Synthesis and antimalarial effects of phenothiazine inhibitors of a plasmodium falciparum cysteine protease

Article abstract of DOI:10.1021/jm970266p

Acridinediones have previously been shown to have potent antimalarial activity. A series of sulfur isosteres of acridinediones have been synthesized and evaluated for their inhibition of the Plasmodium falciparum cysteine protease falcipain and for their antimalarial activity. A number of these phenothiazines inhibited falcipain and demonstrated activity against cultured P. falciparum parasites at low micromolar concentrations. We propose that the compounds exerted their antimalarial effects by two mechanisms, one of which involves the inhibition of falcipain and a consequent block in parasite degradation of hemoglobin. Those compounds and related phenothiazines are worthy of further study as potential antimalarial agents.

Full text of DOI:10.1021/jm970266p

2726
J . Med. Chem. 1997, 40, 2726-2732
Syn th esis a n d An tim a la r ia l Effects of P h en oth ia zin e In h ibitor s of a
P la sm od iu m fa lcipa r u m Cystein e P r otea se
J ose´ N. Dom´ınguez,† Simo´n Lo´pez,† J aime Charris,† Lu´cido Iarruso,† Gricela Lobo,† Andrey Semenov,‡
J ed E. Olson,‡ and Philip J . Rosenthal*,‡
Laboratorio de S´ıntesis Orga´nica, Facultad de Farmacia, Universidad Central de Venezuela, Caracas 1051, Venezuela, and
Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143-0811
Received April 22, 1997X
Acridinediones have previously been shown to have potent antimalarial activity. A series of
sulfur isosteres of acridinediones have been synthesized and evaluated for their inhibition of
the Plasmodium falciparum cysteine protease falcipain and for their antimalarial activity. A
number of these phenothiazines inhibited falcipain and demonstrated activity against cultured
P. falciparum parasites at low micromolar concentrations. We propose that the compounds
exerted their antimalarial effects by two mechanisms, one of which involves the inhibition of
falcipain and a consequent block in parasite degradation of hemoglobin. These compounds
and related phenothiazines are worthy of further study as potential antimalarial agents.
In tr od u ction
pounds had strong activity against murine malaria
parasites in vivo11-17 and a smaller number demon-
strated activity against the human parasite P. falci-
parum in culture16 and in primate models.16-18 In an
effort to develop improved heterocyclic antimalarials
potentially acting against falcipain, we have synthesized
sulfur isosteres of acridinediones19 including phenothi-
azin-4-one (5) and phenothiazin-4-one 5,5-dioxide (6)
derivatives. In this report we describe the synthesis of
5 and 6 and their derivatives and the effectiveness of
these compounds in inhibiting falcipain activity and in
blocking the metabolism and development of cultured
malaria parasites.
Malaria remains one of the most important infectious
disease problems in the world. Hundreds of millions of
cases of malaria occur annually, and Plasmodium
falciparum, the most serious human malaria parasite,
causes over one million deaths per year.1 A significant
and increasing problem in malaria control is the resis-
tance of malaria parasites to available chemotherapeutic
agents.2 There is, therefore, a pressing need to identify
new antimalarial drugs. Ideally, new drugs will act
against targets not previously exploited in antimalarial
drug development.
Potential targets for chemotherapy include falcipain,
a P. falciparum papain family cysteine protease that is
a hemoglobinase required for normal parasite develop-
ment.3,4 Malaria parasites degrade hemoglobin in an
acidic food vacuole to provide free amino acids for
parasite protein synthesis.5 When cultured parasites
are treated with cysteine protease inhibitors directed
against falcipain, hemoglobin degradation is blocked,
parasite food vacuoles fill with undegraded hemoglobin,
and parasite development is halted.6
Inhibitors of falcipain have been studied as potential
antimalarial agents. Selected peptidyl fluoromethyl
ketones inhibited falcipain and blocked hemoglobin
degradation and development by cultured parasites at
nanomolar concentrations.6 One of these compounds
was also effective in vivo against murine malaria.7
Peptidyl vinyl sulfone inhibitors of falcipain have also
demonstrated potent antimalarial effects.8
Ch em istr y
Derivative 5 was easily synthesized by a one-pot
reaction involving condensation and oxidative cycliza-
tion, with quite good yields.20 The preparation of
2-amino-5-halobenzenethiol derivatives21 as key inter-
mediates allowed us to access fluorinated compounds
at position C-7 for structures 5 and 6 (Scheme 1). The
intermediate 1,3-cyclohexanedione derivatives (4) could
readily be prepared in large quantities.22 These struc-
tures allowed the incorporation of phenyl groups sub-
stituted at different positions. Treatment of 2-amino-
5-halobenzenethiol derivatives (3) with 1,3-cyclohexane-
dione derivatives (4) in refluxing dimethyl sulfoxide led
to formation of 5, which was monitored by TLC. The
cyclization reached completion in 45 min. The products
were easier to characterize after the solids were recrys-
tallized and their structures confirmed by spectral and
analytical data, as the phenothiazin-4-ones (5a -p ) could
then be stored under nitrogen atmosphere indefinitely
Nonpeptide inhibitors of falcipain have been devel-
oped through a molecular modeling approach.9
A
number of heterocyclic compounds, including a series
of chalcones (1), have been synthesized and shown to
inhibit falcipain and block the development of cultured
parasites.10 In independent studies, floxacrine (2) and
several series of related acridinediones have been evalu-
ated as potential antimalarials. Many of these com-
* To whom correspondence should be addressed at Box 0811,
University of California, San Francisco, CA 94143-0811. Phone: (415)
206-8845. FAX: (415) 206-6015. E-mail: rosnthl@itsa.ucsf.edu.
† Universidad Central de Venezuela.
‡ University of California.
X Abstract published in Advance ACS Abstracts, J uly 15, 1997.
S0022-2623(97)00266-5 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of a P. falciparum Cysteine Protease
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2727
Sch em e 1a
completion of a cycle of develoment. By each assay,
most of the phenothiazines studied inhibited parasite
development at low micromolar concentrations (Table
2). Two compounds, 6a and 6k , which lacked a phenyl
ring at C-2, were much less effective. Cellular toxicity
of selected compounds was determined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay23 and cultured Madin Darby canine kidney
(MDCK) cells. One compound, 5h , showed cellular
toxicity, with an IC50 for toxicity of 10 µM. None of the
other compounds tested showed measurable toxicity at
concentrations up to 100 µM (Table 2).
a
(a) DMSO, reflux, 45 min; (b) 30% H2O2, AcOH, 2.5 h; R, R1,
and R2 are as in Table 1.
or used directly for the final stage of the synthetic
sequence shown in the scheme.
