Antimalarial activities of new guanidylimidazole and guanidylimidazoline derivatives

Article abstract of DOI:10.1021/jm200503s

A series of new guanidylimidazole derivatives was prepared and evaluated in mice and Rhesus monkeys infected with malarial sporozoites. The majority of the new compounds showed poor metabolic stability and weak in vitro activities in three clones of Plasm

Full text of DOI:10.1021/jm200503s

ARTICLE
pubs.acs.org/jmc
Antimalarial Activities of New Guanidylimidazole and
Guanidylimidazoline Derivatives
Liang Zhang, Ramadas Sathunuru, Diana Caridha, Brandon Pybus, Michael T. O’Neil, Michael P. Kozar, and
Ai J. Lin*
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland
20910, United States
S Supporting Information
b
ABSTRACT: A series of new guanidylimidazole derivatives was
prepared and evaluated in mice and Rhesus monkeys infected with
malarial sporozoites. The majority of the new compounds showed
poor metabolic stability and weak in vitro activities in three clones
of Plasmodium falciparum. Compounds 8a, 8h, 9a, 16a, and 16e
cured the mice infected with sporozoites of P. berghei at 160 and
320 mg/kg/day ꢁ 3 po. Compounds 8a showed better causal
prophylactic activity than primaquine, tafenoquine, and Malarone
in the Rhesus test. In the radical curative test, 8a cured one monkey and delayed relapse of another for 74 days at 30 mg/kg/day ꢁ 7
by im. By oral dosing, 8a delayed relapse 81 days for one and 32 days for other vs 11ꢀ12 days for control monkeys treated with
10 mg/kg of chloroquine by po alone. Compound 8h, which showed superior activity to 8a in mouse test, delayed the relapse of
treated monkeys for 21ꢀ26 days at 30 mg/kg/day ꢁ 7 by oral.
’ INTRODUCTION
showed very weak or no in vitro cell growth inhibition against
blood stage malaria, P. falciparum, and are inactive in the
Thompson mouse test against Plasmodium berghei, a blood stage
rodent malaria.16ꢀ20 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 less likely to develop drug resistance. However, com-
pound 3 showed poor activity by oral administration, delaying
the patency (the first day that parasites can be detected in the
blood smears after infection) of treated monkeys for only 3 days
vs untreated control. Oral efficacy is an essential consideration in
the search of prophylactic/radical curative antimalarial drugs.
Lack of oral activity of carbamates 3 and 4 in Rhesus testing
may be attributed to the hydrolysis of carbamate group in
stomach acid, leading to generation of insoluble parent com-
pounds 1 and 2. To overcome the acid instability of carbamates, a
series of acid stable carboxamide derivatives 5 and 6 were
prepared and again found to be active only by im; no activity
was found by oral dosing in Rhesus.20 Pharmacokinetic studies of
carboxamides 5 and 6 in rats revealed that the former is much
more stable metabolically than the latter, with t1/2 of 2ꢀ3 h and
<20 min, respectively. Both compounds 5 and 6 were converted
to the same s-triazine derivatives as shown in Scheme 1. This
transformation was also observed when both compounds were
Multiple drug resistance and lack of safe drugs to prevent or
radically cure the malaria diseases continue to pose challenges to
the containment of this deadly disease which have threatened the
life of millions of people in the developing world.1ꢀ5 Widespread
drug resistance to chloroquine, the first line antimalarial drug,
was reported in Southeast Asia and South America countries.6ꢀ10
Central nervous system (CNS) toxicity of mefloquine11 and
hemolytic side effects of primaquine and tafenoquine in glucose-
6-phosphate dehydrogenase (G6PD) deficient patients7,8 have
compromised the clinical value of these otherwise highly effec-
tive malaria therapeutic drugs. With the exception of quinolone
esters, only the 8-aminoquinoline drugs such as primaquine (PQ)
or tafenoquine (TQ) have activity against the liver stages of
Plasmodium vivax and Plasmodium falciparum malarias.12ꢀ15
Therefore, there is an eminent need for new and safe malaria
drugs to replace PQ and TQ to treat and protect the tourists
traveling in the endemic areas of the world.
Our malaria drug research teams at the Walter Reed Army
Institute of Research are placing emphasis on developing new
chemical entities with true causal prophylactic and/or radical
curative properties, stopping malaria before blood stages emerge.
Recently, a series of new 2-guanidylimidazolidinedione (IZ)
derivatives (1 and 2, Figure 1) was demonstrated in our labora-
tory to possess causal prophylactic antimalarial activity in Rhesus
monkeys infected with Plasmodium cynomolgi sporozoites.16ꢀ21
Carbamate 3, the most active compound of this class, protects
monkeys infected with P. cynomolgi sporozoites at a dose of
10 mg/kg ꢁ 3 days by im dosing. Nevertheless, the IZ derivatives
Received: April 25, 2011
Published: August 17, 2011
r
2011 American Chemical Society
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incubated in microsomal preparations and in phosphate buffer
solution, indicating that the conversion of carboxamides 5 and 6
to s-triazine 7 is a chemical rather than an enzymatic reaction.20
Because both carboxamides showed causal prophylactic activity
in Rhesus and the corresponding s-triazine are inactive, s-triazine
was ruled out as the possible active metabolite of compounds
5 and 6. As compound 5 is more stable and more efficacious
in Rhesus than compound 6, it was speculated that the parent
compound possesses intrinsic antimalarial activity, not its s-tri-
azine metabolite.
the 5-membered ring highly strained and susceptible to nucleo-
philic assaults. In this study, we report the synthesis and
antimalarial activity of a series of chemically stable imidazole
and imidazoline analogues. The chemically unstable imidazoli-
dinedione ring of the IZ molecules (1ꢀ4) were replaced with a
stable imidazole (8aꢀm) and/or imidazoline (9aꢀe) rings as
shown in Figure 2.
’ CHEMISTRY
The synthesis of carbamates 3 and 4 and later the carbox-
amides 5 and 6 derivatives of imidazolidinediones 1 and 2 were
based on the assumption that carboxamides are more stable
chemically than carbamates. However, as shown in Scheme 1,
although it is an amide, the imidazolidinedione ring is not as
stable as one might think, especially when the 5-membered
ring carries an electron withdrawing dichlorophenyl substituent
(2 and 4). The incorporation of two electron withdrawing
carboxy groups at 4- and 5-positions of the imidazolidine makes
1. Synthesis of Imidazole Analogues (8aꢀm). Two ap-
proaches were used to prepare the new compounds 8aꢀm. The
first approach involved the coupling between N1-substituted-
N2-(3,4-dichlorophenyl)-thiourea (11aꢀc) and 2-aminoimida-
zole (14aꢀd) to prepare compound 8aꢀf under the catalysis of
HgO (Scheme 2). The second approach employed the key inter-
mediate, N1-(3,4-dichlorophenyl)-N2-(2-imidazolyl)-thiourea
(15aꢀd), to react with appropriate substituted amines under
the catalysis of HgCl2 (Scheme 3). The difference between the
two approaches is the sequence of incorporation of 2-aminoimi-
dazole group into the final products. The products prepared by
both methods are identical, but the latter approach gave better
yields than the former.
The key intermediates, thiourea derivatives 11aꢀc, were
prepared in high yields by treatment of 3,4-dichlorophenyl-
isothiocyanate (10) with substituted amines in CH2Cl2 at room
temperature overnight. The other intermediates, 2-aminoimida-
zoles (14aꢀd), were prepared according to a reported pro-
cedure22 by treatment of 2-bromo-1,1-dimethoxyethane (12)
with appropriate amines to give 2-alkylamino-dimethoxyacetals
(13aꢀd). The latter compounds were heated with cyanoamine
in a mixture of 50% AcOH/conc HCl for an hour at 100 °C,
followed by hydrolysis in conc KOH to give 2-aminoimidazoles
(14aꢀd) with yields ranging from 47% to 70%. The coupling of
Figure 1. Guanidylimidazolidinedione derivatives.
Scheme 1. Mechanism of Chemical Transformation from Carboxamides 5 and 6 to s-Triazine 7
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Figure 2. Imidazole 8aꢀm and imidazoline 9aꢀe Analogues.
Scheme 2. Synthesis of Imidazole Analogues 8aꢀfa
a Reagents and conditions: (i) substituted amines, CH2Cl2, rt, 18 h; (ii) substituted amines, sealed tube, 80ꢀ100 °C, neat, 18 h; (iii) (a) NH2CN, HOAc
(50% aq), HCl (10 M), 100 °C, 1 h; (b) KOH (50%).
thioureas (11aꢀc) and 2-aminoimidazoles (14aꢀd) was facili-
tated by mercuric oxide as catalyst to give the desired products
8aꢀf in 30ꢀ54% yields. Without HgO as catalyst, the thioureas
failed to react with the 2-aminoimidazoles. Theoretically, the
coupling of 11aꢀc with compounds 14aꢀd should give, besides
the desired products 8aꢀf, other isomers 80aꢀf.23 However, the
reaction gave exclusively the desired products 8aꢀf.
with 2-aminoimidazoles 14aꢀd to give compounds 15aꢀd
(Scheme 3). The structure of compound 15a was confirmed
by single-crystal X-ray diffraction data as shown in Figure 4
(Supporting Information p S-2: X-ray crystallography of 1-(3,4-
dichlorophenyl)-3-(1-isopropyl-1H-imidazol-2- yl)-thiourea (15a)).
Upon reaction with appropriate amines under the catalysis of
HgCl2, compounds 15aꢀd gave the desired products 8aꢀc and
8gꢀm. Mercuric chloride facilitated the coupling reaction.24
However, it can be replaced with a nontoxic dicyclohexylcarbo-
diimide (DCC), as exemplified by the synthesis of 8m. The
1H and 13C NMR spectrum and the TLC Rf value of 8a prepared
by both methods are identical. It is to be noted that HgO cannot
be used to substitute for HgCl2 as catalyst in the coupling
reaction between thioureas and amines as shown in Scheme 3.
