Unambiguous synthesis and prophylactic antimalarial activities of imidazolidinedione derivatives

Article abstract of DOI:10.1021/jm0504252

WR182393, a guanidinoimidazolidinedione derivatives with potent causal prophylactic antimalarial activity by intramuscular injection, was previouly prepared by treatment of chloroproguanil and diethyl oxalate, yielding a mixture of two closely related iso

Full text of DOI:10.1021/jm0504252

6472
J. Med. Chem. 2005, 48, 6472-6481
Unambiguous Synthesis and Prophylactic Antimalarial Activities of
Imidazolidinedione Derivatives
Quan Zhang,† Jian Guan,† John Sacci,† Arba Ager,‡ William Ellis,† Wilbur Milhous,† Dennis Kyle,† and
Ai J. Lin*,†
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, and
University of Miami, South Campus, Miami, Florida 33177
Received May 4, 2005
WR182393, a guanidinoimidazolidinedione derivatives with potent causal prophylactic anti-
malarial activity by intramuscular injection, was previouly prepared by treatment of chloro-
proguanil and diethyl oxalate, yielding a mixture of two closely related isomers. Poor solubility
of the mixture made the separation and purification impossible. To overcome the separation
problem, new and facile unamgibuous syntheses of the two active components were reported.
The new synthetic methods facilitate the synthesis of not only the active components, but also
their derivatives. To search for compounds with good oral efficacy, a series of carbamate
derivatives of the active components were prepared by the new procedure, many of which
showed profound causal prophylactic antimalarial activity against Plasmodium yoelii in mouse
by oral administration.
Introduction
The current global situation with respect to malaria
indicates that about two billion people are exposed to
the disease. Each year between 100 and 200 million new
cases of infection are reported, and approximately 1-2
million people die due to malaria.1,2 The situation is
rapidly worsening, mainly due to the nonavailability of
effective drugs and development of drug resistance to
Figure 1.
tion. Although WR182393 was one of the few non-8-
aminoquinolines active in the rhesus monkey test
model, an active discovery program based on this hit
was not pursued. Primarily, this was due to the com-
pound’s poor oral bioavailability and the program’s
emphasis on development of tafenoquine (8-amino-
quinoline derivative antimalarial) in the early 1990s.
However, the unique activity of WR182393 observed in
the rhesus monkey model recently led to its inclusion
as a priority within the antimalarial lead optimization
program of Walter Reed Army Institute of Research. To
continue our earlier efforts on in vivo efficacy experi-
ments, the recent priority was characterization of the
chemical properties of WR182393 and the search for
derivatives with good oral prophylactic antimalarial
activity.
Initially, the test material WR182393 was shown to
be a mixture of two products and the starting material,
chlorproguanil, with relative proportions of each com-
ponent in the active mixture estimated to be 54:44:2 of
compound 1, compound 2, and chlorproguanil, respec-
tively. This was not surprising since WR182393 was
prepared by treatment of chlorproguanil with diethyl
oxalate, which could result in six different derivatives.9
Because of the poor solubility, small-scale purification
of the mixture is very difficult, and large-scale purifica-
tion of the biguanide is almost impossible. Conse-
quently, the structure determination of the components
was achieved by carbamate derivatization of the mix-
ture, which enhanced the solubility and facilitated the
separation and structural identification of the compo-
the existing first-line drugs, such as chloroquine and
pyrimethamine.3-5 In addition to the drug resistance
of the first-line antimalarial drugs, the usefulness of
many newer antimalarial drugs was impaired by their
side effects. Multiple drug resistance in Plasmodium
falciparum malaria continues to pose special problems
for targeting the blood stages of malaria. Our product
development teams for malaria prophylaxis are placing
emphasis on developing new chemical entities with true
causal prophylactic or radical curative properties, stop-
ping malaria before blood stages emerge and cause
clinical disease. With the exception of quinoline esters,6
only the 8-aminoquinoline drugs such as primaquine or
tafenoquine7 have activity against the liver stages of
Plasmodium vivax and P. falciparum malarias. How-
ever, the 8-aminoquinoline drugs caused serious lethal
hemolytic side effects in glucose-6-phosphate dehydro-
genase (G6PD) deficient patients. Therefore, there is an
eminent need for new and safe antimalarial drugs to
combat the parasites and protect the tourists traveling
in the epidemic areas of the world.
The imidazolidinedione program has its origins in the
finding that WR182393, a mixture of compounds 1 and
2 (Figure 1), has radical curative and causal prophy-
lactic activity in rhesus monkeys infected with Plasmo-
dium cynomolgi 8 administered by intramuscular injec-
* Corresponding author. Tel: 301-319-9084. Fax: 301-319-9449.
E-mail: ai.lin@na.amedd.army.mil.
† Division of Experimental Therapeutics, Walter Reed Army Insti-
tute of Research, Silver Spring, Maryland 20910.
‡ University of Miami, South Campus, Miami, Florida 33177.
10.1021/jm0504252 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/02/2005

Synthesis of Antimalarial Imidazolidinediones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6473
Scheme 1
nents. The structures of the two key active new chemical
entities are shown in Figure 1. The results of our efforts
to identify components of WR182393 are detailed in a
recent publication.9 During the process of structure
determination of the active components, two carbamate
derivatives, 1a and 2b, were prepared and showed
profound causal prophylactic activity against P. cyno-
molgi in rhesus monkey by intramuscular injection;
however, no oral activity was demonstrated in the same
test.
In this study, we report the development of two new,
facile, and unambiguous methods for the preparation
of compounds 1 and 2, the active components of the
mixture. The new procedures provide facile approaches
to prepare not only the active components 1 and 2, but
also the new derivatives or analogues of these two lead
compounds.
Oral efficacy is an essential consideration in search
of prophylactic drugs. The causal prophylactic antima-
larial activity of WR182393 in animal models was
demonstrated only when the drug was administered by
intramuscular injection. No protection of the malaria-
infected animals was observed when WR182393 was
given orally. To search for compounds with good oral
efficacy, a series of carbamate derivatives of the active
components were prepared by the new procedure, many
of which showed profound causal prophylactic antima-
larial activity against P. yoelii in mouse by oral admin-
istration.
dione (5) and N-(3,4-dichlorophenyl)guanidine (8) in
chloroform overnight to furnish the yellowish product
in 40% yield (Scheme 1).10 The intermediate 5 was
obtained in two steps by methylation of isopropylthio-
urea 3 with methyl iodide to generate S-methylisothio-
uronium 4, which was treated with oxalyl chloride under
the catalysis of triethylamine to give 5 in 56% yield.11,12
Likewise, the synthesis of 3,4-dichlorophenylguanidine
(8) involved methylation of 3,4-dichlorophenylthiourea
6 with CH3I to give the corresponding S-methylisothio-
uronium salt 7, followed by amination to give 8 in 90%
yield. Carbamates of compound 1 were prepared by
treatment of 1 with either alkyl chloroformate or dialkyl
carbonate to give carbamates 1a-h.
Compound 2 was prepared either by reaction of
compound 9 with isopropylguanidine (12) or with t-Boc
isopropylguanidine (11) followed by acid hydrolysis of
the t-Boc carbamate 2a, as shown in Scheme 2. These
two approaches gave a similar total yield, and the
product is identical in melting point, IR, and NMR
spectrum. Since t-Boc guanide 11 is soluble in organic
solvent and isopropylguanidine 12 is much more soluble
in water than in organic solvent, it is easier to handle
the former than the latter during the experiments.
