Malaria causal prophylactic activity of imidazolidinedione derivatives

Article abstract of DOI:10.1021/jm7009815

A series of acid-stable carboxamide derivatives of 2- guanidinoimidazolidinedione (5a-c and 6a-c) were prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate to good causal prophylactic activity in mice infec

Full text of DOI:10.1021/jm7009815

6
226
J. Med. Chem. 2007, 50, 6226-6231
Malaria Causal Prophylactic Activity of Imidazolidinedione Derivatives
†
†
‡
£
§
§
†
Jian Guan, Xihong Wang, Kirsten Smith, Arba Ager, Montip Gettayacamin, Dennis E. Kyle, Wilbur K. Milhous,
†
†
,†
Michael P. Kozar, Alan J. Magill, and Ai J. Lin*
DiVision of Experimental Therapeutics, Walter Reed Army Institute of Research, SilVer Spring, Maryland 20910, UniVersity of Miami,
South Campus, Miami, Florida 33177, USAMC-AFRIMS, Bangkok, Thailand, and UniVersity of South Florida, Tampa, Florida 33612
ReceiVed August 7, 2007
A series of acid-stable carboxamide derivatives of 2-guanidinoimidazolidinedione (5a-c and 6a-c) were
prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate
to good causal prophylactic activity in mice infected with Plasmodium yoelii sporozoites. Three compounds
were further tested for causal prophylactic activity in Rhesus monkeys infected with Plasmodium cynomolgi
sporozoites, and all showed a delay in patency from 13 to 40 days at 30 mg/kg/day × 3 days by IM dosing.
Two out of four compounds tested for radical curative activity in Rhesus showed cure at 30 mg/kg/day ×
3
days. The other two compounds showed delay in relapse from 16 to 68 days. Conversion of new
carboxamides (5 and 6) to s-triazine derivatives (7) was demonstrated in mouse and human microsomal
preparations and in rat plasma. The results suggest the metabolites, s-triazine derivatives 7, may be the
active species of the new carboxamides 5a-c and 6a-c prepared in this study.
Introduction
Scheme 1
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 to 2 million people die due to
malaria.1 The situation is rapidly worsening, mainly due to
unavailability of effective drugs and development of drug
resistance to the existing first line drugs, such as chloroquine
and pyrimethamine.5-10 In addition to the drug resistance of
the first line antimalarials drugs, the usefulness of many newer
antimalarial drugs is 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, stopping
-4
in organic solvents, thus facilitating the purification and
improving the oral absorption, a series of carbamate derivatives
1
6-18
of IZ, such as 3 and 4, was prepared.
Carbamates 3, the
most active compounds of this class, protect monkeys infected
with P. cynomolgi sporozoites at a dose of 10 mg/kg × 3 days
by IM dosing. Nevertheless, the IZ derivatives 1-4 showed
very weak or no in vitro cell growth inhibition against blood
stage malaria P. falciparum and were inactive in the Thompson
mouse test against P. berghei, a blood stage rodent malaria.1
To the best of our knowledge, this is the first class of
antimalarial agents possessing activity against the liver stage
malaria exclusively. From the drug resistant development point
of view, drugs used exclusively for prophylaxis have much less
chance of exposure to parasites than those used for treatment
and, thus, have fewer problems of developing drug resistance.
However, compound 3 showed poor activity by oral administra-
tion and delayed the patency (first day the parasite can be
detected in blood smears after infection) of monkeys treated
for only 3 days versus the untreated control. Oral efficacy is an
essential consideration in the search for prophylactic drugs. The
lack of oral activity of carbamates 3 and 4 in Rhesus testing
may be attributed to the hydrolysis of the carbamate group in
stomach acid, leading to generation of insoluble parent com-
pounds 1 and 2.
malaria before blood stages emerge and cause clinical disease.
With the exception of quinoline esters,11 only the 8-amino-
1
2-14
quinoline drugs such as primaquine or tafenoquine
have
6-18
activity against the liver stages of Plasmodium ViVax and P.
falciparum malarias. However, the 8-aminoquinoline drugs
cause serious lethal hemolytic side effects in glucose-6-
8
phosphate dehydrogenase (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
endemic areas of the world.
