradication. A series of phenothiazinium chlorides were synthesized and evaluated for
in vitro activities against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and
Leishmania donovani. Notably, 3,7-bis(piperidinyl)phenothiazinium chloride showed IC50 of 0.097
against T. cruzi and 3,7-bis(benzyl(methyl)amino)phenothiazinium chloride exhibited IC50 of
0.081
mol Lꢀ1 against L. donovani, although the cytotoxicities of these compounds against L-6 cells were
observed at low concentration.
m
mol Lꢀ1
m
Keywords:
Antiprotozoal
Phenothiazinium chlorides
In vitro
Ó 2010 Elsevier Ltd. All rights reserved.
Plasmodium falciparum
Trypanosoma cruzi
Leishmania donovani
1. Introduction
are used as medicines for Chagas disease, but these drugs were
developed for veterinary use more than 30 years ago, and have low
efficacy and high acute toxicity [4,5]. Phosphinopeptides [6], tipi-
farnib analogues [3] were recently evaluated for anti-T. cruzi
activity; however, there is no effective treatment for the prevalent
chronic form of Chagas disease [7]. Over 10 million patients are
infected by leishmaniasis [8]. Pentostam, amphotericin B, and
miltefosine are used to treat leishmaniasis, but these are either
highly toxic or very expensive compounds. African trypanosomi-
asis, caused by Trypanosoma brucei rhodesiense, has reappeared in
several areas over the past 30 years. Most medicines for this
disease, such as melarsoprol, have been used for 60 years and have
substantial side effects [9,10]. Generally, the presently used drugs
possess severe side effects and do not provide complete eradication
of the disease. In addition, drug resistance has become a major
problem in the treatment of tropical diseases. Therefore, new
promising compounds are urgently needed [11].
Malaria, Chagas disease, Leishmaniasis, and African sleeping
sickness are the main tropical diseases infected by parasitic
protozoa. Even inhabitants of temperate zones are exposed to the
danger of infection owing to global warming.
Malaria, which is caused by the Plasmodium falciparum para-
sites, exists in more than 100 countries. In 2008, there were an
estimated 243 million cases of malaria worldwide, and malaria
accounted for an estimated 863 thousand deaths [1]. In many parts
of the world, parasites have developed resistance to a number of
anti-malarial medicines [2]. Chagas disease, which is caused by
Trypanosoma cruzi, is a very serious public health problem in
several countries, with 18 million people known to be infected with
the parasite and an additional 100 million being at risk of infection.
The lack of interest shown by pharmaceutical companies for
developing anti-T. cruzi drugs makes Chagas one of the major
“neglected” diseases of the world [3]. Benznidazole and nifurtimox
Developing countries have many patients infected by parasitic
protozoa. Thus, the identification of effective, low-toxicity, and
inexpensive antiprotozoal candidates is a challenge for synthetic
chemists, biochemists and medicinal chemists. Our previous
* Corresponding author. Tel.: þ86 512 65880368; fax: þ86 512 65880367.
** Corresponding author. Tel./fax: þ81 03 5498 6391.
work is based on the
p-delocalized lipophilic cation (DLC)
hypothesis [12]. Quaternary ammonium salts of rhodacyanine and
E-mail addresses: gejianfeng@suda.edu.cn (J.-F. Ge), m-ihara@hoshi.ac.jp (M. Ihara).
0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.09.001

Y.-T. Lu et al. / Dyes and Pigments 89 (2011) 44e48
45
(CH), 50.9(NCH2), 50.6(NCH2), 26.2(CH2), 26.1(CH2); m/z(ESIþ)
336.1545 ([M ꢀ Clꢀ]þ, C20H22N3Sþ requires 336.1534); Calcd. for
C20H22ClN3S$2.5H2O: C, 57.61; H, 6.53; N, 10.08; Found: C, 57.84; H,
6.58; N, 9.71.