Compound 9, the key intermediate, was produced in
good yield by treatment of S-methylisothiouronium 7
with methyl chlorooxoacetate (methyl oxalyl chloride)
in CH2Cl2 under Et3N catalysis at -78 °C. The carbam-
ates 2a, 2c, and 13 were prepared by the same proce-
dure, treating intermediate 9 with compound 10, 11,
and 8, respectively, as shown in Scheme 2.
Chemistry
The approach for unambiguous synthesis of 1 involved
heating 1-isopropyl-2-methylsulfanyl-1H-imidazole-4,5-
The key intermediate, N-isopropyl-N′-(tert-butylcar-
bonyl)guanidine 11, was obtained in good yield by

6474 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Zhang et al.
Scheme 2
Scheme 3
(benzylcarbonyl)-N′-isopropylguanidine, was prepared
by treatment of 12 with dibenzyl carbonate.
Although the NMR spectra of compound 1 in CDCl3
showed a pure single compound, the NMR spectra of
compound 2 indicated the existence of two tautomers,
2 and 2′, in ratio of about 3:1, as indicated by the
integration of two sets of doublets at 1.14 and 1.09 ppm
for the methyl protons of the isopropyl group.14
Both imidazolidinediones 1 and 2 are sparingly
soluble in organic solvents and water, especially com-
pound 2. Heating is required to dissolve compound 2 in
DMSO or DMF.
Antimalarial Activity
The causal prophylactic activities of the new imida-
zolidinedione carbamates (1a-h and 2a-f) were as-
sessed in mouse infected with sporozoites of Plasmodi-
um yoelii. The results were shown in Tables 1 and 2.
The lead molecule 1 and its carbamate derivatives are
much less active than lead molecule 2 and its corre-
sponding carbamates. While compound 2 provided 100%
protection (5/5) at 20 mg/kg administered orally and
protected two out of five mice in the 5 mg/kg group,
compound 1 showed no protection activity up to 160 mg/
kg. Likewise, the carbamate derivatives of 2 are much
more active than those of compound 1. Among the
carbamate derivatives of compound 1, only t-Boc (1a),
isobutyl (1b),
treatment of isopropylguanidine (12) with di-tert-butyl
carbonate (Scheme 3). Compound 12 was prepared, in
turn, by amination of a commercially available product,
S-methylisothiouronium sulfate, followed by conversion
of the isopropylguanidine salt to free base 12 by treat-
ment with Ba(OH)2.13 The other intermediate 10, N-

Synthesis of Antimalarial Imidazolidinediones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6475
Table 1. Oral Prophylactic Activity in P. yoelii Sporozoites
Infected Mouse
Table 2. Oral Prophylactic Activity in P. yoelii Sporozoites
Infected Mouse
no. mice
protected/
total
dose
(mg/Kg)
no. mice
protected/
total
compd
Ra
dose
1
160
40
0/5
0/5
0/5
5/5
2/5
0/5
5/5
2/5
0/5
0/5
0/5
5/5
4/5
2/5
0/5
1/5
2/5
0/5
1/5
0/5
0/5
3/5
2/5
2/5
0/5
1/5
0/5
0/5
5/5
2/5
0/5
0/5
0/5
0/5
toxicity
death
5/5
5/5
5/5
3/5
compd
R
(mg/kg)
2
80
40
20
10
5
160
40
10
5
2.5
1.25
160
40
10
5
2.5
1.23
160
40
10
5
5/5
5/5
5/5
3/5
2/5
5/5
4/5a
4/5
4/5
2/5
2/5
5/5
5/5
5/5
5/5
1/5
0/5
3/5
2/5
2/5
3/5
0/5
5/5
5/5
3/5
1/5
1/5
5/5
3/5
4/5
2/5
2/5
10
1a
1b
C(CH3)3
160
40
10
160
40
10
160
40
160
40
10
CH2CH(CH3)2
2a
2b
C(CH3)3
1c
1d
CH2C6H5
CH2CH3
CH2CH(CH3)2
2.5
160
40
1e
1f
(CH2)5CH3
10
CH2CH2CdCH2
160
40
2c
2d
2e
2f
CH2C6H5
10
1g
160
40
2.5
80
40
20
10
5
10
CH2CH3
2.5
160
40
1h
CH2CH2OCH2C6H5
10
160
40
10
160
40
10
160
primaquine
arteether
(CH2)5CH3
80
40
20
10
5
CH2CH2CdCH2
NDb
tafenoquine
a One dead. b ND, not dosed.
40
10
5
keys, a gold standard test. Further, if all of the sporo-
zoites or exoerythrocytic (EE) stage parasites are killed,
then blood stream parasites will not appear. The
number of EE stages that are killed will be reflected in
the subsequent reduction in parasitemia. However, the
strain of P. yoelii (17XNL) used in the model is nonle-
thal. The mice developed high parasitemia in the first
6 days after inoculation with sporozoites and eventually
self-cured, and most survive even without treatment.
To conclude that the mice are cured by the drug may or
may not be true. Therefore, the mouse model can only
be used as a preliminary screen and the results can only
be used as one criterion for selection of candidates for
further tests in the monkey model. This was exemplified
by the fact that, although compound 2b showed superior
efficacy over compound 1a in mouse test, the latter was
more active than the former in tests against P. cyno-
molgi15,16 in rhesus monkey, which has been demon-
strated to give results parallel to that observed in clinics.
Therefore, good activity in the mouse test observed in
this study will be further confirmed in the gold standard
rhesus monkey model. The drug distribution and me-
tabolism of rhesus monkey is generally much closer to
that of human than other commonly used animals.
2.5
and ethyl (1d) carbamates showed 100% protection of
the high dose (160 mg/kg) group. Carbamate 1c showed
no protection (0/5) at 160 mg/kg, and carbamates 1e,
1f, and 1h protected only one out of five treated mice
(1/5) at the same dose. Whereas the isobutyl carbamate
of compound 2 (2b) protected 100% of the treated mice
at a dosage as low as 5 mg/kg, the minimum dose for
100% protection of isobutyl carbamate 1b is >40 mg/
kg. Likewise, while t-Boc 2a protected 4/5 mice at
dosage of 5 mg/kg, t-Boc 1a protected only 2/5 mice at
a higher dose of 40 mg/kg. However, ethyl carbamates
1d and 2d showed comparable causal prophylactic
activity at all dose levels tested, showing 2/5 and 1/5
mice protection, respectively, at 10 mg/kg.
The test in this mouse model against P. yoelii may
yield false-positive results because of the relatively short
pre-erythrocytic stage of the parasite (2 days) and the
unknown biological half-life of the test compounds. It
is, therefore, considered to be a test for presumptive
activity, and a positive result must be confirmed in
another system, such as P. cynomolgi in rhesus mon-

6476 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Table 3. In Vitro Antimalarial Activity against P. falciparum
Zhang et al.
mouse model will be confirmed in rhesus monkey
against P. cynomolgi.