We next sought to determine whether the antima-
larial effects of the phenothiazines were due to the
inhibition of falcipain. In our experience with multiple
other classes of inhibitors, the cellular inhibition of
falcipain is accompanied by the appearance of an
abnormal parasite food vacuole that fills with unde-
graded hemoglobin.3,6,8 Inhibited parasites subse-
quently halt their development, and daughter ring-stage
parasites are not formed. Parasites were incubated
with the phenothiazines for 24 h beginning at the ring
stage and then evaluated microscopically. Typical swol-
len, hemoglobin-containing food vacuoles were identified
after inhibition of parasites with 100 µM concentrations
of most of the phenothiazines studied, indicating that
these compounds had blocked hemoglobin degradation
(Table 3, Figure 1). A block in hemoglobin degradation
was confirmed by SDS-PAGE; proteins isolated from
parasites incubated with 6a and 6k included much more
undegraded globin than did proteins from control para-
sites (Figure 2). In addition to the accumulation of
The analogs 6a -k were easily obtained from com-
pound 5 and its derivatives by treatment with 30%
hydrogen peroxide in glacial acetic acid under reflux for
2.5 h. Aqueous workup resulted in material with, based
on all available evidence, sulfone derivatives (6) as the
sole product. Physicochemical data for these compounds
are displayed in Table 1.
Biologica l Resu lts
Ten of the phenothiazines were fairly potent inhibi-
tors of falcipain. Each of these compounds inhibited the
hydrolysis of the substrate benzyloxycarbonyl-Phe-Arg-
7-amino-4-methylcoumarin (Z-Phe-Arg-AMC) with an
IC50 of 4-5 µM (Table 1). The ability of these com-
pounds to inhibit the development of cultured P. fal-
ciparum parasites was then assessed. Development in
the presence of the phenothiazines was compared with
that in control cultures by measuring the uptake of [3H]-
hypoxanthine and by counting parasites after the
Ta ble 1. Physicochemical and Biochemical Data for Phenothiazine Derivatives
solventa
IC50
b
compd
R
R1
R2
X
mp (°C)
5a
5b
5c
5d
5e
5f
5g
5h
5i
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
H
CH3
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
CH3
H
CH3
C6H5
3-CH3OC6H4
4-CH3OC6H4
2,3-(CH3O)2C6H3
3,4-(CH3O)2C6H3
4-ClC6H4
2,4-Cl2C6H3
CH3
C6H5
3-CH3OC6H4
2,3-(CH3O)2C6H3
2,4-Cl2C6H3
4-ClC6H4
3,4-(CH3O)2C6H3
CH3
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
217-220
272
260-263
265-268
235
270-273
230
228-230
261-264
282-283
250
A
A
B
B
B
B
B
A
B
A
B
B
B
B
A
C
C
C
C
C
B
B
C
B
C
B
C
40
30
10
20
10
10
30
4
10
60
20
20
20
10
5
20
5
4
30
4
50
4
5j
5k
5l
263
5m
5n
5o
5p
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
280-281
240-242
178-180
250-253
340-342
202
C6H5
4-CH3OC6H4
2,3-(CH3O)2C6H3
3,4-(CH3O)2C6H3
4-ClC6H4
2,4-Cl2C6H3
3-CH3OC6H4
3,4-(CH3O)2C6H3
4-ClC6H4
200
144-146
330-331
238
180
296-297
344
30
5
5
5
5
F
F
F
198
220
CH3
a
b
Recrystallization solvents: A, EtOH; B, DMF; C, DMF/H2O. IC50 for inhibition of falcipain activity, measured as the hydrolysis of
Z-Phe-Arg-AMC.

2728 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Dom´ınguez et al.
Ta ble 2. Inhibition of Biological Activities by Phenothiazine
Derivatives
IC50 (µM)a
parasite
parasite
MDCK cell
compd
metabolismb
developmentc
metabolismd
5h
5o
6a
6b
6d
6f
6
4
60
5
2
8
3
3
20
5
2
4
10
>100
>100
NDe
>100
ND
6h
6i
3
3
3
1
>100
ND
6j
4
6
ND
6k
100
30
>100
a
Extrapolated from curves of percent activity vs concentration
for each compound. Uptake of [3H]hypoxanthine by cultured
b
parasites. c Ring-form parasitemia, indicating completion of a
developmental cycle, counted from Giemsa-stained smears after
a 48 h incubation. Uptake of MTT by cultured MDCK cells. e Not
d
done.
Ta ble 3. Abnormal Parasite Morphology Caused by
Phenothiazine Derivatives
F igu r e 1. Morphological effects of phenothiazines. Cultured
P. falciparum parasites were incubated with 1% DMSO (A)
or the phenothiazines 6a (100 µM; B), 6d (100 µM; C), or 6k
(100 µM; D) for 24 h, beginning at the late ring stage. The
parasites were then evaluated on Giemsa-stained smears.
Control cultures (A) contained healthy late trophozoite and
schizont-stage parasites. Cultures incubated with the three
phenothiazines each contained abnormal forms with swollen,
dark-staining food vacuoles indicative of a block in hemoglobin
degradation.
parasite morphologya
food vacuole
cytoplasmic
compd
hemoglobin accumulationb
vacuolizationc
5h
5o
6a
6b
6d
6f
6h
6i
6j
0
+
+
-
+
+
+
+
+
+
-
2+
3+
2+
3+
1+
2+
1+
1+
3+
6k
a
The morphology of trophozoites on Giemsa-stained smears was
b
assessed after
a 24h incubation with the compound. After
incubation with 100 µM compound, parasites were scored based
on the percentage of organisms containing dark, swollen food
vacuoles indicative of a block in hemoglobin degradation as
follows: 0 (<10%); 1+ (10-50%); 2+ (50-90%); 3+ (>90%). c After
incubation with 10 µM compound, parasite cytoplasmic morphol-
ogy was compared to that of controls and scored as abnormal (+)
or normal (-).
hemoglobin in food vacuoles, most of the phenothiazines
studied also caused other morphologic abnormalities in
the cultured parasites. After incubation with 10 µM 5h ,
5o, 6b, 6d , 6f, 6h , 6i, and 6k , the parasites were smaller
than untreated controls, with apparent vacuolization of
parasite cytoplasm and limited development beyond the
trophozoite stage (Table 3). These abnormalities were
most pronounced with 5h , the compound that also was
toxic to MDCK cells (Table 2). Food vacuole accumula-
tion of hemoglobin was particularly pronounced with 6a
and 6k , the two compounds that were significantly less
effective in inhibiting parasite development, but these
compounds did not cause identifiable morphologic ab-
normalities independent of the changes in the food
vacuole noted. The effects of compounds 6a and 6k
therefore appeared to be similar to those of leupeptin,
E-64, and other peptide cysteine protease inhibitors,
which appear to exert antimalarial effects through a
specific inhibition of falcipain.
