Synthesis and in vitro antiprotozoal activities of water-soluble, inexpensive 3,7-bis(dialkylamino)phenoxazin-5-ium derivatives

Article abstract of DOI:10.1021/jm8003619

3,7-Bis(dialkylamino)phenoxazinium salts were synthesized and evaluated for in vitro activities against Plasmodium falciparum, Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania donovani. Notably, the compounds showed potent antiprotozoal activities, especially against P. falciparum and T. cruzi. The compounds with alkyl side chains less than three carbons in length possessed good activities with high selective indices.

Full text of DOI:10.1021/jm8003619

3654
J. Med. Chem. 2008, 51, 3654–3658
Synthesis and in Vitro Antiprotozoal Activities of Water-Soluble, Inexpensive
3,7-Bis(dialkylamino)phenoxazin-5-ium Derivatives
Jian-Feng Ge,†,‡ Chika Arai,† Marcel Kaiser,§ Sergio Wittlin,§ Reto Brun,§ and Masataka Ihara*,†
Institute of Medicinal Chemistry, Hoshi UniVersity, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan, Department of Material and
Chemistry, Soochow UniVersity, Suzhou 215006, China, and Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
ReceiVed February 15, 2008
3,7-Bis(dialkylamino)phenoxazinium salts were synthesized and evaluated for in vitro activities against
Plasmodium falciparum, Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania donoVani. Notably,
the compounds showed potent antiprotozoal activities, especially against P. falciparum and T. cruzi. The
compounds with alkyl side chains less than three carbons in length possessed good activities with high
selective indices.
Introduction
by crystallization.13 In the present study, 3,7-bis(dialkylami-
no)phenoxazinium derivatives were synthesized and tested
against various parasites in in vitro assays. Remarkably, some
of the compounds showed potent activities against the chloro-
quine- and pyrimethamine-resistant strain of P. falciparum and
against T. cruzi.
Tropical diseases that result from infection by parasitic
protozoa, such as malaria, Chagas disease, leishmaniasis, and
African trypanosomiasis, are serious threats to public health.
Malaria, which is caused by the Plasmodium parasites, exists
in 100 countries. Over 300 million patients are infected with
malaria, and two children are killed by the parasite each minute.1
In many parts of the world, the Plasmodium parasites have
developed resistance to a number of antimalarial medicines.2
Chagas disease, which is caused by Trypanosoma cruzi, is
mainly endemic in South and Latin America. Chagas disease
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 at risk of infection. Benznidazole
is used as a medicine for Chagas disease, but the drug has low
efficacy and high acute toxicity. Actually, there is no effective
treatment for the prevalent chronic form of Chagas disease.3
Over 10 million patients are infected by leishmaniasis.4
Pentostam, amphotericin B, and miltefosine are used to treat
leishmaniasis, but these are highly toxic or very expensive
compounds. African trypanosomiasis has reappeared in several
areas over the past 30 years. Most medicines for this disease,
such as melarsoprol, have been used for 60 years but have
substantial side effects.5,6 Developing countries have many
patients infected by parasitic protozoa. Thus, the identification
of effective, low-toxicity, and inexpensive antiprotozoal can-
didates is an important challenge for chemists and pharmacists.
In our previous attempts to develop synthetic antimalarial
compounds, we reported that rhodacyanine and phenoxazinium
derivativesexhibitstronginvitroactivitiesagainstP.falciparum.7–11
Phenoxazinium derivatives showed good efficacies by oral
administration.11 However, the development of 3,7-bis(dialky-
lamino)phenoxazinium derivatives as medicines is difficult
because these compounds typically are purified with the aid of
zinc chloride.12 Therefore, the reactions of m-aminophenols and
p-nitrosoanilines in perchloric acid gave phenoxaziniums in very
poor yields (2-40%).11 Recently, we found that symmetric and
asymmetric 3,7-bis(dialkylamino)phenoxazinium derivatives
with high purity could be obtained by chromatography followed
Results and Discussion
Chemistry. The synthesis of intermediates of 3,7-bis(di-
alkylamino)phenoxaziniums is illustrated in Scheme 1.
3-(Pyrrolidin-1-yl)phenol 3b and 3-(piperidin-1-yl)phenol 3c
were obtained by cleavage of the corresponding ethers 2b,c,14
which can be prepared easily by the palladium-catalyzed
reaction15 or by the reaction of 3-methoxyaniline 4 with
dibromoalkane.16 N,N-Dipropylaminophenol 3a could be
prepared easily by N-alkylation of 3-aminophenol,17 and N,N-
dialkylaminophenols 3f-h were obtained in the same manner.