Conversely, HgCl2 cannot be used to replace HgO as catalyst in a
Because the NMR spectrum of both isomers 8a and 8a0 are too
close to be distinguished, the confirmation of the structure 8a
relied mainly on the single crystal X-ray crystallography tech-
nique. The result is as shown in Figure 3 (Supporting Informa-
tion p S-1: X-ray crystallography of N-(3,4-dichlorophenyl)-N0-
isopropyl-N00-(1-isopropyl-1H-imidazol-2-yl)-guanidine (8a)).
An alternative method to prepare compounds 8gꢀm was
accomplished by treating 3,4-dichlorophenylisothiocyanate (10)
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Scheme 3. Synthesis of 8gꢀm and 16aꢀha
a Reagents and conditions: (i) CH2Cl2, rt, 18 h; (ii) ammonia, or substituted amine, HgCl2, DMF or CH3CN, rt, 30 min to 18 h, or substituted amine,
i-Pr2NEt, DCC, CH3CN, rt, 18 h; (iii) RCOCl, TEA, CHCl3 (for 16e, RCOOCOR was used).
Scheme 4. Synthesis of Imidazolidine Analogues 9aꢀea
a Reagents and conditions: (i) 3,4-dichlorophenylcyanamide, EtOH, reflux, 24 h; (ii) 3,4-dichlorophenyl isothiocyanate, CH2Cl2, rt, 24 h; (iii) ammonia
or substituted amines, HgCl2, CH3CN or DMF, rt, 10 min to 18 h.
coupling reaction between 11aꢀc and 2-aminoimidazoles (14a)
as shown in Scheme 2. A series of carboxamide and carbamate
derivatives 16aꢀh were synthesized from amine 8hꢀj as shown
in Scheme 3.
the preparation of 8gꢀm, with HgCl2 as catalyst in CH3CN or
DMF (Scheme 3). Attempts were made to improve the yield of
18 and minimize the byproduct 180 formation using various
solvents, i.e., hexanes, CHCl3, CH3CN, DMF, THF, Et2O, EtOH,
or Et3N but failed.
Initially, we envisioned that mercuric chloride, lead chloride,
or mercuric oxide which facilitated the synthesis of imidazole
derivatives (8gꢀm) could be used to assemble the imidazoline
analogues (9aꢀe).26 However, coupling reaction between 11a
and imidazoline 17 failed to give the desired compound 9b.
Likewise, heating of 17 with 3,4-dichlorophenylcyanamide in
EtOH under reflux for 24 h also did not yield 9a. Further-
more, treatment of 3,4-dichlorophenylguanidine 20 with 1-
isopropyl-2-methylmercapto-imidazoline 19 did not provide the
desired 9a.
2. Synthesis of Imidazoline Analogues (9aꢀe). Approaches
used for the synthesis of imidazoline analogues 9aꢀe were as
shown in Scheme 4. 3,4-Dichlorophenyl isothiocyanate 10 was
reacted smoothly with 2-aminoimidazoline 17, yielding two iso-
mers 18 (27% yield) and 180 (50% yield), which were separated
via a column chromatography.25 The structure of thiourea 18 was
determined by single crystal X-ray crystallography as shown in
Figure 5 (Supporting Information p S-3: X-ray crystallography of
1-(3,4-dichlorophenyl)-3-(1-isopropyl-4,5-dihydro-1H-imidazol-
2-yl)-thiourea (18)). The conversion of thiourea 18 to the final
products 9aꢀe was carried out by using the same procedure for
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1
’ EXPERIMENTAL SECTION
to give a light-brown liquid product 13a (60.0 g, 96% yield). H NMR
(CDCl3): δ 1.07 (d, J = 6.3 Hz, 6 H), 2.73 (d, J = 5.6 Hz, 2 H), 3.40 (s,
6 H), 4.48 (t, J = 5.6 Hz, 1 H). 13C NMR (CDCl3): δ 22.7, 48.4, 53.7,
53.8, and 103.9. MS (EI): m/z 147 [M]+. The product was pure enough
for next reactions without further purification.
Compounds 13bꢀd were prepared by the same procedure, except
t-butylamine, neopentylamine, and benzylamine were used, respectively,
in place of isopropylamine.
Melting points were determined in open capillary tubes on an
OptiMelt melting point apparatus (Standard Research Systems, USA)
1
and are uncorrected. H NMR and 13C NMR spectra were recorded
using Bruker Avance-300 and Bruker Avance 600 spectrometers (Bruker
Instrument, Inc., Wilmington, DE). Chemical shifts are given in ppm (δ)
relative to tetramethylsilane (TMS) as internal standard. Analytical thin-
layer chromatography (TLC) was performed using HPLC-HLF normal
phase 150 μm silica gel plates (Analtech, Newark, DE). Visualization of
the developed chromatogram was performed with UV absorbance or
iodine chamber. Flash chromatography was conducted with silica gel
60 Å (200ꢀ400 mesh) from Sigma-Aldrich Co. Solvents and reagents
obtained from commercial sources were used without purification unless
noted. Reactions were carried out under an inert atmosphere of nitrogen.
Elemental analysis was 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.
An LC/UVꢀvis/Trap MS was employed for purity analysis and chro-
mophore properties. The system consisted of an Agilent 1100 series LC-
UV/vis system online with a ThermoFinnigan (now Thermo-Fisher
Scientific, Waltham, MA) LCQ MS equipped with an electro spray
ionization (ESI) source. Samples were analyzed using shallow CH3CN:
1% HCOOH/H2O gradients at low flow rate. The purity of the final
products is g95%.
tert-Butyl-(2,2-dimethoxy-ethyl)-amine (13b). The title compound
was obtained by the same method of preparing compound 13a, except,
t-butylamine was used in place of isopropylamine to give as a light-brown
1
liquid in 83% yield. H NMR (CDCl3): δ 1.18 (s, 9 H), 2.71 (d, J =
5.58 Hz, 2 H), 3.40 (s, 6 H), 4.47 (t, J = 5.48 Hz, 1 H). MS (ESI): m/z
162 [M + 1]+.
(2,2-Dimethoxy-ethyl)-(2,2-dimethyl-propyl)-amine (13c). The
title compound was obtained as a light-brown liquid in 87% yield
from 2,2-dimethylpropylamine using the same procedure to prepare
1
compound 13a. H NMR (CDCl3): δ 0.90 (s, 9 H), 2.35 (s, 2 H),
2.72 (d, J = 5.6 Hz, 2 H), 3.36 (s, 6 H), 4.48 (t, J = 5.6 Hz, 1 H). MS
(EI): m/z 175 [M]+.
Benzyl-(2,2-dimethoxy-ethyl)-amine (13d). The title compound was
prepared in 98% yield by the same method of preparing compound 13a,
1
using benzylamine in place of isopropylamine. H NMR (CDCl3): δ
2.75 (d, J = 5.4 Hz, 2 H), 3.37 (s, 6 H), 3.81 (s, 2 H), 4.49 (t, J = 5.4 Hz,
1 H), 7.23ꢀ7.33 (m, 5 H).
1. Synthesis of Compounds 11aꢀc. Synthesis of 1-(3,4-Di-
chlorophenyl)-3-isopropyl-thiourea (11a)27. 3,4-Dichlorophenyl-iso-
cyanate (7.3 mL, 50.0 mmol) in CH2Cl2 (40.0 mL) was added dropwise
with stirring to a solution of isopropylamine (8.6 mL, 0.10 mol) in
CH2Cl2 (20.0 mL) at ambient temperature. After the addition of the
amine was completed, the reaction mixture was further stirred at rt for
18 h. The solvent was removed under reduced pressure. The residue was
suspended in diethyl ether and filtered. The crude product was washed
with diethyl ether, followed by hexanes to give 11a as a white solid
(12.8 g, 97% yield), mp 145.0ꢀ146.5 °C. 1H NMR (CDCl3): δ 1.24 (d,
J = 6.5 Hz, 6 H), 4.55 (m, 1 H), 5.75 (br s, 1 H), 7.08 (dd, J = 2.4 Hz, and
8.5 Hz, 1 H), 7.33 (d, J = 2.4 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1 H), 7.74 (br s,
1 H). MS (ESI): m/z 263 [M + 1]+.
3. Synthesis of Compounds 14aꢀd. Synthesis of 1-Isopropyl-
1H-imidazol-2-ylamine (14a):23. N-Isopropyl substituted aminoacetal
13a (6.2 g, 42 mmol) was added dropwise to a stirring solution of cyan-
amide (5.2 g, 0.124 mol) in aqueous acetic acid (50%, 40 mL) at ambient
temperature. The reaction mixture was then heated at 100 °C for 1 h.
The solvent was removed under reduced pressure. The residue was dis-
solved in concentrated HCl (10 N, 20 mL), and the mixture was heated
at 100 °C for 15 min. The pH of the mixture was adjusted to ∼11 by
addition of conc aqueous KOH solution (50%). The mixture was then
extracted with CH2Cl2 (50 mL ꢁ 3). The CH2Cl2 extracts were
combined and dried over Na2SO4, and the solvent was removed under
reduced pressure to give desired product 14a as light-brown oil which
1
partially solidified on standing (2.5 g, 47% yield). H NMR (CDCl3):
Compounds 11bꢀc were prepared by the same procedure, except
neopentylamine or tert-butylamine was used in replace of isopropyl-
amine.
δ 1.39 (d, J = 6.5 Hz, 6 H), 4.14 (m, 1 H), 6.60 (d, J = 1.6 Hz, 1 H), 6.66
(d, J = 1.6 Hz, 1 H). MS (EI) m/z 125 [M]+.
Compound 14a was pure enough for next step reactions without
further purification. Compounds 14bꢀd were prepared by the same
procedure, except 13bꢀd was used, respectively, in place of 13a.