2.1.2.3. 3,7-Di(piperidinyl)phenothiazinium chloride (1b). Purple
Fig. 1. Structures of phenoxazinium and phenothiazinium.
powder, yield: 17%, mp: >250 ꢁC; UVevis (MeOH), lmax(nm) (l g
3
(L molꢀ1 cmꢀ1)): 667 (4.3); IR (KBr pellet cmꢀ1
) ymax: 2929, 2853
phenoxazinium derivatives were prepared. Rhodacyanines, which
are easily prepared by several steps in high yields, show good in
vitro activity against P. falciparum and L. donovani [13e17]. Phe-
noxaziniums, which are water-soluble, inexpensive and high purity
dyes, exhibit potent antiprotozoal activities, especially against
P. falciparum and T. cruzi [18e21].
Methylene blue having a phenothiazinium skeleton is still
explored as a potential candidate for anti-malarial medicine
[22,23]. On the basis of the structure similarity of phenoxazinium
and phenothiazinium (Fig. 1), we have synthesized several new
derivatives of methylene blue and evaluated their biological
activities. Here, we report our findings, which provide important
information to medicinal chemists working in related areas.
(alkyl-CH), 1595, 1522, 1398 (phenothiazinium skeleton) and 1146
(CeN); 1H NMR(300 MHz; CD3OD; Me4Si) dH: 7.93 (2H, d, J ¼ 9.6,
2 ꢂ CH), 7.60 (2H, d, J ¼ 9.7, 2 ꢂ CH), 7.51 (2H, d, J ¼ 2.6, 2 ꢂ CH),
3.88 (8H, br, 2 ꢂ N(CH2)2), 1.82 (12H, br, 2 ꢂ (CH2)3); 13C NMR
(101 MHz; CD3OD) dC: 154.5(C), 139.7(CH), 137.0(C), 136.2(C), 120.1
(CH), 107.8(CH), 50.6(NCH2), 27.7(CH2), 22.2(CH2); m/z(ESIþ)
364.1854([M ꢀ Clꢀ]þ, C22H26N3Sþ requires 364.1847); Calcd. for
C22H26ClN3S$2.5H2O: C, 59.38; H, 7.02; N, 9.44; Found: C, 59.25; H,
7.26; N, 9.43.
2.1.2.4. 3,7-Bis(methyl(phenyl)amino)phenothiazinium chloride
(1c). Purple powder, yield: 15%, mp: 108 ꢁC; UVevis (MeOH),
lmax(nm) (l g 3 ) ymax:
(L molꢀ1 cmꢀ1)): 652 (4.1); IR (KBr pellet cmꢀ1
1602, 1484, 1385 (phenothiazinium skeleton), 1125(CeN); 1H NMR
(300 MHz; CD3OD; Me4Si) dH: 7.97 (2H, d, J ¼ 8.6, 2 ꢂ CH), 7.61e7.27
(14H, m, AreH), 3.69 (6H, br); 13C NMR (101 MHz; CD3OD) dC: 155.8
(C), 145.6(C), 139.5(CH), 138.1(C), 136.8(C), 131.9(CH), 130.1(CH),
127.6(CH), 121.9(CH), 108.4(CH), 42.6(CH3); m/z(ESIþ) 408.1560
2. Experimental
2.1. Chemistry
2.1.1. General
([M
ꢀ
Clꢀ]þ, C26H22N3Sþ requires 408.1534); Calcd. for
Melting points were measured on an X-4 microscope electro
thermal apparatus (Taike, China) and the results are uncorrected.
Infrared spectra (IR) were recorded on a Nicolet 5200 FT-IR
instrument using solid samples dispersed in KBr pellets. UVevis
C26H22ClN3S$2.5H2O: C, 63.86; H, 5.56; N, 8.59; Found: C, 63.72; H,
5.67; N, 8.49.
2.1.2.5. 3,7-Bis(benzyl(methyl)amino)phenothiazinium chloride
spectra were recorded on a PerkineElmer
l-17 spectrometer using
(1d). Purple powder, yield: 17%, mp: 118 ꢁC; UVevis (MeOH),
a 1 cm square quartz cell. 1H NMR and 13C NMR spectra were
collected on Varian-300 or 400 NMR spectrometer, tetramethylsi-
lane was used as the internal reference for the analyses and CD3OD
as solvent; TMS was used as an internal standard for 1H NMR and
solvent peak was used as an internal standard for 13C NMR. High
resolution mass spectra were recorded on a Finnigan MAT 95 mass
spectrometer (ESIþ). Elementary analyses were conducted on
a Carlo Erba-MOD1106 elementary analysis apparatus. Phenothia-
zinium tetraiodide was obtained by reported method [24].