Recently, a series of new 2-guanidinoimidazolidinedione (IZ)
derivatives (1 and 2, Scheme 1) was demonstrated to possess
causal prophylactic antimalarial activity in Rhesus monkeys
15
infected with P. cynomolgi sporozoites. IZ derivatives 1 and
are sparingly soluble in water or common organic solvents,
2
and the prophylactic activity is demonstrated only when the
a
compounds are administered by subcutaneous (SC ) or intra-
muscular (IM) injection, but not orally. To improve the solubility
To overcome the acid instability of carbamates, a series of
acid-stable carboxamide derivatives of 1 and 2, such as 5a-c
*
To whom correspondence should be addressed. Tel.: 301-319-9084;
Fax: 301-319-9449. E-mail: ai.lin@na.amedd.army.mil.
†
Walter Reed Army Institute of Research.
University of Miami.
USAMC-AFRIMS.
University of South Florida.
‡
a
Abbreviations: Patency: first day the parasite can be detected in blood
£
smears after infection; CQ: chloroquine; PO: oral administration; SC:
subcutaneous injection; IM: intramuscular injection.
§
1
0.1021/jm7009815 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/30/2007

ActiVity of Imidazolidinedione DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6227
Scheme 2
was produced when highly reactive acyl chlorides were used.
As the solubility of the IZ Mixture is sparingly soluble in
chloroform, 5% dimethylformamide (DMF)/chloroform was
used as the solvent to facilitate the reaction. Again, only 6c
was isolated when IZ mixture was treated with butyric
anhydride. Compound 5c was prepared using pure 1 and
trimethylacetic anhydride as the starting materials.
Since 5a-c and 6a-c are derivatives of 1 and 2, the structural
characterization of 5a-c and 6a-c was based on differences
in NMR spectra between compounds 1 and 2. The chemical
shifts of the methylene proton (-N-CH-) of the isopropyl
group in 5a-c and 6a-c are distinctively different, with the
former being more downfield shifted than the latter by ∼0.3
1
6
ppm. Furthermore, the Ha proton signal of compounds 5a-c
(
Scheme 1) also resonances ∼0.30 ppm downfield to that of
the corresponding proton of 6a-c. Detailed chemical shift
differences between derivatives of compound 1 and 2 were
discussed in the earlier publications of this series.
1
6,17
Experimental Section
Melting points were determined on a Mettler FP62 melting point
apparatus and are uncorrected. Analytical thin-layer chromatography
(TLC) was performed using HPTLC-HLF normal phase 150 micron
silica gel plates (Analtech, Newark, DE). Visualization of the
developed chromatogram was performed by UV absorbance or by
staining with iodine vapor. Liquid chromatography was performed
using a Horizon HPFC System (Biotage, Charlottesville, VA) with
Flash 25M or 40M cartridges (KP-Sil Silica, 32-63 µm, 60 Å).
Preparative TLC was performed using silica gel GF tapered
1
13
uniplates (Analtech, Newark, DE). H NMR and C NMR spectra
were recorded in deuteriochloroform, unless otherwise noted, on a
Bruker Avance 300 and 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 analytical results obtained were within
(0.4% of the theoretical values.
and 6a-c, was prepared in this study. The new compounds were
tested against exoerythrocytic P. yoelii in mice and P. cynomolgi
in Rhesus monkeys. In vitro metabolic stability of the new com-
pounds in human and mouse microsomal preparations were
performed.