1-tert-Butyl-1H-imidazol-2-ylamine (14b). A brown crystalline solid
prepared from 13b and cyanamide in 69% yield, mp 100.8ꢀ101.5 °C. 1H
NMR (CDCl3): δ 0.98 (s, 9 H), 6.50 (d, J = 2.4 Hz, 1 H), 6.62 (d, J =
2.4 Hz, 1 H). MS (EI): m/z 153 [M]+.
1-(3,4-Dichlorophenyl)-3-(2,2-dimethyl-propyl)-thiourea (11b).
The title compound was prepared according to the same procedure of
making 11a, using neopentylamine in place of isopropylamine, to give a
1
white crystalline solid in 93% yield, mp 148.0ꢀ149.5 °C. H NMR
(CDCl3): δ 0.99 (s, 9 H), 3.53 (d, J = 5.0 Hz, 2 H), 6.06 (br s, 1 H), 7.15
(dd, J = 2.2 and 8.5 Hz, 1 H), 7.42 (d, J = 2.2 Hz, 1 H), 7.56 (d, J = 8.5 Hz,
1 H), 7.62 (br s, 1 H). 13C NMR (CDCl3): δ 27.5, 32.0, 60.5, 124.0,
126.6, 131.6, 134.0, 135.9, 180.9. MS (EI): m/z 290 [M]+.
1-(2,2-Dimethyl-propyl)-1H-imidazol-2-ylamine (14c).
A light-
brown liquid prepared from 13c and cyanamide in 70% yield. 1H
NMR (CDCl3): δ 1.59 (s, 9 H), 3.93 (br s, 2 H), 6.59 (d, J = 2.4 Hz,
1 H), 6.69 (d, J = 2.4 Hz, 1 H). MS (EI): m/z 139 [M]+.
1-tert-Butyl-3-(3,4-dichlorophenyl)-thiourea (11c). The title com-
pound was prepared according to the same procedure of making 11a,
using t-butylamine in place of isopropylamine, to give a brown crystalline
solid in 92% yield, mp 168.4ꢀ169.2 °C. 1H NMR (CDCl3): δ 1.54 (s,
9 H), 6.00 (br s, 1 H), 7.10 (d, J = 8.4 Hz, 1 H), 7.35 (s, 1 H), 7.49 (d, J =
8.4 Hz, 1 H). 13C NMR (CDCl3): δ 29.0, 54.4, 123.9, 126.3, 130.3,
131.4, 133.6, 136.6, 179.0. MS (EI): m/z 276 [M]+.
2. Synthesis of Compounds 13aꢀd. N-(2,2-Dimethoxy)-ethyl-
N-isopropyl-amine (13a)22. A mixture of 2-bromo-1,1-dimethoxyethane
(50.0 mL, 0.423 mol) and isopropylamine (150 mL, 1.74 mol) was heated
in a sealed tube at 80 °C for 18 h. The crystals of isopropylamine HBr salt
were filtered off. The filtrate was concentrated under reduced pressure.
Water (50 mL) was added to the gummy residue, and the mixture was
extracted with CH2Cl2 (150 mL ꢁ 2). The organic extracts were
combined, dried over Na2SO4, and concentrated under reduced pressure
1-Benzyl-1H-imidazol-2-ylamine (14d). Brown oil prepared from
13d and cyanamide in 82% yield. 1H NMR (CDCl3): δ 3.81 (br s, 2 H),
4.93 (s, 2 H), 6.60 (s, 1 H), 6.68 (s, 1 H), 7.13ꢀ7.70 (m, 5 H). MS (EI):
m/z 173 [M]+.
4. Synthesis of Compounds 15aꢀd. Synthesis of 1-(3,4-
Dichlorophenyl)-3-(1-isopropyl-1H-imidazol-2-yl)-thiourea (15a). Amine
14a (20g, 0.16 mol) inCH2Cl2 (100 mL) was added dropwise to a solution
of 3,4-dichlorophenylisothiocyanate (10, 32.0 g, 0.16 mol) in CH2Cl2
(75 mL). The mixture was stirred at rt for 18 h, and the solvent was removed
under reduced pressure. The residue was suspended in Et2O and filtered
to give a brown solid (22.6 g, 46%), mp 164ꢀ165 °C. 1H NMR (CDCl3):
δ 1.45 (d, J = 6.7 Hz, 6 H), 4.75 (m, 1 H), 6.73 (s, 1 H), 6.78 (s, 1 H),
7.03 (dd, J = 2.2 Hz, and 8.6 Hz, 1 H), 7.31 (d, J = 8.6 Hz, 1 H), 7.64 (br s,
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1 H), 8.20 (d, J = 2.2 Hz, 1 H), 13.6 (br s, 1 H). MS (ESI): m/z 329
[M + 1]+. Anal. (C13H14Cl2N4S): C, H, N, Cl, S.
129.9, 132.2, 133.0, 138.0, 173.6. MS (ESI): m/z 430 [M + 1]+. Anal.
(C21H21Cl2N5O): C, H, N, Cl.
Compounds 15bꢀd were prepared by the same procedure, except,
N-(3,4-Dichlorophenyl)-N0-(t-butyloxycarbonyl)-N00-(1-isopropyl-1H-
imidazol-2-yl)-guanidine (16e). A solution of di-tert-butyl dicarbonate
(9.3 g, 42.6 mmol) in CHCl3 (80 mL) was added dropwise with stirring to
a mixture of 8h (12.4 g, 35.5 mmol), triethylamine (14 mL, 0.10 mol), and
DMAP (0.10 g, 0.8 mmol) in CHCl3 (250 mL) at 0 °C. The reaction
mixture was further stirred at rt for 18 h, and saturated aqueous Na2CO3
solution (100 mL) was added. The organic layer was separated, and the
aqueous layer was extracted with CHCl3 (80 mL ꢁ 2). The CHCl3
extracts were combined, washed with brine, dried over Na2SO4, and
evaporated to dryness under the reduced pressure to give a light-brown
gum. Purification was achieved by the use of flash chromatography (0ꢀ
15% ethyl acetate in hexanes), followed by recrystallization from MeOH
to give 16e as a white solid (10 g, 68% yield), mp 101ꢀ102 °C. 1H NMR
(CDCl3): δ 1.43 (d, J = 6.6 Hz, 6 H), 1.55 (s, 9 H), 4.74 (m, 1 H), 6.76 (d,
J = 1.5 Hz, 1 H), 6.86 (d, J = 1.5 Hz, 1 H), 7.25 (dd, J = 2.4 and
8.7 Hz, 1 H), 7.35 (d, J = 8.7 Hz, 1 H), 8.32 (d, J = 2.4 Hz, 1 H), 10.2 (s,
1 H), 12.9 (br s, 1 H). 13C NMR (CDCl3): δ 22.8, 28.1, 45.9, 82.8, 111.6,
119.3, 121.9, 123.7, 125.5, 129.9, 132.2, 138.2, 144.9, 148.2, 153.9. MS
(ESI): m/z 412 [M + 1]+. Anal. (C18H23Cl2N5O2): C, H, N, Cl.
N-(1-tert-Butyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-N00-(2,2-
dimethylpropionyl)-guanidine (16f). The title compound was pre-
pared from 8i and 2,2-dimethylpropionyl chloride as that for the
preparation of 16a to give a white crystalline solid (0.95 g, 42%), mp
193.1ꢀ193.8 °C. 1H NMR (CD3OD): δ 1.34 (s, 9 H), 1.67 (s, 9 H),
6.74 (d, J = 1.3 Hz, 1 H), 6.94 (d, J = 1.3 Hz, 1 H), 7.24 (dd, J = 2.4 and
8.7 Hz, 1 H), 7.44 (d, J = 8.7 Hz, 1 H), 8.32 (d, J = 2.4 Hz, 1 H). 13C
NMR (CD3OD): δ 27.2, 29.6, 56.0, 115.0, 121.8, 121.9, 123.2, 125.4,
130.9, 131.5, 138.4, 145.4, 148.4, 181.1. MS (ESI): m/z 410 [M + 1]+.
Anal. (C19H25Cl2N5O): C, H, N, Cl.
different amines 14bꢀd were used in place of 14a.
1-(3,4-Dichlorophenyl)-3-(1-tert-butyl-1H-imidazol-2-yl)-thiourea
(15b). A brown crystalline solid prepared from compound 10 and amine
14b in 74% yield, mp 175.4 °C (dec). 1H NMR (CDCl3): δ 1.62 (s, 9 H),
6.71 (br s, 1 H), 6.78 (br s, 1 H), 7.03 (d, J = 8.3 Hz, 1 H), 7.33 (d, J =
8.5 Hz, 1 H), 7.56 (br s, 1 H), 7.98 (s, 1 H). MS (ESI): m/z 343 [M + 1]+.
1-(1-Benzyl-1H-imidazol-2-yl)-3-(3,4-dichlorophenyl)-thiourea (15c).
An off-white solid prepared from compound 10 and amine 14c in 23%
yield, mp 163.5ꢀ164.5 °C. 1H NMR (CDCl3): δ 5.11 (s, 2 H), 6.60 (s,
1 H), 6.79 (s, 1 H), 7.14ꢀ7.38 (m, 6 H), 7.67 (s, 1 H), 7.96 (s, 1 H). MS
(ESI): m/z 377 [M + 1]+.
1-(3,4-Dichlorophenyl)-3-(1H-imidazol-2-yl)-thiourea (15d). A
light-brown solid prepared from compound 10 and amine 14d in
42% yield. 1H NMR (CD3OD): δ 6.90 (s, 2 H), 7.35 (d, J = 8.7 Hz,
1 H), 7.56 (dd, J = 2.4 and 8.7 Hz, 1 H), 8.04 (d, J = 2.4 Hz, 1 H).
LC-MS (ESI): m/z 287 [M + 1]+.
