Structure identification and prophylactic antimalarial efficacy of 2-guanidinoimidazolidinedione derivatives

Article abstract of DOI:10.1016/j.bmc.2004.10.052

The reported synthetic procedure of WR182393, a 2- guanidinoimidazolidinedione derivative with high prophylactic antimalarial activity, was found to be a mixture of three closely related products. Poor solubility of WR182393 in both water and organic solvents and its impractical synthetic method have made the purification and structure identification of the reaction mixture a highly challenging task. The problems were circumvented by prodrug approach involving carbamate formation of the mixture, which enhances the solubility of the mixture in common organic solvents and facilitates the separation and structure determination of the two products. The structures of the two components were determined by X-ray crystallography and NMR of their corresponding carbamates 3a and 4a. Additional alkyl carbamates were prepared according to the same approach and two new carbamates 3b and 4d were found to possess higher intramuscular (im) efficacy than the parent compound WR182393 against Plasmodium cynomolgi in Rhesus monkey.

Full text of DOI:10.1016/j.bmc.2004.10.052

Bioorganic & Medicinal Chemistry 13 (2005) 699–704
Structure identification and prophylactic antimalarial efficacy
of 2-guanidinoimidazolidinedione derivatives
Jian Guan,a,* Quan Zhang,a Gettayacamin Montip,b Jean M. Karle,a Charles A. Ditusa,a
Wilbur K. Milhous,a Donald R. Skillmana and Ai J. Lina
aDivision of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring,
MD 20910, USA
bArmed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand
Received 30 July 2004; revised 20 October 2004; accepted 25 October 2004
Abstract—The reported synthetic procedure of WR182393, a 2-guanidinoimidazolidinedione derivative with high prophylactic anti-
malarial activity, was found to be a mixture of three closely related products. Poor solubility of WR182393 in both water and
organic solvents and its impractical synthetic method have made the purification and structure identification of the reaction mixture
a highly challenging task. The problems were circumvented by prodrug approach involving carbamate formation of the mixture,
which enhances the solubility of the mixture in common organic solvents and facilitates the separation and structure determination
of the two products. The structures of the two components were determined by X-ray crystallography and NMR of their corre-
sponding carbamates 3a and 4a. Additional alkyl carbamates were prepared according to the same approach and two new carba-
mates 3b and 4d were found to possess higher intramuscular (im) efficacy than the parent compound WR182393 against Plasmodium
cynomolgi in Rhesus monkey.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
WR182393 was synthesized first by Werbel et al. in 1972
and later by Starks Co.4 The procedure used to synthe-
size WR182393 involved treatment of chlorproguanil (1)
with diethyl oxalate (2). The structure of the reaction
product was assumed by the manufacture to be com-
pound 3 (Scheme 1). However, this reaction could pos-
sibly give a mixture of six structurally closely related
products as shown in Scheme 1. There was no convinc-
ing analytical data to support the structural assign-
ments. In this study, it was found that WR182393
prepared by Starks Co. comprised at least three com-
pounds as indicated by HPLC and NMR, the starting
material 1 plus two compounds out of the six proposed
products 3–8. Thus, the biological data derived from
WR182393 is the result of a mixture, not a single pure
compound. Nevertheless, the active components of
WR182393 are potentially valuable leads for medici-
nal chemists to search for new malaria prophylactic
drugs.
Chlorproguanil (1) is highly active against primary exo-
erythrocytic forms of Plasmodium falciparum and P.
vivax. WR182393, a cyclic dicarboxamide derivative of 1
was found to possess prophylactic activity in the mouse
Rane assay comparable to that of tafenoquine via sub-
cutaneous administration, where WR182393 demon-
strated radical cure (complete elimination of malaria
parasites from the body so that relapses cannot occur)
at 10mg/kg.1,2 It also showed radical cure at 160mg/
kg when dosed orally. In Rhesus monkeys, WR182393
demonstrated both radical curative and causal prophy-
lactic (complete prevention of erythrocytic infection by
the administration of drugs that destroy either the
sporozoites or the tissue forms of the malaria parasite)
activity against P. cynomolgi via intramuscular
administration (im).3
Due to the poor solubility of WR182393 in both water
and organic solvents, the purification and structure
identification of the reaction mixture have been a frus-
trating task to medicinal chemists. Because prodrug
Keywords: Antimalarial; Guanidinoimidazolidinediones; Carbamates;
WR182398.