IC50
IC90
drug
parasites
(ng/mL)
(ng/mL)
Experimental Section
mefloquine
W2
D6
TM91C235
W2
D6
TM91C235
W2
D6
TM91C235
W2
D6
1.86
4.90
21.63
125.71
4.1754
34.57
2664.69
2971.72
2589.26
2380.95
1627.04
2541.89
6663.44
7182.39
5384.93
4.01
13.44
Chemistry. Melting points were determined on a Mettler
FP62 melting point apparatus and are uncorrected. Analytical
thin-layer chromatography (TLC) was performed using EM
Reagents 0.25 mm HPTLC-HLF normal-phase 150 µm silica
gel 60-F plates (Analtech, Newark, DE). Liquid chromatogra-
phy was performed using a force flow (flash chromatography)
Horizon HPFC system (Biotage, Charlottesville, VA) with
Flash 25M or 40M cartridges (KP-Siltm Silica, 32-63 µm,
60 Å). Preparative TLC was performed using silica gel GF
Tapered Uniplates (Analtech, Newark, DE). Infrared spectra
were recorded on a Bio-Rad FTS 3000 spectrophotometer (Bio-
Rad Laboratories, Cambridge, MA) and are reported in
44.01
chloroquine
198.69
5.33
55.24
WR182393
10894.01
12066.59
11351.15
11918.96
9365.79
11874.15
12282.73
9631.66
9981.42
1
2
TM91C235
W2
D6
TM91C235
reciprocal centimeters (cm-1). H NMR and 13C NMR spectra
1
were recorded in deuteriochloroform, unless otherwise noted,
on a Bruker AC300 or Bruker Avance 600 spectrometer
(Bruker Instruments, Inc., Wilmington, DE). Chemical shifts
are reported in parts per million on the δ scale from an internal
standard of tetramethylsilane. Combustion analyses were
performed by Atlantic Microlab, Inc. (Norcross, GA). Where
analyses are indicated by symbols of the elements, the analyti-
cal results obtained were within (0.4% of the theoretical
values.
It is interesting to note that while carbamates 1a and
2b possess high oral prophylactic activity against P.
yoelii in mouse model, no oral causal prophylactic
activity was observed in rhesus monkey model against
liver stage P. cynomolgi at a dose up to 60 mg/kg.9 The
cause of differences in oral efficacy observed in rhesus
monkey and mouse may relate to the difference in their
stomach acidity. The facts that the pH of monkey
stomach (1-3) is lower than that of mouse (pH 3.1-
4.5) and that the half-life of carbamates 1a and 2b in
simulated stomach acid (0.01 N HCl in ethanol) is only
about 30 min may account for the species differences
in oral activity observed.17,18 The major hydrolysis
product of 1a and 2b in simulated stomach acid was
identified as the lead compounds 1 and 2, respectively,
both of which are sparingly soluble in organic solvents
and water. The difference in stomach acidity of mouse
and monkey leads to the difference in rate of hydrolysis
of carbamates. The higher pH of mouse stomach acid
may account for the lower rate of carbamate hydrolysis
and thus better bioavailability and higher oral efficacy.
It is further noted that neither the lead compounds 1
and 2 nor their carbamates showed activity against the
blood stage parasites in the in vitro assay against P.
falciparum (Table 3). Likewise, compounds 1a, 1b, 1c,
1d, 1e, and 1f were inactive in the Thompson test
against blood stage parasite Plasmodium berghei from
20 to 320 mg/kg by sc or po, although moderate activity
in EE tests against P. yoelii (liver stage model) were
observed (Table 1). To the best of our knowledge, this
is the first class of chemoprophylaxis antimalarial agent
that is totally devoid of activity against blood stage
malaria. Although it is a general belief that an ideal
therapeutic drug should be effective for both prophylaxis
and treatment, drugs with only prophylactic activity,
such as the compounds under this study, will have much
less exposure to the parasites in the field than the
treatment drugs. Thus, development of drug resistant
parasites to drugs with prophylaxis only is less likely
to be a problem.
1-Isopropyl-2-methylsulfanyl-1H-imidazole-4,5-dione
(5). To isopropyl thiourea 3 (2.364 g, 20.0 mmol) in 40 mL of
dry acetone was added iodomethane (1.87 mL, 30.0 mmol)
dropwise at room temperature. After the addition was com-
pleted, the mixture was refluxed for 1 h, and the solvent was
evaporated to dryness under the reduced pressure to give a
gum. The gummy HI salt was suspended in 200 mL of dry
CH2Cl2 and to the suspension was added triethylamine (11
mL, 80.0 mmol). The mixture was stirred for 30 min at room
temperature and cooled to -78 °C with a dry ice/acetone bath.
To the mixture was added dropwise oxalyl chloride (12 mL of
2 M solution, 24 mmol), and the mixture stirred at -78 °C for
1 h. The reaction was quenched by addition of water to
decompose the excess oxalyl chloride; the solvent was dried
over Na2SO4 and evaporated to dryness in vacuo. Recrystal-
lization of the solid product with CHCl3/hexanes mixed solvent
yielded intermediate 5 as a yellow needle (1.62 g, 44%), mp
1
193.5 °C. H NMR (600 MHz, CDCl3): δ 4.19 (m, 1H), 2.76(s,
3H), 1.48 (d, J ) 6.6 Hz, 6H). 13C NMR: δ 191.64, 167.18,
158.49, 48.62, 20.06, 14.64; IR: 1759, 1740, 1450, 1303, 1050
cm-1. MS (m/z): 172, 144, 83. Anal. (C7H10N2O2S): C, H, N,
S.
1-(3,4-Dichlorophenyl)-2-methylisothiourea HI Salt
(7). To a solution of 3,4-dichlorophenylthiourea (6) (5.00 g,
22.61 mmol) in 80 mL of anhydrous acetone was added
iodomethane (2.11 mL, 33.92 mmol), and the resultant solution
was heated at reflux for 1 h. The solvent was removed under
reduced pressure, and the residue was suspended in EtOAc.
The white solid product was collected, washed with fresh
solvent, and dried in vacuo to give HI salt (7) (8.119 g, 22.37
1
mmol, 99%) as a white solid, mp 160 °C. H NMR (600 MHz,
DMSO-d6): δ 7.80 (d, J ) 8.6 Hz, 1H), 7.75 (d, J ) 2.4 Hz,
1H), 7.37 (dd, J ) 8.6 and J ) 2.4 Hz, 1H), 2.78 (s, 3H). 13C
NMR: δ 170.64, 136.24, 132.94, 132.63, 131.68, 128.95, 123.19.
IR: 1628, 1576, 1541, 1464, 1306, and 1124 cm-1. Anal. (C8H9-
Cl2IN2S): C, H, Cl, N, S,
To the water solution of 1-(3,4-dichlorophenyl)-2-methyl-
isothiourea hydroiodide (7) was added saturated Na2CO3
solution at ice-cold temperature till the pH reached about
9-10. The reaction mixture was extracted with CHCl3 three
times. The chloroform extracts were combined and dried over
Na2SO4. The solvent was removed in vacuo to give crude free
base of 7 as a white solid in quantitative yield, mp 166 °C.