5. Synthesis of Compounds 16aꢀ16h. Synthesis of N-(3,4-
dichlorophenyl)-N0-isobutyryl-N00-(1-isopropyl-1H-imidazol-2-yl)-
guanidine (16a). A solution of isobutyryl chloride (0.63 mL, 6.0 mmol)
in CHCl3 (10 mL) was added dropwise to a suspension of 8h (1.05 g,
3.0 mmol) and triethylamine (1.4 mL, 10.0 mmol) in CHCl3 (30 mL) at
0 °C. The reaction mixture was stirred at rt for 18 h. The solvent was
removed under reduced pressure. The residue was purified via flash
column chromatography (0ꢀ5% ethyl acetate in hexanes), followed by
recrystallization in MeOH to give 16a as a white crystalline solid (0.57 g,
73%), mp 9ꢀ92 °C. 1H NMR (CD3OD): δ 1.30 (d, J = 6.9 Hz, 6 H),
1.45 (d, J = 6.9 Hz, 6 H), 2.69 (m, 1 H), 4.76 (m, 1 H), 6.84 (s, 1 H), 6.94
(s, 1 H), 7.33 (dd, J = 2.1 and 8.7 Hz, 1 H), 7.44 (d, J = 8.7 Hz, 1 H), 8.39
(d, J = 2.1 Hz, 1 H). 13C NMR (CDCl3): δ 18.2, 19.0, 22.7, 24.3, 29.6,
34.0, 37.1, 46.0, 111.6, 112.8, 114.3, 119.3, 122.0, 123.7, 125.6, 129.9,
132.2, 138.0, 145.3, 148.1, 179.3. MS (ESI): m/z 382 [M + 1]+. Anal.
(C17H21Cl2N5O): C, H, N, Cl.
N-(1-tert-Butyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-N00-(2-
ethylbutyryl)-guanidine (16g). The title compound was prepared
from 8i and 2-ethylbutyryl chloride as colorless oil (1.4 g, 60% yield),
using the same procedure for the preparation of 16a. 1H NMR
(CD3OD): δ 0.99 (t, J = 7.4 Hz, 6 H), 1.67 (s, 9 H), 1.77 (m, 4 H),
2.26 (m, 1 H), 6.75 (d, J = 1.6 Hz, 1 H), 7.79 (d, J = 1.6 Hz, 1 H), 7.20
(dd, J = 2.5 and 8.7 Hz, 1 H), 7.34 (d, J = 8.7 Hz, 1 H), 8.19 (d, J =
2.5 Hz, 1 H). 13C NMR (CD3OD): δ 11.8, 25.1, 29.6, 52.6, 55.9, 113.5,
120.5, 121.8, 123.2, 126.3, 130.0, 132.4, 138.0, 145.0, 149.0, 178.6. MS
(ESI): m/z 424 [M + 1]+. Anal. (C20H27Cl2N5O): C, H, N, Cl.
N-(1-Benzyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-N00-isobutyryl-
guanidine (16h). The title compound was prepared by treatment of 8j
with isobutyryl chloride in 33% yield as white needles (0.50 g, 33%), mp
117.5ꢀ118.5 °C. 1H NMR (CDCl3): δ 1.33 (d, J = 6.9 Hz, 6 H), 2.71 (d,
J = 6.9 Hz, 6 H), 5.18 (s, 2 H), 6.72 (d, J = 1.2 Hz, 1 H), 6.88 (d, J = 1.2 Hz,
1 H), 7.18ꢀ7.34 (m, 7 H), 8.09 (s, 1 H), 10.85 (s, 1 H), 13.84 (s, 1 H). 13C
NMR (CDCl3): δ 19.1, 37.3, 48.2, 116.0, 119.7, 122.1, 124.0, 125.9, 127.0,
127.6, 128.7, 130.0, 132.2, 137.2, 137.8, 145.8, 149.2, 179.5. MS (ESI): m/z
430 [M + 1]+. Anal. (C21H21Cl2N5O2): C, H, N, Cl.
6. Synthesis of 1-Isopropyl-4,5-dihydro-1H-imidazol-2-
ylamine (17)26. A solution of cyanogen bromide (10.6 g, 0.10 mol)
in MeOH (20 mL) was added dropwise to a solution of N-isopropyl-
ethylenediamine (10.3 g, 0.10 mol) in MeOH (10 mL) at 0 °C. The
reaction mixture was then heated under reflux for 1 h. The solvent was
removed under reduced pressure. The crude product was suspended in
diethyl ether, filtered, and washed with diethyl ether to give 17 as a
yellow solid (20.0 g, 96% yield), mp 150.5ꢀ152.5 °C. 1H NMR
(CDCl3): δ 1.26 (d, J = 6.6 Hz, 6 H), 3.59ꢀ3.71 (m, 4 H), 4.39 (m,
1 H), 7.57 (br s, 1 H), 7.71 (br s, 2 H). MS (EI): m/z 127 [M+].
7. Synthesis of 1-(3,4-dichlorophenyl)-3-(1-isopropyl-4,5-
dihydro-1H-imidazol-2-yl)-thiourea (18) and 2-Imino-3-
isopropyl-imidazolidine-1-carbothioic Acid (3,4-Dichloro-
phenyl)-amide (180). Amine 17 (5.2 g, 25 mmol) was added to a
Compounds 16bꢀh were prepared by the same procedure by
treatment of 8h, 8i, or 8j with appropriate acyl chloride or dialkyl-
dicarbonate.
N-(3,4-Dichlorophenyl)-N0-(2-ethyl-butyryl)-N00-(1-isopropyl-1H-imi-
dazol-2-yl) guanidine (16b). The title compound was prepared from 8h
and 2-ethylbutyryl chloride using the same procedure for the preparation
of 16a to give pale-brown solid (0.9 g, 75%), mp 57.5ꢀ59.0 °C. 1H NMR
(CDCl3): δ 0.98 (t, J = 4.7 Hz, 6 H), 1.45 (d, J = 6.6 Hz, 6 H), 1.59ꢀ1.83
(m, 4 H), 2.23ꢀ2.32 (m, 1 H), 4.78 (m, 1 H), 6.77 (s, 1 H), 6.85 (s, 1 H),
7.24 (dd, J = 2.4 and 8.7 Hz, 1 H), 7.35 (d, J = 8.7 Hz, 1 H), 8.35 (d, J =
2.4 Hz, 1 H). 13C NMR (CDCl3): δ 11.4, 11.7, 22.7, 24.3, 25.2, 46.0, 52.5,
53.3, 111.5, 119.4, 122.0, 123.8, 125.6, 129.9, 132.2, 138.1, 145.2, 148.2,
178.5. MS (ESI): m/z 410 [M + 1]+. Anal. (C19H25Cl2N5O): C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-isobutyryl-N00-(1-isopropyl-1H-imidazol-
2-yl)-guanidine (16c). The title compound was prepared from 8h and
isobutyryl chloride using the same procedure for the preparation of 16a
in 84% yield, mp 101ꢀ102 °C. 1H NMR (CDCl3): δ 1.37 (s, 9 H), 1.44
(d, J = 6.6 Hz, 6 H), 4.76 (m, 1 H), 6.77 (s, 1 H), 6.85 (s, 1 H), 7.24 (br d,
J = 8.4 Hz, 1 H), 7.34 (d, J = 8.4 Hz, 1 H), 8.34 (br s, 1 H). 13C NMR
(CDCl3): δ 22.7, 27.2, 40.7, 46.0, 111.6, 119.4, 122.1, 123.7, 125.6, 129.9,
132.2, 138.2, 145.6, 148.2, 181.5. MS (ESI): m/z 396 [M + 1]+. Anal.
(C18H23Cl2N5O): C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-1H-imidazol-2-yl)-N00-phenyl-
acetyl-guanidine (16d). The title compound was prepared from 8h and
phenylacetyl chloride as that for the preparation of 16a, giving a white solid
in 85% yield, mp 91ꢀ92 °C. 1H NMR (CDCl3): δ 1.41 (d, J = 6.6 Hz,
6 H), 3.81 (s, 2 H), 4.73 (m, 1 H), 6.69 (s, 1 H), 6.72 (s, 1 H), 7.19 (dd, J =
1.9 and 8.8 Hz, 1 H), 7.30ꢀ7.40 (m, 6 H), 8.29 (br s, 1 H). 13C NMR
(CDCl3): δ 22.7, 45.2, 46.0, 119.4, 122.1, 123.4, 125.7, 127.6, 128.8, 129.8,
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stirring solution of 3,4-dichlorophenyl isothiocyanate (4.08 g, 20.0 mmol)
in CH2Cl2 (80 mL), followed by addition of triethylamine (4.2 mL,
30 mmol) at ambient temperature. The reaction mixture was further
stirred at rt for 72 h. The reaction mixture was basified with aqueous
saturated Na2CO3 solution. The organic phase was separated, washed
with brine, dried over Na2SO4, and filtered, and the solvent was removed
under reduced pressure to give a yellow solid. The crude product was
purified via column chromatography (silica gel, CH2Cl2) to give two
white crystalline solids 18 and 180. The former is a less polar isomer
(27% yields, mp 184.0ꢀ185.0 °C), and the latter is a more polar isomer
(50% yields, mp 134.0ꢀ135.0 °C). The structure of 18 was confirmed
by single crystal X-ray diffraction spectrum. Both isomers gave the same
MS (ESI): m/z 331 [M + 1]+. 1H NMR (CDCl3) of 18: δ 1.21 (d, J =
6.6 Hz, 6 H), 3.48ꢀ3.55 (m, 2 H), 3.70ꢀ3.76 (m, 2 H), 4.39 (m, 1 H),
J = 2.4 Hz, 1 H), 7.06 (d, J = 8.5 Hz, 1 H), 7.29 (s, 1 H), 7.47 (d, J =
8.5 Hz, 1 H). 13C NMR (CDCl3): δ 27.9, 29.7, 33.1, 51.4, 55.0, 116.0,
122.2, 123.0, 125.4, 127.7, 130.9, 133.1, 138.7, 149.8, 152.1. MS (ESI):
m/z 396 [M + 1]+. Anal. (C19H27Cl2N5): C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-[1-(2,2-dimethyl-propyl)-1H-imidazol-
2-yl]-N00-isopropyl-guanidine (8e). Title compound was prepared from
1
14c and11a as a white crystalline solid in 36% yield, mp 89.5 °C. H
NMR (CDCl3): δ 0.99 (s, 9 H), 1.23 (s, 6 H), 3.77 (s, 2 H), 4.04 (br s,
1 H), 4.20 (m, 1 H), 6.63 (d, J = 2.4 Hz, 1 H), 6.97 (d, J = 2.4 Hz, 1 H),
7.20 (d, J = 8.4 Hz, 1 H),7.40 (d, J = 8.4 Hz, 2 H). 13C NMR (CDCl3): δ
23.0, 28.0, 33.3, 42.9, 55.2, 116.1, 122.2, 123.3, 125.8, 128.1, 131.0, 133.2,
138.3, 149.9, 152.1. MS (ESI): m/z 382 [M + 1]+. Anal. (C18H25Cl2N5):
C, H, N, Cl.