*
Corresponding author. Tel.: +1 301 319 9906; fax: +1 301 319
9449; e-mail: jian.guan@na.amedd.army.mil
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.052

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J. Guan et al. / Bioorg. Med. Chem. 13 (2005) 699–704
O
C
NH
C NH
NH
NH C NH
O
C
OCH CH
2 3
+
Cl
CH(CH )
3 2
OCH CH
2
3
Cl
1
2
O
O
O
O
NH
N
Cl
Cl
NH C
C N
NH
Cl
NH
N
N
+
N
N
H
+
NHCH(CH )
Cl
Cl
3 2
Cl
N
HN C N CH(CH )
3 2
H
(H C) HC
3
2
NH
O
O
5
3
4
O
NH
HN
N
O
N
N
Cl
NH
C
HN
N
NH
Cl
NH
N
NHCH(CH )
3 2
+
N
Cl
Cl
N
O
+
H
Cl
CH(CH )
3 2
O
NHCH(CH )
3 2
O
Cl
O
8
7
6
Scheme 1.
strategy is usually adapted to improve drugꢀs solubility,
and hence bioavailability, we have successfully utilized
this strategy to increase the solubility of WR182393 by
carbamate formation, which facilitates the separation,
purification, and structure determination of its
components.
products with an estimated ratio of 2:1 as indicated by
the NMR spectrum. The resulting two carbamates mix-
ture had much better solubility than WR182393, but
had identical Rf values on TLC under various solvent
systems and thus, could not be separated by chromato-
graphy. However, the differences in their solubility in
ethyl acetate facilitated the fractional crystallization to
yield, first, the white crystal component 4a, followed
by yellow crystal component 3a. Since the structures
of the two carbamate products are closely related to four
other possible products, unambiguous structure identifi-
cation based solely on NMR data of the carbamate
products is very difficult.
2. Results and discussion
Initially, WR182393 was treated with ethyl chlorofor-
mate in the presence of triethylamine (Scheme 2). The
ethylcarbamate obtained was a combination of two
O
O
O
N
O
NH
N
O
O
H
N
O
Cl
N
Cl
N
H
N
N
N
Cl
O
NH
Cl
4a
3a
O
O
NH
a
N
O
O
Cl
N
N
O
H
N
N
O
H
N
O
O
Cl
N
Cl
b
d
e
3b
N
WR182393
3 & 4
Cl
NH
O
O
N
4d
O
O
H
c
Cl
N
N
N
Cl
NH
O
O
O
NH
O
N
4b
O
O
H
Cl
N
N
N
O
N
Cl
Cl
N
H
N
NH
N
O
Cl
3c
4c
Scheme 2. Reagents: (a) CIOCOEt, Et3N, CHCI3; (b) di-t-butyl-dicarbonate, DMAP, CHCI3; (c) dibenzyldicarbonate, DMAP, DMF; (d)
CIOCOCH2CH(CH3)2, Et3N, CHCI3; (e) 3N HCI/EtOAc.

J. Guan et al. / Bioorg. Med. Chem. 13 (2005) 699–704
701
The structures of the two carbamates 3a and 4a were,
therefore, determined by X-ray crystallography as de-
picted in Figures 1 and 2. In the ethyl carbamate 3a,
the imidazolidinedione and 3,4-dichlorophenyl rings
are separated by an N–C–N chain with the isopropyl
group attached to the imidazolidinedione ring. Whereas,
in the ethyl carbamate 4a, the imidazolidinedione and
3,4-dichlorophenyl rings are bonded directly to each
other with the isopropyl group remaining part of the
side chain. However, many features of their crystalline
structures are similar. Both 3a and 4a possess large
nearly planar portions with the 3,4-dichlorophenyl ring
angled around 52ꢁ to the planar portion. Besides the
3,4-dichlorophenyl ring, the only nonhydrogen atoms
that are not essentially co-planar are the terminal car-
bon atoms of the isopropyl groups. Both 3a and 4a have
two intramolecular hydrogen bonds between hydrogen
atoms residing on acyclic nitrogen atoms and the imid-
azolidinedione ring nitrogen and the carbamate carbo-
nyl oxygen atom. Neither structure contained any
intermolecular hydrogen bonds.