In summary, two facile and unambiguous synthetic
procedures were developed to prepare both active com-
ponents of WR182393. The new methods were also
utilized to make a series of carbamate derivatives, some
of which showed good oral efficacy in mouse infected
with sporozoites of P. yoelii. The causal prophylactic
activity of the best compound demonstrated in the
1
The product was pure enough for further reaction. H NMR
(600 MHz, CDCl3) δ 7.34(d, J ) 8.4 Hz, 1H), 7.02 (d, J ) 2.4
Hz, 1H), 6.75 (dd, 8.4 and 2.4 Hz, 1H), 4.58 (s, 2H), 2.42 (s,
3H). 13C NMR: δ 157.14, 148.74, 133.24, 131.31, 126.80,
124.23, 122.09, and 14.16.

Synthesis of Antimalarial Imidazolidinediones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6477
N-(3,4-Dichlorophenyl)guanidine (8). To the solution of
7 (2.29 g, 6.31 mmol) in 23 mL of ethanol was added 6.87 mL
of NH4OH. The reaction mixture was stirred at room temper-
ature for 10 min, and the clear solution was refluxed for 12 h.
The solvent was removed under reduced pressure and the
residue was suspended in saturated Na2CO3 solution. The solid
was collected, washed sequentially with H2O and Et2O and
dried to give the title compound as a white solid (1.16 g, 90%),
mp 208 °C. 1H NMR (600 MHz, DMSO-d6): δ 7.39 (d, J ) 8.6
Hz, 1H), 7.02 (d, J ) 2.4 Hz, 1H), 6.79 (dd, J ) 8.6 and 2.4
Hz, 1H), 5.79 (br s, 3H). 13C NMR: δ 155.38, 130.69, 130.28,
124.30, and 123.50. IR: 1684, 1624, 1559, 1472, 1380, 1328,
869, 818, 671 cm-1. MS (m/z): 204 (M+), 187, 162, 127, and
90. The compound was pure enough to be used for further
reactions without purification.
The isopropylguanidine sulfate was converted to its free
base by addition of Ba(OH)2‚H2O, and the barium sulfate
formed was removed by centrifugation to give free base 12 as
a colorless viscous gel in 65% yield. 1H NMR (300 MHz, D2O):
δ 3.64 (m, 1H), 1.17 (d, J ) 6.5 Hz, 6H). 13C NMR: δ 46.34,
24.10.
N-(3,4-Dichlorophenyl)-N′-[1-(3,4-dichlorophenyl)-4,5-
dioxoimidazolidin-2-ylidene]guanidine (13). To the solu-
tion of the crude dione 9 (0.551 g, 1.907 mmol) in 25 mL of
dry CHCl3 was added N-(3,4-dichlorophenyl)guanidine (8)
(0.389 g, 1.907 mmol) at room temperature. The reaction
mixture was heated at 50 °C overnight. The solid was collected
and washed sequentially with CHCl3 and H2O to afford the
compound 13 as a light yellow solid (0.573 g, 1.288 mmol, 68%),
1
mp 220 °C. H NMR (300 MHz, DMSO-d6): δ 7.75(d, J ) 8.3
Hz, 1H), 7.69 (s, 1H), 7.55-7.40 (m, 3H), 7.15 (d, 7.9 Hz, 1H).
13C NMR:δ 170.54, 169.33, 160.26, 156.74, 136.75, 132.72,
131.15, 131.00, 130.73, 130.52, 129.64, 128.04. MS (m/z) 444
(MH+), 256, 227, 185. Anal. (C16H9Cl4N5O2) C, H, Cl, N.
1-(3,4-Dichlorophenyl)-2-methylsulfanyl-1H-imidazole-
4,5-dione (9). The reaction solution of 1-(3,4-dichlorophenyl)-
2-methylisothiourea (7) (6.6 g, 28.3 mmol) and triethylamine
(7.5 mL, 56.5 mmol) in 200 mL of dry CH2Cl2 was cooled to
-78 °C with an acetone/dry ice bath. To the solution was added
dropwise methyl oxalyl chloride (5.2 mL, 56.5 mmol). The
resulting brown reaction mixture was stirred at -78 °C for 2
h and quenched with water. The mixture was extracted with
CHCl3 three times and the CHCl3 extracts were combined,
dried over Na2SO4, and evaporated to dryness under the
reduced pressure. The crude product was purified by recrys-
tallization with hexanes/CHCl3 mixed solvent to yield 9 as
N-(3,4-Dichlorophenyl)-N′-(1-isopropyl-4,5-dioxoimi-
dazolidin-2-ylidene)guanidine (1). To the solution of dione
5 (0.712 g, 3.83 mmol) in 80 mL of dry CHCl3 was added
guanidine 8 (0.74 g, 3.64 mmol), and the reaction mixture was
heated in an oil bath at 50 °C for 16 h. The solid precipitates
were collected and washed sequentially with H2O, CH3OH, and
CHCl3 to afford the compound 1 as a light yellow solid (0.497
g, 40%), mp 233 °C. 1H NMR (600 MHz, DMSO-d6): δ 7.77 (d,
J ) 2.2 Hz, 1H), 7.68 (d, J ) 8.6 Hz, 1H), 7.31 (dd, J ) 8.6
and 2.2 Hz, 1H), 4.38 (m, 1H), 1.33 (d, J ) 6.7 Hz, 6H). 13C
NMR: δ 172.24, 170.65, 162.14, 160.08, 132.13, 131.76, 126.96,
125.00, 44.44, 20.54. IR: 3277, 3140, 1752, 1725, 1553, 1464,
1317, 996, 808, 754 cm-1. Anal. (C13H13Cl2N5O2): C, H, N.
1
yellow crystals (4.0 g, 16.12 mmol, 48%). H NMR (300 MHz,
CDCl3): δ 7.63 (d, J ) 8.7 Hz, 1H), 7.46 (d, J ) 2.4 Hz, 1H),
7.20 (dd, J ) 8.7 and 2.4 Hz, 1H), 2.76 (s, 3H). 13C NMR: δ
179.00, 158.00, 135.14, 134.19, 129.97, 131.67, 129.07, 126.36,
14.44. MS (m/z): 289 (M+), 286, 187, 159, and 124. Anal.
(C10H6Cl2O2S) C. H. Cl. N.
N-(3,4-Dichlorophenyl)-N′-(1-isopropyl-4,5-dioxoimi-
dazolidin-2-ylidene)-N′′-(tert-butylcarbonyl)guanidine
(1a). To the mixture of N-(3,4-dichlorophenyl)-N′-(1-isopropyl-
4,5-dioxoimidazolidin-2-ylidene)guanidine (1) (50 mg, 0.146
mmol) and DMF (5 mL) was added DMAP (1.8 mg), followed
by di-tert-butyl dicarbonate (127 mg, 0.58 mmol). The reaction
mixture was further stirred at room temperature overnight.
After being quenched with water, the mixture was extracted
with CHCl3, dried over Na2SO4, and concentrated. The crude
product was purified by recrystallization from EtOAc/CHCl3
to furnish 1a as a yellow needle (15.5 g, 24%), mp 226 °C. 1H
NMR (600 MHz, CDCl3): δ 7.81 (d, J ) 2.3 Hz, 1H), 7.50 (d,
J ) 8.6 Hz, 1H), 7.24-7.7.22 (dd, J ) 8.6 and 2.3 Hz, 1H),
4.42 (m, 1 H), 1.57 (s, 9H), 1.37 (d, J ) 4.6 Hz, 6H). 13C NMR:
δ 156.32, 134.82, 133.51, 131.45, 131.03, 126.77, 126.68,
123.55, 86.72, 45.69, 28.42, 20.35. MS m/z: 442 (M+), 390, 387,
331, 288, 248, 204. The compound was identical to that
prepared from the mixture WR182393 in melting point and
NMR.9,10
N-(3,4-Dichlorophenyl)-N′-(isobutylcarbonyl)-N′′-(1-
isopropyl-4,5-dioxoimidazolidin-2-ylidene)guanidine (1b).
