New imidazolidinedione derivatives as antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2010.12.028

A series of new N-alky- and N-alkoxy-imidazolidinediones was prepared and assessed for prophylactic and radical curative activities in mouse and Rhesus monkey models. New compounds are generally metabolically stable, weakly active in vitro against Plasmodium falciparum clones (D6 and W2) and in mice infected with Plasmodium berghei sporozoites. Representative compounds 8e and 9c showed good causal prophylactic activity in Rhesus monkeys dosed 30 mg/kg/day for 3 consecutive days by IM, delayed patency for 19-21 days and 54-86 days, respectively, as compared to the untreated control. By oral, 9c showed only marginal activity in causal prophylactic and radical curative tests at 50 mg/kg/day × 3 and 30 mg/kg/day × 7 plus chloroquine 10 mg/kg for 7 days, respectively.

Full text of DOI:10.1016/j.bmc.2010.12.028

Bioorganic & Medicinal Chemistry 19 (2011) 1541–1549
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New imidazolidinedione derivatives as antimalarial agents
⇑
Liang Zhang, Ramadas Sathunuru, ThuLan Luong, Victor Melendez, Michael P. Kozar, Ai J. Lin
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new N-alky- and N-alkoxy-imidazolidinediones was prepared and assessed for prophylactic
and radical curative activities in mouse and Rhesus monkey models. New compounds are generally
metabolically stable, weakly active in vitro against Plasmodium falciparum clones (D6 and W2) and in
mice infected with Plasmodium berghei sporozoites. Representative compounds 8e and 9c showed good
causal prophylactic activity in Rhesus monkeys dosed 30 mg/kg/day for 3 consecutive days by IM,
delayed patency for 19–21 days and 54–86 days, respectively, as compared to the untreated control.
By oral, 9c showed only marginal activity in causal prophylactic and radical curative tests at 50 mg/kg/
day ꢀ 3 and 30 mg/kg/day ꢀ 7 plus chloroquine 10 mg/kg for 7 days, respectively.
Received 14 October 2010
Revised 7 December 2010
Accepted 13 December 2010
Available online 16 December 2010
Keywords:
Antimalarials
Imidazolidinedione
Preparation
Published by Elsevier Ltd.
Metabolic stability
1. Introduction
Carbamate 3, the most active compound of this class, protects
monkeys at a dose of 10 mg/kg/day for 3 days by im. Nevertheless,
8-Aminoquinoline (8-AQ) antimalarials remain the most potent
class of malaria drugs with radical curative activity. The therapeutic
value of the 8-AQ as first line antimalarial drug, nevertheless, is com-
promised by the lethal hemolysis side effect in glucose-6-phosphate
dehydrogenase (G6PD) deficient patients. Search of non-8-AQ new
antimalarial drugs with prophylactic and/or radical curative activity
to replace 8-AQ antimalarial drugs, such as primaquine (PQ) and
tafenoquine (TQ), is the paramount interest of this laboratory in re-
cent years.1–4 Imidazolidinedione derivatives, such as compound
WR182393 (IZ), is one of the very few classes of compounds with
liver stage antimalarial activity. It was first prepared and tested in
exoerythrocytic (EE) mouse model in early 1970s and was later
found to have radical curative and causal prophylactic activity in
Rhesus infected with sporozoites of Plasmodium cynomolgi.5 The test
material was later shown to be a mixture of the biguanide starting
material, chloroproguanil, and two imidazolidinedione derivatives
(1 and 2). The results of our efforts to identify each of the
components in the mixture are detailed in a recent publication.1
Subsequently, unambiguous new methods for synthesis of the pure
imidazolidinediones 1, and 2 were developed which facilitated the
analogs synthesis and structure activity relationship (SAR) studies.
The efforts lead to the discovery of carbamates 3 and 4 which
showed superior prophylactic activity to the lead compounds in
Rhesus monkeys.2–4 The structures of the two key active
components (1 and 2) and their carbamate derivatives (3 and 4)
are shown in Figure 1.
the IZ derivatives showed very weak or no activity against cell
growth of Plasmodium falciparum clones (W-2 and D-6) and
Plasmodium berghei in mice. To the best of our knowledge, this is
the first class of antimalarial agents possessing activity against
liver stage malaria exclusively. Given by oral, however, compound
3 only delayed patency (first day the parasite can be detected in
blood smears after infection) of the treated monkeys for 3 days
as compared to the untreated animals. The lacking of oral activity
of carbamates 3 and 4 in Rhesus tests was first thought due to their
poor bioavailability as a result of hydrolysis of carbamate group in
stomach acid leading to generation of insoluble parent compounds
1 or 2. Chemically stable IZ-carboxamide derivatives (5 and 6)
were then prepared. However, the electron inductive effects of
the carboxamide groups made the imidazolidinedione ring of 5
and 6 liable to hydrolysis and consequently leading to formation
of s-triazines 7.4 The s-triazine formation was first thought to be
a bioactivation process of the IZ-carboxamide derivatives, but
was later found to be a chemical inactivation route. As a result,
the carboxamide derivatives (5 and 6) are in general not as active
as the carbamate analogs 3 and 4.4
In this study, a series of chemically stable N-alkoxyl- (8) and
N-alkyl- (9) derivatives of compound 1 (Fig. 2) was synthesized.
Alkylamino- and alkoxylamine- groups are chemically stable
though may be subjected to metabolic dealkylation. Alkoxylamino
group has been used as a masking group in the design of amidine
pro-drugs.6 The new compounds prepared in this study are ex-
pected to be more stable chemically and/or metabolically, resulting
in better antimalarials than carbamates (3 and 4) and carboxam-
ides (5 and 6).
⇑
Corresponding author. Tel.: +1 301 319 9084; fax: +1 301 319 9449.
E-mail address: ai.lin@us.army.mil (A.J. Lin).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.12.028

1542
L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
O
NH
O
N
CH(CH3)2
R'
NHR
N
O
Cl
N
N
N
N
Cl
Cl
NH C N
N
NCH(CH3)2
O
N
H
Cl
HN
Cl
NHR
Cl
R =
R =
H
2
4
R =
R =
1
3
7
-COOC(CH3)3
-CONHR'
-COOCH2CH(CH3)2
R =
R = -CONHR'
6
5
Figure 1. Active components of WR182393 (1 and 2) and derivatives (3–6).
X-ray crystallography on one of the representative compound 9m
was conducted and the result is shown in Supplementary data
[S3, Fig. 4].
R
OR
NH
NH-C N
NH
NH-C N
N
N
O
O
Cl
Cl
N
N
O
O
Cl
Cl
3. Results and discussion
9
8
R = alkyl, aryl, alkylaryl, aminoalkyl et al
Antimalarial activities of the new compounds were assessed in
clones of P. falciparum (W2 and D6), and in mice challenged with
sporozoites of P. berghei. The results are shown in Table 1. Like
the imidazolidinedione analogs reported earlier, the new
compounds showed only weak in vitro activity against clones of
Figure 2. N-Alkyl and N-alkoxyl derivatives of compound 1.
2. Chemistry
P. falciparum, with IC50 in the range of 1 to >6 lg/ml. As was ex-
The preparation of the new compounds for this study is
straightforward. Compounds 8a–e and 9a–p were prepared by
heating of 1-isopropyl-2-methylsulfanyl-1H-imidazole-4,5-dione
(10) with N-alkoxyl-3,4-dichlorophenylguanidine (12a–e) or N-al-
kyl-3,4-dichlorophenylguanidine (13a–n) in chloroform in a sealed
tube at 100 °C for 18–72 h as shown in Scheme 1. The N-alkyl- and
N-alkoxyguanidines were prepared in turn by either treating
1-(3,4-dichlorophenyl)-2-methylisothiourea hydroiodide (11)
(method 1), or (3,4-dichlorophenyl)-cyanamide (14) (method 2)
with alky- or alkoxyl-amines.2,7–13 Synthesis of the starting mate-
rials, compound 1 and isopropyl-2-methylsulfanyl-1H-imidazole-
4, 5-dione (10), were described in our previous publications of this
series.1–4 The 1H NMR spectra of the final products 8 and 9 in CDCl3
indicated the formation of tautomers 80 and 90, respectively. Two
sets of proton NMR spectra were observed in analogs of 8 and 9,
which coalesced into a single spectrum upon addition of a drop
of CF3COOD. The ratio of the two tautomers is concentration and
temperature dependent and is roughly 4:1 at room temperature.
Attempts were also made to prepare the N-alkyl analogs 9 by
direct alkylation of compound 1 using relatively reactive benzyl
bromide. As compound 1 is a biguanide derivative, direct benzyla-
tion of which could lead to a mixture of products with the benzyl
group randomly located at N1 through N4 as shown in Scheme 2.