F igu r e 2. Inhibition of hemoglobin degradation by phenothi-
azines. Cultured P. falciparum parasites were incubated with
1% DMSO (lane A) as a control or with 100 µM 6a (B) or 6k
(C) for 24 h beginning at the ring stage. Trophozoite-infected
erythrocytes were then collected, lysed with saponin, and
washed extensively, and parasite proteins were electropho-
resed in 15% SDS-polyacrylamide gels and stained with Coo-
massie blue. The contents of about 107 parasites were included
in each lane. Parasites incubated with the phenothiazines
accumulated much more undegraded globin (arrow) than did
controls incubated with the DMSO solvent. The locations of
molecular weight standards are indicated in kilodaltons.
not appear to be as potent as the most effective
acridinediones. A direct comparison of results with the
two classes of compounds is difficult, however, as the
acridinediones have mostly been studied against murine
malaria in vivo, and our studies of phenothiazines were
against the human malaria parasite P. falciparum in
vitro. In any event, the antimalarial effects of the small
group of phenothiazines studied to date suggest that
additional evaluation of this class is warranted.
Discu ssion
We have shown that phenothiazine isosteres of previ-
ously evaluated acridinediones are effective antimalarial
agents, although the phenothiazines studied to date do

Inhibitors of a P. falciparum Cysteine Protease
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2729
Of greatest biological interest, the phenothiazines
studied inhibited the malarial cysteine protease falci-
pain and blocked the hydrolysis of hemoglobin, the
natural substrate of falcipain. This inhibition may have
been due to the binding of the phenothiazines to the
falcipain active site in a manner analogous to that
predicted for chalcones and other heterocyclic com-
pounds that inhibit falcipain at similar concentrations.10
Of particular interest, compounds 6a and 6k were
considerably less potent antimalarials than compounds
containing a C-2 phenyl group, but they inhibited
falcipain activity and parasite hemoglobin degradation
at concentrations similar to those at which the other
phenothiazines inhibited these processes.
resembling previously evaluated acridinediones may be
of interest as antimalarial drugs for two reasons. First,
the phenothiazines share the antimalarial efficacy of the
acridinediones, and this class of compounds may provide
agents with improved solubility and toxicity profiles.
Second, the phenothiazines inhibited the hemoglobinase
falcipain and blocked parasite development through the
inhibition of hemoglobin degradation. While this latter
effect required quite high concentrations of the phe-
nothiazines, modifications of these compounds, particu-
larly with consideration of the predicted structure of
falcipain,9 should provide improved efficacy. Thus,
further evaluation of phenothiazines related to the
acridinedione antimalarials is likely to identify com-
pounds with improved antimalarial efficacy.
We propose that the phenothiazines we evaluated eli-
cit antimalarial effects through two independent mech-
anisms. The first mechanism is probably the same as
that of the acridinediones that have been previously
identified as promising antimalarial drugs. For both
acridinediones12,15,17 and phenothiazines (compounds 6a
and 6k ), compounds lacking a phenyl ring at C-2 had
decreased antimalarial efficacy. The mechanism of
action of the acridinediones is unknown, but a number
of the compounds have demonstrated efficacy in murine
models of malaria and selected compounds cured P.
falciparum-infected primates at doses of 20 mg/kg or
less.16,17 However, limited solubility and significant tox-
icity of floxacrine and perhaps other acridinediones are
concerns. Phenothiazines should offer improved solubil-
ity, and the extensive use of other phenothiazines in
clinical medicine suggests that toxicity may not be a
problem with these compounds. The optimal phenothi-
azine in our studies, 6d , may thus be considered a lead
compound for the development of phenothiazine anti-
malarials.
Exp er im en ta l Section
Eva lu a tion of Biologica l Effects. Assays of the hydroly-
sis of the fluorogenic substrate Z-Phe-Arg-AMC by soluble
parasite extracts containing falcipain were performed as
previously described.8 For all assays, 30 nM falcipain was
incubated with 10 mM dithiothreitol and phenothiazines added
from 100× stocks (in DMSO) in 0.1 M sodium acetate, pH 5.5,
for 30 min at room temperature before the Z-Phe-Arg-AMC
substrate (final concentration 50 µM) was added. Fluorescence
caused by the cleavage of the substrate (excitation 380 nm,
absorbance 460 nm) was then monitored continuously over 30
min. The rate of hydrolysis of substrate (increase in fluores-
cence over time) in the presence of phenothiazine inhibitors
was compared with the rate of hydrolysis in controls incubated
with an equivalent volume of DMSO. In each experiment,
multiple concentrations of phenothiazines were evaluated in
duplicate or triplicate, and IC50 values were extrapolated from
curves of % control activity over concentration.
Parasite metabolic activity and development were evaluated
by standard assays as previously described.8 Parasites were
of the Itg2 chloroquine-resistant P. falciparum strain. For the
[3H]hypoxanthine uptake assay,24 300 µL cultures of ring stage
parasites in 96-well plates were incubated with phenothiazines
(from 100× stocks in DMSO) for 24 h, [3H]hypoxanthine was
added (0.0144 µg per well; 1.2 µCi per well), and the cultures
were maintained for an additional 18 h. The cells were then
harvested and deposited on glass-fiber filters that were washed
with water and dried with ethanol. [3H]Hypoxanthine uptake
by the parasites was quantitated by scintillation counting of
the filters, the uptake by treated cultures was compared with
that by control cultures containing 1% DMSO, and IC50 values
were extrapolated from curves of percent control activity over
concentration. To assess effects on parasite development, 2
mL cultures containing ring-stage parasites were incubated
with phenothiazines from 100× stocks in DMSO. Medium was
changed after 24 h, with maintenance of the appropriate
inhibitor concentration. Parasites were collected after 24 h,
when control cultures contained trophozoites, and 48 h, when
controls contained nearly all new ring-stage parasites. Gi-
emsa-stained smears were made at each time point. Parasites
were evaluated microscopically for abnormal trophozoite mor-
phology at 24 h. For evaluation of development, new ring
forms per 1000 erythrocytes were counted in the 48 h smears,
and the counts were compared with those of controls cultured
in 1% DMSO. To quantitate the accumulation of hemoglobin
in treated parasites, trophozoite-infected erythrocytes incu-
bated with DMSO or phenothiazines as described above were
collected after 24 h, treated with saponin as described previ-
ously25 to lyse erythrocyte membranes, washed extensively,
and centrifuged, and supernatants were electrophoresed in
15% SDS-polyacrylamide gels.