Figure 2. Thermal ellipsoid plot of crystalline 4a, N-[1-(3,4-dichloro-
phenyl)-4,5-dioxo-imidazolidin-2-ylidene]-N0-isopropyl-N00-(ethylcar-
bonyl)guanidine, showing the atom numbering scheme used for the X-
ray crystallographic data. Atoms N7, C8, N9, C10, C11, N12, C13,
N14, C15, O16, O21, O22, N23, and O26 are essentially co-planar with
˚
atoms O16 and O22 showing the largest deviations (0.14A) off a least-
squares plane through these atoms. The phenyl ring is angled such that
the C3–C4–N7–C8 torsion angle is 50.7ꢁ. The crystal structure
contained only intramolecular hydrogen bonds, between N14–H14
and N7 and N23–H23 and O26. The N14–H14ꢀ ꢀ ꢀN9 angle is 148.7ꢁ,
The structures of 3a and 4a were further confirmed by
NMR as shown in Figure 3. The chemical shifts of
methlyene and methyl protons of the isopropyl group
from 3a and 4a are distinctively different, with the for-
mer (4.45ppm for –CH– and 1.40ppm for –CH3) being
more down shift than the latter (4.16ppm for –CH– and
1.25ppm for –CH3). Molecular model of 4a indicated
that the imidazolidinedione and the 3,4-dichlorophenyl
rings are not in the same plane, but rather perpendicular
to each other because of the steric hindrance of the
bulky guanidino substituents and the isopropyl group
˚
˚
H14ꢀ ꢀ ꢀN9 distance is 1.966A, and N14ꢀ ꢀ ꢀN9 distance is 2.653A. The
˚
N23–H23ꢀ ꢀ ꢀO26 angle is 129.8ꢁ, the H23ꢀ ꢀ ꢀO26 distance is 2.077A,
˚
and N23ꢀ ꢀ ꢀO26 distance is 2.679A. The large thermal ellipsoids for the
terminal methyl groups indicate that these methyl groups are not
tightly sterically restricted in the crystalline packing.
is sitting above the 3,4-dichlorophenyl ring. Therefore,
all seven isopropyl protons in 4a are shielded by the phe-
nyl ring electrons, resulting in the observed up field
chemical shifts. Whereas both 3,4-dichlorophenyl and
the imidazolidinedione rings of 3a are separated by a
guanidine side chain, no electronic interactions between
the isopropyl group and the aromatic ring are expected.
Other distinct difference in chemical shifts between 3a
and 4a was that the aromatic proton Ha of 4a
(7.56ppm) is 0.3ppm up field shifted relative to the cor-
responding proton of 3a (7.84ppm), possibly due to
shielding effects of the carbonyl oxygen of the imidazol-
idinedione ring in 4a. NMR data is clearly in agreement
with the structure assignment of 3a and 4a by X-ray
crystallography.
Several new carbamates (3b, 4b–d) were prepared and
separated by the same approach. The differences in
chemical shifts between 3a and 4a were, likewise, ob-
served in the new carbamates 3b,c and 4b–d. The t-
Boc groups of the t-Boc carbamates 3b and 4b were
hydrolyzed in 3N HCl/ethyl acetate5 resulting in regen-
eration of the parent components 3 and 4, respectively.
The structures of 3 and 4 were further confirmed by
NMR, MS, and elemental analysis.
Figure 1. Thermal ellipsoid plot of crystalline 3a, N-(3,4-dichlorophen-
yl)-N0-ethylcarbonyl-N00-(1-isopropyl-4,5-dioxo-imidazolidin-2-ylide-
ne)-guanidine, showing the atom numbering scheme used for the X-ray
crystallographic data. Atoms N7, C8, N9, C10, N11, C12, C13, N14,
N15, C16, O17, O25, O26, and O27 are essentially co-planar with atom
˚
N7 showing the largest deviation (0.13A) off a least-squares plane
through these atoms. The phenyl ring is angled such that the C3–C4–
N7–C8 torsion angle is 52.8ꢁ. The crystal structure contained only
intramolecular hydrogen bonds, between N7–H7 and O27 and N15–
H15 and N14. The N7–H7ꢀ ꢀ ꢀO27 angle is 131.1ꢁ, H7ꢀ ꢀ ꢀO27 distance is
The causal prophylactic antimalarial activity of two car-
bamates 3b and 4d were assessed against P. cynomolgi in
Rhesus monkey according to the methodology described
by Schmidt for the evaluation of causal prophylaxis and
radical cure.6–8 Two monkeys were used in each test
group. The control animals developed parasitemia in
˚
˚
2.029A, and N7ꢀ ꢀ ꢀO27 distance is 2.674A. The N15–H15ꢀ ꢀ ꢀN14 angle
˚
is 139.8ꢁ, H15ꢀ ꢀ ꢀN14 distance is 1.879A, and N15ꢀ ꢀ ꢀN14 distance is
˚
2.660A.