Surprisingly, under the reaction conditions employed, only one
benzylation product was isolated. Based on NMR data alone, it is
difficult to pinpoint the location of the benzyl group in the new
product without using X-ray crystallography technique. The single
crystal X-ray diffraction data indicated that the benzylation
occurred at N1 instead of the desired N2 position, giving
exclusively N-benzyl-N-(3,4-dichloro-phenyl)-N0-(1-isopropyl-4,5-
dioxo-4,5-dihydro-1H-imidazol-2-yl)-guanidine (IZB) as shown in
Supplementary data [S1, Fig. 3].
pected, most of the compounds are metabolically stable in mouse
and human microsomal preparations with t1/2 >60 min, except
analogs 8e, 9h, 9i and 9p which showed a half-life (t1/2) of
10 min or less in mice microsomal preparations. Chemically, these
four compounds showed no substituents which are particularly
vulnerable to enzymatic or chemical hydrolysis. Nevertheless, the
c-Log P values of these four analogs are all greater than 5, a log
value higher than the other compounds, indicating poorer water
solubility. Most likely, compounds 8e, 9h, 9i and 9p may be precip-
itated out from the assay medium during the dilution process and
thus giving a false high rate of hydrolysis in metabolic stability as-
say. In addition to the poor solubility, since 9P is a t-Boc derivative,
chemical hydrolysis of the t-Boc group during the experiment may
also contribute to the observed instability results.
Like other IZ derivatives, the new compounds also showed weak
or no activity in mice infected with sporozoites of P. berghei, dosed
up to 320 mg/kg/day ꢀ 3 PO, except compounds 8a–c, 9c and 9e
which showed cures at 160 mg/kg/day ꢀ 3. Severe restless side ef-
fects and early death were observed among the mice received high
dose of N-alkoxyl analogs, 8a–c. Among the N-alkyl analogs tested,
only 9c and 9e showed cures administered 160 mg/kg/day for
three consecutive days. Unlike N-alkoxy derivatives, no sign of tox-
icity was observed among the mice treated with N-alkyl analogs
9a–p up to 320 mg/kg.
Good in vitro, rodent and/or non-human primate test models
are essential for lead optimization projects such as this study.
Our efforts have been hampered by lacking of a good predictive
exoerythrocytic (EE) mouse model and liver cell culture to conduct
proper SAR studies to prioritize compounds for further testing in
Rhesus. Two mouse models infected with, P. yoelli or P. berghei
sporozoites, respectively, have been used to assess the liver stage
antimalarial activities of the imidazolidinedione (IZ) deriva-
tives.1–4 Neither of these two models gave good predictive results
in Rhesus. The former gave too many false positive and the latter
gave too many false negative results. Lacking of good EE mouse
model, the selection of new candidates for further test in Rhesus
was based mainly on the previous observations that compounds
of this class generally showed moderate to good IM activity, even
though they showed poor activity in mice model. Therefore, only
two new compounds, 8e and 9c, which represent alkoxy and alkyl
Likewise, although 3 possible isomers of alkylation products can
be formed by heating N-alkoxyl or N-alkyl-3,4-dichlorophenylgua-
nidine (12 or 13) and 1-isopropyl-2-methylsulfanyl-1H-imidazole-
4,5-dione (10) under the conditions stated above, only one product
was isolated. Contrary to the direct alkylation method described
above which formed undesirable isomer IZB, reaction of 10 and
13a–p provided the desired products in good yields. To unequivo-
cally confirm the structure as the desired product, a single crystal

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
1543
H
H
H
N
Cl
Cl
N
NH2
Cl
Cl
N
SCH3
.
Cl
Cl
N
N
i
iii
O
N
HI
NH
N
O
R
NH
O
O
R
R
-CH3
-CH2CH3
-CH2CH=CH2
-C(CH3)3
R
-CH3
-CH2CH3
-CH2CH=CH2
-C(CH3)3
-CH2C6H5
11
12a
b
c
d
e
8a
b
c
d
e
ii
i
-CH2C6H5
H
H
N
Cl
Cl
NHCN
N
Cl
N
NH2
N
O
Cl
Cl
ii
N
Cl
HN
N
R
R O
O
14
8'
R
R
CH3
base
13a
b
c
d
e
f
g
h
i
2-adamantyl
(CH2)2N(CH3)2
(CH2)2N(CH2CH3)2
(CH2)2N(CH2)4
CH2CH3
CH(CH3)2
C(CH3)3
neopentyl
C6H5
j
k
l
Cl
Cl
NH2
+
or
ClCN
BrCN
m (CH2)3N(CH3)2
n
p
(CH2)4N(CH2)4
(CH2)6NH-t-Boc
CH2C6H5
(CH2)5 CH3
iii
H
H
N
O
O
N
N
N
N
Cl
Cl
N
N
N
O
Cl
Cl
O
O
CH3S
N
NH
HN
N
R
O
R
R
R
CH3
10
9'
9a
b
c
d
e
9i
j
k
l
2-adamantyl
(CH2)2N(CH3)2
(CH2)2N(CH2CH3)2
(CH2)2N(CH2)4
CH2CH3
CH(CH3)2
C(CH3)3
neopentyl
C6H5
m (CH2)3N(CH3)2
f
n
p
(CH2)4N(CH2)4
(CH2)6NH-t-Boc
g
h
CH2C6H5
(CH2)5CH3
Scheme 1. Synthesis of N-alkyl and N-alkoxyl derivatives of compound 1. Reagents and conditions: (i) NH2OR, EtOH, reflux, 18 h; (ii) RNH2, MeOH, 80–100 °C/sealed tube,
18 h; (iii) 10, CHCl3, 100 °C/sealed tube, 18–72 h.
derivatives, respectively, were assessed for causal prophylactic and
H
radical curative activities in Rhesus. Prior to the monkey test, both
compounds were confirmed no acute toxicity at 300 mg/kg/day for
three consecutive days by sc administration, and lost no more than
5% body weight within 30 days after receiving the test compound.
Benzyloxy (8e) and isopropyl (9c) derivatives showed promis-
ing causal prophylactic activity in Rhesus test by IM. The former
delayed patency of the treated monkeys for 19 days in one monkey
and 21 days in the other at a dose of 30 mg/kg/day ꢀ 3 by IM
(Table 2). In the same test, compound 9c exhibited superior effi-
cacy to 8e, delayed patency for 54 days in one monkey and 86 days
for the others. By oral administration, compound 9c delayed the
patency of the treated monkeys for only 3 days at 50 mg/kg/
day ꢀ 3. Due to limited availability of naïve monkeys and modest
IM activity, no attempt was made to evaluate 8e for oral activity
in Rhesus. In the same experiment, three clinical antimalarial
drugs, primaquine, tafenoquine and malarone, were used as posi-
tive control at a human equivalent PO dose. To our surprise, none
of these three first line antimalarial drugs protected the monkeys
and showed only comparable activity to 9c, delayed the patency
of the treated monkeys for only 2–5 days (Table 2). The results
Cl
Cl
N
1
N
3
H
N
N
Cl
N
N
O
O
N
4
NH2
NH
N
O
Cl
2
1a
O
Not observed
Br
Bn
3
N
Cl
Cl
N
1
N
O
O
i
Cl
Cl
N
N
N
N
NH2
N
2
O
O
4
O
NH2
N
N
H
1b
O
IZB
H
N
1
H
N
1
H
Cl
Cl
N
3
N
Cl
Cl
N
3
NH HN
NH
2
2
4
4
O
O
1c
1d
Scheme 2. Benzylation of compound 1. Reagents: (i) BnBr, TBAI, DMF.

1544
L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
Table 1
Prophylactic activity, metabolic stability and in vitro efficacy
Compd #
Met. stability t1/2 (min)
EE P. berghei (mg/k/day) ꢀ 3
P. falciparum IC50
(lg/ml)
c-Log P
Mouse
Human
MADa
MTDb
D6
TMc
W2
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
>60
>60
>60
>60
12.87
>60
>60
>60
>60
>60
>60
>60
7.84
7.42
>60
17.65
53.17
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
34.74
5.58
>60
31.51
>60
>60
>60
12.47
ꢁ40
<40
<40
120
>5.0
>5.0
8.5
2.75
>5.0
>5.0
>5.0
>6.0
0.72
1.1
2.16
2.36
1.35
0.81
>5.0
>5.0
>5.0
>5.0
1.55
>5.0
>5.0
>5.0
>15
3.36
>5.0
>5.0
>5.0
-
1.36
0.95
2.60
>5.0
2.80
2.07
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
6.7
3.37
3.90
4.20
4.60
5.14
—
3.45
3.80
4.14
4.68
4.67
—
5.57
5.81
3.07
4.13
3.77
3.29
4.29
5.41
160
160
>160
>160
>320
>160
160
ꢂ160
ꢂ160
ꢂ320
ꢂ160
ꢂ160
ꢂ320
ꢂ320
ꢂ160
ꢂ320
ꢂ160
ꢂ320
ꢂ160
ꢂ160
ꢂ160
ꢂ160
ꢂ160
ꢂ160
1.14
>5.0
>5.0
1.67
>6.0
1.15
0.37
0.88
1.30
2.62
2.68
>5.0
>5.0
1.32
>5.0
2.30
1.46
>320
160
>160
>320
>160
>320
>160
>160
>160
>160
>160
>160
9m
9n
9p
>60
10.93
a
b
c
MAD = minimum active dose.