Chemistry
Preparation of Compound 5a. To a suspension of compound
A series of carboxamide derivatives 5a-c and 6a-c were
prepared as acid-stable analogues of 3 and 4, respectively
1
(5 g, 14.6 mmol) in 100 mL of CHCl
Et N (4.1 mL, 2 equiv) and DMAP (179 mg, 0.1 equiv). The
reaction mixture was cooled to 0 °C with an ice bath, and
-ethylbutyryl chloride (3 mL, 1.5 equiv) was added dropwise. The
reaction mixture was stirred at room temperature overnight and
washed with water, and the chloroform layer was dried over Na
3
was added, with stirring,
3
(
Scheme 2). Carboxamide derivatives 5a-c and 6a-c were
prepared according to the same method as that used for the
2
preparation of carbamate derivatives 3 and 4,1
6-18
using acyl
chloride or anhydride instead of alkyl chloroformates as
2
-
reagents. IZ mixture (mixture of compounds 1 and 2,
SO and evaporated to dryness under the reduced pressure. The
4
16
WR182393) was obtained by treatment of chloroproguanil with
residue was applied to a silica gel flash column and eluted with
diethyloxalate using NaOCH2CH3 as the catalyst. Compounds
2% EtOAc /CHCl
from hexane/CHCl
3
to give a white solid, which was recrystallized
to get the desired compound as a light-yellow
5
a and 6a were obtained by treatment of IZ mixture with 1.1
3
solid (2.6 g, 40%); mp 206.5-208.4 °C. MS, NMR, and elemental
analysis data are available in the Supporting Information, S-1.
Compound 5a was also prepared using IZ Mixture as the starting
material, with a low yield (15%); mp 206-208 °C. The NMR and
MS spectra of the product are identical to those of 5a prepared
from pure compound 1.
Compound 5b was prepared according to the same procedure
as that for the preparation of compound 5a, using pure compound
1 and isobutyryl chloride as staring materials to give a 26% yield
of pale-yellow crystals; mp 188-190 °C. MS, NMR, and elemental
analysis data of 5b are available in the Supporting Information,
S-1.
Compound 5c was prepared using trimethylacetic anhydride as
the acylating agent to yield 23% of the desired product after
purification; mp 183.3-190.7 °C. MS, NMR, and elemental analysis
data are available in the Supporting Information, S-1.
Preparation of Compound 6a. To the suspension of IZ mixture
molar equiv of ethylbutyryl chloride under the catalysis of
triethylamine and 4-N,N-dimethylaminopyridine (DMAP). The
crude product was purified using a silica gel column to give a
mixture of 5a and 6a. Due to the close structural similarity of
5
a and 6a, their Rf values in various solvent systems are almost
identical. Thus, separation of 5a and 6a by column chroma-
tography was an impractical option. NMR spectra indicated that
the product ratio of 5a and 6a is 1 to 2. Since compound 6a is
more soluble than 5a in organic solvents, the separation was
achieved by washing the mixture with an EtOAc/CHCl3 (2:1
v/v) mixed solvent.
The same procedure was employed to prepare isobutyryl
carboxamide 5b and 6b using IZ mixture as the starting
material. However, only 6b was obtained as a major product,
and 5b had to be prepared from pure compound 1, the synthesis
1
7,18
of which was described in our previous publication.
(2.0 g, 5.85 mmol) in 20 mL of dry chloroform were added
Anhydrides instead of acyl chlorides were used in the prepara-
tion of 5c and 6c because a spectrum of unknown byproducts
triethylamine (2.5 mL), DMAP (50 mg, 0.4 mmol), and 2-ethyl-
butyryl chloride (2.52 mL, 18.3 mmol), successively under the ice

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228 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Guan et al.
Table 1. Causal Prophylactic Activity in P. Yoelii
Sporozoite-Challenged Mouse and Metabolic Stability in Human and
Mouse Microsomal Preparations
To evaluate the radical curative properties, daily blood samples
were followed for 21 days, 3 times per week for 4 weeks, and then
2
times weekly until 100 days after the last day of test compound
metabolic stability
administration. Parasite clearance should occur in all animals treated
with chloroquine. Relapse was expected in the control group.
Relapse in the treated group indicates failure of the test compounds.