702
J. Guan et al. / Bioorg. Med. Chem. 13 (2005) 699–704
31 Mar 2004
Acquisition Time (sec) 5.3084
Comment
Imported from UXNMR.
Date
31 Mar 2004 18:01:36
300.13
32768
File Name
Nucleus
D:\Bruker\Xwin-nmr\data\ajl\nmr\Ethyl of 1st Component\Ethyl of 1st Component_001000fid
1H
Frequency (MHz)
Points Count
Sweep Width (Hz)
Number of Transients 16
Solvent
CHLOROFORM-D
Original Points Count 32768
Pulse Sequence
zg30
6172.84
O
O
Hc
Cl
Hb
Ha
HN
O
O
N
Cl
N
H
N
N
3a
Hc
Ha
Hb
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
ppm
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
O
Hc
Cl
Hb
Ha
O
Cl
N
N
H
N
N
O
Hc
Ha
HN
Hb
O
4a
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
ppm
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Figure 3. NMR spectrum of 3a and 4a.
about 10days after inoculation of about 1 · 106 P. cyno-
molgi sporozoites harvested from Anopheles dirus. All
the monkeys received three treatments, once a day for
monkey and delayed parasitemia formation in the other
monkey for 75days, which confirmed a previous report
that this compound possessed im causal prophylactic
and radical curative activity against P. cynomologi. Both
monkeys receiving 30mg/kg by im of compound 4d were
protected and stayed parasite free 100days after treat-
ment. At lower dose of 10mg/kg, one monkey remained
parasite free after day 100, but the other monkey devel-
oped parasitemia after day 47. Compound 3b showed
the best prophylactic efficacy among the three com-
three consecutive days beginning
a
day before
(ꢁ1,0,+1) the sporozoites challenge. The control mon-
keys (group 1) received HECT and the experimental ani-
mals received testing compound by using 8 Fr., 15-in.
long nasogastric tubes. The results are shown in Table
1. The positive control drug, WR182393, at 30mg/kg
given via intramuscular (im) injection, protected one
Table 1. Causal prophylactic activities of 3b and 4d
Monkey no.
Group
Drug
Day of parasitemia
Resultsa
DA889
DA891
Control
None (DMSO)
Parasitemia on day 10
Parasitemia on day 13
Valid control
Valid control
DA888
DA895
1
2
3
4
5
WR182393, 30mg/kg
4d, 30mg/kg
Parasitemia on day 75
Parasite free on day 100
Delay the parasitemia
Causal prophylaxis
DA874
DA894
Parasite free on day 100
Causal prophylaxis
DA880
DA883
4d, 10mg/kg
Parasitemia on day 47
Parasite free on day 100
Delay the parasitemia
Causal prophylaxis
DA873
DA877
3b, 30mg/kg
Parasite free on day 100
Causal prophylaxis
DA875
DA878
3b, 10mg/kg
a Treated monkeys remained parasite free for 100days after the treatment were considered causal prophylaxis or protected.

J. Guan et al. / Bioorg. Med. Chem. 13 (2005) 699–704
703
pounds tested, protecting all four monkeys dosing with
either 10 or 30mg/kg of the compound. Criteria used
to define ꢁcureꢀ in the test is that the treated monkeys re-
mained parasite free for 100 days after the treatment
were considered cured or protected.
ture was stirred at room temperature overnight, washed
with water, and the chloroform layer was dried over
Na2SO4 and concentrated. The residue was applied to
a silica gel flash column chromatography and eluted
with 2.5% EtOAc/CHCl3. The ethyl carbamate obtained
(2.8g) was a mixture of two compounds with a ratio of
approximately 2:1 (A:B) as indicated by NMR. Frac-
tional crystallization of the mixture from ethyl acetate
gave first isomer B (850mg, 18%), followed by isomer
A (1.3g, 27%). The structures of the two products were
determined by NMR and X-ray crystallography as 3a
(isomer A) and 4a (isomer B).