lmax(nm) (l g 3 ) ymax:
(L molꢀ1 cmꢀ1)): 655 (4.6); IR (KBr pellet cmꢀ1
1595, 1489, 1392 (phenothiazinium skeleton), 1144(CeN); 1H NMR
(400 MHz; CD3OD) dH: 7.98 (2H, d, J ¼ 9.6, 2 ꢂ CH), 7.58 (2H, d,
J ¼ 9.7, 2 ꢂ CH), 7.45 (2H, d, J ¼ 2.5, 2 ꢂ CH), 7.42e7.28 (10H, m,
2 ꢂ C6H5), 5.03 (4H, s, 2 ꢂ CH2), 3.45 (6H, s, 2 ꢂ CH3); 13C NMR
(101 MHz; CD3OD) dC: 155.8(C), 139.8(C), 137.3(CH), 136.9(C), 136.4
(C), 130.3(CH), 129.2(CH), 127.9(CH), 120.5(CH), 107.7(CH), 57.5
(CH2), 40.7(CH3); m/z(ESIþ) 436.1864 ([M ꢀ Clꢀ]þ, C28H26N3Sþ
requires 436.1842); Calcd. for C28H26ClN3S$2.5H2O: C, 65.04; H,
6.04; N, 8.13; Found: C, 64.95; H, 6.21; N, 8.12.
2.1.2. Synthesis
2.1.2.1. General procedure. Phenothiazinium tetraiodide (1.45 g,
2.0 mmol) was slowly added to a solution of the requisite dialkyl-
amine (12 mmol) in methanol (50 mL) during a time of 30 min. The
resulting solution was allowed to stir at room temperature for 8 h.
The mixture was concentrated to approximate 20 mL and precipi-
tated by addition of diethyl ether (100 mL). The crude product was
collected by filtration, and purified by column chromatography
(CHCl3:CH3OH ¼ 10:1, silica gel (200e300 mesh)). The solvent was
removed by evaporator under reduced pressure, then the residue
obtained was dissolved in 1:1 CH3OH:CHCl3 and passed through an
IRA-400 (Cl) resin exchange column to obtain phenothiazinium
chloride. The product was finally further purified by column
chromatography (CHCl3:CH3OH ¼ 10:1, silica gel (300e400 mesh)).
2.1.2.6. 3,7-Bis(dibenzylamino)phenothiazinium chloride (1e). Purple
powder, yield: 16%, mp: 110 ꢁC; UVevis (MeOH), lmax(nm) (l g
3
(L molꢀ1 cmꢀ1)): 654 (5.0); IR (KBr pellet cmꢀ1
) ymax: 1595, 1487,
1393 (phenothiazinium skeleton), 1140(CeN); 1H NMR(400 MHz;
CD3OD) dH: 7.98 (2H, d, J ¼ 9.4, 2 ꢂ CH), 7.56 (2H, d, J ¼ 9.7, 2 ꢂ CH),
7.47e7.36 (22H, m, AreH), 5.09 (4H, br, 2 ꢂ CH2), 5.09 (4H, br,
2 ꢂ CH2); 13C NMR (101 MHz; CD3OD; Me4Si) dC: 156.1(C), 140.1(C),
137.8(CH), 136.7(C), 136.6(C), 130.3(CH), 129.2(CH), 127.9(CH), 121.2
(CH), 108.0(CH), 56.3(CH2); m/z(ESIþ) 588.2515 ([M
ꢀ
Clꢀ]þ,
C40H34N3Sþ requires 588.2473); Calcd. for C40H34ClN3S$2.5H2O: C,
71.78; H, 5.87; N, 6.28; Found: C, 71.65; H, 5.92; N, 6.25.