The second mechanism of action of the phenothiazines
against P. falciparum appears to involve the inhibition
of the hemoglobinase falcipain. The phenothiazines
effectively inhibited falcipain in an assay utilizing a
standard peptide substrate. As seen with peptidyl
inhibitors of falcipain, considerably higher concentra-
tions of the phenothiazines were required for biological
effects, presumably due to limitations on transport into
the parasite food vacuole, the organelle in which falci-
pain resides and hemoglobin degradation takes place.
At these higher concentrations, the phenothiazines
clearly blocked hemoglobin degradation and parasite
development. Antimalarial effects due to falcipain
inhibition appeared to require about 10-20 times the
concentration of phenothiazine required for effects due
to the acridinedione-related mechanism. For com-
pounds that elicited antimalarial effects by both of the
mechanisms, the inhibition of hemoglobin degradation
was overshadowed by the acridinedione-related mech-
anism; most inhibitors caused food vacuole hemoglobin
accumulation, but parasites were clearly altered due to
other effects. For compounds 6a and 6k , however,
which lacked a phenyl ring at C-2, the only apparent
antimalarial effect was that due to the inhibition of
falcipain and subsequent blocks in hemoglobin degrada-
tion and parasite development. Compounds 6a and 6k
may thus be considered lead compounds for the devel-
opment of nonpeptide antimalarial agents that act
specifically via the inhibition of falcipain.
The cellular toxicity of phenothiazines was evaluated with
a standard assay that evaluates the reduction of MTT, which
requires the mitochondrial enzyme succinate dehydrogenase.23
MDCK cells were cultured overnight in phenyl red-free RPMI
with 1-100 µM concentrations of selected phenothiazines
(from 100× stocks in DMSO). The culture medium was then
In summary, our studies suggest that phenothiazines

2730 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Dom´ınguez et al.
changed to phenyl red-free RPMI containing MTT (1 mg/mL),
and after 2 h culture supernatants were removed, 50 µL of
ethanol was added to each well, and MTT reduction, as
indicated by absorbance at 560 nm, was compared with that
of control cells that had been incubated overnight with 1%
DMSO.
(d, 2H, 2′,6′-H, J ) 5.32 Hz), 9.0 (s, 1H, NH); LSIMS m/z (M
+ H) 358.05. Anal. (C19H16ClNSO2) C, H, N.
7-Ch lor o-1,2-d ih yd r o-2-(2,3-d im et h oxyp h en yl)-4(3H ,-
10H)-p h en oth ia zin on e (5f) (1.20 g, 87% yield): IR 1603
(CdO), 3456 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.32-2.46 (m,
2H, CH2, 1-H), 2.54-2.69 (m, 2H, CH2, 3-H), 3.52-3.61 (m,
1H, 2-H), 3.74 (s, 3H, OCH3), 3.8 (s, 3H, OCH3), 6.52 (d, 1H,
9-H, J ) 8.4 Hz), 6.85-6.94 (m, 3H, Ar), 6.97 (d, 1H, 6-H, J )
1.5 Hz), 7.05 (dd, 1H, 5′-H, J ) 7.9, 7.9 Hz), 9.0 (s, 1H, NH);
LSIMS m/z (M + H) 388.07. Anal. (C20H18ClNSO3) C, H, N.
7-Ch lor o-1,2-d ih yd r o-2-(3,4-d im et h oxyp h en yl)-4(3H ,-
10H)-p h en oth ia zin on e (5g) (1.16 g, 84% yield): IR 1600
(CdO), 3453 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.36-2.71 (m,
4H, CH2, 1,3-H), 3.72 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 6.52
(d, 1H, 9-H, J ) 8.4 Hz), 6.87 (m, 1H, 6′-H), 6.9 (s, 1H, 2′-H),
6.93-6.96 (m, 2H, 6,8-H), 7.05 (d, 1H, 5′-H, J ) 8.4 Hz), 9.0
Ch em istr y. Reagent grade solvents were used without
further purification. All starting materials were purchased
from Aldrich. All organic extracts were dried over MgSO4.
TLC plates coated with silica gel 60 F254 (Merck) were used;
detection by UV light (254 nm) or I2 vapor was applied.
Melting points were determined with a Thomas micro hot
stage apparatus and are uncorrected. Proton NMR spectra
(NMR) were recorded on a J EOL GSX (270 MHz) spectrometer;
δ values in ppm relative to tetramethylsilane are given. IR
spectra (IR) were recorded as KBr pellets using a Shimadzu
model 470 spectrometer. Elemental analyses were performed
by Dr. H. Daniel Lee, Purdue University; results were within
0.4% of predicted values for all compounds. Liquid secondary
ion mass spectrometry (LSIMS) spectra were obtained at the
Instituto Venezolano de Petroleo mass spectrometry facility.
Gen er a l Meth od for th e Syn th esis of P h en oth ia zin -4-
on e Der iva tives (5a -p ). A mixture of 2-amino-5-haloben-
zenethiol (3; 3.6 mmol) and the appropriate 1,3-dicyclohex-
anedione (4; 3.6 mmol) was dissolved in DMSO (10 mL) and
heated under reflux for 45 min. The mixtures were left to cool
at ambient temperature and poured into water. The solids
thus obtained were collected and recrystallized (see Experi-
mental Section 5a -p ). In the experimental section, when R2
in 5 or 6 includes a phenyl ring, the positions are numbered
1′-6′.
Gen er a l P r oced u r e for th e Syn th esis of P h en oth ia zin -
4-on e 5,5-Dioxid e Der iva tives (6a -k ). Hydrogen peroxide
(30%; 0.7 mL) was added to a solution of phenothiazin-4-one
(5; 0.7 mmol) in glacial acetic acid (7 mL) and refluxed for 15
min. Additional 30% hydrogen peroxide (0.7 mL) was added.