MTD = maximum tolerated dose.
TM = TM91C235 (a clone of P. falciparum).
Table 2
Causal prophylactic activity of 8e, and 9c in P. cynomolgi sporozoites challenged Rhesus monkeys
Compd #
Dosea
(mg/kg/day ꢀ 3)
Route
Delayed Patency
(Day)
Patencyb
(Days Post-inoculation) (Day)
Control
8e
N/A
IMc
POd
IM
Valid control
8
8
30
19
21
54
86
3
3
5
5
3
27
29
62
94
11
11
13
13
11
10
10
11
9c
30
IM
PO
PO
PO
PO
50
PQe
TQf
1.78
0.316
14
2
2
3
MAg
a
b
c
d
e
f
Two monkeys per dose group.
Patency = first day parasites can be detected in blood smears after infection.
IM = intramuscular injection.
PO = oral administration.
PQ = primaquine.
TQ = tafenoquine.
MA = malarone.
g
indicated that 9c cannot be ruled out as a potential prophylaxis for
malaria infection and that the causal prophylactic protocol in Rhe-
sus is in need of revalidation.
treatment (7–14 days) and higher dosing regimen (twice a day at
30–50 mg/kg/day) may be necessary to determine the oral activity
of this class of compounds in Rhesus model.
In a combined causal prophylactic/radical curative Rhesus mod-
el, compound 9c exhibited no significant radical curative activity
by oral administration, delayed the relapse time of treated mon-
keys for only 2–3 days versus untreated control at a dose of
30 mg/kg/day ꢀ 7 PO plus CQ 10 mg/kg/day ꢀ 7 PO (Table 3). In
the same test, PQ at 1.78 mg/kg/day ꢀ 7 PO plus CQ at 10 mg/kg/
day ꢀ 7 PO radically cured the infected monkeys.
4. Experimental
Melting points were determined on a Mettler FP62 melting
point apparatus and are uncorrected. Analytical thin-layer chroma-
tography (TLC) was performed using HPTLC-HLF normal phase
150
l silica gel plates (Analtech, Newark, DE). Visualization of
As the life cycle of liver stage malaria in Rhesus is about 8 days
and the plasma t1/2 of the IZ compounds by oral administrations
are generally less than 5 h. Thus, very low or no drug will
remain in the plasma a day after dosing. Therefore, a 3–7 day
treatment regimen is obviously insufficient to eradicate the
hypnozoites unless the drug is very potent or long acting. Longer
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 25 M or 40 M cartridges (KP-Silica, 32–63 lm,
60 Å). Preparative TLC was performed using silica gel GF tapered
uniplates (Analtech, Newark, DE). 1H NMR and 13C NMR spectra

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
1545
Table 3
Radical curative activity of compound 9c in relapsed Rhesus monkeys
Drug 1
Drug 2
None
Dose (mg/kg)
None
# Days treated
Route
POa
Results
Relapsedc (Day)
Chloroquine (CQ) 10 mg/kg ꢀ 7, POa
1 daily for 7 days
Relapsed
11
12
9c
30
1 daily for 7 days
1 daily for 7 days
PO
PO
Relapsed
15
14
>100
PQb
1.78
Radical cure
a
b
c
PO = oral administration.
PQ = primaquine.
Relapsed = first day parasitemia detected post treatment.
were recorded in deuterochloroform, unless otherwise noted, on a
Bruker Avance 300 spectrometer (Bruker Instruments, Inc.,
Wilmington, DE). Chemical shifts are reported in parts per million
on the d scale from an internal standard of tetramethylsilane.
When the compounds formed two tautomers, the chemical shifts
with * indicated the peaks were shared by both tautomers in
NMR spectra. Combustion analyses were performed by Atlantic
Microlab, Inc. (Norcross, GA). Where analyses are indicated by
symbols of the elements, the analytical results obtained were with-
in 0.4% of the theoretical values. Mass analysis based on electron
impact (EI) was done on a Hewlett Packard (Agilent; Santa Clara,
CA) 5973 Mass Selector adapted with a HPP7 Direct Insertion Probe
(Scientific Instrument Services; Ringoes, NJ) using a steep temper-
ature gradient at either low voltage (5 eV) to identify masses
present or high voltage (70 eV) to obtain fragmentation data. An
LC/UV–vis/Trap MS was also employed for purity analysis and
chromophore properties. The system consisted of an Agilent 1100
Series LC–UV/vis system online with a ThermoFinnigan (now
ThermoFisher; Waltham, MA) LCQ MS equipped with electrospray
ionization (ESI) source. Samples were analyzed using shallow
acetonitrile buffer gradients at low flow rate. Purity of the final
products are >95%. Single crystal X-ray diffraction data was
collected by Dr. Maren Pink, Director of the Molecular Structure
Center, Indiana University, Bloomington, IN.
4.1.3. N-Allyloxy-N0-(3,4-dichlorophenyl)-guanidine (12c)
Following the general procedure, the title compound was ob-
tained from O-allylhydroxylamine HCl. Yield: 72%, mp 90 °C. 1H
NMR (CDCl3): d 4.39–4.45 (m, 2H), 4.55 (br s, 2H), 5.78 (br s, 1H),
5.24 (dd, J = 1.4 Hz, and 10.4 Hz, 1H), 5.33 (dd, J = 1.5 Hz,
and17.2 Hz, 1H), 5.98–6.09 (m, 1H), 6.95 (dd, J = 2.7 Hz, and
8.7 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.33 (d, J = 2.7 Hz, 1H). MS
(EI): m/z 259 [M]+.
4.1.4. N-(t-Butyloxy)-N0-(3,4-dichlorophenyl)-guanidine (12d)
Following the general procedure, the title compound was ob-
tained from O-tert-butylhydroxylamine HCl. Yield: 41%, mp
126 °C. 1H NMR (CDCl3): d 1.30 (s, 9H), 6.96 (dd, J = 2.6 Hz, and
8.8 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 2.6 Hz, 1H). MS
(EI): m/z 275 [M]+.
4.1.5. N-Benzyloxy-N0-(3,4-dichlorophenyl)-guanidine (12e)
Following the general procedure, the title compound was ob-
tained from O-benzylhydroxylamine HCl. Yield: 55%, mp 105 °C.
1H-NMR (CDCl3): d 4.93 (s, 2H), 6.89 (dd, J = 2.3 Hz, and 8.6 Hz,
1H), 7.23–7.42 (m, 7H). MS (EI): m/z 309 [M]+.
4.2. General procedure for the synthesis of N-alkyl-N0-(3,4-dich-
lorophenyl)-guanidines (13a–p)
4.1. General procedure for the synthesis of N-alkoxy-N0-(3,4-
dichlorophenyl)-guanidines 12a–e
Method 1: A mixture of 1-(3,4-dichlorophenyl)-2-methyliso-
thiourea HI salt (11, 1.0 equiv) and alkylamine (5–10 equiv) in
methanol was heated in a sealed tube at 80–100 °C for 18 h. The
solvent and the excess amine were removed under reduced pres-
sure. Saturated aqueous Na2CO3 solution was added to the
residue and the mixture was extracted with EtOAc. The organic
extracts were combined, washed with brine, dried over Na2SO4,
filtered, and the solvent was removed under reduced pressure.
The residue was suspended in cold (ꢃ4 °C) CH2Cl2, filtered and
washed with fresh CH2Cl2 to give a white solid. The product was
used for next step reactions without further purification.
Method 2: A mixture of 3,4-dichlorophenylcyanamide (1.0
equiv) (14) and alkyl or alkoxylamine (2.0 equiv) in absolute
methanol was heated in a sealed tube at 100 °C for 18 h. The
solvent and the excess amine were removed under the reduced
pressure. The residue was purified by recrystallization in hexane/
CH2Cl2 to give light brown solid.