Monkeys that showed no relapse after 100 days were considered
radically cured. Relapses of the control monkeys were treated with
chloroquine once daily for 7 days, and they were observed for the
second relapse. Relapse in experimental animals and the second
relapse of the control monkeys were treated with the standard 7
day oral CQ and primaquine (1.78 mg base/kg). After standard
treatment, blood smears were monitored daily for 4 consecutive
days of negative results and 2 times weekly for 2 weeks. The results
are shown in Table 3.
t
1/2 (min)
dose
total
# protected/
# mice used
at day 6
compound (mg/kg)/ # days dose
#
day
treated (mg) route
human mouse
5
a
b
c
10
5
3
3
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
3
3
3
30
15
7.5
480
120
30
480
120
30
160
40
10
160
40
10
160
40
10
5
2.5
480
120
30
PO
PO
PO
PO
PO
PO
4/5
1/5
2/5
1/5
2/5
2/5
0/5
0/5
1/5
5/5
4/5
2/5
5/5
5/5
2/5
5/5
5/5
5/5
3/5
1/5
2/5
2/5
3/5
>60
>60
>60
<10
<10
<10
>60
2
.5
5
5
6
6
6
160
40
4
1
0
0
160
>60
<10
<10
<10
4
1
0
0
4. Metabolic Stability Study. The metabolic stability assay
a
b
c
160
sample preparation was performed in a 96 well plate on a TECAN
4
1
0
0
Genesis robotic sample processor following WRAIR SOP SP 01-
02. All incubations were carried out in 0.1 M sodium phosphate
160
4
1
0
0
buffer (pH 7.4) in the presence of a NADPH-regenerating system
(NADP sodium salt, MgCl ‚6H O, and glucose-6-phosphate). The
2
2
160
test drug (1 µM), microsomes (0.5 mg/mL total protein), buffer,
and the NADPH-regenerating system were warmed to 37 °C, and
the reaction was initiated by the addition of glucose-6-phosphate
dehydrogenase (G6PD). Samples were quenched at 0, 10, 30, and
60 min using an equal volume of cold methanol. Samples were
centrifuged to pellet the proteins, and the supernatant was analyzed
by LC-MS/MS using fast LC gradient or isocratic methods.
Chromatograms were analyzed using the mass spectrometry
software Xcalibur QuanBrowser. Concentrations of the parent drug
remaining at each time point were calculated using the unknown
peak areas and corresponding calibration curves. If sufficient
sensitivity was not obtained for the test drugs at 1 µM, 10 µM
drug and 1 mg/mL total microsomal protein were used. In order to
calculate the half-life, a first-order rate of decay was assumed. A
plot of the natural log (ln) of the drug concentration versus time
was generated, where the slope of that line was -k. The half-life
was calculated as 0.693/k.
4
1
0
0
5
2
.5
7
a
160
PO
>60
>60
4
1
0
0
bath temperature. The reaction mixture was gradually warmed up
to room temperature. After stirring overnight, the mixture was
quenched with water and extracted with CHCl
chloroform extracts were combined, washed with water three times,
and dried over Na SO4. The solvent was filtered and evaporated
3
three times. The
2
to dryness under reduced pressure. The crude product was purified
by flash silica gel column chromatography using 35% EtOAc/
hexane as the eluent to give a white product. Recrystallization from
EtOAc/CHCl yielded pure compound 6a as white needle crystals
3
700 mg, 26%); mp 160 °C. MS, NMR, and elemental analysis
For metabolite identification, samples were prepared as described
above with human liver microsomes. Additional samples were
prepared with each drug using mouse, rat, and Rhesus monkey liver
microsomes. Samples were separated using a LC gradient method
and analyzed by full-scan LC-MS and LC-MS/MS. Parent com-
pounds and putative metabolites were all fragmented, and these
MS/MS experiments were used in combination with the no-NADPH
control experiments to confirm the assignment of peaks as
metabolites. These MS/MS data were also used to do preliminary
structural elucidation.
(
data of 6a are available in the Supporting Information, S-1 and
S-2.
Compound 6b and 6c were obtained following the same
procedure as that for 6a by treatment of IZ mixture with isobutyryl
and butyryl anhydride, respectively. MS, NMR, and elemental
analysis data of 6b and 6c are available in the Supporting
Information, S-1 and S-2.
Antimalarial Studies. 1. Plasmodium yoelii Sporozoite-
Induced Test in Mice (SM Test). The causal prophylactic
antimalarial activity of the new derivatives was assessed in a P.
yoelli sporozoite-challenged mouse model by Dr. Arba Ager of the
University of Miami, Miami, FL. The detailed procedure of
sporozoites harvest and drug tests are disclosed in the previous
The results are shown in Table 1.