The reaction mixture was again refluxed for 2.5 h. The excess
of solvent was removed under reduced pressure, and the
mixture was poured into ice-water. The residue obtained was
filtered off, washed with water repeatedly, and recrystallized
from the appropriate solvent (see Experimental Section 6a -
k ).
7-Ch lor o-1,2-d ih yd r o-4(3H,10H)-p h en oth ia zin on e (5a )
(0.46 g, 52% yield): IR 1578 (CdO), 3488 (NH) cm-1; 1H NMR
(DMSO-d6) δ 1.76-1.85 (m, 1H, CH2, 2-H), 2.25 (t, 2H, CH2,
1-H), 2.31 (t, 2H, CH2, 3-H), 6.51 (d, 1H, 9-H, J ) 8.7 Hz),
6.84 (d, 1H, 6-H, J ) 1.48 Hz), 6.9 (dd, 1H, 8-H, J ) 9.4, 1.0
Hz), 9.0 (s, 1H, NH); LSIMS m/z (M + H) 252.02. Anal.
(C12H10ClNSO) C, H, N.
(s, 1H, NH); LSIMS m/z (M + H) 388.07. Anal. (C20H18
ClNSO3) C, H, N.
-
7-Ch lor o-1,2-dih ydr o-2-(4-ch lor oph en yl)-4(3H,10H)-ph e-
n oth ia zin on e (5h ) (1.02 g, 79% yield): IR 1594 (CdO), 3440
(NH) cm-1; 1H NMR (DMSO-d6) δ 2.38-2.71 (m, 4H, CH2, 1,3-
H), 3.38 (m, 1H, 2-H), 6.52 (d, 1H, 9-H, J ) 8.7 Hz), 6.86 (s,
1H, 6-H), 6.91 (d, 1H, 8-H, J ) 8.2, 1.6 Hz), 7.35 (d, 2H, 2′,6′-
H, J ) 9.1 Hz), 7.38 (d, 2H, 3′,5′-H, J ) 9.3 Hz), 9.1 (s, 1H,
NH); LSIMS m/z (M + H) 362.03. Anal. (C18H13Cl2NSO) C,
H, N.
7-Ch lor o-1,2-dih ydr o-2-(2,4-dich lor oph en yl)-4(3H,10H)-
p h en oth ia zin on e (5i) (0.99 g, 70% yield): IR 1578 (CdO),
3440 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.39-2.53 (m, 2H, CH2,
1-H), 2.6-2.74 (m, 2H, CH2, 3-H), 3.57-3.66 (m, 1H, 2-H), 6.50
(d, 1H, 9-H, J ) 8.4 Hz), 6.52 (d, 1H, 6-H, J ) 2.2 Hz), 6.89
(dd, 1H, 8-H, J ) 6.2, 2.5 Hz), 7.48 (m, 2H, 5′,6′-H), 7.63 (s,
1H, 3′-H), 9.2 (s, 1H, NH); LSIMS m/z (M + H) 395.97. Anal.
(C18H12Cl3NSO) C, H, N.
7-Flu or o-1,2-dih ydr o-2,2-dim eth yl-4(3H,10H)-ph en oth i-
a zin on e (5j) (0.71 g, 76% yield): IR 1613 (CdO), 3472 (NH)
cm-1 1H NMR (DMSO-d6) δ 0.96 (s, 6H, CH3), 2.12 (s, 2H,
;
CH2, 1-H), 2.16 (s, 2H, CH2, 3-H), 6.51 (dd, 1H, 9-H, J ) 9.3
Hz, J ) 5.2 Hz), 6.63-6.7 (m, 2H, 6,8-H), 8.9 (s, 1H, NH);
LSIMS m/z (M + H) 264.08. Anal. (C14H14FNSO) C, H, N.
7-F lu or o-1,2-d ih yd r o-2-p h en yl-4(3H,10H)-p h en oth ia zi-
n on e (5k ) (0.92 g, 83% yield): IR 1600 (CdO), 3472 (NH)
cm-1 1H NMR (DMSO-d6) δ 2.16 (m, 2H, CH2, 3-H), 2.38-
;
2.45 (m, 2H, CH2, 1-H), 6.68-6.76 (m, 2H, 6,8-H), 6.54 (dd,
1H, 9-H, J ) 8.3 Hz, 4.9 Hz), 7.22-7.34 (m, 5H, Ar), 9.1 (s,
1H, NH); LSIMS m/z (M + H) 312.08. Anal. (C18H14FNSO)
C, H, N.
7-F lu or o-1,2-d ih yd r o-2-(3-m eth oxyp h en yl)-4(3H,10H)-
p h en oth ia zin on e (5l) (0.95 g, 78% yield): IR 1597 (CdO),
3456 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.37-2.73 (m, 4H, CH2,
1,3-H), 3.29 (m, 1H, 2-H), 3.74 (s, 3H, OCH3), 6.54 (dd, 1H,
9-H, J ) 8.2, 5.2 Hz), 6.71-6.9 (m, 5H, Ar), 7.24 (dd, 1H, 5′-
H, J ) 8.2, 8.2 Hz), 9.1 (s, 1H, NH); LSIMS m/z (M + H)
342.09. Anal. (C19H16FNSO2) C, H, N.
7-Ch lor o-1,2-dih ydr o-2,2-dim eth yl-4(3H,10H)-ph en oth i-
a zin on e (5b) (0.80 g, 85% yield): IR 1581 (CdO), 3536 (NH)
cm-1 1H NMR (DMSO-d6) δ 0.98 (s, 6H, CH3), 2.14 (s, 2H,
;
CH2), 2.15 (s, 2H, CH, 1-H), 6.84 (s, 1H, 6-H), 6.89 (d, 1H, 8-H,
J ) 8.4 Hz), 6.5 (d, 1H, 9-H, J ) 8.4 Hz), 8.96 (s, 1H, NH);
LSIMS m/z (M + H) 280.04. Anal. (C14H14ClNSO) C, H, N.
7-F lu or o-1,2-d ih yd r o-2-(2,3-d im et h oxyp h en yl)-4(3H ,-
10H)-p h en oth ia zin on e (5m ) (1.06 g, 80% yield): IR 1613
1
(CdO), 3504 (NH) cm-1; H NMR (DMSO-d6) δ 2.3-2.69 (m,
7-Ch lor o-1,2-d ih yd r o-2-p h en yl-4(3H,10H)-p h en oth ia zi-
n on e (5c) (1.03 g, 88% yield): IR 1594 (CdO), 3424 (NH) cm-1
;
4H, CH2, 1,3-H), 3.47-3.59 (m, 1H, 2-H), 3.73 (s, 3H, OCH3),
3.79 (s, 3H, OCH3), 6.53 (dd, 1H, 9-H, J ) 8.2, 5.2 Hz), 6.68-
6.75 (m, 2H, 6,8-H), 6.9 (d, 1H, 4′-H, J ) 7.7 Hz), 6.95 (d, 1H,
6′-H, J ) 7.7 Hz), 7.05 (m, 1H, 5′-H), 9.1 (s, 1H, NH); LSIMS
m/z (M + H) 372.10. Anal. (C20H18FNSO3) C, H, N.