N,N-Dialkyl-3-methoxy-4-nitrosoanilines 4a-e were prepared
by the reaction of 2a-e with sodium nitrate in the presence
of hydrochloric acid. 3-Methoxy-N,N-dipropylaniline 2a and
4-(3-methoxyphenyl)morpholine 2d were obtained by the
reactions of the corresponding N,N-dialkylaminophenols and
methyl p-toluenesulfonate in the presence of sodium hydrox-
ide in acetonitrile.13
3,7-Bis(dialkylamino)phenoxazinium salts 5a-s were af-
forded by the reaction of N,N-dialkylaminophenols 3a-h and
N,N-dialkyl-3-methoxy-4-nitrosoanilines 4a-e in acidic solu-
tions (Scheme 2).13 5a-s were purified by chromatography with
a short silica gel column and eluted with a CHCl3/MeOH
gradient from 10:1 to 10:3 (v/v). After evaporation of the
solvents, the remaining crude products were crystallized by
treatment with ethyl alcohol and then with ethyl acetate to
remove impurities.
Biology. Antiprotozoal and cytotoxic activities of the 3,7-
bis(dialkylamino)phenoxazinium salts are shown in Table 1.
Comparison of the IC50 values of 5d, 5h, 5i, and 5j with the
IC50 values of the other compounds suggests that the presence
of a morpholino group in 5d, 5h, 5i, and 5j might increase
the polarity and hydrophilicity of those compounds, thereby
decreasing their activities. However, the introduction of a
morpholino group substantially reduced the toxicity, which
suggests that increased polarity and hydrophilicity result in
decreased toxicity. This phenomenon was indicated by the
high toxicity and low selectivity of 5p-s, in which long alkyl
chains led to increased lipophilicity of the compounds.
* To whom correspondence should be addressed. Phone and fax: +81-
03-5498-6391. E-mail: m-ihara@hoshi.ac.jp.
†
Hoshi University.
Soochow University.
Swiss Tropical Institute.
‡
§
10.1021/jm8003619 CCC: $40.75
2008 American Chemical Society
Published on Web 05/14/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3655
Scheme 1. Synthesis of N,N-Dialkylaminophenol and N,N-Dialkyl-3-methoxy-4-nitrosoaniline
Scheme 2. Synthesis of 3,7-Bis(dialkylamino)phenoxazinium Salts
Compounds with a short alkyl chain (e.g., methyl or ethyl)
had high selectivity, low toxicity, and high activity. However,
when the number of carbon atoms exceeded three (propyl or
longer chains), the selectivity decreased and the toxicity
increased dramatically. Taking into consideration activity and
toxicity, the compounds with a short alkyl chain (methyl and
ethyl) are good antiprotozoal candidates, whereas the intro-
duction of long alkyl chains or morpholino groups is not
recommended. Symmetrical and asymmetrical structures did
not have different activities, but a bulky wing structure
affected toxicity and efficacy. We hypothesize that the active
center of these drugs might be the central tricyclic moiety
and the flattened structure is essential. Thus, the introduc-
tion of bulky groups might increase toxicity or decrease
activity.
Most 3,7-bis(dialkylamino)phenoxaziniums, with the excep-
tions of 5d, 5h, 5i, and 5p-s, showed good activities (in the
nanomolar region) against P. falciparum. Compounds 5j-l, 5n,
and 5o had good selective indices. Phenoxazinium derivatives
had particularly potent activity against T. cruzi. Nine compounds
including basic blue 3, 5a-c, 5f, 5g, and 5l-o showed good
activities against T. cruzi, with IC50 of 0.036-0.174 µM and
selectivity of 32-185. In the in vitro assays against T. brucei

3656 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Brief Articles
Table 1. Antiprotozoal and Cytotoxic Activities (IC50, µM) of 3,7-Bis(dialkylamino)phenoxazinium salts (5a-s)a
P. falc. K1
T. cruzi
T.b. rhod.