. Plasmodium cynomolgi Sporozoite-Induced Test in Rhesus
Monkeys. The causal prophylactic and radical curative antimalarial
activity of the new derivatives 5a and 6a was assessed in a P.
cynomolgi sporozoite-challenged Rhesus monkey model. The de-
tailed procedure of sporozoites harvest and drug tests are described
in the previous reports.16 The results are shown in Table 2.
Results and Discussion
By single-dose oral treatment, all three carboxamide deriva-
tives 6a-c exhibited profound activity in the P. yoelii sporo-
zoite-challenged mouse model with a minimum active dose
16-18
reports of this series.
(MAD) of <10 mg/kg. In contrast, 5a-c showed much weaker
2
causal prophylactic activity than that of 6a-c in the same test,
with MAD > 160 mg/kg. However, when 5a-c were admin-
istered once a day for 3 consecutive days (-1, 0, +1 day), start-
ing a day before the sporozoites challenge, the MAD of 5a,
5b, and 5c improved to 7.5, <30, and >480 mg/kg, respectively
3
. Radical Cure Test in Rhesus Monkeys. Assessment of
(Table 1). Likewise, triazine 7a showed weak single-dose oral
activity, but substantially better efficacy was observed when
the mice were treated for 3 consecutive days (Table 1).
Selected new compounds 5a, 5c, and 6a were further tested
for causal prophylactic (CP) and radical cure (DB) activities in
Rhesus moneys infected with P. cynomolgi sporozoites. For a
causal prophylactic test, the drug was administered by IM once
a day for three consecutive days, starting a day before the
parasites’ inoculation. Blood smears were taken to monitor the
development of parasitemia formation during the course of
radical curative activity of the test compounds was carried out using
the monkeys’ developed parasitemia during the causal prophylactic
experiments when the test compounds showed no or weak activity.
Monkeys were treated with chloroquine (10 mg/kg/day) orally for
7
consecutive days, and the test compounds by IM for 3 consecutive
3
days after the parasitemia level reached 5,000 parasites/mm .
Chloroquine at 10 mg/kg/day × 7 days eliminated the blood stage
parasites but not the liver stage hypnozoites. Compounds with
antihypnozoite activity will delay the relapse or radically cure the
infection.

ActiVity of Imidazolidinedione DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6229
Table 2. Causal Prophylactic Activity of 3, 5a, 5c, and 6a in P. Cynomolgi Sporozoite-Challenged Rhesus Monkeys
parasite patency
(days post-
inoculation)
monkey
group #
dose
(mg/kg)
a
days treatedc
compound #
control
route
IM
results
1
N/Ab
1 daily for 3 days
valid control
8 days
8
days
47 days
8 days
17 days
3 days
40 days
1 days
13 days
5 days
d
2
3
4
5
3
30
1 daily for 3 days
1 daily for 3 days
1 daily for 3 days
1 daily for 3 days
IM
IM
IM
IM
delayed patency for 39-60 days
delayed patency for 4-9 days
delayed patency for 13-32 days
delayed patency for 5-7 days
6
5a
5c
6a
30
1
30
2
30
1
a
b
Two monkeys per dose group. At the same volume as other experimental groups. Maximum DMSO volume per site is 1 mL for 2 injection sites (one
c
d
in each thigh). Drugs were dissolved in DMSO and given on the day before, on the day, and a day after (-1, 0, +1 day) sporozoite inoculation. First day
the parasite can be detected in blood smears after infection.
Table 3. Radical Curative Activity of Compounds 3, 5a, 5c, and 6a in Relapsed Rhesus Monkeys
dose
(mg/kg)
# doses
per day
days post-
treatment
group no.
drug 1
drug 2
none
30
route
results
CQ, 10 mg/ kg
7, PO
9 days
9 days
1
oral
relapse
×
5a
no relapse
1 daily for 3 days
IMradical curative
1 daily for 3 days
1 daily for 3 days
1 daily for 3 days
1 daily for 3 days
delayed relapse
delayed relapse
16 days
38days
3
5
6
3
3
c
30
30
30
30
IM
IM
IM
IM
4
1 days
CQ, 10 mg/ kg
7, PO
4
5
6
26days
16 days
53 days
a
delayed relapse
delayed relapse
×
6
8 days
delayed relapse
radical curative
55 days
no relapse
Scheme 3
experiment, first on day 6 after parasite inoculation and
continued daily for 21 days. After that, the parasite count was
checked 3 times per week for 4 weeks and then 2 times weekly
until 100 days after the last day of testing compound treatment.