N-(1-tert-Butyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-N00-isopro-
pyl-guanidine Hydrochloride (8f). Prepared from 14b and 11a as a white
crystalline solid in 54% yield, mp 180.7 °C. 1H NMR (CD3OD): δ 1.26
(d, J = 6.5 Hz, 6 H), 1.67 (s, 9 H), 4.07 (m, 1 H), 6.80 (d, J = 1.8 Hz, 1 H),
7.06 (d, J = 1.8 Hz, 1 H), 7.09 (dd, J = 2.1 and 8.7 Hz, 1 H), 7.34 (d, J = 2.1
Hz, 1 H), 7.45 (d, J = 8.7 Hz, 1 H). 13C NMR (CDCl3): δ 22.7, 28.3, 44.6,
57.8, 112.1, 112.3, 121.1, 122.0, 126.8, 130.2, 131.9, 138.1, 145.1, 154.4.
MS (ESI): m/z 368 [M + 1]+. Anal. (C17H24Cl3N5): C, H, N, Cl.
9. General Procedure for the Synthesis of Compound
8gꢀm. N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-1H-imidazol-2-yl)-
piperidinecarboxamidine Hydrochloride Salt (8g). HgCl2 (1.6 g,
6.0 mmol) was added at rt to a stirring solution consisting of 15a (1.0 g,
3.0 mmol), piperidine (2.0 mL, 20.0 mmol), and acetonitrile (50 mL).
After the addition, the reaction mixture was further stirred at rt for 18 h
and then filtered through a pad of Celite. The filtrate was concentrated
under reduced pressure. The crude product was purified by a silica gel
column and eluted with CHCl3 to give a gummy yellow solid (0.80 g,
64% yield). The pure 8g was obtained as a yellow crystalline solid by
recrystallization from MeOH, mp 99.0ꢀ101.0 °C. 1H NMR (CD3OD):
δ 1.40 (d, J = 6.6 Hz, 6 H), 1.74 (br s, 6 H), 3.67 (br s, 4 H), 4.56 (m,
1 H), 6.69 (s, 1 H), 6.92 (s, 1 H), 6.95 (dd, J = 2.4 and 8.5 Hz, 1 H), 7.16
(d, J = 2.4 Hz, 1 H), 7.35 (d, J = 8.5 Hz, 1 H). 13C NMR (CD3OD): δ
22.3, 25.5, 27.2, 113.9, 114.5, 121.5, 122.9, 127.8, 132.1, 133.6, 140.7,
146.9, 156.1. MS (ESI): m/z 380 [M + 1]+. Anal. (C18H24Cl3N5): C,
H, N, Cl.
1
7.05 (br s, 1 H), 7.32 (d, J = 8.7 Hz, 1 H), 8.03 (s, 1 H). H NMR
(CDCl3) of 180: δ 1.24 (d, J = 6.6 Hz, 6 H), 3.45 (t, J = 7.8 Hz, 2 H), 3.72
(m, 1 H), 4.28 (t, J = 7.8 Hz, 2 H), 7.39 (d, J = 8.7 Hz, 1 H), 7.54 (dd, J =
1.8 and 8.7 Hz, 1 H), 7.86 (d, J = 1.8 Hz, 1 H).
8. Synthesis of Compounds 8aꢀf. Synthesis of N-(3,4-
dichlorophenyl)-N0-isopropyl-N00-(1-isopropyl-1H-imidazol-2-yl)-
guanidine (8a)28. Triethylamine (35.0 mL, 0.24 mol) was added
dropwise to a stirring suspension of amine 14a (3.0 g, 24.0 mmol), thiourea
11a (6.3 g, 24 mmol), and HgO (7.8 g, 36.0 mmol) in DMF (∼100 mL) at
ambient temperature. The mixture was further stirred at rt for 60 h. The
precipitate was filtered through a pad of Celite. The filtrate was concentrated
under reduced pressure. Water was added to the residue, and the mixture
was extracted with chloroform. The organic extracts were combined,
washed with brine, dried over Na2SO4, and filtered, and the solvent was
removed under reduced pressure. The crude product was purified via
column chromatography (silica gel, 0ꢀ33% ethyl acetate in hexanes) to give
a light-brown oil, which was recrystallized in MeOH/CHCl3 mixed solvent
to give 8a as a light-yellow crystalline solid (1.3 g, 31% yield), mp
111ꢀ112 °C. 1H NMR (CDCl3 w/TFA-d): δ 1.34 (d, J = 6.6 Hz, 6 H),
1.41 (d, J = 6.6 Hz, 6 H), 4.05 (m, 1 H), 4.52 (m, 1 H), 6.92 (d, J = 2.4 Hz,
1 H), 7.05 (dd, J= 2.4 and 8.4 Hz, 1 H), 7.13 (d, J= 2.4 Hz, 1 H), 7.32 (d, J=
2.4 Hz, 1 H), 7.41 (d, J = 8.4 Hz, 1 H). 13C NMR (CDCl3): δ 22.7, 42.9,
45.1, 110.4, 122.7, 123.2, 125.6, 128.0, 130.9, 133.0, 138.0, 149.8, 150.5. MS
[ESI]: m/z 354 [M + 1]+. Anal. (C16H21Cl2N5): C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-1H-imidazol-2-yl)-guanidine
Hydrochloride Salt (8h). Mercuric chloride (HgCl2, 2.4 g, 9.0 mmol)
was added with stirring to a solution of 15a (1.5 g, 4.5 mmol) in
methanolic NH3 (7 N NH3 in MeOH, 5.0 mL, 35.0 mmol) and DMF
(70 mL) at ambient temperature. The reaction mixture was further
stirred at rt for 18 h. The precipitate was filtered off through a pad of
Celite. The filtrate was concentrated under reduced pressure. The
residue was suspended in CH2Cl2, filtered, and washed with CH2Cl2
to give a pale-brown solid, which was recrystallized from MeOH to give
8h as a white crystalline solid (0.70 g, 50% yield), mp 277.0ꢀ277.9 °C.
1H NMR (CD3OD): δ 1.44 (d, J = 6.6 Hz, 6 H), 4.56 (m, 1 H), 7.04 (d,
J = 2.4 Hz, 1 H), 7.18 (d, J = 2.4 Hz, 1 H), 7.30 (dd, J = 2.7 and
8.7 Hz, 1 H), 7.45 (d, J = 8.7 Hz, 1 H), 7.80 (d, J = 2.7 Hz, 1 H). 13C
NMR (CD3OD): δ 22.3, 115.0, 115.6, 122.0, 124.0, 128.0, 131.7, 133.4,
140.1, 147.4, 156.7. MS (ESI): m/z 312 [M + 1]+. Anal. (C13H16Cl3N5):
C, H, N, Cl.
Compounds 8bꢀf was prepared by the same procedure except
different compounds 11 and 14 were used.
N-tert-Butyl-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-1H-imidazol-
2-yl)-guanidine (8b). Compound 8b was prepared by the same proce-
dure from 14a and 11c to give a light-yellow crystalline solid in 40%
yield, mp 57.2 °C. 1H NMR (CDCl3): δ 1.38 (d, J = 6.7 Hz, 6 H), 1.44 (s,
9 H), 4.10 (br s, 1 H), 4.76 (m, 1 H), 6.68 (d, J = 2.4 Hz, 1 H), 6.75 (d, J =
2.4 Hz, 1 H), 7.06 (d, J = 8.4 Hz, 1 H), 7.32 (s,1 H), 7.38 (d, J = 8.4 Hz,
1 H), 11.67 (br s, 1 H). 13C NMR (CDCl3): δ 22.1, 29.3, 46.7, 52.6,
110.4, 113.5, 121.3, 122.2, 126.8, 130.3, 132.1, 138.4, 144.5, 155.6. MS
(ESI): m/z 368 [M + 1]+. Anal. (C17H23Cl2N5): C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-(2,2-dimethylpropyl)-N00-(1-isopropyl-
1H-imidazol-2-yl)-guanidine Hydrochloride (8c). Compound 8c was
prepared by the same procedure from 14a and 11b to give a white
crystalline solid in 31% yield, mp 139.8 °C. 1H NMR (CDCl3): δ 1.05 (s,
9 H), 1.46 (d, J = 6.7 Hz, 6 H), 3.14 (d, J = 5.9 Hz, 2 H), 4.64 (m, 1 H),
6.55 (d, J = 2.4 Hz, 1 H), 6.72 (d, J = 2.4 Hz, 1 H), 7.23 (d, J = 8.1 Hz,
2 H), 7.37 (s, 1 H), 7.60 (br s, 1 H), 10.16 (br s, 1 H). 13C NMR
(CDCl3): δ 22.1, 27.4, 32.3, 46.9, 54.1, 110.8, 113.7, 121.2, 122.3, 127.0,
130.3, 132.1, 138.2, 144.7, 156.64. MS (ESI): m/z 382 [M + 1]+. Anal.