L. don., axenic
cytotox L6
IC50
compd
IC50
SIb
IC50
SIb
IC50
SIb
IC50
SIb
standardc
0.148
0.003
0.002
0.003
0.005
0.431
0.006
0.006
0.003
0.023
0.029
0.005
0.006
0.008
0.007
0.003
0.003
0.008
0.007
0.002
0.004
NTd
0.866
0.036
0.017
0.121
0.081
7.090
0.258
0.056
0.036
5.060
2.942
0.406
0.608
0.154
0.163
0.174
0.048
0.023
0.058
0.015
0.021
NTd
185.4
23.6
32.6
67.7
4.0
51.0
139.0
25.4
9.8
13.6
47.6
36.6
141.1
43.1
129.0
132.8
35.1
1.5
0.006
0.228
0.023
0.160
0.154
8.735
0.091
0.168
0.016
2.594
4.065
0.871
5.471
1.613
1.510
0.843
0.245
0.238
0.269
0.022
0.014
NTd
29.4
17.4
24.6
35.6
3.3
144.4
46.3
59.2
19.2
9.9
22.2
4.1
13.5
4.6
26.7
26.3
3.3
0.280
0.214
0.002
0.037
1.136
5.182
1.710
0.654
0.016
1.055
16.315
0.754
14.369
1.005
1.170
1.655
1.439
0.098
0.002
0.002
0.014
NTd
31.3
165.0
107.7
4.8
00.02
06.70
00.40
03.93
05.47
28.62
13.14
07.77
00.92
49.83
40.12
19.32
22.23
21.71
07.01
22.51
06.43
00.79
00.09
00.14
00.20
basic blue 3e
2410.0
165.0
1400.0
1050.0
66.5
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5.5
7.7
2166.5
1390.0
355.0
2153.9
1363.6
3883.0
4022.5
2822.0
976.7
7740.0
2390.0
105.3
12.3
11.9
59.2
47.2
2.5
25.6
1.5
21.6
6.0
13.6
4.5
8.1
0.3
6.3
13.7
37.0
63.0
13.7
63.0
48.0
9.0
9.6
a Values indicate the inhibitory concentration of a compound or standard in µM that is necessary to achieve 50% growth inhibition (IC50). Data shown
are values from two replicate experiments. b Selectivity index ) (IC50 for L6)/(IC50 for P. falciparum, T.b. rhodesiense, T. cruzi, or L. donoVani). c Standards:
chloroquine (P. falciparum), melarsoprol (T.b. rhodesiense), benznidazole (T. cruzi), miltefosine (L. donoVani), and podophyllotoxin (L6 cells, cytotoxicity).
d Not tested. e 3,7-Bis(diethylamino)phenoxazinium chloride, 99% purity.13
3,7-Di(piperidin-1-yl)phenoxazin-5-ium Chloride (5c). Yield
60%, mp >250 °C. IR ν (neat, cm-1): 2933, 2856, 1595, 1488,
1399, 1157. UV-vis (CHCl3), λ (nm) (log ε/L mol-1 cm-1): 659
rhodense and Leishmania donoVani, only 5b showed reasonable
activity against L. donoVani.
1
(4.99), 265 (4.44). H NMR (270 MHz, CD3OD) δppm: 1.82 (br,
Conclusion
12H), 3.87 (br, 8H), 7.05 (d, J ) 2.6 Hz, 2H), 7.47 (dd, J ) 9.6,
2.6 Hz, 2H), 7.68 (d, J ) 9.6 Hz, 2H). 13C NMR (68 MHz, CD3OD)
3,7-Bis(dialkylamino)phenoxazinium salts were synthesized
and evaluated for effectiveness against various parasitic protozoa
by in vitro assays. A number of derivatives showed substantial
activities against the chloroquine- and pyrimethamine-resistant
strain of P. falciparum as well as against T. cruzi.
δ
ppm: 25.2, 27.6, 50.9, 97.9, 118.6, 135.4, 135.7, 150.9, 157.9. MS
(ESI+), m/z: 348.1 [M - Cl- ]+. Anal. Calcd. for C22H26ClN3O·
2.25H2O: C, 62.25; H, 7.24; N, 9.90. Found: C, 62.08; H, 7.21; N,
9.83.
3-(Dimethylamino)-7-(pyrrolidin-1-yl)phenoxazin-5-ium Chlo-
ride (5e). Yield 70%, mp >250 °C. IR ν (neat, cm-1): 2975, 2873,
1603, 1489, 1397, 1151. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
Experimental Section
cm-1): 647 (5.02), 261 (4.49). 1H NMR (270 MHz, CD3OD) δppm
:
Chemistry. General Details. Starting materials were obtained
from Wako and Aldrich Company and used as received. Melting
points were determined on a Yanaco apparatus and are uncorrected.
NMR spectra were recorded on a Jeol-270, Bruker-400, or Vaian-
2.15-2.20 (m, 4H), 3.41 (s, 6H), 3.73-3.82 (m, 4H), 6.83 (d, J )
2.5 Hz, 1H), 6.94 (d, J ) 2.6 Hz, 1H), 7.29 (dd, J ) 9.5, 2.5 Hz,
1H), 7.37 (dd, J ) 9.6, 2.6 Hz, 1H), 7.79 (d, J ) 9.6 Hz, 1H), 7.80
1
(d, J ) 9.5 Hz, 1H).