7-Flu or o-1,2-dih ydr o-2-(2,4-dich lor oph en yl)-4(3H,10H)-
p h en oth ia zin on e (5n ) (0.60 g, 44% yield): IR 1590 (CdO),
3456 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.35-2.7 (m, 4H, CH2,
1,3-H), 3.6 (m, 1H, 2-H), 6.67-6.74 (m, 2H, 6,8-H), 6.51-6.54
(m, 1H, 9-H), 7.46 (m, 2H, 5′,6′-H), 7.61 (s, 1H, 13-H), 9.12 (s,
1H, NH); LSIMS m/z (M + H) 380.05. Anal. (C18H12FCl2NSO)
C, H, N.
1H NMR (DMSO-d6) δ 2.38-2.45 (m, 2H, CH2, 1-H), 2.53-
2.68 (m, 2H, CH2, 3-H), 3.4-3.6 (m, 1H, 2-H), 6.52 (d, 1H, 9-H,
J ) 8.4 Hz), 6.87 (s, 1H, 6-H), 6.91 (dd, 1H, 8-H, J ) 8.15, 1.2
Hz), 7.32 (m, 4H, Ar), 7.2-7.26 (m, 1H, 4′-H), 9.1 (s, 1H, NH);
LSIMS m/z (M + H) 328.82. Anal. (C18H14ClNSO) C, H, N.
7-Ch lor o-1,2-d ih yd r o-2-(3-m eth oxyp h en yl)-4(3H,10H)-
p h en oth ia zin on e (5d ) (0.98 g, 77% yield): IR 1600 (CdO),
3440 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.37-2.72 (m, 4H, CH2,
1,3-H), 3.73 (s, 3H, OCH3), 6.52 (d, 1H, 9-H, J ) 8.2 Hz), 6.80
(dd, 1H, 4′-H, J ) 8.6, 1.2 Hz), 6.86-6.92 (m, 4H, Ar), 7.23
(dd, 1H, 5′-H, J ) 7.2, 7.2 Hz), 9.1 (s, 1H, NH); LSIMS m/z (M
+ H) 358.05. Anal. (C19H16ClNSO2) C, H, N.
7-Ch lor o-1,2-d ih yd r o-2-(4-m eth oxyp h en yl)-4(3H,10H)-
p h en oth ia zin on e (5e) (1.10 g, 85% yield): IR 1600 (CdO),
3472 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.37-2.44 (m, 2H, CH2,
1-H), 2.57-2.67 (m, 2H, CH2, 2-H), 3.73 (s, 3H, OCH3), 6.52
(d, 1H, 9-H, J ) 8.4 Hz), 6.84 (d, 1H, 6-H, J ) 2.2 Hz), 6.88 (d,
1H, 8-H, J ) 3.4 Hz), 6.91 (d, 2H, 3′,5′-H, J ) 5.32 Hz), 7.23
7-F lu or o-1,2-d ih yd r o-2-(4-ch lor op h en yl)-4-(3H ,10H )-
p h en oth ia zin on e (5o) (0.94 g, 76% yield): IR 1581 (CdO),
3472 (NH) cm-1; 1H NMR (DMSO-d6) δ 2.35-2.7 (m, 4H, CH2,
1,3-H), 6.5-6.56 (m, 1H, 9-H), 6.66-6.73 (m, 2H, 6,8-H), 7.38
(d, 2H, 3′,5′-H, J ) 8.9 Hz), 7.35 (d, 2H, 2′,6′-H, J ) 8.9 Hz),
9.04 (s, 1H, NH); LSIMS m/z (M + H) 346.04. Anal. (C18H13
FClNSO) C, H, N.
-

Inhibitors of a P. falciparum Cysteine Protease
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2731
7-F lu or o-1,2-d ih yd r o-2-(3,4-d im et h oxyp h en yl)-4(3H ,-
10H)-p h en oth ia zin on e (5p ) (0.99 g, 75% yield): IR 1594
3.74 (s, 3H, OCH3), 6.84 (d, 1H, 5′-H, J ) 8.15 Hz), 6.90 (d,
1H, 6′-H, J ) 7.67 Hz), 7.40 (dd, 1H, 9-H, J ) 3.8, 8.53 Hz),
7.56 (m, 1H, 8-H), 7.72 (d, 1H, 6-H, J ) 7.4 Hz), 11.51 (s, 1H,
NH); LSIMS m/z (M + H) 404.07. Anal. (C20H18FNSO5) C,
H.
1
(CdO), 3520 (NH) cm-1; H NMR (DMSO-d) δ 2.37-2.71 (m,
4H, CH2, 1,3-H), 3.21 (m, 1H, 2-H), 3.71 (s, 3H, OCH3), 3.73
(s, 3H OCH3), 6.67-6.73 (m, 2H, 7,8-H), 6.51-6.56 (m, 1H,
9-H), 6.80 (d, 1H, 6′-H, J ) 8.15 Hz), 6.88 (d, 1H, 5′-H, J )
8.15 Hz), 6.95 (s, 1H, 2′-H), 9.05 (s, 1H, NH); LSIMS m/z (M
+ H) 372.10. Anal. (C20H18FNSO3‚H2O) C, H, N.
7-Flu or o-1,2-dih ydr o-2-(4-ch lor oph en yl)-4(3H,10H)-ph e-
n oth ia zin on e 5,5-d ioxid e (6j) (0.21 g, 79% yield): IR 1706
1
(CdO), 3472 (NH) 1306, 1123 (SO2) cm-1; H NMR (DMSO-
7-Ch lor o-1,2-dih ydr o-2,2-dim eth yl-4(3H,10H)-ph en oth i-
a zin on e 5,5-d ioxid e (6a ) (0.19 g, 66% yield): IR 1635 (CdO),
3536 (NH) 1312, 1270 (SO2) cm-1; 1H NMR (DMSO-d6) δ 1.04
(s, 6H, CH3), 2.32 (s, 2H, CH2, 1-H), 2.72 (s, 2H, CH2, 3-H),
7.38 (d, 1H, 9-H, J ) 8.91 Hz), 7.70 (d, 1H, 8-H, J ) 1.49, 8.78
Hz), 7.85 (d, 1H, 6-H, J ) 2.24 Hz), 11.48 (s, 1H, NH); LSIMS
m/z (M + H) 312.04. Anal. (C14H14ClNSO3) C, H, N.