Compound 1b was prepared by the same method as for
preparation of 1a, using pure compound 1 (1.400 g, 4.094
mmol), to yield a pale yellow solid (1.162 g, 2.629 mmol, 64%).
1H NMR (300 MHz, CDCl3) δ 13.10 (s, 1H), 11.13 (s, 1H), 7.81
(d, J ) 2.5 Hz, 1H), 7.50 (d, J ) 8.6 Hz, 1H), 7.21 (dd, J ) 8.6
and 2.5 Hz, 1H), 4.07 (d, J ) 6.9 Hz, 1H), 2.09-2.03 (m, 1H),
1.37 (d, J ) 6.9 Hz, 6H),1.00 (d, J ) 6.7 Hz, 6H). 13C NMR: δ
173.40, 168.24, 160.28, 155.91, 154.85, 134.28, 133.06, 131.12,
130.67, 126.45, 123.23, 73.97, 45.34, 27.64, 19.95, 18.91. MS
m/z: 442 (M+), 386, 352, 341, 314, 248, 179, 156, 114. Anal.
(C18H21Cl2N5O4): C, H, N.
N-(Benzylcarbonyl)-N′-isopropylguanidine (10). To the
solution of isopropylguanidine (12) (0.50 g, 4.95 mmol) in DMF
(20 mL) was added with stirring at -20 °C a solution of
dibenzyl dicarbonate (0.644 g, 2.25 mmol) in 20 mL of CHCl3.
The mixture was stirred for an additional 2 h after the addition
was completed and allowed to stir at room temperature
overnight. After being quenched with water, the mixture was
extracted with CHCl3, washed with water (4 × 100 mL), and
dried over Na2SO4. The solvent was removed under reduced
pressure to afford the product 10 as a colorless crystal (0.400
g, 1.702 mmol, 76%). 1H NMR (300 MHz, CDCl3) δ 7.40-7.25
(m, 5H), 5.08 (s, 2H), 3.57 (m, 1H), 1.22 (d, J ) 6.0 Hz, 6H).
13C NMR: δ 163.80, 160.47, 137.33, 128.17, 127.62, 127.45,
66.05, 43.20, and 22.63.
N-Isopropyl-N′-(tert-butylcarbonyl)guanidine (11). To
the solution of isopropylguanidine (12) (1.33 g, 13.17 mmol)
in DMF (30 mL) was added, in small portions, at room
temperature a solution of di-tert-butyl dicarbonate (1.435 g,
6.584 mmol) in 40 mL of CHCl3, and the reaction mixture was
stirred at room temperature overnight. After quenching with
water (20 mL), the mixture was extracted with CHCl3 three
times. The chloroform extracts were combined, washed with
water (4 × 50 mL), and dried over Na2SO4. The solvent was
evaporated to dryness under reduced pressure to afford the
pure crude product 11 as a colorless liquid (1.32 g, 6.58 mmol,
100%). The crude product was used for further reactions
1
without purification. H NMR (300 MHz, CDCl3): δ 3.51 (m,
J ) 6.3 Hz, 1H), 1.43 (s, 9H), 1.20 (d, 5.3 Hz, 6H). 13C NMR:
δ 63.81, 160.49, 77.84, 43.16, 29.70, and 22.88.
Isopropylguanidine (12). A solution of S-methylisothio-
urea sulfate (65.9 g, 250 mmol) in water (100 mL) was cooled
with an ice/salt bath. To the solution was added isopropyl-
amine (42.5 mL, 500 mmol) dropwise with stirring. The
reaction mixture was allowed to stir at room temperature for
16 h after the addition of amine was completed and was then
refluxed for 4-5 h. The solution was evaporated to dryness
under the reduced pressure and the residue was crystallized
from 95% EtOH, to give isopropylguanidine sulfate as colorless
needles (16.12 g, 81.01 mmol, 32%). 1H NMR (300 MHz,
D2O): δ 3.72 (m, 1H), 1.27 (d, J ) 6.4 Hz, 6H). 13C NMR: δ
N-(3,4-Dichlorophenyl)-N′-(benzylcarbonyl)-N′′-(1-iso-
propyl-4,5-dioxoimidazolidin-2-ylidene)guanidine (1c).
Compound 1c was prepared by the same method as the
preparation of 1a, using mixture WR182393 as starting
material to yield compound 1c as a pale yellow solid (20%),9
mp 235 °C. 1H NMR (CDCl3, 600 Hz) δ 7.81 (1H, s), 7.53 (1H,
d, J ) 8.6), 7.42-7.45 (5H, m), 7.23 (1H, d, J ) 8.6), 5.32 (2H,
s), 4.43 (1H, m), 1.39 (6H, d).
N-(3,4-Dichlorophenyl)-N′-ethylcarbonyl-N′′-(1-isopro-
pyl-4,5-dioxoimidazolidin-2-ylidene)guanidine (1d). To
158.45, 46.50, 24.11; IR: 1675, 1623, 1071 cm-1
.

6478 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Zhang et al.
the mixture of N-(3,4-dichlorophenyl)-N′-(1-isopropyl-4,5-di-
oxoimidazolidin-2-ylidene)guanidine (1) (0.050 g, 0.146 mmol)
in CHCl3 (5 mL) was added, at room temperature, first Et3N
(63 µL, 0.045 mmol) and then ethyl chloroformate (42 µL, 0.44
mmol). The reaction mixture was further stirred at room
temperature overnight. After being quenched with water, the
mixture was extracted with CHCl3, dried over Na2SO4, and
evaporated to dryness. The residue was purified by flash silica
gel column chromatography using 10% ethyl acetate in hex-
anes as eluent to furnish 1d as a white solid (0.0121 g, 20%),9
N-[1-(3,4-Dichlorophenyl)-4,5-dioxo-4,5-dihydro-1H-
imidazol-2-yl]-N′-isopropylguanidine (2). 1-(3,4-Dichlo-
rophenyl)-2-methylsulfanyl-1H-imidazole-4,5-dione (9) (1.89 g,
6.54 mmol) was dissolved in 10 mL of dried DMF and cooled
with an ice bath to 0 °C. To the solution was added dropwise
40 mL of DMF solution containing guanidine 12 (1.369 g,
13.554 mmol). The solution was stirred at room temperature
for 2 h after addition and then further stirred at 50 °C for 24
h. The solvent DMF was dried under vacuum and the crude
product was suspended in 30 mL of chloroform. The yellow
solid was collected and washed sequentially with 3 × 20 mL
of CHCl3 and 10 mL of methanol to afford compound 2 as a
white powder (1.588 g, 6.54 mmol, 71%), mp 245 °C. NMR
indicates that the compound exists in two tautomeric forms
in the CDCl3 solution. Major tautomer 1H NMR (600 MHz,
DMSO-d6): δ 8.74-8.69 (m, 1H), 7.79 (d, J ) 8.6 Hz, 1H), 7.76
(d, J ) 2.2 Hz, 1H), 7.44 (dd, J ) 8.6 and 2.2 Hz, 1H), 3.73 (m,
1H), 1.14 (d, J ) 6.2 Hz, 6H). 13C NMR: δ 168.60, 162.45,
161.15, 161.09, 134.01, 131.84, 131.48, 131.20, 130.29, 128.86.