13C NMR (68 MHz, CD3OD) δppm: 26.0, 26.3,
500 spectrometer. TMS was used as an internal standard for H
41.7, 51.0, 51.3, 97.5, 98.1, 118.1, 119.9, 134.7, 135.0, 135.4, 136.0,
NMR, and the solvent peak was used as an internal standard for
13C NMR. Absorption spectra were taken on Jasco V-550 UV-vis
spectrophotometer. IR spectra were determined on a JASCO FT/
IR-4100. LC-MS (ESI+) spectra were recorded with a Shimadzu
LCMS-2010EV spectroscope. The elemental analysis was per-
formed with Yanano CHN Corder MT-5 element analyzer.
+
150.1, 150.3, 156.7, 158.9. MS (ESI+
), m/z: 294.1 [M - Cl-
] .
Anal. Calcd for C18H20ClN3O·1.25H2O: C, 61.36; H, 6.44; N,
11.93. Found: C, 61.50; H, 6.26; N, 11.68.
3-(Dipropylamino)-7-(pyrrolidin-1-yl)phenoxazin-5-ium Chlo-
ride (5g). Yield 63%, mp >250 °C. IR ν (neat, cm-1): 2964, 2873,
1599, 1488, 1398, 1149. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
General Method of Preparation of 3,7-Bis(Dialkylamino)phe-
noxazin-5-iums 5a, 5c, 5e, 5g-s. A mixture of N,N-dialkylami-
nophenol (3a-g, 1 mmol) and 90% i-PrOH (20 mL) was stirred at
70 °C in a 50 mL two-neck bottle with distilling apparatus filled
with argon. A suspended solution of N,N-dialyl-3-methoxy-4-
nitrosoaniline (4a-e, 1 mmol) and acid (1 mmol) in 90% i-PrOH
(20 mL) was injected with syringe into the above mixture in four
portions during 45 min. The temperature rose to reflux. When about
20 mL of the solvent was distilled out, 20 mL of 90% i-PrOH was
added to the reaction mixture. This procedure was repeated three
times during 3-4 h. The dark-blue solution was evaporated, and
the residue was purified by column chromatography with silica gel,
eluting with CHCl3/MeOH from 10:1 to 10:3 (v/v). The dark-blue
solution was evaporated. To a solution of the residue in EtOH or
MeOH (2 mL) was added AcOEt (20 mL). After ultrasonication
for 10 min, the mixture was filtrated. The powder was washed by
AcOEt and Et2O and then dried in vacuum.
cm-1): 653 (5.10), 262 (4.52). 1H NMR (270 MHz, CD3OD) δppm
:
1.06 (t, J ) 7.3 Hz, 6H), 1.72-1.86 (m, 4H), 2.15-2.20 (m, 4H),
3.64-3.70 (m, 8H), 6.77 (br, 1H), 6.89 (d, J ) 2.2 Hz, 1H), 7.23
(dd, J ) 9.4, 1.4 Hz, 1H), 7.34 (dd, J ) 9.7, 2.5 Hz, 1H), 7.69-7.75
(m, 2H). 13C NMR (68 MHz, CD3OD) δppm: 11.4, 22.1, 26.0, 26.3,
51.0, 51.2, 55.0, 97.6, 98.0, 118.4, 119.7, 135.0, 135.2, 135.3, 136.0,
150.4, 150.5, 156.6, 157.9. MS (ESI+), m/z: 350.1 [M - Cl- ]+.
Anal. Calcd for C22H28ClN3O·2.25H2O: C, 61.96; H, 7.68; N, 9.85.
Found: C, 61.83; H, 7.45; N, 9.68.
3-(Dimethylamino)-7-morpholinophenoxazin-5-ium Chloride
(5h). Yield 40%, mp >250 °C. IR ν (neat, cm-1): 3032, 2869,
1603, 1490, 1400, 1159. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
cm-1): 648 (4.92), 263 (4.39). 1H NMR (270 MHz, CD3OD) δppm
:
3.44 (s, 6H), 3.83-3.90 (m, 8H), 6.91 (d, J ) 2.6 Hz, 1H), 7.09
(d, J ) 2.6 Hz, 1H), 7.41 (dd, J ) 9.6, 2.6 Hz, 1H), 7.48 (dd, J )
9.6, 2.6 Hz, 1H), 7.70-7.76 (m, 2H). 13C NMR (68 MHz, CD3OD)

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3657
308.1 [M - Cl- +. Anal. Calcd for C19H22ClN3O·1.5H2O: C,
δ
ppm: 42.1, 49.2, 67.5, 97.7, 98.2, 118.0, 119.5, 135.0, 135.2, 135.4,
]
136.8, 150.47, 150.5, 158.4, 159.7. MS (ESI+), m/z: 310.1 [M -
Cl- ]+. Anal. Calcd for C18H20ClN3O2 ·2.75H2O: C, 54.68; H, 6.50;
N, 10.63. Found: C, 54.62; H, 6.37; N, 10.48.