A mixture of 3,4-dichlorophenyl-cyanamide (1.0 equiv) (14)
and alkoxylamine HCl salt (2.0 equiv) in absolute ethanol was
heated under reflux for 18 h. The solvent was removed under re-
duced pressure. Saturated aqueous Na2CO3 was added to the resi-
due. The mixture was extracted with ethyl acetate three times. The
organic extracts were combined, washed with brine, dried over
Na2SO4, filtered, and the solvent was evaporated to dryness under
reduced pressure. The residue was suspended in small amount of
cold (ꢃ4 °C) CH2Cl2, filtered and washed with fresh CH2Cl2 to give
white solid. Compounds 12a–e was used for reactions without fur-
ther purification.
4.1.1. N-Methoxy-N0-(3,4-dichlorophenyl)-guanidine (12a)
Following the general procedure, the title compound was ob-
tained from methoxyamine HCl. Yield: 46%, mp 134 °C. 1H NMR
(CD3OD): d 3.69 (s, 3H), 7.11 (dd, J = 2.6 Hz and 8.8 Hz, 1H), 7.28
(d, J = 8.8 Hz, 1H), 7.56 (d, J = 2.6 Hz, 1H). MS (EI): m/z 233 [M]+.
4.2.1. N-Methyl-N0-(3,4-dichlorophenyl)-guanidine (13a)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with methylamine in absolute
EtOH. Yield: 77%, mp 156 °C. 1H NMR (CDCl3): d 2.87 (s, 3H), 3.72
(br s, 3H), 6.78 (d, J = 8.5 Hz, 1H), 7.05 (s, 1H), 7.32 (d, J = 8.5 Hz,
1H). Major tautomer: 13C NMR (DMSO-d6) d 20.0, 29.5, 43.9,
125.7, 127.8, 130.7, 131.8, 136.3, 137.9, 159.0, 161.7, 168.2,
170.3; minor tautomer: 13C NMR (DMSO-d6) d 20.3, 29.3, 127.2,
128.1, 131.0, 132.2, 160.3, 162.1, and 170.1. MS (EI): m/z 217 [M]+.
4.1.2. N-Ethoxy-N0-(3,4-dichlorophenyl)-guanidine (12b)
Following the general procedure, the title compound was ob-
tained from O-ethylhydroxylamine HCl. Yield: 54%, mp 124 °C. 1H
NMR (CDCl3): d 1.28 (t, J = 7.0 Hz, 3H), 3.96 (q, J = 6.8 Hz, 2H),
4.00 (br s, 2H), 6.97 (br d, J = 7.4 Hz, 1H), 7.30 (s, 1H), 7.34 (d,
J = 2.3 Hz, 1H). MS (EI): m/z 247 [M]+.

1546
L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
4.2.2. N-Ethyl-N0-(3,4-dichlorophenyl)-guanidine (13b)
4.2.11. N-(2-Diethylaminoethyl)-N0-(3,4-dichlorophenyl)-guani-
dine (13k)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with ethylamine in MeOH. Yield:
76%, mp 129 °C. 1H NMR (CDCl3): d 1.22 (t, J = 7.2 Hz, 3H), 3.27 (q,
J = 7.2 Hz, 2H), 6.79 (dd, J = 2.4 Hz and 8.4 Hz, 1H), 7.05 (d,
J = 2.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H). MS (EI): m/z 231 [M]+.
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with N,N-diethylethylenediamine.
Yield: 77%, yellow oil. 1H NMR (CDCl3): d 1.00 (t, J = 7.2 Hz, 6H),
2.50 (m, 6H), 3.20 (t, J = 4.0 Hz, 2H), 5.17 (br s, 1H), 6.75 (dd,
J = 2.0 Hz, and 8.5 Hz, 1H), 7.01 (d, J = 2.0 Hz, 1H), 7.27 (d,
J = 8.5 Hz, 1H). MS (EI): m/z 303 [M]+.
4.2.3. N-(Isopropyl)-N0-(3,4-dichlorophenyl)-guanidine (13c)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with isopropylamine. Yield: 92%,
mp 179 °C. 1H NMR (acetone-d6): d 1.35 (d, J = 6.3 Hz, 6H), 2.89 (br
s, 3H), 4.18 (m, 1H), 7.41 (dd, J = 2.4 Hz, and 8.7 Hz, 1H), 7.64–7.68
(m, 2H). MS (EI): m/z 245 [M]+.
4.2.12. N-(2-Pyrrolinoethyl)-N0-(3,4-dichlorophenyl)-guanidine
(13l)
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with 1-(2-aminethyl)pyrrolidine.
Yield: 46%, mp 146 °C. 1H NMR (CDCl3): d 1.77 (br s, 4H), 2.56 (br s,
4H), 2.66 (t, J = 5.2 Hz, 2H), 3.28 (t, J = 5.2 Hz, 2H), 6.77 (dd,
J = 2.2 Hz, and 8.4 Hz, 1H), 7.03 (d, J = 2.2 Hz, 1H), 7.34 (d,
J = 8.4 Hz, 1H). MS (EI): m/z 301 [M]+.
4.2.4. N-(t-Butyl)-N0-(3,4-dichlorophenyl)-guanidine (13d)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with tert-butylamine. Yield: 74%,
mp 149 °C. 1H NMR (CDCl3): d 1.40 (s, 9H), 3.09 (br s, 3H), 6.75 (dd,
J = 2.4 Hz, and 8.4 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.31 (d,
J = 8.4 Hz, 1H). MS (EI): m/z 259 [M]+.
4.2.13. N-(3-Dimethylaminopropyl)-N0-(3,4-dichlorophenyl)-gu-
anidine (13m)
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with 3-(dimethylamino)-1-pro-
pylamine. Yield: 52%, mp 90 °C. 1H NMR (CDCl3): d 1.65 (br s,
2H), 2.16 (s, 6H), 2.37 (t, J = 5.5 Hz, 2H), 3.26 (t, J = 5.5 Hz, 2H),
6.77 (br d, J = 8.1 Hz, 1H), 7.03 (br s, 1H), 7.29 (br d, J = 8.1 Hz,
1H). MS (EI): m/z 289 [M]+.
4.2.5. N-Neopenty-N0-(3,4-dichlorophenyl)-guanidine (13e)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with neopentylamine. Yield: 78%,
mp 107 °C. 1H NMR (CDCl3): d 0.95 (s, 9H), 3.00 (s, 2H), 4.11 (br s,
3H), 6.76 (br d, J = 8.4 Hz, 1H), 7.02 (s, 1H), 7.31 (br d, J = 8.4 Hz,
1H). MS (EI): m/z 274 [M]+.
4.2.14. N-(4-Pyrrolinobutyl)-N0-(3,4-dichlorophenyl)-guanidine
(13n)
4.2.6. N-Phenyl-N0-(3,4-dichlorophenyl)-guanidine (13f)
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with 2 equiv of aniline HCl. Yield:
73%, mp. 192 °C. 1H NMR (CD3OD): d 6.98–7.03 (m, 2H), 7.21–7.37
(m, 6H). MS (EI): m/z 279 [M]+.
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with 4-(1-pyrrolidino)-butyl-
amine. Yield: 68%, brown oil. 1H NMR (CDCl3): d 1.60 (m, 4H),
1.76 (br s, 4H), 2.48 (br s, 6H), 3.21 (t, J = 6.6 Hz, 2H), 4.05 (br s,
2H), 6.75 (dd, J = 2.3 Hz, and 8.5 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H),
7.28 (d, J = 8.5 Hz, 1H). MS (EI): m/z 329 [M]+.
4.2.7. N-Benzyl-N0-(3,4-dichlorophenyl)-guanidine (13g)
Following the general procedure, Method 1, the title compound
was obtained by treatment of 11 with benzylamine. Yield: 88%, mp
126 °C. 1H NMR (CDCl3): d 4.40 (s, 2H), 6.76 (dd, J = 2.4 Hz, and
8.5 Hz, 1H), 7.03 (d, J = 2.5 Hz, 1H), 7.26–7.37 (m, 6H). MS (EI):
m/z 293 [M]+.
4.2.15. N-(6-t-Butoxycarbonylaminohexyl)-N0-(3,4-dichloro-
phenyl)-guanidine (13p)
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with N-Boc-1,6-hexanediamine.
Yield: 67%, brown oil. 1H NMR (CDCl3): d 1.30–1.60 (m, 8H), 1.44
(s, 9H), 3.09–3.16 (m, 2H), 3.21 (t, J = 6.9 Hz, 2H), 6.77 (dd,
J = 2.4 Hz, and 8.5 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.30 (d,
J = 8.4 Hz, 1H). MS (EI): m/z 402 [M]+.