7-Ch lor o-1,2-d ih yd r o-2-p h en yl-4(3H,10H)-p h en oth ia zi-
n on e 5,5-d ioxid e (6b) (0.18 g, 71% yield): IR 1648 (CdO),
3488 (NH) 1319, 1296 (SO2) cm-1; 1H NMR (DMSO-d6) δ 2.49-
2.59 (m, 2H, CH2, 1-H), 2.78-2.98 (m, 2H, CH2, 3-H), 3.27 (m,
1H, 2-H), 7.37 (m, 5H, Ar), 7.62-7.68 (m, 1H, 4′-H), 7.87 (s,
1H, 6-H), 11.54 (s, 1H, NH); LSIMS m/z (M + H) 360.04. Anal.
(C18H14ClNSO3) C, H, N.
d6) δ 2.76-2.95 (m, 2H, CH2, 1-H), 7.30 (m, 1H, 9-H), 7.41 (d,
2H, 2′,6′-H, J ) 7.7 Hz), 7.44 (d, 2H, 3′,5′-H, J ) 7.7 Hz), 7.56
(m, 1H, 8-H), 7.73 (dd, 1H, 6-H, J ) 2.9, 8.02 Hz), 11.53 (s,
1H, NH); LSIMS m/z (M + H) 378.81. Anal. (C18H13FClNSO3)
C, H, N.
7-Flu or o-1,2-dih ydr o-2,2-dim eth yl-4(3H,10H)-ph en oth i-
a zin on e 5,5-d ioxid e (6k ) (0.1 g, 50% yield): IR 1699 (CdO),
3504 (NH), 1312, 1155 (SO2) cm-1; 1H NMR (DMSO-d6) δ 1.04
(s, 6H, CH3), 2.31 (s, 1H, 1-H), 2.72 (s, 1H, 3-H), 7.41 (dd, 1H,
9-H, J ) 9, 4.5 Hz), 7.55 (m, 1H, 8-H), 7.7 (dd, 1H, 6-H, J )
7.70, 2.73 Hz), 11.43 (s, 1H, NH); LSIMS m/z (M + H) 296.07.
Anal. (C14H14FNSO3) C, H, N.
Ack n ow led gm en t. This work was supported by
grants from the National Institutes of Health, the
UNDP/World Bank/WHO Special Programme for Re-
search and Training in Tropical Diseases, the American
Heart Association, and Consejo de Desarrollo Cient´ıfico
y Human´ıstico (CDCH-UCV # 06.30-3283-94). P.J .R.
is an Established Investigator of the American Heart
Association. This paper is dedicated to Dr. Yoshito
Kishi, Department of Chemistry, Harvard University,
on the occasion of his 60th birthday.
7-Ch lor o-1,2-d ih yd r o-2-p h en yl-2-(4-m et h oxyp h en yl)-
4(3H,10H)-p h en oth ia zin on e 5,5-d ioxid e (6c) (0.20 g, 78%
1
yield): IR 1699 (CdO), 3488 (NH) 1299, 1180 (SO2) cm-1; H
NMR (DMSO-d6) δ 2.74-2.94 (m, 2H, CH2, 1-H), 3.73 (s, 3H,
OCH3), 6.91 (d, 2H, 3′,5′-H, J ) 8.64 Hz), 7.28 (d, 2H, 2′,6′-H,
J ) 8.64 Hz), 7.37 (d, 1H, 9-H, J ) 8.9 Hz), 7.69 (dd, 1H, 8-H,
J ) 8.8, 2.21 Hz), 7.87 (d, 1H, 6-H, J ) 2.21 Hz), 11.54 (s, 1H,
NH); LSIMS m/z (M + H) 390.05. Anal. (C19H16ClNSO4) C,
H, N.
7-Ch lor o-1,2-d ih yd r o-2-(2,3-d im et h oxyp h en yl)-4(1H ,-
10H )-p h en ot h ia zin on e 5,5-d ioxid e (6d ) (0.18 g, 62%
yield): IR 1712 (CdO), 3504 (NH), 1290, 1126 (SO2) cm-1; 1H
NMR (DMSO-d6) δ 2.74-2.90 (m, 2H, CH2, 1-H), 3.76 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 6.95-7.1 (m, 2H, 6,4′-H), 7.72 (d,
1H, 8-H, J ) 7.4 Hz), 6.92 (d, 1H, 9-H, J ) 7.43 Hz), 7.4 (m,
1H, 6′-H), 7.56 (m, 1H, 5′-H), 11.49 (s, 1H, NH); LSIMS m/z
(M + H) 420.10. Anal. (C20H18ClNSO5) C, H, N.
Refer en ces
(1) Walsh, J . A. Disease problems in the Third World. Ann. N.Y.
Acad. Sci. 1989, 569, 1-16.
(2) Oaks, S. C., Mitchell, V. S., Pearson, G. W., Carpenter, C. C. J .,
Eds. Malaria: Obstacles and Opportunities; National Academy
Press: Washington, D.C., 1991.
(3) Rosenthal, P. J .; McKerrow, J . H.; Aikawa, M.; Nagasawa, H.;
Leech, J . H. A malarial cysteine proteinase is necessary for
hemoglobin degradation by Plasmodium falciparum. J . Clin.
Invest. 1988, 82, 1560-1566.
(4) Salas, F.; Fichmann, J .; Lee, G. K.; Scott, M. D.; Rosenthal, P.
J . Functional expression of falcipain, a Plasmodium falciparum
cysteine proteinase, supports its role as a malarial hemoglobi-
nase. Infect. Immun. 1995, 63, 2120-2125.
(5) Rosenthal, P. J .; Meshnick, S. R. Hemoglobin catabolism and
iron utilization by malaria parasites. Mol. Biochem. Parasitol.
1996, 83, 131-139.
(6) Rosenthal, P. J .; Wollish, W. S.; Palmer, J . T.; Rasnick, D.
Antimalarial effects of peptide inhibitors of a Plasmodium
falciparum cysteine proteinase. J . Clin. Invest. 1991, 88, 1467-
1472.