61.53; H, 6.79; N, 11.33. Found: C, 61.85; H, 6.61; N, 11.14.
3-(Diethylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5o). Yield 28%, mp >250 °C. IR ν (neat, cm-1): 2936, 2862,
1596, 1490, 1401, 1155. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
3-(Diethylamino)-7-morpholinophenoxazin-5-ium Chloride
(5i). Yield 58%, mp >250 °C. IR ν (neat, cm-1): 2977 (alkyl-
CH), 1597, 1488, 1400, 1153. UV-vis (CHCl3), λ (nm) (log ε/L
mol-1 cm-1): 651 (5.06), 263 (4.51). 1H NMR (270 MHz, CD3OD)
cm-1): 655 (5.05), 263 (4.51). 1H NMR (270 MHz, CD3OD) δppm
:
1.36 (t, J ) 7.2 Hz, 6H), 1.83 (br, 6H), 3.77 (q, J ) 7.0, 4H), 3.88
(br, 4H), 6.92 (d, J ) 2.7 Hz, 1H), 7.08 (d, J ) 2.6 Hz, 1H), 7.36
(dd, J ) 9.7, 2.7 Hz, 1H), 7.49 (dd, J ) 9.7, 2.7 Hz, 1H), 7.71
(d, J ) 9.7 Hz, 1H), 7.73 (d, J ) 9.6 Hz, 1H). 13C NMR (68 MHz,
DMSO-d6) δppm: 13.1, 25.2, 27.6, 47.7, 50.9, 97.4, 97.9, 118.5,
118.7, 135.46, 135.5, 135.6, 150.7, 151.0, 157.6, 158.0. MS (ESI+),
m/z: 336.1 [M - Cl- ]+. Anal. Calcd for C21H26ClN3O·H2O: C,
64.69; H, 7.24; N, 10.78. Found: C, 64.94; H, 7.34; N, 10.76.
3-(Dipropylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5p). Yield 51%, mp 154-155 °C. IR ν (neat, cm-1): 2936,
2873, 1595, 1490, 1400, 1153. UV-vis (CHCl3), λ (nm) (log ε/L
mol-1 cm-1): 657 (5.00), 264 (4.48). 1H NMR (270 MHz, CD3OD)
δppm: 1.38 (t, J ) 7.2 Hz, 6H), 3.78-3.86 (m, 12H), 7.00 (d, J )
2.6 Hz, 1H), 7.12 (d, J ) 2.6 Hz, 1H), 7.44-7.51 (m, 2H), 7.78
(d, J ) 9.5 Hz, 1H), 7.79 (d, J ) 9.5 Hz, 1H). 13C NMR (68 MHz,
CD3OD) δppm: 12.9, 46.4, 47.7, 65.9, 96.2, 96.9, 117.0, 118.5, 133.1,
133.6, 134.3, 135.2, 148.6, 149.1, 156.1, 156.3. MS (ESI+), m/z:
338.1 [M - Cl- ]+. Anal. Calcd for C20H24ClN3O2 ·1.25H 2O: C,
60.60; H, 6.74; N, 10.60. Found: C, 60.33; H, 6.63; N, 10.49.
3-(Dipropylamino)-7-morpholinophenoxazin-5-ium Chloride
(5j). Yield 35%, mp 133-135 °C. IR ν (neat, cm-1): 2964, 2873,
1595, 1490, 1400, 1151. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
cm-1): 654 (5.01), 264 (4.47). 1H NMR (270 MHz, CD3OD) δppm
:
δppm: 1.05 (t, J ) 7.4 Hz, 6H), 1.72-1.86 (m, 10H), 3.65-3.71
(m, 4H), 3.89 (br, 4H), 6.91 (d, J ) 2.6 Hz, 1H), 7.11 (d, J ) 2.6
Hz, 1H), 7.37 (dd, J ) 9.7, 2.6 Hz, 1H), 7.52 (dd, J ) 9.7, 2.6 Hz,
1H), 7.76 (br, 1H), 7.79 (br, 1H). 13C NMR (68 MHz, CD3OD)
1.06 (t, J ) 7.3 Hz, 6H), 1.74-1.88 (m, 4H), 3.73 (t, J ) 7.7
Hz, 4H), 1.86 (br, 8H), 6.97 (d, J ) 2.6 Hz, 1H), 7.12 (d, J ) 2.5
Hz, 1H), 7.46 (dd, J ) 9.7, 2.6 Hz, 1H), 7.49 (dd, J ) 9.7, 2.6 Hz,
1H), 7.82 (br, 2H). 13C NMR (126 MHz, DMSO-d6) δppm: 10.8,
20.7, 47.7, 53.3, 65.9, 96.4, 96.8, 117.0, 118.7, 133.2, 133.6, 134.2,
135.1, 148.6, 149.0, 156.3, 156.6. MS (ESI+), m/z: 366.1 [M -
Cl- ]+. Anal. Calcd for C22H28ClN3O2 ·1.25H2O: C, 62.25; H, 7.24;
N, 9.90. Found: C, 62.09; H, 7.41; N, 9.64.