4.2.8. N-(n-Hexyl)-N0-(3,4-dichlorophenyl)-guanidine (13h)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with n-hexylamine. Yield: 95%,
oil. 1H NMR (CDCl3): d 0.89 (t, J = 6.5 Hz, 3H), 1.30 (br s, 6H), 1.52
(br s, 2H), 3.11–3.20 (m, 2H), 6.77 (dd, J = 2.7 Hz, and 8.5 Hz, 1H),
7.02 (d, J = 2.7 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H). MS (EI): m/z 287
[M]+.
4.3. General procedure for the synthesis of N-alkoxy (8a–e) and
N-alkyl (9a–p) derivatives of N0-(3,4-dichlorophenyl)-N00-(1-iso-
propyl-4,5-dioxoimidazolidine-2-ylidene)guanidine (1)
A mixture of guanidine 12 or 13 (1.0 equiv) and 1-isopropyl-2-
methylsulfanyl-1H-imidazole-4,5-dione (10, 1.1–1.5 equiv) in
anhydrous CHCl3 was heated in a sealed tube at 100 °C for
18–72 h. The solvent was removed under reduced pressure. The
residue was purified via a silica gel column chromatography.
Chloroform and CHCl3/EtOAc were used as eluent for the purifica-
tion of 8a–e and 9a–p, respectively. The former was further
purified by recrystallization from CH2Cl2/hexane and the latter
was purified by recrystallization from EtOAc.
4.2.9. N-(2-Adamantyl)-N0-(3,4-dichlorophenyl)-guanidine (13i)
Following the general procedure, method 1, the title compound
was obtained by treatment of 11 with 2-adamantylamine HCl.
Yield: 55%, mp 212 °C. 1H NMR (CDCl3): d 1.62–2.05 (m, 15H),
3.73 (s, 1H), 6.79 (dd, J = 2.4 Hz, and 8.5 Hz, 1H), 7.06 (d,
J = 2.4 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H). MS (EI): m/z 337 [M]+.
4.2.10. N-(2-Dimethylaminoethyl)-N0-(3,4-dichlorophenyl)-gua-
nidine (13j)
4.3.1. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-methoxyguanidine (8a)
Following the general procedure, the title compound was
obtained from 12a. Yield: 20%; mp 156 °C. 1H NMR (CDCl3): d 1.45
(d, J = 7.0 Hz, 6H), 3.88 (s, 3H), 4.55 (m, 1H), 6.94 (dd, J = 2.5 Hz,
and 8.6 Hz, 1H), 7.12 (br s, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.46 (s,
2.5 Hz, 1H); 13C NMR (CDCl3): d 19.5, 45.8, 62.3, 121.1, 124.1,
Following the general procedure, method 2, the title compound
was obtained by treatment of 14 with N,N-dimethylethylenedi-
amine. Yield: 61%, brown oil. 1H NMR (CDCl3): d 2.25 (s, 6H),
2.46 (t, J = 5.2 Hz, 2H), 3.25 (t, J = 5.2 Hz, 2H), 4.74 (br s, 1H), 6.76
(dd, J = 2.40 Hz, and 8.49 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.29 (d,
J = 8.5 Hz, 1H). MS (EI): m/z 275 [M]+.

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
1547
127.1, 130.2, 132.4, 137.6, 145.5, 149.6, 155.8, 155.9; MS (EI): m/z
371 [M]+. Anal. Calcd for C14H15Cl2N5O3: C, 45.18; H, 4.06; N,
18.82; Cl, 19.05. Found: C, 45.46; H, 4.10; N, 18.80; Cl, 19.21.
J = 7.0 Hz, 3H), 1.47 (d, J = 6.9 Hz, 6H), 3.57 (q, J = 7.4 Hz, 2H), 4.55
(m, 1H), 7.12 (d, J = 8.74, Hz, 1H), 7.39 (br s, 1H), 7.55 (d, J = 8.6 Hz,
1H), 10.5 (br s, 1H); 13C NMR (DMSO-d6): d 13.6, 19.2, 39.0, 47.4,
119.9, 124.2, 127.0, 131.3, 131.8, 155.6, 158.3; MS (EI): m/z 369
[M]+. Anal. Calcd for C15H17Cl2N5O2: C, 48.66; H, 4.63; N, 19.15; Cl,
18.92. Found: C, 48.48; H, 4.47; N, 19.25; Cl, 18.72.
4.3.2. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-ethoxyguanidine (8b)
Following the general procedure, the title compound was ob-
tained from 12b. Yield: 48%; mp 155 °C. 1H NMR (CDCl3): d 1.31
(t, J = 7.0 Hz, 3H), 1.45 (d, J = 7.0 Hz, 6H), 4.10 (q, J = 7.0 Hz, 2H),
4.54 (m, 1H), 6.95 (dd, J = 8.7, and 2.6 Hz, 1H), 7.13 (br s, 1H),
7.34 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 2.5 Hz, 1H), 10.52 (br s, 1H);
13C NMR (CDCl3): d 14.6, 19.5, 45.8, 70.1, 121.2, 124.1, 127.0,
130.2, 132.4, 137.7, 145.4, 149.5, 155.8, 156.9; MS (EI): m/z 385
[M]+. Anal. Calcd for C15H17Cl2N5O3: C, 46.65; H, 4.44; N, 18.36;
Cl, 18.13. Found: C, 46.49; H, 4.35; N, 18.54; Cl, 17.83.
4.3.8. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-isopropylguanidine (9c)
Following the general procedure, the title compound was ob-
tained from 13c. Yield: 20%; mp 191 °C. 1H NMR (CDCl3): d 1.27
(d, J = 6.6 Hz, 6H), 1.47 (d, J = 6.9 Hz, 6H), 4.39 (m, 1H), 4.53 (m,
1H), 7.11 (dd, J = 2.5 Hz, and 8.4 Hz, 1H), 7.38 (d, J = 2.5 Hz, 1H),
7.57 (d, J = 8.4 Hz, 1H); 13C NMR (CDCl3): d 20.2, 22.7, 44.6, 45.1,
125.1, 127.8, 132.0, 132.4, 134.1, 134.3, 157.6, 161.1, 169.9,
171.9; MS (EI): m/z 383 [M]+. Anal. Calcd for C16H19Cl2N5O2: C,
50.01; H, 4.98; N, 18.45; Cl, 18.23. Found: C, 49.86; H, 5.05; N,
17.99; Cl, 18.58.
4.3.3. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-allyloxyguanidine (8c)
Following the general procedure, the title compound was
obtained from 12c. Yield: 10%; mp 266 °C. 1H NMR (CDCl3): d
1.45 (d, J = 7.0 Hz, 6H), 4.52–4.57 (m, 3H), 5.32–5.41 (m, 2H),
5.96–6.09 (m, 1H), 6.95 (dd, J = 2.5 Hz, and 8.6 Hz, 1H), 7.14 (br s,
1H), 7.33 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 2.5 Hz, 1H); 13C NMR
(CDCl3): d 19.5, 45.8, 75.3, 77.2, 118.8, 121.2, 124.1, 127.1, 130.2,
132.4, 134.0, 137.6, 145.7, 149.7, 155.7, 155.9; MS (EI) m/z 397
[M]+. Anal. Calcd for C16H17Cl2N5O3: C, 48.25; H, 4.30; N, 17.59;
Cl, 17.80. Found: C, 48.31; H, 4.36; N, 17.55; Cl, 18.03.
4.3.9. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-t-butylguanidine (9d)
Following the general procedure, the title compound was ob-
tained from 13d. Yield: 26%; mp 243 °C. 1H NMR (CDCl3): d 1.42
(s, 9H), 1.51 (d, J = 6.9 Hz, 6H), 4.57 (m, 1H), 5.19 (br s, 1H), 7.10
(dd, J = 2.2 Hz, and 8.4 Hz, 1H), 7.37 (br s, 1H), 7.56 (d, J = 8.5 Hz,
1H); 13C NMR (CDCl3): d 19.8, 29.6, 44.9, 54.1, 124.8, 127.6,
131.9, 134.3, 158.1, 161.2, 169.9, 171.2; MS (EI): m/z 397 [M]+.
Anal. Calcd for C17H21Cl2N5O2: C, 51.27; H, 5.31; N, 17.80; Cl,
17.58. Found: C, 51.30; H, 5.32; N, 17.99; Cl, 17.29.
4.3.4. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-t-butoxyguanidine (8d)
Following the general procedure, the title compound was ob-
tained from 12d. Yield: 70%; mp 250 °C. 1H NMR (CDCl3): d 1.36 (s,
9H), 1.46 (d, J = 7.0 Hz, 6H), 4.56 (m, 1H), 6.97 (dd, J = 2.5 Hz, and
8.6 Hz, 1H), 7.15 (br s, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.47 (d,
J = 2.5 Hz, 1H); 13C NMR (CDCl3): d 19.5, 27.7, 45.7, 79.4, 121.1,
124.0, 126.7, 130.1, 132.3, 137.9, 145.3, 149.1, 155.8, 156.0; MS
(EI): m/z 413 [M]+. Anal. Calcd for C17H21Cl2N5O3: C, 49.29; H, 5.11;
N, 16.90; Cl, 17.12. Found: C, 49.14; H, 5.14; N, 16.67; Cl, 17.32.