Carbamate derivative 3, the most active compound of the series,
was used as the positive control.16
Results (Table 2) indicated that all three new compounds
delayed patency of treated monkeys from 4 days (5a) to more
than a month (5c) as compared with the untreated control
monkeys. Although compound 3, the positive control, showed
1
×
00% protection (parasite free for >100 days) at 10 mg/kg/day
3 in previous experiments,16 the results of this experiment
showed compound 3 only delayed patency of the treated
monkeys for 39 days for one monkey and 60 days for other, as
compared with untreated monkeys. The discrepancy in prophy-
lactic efficacy from one experiment to the other may be the
result of differences in viability of the sporozoites used.
Compounds 5a and 6a are 2-ethybutyrylamide derivatives of 1
and 2, respectively, and are about equal in efficacy, with delayed
patency for 4-9 days. Compound 5c, a carboxamide analogue
of 3, showed superior protective activity to that of 5a and 6a,
with delayed patency 13 days for one of the two treated monkeys
and 32 days for another.
The data from the liver stage malaria mouse model used in
this study appear to provide poor prediction of the monkey test
results. Compound 6a showed superior efficacy to 5a and 5c
in the mouse test, yet the latter compounds (5a and 5c) showed
equal or better causal prophylactic activity than that of the
former compound 6a. Especially, compound 5c, which showed
no activity in the mouse test either by single or 3 day treatment,
exhibited superior activity in Rhesus monkeys to that of 6a.
Monkeys which developed parasitemia in the causal prophy-
lactic test were used for the radical curative test. Test compounds
were co-administered with chloroquine for 7 days to eliminate
the blood stage parasites. One out of two monkeys that were
treated with 5a and one out of four monkeys that received
compound 3 were cured. Other treated monkeys all showed a
delay in relapse from 16 to 68 days, as compared to 9 days for
untreated control animals (Table 3).
These preliminary monkey results are rather encouraging
since monkeys were only treated for 3 days in this experiment,
instead of 7 days for 8-aminoquinoline antimalarials, such as
primaquine and tafenoquine, in the reported protocols.19 Longer
treatment with the test compounds may lead to a higher rate of
cures. Compounds 3 and 5c were again more active than 5a
and 6a in the curative test, as in the prophylactic experiments
(Table 2). The results indicated that compounds with better
causal prophylactic activity also possess superior radical curative
efficacy. Since no adverse side effects were observed in the
monkeys treated with the test compounds at the level of 30 mg/
kg × 3 days, further causal prophylactic and radical curative
studies with treatment extended from 3 days to 7 consecutive
days will be carried out.
In metabolic stability studies using mouse and human liver
microsomal preparations, carboxamides 5a-c were stable, with
t1/2 > ∼60 min, whereas the carboxamides 6a-c were highly

6
230 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Guan et al.
Scheme 4. Mechanism of Chemical Transformation from Carboxamides 5a and 6a to Triazine 7a
to possess antimalarial activity in rodents.2
0-22
However, the
unstable, with a half-life (t1/2) of less than 10 min (Table 1).
The LC mass spectrum analysis of the metabolites indicated
conversion of compounds 6a to new substances, the molecular
weight of which is 72 mass units (C2O3) less than that of the
parent compound. The same conversion was also observed when
the carboxamides were incubated in rat plasma. The observation
suggests that carboxamides 6a-c are the prodrugs of s-triazine
activities are generally confined to triazines with two unsub-
stituted free amino groups, an essential structure requirement
for antifolate drugs. The triazine derivatives 7 are substituted
2,4-diaminotriazine derivatives, which showed no activity
against both pyrimethamine-sensitive (D-6) and pyrimidine-
resistant (W-2) clones of blood stage malarias P. falciparum in
vitro and P. berghei in vivo, suggesting the causal prophylactic
activity of s-triazines 7 in rodents is not due to inhibitory activity
against dihydrofolate reductase, a key enzyme for parasite
growth. Antimalarial activity of triazines 7 and the analogues
will be discussed in a separate article.