δ
ppm: 11.4, 22.1, 25.2, 27.6, 50.9, 55.0, 97.6, 97.9, 118.6, 118.7,
135.3, 135.5, 135.8, 150.7, 151.0, 158.0, 158.1. MS (ESI+), m/z:
364.1 [M - Cl- ]+. Anal. Calcd for C23H30ClN3O·2H2O: C, 63.36;
H, 7.86; N, 9.64. Found: C, 63.39; H, 7.62; N, 9.42.
3-(Dibutylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5q). Yield 50%, mp 60-61 °C. IR ν (neat, cm-1): 2931,
2858, 1595, 1490, 1400, 1153. UV-vis (CHCl3), λ (nm) (log ε/L
mol-1 cm-1): 659 (5.03), 264 (4.48). 1H NMR (270 MHz, CD3OD)
3-(Dimethylamino)-7-thiomorpholinophenoxazin-5-ium Chlo-
ride (5k). Yield 44%, mp >250 °C. IR ν (neat, cm-1): 3031, 1598,
1490, 1397, 1148. UV-vis (CHCl3), λ (nm) (log ε/L mol-1 cm-1):
δ
ppm: 1.03 (t, J ) 7.3 Hz, 6H), 1.42-1.55 (m, 4H), 1.69-1.83
651 (4.94), 239 (4.44). 1H NMR (400 MHz, CD3OD) δppm
:
(m, 10H), 3.68-3.74 (m, 4H), 3.89 (br, 4H), 6.87 (d, J ) 2.3 Hz,
1H), 7.11 (d, J ) 2.1 Hz, 1H), 7.34 (dd, J ) 9.6, 2.6 Hz, 1H), 7.51
(dd, J ) 9.6, 2.6 Hz, 1H), 7.73 (br, 1H), 7.76 (br, 1H). 13C NMR
(68 MHz, CD3OD) δppm: 14.2, 21.1, 25.2, 27.6, 30.9, 50.9, 53.3,
97.5, 97.9, 118.6, 118.7, 135.3, 135.5, 135.6, 135.8, 150.7, 151.1,
157.9, 158.1. MS (ESI+), m/z: 392.2 [M - Cl- ]+. Anal. Calcd
for C25H34ClN3O·0.75H 2O: C, 68.01; H, 8.10; N, 9.52. Found: C,
67.99; H, 8.28; N, 9.36.
2.80-2.82 (m, 4H), 3.36 (s, 6H), 4.43 (br, 4H), 6.95 (br, 1H), 7.22
(br, 1H), 7.47-7.59 (m, 2H), 7.81 (br, 2H). 13C NMR (126 MHz,
DMSO-d6) δppm: 26.9, 41.6, 51.1, 96.4, 97.3, 117.3, 118.6, 133.2,
133.8, 133.9, 135.2, 148.7, 148.9, 155.7, 157.6. MS (ESI+), m/z:
326.0 [M - Cl- ]+. Anal. Calcd for C18H20ClN3OS·3H O: C,
2
51.98; H, 6.30; N, 10.10. Found: C, 51.70; H, 6.12; N, 9.97.