(C18H26 Cl3N5): C, H, N, Cl.
N-(1-tert-Butyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-guanidine
HCl Salt (8i). The title compound was prepared by the same procedure
except 15b was used in place of 15a to give a bright-white crystalline solid
(1.2 g, 63%), mp 264.7 °C. 1H NMR (CD3OD): δ 1.63 (s, 9 H), 7.00 (d,
J= 2.5 Hz, 1 H), 7.17 (d, J= 2.5 Hz, 1 H), 7.29 (dd, J=2.4, and8.7Hz, 1H),
7.45 (d, J= 8.7 Hz, 1H), 7.81 (d, J= 2.4 Hz, 1 H). 13C NMR (DMSO-d6):δ
28.8, 58.2, 113.9, 115.9, 119.9, 121.0, 124.4, 131.0, 131.3, 140.0, 146.5,
154.5. MS (ESI): m/z 326 [M + 1]+. Anal. (C14H18Cl3N5): C, H, N, Cl.
N-(Benzyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)guanidine Hydro-
chloride (8j). The title compound was prepared by the same procedure,
N-tert-Butyl-N0-(3,4-dichlorophenyl)-N00-[1-(2,2-dimethyl-propyl)-
1H-imidazol-2-yl]-guanidine (8d). Compound 8d was prepared by the
same procedure from 14c and 11c to give a light-brown crystalline solid
in 44% yield, mp 124.8 °C. 1H NMR (CDCl3): δ 0.97 (s, 9 H), 1.45 (s,
9 H), 3.77 (s, 2 H), 4.14 (br s, 1 H), 6.62 (d, J = 2.4 Hz, 1 H), 6.74 (d,
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except 15c was used in place of 15a to give an off-white solid (1.8 g, 85%
yield), mp 237ꢀ238 °C. 1H NMR (CD3OD): δ 5.10 (s, 2 H), 7.04 (d,
J = 2.4 Hz, 1 H), 7.10 (d, J = 2.4 Hz, 1 H), 7.24 (dd, J = 2.4 and 8.7 Hz,
1 H), 7.25ꢀ7.40 (m, 6 H), 7.71 (d, J = 2.4 Hz, 1 H). 13C NMR (CD3-
OD): δ 115.4, 118.6, 122.0, 124.0, 128.0, 129.0, 129.5, 130.2, 131.6,
133.4, 137.1, 139.9, 148.4, 156.8. MS (ESI): m/z 360 [M + 1]+. Anal.
(C17H16Cl3N5): C, H, N, Cl.
Synthesis of N-(3,4-dichlorophenyl)-N0-isopropyl-N00-(1-isopropyl-
4,5-dihydro-1H-imidazol-2-yl)-guanidine Hydrochloride (9b). Mercuric
chloride (2.0 g, 7.4 mmol) was added with stirring to a mixture of 18
(1.25 g, 3.7 mmol) and isopropylamine (0.80 mL, 9.3 mmol) in CH3CN
(80 mL) at ambient temperature. The reaction mixture was stirred at rt
for additional 5 min, filtered through Celite, and concentrated under
reduced pressure to dryness. The product was purified via column
chromatography (silica gel, 0ꢀ5% CH3OH in CH2Cl2), followed
by recrystallization from CH2Cl2/hexanes to give 9b as a white solid
(0.85 g, 58% yield), mp 156.5ꢀ157.5 °C. 1H NMR (CDCl3): δ 1.18 (d,
J = 6.9 Hz, 6 H), 1.27(d, J = 6.6 Hz, 6 H), 3.37 (s, 4 H), 3.87 (m, 1 H), 4.24
(m, 1 H), 7.10 (dd, J = 2.1 and 8.4 Hz, 1 H), 7.24 (d, J = 8.4 Hz,
1 H), 7.30 (d, J = 2.1 Hz, 1 H). 13C NMR (CDCl3): δ 19.6, 22.4, 40.2,
40.4, 44.5, 44.7, 121.4, 122.9, 127.1, 130.0, 132.0, 137.6, 157.1, 157.9. MS
(ESI): m/z 356 [M + 1]+. Anal. (C16H24Cl3N5): C, H, N, Cl.
N-(1-Benzyl-1H-imidazol-2-yl)-N0-(3,4-dichlorophenyl)-N00-isopropyl-
guanidine (8k). The title compound was prepared by the same pro-
cedure, except 15c and isopropylamine instead of ammonia were used to
give an off-white solid (2.5 g, 61%), mp 87ꢀ89 °C. 1H NMR (CDCl3
w/TFA): δ 1.23 (d, J = 6.6 Hz, 6 H), 4.03 (m, 1 H), 5.02 (s, 2 H), 6.39 (s,
1 H), 6.55 (s, 1 H), 6.77 (dd, J = 2.4 and 8.7 Hz, 1 H), 7.04ꢀ7.13 (m,
2 H), 7.26ꢀ7.29 (m, 2 H), 7.35ꢀ7.44 (m, 3 H). 13C NMR (CDCl3): δ
22.9, 43.0, 47.8, 114.5, 123.2, 123.5, 126.0, 127.4, 127.6, 128.6, 131.1,
133.3, 138.0, 138.4, 150.3, 151.6. MS (ESI): m/z 402 [M + 1]+. Anal.
(C20H21Cl2N5): C, H, N, Cl.
Compounds 9cꢀe was synthesized by the same procedure for the
preparation of compound 9b, except t-butylamine, 1-adamantylamine,
and piperidine were used in place of isopropylamine.
Synthesis of N-(3,4-dichlorophenyl)-N0-(1H-imidazol-2-yl)-guani-
dine Hydrochloride (8l). Mercuric chloride (1.6 g, 6.0 mmol) was added
with stirring at rt to a mixture of 15d (1.15 g, 4.0 mmol) and methanolic
ammonia (7 N, 5 mL, 35 mmol) in DMF (40 mL). The mixture was
further stirred at rt for 24 h and filtered through Celite. The filtrate was
evaporated to dryness under reduced pressure. The crude product was
suspended in ethyl acetate and filtered to give a light-brown solid, which
was recrystallized successively in MeOH and water to give 8l as an off-
white solid (0.44 g, 21% yield), mp 240 °C (decd). 1H NMR (CD3OD):
δ 6.97 (s, 2 H), 7.31 (dd, J = 2.4 and 8.7 Hz, 1 H), 7.43 (d, J = 8.7 Hz,
1 H), 7.75 (d, J = 2.4 Hz, 1 H). 13C NMR (CD3OD): δ 115.9, 122.0,
123.9, 127.9, 131.5, 133.3, 139.9, 148.9, 156.6. MS (ESI): m/z 270
[M + 1]+. Anal. (C10H10Cl3N5): C, H, N, Cl.
N-tert-Butyl-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4,5-dihydro-1H-
imidazol-2-yl)-guanidine Hydrochloride (9c). A white solid in 49% yield,
mp 212.0ꢀ213.5 °C. 1H NMR (CDCl3): δ 1.21 (d, J = 6.6 Hz, 6 H), 1.46
(s, 9 H), 3.10ꢀ3.20 (m, 2 H), 3.28ꢀ3.34 (m, 2 H), 4.27 (m, 1 H), 7.17
(d, J = 8.4 Hz, 1 H), 7.25 (br s, 1 H), 7.31 (d, J = 8.4 Hz, 1 H). 13C NMR
(CDCl3): δ 19.8, 29.2, 40.3, 40.4, 44.6, 53.1, 121.9, 123.1, 127.4, 130.4,
132.2, 137.8, 156.6, 158.4. MS (ESI): m/z 370 [M + 1]+. Anal. (C17H26-
Cl3N5): C, H, N, Cl.
N-Adamantan-1-yl-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4,5-
dihydro-1H-imidazol-2-yl)-guanidine Hydrochloride (9d). A white
1
solid in 55% yield, mp 240.5ꢀ241.5 °C. H NMR (CD3OD): δ 1.25
(d, J = 6.6 Hz, 6 H), 1.68ꢀ1.79 (m, 6 H), 2.11 (br s, 1 H), 3.22ꢀ3.34 (m,
2 H), 3.46 (t, J = 8.7 Hz, 2 H), 4.28 (m, 1 H), 7.02 (br d, J = 8.4 Hz, 1 H),
7.25 (s, 1 H), 7.51 (d, J = 8.4 Hz, 1 H). 13C NMR (CD3OD): δ 20.2,
31.1, 37.5, 41.6, 41.9, 42.7, 46.6, 54.8, 123.0, 124.5, 129.0, 132.3, 133.8,
139.4, 157.5, 159.1. MS (ESI): m/z 448 [M + 1]+. Anal. (C23H32Cl3N5):
C, H, N, Cl.
Synthesis of N-(3,4-dichlorophenyl)-N0-(1H-imidazol-2-yl)-N00-iso-
propyl-guanidine (8m). Thiourea 15d (1.0 g, 3.5 mmol) was suspended
in 10.0 mL of acetonitrile and to the suspension, dicyclohexylcarbodi-
imide (DCC) (1.0 g, 5.2 mmol), isopropyl amine (0.60 mL, 6.9 mmol),
and a catalytic amount of i-Pr2EtN (0.12 mL) were added successively.
The reaction mixture was then stirred at ambient temperature for 5 h.