Preliminary data indicated that none of the three com-
pounds showed significant oral protective activity up
to 60mg/kg in Rhesus monkey model. However, good
oral activity was observed in mouse model. The mouse
oral efficacy data will be published later in a separate
report.
Ethyl carbamate 3a: mp 181ꢁC. 1H NMR (CDCl3,
600Hz): d 13.20 (s, 1H, –NH), 11.17 (s, 1H, –NH),
7.84 (d, J = 2.4Hz, 1H), 7.53 (d, J = 8.6Hz, 1H), 7.24
(dd, J = 8.6, 2.4Hz, 1H), 4.45 (m, 1H), 4.38 (q,
J = 7.1Hz, 2H), 1.43 (t, J = 7.1Hz, 3H), 1.40 (d,
J = 7.0Hz, 6H). Anal. (C16H17N5O4Cl2) C, H, N.
3. Conclusion
In summary, we successfully separated, purified, and
identified the components of WR182393 through carba-
mate prodrug approach. Pure carbamates 3b and 4d
possessed superior prophylactic efficacy to that of
WR182393 mixture in tests against P. cynomolgi in Rhe-
sus monkey by im. Due to promising prophylactic effi-
cacy, an unambiguous synthesis for 3 and 4 from
commercially available chemicals had been accom-
plished in our laboratory. A series of new carbamates
derived from pure compounds 3 and 4 were prepared
and tested in a sporozoites challenged mouse model.
The new synthetic methodologies of compound 3 and
4 and the biological results of new derivatives will be re-
ported separately elsewhere.
Ethyl carbamate 4a: mp 257ꢁC. 1H NMR (CDCl3,
600Hz): d 7.56 (d, J = 2.4Hz, 1H), 7.56 (d, J = 8.6Hz,
1H), 7.29 (dd, J = 2.4 and 8.6Hz, 1H), 4.32 (q,
J = 7.1Hz, 2H), 4.16 (m, 1H), 1.39 (t, J = 7.1Hz, 3H),
1.25 (d, J = 6.6Hz, 6H). Anal. (C16H17N5O4Cl2) C,
H, N.
Benzyl carbamates 3c and 4c and isobutyl carbamate 4d
were prepared using the same procedure.
Benzyl carbamate 3c: 25%, mp 235ꢁC. 1H NMR
(CDCl3, 600Hz): d 7.81 (s, 1H), 7.52 (d, J = 8.70Hz,
1H), 7.44 (m, 5H), 7.23 (d, J = 8.70Hz, 1H), 5.32 (s,
2H), 4.42 (m, 1H), 1.39 (d, J = 7.0Hz, 6H). Anal.
(C21H19N5O4Cl2) C, H, N.
4. Experimental
Melting points were determined on a Mettler FP62 melt-
ing point apparatus and are uncorrected. Analytical thin
layer chromatography (TLC) was performed using
HPTLC-HLF normal phase 150 microns silica gel plates
(Analtech, Newark, DE). Visualization of the developed
chromatogram was performed by UV absorbance, or
spreading with aqueous potassium permanganate, or
ethanolic anisaldehyde. Liquid chromatography was
performed using a Horizon HPFC System (Biotage,
Charlottesville, VA) with Flash 25 or 40M cartridges
Benzyl carbamate 4c: 16%, mp 229ꢁC. 1H NMR (d-
DMSO, 600Hz): d 7.79 (d, J = 8.61Hz, 1H), 7.75 (d,
J = 2.30Hz, 1H), 7,48–7.34 (m, 6H), 5.30 (s, 2H), 3.97
(m, 1H), 1.17 (d, J = 6.5Hz, 6H). Anal.
(C21H19N5O4Cl2) C, H, N.
Isobutyl carbamate 4d: 31%, mp 260ꢁC. 1H NMR
(600MHz, CDCl3): d 12.44 (s, 1H, –NH), 9.24 (d,
J = 7.2Hz, 1H, –NH), 7.55 (d, J = 2.4Hz, 1H), 7.54
(d, J = 8.6Hz, 1H), 7.28 (dd, J = 8.6 and 2.4Hz, 1H),
4.12 (m, 1H), 4.02 (d, J = 6.8Hz, 2H), 2.05 (m, 1H),
1.24 (d, J = 6.6Hz, 6H), 0.99 (d, J = 6.7Hz, 6H). Anal.