2.2. Antiprotozoal activities
2.1.2.2. 3,7-Di(pyrrolidinyl)phenothiazinium chloride (1a). Purple
powder, yield: 15%, mp: >250 ꢁC; UVevis (MeOH), lmax(nm) (l g
3
The tests were performed at pH 7.4 as microplate assays using
T. b. rhodesiense (STIB900), T. cruzi (Tulahuen C4), L. donovani
(MHOM-ET-67/L82), and P. falciparum K1 (resistant to chloroquine
and pyrimethamine), and the cytotoxicity was assessed with rat
skeletal myoblasts (L-6 cells) as described previously [20]. The
following substances were used as reference standards: melarso-
prol (T. b. rhodesiense), benznidazole (T. cruzi), miltefosine
(L molꢀ1 cmꢀ1)): 660 (5.1); IR (KBr pellet cmꢀ1
) ymax: 2936, 2867
(alkyl-CH), 1590, 1523, 1398 (phenothiazinium skeleton), 1146
(CeN); 1H NMR(300 MHz; CD3OD; Me4Si) dH: 7.91 (2H, d, J ¼ 9.4,
2 ꢂ CH), 7.32 (2H, d, J ¼ 9.4, 2 ꢂ CH), 7.21 (2H, s, 2 ꢂ CH), 3.72 (8H,
br, 2 ꢂ N(CH2)2), 2.16 (8H, br, 2 ꢂ (CH2)2); 13C NMR (101 MHz;
CD3OD) dC: 153.0(C), 139.5(CH), 136.7(C), 135.5(C), 120.6(CH), 108.0

46
Y.-T. Lu et al. / Dyes and Pigments 89 (2011) 44e48
H
N
N
N
HNR1R2, MeOH
I2
R1
R1
CH2Cl2
I4
I
N
R2
S
N
R2
S
3
S
2
4a-e
N
1a: R1,R2=(CH2)4;
R1
N
R2
R1
IRA-400 (Cl)
1b: R1,R2=(CH2)5;
Cl
S
N
R2
1c: R1=CH3, R2=C6H5;
1d: R1=CH3, R2=CH2C6H5;
1e: R1=R2=CH2C6H5.
1a-e
Fig. 2. Synthesis of phenothiazinium chlorides (1aee).
(L. donovani), chloroquine (P. falciparum), and podophyllotoxin
(cytotoxicity assay).
1590e1398 cmꢀ1 in their IR spectra. It was difficult to obtain good
NMR spectra of the phenothiazinium iodides (4aee), indeed
several papers reporting such compounds did not report their NMR
spectra [24,26]. The nitrate salt of phenothiazinium, which can be
prepared by the reported method, might be suitable for charac-
terization [30]; however, traces of silver ions might affect the result
of the biological assay results. So, IRA-400 (Cl) exchange resin was
used to obtain the phenothiazinium chlorides. After the purifica-
tion by column chromatography, the NMR spectra of phenothiazi-
nium chlorides were easily obtained in deuterated methanol
solution. The NMR spectra of 1a are shown in Fig. 3. The chemical
shift of tertiary carbons is located at 105.4,118.0,136.9 ppm, and the
chemical shift of quaternary carbon atom is located at 132.8, 134.0,
150.3 ppm. The peaks of secondary carbon were shown by four
peaks. Both 1H NMR and 13C NMR are clearly demonstrated that
phenothiazinium chlorides are symmetric structures in methanol
solution.
3. Results and discussion
3.1. Chemistry
Several synthetic routes have been reported for the preparation
of phenothiazinium dyes [25], and only a few were suitable for our
experiment since they had multi-step reactions and gave mixed
products, while the phenothiazinium dyes are notoriously difficult
to separate and purify [26]. As phenothiazinium compounds had
been obtained for biological evaluation by Brown et al. [27], it was
decided therefore to use the same synthetic route [28,29], which
allows the reaction to progress to completion under ambient
temperature and pressure in air. Thus, symmetrically disubstituted
phenothiaziniums were obtained (Fig. 2).
In order to obtain specific structures of phenothiazinium salts,
pyrrolidine, piperidine, N-methylaniline, N-methylbenzylamine
and dibenzylamine were selected for the amines in the reaction.
The compounds (1aee) exhibit absorption maxima in the range
652e667 nm in methanol solution, and the characteristic absorp-
tion peaks of phenothiazinium skeleton are located at ca.
Fig. 3. NMR spectra of 1a.

Y.-T. Lu et al. / Dyes and Pigments 89 (2011) 44e48
47
Table 1
Antiprotozoal and cytotoxic activities (IC50 values,
m
mol Lꢀ1) of title compounds (1aee)a.
T. cruzi
IC50
Compd.
P. falc.K1
T.b. rhod.