IR: 1757, 1722, 1645, 1595, 1551, 1535, 1480, 1304, 997, 754
cm-1; MS (m/z): 342 (M+), 271, 229, 187, 127. Anal. (C13
H13Cl2N5O2‚0.25H2O) C, H, Cl, N.
1
mp 166.4 °C: H NMR (300 MHz, CDCl3): δ 7.80 (d, J ) 2.5
Hz, 1H), 7.54 (d, J ) 8.6 Hz, 1H), 7.23-7.7.19 (dd, J ) 8.6
and 2.5 Hz, 1H), 4.47-4.28(m, 3 H), 1.39 (t, J ) 7.1 Hz, 3H),
1.36 (d, J ) 7.0 Hz, 6H). 13C NMR: δ 173.40, 168.26, 160.28,
155.89, 154.65, 134.25, 133.06, 131.12, 130.67, 126.44, 123.22,
64.16, 45.35, 19.95, and 14.14. The compound was identical
to that prepared from the mixture WR182393 in melting point
and NMR.9,10
N-(3,4-Dichlorophenyl)-N′-(3-hexylcarbonyl)-N′′-(1-iso-
propyl-4,5-dioxoimidazolidin-2-ylidene)guanidine (1e).
Compound 1e was prepared by the same method as for 1a to
give yellow crystals (0.6 g, 31%). 1H NMR (300 MHz, CDCl3):
δ 7.84 (d, J ) 2.4 Hz, 1H), 7.53 (d, J ) 8.6 Hz, 1H), 7.23 (dd,
J ) 8.6 and 2.4 Hz, 1H), 4.49-4.40 (m, 1H), 4.32 (t, J ) 6.7
Hz, 2H), 1.82-1.72 (m, 3H), 1.58 (s, 5H), 1.40 (d, J ) 6.9 Hz,
6H), 0.93 (t, J ) 6.4 Hz, 3H). 13C NMR δ 173.10, 168.10, 160.50,
155.89, 154.82, 134.27, 133.07, 131.30, 130.67, 126.44, 123.20,
68.29, 45.35, 31.31, 28.38, 25.31, 22.48, 19.95, 13.99. MS (m/
z): 472, 470 (M+), 416, 368, 316, 260, 186, and 156. Anal.
(C20H25Cl2N5O4): C, H, N.
N-(3,4-Dichlorophenyl)-N′-(3-butenylcarbonyl)-N′′-(1-
isopropyl-4,5-dioxoimidazolidin-2-ylidene)guanidine (1f).
Compound 1f was also prepared by the same method as the
preparation of 1a, using pure compound 1 (100 mg) as starting
material to yield compound 1f as a pale yellow solid (66 mg,
52%). 1H NMR (300 MHz, CDCl3): δ 7.83 (d, J ) 2.5 Hz, 1H),
7.53 (d, J ) 8.6 Hz, 1H), 7.23 (dd, J ) 8.6 and 2.5 Hz, 1H),
5.89-5.78 (m, 1H), 5.24-5.16 (m, 2H), 4.49-4.42 (m, 1H), 4.40
(t, J ) 7.1 Hz, 2H), 2.54 (q, J ) 6.8 Hz, 2H),1.40 (d, J ) 6.9
Hz, 6H). 13C NMR δ 173.10, 168.10, 160.18, 155.95, 154.81,
134.27, 133.07, 132.57, 131.14, 130.68, 126.45, 123.22, 118.44,
66.90, 45.37, 32.78, 19.95. MS (m/z): 442, 440 (M+), 351, 267,
191, 155. Anal. (C18H19Cl2N5O4): C, H, N.
Preparation of N-(3,4-Dichlorophenyl)-N′-(5-methyl-
2-oxo-1,3-dioxol-4-yl)methyl-N′′-(1-isopropyl-4,5-dioxoim-
idazolidin-2-ylidene)guanidine (1g). A dimethylformamide
suspension of compound 1 (200 mg, 0.58 mmol) was treated
with (5-methyl-1,3-dioxolene-2-oxo-4-yl)methyl p-nitrophenyl
carbonate19-22 (204 mg, 0.69 mmol) and DMAP (14 mg, 0.11
mmol). The reaction mixture was stirred at room temperature
overnight and partitioned in CHCl3/H2O. The water layer was
extracted several times with chloroform. The chloroform
extracts were combined, washed successively with brine and
water, dried over Na2SO4, filtered, and concentrated to yield
crude product as yellow gum (258 mg). The crude product
solidified upon treatment in ethyl acetate/hexane (1:1) to give
yellow solid 1g (91 mg, 31%), mp 144 °C. 1H NMR (CDCl3,
300 Hz) δ 7.73 (d, J ) 2.4 Hz, 1H), 7.50 (d, J ) 8.6 Hz, 1H),
7.20 (dd, J ) 8.6, J ) 2.4 Hz, 1H), 5.01 (s, 2H), 4.41 (m, 1H),
2.23 (s, 3H), 1.36 (d, J ) 6.9 Hz, 6H). MS (m/z): 497 (M+).
Anal. (C19H17N5O7Cl2) C, H, N.
1
Minor tautomer H NMR (600 MHz, DMSO-d6): δ 8.35 (s,
1H), 7.99 (s, 1H), 7.88 (d, J ) 7.3 Hz, 1H), 7.42 (dd, J ) 8.6
and 2.2 Hz, 1H), 1.09 (d, J ) 6.4 Hz, 6H).
The signals of one of the aromatic protons and the methyl-
ene proton of the isopropyl function cannot be identified. The
former was overlapping with the signals of the major tautomer
and the latter was too weak and too broad to be identified.
Compound 2 was also prepared by an alternative method:
A solution of 2a (0.424 g, 0.959 mmol) in dry CHCl3 (60 mL)
was cooled with an ice bath. To the solution was added
dropwise concentrated HCl (2 mL), and the mixture stirred
at 0 °C for 1 h and then at room temperature overnight.
Saturated Na2CO3 was added to basicify the mixture. The
white solid was collected, washed successively with CHCl3 and
water, and dried under reduced pressure to afford 2 as a white
solid (0.292 g, 0.854 mmol, 89%), mp 244 °C. The NMR spectra
of compound 2 made by the two methods are identical.