7
a-c, as shown in Scheme 3. The structure of the metabolite
a was confirmed by comparing it with authentic material of
7
their mass spectra, LC retention times, and the spiking technique.
The conversion of carboxamide 5a to triazine 7a was also
demonstrated in rat PK studies, though at a slower rate.
Likewise, carboxamides 6b and 6c were transformed to 7b and
Conclusion
7c, respectively. The rat plasma half-life of 6a after intravenous
A series of acid-stable carboxamide derivatives of 2-guani-
dinoimidazolidinediones (5a-c and 6a-c) were prepared as
potential malaria prophylactic and radical cure agents. Car-
boxamides 5a-c are stable in human and mice liver microsomal
preparations, but 6a-c are unstable under the same test
conditions with t1/2 < 10 min and were converted to s-triazine
derivatives 7a-c. Carboxamides 5a-c also formed s-triazines
but at a much slower rate than that of carboxamides 6a-c. The
conversion is a chemical transformation, not an enzyme-
catalyzed reaction. The results suggest that the new carboxa-
mides are prodrugs of s-triazine derivatives. Carboxamides 6a-c
are more active than carboxamides 5a-c in the exoerythrocytic
mouse model, but the latter showed superior causal prophylactic
and radical curative activity in Rhesus monkeys. Therefore, the
test results from mice infected with sporozoites of P. yoelii
provide poor predictability of the efficacy in the Rhesus monkey
test. As both amino groups of the triazines 7 are secondary, not
primary, amines and that they showed no activity against the
pyrimethamine-sensitive D-6 clone of P. falciparum, triazines
injection (IV) was less than 10 min. The only metabolite that
could be detected was compound 7a. Detailed pharmacokinetics
and metabolite identification of 5a-c and 6a-c will be
published in a separate paper.
The mechanism of conversion of carboxamides to s-triazine
is proposed as shown in Scheme 4. The mechanism involves
hydrolysis of the imidazolidinedione ring of 6a and 5a,
generating intermediates 5a′ and 6a′. Cyclization of the
intermediates and elimination of a water molecule yielded the
s-triazine 7a. Due to the electron-withdrawing effect of the
dichlorophenyl ring, the carbonyl group of the imidazolidinedi-
one ring of 6a is more electron deficient than that of 5a. Thus,
the former is more susceptible to hydrolysis and form s-triazine
7
a at a faster rate than the latter.
The fact that transformation of 6a-c to 7a-c also took place
when 6a-c was incubated in buffer solution or plasma indicates
that the conversion is a chemical transformation and not an
enzymatic reaction. Furthermore, triazines 7a-c are the only
metabolites detected in the rat after oral or IM dosing with 6a-
c. The results suggest that 6a-c may be the prodrugs of
s-triazines 7a-c. This possibility is substantiated by the
observation that s-triazine 7a also showed moderate causal
prophylactic activity in the P. yoelii sporozoite-challenged
mouse test (Table 1). Exploration of s-triazine derivatives as
potential malaria prophylactic agents is currently in progress.
s-Triazine and dihydrotriazine derivatives have been reported
7
are not antifolate.
Acknowledgment. Material 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 views of the author and are not
to be construed as official or as reflecting true views of the
Department of the Army or the Department of Defense.

ActiVity of Imidazolidinedione DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6231
Acknowledgment. This research is supported by funding
from Military Infectious Diseases Research Program
A40096_06_WR_CSPP), U.S. Army Medical Research and
Material Command, Department of Defense, U.S.A., Peer
ReviewedMedicalResearchProgram(PRMRP)(Grant#PR054609),
and Malaria and Medicine Venture (MMV), Geneva, Switzer-
land, (Grant #MMV04/0013).
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at http://pubs.acs.org.
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