4.3.10. N-(3,4-Dichloro-phenyl)-N0-(2,2-dimethyl-propyl)-N00-(1-
isopropyl-4,5-dioxo-4,5-dihydro-1H-imidazol-2-yl)-guanidine (9e)
Following the general procedure, the title compound was ob-
tained from 13e. Yield: 37%; mp 203 °C. Major tautomer: 1H
NMR (CDCl3): d 0.94 (s, 9H), 1.48 (d, J = 6.5 Hz, 6H), 3.38 (br d,
J = 5.8 Hz, 2H), 4.55 (m, 1H), 7.14 (br d, J = 8.3 Hz, 1H), 7.41 (br s,
1H), 7.58 (br d, J = 8.3 Hz, 1H); 13C NMR (CDCl3): d 20.1, 27.4,
31.7, 44.7, 54.1, 124.4, 127.5, 127.5, 129.9, 131.9, 136.4, 157.8,
161.6, 170.7, 171.5; Minor tautomer: 1H NMR (CDCl3) d 1.07 (s,
9H), 1.30 (d, J = 7.1 Hz, 6H), 3.23 (br s, 2H), 4.34 (m, 1H), 7.20 (br
d, J = 8.3 Hz, 1H), 7.55 (br s, 1H), 7.64 (br s, 1H); 13C NMR (CDCl3):
d.19.8, 27.4, 32.6, 44.6, 53.3, 125.1, 127.8, 129.4, 131.8, 134.1,
159.2, 161.5, 170.3, 171.5. MS (EI): m/z 411 [M]+. Anal. Calcd for
4.3.5. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-benzyloxyguanidine (8e)
Following the general procedure, the title compound was ob-
tained from 12e. Yield: 20%; mp 174 °C. 1H NMR (CDCl3): d 1.43
(d, J = 7.0 Hz, 6H), 4.50 (m, 1H), 5.05 (s, 2H), 6.93 (dd, J = 2.7 Hz,
and 8.6 Hz, 1H), 7.14 (br s, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.39–7.44
(m, 5H); 13C NMR (CDCl3): d 19.7, 46.0, 76.8, 121.4, 124.3, 124.4,
127.4, 128.8, 129.0, 130.4, 132.6, 137.2, 137.7, 146.0, 150.0,
155.8, 156.2; MS (EI): m/z 447 [M]+. Anal. Calcd for C20H19Cl2N5O3:
C, 53.58; H, 4.27; N, 15.62; Cl, 15.82. Found: C, 53.47; H, 4.23; N,
15.55; Cl, 15.68.
C18H23Cl2N5O2: C, 52.43; H, 5.62; N, 17.20; Cl, 16.99. Found: C,
52.66; H, 5.71; N, 16.91; Cl, 16.81.
4.3.11. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-phenylguanidine (9f)
Following the general procedure, the title compound was
obtained from 13f. Yield: 25%; mp 189 °C. 1H NMR (CDCl3): d
1.34 (d, J = 6.9 Hz, 6H), 4.40 (m, 1H), 7.10 (br d, J = 8.1 Hz, 1H),
7.32–7.60 (m, 7H); 13C NMR (CDCl3): d 19.8, 44.7, 123.8, 125.6,
127.1, 128.2, 130.4, 132.6, 134.2, 135.0, 157.1, 161.0, 169.8,
172.4; MS (EI): m/z 417 [M]+; Anal: Calcd for C19H17Cl2N5O2: C,
54.56; H, 4.10; N, 16.74; Cl, 16.95. Found: C, 54.39; H, 3.98; N,
16.60; Cl, 16.90.
4.3.6. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-methylguanidine (9a)
Following the general procedure, the title compound was
obtained from 13a. Yield: 40%; mp 229 °C. 1H NMR (CDCl3): d
1.48 (d, J = 6.9 Hz, 6H), 3.10 (s, 3H), 4.58 (m, 1H), 7.14 (br d,
J = 8.6 Hz, 1H), 7.40 (s, 1H), 7.56 (d, J = 8.6 Hz, 1H); 13C NMR
(DMSO-d6): d 170.29, 168.24, 161.73, 159.02, 137.87, 136.26,
131.82, 130.67, 127.75, 125.68, 43.89, 29.51, 20.04; MS (EI): m/z
355 [M]+. Anal. Calcd for C14H15Cl2N5O2: C, 47.21; H, 4.24; N,
19.91; Cl, 19.66. Found: C, 47.28; H, 4.22; N, 20.07; Cl, 19.52.
4.3.12. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-benzylguanidine (9g)
Following the general procedure, the title compound was
obtained from 13g. Yield: 34%; mp 190 °C. 1H NMR (CDCl3): d
1.40 (d, J = 6.8 Hz, 6H), 4.52 (m, 1H), 4.74 (br d, J = 5.4 Hz, 2H),
5.97 (br s, 1H), 7.13 (br d, J = 8.5 Hz, 1H), 7.25–7.40 (m, 6H), 7.53
(d, J = 8.5 Hz, 1H); Major tautomer: 13C NMR (CDCl3) d 19.6, 43.5,
45.5, 125.2, 127.5, 127.8, 128.4, 129.4, 130.3, 131.5, 136.1, 138.1,
159.0, 161.3, 169.5; Minor tautomer: 13C NMR (CDCl3) d 19.6,
4.3.7. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-ethylguanidine (9b)
Following the general procedure, the title compound was ob-
tained from 13b. Yield: 26%; mp 217 °C. 1H NMR (CDCl3): d 1.24 (t,

1548
L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
43.5, 45.3, 125.8, 127.1, 127.8, 128.4, 129.4, 130.6, 131.8, 136.1,
(d, J = 6.9 Hz, 6H), 1.91 (br s, 4H), 2.85 (br s, 4H), 2.92–2.95 (m,
2H), 3.55 (br s, 2H), 4.38 (m, 1H), 6.98 (d, J = 8.6 Hz, 1H), 7.41 (d,
J = 8.6 Hz, 1H), 7.49 (s, 1H); 13C NMR (CDCl3): d 19.7, 23.6, 41.9,
44.3, 53.6, 57.0, 122.3, 125.7, 128.1, 130.1, 132.2, 138.2, 159.6,
161.3, 170.4; LC–MS (ESI): m/z 439 [M+1]+. Anal. Calcd for
137.2, 155.5, 161.2, 168.9; MS (EI): m/z 431 [M]+. Anal. Calcd for
C20H19Cl2N5O2: C, 55.57; H, 4.43; N, 16.40; Cl, 16.20. Found: C,
55.38; H, 4.43; N, 16.52; Cl, 16.01.
4.3.13. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-N-hexyl-guanidine (9h)
C19H24Cl2N6O2: C, 51.94; H, 5.51; N, 19.13; Cl, 16.14. Found: C,
51.78; H, 5.59; N, 18.84; Cl, 15.89.
Following the general procedure, the title compound was
obtained from 13h. Yield: 21%; mp 147 °C. 1H NMR (CDCl3): d
0.86–0.91 (m, 3H), 1.30 (br s, 6H), 1.47 (d, J = 6.9 Hz, 6H), 1.58–
1.63 (m, 2H), 3.49–3.54 (m, 2H), 4.55 (m, 1H), 7.12 (dd, J = 2.2 Hz,
and 8.5 Hz, 1H), 7.39 (d, J = 2.2 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H); Major
tautomer: 13C NMR (CDCl3) d 14.0, 20.1, 22.5, 26.4, 29.6, 31.4, 42.7,
44.7, 124.5, 127.6, 129.9, 131.6, 133.8, 136.4, 157.4, 161.5, 170.4,
170.9; Minor tautomer: 13C NMR (CDCl3) d 14.0, 19.8, 22.5, 26.5,
28.6, 31.3, 42.5, 44.7, 125.1, 127.6, 129.4, 131.9, 134.3, 136.4,
158.6, 161.5, 170.4, 171.4; LC–MS (ESI): m/z 426 [M+1]+. Anal. Calcd
for C19H25Cl2N5O2: C, 53.53; H, 5.91; N, 16.43; Cl, 16.63. Found: C,
53.47; H, 5.84; N, 16.27; Cl, 16.74.