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(tert-butylcarbonyl)guani-
dine (2a). To the solution of the crude dione 9 (0.7 g, 2.5 mmol)
in 25 mL of dry CHCl3 was added N-isopropyl-N′-(tert-
butylcarbonyl)guanidine (11) (0.5 g, 2.5 mmol). The reaction
mixture was heated at 50 °C for 16 h. The reaction was
quenched with water, extracted with CHCl3, and dried over
Na2SO4. The solvent was removed in vacuo and the crude
product was purified with a silica gel column using 3% EtOAc/
CHCl3 as eluent to yield 2a as a white solid (0.25 g, 22%), mp
227 °C. 1H NMR (300 MHz, CDCl3): δ 7.55 (d, J ) 2.4 Hz,
1H), 7.53(d, J ) 8.6 Hz, 1H), 7.27 (dd, J ) 8.6 and 2.4 Hz,
1H), 4.10 (m, 1H), 1.54 (s, 9H), 1.24 (d, J ) 6.6 Hz, 6H). 13C
NMR: δ 170.50, 168.40, 159.34, 156.09, 153.00, 132.51, 132.09,
131.34, 130.38, 128.62, 125.62, 85.59, 45.05, 29.70, 22.34. IR:
2979, 2930, 1766, 1729, 1532, 1474, 1142, 775, 734 cm-1. MS
(m/z): 444 (M++1). Anal. (C18H21N5O4Cl2) C, H, N.
An alternative method also has been successfully achieved
by the following procedure: To the suspension of N-[1-(3,4-
dichlorophenyl)-4,5-dioxo-4,5-dihydro-1H-imidazol-2-yl]-N′-iso-
propylguanidine (2) (27 mg, 0.079 mmol) in CHCl3 (5 mL) was
added first p-(dimethylamino)pyridine (DMAP, 0.964 mg,
0.0079 mmol), followed by the addition of di-tert-butyl dicar-
bonate (19 mg, 0.087 mmol)/CHCl3 solution (10 mL), and the
reaction mixture was stirred at room temperature overnight.
The reaction was quenched with water and extracted with
CHCl3 three times. The chloroform extracts were combined,
dried over Na2SO4, and concentrated. The residue was purified
by flash silica gel column chromatography and eluted with 10-
30% diethyl ether in hexanes to furnish 2a as crystals (11 mg,
31%). The NMR spectrum of the compound prepared by the
alternative method is identical to the sample prepared by the
condensation of compounds 9 and 11.
N-(3,4-Dichlorophenyl)-N′-(benzyloxyethylcarbonyl)-
N′′-(1-isopropyl-4,5-dioxoimidazolidin-2-ylidene)guani-
dine (1h). Compound 1h was prepared by the same method
of 1a to yield compound 1h as a white crystal (0.4 g, 22%). 1H
NMR (300 MHz, CDCl3) δ 13.17 (s, 1H), 11.08 (s, 1H), 7.81 (d,
J ) 2.4 Hz, 1H), 7.51 (d, J ) 8.6 Hz, 1H), 7.39-7.29 (m, 5H),
7.21 (dd, J ) 8.6 and 2.4 Hz, 1H), 4.61 (s, 2H), 4.49-4.42 (m,
3H), 3.78 (t, J ) 4.7 Hz, 2H), 1.39 (d, J ) 6.9 Hz, 6H). 13C
NMR δ 172.50, 167.60, 166.10, 156.04, 155.30, 155.00, 138.00,
137.47, 134.31, 133.04, 131.21, 130.66, 128.54, 128.48, 127.94,
127.85, 127.80, 126.47, 123.25, 73.30, 71.35, 67.16, 66.66,
64.56, 61.92, 45.40, 19.94,. MS (m/z): 522, 520 (M+), 492, 432,
370, 368, 298, 232, and 181.
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(3-isobutylcarbonyl)guani-

Synthesis of Antimalarial Imidazolidinediones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6479
dine (2b). Compound 2 (25 mg, 0.073 mmol) in 4 mL of dry
CHCl3 was added dry triethylamine (45.8 µl, 0.329 mmol), and
the mixture was stirred at room temperature for 30 min. The
suspension was cooled with an ice bath, and isobutyl chloro-
formate (38 µL, 0.292 mmol) was added dropwise with stirring.
After the addition, the ice bath was removed and the reaction
mixture was further stirred at room temperature for 2 h. The
clear CHCl3 solution was washed with H2O three times, dried
over Na2SO4, and evaporated under reduced pressure to small
volume. The crude product was collected and purified by silica
gel flash chromatography (2% ethyl acetate/chloroform) to give
the pure isobutyl derivative (2b) as a white solid (15 mg, 47%
yield), mp 260 °C. 1H NMR (600 MHz, CDCl3): δ12.44 (s, 1H),
7.55 (d, J ) 2.4 Hz, 1H), 7.54 (d, J ) 8.6 Hz, 1H), 7.28 (dd, J
) 8.6 and 2.4 Hz, 1H), 4.12 (m, 1H), 4.02 (d, J ) 6.8 Hz, 2H),
2.05 (m, 1H), 1.24 (d, J ) 6.6 Hz, 6H), 0.99 (d, J ) 6.7 Hz,
6H). 13C NMR: δ 170.73, 168.08, 159.18, 155.88, 154.10,
132.49, 132.10, 131.26, 130.35, 128.58, 125.6173.91, 45.15,
27.57, 22.20, 18.84. IR: 1769, 1723, 1624, 1546, 1472, 1387,
1308, 1242, 1205, 1059, 754 cm-1. MS (m/z): 442 (M+), 376,
316, 314, 272, 202. Anal. (C18H21Cl2N5O4): C, H, N, Cl. The
compound was identical to that prepared from the mixture of
WR182393 in melting point and NMR.9
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(3-butenylcarbonyl)guani-
dine (2f). Compound 2f was prepared by the same method
for the preparation of 2a, using pure compound 2 (0.200 g,
585 mmol) and butenyl chloroformate as starting materials
1
to yield compound 2f as a white color solid (98 mg, 38%). H
NMR (300 MHz, CDCl3) δ 12.46 (s, 1H), 9.22 (s, 1H), 7.56-
7.53 (m, 2H), 7.30-7.26 (dd, J ) 8.6 and 2.4 Hz, 1H), 5.85-
5.72 (m, 1H), 5.20-5.13 (m, 2H), 4.29 (t, J ) 6.8 Hz, 2H), 4.16
(m, 1H), 2.47 (q, J ) 6.8 Hz, 2H), 1.24 (d, J ) 6.6 Hz, 6H). 13
C
NMR: δ170.76, 168.08, 159.19, 155.88, 153.96, 132.63, 132.53,
131.26, 130.40, 128.61, 125.65, 118.32, 66.60, 45.23, 32.74,
22.27; MS (m/z): 440 (M+), 388, 338, 269, 245, 180, 159. Anal.
(C18H19Cl2N5O4‚H2O) C. H. N.
Antimalarial Studies. (A) Sporozoite-Induced Test in
Mouse.23 The causal prophylactic antimalarial activity of the
new carbamate derivatives were assessed in a sporozoite-
challenged mouse model according to the following protocol,
and the results are shown in Tables 1 and 2.
(1) Raising and Infecting the Mosquitoes. Cages of
noninfected mosquitoes (Anopheles stephensi) are kept in a
room maintained at 26.6 °C. They are allowed to feed on
noninfected mice to obtain enough blood needed to produce
eggs. Jars with wet cotton and moist paper towels are placed
in the mosquito cages. Female mosquitoes lay their eggs on
the moist paper towels. The eggs are collected and placed in
enamel pans containing water. The eggs hatch and develop
into larvae and then pupae. These stages are fed a liver powder
suspension (2.5% liver powder in water) until the adults
emerge. When the pupae have fully developed they are placed
in empty jars, which are then placed in empty mosquito cages.