3-(Diethylamino)-7-thiomorpholinophenoxazin-5-ium Chlo-
ride (5l). Yield 68%, mp 145-147 °C. IR ν (neat, cm-1): 2975,
1594, 1489, 1399, 1151. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
3-(Dipentylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5r). Yield 54%, mp 83-84 °C. IR ν (neat, cm-1): 2933, 2859,
1595, 1490, 1399, 1153. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
cm-1): 654 (5.06), 239 (4.59). 1H NMR (400 MHz, CD3OD) δppm
:
cm-1): 659 (5.06), 264 (4.51). 1H NMR (270 MHz, CD3OD) δppm
:
1.27 (t, J ) 7.1 Hz, 6H), 2.80-2.81 (m, 4H), 3.78-3.84 (m, 4H),
4.19-4.20 (m, 4H), 6.98 (d, J ) 2.3 Hz, 1H), 7.20 (d, J ) 2.3 Hz,
1H), 7.51 (dd, J ) 9.7, 2.4 Hz, 1H), 7.58 (dd, J ) 9.6, 2.5 Hz,
0.94-1.00 (m, 6H), 1.40-1.47 (m, 8H), 1.71-1.83 (m, 10H),
3.67-3.73 (m, 4H), 3.89 (br, 4H), 6.87 (d, J ) 2.6 Hz, 1H), 7.11
(d, J ) 2.5 Hz, 1H), 7.34 (dd, J ) 9.6, 2.5 Hz, 1H), 7.51 (dd, J )
9.6, 2.6 Hz, 1H), 7.74 (d, J ) 9.6 Hz, 1H), 7.75 (d, J ) 9.5 Hz,
1H). 13C NMR (68 MHz, CD3OD) δppm: 14.4, 23.6, 25.2, 27.6,
28.5, 30.0, 50.9, 53.5, 97.5, 98.0, 118.6, 118.7, 135.4, 135.5, 135.6,
135.8, 150.7, 151.1, 157.9, 158.1. MS (ESI+), m/z: 420.2 [M -
Cl- ]+. Anal. Calcd for C27H38ClN3O·1.75H 2O: C, 66.51; H, 8.58;
N, 8.62. Found: C, 66.23; H, 8.50; N, 8.55.
1H), 7.80-7.82 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δppm
:
12.8, 26.9, 46.4, 51.1, 96.1, 97.3, 117.2, 118.5, 133.1, 133.8, 134.3,
135.2, 148.8, 149.1, 155.6, 156.1. MS (ESI+), m/z: 354.0 [M -
Cl- ]+. Anal. Calcd for C20H24ClN3OS·H2O: C, 58.88; H, 6.42;
N, 10.30. Found: C, 58.62; H, 6.67; N, 10.15.
3-(Dipropylamino)-7-thiomorpholinophenoxazin-5-ium Chlo-
ride (5m). Yield 55%, mp 126-127 °C. IR ν (neat, cm-1): 2964,
2873, 1593, 1490, 1399, 1150. UV-vis (CHCl3), λ (nm) (log ε/L
mol-1 cm-1): 658 (4.98), 264 (4.44). 1H NMR (400 MHz, CD3OD)
3-(Dihexylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5s). Yield 51%, mp 90-91 °C. IR ν (neat, cm-1): 2954, 2869,
1595, 1490, 1399, 1152. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
δppm: 1.06 (t, J ) 7.4 Hz, 6H), 1.77-1.86 (m, 4H), 2.83-2.87 (m,
cm-1): 658 (4.84), 264 (4.29). 1H NMR (270 MHz, CD3OD) δppm
:
4H), 3.70-3.78 (m, 4H), 4.18-4.22 (m, 4H), 6.96 (d, J ) 2.6
Hz, 1H), 7.12 (d, J ) 2.6 Hz, 1H), 7.43-7.51 (m, 2H), 7.78 (br,
1H), 7.81 (br, 1H). 13C NMR (126 MHz, DMSO-d6) δppm: 10.8,
20.5, 26.8, 51.1, 53.3, 96.3, 97.3, 117.2, 118.7, 133.2, 133.8, 134.2,
135.2, 148.8, 149.0, 155.6, 156.6. MS (ESI+), m/z: 382.1 [M -
0.94-0.97 (m, 6H), 1.38-1.49 (m, 12H), 1.70-1.83 (m, 10H),
3.67-3.73 (m, 4H), 3.89 (br, 4H), 6.87 (d, J ) 2.5 Hz, 1H), 7.11
(d, J ) 2.6 Hz, 1H), 7.34 (dd, J ) 9.6, 2.5 Hz, 1H), 7.51 (dd, J )
9.7, 2.6 Hz, 1H), 7.74 (d, J ) 9.7 Hz, 1H), 7.75 (d, J ) 9.6 Hz,
1H). 13C NMR (68 MHz, CD3OD) δppm: 14.4, 23.7, 25.2, 27.5,
27.6, 28.8, 32.8, 50.9, 53.5, 97.5, 98.0, 118.6, 118.7, 135.4, 135.5,
135.6, 135.8, 150.7, 151.1, 157.9, 158.1. MS (ESI+), m/z: 448.3
Cl-
]
+. Anal. Calcd for C22H28ClN3OS·2.25H2O: C, 57.63; H,
7.14; N, 9.16. Found: C, 57.59; H, 7.06; N, 9.01.