(7) Rosenthal, P. J .; Lee, G. K.; Smith, R. E. Inhibition of a
Plasmodium vinckei cysteine proteinase cures murine malaria.
J . Clin. Invest. 1993, 91, 1052-1056.
(8) Rosenthal, P. J .; Olson, J . E.; Lee, G. K.; Palmer, J . T.; Klaus,
J . L.; Rasnick, D. Antimalarial effects of vinyl sulfone cysteine
proteinase inhibitors. Antimicrob. Agents Chemother. 1996, 40,
1600-1603.
(9) Ring, C. S.; Sun, E.; McKerrow, J . H.; Lee, G. K.; Rosenthal, P.
J .; Kuntz, I. D.; Cohen, F. E. Structure-based inhibitor design
by using protein models for the development of antiparasitic
agents. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3583-3587.
(10) Li, R.; Kenyon, G. L.; Cohen, F. E.; Chen, X.; Gong, B.;
Dominguez, J . N.; Davidson, E.; Kurzban, G.; Miller, R. E.;
Nuzum, E. O.; Rosenthal, P. J .; McKerrow, J . H. In vitro
antimalarial activity of chalcones and their derivatives. J . Med.
Chem. 1995, 38, 5031-5037.
(11) Raether, W.; Fink, E. Antimalarial activity of floxacrine (HOE
991) I: studies on blood schizontocidal action of floxacrine
against Plasmodium berghei, P. vinckei, and P. cynomolgi. Ann.
Trop. Med. Parasitol. 1979, 73, 505-526.
(12) Du¨rckheimer, W.; Raether, W.; Seliger, H.; Seidnath, H. 10-
Hydroxy-3,4-dihydroacridine-1,9(2H,10H)-diones, a new group
of malricidal and coccidiostatic compounds. Arzneim.-Forsch./
Drug Res. 1980, 30, 1041-1046.
7-Ch lor o-1,2-d ih yd r o-2-(3,4-d im eth oxyp h en yl)-4-(3H,-
10H)-p h en oth ia zin on e 5,5-d ioxid e (6e) (0.17 g, 58% yield):
1
IR 1645 (CdO), 3472 (NH), 1306, 1146 (SO2) cm-1; H NMR
(DMSO-d6) δ 2.72-2.95 (m, 2H, CH2, 1-H), 3.73 (s, 3H, OCH3),
3.74 (s, 3H, OCH3), 6.84 (d, 1H, 5′-H, J ) 8.15 Hz), 6.91 (d,
1H, 6′-H, J ) 8.15 Hz), 7.01 (s, 1H, 2′-H), 7.38 (d, 1H, 9-H, J
) 8.9 Hz), 7.71 (d, 1H, 8-H, J ) 8.4 Hz), 7.89 (s, 1H, 6-H),
11.57 (s, 1H, NH); LSIMS m/z (M + H) 420.06. Anal. (C20H18
ClNSO5) C, H, N.
-
7-Ch lor o-1,2-dih ydr o-2-(4-ch lor oph en yl)-4(3H,10H)-ph e-
n oth ia zin on e 5,5-d ioxid e (6f) (0.18 g, 66% yield): IR 1645
1
(CdO), 3504 (NH) 1302, 1149 (SO2) cm-1; H NMR (DMSO-
d6) δ 2.77-2.95 (m, 2H, CH2, 1-H), 3.17-3-31 (m, 2H, CH2,
3-H), 3.48-3.58 (m, 1H, 2-H), 7.38 (d, 1H, 9-H, J ) 8.9 Hz),
7.41 (d, 2H, 2′,6′-H, J ) 8.4 Hz), 7.42 (d, 2H, 3′,5′-H, J ) 8.4
Hz), 7.8 (d, 1H, 6-H, J ) 2.5 Hz), 11.57 (s, 1H, NH); LSIMS
m/z (M + H) 394.27. Anal. (C18H13Cl2NSO3) C, H, N.
7-Ch lor o-1,2-dih ydr o-2-(2,4-dich lor oph en yl)-4(3H,10H)-
p h en oth ia zin on e 5,5-d ioxid e (6g) (0.23 g, 78% yield): IR
1702 (CdO), 3472 (NH) 1393, 1152 (SO2) cm-1 1H NMR
;
(DMSO-d6) δ 2.79-2.99 (m, 2H, CH2, 1-H), 3.76-3-87 (m, 1H,
2-H), 7.38 (d, 1H, 9-H, J ) 8.67 Hz), 7.50 (m, 2H, 5′,6′-H),
7.65-7.72 (m, 2H, 8,3′-H), 7.88 (s, 1H, 6-H), 11.64 (s, 1H, NH);
LSIMS m/z (M + H) 427.96. Anal. (C18H12Cl3NSO3) C, H, N.
7-F lu or o-1,2-d ih yd r o-2-(3-m eth oxyp h en yl)-4(3H,10H)-
p h en oth ia zin on e 5,5-d ioxid e (6h ) (0.17 g, 66% yield): IR
1645 (CdO), 3520 (NH) 1312, 1146 (SO2) cm-1 1H NMR
;
(DMSO-d6) δ 2.77-2.97 (m, 2H, CH2, 1-H), 3.18-3.28 (m, 2H,
CH2, 3-H), 3.74 (s, 3H, OCH3), 6.81-6.85 (m, 1H, 6′-H), 6.92-
6.95 (m, 2H, 2′,4′-H), 7.41 (dd, 1H, 9-H, J ) 9.03, 4.5 Hz), 7.56
(m, 1H, 8-H), 7.73 (dd, 1H, 6-H, J ) 2.73, J ) 7.9 Hz), 11.51
(s, 1H, NH); LSIMS m/z (M + H) 373.08. Anal. (C19H16FNSO4)
C, H, N.
(13) Raether, W.; Fink, E. Antimalarial activity of floxacrine (HOE
991) II: studies on causal prophylactic and blood schizontocidal
action of floxacrine and related dihydroacridinediones against
Plasmodium yoelii and P. berghei. Ann. Trop. Med. Parasitol.
1982, 76, 507-516.
7-F lu or o-1,2-d ih yd r o-2-(3,4-d im et h oxyp h en yl)-4(3H ,-
10H)-p h en oth ia zin on e 5,5-d ioxid e (6i) (0.13 g, 46% yield):
1
IR 1642 (CdO), 3472 (NH), 1306, 1146 (SO2) cm-1; H NMR
(DMSO-d6) δ 2.75-2.94 (m, 2H, CH2, 1-H), 3.72 (s, 3H, OCH3),

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