The thickened suspension was evaporated under reduced pressure to
dryness and extracted with EtOAc 100 mL ꢁ 3. The organic extracts
were combined, washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified via flash chro-
matography (silica gel, 5% ethyl acetate in hexanes) to give a white solid,
which was recrystallized from methanol to give 8m as a white crystalline
solid (0.70 g, 64%), mp 163.0 °C. 1H NMR (CD3OD): δ 1.32 (d, J =
6.6 Hz, 6 H), 4.13 (m, 1 H), 6.97 (s, 2 H), 7.14 (dd, J = 2.4 and 8.7 Hz,
2 H), 7.38 (d, J = 2.4 Hz, 1 H), 7.49 (d, J = 8.7 Hz, 1 H). 13C NMR
(CD3OD): δ 23.6, 45.0, 118.4, 122.5, 124.3, 126.8, 131.6, 133.3, 141.4,
152.4, 153.3. MS (ESI): m/z 312 [M + 1]+. Anal. (C13H15Cl2N5): C,
H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-4,5-dihydro-1H-imidazol-2-
yl)-piperidine-1-carboxamidine Hydrochloride (9e). A white solid in
56% yield, mp 110.0ꢀ110.6 °C. 1H NMR(CDCl3): δ 1.13 (d, J = 6.6 Hz,
6 H), 1.78 (br s, 6 H), 3.22 (br s, 4 H), 3.76 (br s, 4 H), 4.16 (m, 1 H),
7.27 (d, J = 2.4 Hz, 1 H), 7.31 (d, J = 8.4 Hz, 1 H), 7.38 (dd, J = 2.4 and
8.4Hz, 1 H). 13C NMR(CDCl3): δ 19.7, 23.9, 25.6, 40.1, 40.4, 44.4, 47.9,
121.2, 122.1, 126.5, 130.2, 131.8, 139.0, 158.0, 158.1. MS (ESI): m/z 382
[M + 1]+. Anal. (C18H25Cl2N5): C, H, N, Cl.
Antimalarial Studies. 1. Assessment of Metabolic Stability. All
samples were tested in human and mouse liver microsomal preparations,
and selected samples were further tested in Rhesus monkey microsomes.
Sample stocks at 10 or 20 mM (depending on solubility) in DMSO
were diluted to a final concentration of 1 μM into a mixture containing
0.5 mg/mL of prewarmed pooled human or mouse liver microsomes
(BD Gentest), 1.3 mM NADP (Sigma), 3.3 mM MgCl2 (Sigma), and
0.1 M pH 7.4 PBS using a TECAN Genesis robotic liquid handler. The
reaction was started with the addition of 1U/mL glucose-6-phosphate
dehydrogenase G6PD. The mixture was incubated on a shaking platform
at 37 °C, and aliquots were taken and quenched with the addition of an
equal volume of cold acetonitrile at 0, 10, 20, 30, and 60 min. Samples
were centrifuged at 3700 rpm for 10 min at 20 °C to remove debris.
Sample quantification was carried out by LC/MS, and metabolic half-
life was calculated by log plots of the total ion chromatograph area
remaining. The results are shown in Table 1.
10. General Procedure for the Synthesis of Compounds
9aꢀ9e. Synthesis of N-(3,4-dichlorophenyl)-N0-(1-isopropyl-4,5-
dihydro-1H-imidazol-2-yl)-guanidine Hydrochloride (9a). Mercuric
chloride (0.49 g, 1.8 mmol) was added in small portions at rt to a
stirring solution of 18 (0.41 g, 1.2 mmol) in methanolic NH3 (7 N
NH3 in MeOH, 1.0 mL, 7.0 mmol) and DMF (15 mL) mixed solvents.
After the addition, the mixture was stirred at rt for additional 30 min.
The precipitate was filtered off through a pad of Celite. The filtrate was
concentrated in vacuo, and the solid material was suspended in CH2Cl2,
filtered, and washed with CH2Cl2 to give 9a as a white solid which was
recrystallized from MeOH to give a colorless crystalline solid (0.75 g,
1
71% yield), mp 259.5ꢀ260.5 °C. H NMR (CD3OD): δ 1.21 (d, J =
2. Metabolites Identification. Good antimalarial activity in mouse and
Rhesus test models coupled with metabolic instability of 8a and 8h
suggested that metabolite(s), not the parent drug, is the active species of
the imidazole antimalarial. The metabolite identification of compound
8a was performed in mice given a single oral dose of 50 mg/kg. The
6.6 Hz, 6 H), 3.66 (s, 4 H), 4.21 (m, 1 H), 7.23 (dd, J = 2.4 and 8.5 Hz, 1
H), 7.47 (d, J = 8.5 Hz, 1 H), 7.70 (d, J = 2.4 Hz, 1 H). 13C NMR (CD3-
OD): δ 19.3, 44.7, 120.2, 121.4, 124.8, 130.5, 130.9, 138.7, 156.1, 160.0.
MS (ESI): m/z 314 [M + 1]+. Anal. (C13H18Cl3N5): C, H, N, Cl.
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Table 1. Metabolic Stability and Antimalarial Activities of the New Compounds
metabolic stability (t1/2, min)
P. falciparum IC50 (μg/mL)
EE P. berghei, dose (mg/kg/day)b
compd no.
mouse
human
Rhesus
D6
TMa
2.4
W2
160
320
8a
5.9
22.5
40.4
32.9
12.1
20.3
48
7.8
2.6
0.8
CP (1/5), DPd (3/5)
CPc (4/5), DP (1/5)
8b
8c
18.2
10.8
11.7
1.1
1.0
IA
IA
1.4
0.9
IA
IA
8d
8e
0.8
0.8
IA
IA
1.1
1.5
IA
IA
8f
9
1.4
0.9
0.3
2.4
1.2
>2.0
2.5
2.5
>5.0
1.2
0.7
0.4
toxic
toxic
8g
19.7
27.7
48
27
7.8
>60
45
0.5
0.3
IA
IA
8h
8i
>60
>60
>60
16
2.8
0.7
CP (5/5)
CP (5/5)
1.6
0.4
IA
IA
8j
37
>2.0
1.3
>2.0
1.6
IA
IA
8k
18
IA
IA
8l
23.6
37.6
>60
13.6
16.6
>60
21.5
>60
>60
>60
50.8
>60
>60
12.5
11
42.5
>60
>60
50.7
>60
>60
>60
>60
>60
>60
>60
>60
>60
26
0.5
1.4
IA
IA
8m
9a
>5.0
1.4
0.6
toxic death (1/5)
CP (4/5)
toxic and IA
toxic and IA
toxic death (4/5)
36.3
0.7
9b
9c
0.5
0.4
toxic death
toxic death
0.4
0.4
9d
9e
0.3
0.2
0.2
0.4
0.3
0.2
toxic
toxic death
16a
16b
16c
16d
16e
16f
16g
16h
2.5
2.2
1.0
DP (2/5) IA (3/5)
CP (4/5), DP (1/5)
1.0
2.9
0.3
IA
IA
>5.0
>5.0
>5.0
>5.0
>5.0
>2.0
>5.0
>5.0
>5.0
>5.0
>5.0
>2.0
>5.0
>5.0
>5.0
>5.0
>5.0
>2.0
IA
IA
IA
IA
DP
DP
IA
CP (3/5), DP (2/5)
DP
DP
IA
19.7
IA
a TM = TM91C235. b Treatment for 3 days, on day before (ꢀ1), the day (0), and day after (1) sporozoites inoculation; CP = causal prophylaxis; DP =
delayed patency; IA = inactive, c causal prophylaxis, d delay parasitemia patency.
blood and liver samples were collected from hours 1 to 32, and the
parent and putative metabolites were analyzed by LC/MS in full scan
mode (m/z 150ꢀ650) on an LTQ (Thermo Fisher Scientific, Bremen,
Germany), with a data dependent MS2 and MS3 scan event on the most
intense ions. A total of 19 and 17 putative metabolites were detected in
mouse liver and blood samples, respectively. All metabolites detected
in both blood and liver samples have the same retention time in LC
chromatogram and m/z, except peak m/z 349 was observed only in
blood samples not in liver homogenate. On the contrary, peak m/z 339
was detected only in liver samples not in blood. The major metabolites in
both blood and liver samples are m/z 370, 384, 386, and 388.
prepared at different dose levels. Compounds were administered either
po or sc to mice once a day for three consecutive days, to mice on the day
before, 4 h before, and the day after being inoculated with sporozoites of
P. berghei intravenously. Whole body weights were taken on day 0 and
day 6 and 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
losing 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.
3. In Vitro Antimalarial Activity against P. falciparum. The in vitro
assays were conducted by using a modification of the semiautomated
microdilution techniques of Desjardins et al. and Chulay et al.29,30 Three
P. falciparum malaria parasite clones from CDC Indochina III (W-2),
CDC Sierra Leone I (D-6), and Southeast Asia isolates (TM91C235)
were utilized in susceptibility testing. They were derived by direct
visualization and micromanipulation from patient isolates.31 The results
are shown in Table 1.
4. Assessment of Causal Prophylactic Activity in Mice. New com-
pounds were assessed for their causal prophylactic activity in exoery-
throcytic (EE) mouse model using sporozoites of P. berghei. The
procedures have been previously described.16ꢀ21 Briefly, each com-
pound was ground with a mortar and pestle and suspended in hydroxy-
ethylcellulose and Tween 80 for compounds to be administered po,
and those given sc were suspended in peanut oil. Each compound was
5. Assessment of Causal Prophylactic/Radical Curative Activities in
Rhesus Monkeys. The causal prophylactic and radical curative antim-
alarial activity of the new derivatives 8a and 8h were assessed in a
P. cynomolgi sporozoites challenged Rhesus monkey model. Detailed
procedure of sporozoites harvest and drug tests are described in the
previous reports.16ꢀ21 The results are shown in Tables 2 and 3. The
protocol for assessing causal prophylactic activity of test compounds
involved 3 consecutive day treatment of Rhesus monkeys by oral
administration, one day before the inoculation of sporozoites, on the
day the sporozoites inoculated and a day after the inoculation. Com-
pound 8a was evaluated side by side with 3 clinical drugs, primaquine,
tafenoquine, and Malarone (a fixed combination of atovaquone (10 mg)
and proguanil (4 mg)) in the same experiment. Assessment of radical
curative activity of the test compounds were carried out using the
monkeys that developed parasitemia during the causal prophylactic
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Table 2. Causal Prophylactic Activity in Rhesus Monkey Model
groupa
drug
doseb (mg/kg/day ꢁ 3)
vehicle
HECT
route
po
first patency day after last treatment
delay patency (days)
1
vehicle
N/A
8
8
valid control
valid control
2
3
4
5
PQ
1.78
0.316
14
MC
po
po
po
po
13
13
11
10
11
12
19
17
5
5
TQ
MC
3
2
Malarone
8a
HECT
HECT
3
4
50
11
9
a Two monkeys per group. b Drug was administered on the day before (ꢀ1), the day (0), and the day after (+1) sporozoites inoculation; Malarone: a
fixed combination of atovaquone (10 mg) plus proguanil (4 mg).