(C18H21Cl2N5O4) C, H, N, Cl.
TM
˚
(KP-Sil Silica, 32–63lm, 60A). Preparative TLC was
performed using silica gel GF Tapered Uniplates (Anal-
1
tech, Newark, DE). H NMR and 13C NMR spectra
were recorded in deuteriochloroform, unless otherwise
noted, on a Bruker Avance 300 and Bruker Avance
600 spectrometer (Bruker Instruments, Inc., Wilming-
ton, DE). Chemical shifts are reported in parts per mil-
lion on the d scale from an internal standard of
tetramethylsilane. Combustion analyses were performed
by Atlantic Microlab, Inc. (Norcross, GA). Where anal-
yses are indicated by symbols of the elements, the ana-
lytical results obtained were within 0.4% of the
theoretical values.
4.2. Procedure for preparation of t-butyl carbamates
To a suspension of WR182393 (500mg, 1.46mmol) in
DMF (10mL) was added DMAP (18mg, 0.2equiv)
and di-tert-butyl-dicarbonate (1.27g, 4equiv). The reac-
tion mixture was stirred at room temperature overnight
and partitioned in CHCl3/H2O. The organic layer was
successively washed with brine and water, dried over
Na2SO4, filtered, and concentrated. The crude products
were purified by silica gel flash column chromatography
(2.5% EtOAc/CHCl3) and crystallized from EtOAc to
give pure isomer A (3b) (54mg, 26%) and isomer B
(4b) in about equal yield.
4.1. Procedure for preparation of ethyl carbamates
To a suspension of WR182393 (4g, 11.7mmol) in 80mL
of CHCl3 was treated with Et3N (6.2mL, 4equiv) and
ethyl chloroformate (4.4mL, 4equiv). The reaction mix-

704
J. Guan et al. / Bioorg. Med. Chem. 13 (2005) 699–704
t-Boc carbamate 3b: mp 223ꢁC. 1H NMR (CDCl3,
600Hz): d 7.84 (d, J = 2.3Hz, 1H), 7.52 (d, J = 8.6Hz,
1H), 7.25 (dd, J = 8.6, 2.3Hz, 1H), 4.44 (m, 1H), 1.60
(s, 9H), 1.39 (d, J = 7.0Hz, 6H). Anal. (C18H21N5O4Cl2)
C, H, N.
controls every 97 reflections and remained constant
within 3.2% for 3a, 3.5% for 4a. No absorption correc-
tion was applied. The structures were solved using direct
methods.9,10 Full matrix least-squares refinement11 was
performed on coordinates and anisotropic thermal
parameters for the nonhydrogen atoms, isotropic ther-
mal parameters for the hydrogen atoms using reflections
for which jFOj > 4r(FO). Hydrogen atoms H7 and H15
for 3a and H14 and H23 for 4a were located in the dif-
ference maps. The remaining hydrogen atoms were
placed in idealized positions, and during refinement,
the coordinates of these hydrogen atoms rode with the
coordinates of the carbon to which they are attached.
Final bond distances and angles were all within expected
and acceptable limits.
t-Boc carbamate 4b: mp 227ꢁC. 1H NMR (CDCl3,
300Hz):
d
7.55 (d, J = 2.3Hz, 1H), 7.54 (d,
J = 7.50Hz, 1H), 7.27 (dd, J = 2.3, 7.50Hz, 1H), 4.15
(m, 1H), 1.53 (s, 9H), 1.22 (d, J = 6.6Hz, 6H). Anal.
(C18H21N5O4Cl2) C, H, N.
4.3. Hydrolysis of carbamate
t-Boc carbamate (3b) (196mg, 0.44mmol) was dissolved
in 3N HCl/EtOAc (3mL). After 2h, the mixture was
neutralized with satd NaHCO3 and filtered. The yellow
solid was washed successively with water and chloro-
form to yield pure 3 (140mg, 93%).
Acknowledgements
Material has been reviewed by the Walter Reed Army
Institute of Research. There is no objection to its presen-
tation and/or publications. 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 Defence.
Compound 3: mp 227ꢁC. 1H NMR (DMSO-d6, 600Hz)
d 7.78 (s, 1H), 7.69 (d, J = 8.4Hz, 1H), 7.31 (d,
J = 8.4Hz, 1H), 4.39 (s, 1H), 1.33 (d, J = 6.7Hz, 6H).