3-(Dimethylamino)-7-(piperidin-1-yl)phenoxazin-5-ium Chlo-
ride (5n). Yield 27%, mp >250 °C. IR ν (neat, cm-1): 2940, 2862,
1600, 1490, 1397, 1153. UV-vis (CHCl3), λ (nm) (log ε/L mol-1
[M - Cl- ]+. Anal. Calcd for C29H42ClN3O·2.75H O: C, 65.27;
2
H, 8.97; N, 7.87. Found: C, 65.05; H, 8.88; N, 7.90.
cm-1): 652 (5.04), 263 (4.50). 1H NMR (270 MHz, CD3OD) δppm
:
Antiprotozoal Activity. General Procedure for Preparing
the Stock Drug Solutions. Stock drug solutions were prepared in
100% DMSO at 10 mg/mL and heated or sonicated if necessary to
dissolve the sample. For the assay, the compound was further diluted
in serum-free culture medium and finally to the appropriate
concentration in complete medium without hypoxanthine. The
1.83 (br, 6H), 3.41 (s, 6H), 3.90 (br, 4H), 6.93 (d, J ) 2.5 Hz,
1H), 7.13 (d, J ) 2.3 Hz, 1H), 7.37 (dd, J ) 9.5, 2.6 Hz, 1H), 7.53
(dd, J ) 9.7, 2.6 Hz, 1H), 7.77 (br, 2H). 13C NMR (68 MHz,
CD3OD) δppm: 25.1, 27.7, 41.7, 51.0, 97.5, 97.9, 118.3, 118.9, 135.0,
135.2, 135.5, 135.8, 150.2, 151.0, 158.0, 159.0. MS (ESI+), m/z:

3658 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Brief Articles
(6) Croft, S. L. The Current Status of Antiparasite Chemotherapy.
Parasitology 1997, 114, S3–S15.
(7) Takasu, K.; Inoue, H.; Kim, H.-S.; Suzuki, M.; Shishido, T.; Wataya,
Y.; Ihara, M. Rhodacyanine Dyes as Antimalarials. 1. Preliminary
Evaluation of Their Activity and Toxicity. J. Med. Chem. 2002, 45,
995–998.
(8) Takasu, K.; Shimogama, T.; Saiin, C.; Kim, H.-S.; Wataya, Y.; Brun,
R.; Ihara, M. Synthesis and Evaluation of ꢀ-Carbolinium Cations as
New Antimalarial Agents Based on π-Delocalized Lipophilic Cation
(DLC) Hypothesis. Chem. Pharm. Bull. 2005, 53, 653–661.
(9) Takasu, K.; Pudhom, K.; Kaiser, M.; Brun, R.; Ihara, M. Synthesis
and Antimalarial Efficacy of Aza-Fused Rhodacyanines in Vitro and
in the P. berghei Mouse Model. J. Med. Chem. 2006, 49, 4795–4798.
(10) Pudhom, K.; Kasai, K.; Terauchi, H.; Inoue, H.; Kaiser, M.; Brun,
R.; Ihara, M.; Takasu, K. Synthesis of Three Classes of Rhodacyanine
Dyes and Evaluation of Their in Vitro and in Vivo Antimalarial
Activity. Bioorg. Med. Chem. 2006, 14, 8550–8563.
(11) Takasu, K.; Shimogama, T.; Satoh, C.; Kaiser, M.; Brun, R.; Ihara,
M. Synthesis and Antimalarial Property of Orally Active Phenox-
azinium Salts. J. Med. Chem. 2007, 50, 2281–2284.
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Chem. 1969, 6, 755–758.
(13) Ge, J.; Arai, C.; Ihara, M. The Convenient Synthesis of Zinc Chloride-
Free 3,7-Bis(dialkylamino)phenoxazinium Salts. Dyes Pigm. 2008, 79,
33–39.
(14) Gompel, J. V.; Schuster, G. B. Chemiluminescence of Organic
Peroxides: Intramolecular Electron-Exchange Luminescence from a
Secondary Perester. J. Org. Chem. 1987, 52, 1465–1468.
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DMSO concentration in the wells with the highest drug concentra-
tion did not exceed 1%.
In Vitro Assay. P. falciparum K1 (resistant to chloroquine and
pyrimethamine), T. cruzi (Tulahuen C2C4), L. donoVani (MHOM-
ET-67/L82), and T. b. rhodesiense (STIB900) were used for the in
vitro assay. The cytotoxicity was assessed with rat skeletal
myoblasts (L6 cells). IC50 values were obtained by the earlier
described methods.18 The following compounds were used as
reference standards: chloroquine (P. falciparum), melarsoprol (T.b.
rhodesiense), benznidazole (T. cruzi), miltefosine (L. donoVani),
and podophyllotoxin (L6 cells, cytotoxicity).
Acknowledgment. This work was financially supported by
a creation and support program for start-ups from University,
Japan Science Technology Agency.
Supporting Information Available: Additional experimental
details and combustion data for new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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