After the adult mosquitoes emerge from the pupae stage the
jars are removed. The cages containing mosquitoes to be
infected are transferred to a room maintained at 21 °C.
Donor mice used to infect the mosquitoes are infected with
25,000 P. yoelii parasitized erythrocytes. The mosquitoes are
allowed to feed on these malaria-infected mice on day 4 of their
infection, when the parasitemia is low. The mice are anesthe-
tized with Ketamine and placed on top of the mosquito cage,
allowing the females to take an infected blood meal. The
mosquitoes are maintained in this cool room for 17 days, when
they are then taken for sporozoite isolation. During the last 4
days, a solution of PenStrep is fed to the mosquitoes to kill
the bacteria in their gut. These mosquitoes are vacuumed into
a plastic baggie that is heat-sealed. This bag of mosquitoes is
placed on a freezing table to stun the mosquitoes. The bag is
opened and the female mosquitoes are collected while the
males are discarded. These infected females are ground with
a mortar and pestle that has 0.1 mL of saline added. An
additional 20 mL of saline is added to the mortar, and the
suspension is filtered to remove large pieces of mosquitoes.
The sporozoites in the saline suspension are then counted and
diluted to get an inoculum of 250 000 sporozoites per 0.1 mL.
This is then inoculated intraperitoneally into the test mice.
(2) Test Procedure. Mice are inoculated intraperitoneally
with 250 000 sporozoites of P. yoelii on day 0. Blood examina-
tions and weights are taken at frequent intervals, at least
twice a week. The days of examination relative to day 0 vary
to avoid weekend work. The test is run for a minimum of 31
days. All mice alive on the last day with negative blood films
are considered cured. Mice losing >20% of their body weight
at any time are sacrificed.
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(benzylcarbonyl)guanidine (2c).
To the solution of the crude dione 9 (0.10 g, 0.346 mmol) in 25
mL of dry CHCl3 was added N-(benzylcarbonyl)-N′-isopropyl-
guanidine (10) (0.122 g, 0.519 mmol) at room temperature. The
reaction mixture was heated at 50 °C for 18 h and then
quenched with water. The mixture was extracted with chlo-
roform and dried over Na2SO4. The CHCl3 was removed in
vacuo and the crude product was recrystallized from hexanes/
CHCl3 to yield compound 2c as light yellow crystals (51 mg,
1
0.11 mmol, 31%), mp 211 °C. H NMR (300 MHz, CDCl3): δ
7.54-7.51(m, 2H), 7.41 (s, 5H), 7.27-7.23 (dd, J ) 8.6 and
2.4 Hz, 1H), 5.25 (s, 2H), 4.12 (m, 1H), 2.46 (d, J ) 6.6 Hz,
6H). 13C NMR: δ 170.83, 155.86, 153.87, 133.89, 132.56,
132.19, 131.22, 130.42, 129.21, 128.93, 128.65, 128.61, 125.60,
69.24, 45.25, 22.28. MS (m/z): 476 (M+), 370, 316, 314, 236.
Anal. (C21H19Cl2N5O4) C, H, N, Cl.
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(ethylcarbonyl)guanidine (2d).
Compound 2d was prepared by two methods. Method A follows
the same procedure for the preparation of 2b, using pure
compound 2 (0.788 g, 2.304 mmol) as starting material to yield
1
compound 2d as a white solid (0.632 g, 66%), mp 257 °C. H
NMR (CDCl3, 600 Hz) δ 7.56 (d, J ) 2.4 Hz, 1H), 7.55 (d, J )
8.3 Hz, 1H), 7.29 (dd, J ) 2.4 Hz, J ) 8.3 Hz, 1H), 4.32 (q, J
) 7.1 Hz, 2H), 4.16 (m, 1H), 1.39 (t, J ) 7.1 Hz, 3H), 1.25 (d,
J ) 6.6 Hz, 6H). Anal. (C16H17N5O4Cl2) C, H, N.
Method B adapted an alternative method for the preparation
of 2c as follows. To the solution of the crude dione 9 (0.413 g,
1.429 mmol) in 50 mL of dry CHCl3 was added N-isopropyl-
N′-(ethylcarbonyl)guanidine (0.206 g, 1.191 mmol). The reac-
tion mixture was heated at 50 °C for 48 h. The reaction was
quenched with water, extracted with chloroform, and dried
over Na2SO4. The solvent was removed in vacuo and the crude
product was purified with a silica gel column using 3% EtOAc/
CHCl3 as eluent to yield 2d as a white solid (0.115 g, 20%).
The melting point and NMR of compound 2d made by both
procedures and from the mixture WR182393 9 are identical.
N-[1-(3,4-Dichlorophenyl)-4,5-dioxoimidazolidin-2-
ylidene]-N′-isopropyl-N′′-(1-hexylcarbonyl)guanidine (2e).
Compound 2e was prepared by the same method for the
preparation of 2a, using pure compound 2 (0.800 g, 2.34 mmol)
and hexyl chloroformate as starting materials to yield com-
pound 2e as a white color crystal (0.354 g, 32%). 1H NMR (300
MHz, CDCl3) δ 12.45 (s, 1H), 7.57-7.54 (m, 2H), 7.31-7.27
(dd, J ) 8.5 and 2.4 Hz, 1H), 4.25 (t, J ) 6.8 Hz, 2H), 4.12 (m,
1H),1.74 (m, 2H), 1.35 (m, 6H), 1.25 (d, J ) 6.6 Hz, 6H), 0.93
(t, J ) 6.4 Hz, 3H). 13C NMR: δ170.78, 168.50, 159.20, 155.93,
154.15, 132.56, 132.18, 131.50, 130.41, 128.62, 125.61, 31.31,
28.38, 25.32, 22.49, 22.30, 14.00. MS (m/z): 470 (M+), 418, 416,
368, 342, 314, 272, 230, 174.
Nontreated, negative controls are run with every experiment
to validate the infectivity of the sporozoites. While these
sporozoites usually produce patent infections, a small number
of mice remain blood negative. Caution must be taken when
judging a compound as prophylactic when the patency rate in
the negative controls is less than 100%.
Positive control groups are included occasionally, and they
are treated with either primaquine or tafenoquine.
Routinely, 20 000 red blood cells (RBCs) are examined in a
thin blood film before an animal is judged negative. The
sensitivity of this technique is 0.01% infected RBCs. Each
compound was ground with a mortar and pestle and suspended

6480 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Zhang et al.
Table 4. In Vitro Antimalarial Activity against
Exoerythrocytic P. yoelii in Hepatocytes
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
dose
compd
(µg/mL) schizonts/well means ( SD % inhibn
References
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
8, 9, 14
100, 80, 87
1, 1, 0
48, 37, 53
96, 85, 71
106, 124, 103
0, 2, 3
60, 49, 53
3, 2, 4
10 ( 3.2
89 ( 4
93
40
99
69
43
25
99
63
98
10
87
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0.66 ( 0.5
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Four-week-old male CD-1 mice, purchased from Charles
River and weighing about 16-17 g were placed five per cage
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Acknowledgment. This paper has been reviewed
by the Walter Reed Army Institute of Research. There
is no objection to its presentation and/or publication. The
opinions or assertions contained herein are the private
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