Anal. (C13H13N5O2Cl2Æ1/4CH3CO2C2H5) C, H, N.
Compound 4 was also prepared by the hydrolysis of 4b
under the same conditions as that of compound 3. Yield
1
89%, mp 244ꢁC. H NMR (DMSO-d6, 600Hz) d 8.72–
References and notes
8.67 (m, 1H), 7.76 (d, J = 8.5Hz, 1H), 7.72 (d,
J = 2.0Hz, 1H), 7.42 (dd, J = 8.5, 2.0Hz, 1H), 3.76 (m,
1H), 1.14 (d, J = 6.1Hz, 6H). Anal. (C13H13N5O2Cl2)
C, H, N.
1. Curd, F. S. H.; Davey, D. G.; Rose, F. L. Ann. Trop. Med.
Parasitol. 1945, 39, 208.
2. Shanks, G. D.; Oloo, A. J.; Aleman, G. M.; Ohrt, C.;
Klotz, F. W.; Braitman, D.; Horton, J.; Brueckner, R.
Clin. Infect. Dis. 2001, 33, 1968.
4.4. X-ray crystal structure determination
3. Corcoran, K. D.; Hansukjariya, P.; Sattabongkot, J.;
Ngampochjana, M.; Edstein, M. D.; Smith, C. D.; Shanks,
G. D. Am. J. Trop. Med. Hyg. 1993, 49, 473.
4. Contractors of Walter Reed Army Institute of Research,
U.S. Army.
The X-ray samples were colorless plates (3a:
0.7 · 0.35 · 0.12mm; 4a: 0.45 · 0.42 · 0.06mm) crystal-
lized from methanol. Data collection was performed at
room temperature (293 2K) on a Bruker P4 diffrac-
tometer using CuKa radiation and a graphite mono-
chrometer in the incident beam. Reflections used to
refine the unit cell parameters by least-squares methods
are as follows: 3a, 31 reflections in the range of
12ꢁ 6 h 6 40ꢁ; 4a, 27 reflections in the range of
5. Stahl, G. L.; Walter, R.; Smith, C. W. J. Org. Chem. 1978,
43, 2285.
6. Schmidt, L. H.; Fradkin, R.; Genther, C. S.; Rossan, R.
N.; Squires, W. Am. J. Trop. Med. Hyg. 1982, 31, 612.
7. Schmidt, L. H.; Fradkin, R.; Genther, C. S.; Rossan, R.
N.; Squires, W., II. Am. J. Trop. Med. Hyg. 1982, 31, 646.
8. Schmidt, L. H.; Fradkin, R.; Genther, C. S.; Hughes, H.
B., III. Am. J. Trop. Med. Hyg. 1982, 31, 666.
9. Karle, J.; Karle, I. L. Acta Crystallogr. 1966, 21, 849–
859.
14ꢁ 6 h 6 26ꢁ. Crystal data 3a: C16H17C12N5O4, FW =
˚
a = 14.919(2)A,
414.25,
monoclinic,
˚
P2(1)/n,
˚
b = 7.613(1)A, c = 17.504(3)A, b = 102.79(2)ꢁ, V =
1938.7(5)A , Z = 4. Crystal data 4a: C16H17C12N5O4,
˚
FW = 414.25, triclinic, P-1, a = 7.924(2)A, b =
3
˚
10. Sheldrick, G. M. Crystallographic Algorithms for Mini
and Maxi Computers. In Crystallographic Computing;
Sheldrick, G. M., Kruger, C., Goddard, R., Eds.; Oxford
¨
˚
9.704(2)A,
˚
c = 13.375(2)A,
a = 102.90(1)ꢁ,
b =
3
˚
96.18(1)ꢁ, c = 97.39(2)ꢁ, V = 984.2(4)A , Z = 4. The
data were collected using the x scan technique with a
variable scan rate ranging from 3ꢁ/min minimum to
60ꢁ/min maximum depending upon the intensity of the
reflection. Three reflections were checked as intensity
University Press: Oxford, 1985; Vol. 3, pp 175–179,
Sheldrick, G. M. SHELXTL software; University of
Go¨ttingen, Federal Republic of Germany, 1990.
11. Sheldrick, G. M. SHELX-97; University of Go¨ttingen,
Federal Republic of Germany, 1997.