4.3.18. N-(3,4-Dichloro-phenyl)-N0-(3-dimethylamino-propyl)-
N00-(1-isopropyl-4,5-dioxo-4,5-dihydro-1H-imidazol-2-yl)-
guanidine (9m)
Following the general procedure, the title compound was ob-
tained from 13m. Yield: 31%; mp 110 °C. Major tautomer: 1H
NMR (CDCl3) d 1.33 (d, J = 6.9 Hz, 6H), 1.70 (br s, 2H), 1.89 (s,
6H), 2.41–2.44 (m, 2H), 3.64 (br s, 2H), 4.37 (m, 1H), 7.06 (dd,
J = 2.4 Hz, and 8.6 Hz, 1H), 7.40 (d, J = 2.6 Hz, 1H), 7.42 (d,
J = 8.6 Hz, 1H); 13C NMR (CDCl3): d 20.8, 27.9, 40.8, 45.4, 45.6,
54.7, 123.9, 127.2, 131.2, 132.4, 133.5, 138.6, 160.8, 162.2,
171.6, 172.7; Minor tautomer: 1H NMR (CDCl3)
d 1.47 (d,
J = 6.9 Hz, 6H), 1.89 (br s, 2H), 2.22 (s, 6H), 2.47–2.52 (m, 2H),
3.66–3.68 (m, 2H), 4.56 (m, 1H), 7.13 (dd, J = 2.6 Hz, and 8.5 Hz,
1H), 7.54 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 2.8 Hz, 1H); 13C NMR
(CDCl3): d 21.1, 24.4, 40.8, 45.3, 45.8, 60.6, 126.9, 129.3, 129.8,
132.8, 134.6, 136.0, 159.2, 162.2, 171.0, 172.5; LC–MS (ESI):
m/z 427 [M+1]+. Anal. Calcd for C18H24Cl2N6O2: C, 50.59; H,
5.66; N, 19.67; Cl, 16.59. Found: C, 50.46; H, 5.66; N, 19.76; Cl,
16.48.
4.3.14. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-(2-adamantyl)-guanidine (9i)
Following the general procedure, the title compound was ob-
tained from 13i. Yield: 32%; mp 251 °C. 1H NMR (DMSO-d6 with
D2O): d 1.17 (d, J = 6.7 Hz, 6H), 1.64–1.74 (m, 4H), 1.87–2.03 (m,
10H), 4.07 (s, 1H), 4.17 (m, 1H), 7.34 (br d, J = 8.6 Hz, 1H), 7.66
(br d, J = 8.6 Hz, 1H), 7.74 (s, 1H); Major tautomer: 13C NMR (CDCl3)
d 20.3, 26.7, 31.9, 36.8, 37.0, 44.6, 56.5, 124.4, 127.3, 129.9, 132.2,
134.0, 136.3, 157.3, 161.1, 169.9, 171.6; Minor tautomer: 13C NMR
(CDCl3) d 19.8, 26.6, 31.9, 36.6, 37.1, 44.5, 56.8, 124.5, 127.7, 129.5,
132.5, 134.6, 136.3, 156.1, 161.5, 170.7, 172.2; MS (EI): m/z 475
[M]+. Anal. Calcd for C23H27Cl2N5O2: C, 57.99; H, 5.71; N, 14.70;
Cl, 14.88. Found: C, 57.86; H, 5.66; N, 14.62; Cl, 14.80.
4.3.19. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-(4-pyrrolidin-1-yl-butyl)-
guanidine (9n)
Following the general procedure, the title compound was ob-
tained from 13n. Yield: 23%; mp 80 °C. Major tautomer: 1H NMR
(CDCl3): d 1.44 (d, J = 6.9 Hz, 6H), 1.83 (br s, 4H), 2.18 (br s, 4H),
2.98 (br s, 2H), 3.18 (br s, 2H), 3.37 (br s, 1H), 3.52 (br s, 1H),
3.78 (br s, 2H), 4.56 (m, 1H), 7.05 (dd, J = 2.4 and 8.6 Hz, 1H),
7.36 (d, J = 2.4 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H); 13C NMR (CDCl3):
d 20.1, 23.3, 26.3, 27.6, 42.7, 44.5, 54.1, 55.8, 124.0, 126.3, 131.4,
133.6, 135.4, 139.3, 158.5, 161.5, 168.6, 170.5; Minor tautomer:
1H NMR (CDCl3) d 1.32 (d, J = 6.9 Hz, 6H), 1.74 (br s, 4H), 2.15 (br
s, 4H), 2.99 (br s, 2H), 3.17 (br s, 2H), 3.39 (br s, 1H), 3.54 (br s,
1H), 3.79 (br s, 2H), 4.42 (m, 1H), 7.07 (dd, J = 2.4 and 8.5 Hz,
1H), 7.32 (d, J = 2.4 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H); 13C NMR
(CDCl3): d 19.8, 24.0, 29.6, 31.6, 40.5, 43.7, 55.2, 60.4, 125.3,
128.1, 129.1, 129.9, 130.7, 132.1, 158.1, 161.5, 168.5, 171.1; LC–MS
(ESI): m/z 467 [M+1]+. Anal. Calcd for C21H28Cl2N6O2ꢄ0.1H2O: C,
53.76; H, 6.06; N, 15.11; Cl, 17.91. Found: C, 53.43; H, 6.03; N,
14.93; Cl, 17.80.
4.3.15. N-(3,4-Dichloro-phenyl)-N0-(2-dimethylamino-ethyl)-
N00-(1-isopropyl-4,5-dioxo-4,5-dihydro-1H-imidazol-2-yl)-
guanidine (9j)
Following the general procedure, the title compound was ob-
tained from 13j. Yield: 18%; mp 198 °C. 1H NMR (CDCl3): d 1.34
(d, J = 6.9 Hz, 6H), 2.46 (s, 6H), 2.78 (t, J = 4.1 Hz, 2H), 3.51 (br s,
2H), 4.39 (m, 1H), 6.95 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 7.40 (d,
J = 8.6 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 10.61 (br s, 1H); 13C NMR
(CDCl3): d 19.9, 41.7, 44.6, 45.0, 60.6, 121.9, 125.4, 128.4, 130.3,
132.6, 137.5, 160.0, 161.3, 170.6, 171.7; LC–MS (ESI): m/z 413
[M+1]+. Anal. Calcd for C17H22Cl2N6O2: C, 49.40; H, 5.37; N,
17.16; Cl, 20.33. Found: C, 49.60; H, 5.53; N, 17.03; Cl, 19.97.
4.3.16. N-(3,4-Dichloro-phenyl)-N0-(2-diethylamino-ethyl)-N00-
(1-isopropyl-4,5-dioxo-4,5-dihydro-1H-imidazol-2-yl)-
guanidine (9k)
4.3.20. {6-[N0-(3,4-Dichloro-phenyl)-N00-(1-isopropyl-4,5-dioxo-
4,5-dihydro-1H-imidazol-2-yl)-guanidino]-hexyl}-carbamic
acid tert-butyl ester (9p)
Following the general procedure, the title compound was ob-
tained from 13k. Yield: 17%; mp 139 °C. 1H NMR (CDCl3): d 1.10
(t, J = 7.2 Hz, 6H), 1.32 (d, J = 7.0 Hz, 6H), 2.73 (q, J = 7.8 Hz, 4H),
2.78 (t, J = 3.9 Hz, 2H), 3.52 (br s, 2H), 4.36 (m, 1H), 6.97 (dd,
J = 2.4 Hz, and 8.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.52 (d,
J = 2.4 Hz, 1H), 10.60 (br s, 1H); 13C NMR (CDCl3): d 11.0, 19.9,
42.7, 44.5, 47.2, 55.2, 122.5, 126.1, 128.8, 130.3, 132.6, 137.1,
160.4, 161.3, 170.6, 171.9; LC–MS (ESI): m/z 441 [M+1]+. Anal.
Calcd for C19H26Cl2N6O2: C, 51.71; H, 5.94; N, 16.07; Cl, 19.04.
Found: C, 51.69; H, 5.95; N, 16.19; Cl, 18.91.
Following the general procedure, the title compound was
obtained from 13p. Yield: 39%, mp 142 °C. 1H NMR (CDCl3):
d 1.30–1.80 (m, 8H), 1.36 (s, 9H), 1.47 (d, J = 6.9 Hz, 6H), 3.06–
3.19 (m, 2H), 3.43–3.55 (m, 2H), 4.55 (m, 1H), 7.13 (dd,
J = 2.2 Hz, and 8.5 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.52 (d,
J = 8.5 Hz, 1H); Major tautomer: 13C NMR (CDCl3) d 20.0, 25.5,
26.0, 28.2, 29.3, 29.9, 39.6, 42.2, 44.5, 79.0, 124.4, 127.5, 129.8,
131.4, 133.6, 136.3, 156.0, 161.4, 170.7, 171.2; Minor tautomer:
13C NMR (CDCl3) d 19.7, 25.4, 26.3, 28.3, 29.7, 29.9, 40.2, 41.8,
44.5, 125.2, 127.7, 129.3, 131.8, 134.2, 136.3, 157.4, 158.3, 170.2,
171.2; LC–MS (ESI): m/z 541 [M+1]+. Anal. Calcd for C24H34Cl2N6O4:
C, 53.24; H, 6.33; N, 15.52; Cl, 13.10. Found: C, 53.23; H, 6.40; N,
15.47; Cl, 13.05.