Table 3. Radical Curative Activity in Rhesus Monkey Model
test compd
groupa
1
drug 1
drug 2
none
drug 2 dose (mg/kg/day)
drug 2 (route)
relapse resultb (days)
chloroquine 10 mg/kg/day, po for 7 days
11
12
2
3
4
5
chloroquine 10 mg/kg/day, po for 7 days
chloroquine 10 mg/kg/day, po for 7 days
chloroquine 10 mg/kg/day, po for 7 days
chloroquine 10 mg/kg/day, po for 7 days
8a
30 ꢁ 3 days
30 ꢁ 7 days
30 ꢁ 7 days
1.78 ꢁ 7 days
im
po
po
po
radical cure (>100)
74
8a
81
32
8h
PQ
26
21
radical cure (>100)
radical cure (>100)
a Two monkeys per group. b Number of days relapse occurred after the last day of treatment.
experiments when the test compounds showed no or weak activity.
Monkeys were treated with chloroquine (10 mg/kg/day) by oral for 7
consecutive days to clear the blood stage parasites. Relapse occurred
after 11ꢀ12 days. Test compounds were administered by im or oral for
3 or 7 consecutive days, respectively, after the parasitemia level reached
5000 parasites/mm3. Chloroquine at 10 mg/kg/day ꢁ 7 days eliminated
the blood stage parasites, but not the liver stage hypnozoites. Com-
pounds with antihypnozoite activity will delay the relapse or radically
cure the infection.
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 administration.
Parasite clearance should occur in all animals treated with chloroquine.
Relapse was expected in the control group. Relapse in the treated group
indicated 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
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 daily for 4 consecutive days and 2 times weekly
for 2 weeks were monitored. The results were shown in Table 3.
also tested in Rhesus microsomes. Majority of the new imidazole
analogues showed poor metabolic stability in mice microsomal
preparations, with t1/2 < 60 min, but are much more stable in the
human microsomal preparation. It is to be noted that the rate of
drug metabolism in Rhesus microsome preparations is faster than
or about equal to that in mice. This is in sharp contrast to the IZ
derivatives, such as compounds 1, 3, and 5, which are metabo-
lically stable in both human and mouse. As was observed with
IZ derivatives, the new compounds (8aꢀm and 16aꢀh) also
showed weak to moderate in vitro antimalarial activities in three
clones of P. falciparum (D6, W2, and TM91C235) with IC50 in
the range of 0.3 to >5 μg/mL, with the exception of 8g (0.3ꢀ
0.5 μg/mL) and imidazoline analogues 9aꢀe (0.2ꢀ1.4 μg/mL).
Although moderate in vitro activity against P. falciparum was
observed, compounds 9bꢀe also caused toxic deaths in the
treated mice.
Among the new compounds tested, only compounds 8a, 8h,
9a, 16a, and 16e protected the mice infected with sporozoites of
P. berghei, at 160 and 320 mg/kg/day by oral dosing. The other
compounds, although inactive, showed some degree of growth
inhibitory activity against liver stage parasite in the in vivo
imaging system (IVIS) test during the first 3 days after the mice
were infected with luciferase-labeled sporozoites.32,33 The results
suggest that the imidazole analogues have much smaller bulk
tolerance than IZ analogues on the size of substituents R1 and R2.
For example, the causal prophylactic activity of compounds 8bꢀf
’ RESULTS AND DISCUSSION
All new compounds were assessed for metabolic stability in
human and mouse microsomal preparations with some analogues
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with bulky substituents, t-butyl or ꢀCH2C(CH3)3, at either R1
or R2, decrease considerably or totally devoid activity in mice
tests. The high efficacy in mice test and poor metabolic stability
of 8a and 8h plus the unusual SAR results among the tested
compounds in Table 1 led us to investigate the metabolite profile
of the two active compounds.
Compound 8a produced a host of metabolites in mouse liver
microsome preparations, as well as in blood and in liver homo-
genate of the treated mice. The mass of major metabolites in LC-
mass spectra are m/z 370, 386, and 384 [M + 1]+, which suggest
the parent drug 8a (m/z 354) was metabolized to either 4- or
5-hydroxyl-imidazole derivative (A or A0), which tautomerized
to compound B or B0 (m/z 370) as proposed in Scheme 5.
Compound B is an unknown compound, but compound B0 was
reported earlier from the authors0 laboratory.34 Hydroxylation of
B or B0 led to formation of C or C0 (m/z 386), which was further
oxidized to the stable and known compound WR301855 (D, m/z
384). Spiking with authentic sample, metabolite m/z 370 has a
different retention time (RT) and ms fragmentation pattern from
the known compound B0. The RT of metabolite m/z 370 is
10.2 min vs 9.9 min for standard B0. Further, the MS2 spectra of
the m/z 370 metabolite in the PK samples consistently show a
peak at m/z 328, which is not present in the standard B0. The
peak m/z 328 [M+ ꢀ 42] is the result of losing a CH3CHdCH2
(m/z 42) fragment from the metabolite m/z 370, a fragmenta-
tion pattern that can only occur with compound B, but not B0, as
shown in Scheme 6. Thus, the primary hydroxylation metabolite
of 8a is compound A, not A0, and the secondary hydroxylation
metabolite m/z 386 is compound C, not C0. Finally, the meta-
bolite m/z 384 has the same m/z, RT, and MS fragmentation
pattern as that of the known compound D (WR301855).21
Spiking technique with standard sample further confirmed the
structure of metabolite m/z 384 as D and established the meta-
bolic pathway of compound 8a to D shown in Scheme 5. As
expected, the metabolic pathway of 8h follows the same pattern
as 8a.
Scheme 5. Metabolic Pathway of Compound 8a
Realizing the stepwise metabolic transformation of compound
8a to active metabolite D (m/z 384) helps to explain the unusual
SAR results in Table 1. All compounds 8aꢀm are metabolically
unstable in mouse microsomal preparations, yet only 8a and 8h
with substituent R1 = isopropyl are highly active in mice infected
with sporozoites. Compounds with bulky substituent such as
ꢀC(CH3)3 or ꢀCH2C(CH3)3 at either R1 (8dꢀf, 8iꢀk), or R2
(8bꢀc) or both (8d), are inactive or activity drastically de-
creased. The plausible explanation of the observation is that
compound 8a acts as a prodrug of metabolite D, which was found
highly active in causal prophylactic and radical curative tests in
Rhesus by im injection.21 If the contention is correct, the rate of
primary hydroxylation of parent compound and/or the second-
ary hydroxylation of metabolite B to C constitute the rate
limiting step for the generation of active metabolite D. Placement
of a bulky group at R1 or R2 results in steric hindrance to the
enzymatic hydroxylation at the 4- or 5-position of the imidazole
ring. Consequently, generation of metabolites B, C, or D of
analogues with bulky substituent at R1 or R2 will be substantially
depressed and the antimalarial activity gravely affected.
In a combined causal prophylactic/radical curative Rhesus
model, 8a delayed the patency of treated monkeys for 9ꢀ11 days
at oral dose of 50 mg/kg/day ꢁ 3. In the same test, the first line
clinical drugs, primaquine, tafenoquine and Malarone, delayed
the patency of treated Rhesus for only 2ꢀ5 days at human
equivalent dose of 1.78, 0.32, and 14 mg/kg/day ꢁ 3, respec-
tively (Table 2). In radical curative test, 8a cured one monkey
and delayed relapse of another for 74 days at 30 mg/kg/day ꢁ 7
Scheme 6. MS2 Fragmentation Pattern of Metabolite B
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by im, plus 10 mg/kg of chloroquine by oral. By oral adminis-
tration, 8a also showed promising activity at the same dose,
delayed relapse 81 days for one monkey and 32 days for the
other. Control monkeys treated with chloroquine alone relapsed
11ꢀ12 days after parasite free. In contrast, compound 8h which
showed superior activity to 8a in mouse test delayed the relapse
of treated monkeys for only 21ꢀ26 days at 30 mg/kg/day ꢁ 7 by
oral (Table 3). The efficacy discrepancy of 8h observed in mouse
and Rhesus tests can be related to the species difference in
metabolic stability. The half-life (t1/2) of 8h in mouse is only
27.7 min vs >60 min in Rhesus.
’ REFERENCES
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’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis and X-ray
b
crystallography results. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
*Phone: 301-319-9084. Fax: 301-319-9449. E-mail: ai.lin@
us.army.mil.
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’ ACKNOWLEDGMENT
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. Research was conducted in
compliance with the Animal Welfare Act and other federal
statutes and regulations relating to animals and experiments
involving animals and adheres to principles stated in the Guide
for the Care and Use of Laboratory Animals, NRC Publication,
1996 edition. This research is supported in part by funding from
Military Infections Diseases Research Program (A40191_09_
WR), U.S. Army Medical Research and Material Command,
Department of Defense, USA., Peer Reviewed Medical Research
Program (PRMRP) (grant no. PR054609), and Medicines for
Malaria Venture (MMV 04/0013), Geneva, Switzerland.
’ ABBREVIATIONS USED
PQ, Primaquine; TQ, tafenoquine; DP, Delayed Parasitemia
Patency; IZ, guanidylimidazolinedione derivatives; G6PD, glu-
cose-6-phosphate dehydrogenase
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