L. don., axenic
Cytotox. L6
IC50
SIb
SIb
IC50
SIb
1.06
5.05
22.04
4.82
IC50
SIb
0.62
0.49
11.68
33.77
19.14
IC50
1a
1b
1c
1d
0.005
0.010
2.500
0.084
0.188
0.148
18.40
53.12
3.49
32.30
21.02
NDc
NDc
5.31
1.10
4.53
5.55
0.089
0.103
0.396
0.566
0.266
0.150
1.055
0.748
0.081
0.207
0.094
0.517
8.739
2.731
3.957
0.097
7.905
0.603
0.713
1e
14.88
Chloroquined
Benznidazoled
Melarsoprold
Miltefosined
Podophyllotoxind
0.886
0.006
0.280
0.02
a
Values indicate the inhibitory concentration of a compound or standard in mM, which is necessary to achieve 50% growth inhibition (IC50). Data shown are values from two
replicate experiments.
b
Selectivity index ¼ IC50 value for L6/IC50 value for P. falciparum, T. b. rhodesiense, T. cruzi, or L. donovani.
c
Not determined due to insufficient activity.
d
Standard samples.
Diphenylamine was also used for the reaction, but no product can
be obtained owing to steric hindrance and weak nucleophilicity.
Acknowledgements
We thank Professor Terumi Nakajima and Professor Toshio
Honda, Hoshi University for their encouragement. This study was
supported by the Creation and Support Program for Start-ups from
Universities, Adaptable and Seamless Technology Transfer Program
through Target-driven R&D, Japan Science Technology Agency (JST)
and the Program for Promotion of Fundamental Studies in Health
Sciences of the National Institute of Biomedical Innovation (NIBIO).
Synthetic part was partly support by Natural Science Fund
(SBK200930379) and Natural Science Fund for Colleges and
Universities (08KJB430013) in Jiangsu Province, China.
3.2. Biological evaluations
3.2.1. Antiprotozoal activities
Antiprotozoal and cytotoxic activities of the phenothiazinium
chlorides (1aee) are shown in Table 1. All compounds show
insufficient activities against T. b. rhodesiense. 3,7-Di(pyrrolidinyl)
phenothiazinium chloride (1a) shows median inhibitory concen-
tration (IC50) of 0.005 m
mol Lꢀ1 against P. falciparum K1 strain, and it
is moderately active compared with rhodacyanine [13e17] and
phenoxazinium [18e21] derivatives.
References
Notably, 3,7-di(piperidinyl)phenothiazinium chloride (1b)
shows IC50 of 0.097 m
mol Lꢀ1 against T. cruzi, which reveals that it
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might be a lead compound for the Chagas disease, since there are
no good candidates for the treatment of this type disease at present.
3,7-Bis(benzyl(methyl)amino)phenothiazinium
chloride
(1d)
exhibits IC50 of 0.081
m
mol Lꢀ1 against L. donovani.
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3.2.2. Cytotoxicity
The IC50 value to the normal L6 cell, 3,7-di(pyrrolidinyl)pheno-
thiazinium chloride (1a, IC50 0.094
¼
m
mol Lꢀ1) with five-
membered ring shows more than five times toxicity comparing with
the 3,7-di(piperidinyl)phenothiazinium chloride (1b, IC50 ¼ 0.517
m
mol Lꢀ1). From the IC50 values of 1d and 1e, it is clearly
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toxicity, and 3,7-bis(dibenzylamino)phenothiazinium chloride (1e,
IC50 ¼ 3.957
amino)phenothiazinium chloride (1d, IC50 ¼ 2.731
mol Lꢀ1) of 3,7-bis(methyl(phenyl)
m
mol Lꢀ1) is less toxic than 3,7-bis(benzyl(methyl)
m
mol Lꢀ1). The
cytotoxicity (IC50 ¼ 8.739
m
amino)phenothiazinium chloride(1c) was lowest among the tested
compounds.
4. Conclusions
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A series of phenothiazinium chlorides were synthesized and
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rhodesiense, and L. donovani. 3,7-Di(piperidinyl)phenothiazinium
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[13] Takasu K, Inoue H, Kim HS, Suzuki M, Shishido T, Wataya Y, et al. Rhoda-
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chloride shows IC50 of 0.097
(benzyl(methyl)amino)phenothiazinium chloride exhibits IC50 of
0.081
mol Lꢀ1 against L. donovani. The N-Aromatic and N-benzyl
m
mol Lꢀ1 against T. cruzi; and 3,7-bis
m
substituted phenothiazinium chlorides are less toxic compounds
than the N-alkyl substituted compounds.

48
Y.-T. Lu et al. / Dyes and Pigments 89 (2011) 44e48
[15] Takasu K, Pudhom K, Kaiser M, Brun R, Ihara M. Synthesis and antimalarial
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