4.3.17. N-(3,4-Dichloro-phenyl)-N0-(1-isopropyl-4,5-dioxo-4,5-
dihydro-1H-imidazol-2-yl)-N00-(2-pyrrolidin-1-yl-ethyl)-
guanidine (9l)
Following the general procedure, the title compound was ob-
tained from 13l. Yield: 16%; mp 196 °C. 1H NMR (CDCl3): d 1.33

L. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 1541–1549
1549
4.4. Biological studies
antihypnozoite activity will delay the relapse or radically cure
the infection. To evaluate the radical curative properties, daily
blood samples are followed for 21 days, three times per week for
4 weeks, and then two times weekly until 100 days after the last
day of test compound administration. Parasite clearance should oc-
cur in all animals treated with chloroquine. Relapse is expected in
the control group. Relapse in the treated group indicates failure of
the test compounds. Monkeys showed no relapse after 100 days
are considered radically cured. Relapses of the control monkeys
are treated with chloroquine once daily for 7-days and observe
for the second relapse. Relapse in experimental animals and the
second relapse of the control monkeys are treated with the stan-
dard 7-day oral CQ and primaquine (1.78 mg base/kg). After stan-
dard treatment, blood smears were monitored daily for four
consecutive days and two times weekly for 2 weeks. The results
were shown in Table 3.
4.4.1. Assessment of metabolic stability
The in vitro half-life of each compound was measured in the
presence of human and mouse liver microsomes. The metabolic
stability assay sample preparation was performed in a 96-well
plate on a TECAN Genesis robotic sample processor following
WRAIR SOP SP 01-02. Samples were analyzed by LC–MS/MS using
fast LC gradient or isocratic methods. Parent drug was quantified
using external calibration, using plots of parent drug response ver-
sus amount. Chromatograms were analyzed using the mass spec-
trometry software Xcalibur QuanBrowser (for ThermoFinnigan
instruments) or MassLynx (for Waters instruments). Concentra-
tions of parent drug remaining at each time point were calculated
using the unknown peak areas and corresponding calibration
curves. 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 concentra-
tion versus time was generated, where the slope of that line was
ꢅk. Half-life was calculated as 0.693/k. The results are shown in
Table 1.
Acknowledgments
Material has been reviewed by the Walter Reed Army Institute
of Research. There is no objection to its presentation and/or
publications. The opinions or assertions contained herein are the
private views of the authors, and are not to be construed as official,
or as reflecting true views of the Department of the Army or the
Department of Defense. This research is supported in part by
funding from Military Infections Diseases Research Program
(A40191_09_WR), US Army Medical Research and Material
Command, Department of Defense, USA., Peer Reviewed Medical
Research Program (PRMRP) (Grant # PR054609), and Medicines
for Malaria Venture (MMV 04/0013), Geneva, Switzerland.
ChemMatCARS Sector 15 is principally supported by the National
Science Foundation/Department of Energy under grant number
CHE-0535644. Use of the Advanced Photon Source was supported
by the US Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. DE-AC02-06CH11357.
4.4.2. In vitro antimalarial studies
The in vitro assays were conducted by using a modification of
the semi automated microdilution technique of Desjardins et al
and Chulay et al.14,15 Two P. falciparum malaria parasite clones,
from CDC Indochina III (W-2), and CDC Sierra Leone I (D-6), were
utilized in susceptibility testing. They were derived by direct visu-
alization and micromanipulation from patient isolates.16 The re-
sults are shown in Table 1.
4.4.3. Assessment of prophylactic activity in mice
New compounds were assessed for their prophylactic activity in
exoerythrocytic (EE) mouse model using sporozoites of P. berghei.
The procedures have been previously described.1–4 Briefly, each
compound was ground with a mortar and pestle and suspended
in hydroxyethylcellulose and Tween 80 for compounds to be
administered PO and those given SC were suspended in peanut
oil. Each compound was prepared at different dose levels. Com-
pounds were administered either PO or SC to mice, once a day
for three consecutive days, on the day before, 4 h before, and the
day after being inoculated with sporozoites of P. berghei intrave-
nously. Whole body weights were taken on Day 0 and Day 6 then
approximately twice a week for 31 days. A blood film was taken on
Day 5 and then approximately twice a week for 31 days. Mice los-
ing greater than about 20% of their body weight were sacrificed. A
compound was considered active if only low levels of parasites
were found (less than about 10%) in blood films taken on Day 5
or any biweekly for 31 days. Mice alive on Day 31 with no parasites
found in any blood films were considered protected. Table 1 sum-
marizes the test results of the new compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.028.
References and notes
1. Guan, J.; Zhang, Q.; Gettayacamin, M.; Karle, J. M.; Ditusa, C. A.; Milhous, W. K.;
Skillman, D. R.; Lin, A. J. Bioorg. Med. Chem. 2005, 23, 699.
2. Zhang, Q.; Guan, J.; Sacci, J.; Ager, A.; Ellis, W. Y.; Milhous, W. K.; Kyle, D. E.; Lin,
A. J. J. Med. Chem. 2005, 48, 6472.
3. Lin, A. J.; Zhang, Q.; Guan, J.; Milhous, W. K. U.S. Patent No. 7101,902, 2006,
September 5.
4. Guan, J.; Wang, X.; Smith, K.; Ager, A.; Gettayacamin, M.; Kyle, D. E.; Milhous,
W. K.; Kozar, M. P.; Magill, A. J.; Lin, A. J. J. Med. Chem. 2007, 50, 6226.
5. Corcoran, K. D.; Hansukjariya, P.; Sattabongkot, J.; Ngampochjana; Edstein, M.
D.; Smith, C. D.; Shanks, G. D.; Milhous, W. K. Am. J. Trop. Med. Hyg. 1993, 49,
473.
4.4.4. Assessment of causal prophylactic/radical curative
activities in Rhesus monkeys
6. Sturk, L. M.; Brock, J. L.; Bagnell, C. R.; Hall, J. E.; Tidwell, R. R. Acta Trop. 2004,
91, 131.
The causal prophylactic antimalarial activity of the new deriva-
tives 8e and 9c were assessed in a P. cynomolgi sporozoites chal-
lenged Rhesus monkey model. Detailed procedure of sporozoites
harvest and drug tests are described in the previous reports.1–4
The results are shown in Table 2. Assessment of radical curative
activity of the test compounds were 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) by oral for seven
consecutive days and the test compounds by im for three consec-
utive days after the parasitemia level reached 5000 parasites/
mm.3 Chloroquine at 10 mg/kg/day ꢀ 7 eliminates the blood stage
parasites, but not the liver stage hypnozoites. Compounds with
7. Linney, I. D.; Buck, I. M.; Harper, E. A.; Kalindjian, S. K.; Pether, M. J.; Shankley,
N. P.; Watt, G. F.; Wright, P. T. J. Med. Chem. 2000, 43, 2362.
8. Fauss, R.; Jochem, R. U.S. Patent No. 4791,229, 1988, December 13.
9. Corniere, A. R.; Dijols, S.; Perollier, C.; Groboillot, D. L.; Boucher, J. L.; Attias, R.;
Sari, M. A.; Stuehr, D.; Mansuy, D. J. Med. Chem. 2002, 45, 944.
10. Wallace, G. C.; Fukuto, J. M. J. Med. Chem. 1991, 34, 1746.
11. Schantl, J. G.; Tuerk, W. Sci. Pharm. 1989, 57, 375.
12. Bailey, D. M.; DeGrazia, C. G.; Lape, H. E.; Frering, R.; Fort, D.; Skulan, T. J. Med.
Chem. 1973, 16, 151.
13. DeCosta, B. R.; He, X. S.; Dominguez, C.; Cutts, J.; Williams, W.; Bowen, W. D. J.
Med. Chem. 1994, 37, 314.
14. Desjardins, R. E.; Canfield, C. J.; Haynes, D. E.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710.
15. Chulay, J. D.; Haynes, J. D.; Diggs, C. L. Exp. Parasitol. 1983, 55, 138.
16. Oduola, A. M.; Weatherly, N. F.; Bowdre, J. H. Exp. Parasitol. 1988, 66,
86.