Synthesis and antimalarial activity of 2-guanidino-4-oxoimidazoline derivatives

Article abstract of DOI:10.1021/jm200111g

A series of 2-guanidino-4-oxoimidazoline (deoxo-IZ) derivatives was prepared and showed potent antimalarial activities in rodent and Rhesus models. Compound 8e, the most potent analogues of this series, is the first non-8-aminoqinoline antimalarial that demonstrated radical curative activity in non-human primate by oral route and showed causal prophylactic activity comparable to that of the commonly used clinical drugs in Rhesus monkeys infected with sporozoites of Plasmodium cynomolgi. The metabolic stability and metabolites profile indicated that the new deoxo-IZ derivatives (8) may act as prodrugs of the corresponding IZ (1 and 2) derivatives.

Full text of DOI:10.1021/jm200111g

ARTICLE
pubs.acs.org/jmc
Synthesis and Antimalarial Activity of 2-Guanidino-4-
oxoimidazoline Derivatives
Xianjun Liu, Xihong Wang, Qigui Li, Michael P. Kozar, Victor Melendez, Michael T. O’Neil, 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 2-guanidino-4-oxoimidazoline (deoxo-IZ) derivatives was
prepared and showed potent antimalarial activities in rodent and Rhesus models.
Compound 8e, the most potent analogues of this series, is the first non-8-aminoqino-
line antimalarial that demonstrated radical curative activity in non-human primate by
oral route and showed causal prophylactic activity comparable to that of the
commonly used clinical drugs in Rhesus monkeys infected with sporozoites of
Plasmodium cynomolgi. The metabolic stability and metabolites profile indicated that
the new deoxo-IZ derivatives (8) may act as prodrugs of the corresponding IZ (1 and 2)
derivatives.
’ INTRODUCTION
blood stage malaria P. falciparum and are inactive in Thompson
mouse test against P. berghei, a blood stage rodent malaria.17,18
To the best of our knowledge, this is the first class of antimalarial
agents possessing activity against the liver stage malaria exclu-
sively. From the drug resistant development point of view, drugs
used exclusively for prophylaxis have much less chance of
exposure to parasites than those used for treatment and thus
have fewer problems of developing drug resistance. However,
carbamate 3 showed poor activities by oral administration; it
protects the treated monkeys for only 3 days vs untreated
control. The acid sensitive carbamate group of 3 and 4 was
considered the cause of poor oral activity. To overcome the acid
stability problem, chemically more stable carboxamide analogues
5 and 6 were prepared and again found to be active only by
intramuscular injection (im), not by oral (po) dosing.20 Pharma-
cokinetic 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, and both were converted to
the same s-triazine derivatives.20 This transformation was also
observed when both compounds were incubated in microsomal
preparations and in phosphate buffer solution, indicating that
conversion of carboxamides 5 and 6 to s-triazine 7 is a chemical
rather than an enzymatic reaction.20 The facile transformation of
IZ molecules 5 and 6 to s-triazine 7 revealed that the five-
membered imidazolinedione ring, though an amide, is rather
labile chemically, especially, when the five-membered imidazoli-
nedione ring carries electron withdrawing substituents, such as
3,4-dichlorophenyl and carboxamide, as in 5 and 6. The imida-
zolinedione rings of both 5 and 6 are readily hydrolyzed to
biguanides which cyclized to form s-triazine derivatives.20 In this
study, we report the synthesis and antimalarial activity of a series
Multiple drug resistance and lack of safe drugs to prevent or to
radically cure malaria diseases continue to pose challenges to the
containment of this deadly disease that has threatened the life of
millions of people in the underdeveloped world.1ꢀ5 Widespread
drug resistance to chloroquine, the first line antimalarial drug,
was reported in Southeast Asia and South America.6ꢀ10 Central
nervous system (CNS) toxicity of mefloquine11 and hemolytic
side effects of primaquine (PQ) and tafenoquine (TQ) in glucose
6-phosphate dehydrogenase (G6PD) deficiency patients7,8 have
compromised the clinical value of these otherwise highly effective
malaria therapeutic drugs. Multiple drug resistance in Plasmo-
dium falciparum malaria continues to pose special problems for
targeting the blood stages of malaria. 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 and cause clinical disease. With the
exception of quinoline esters,12 only the 8-aminoquinoline drugs
such as PQ or TQ13ꢀ15 have activity against the liver stages of
Plasmodium vivax and P. falciparum malarias. However, the
8-aminoquinoline drugs can cause serious lethal hemolytic side
effects in G6PD deficient patients.7,8,13 Therefore, there is an
eminent need for new and safe antimalarial drugs to combat the
parasites and protect the tourists traveling in the endemic areas of
the world.
Recently, a series of new 2-guanidinoimidazolidinedione (IZ)
derivatives (1 and 2, Figure 1) were demonstrated in our
laboratory to possess causal prophylactic antimalarials activity
in Rhesus monkeys infected with P. cynomolgi sporozoites.16ꢀ20
Carbamates 3, the most active compounds of this class, protect
monkeys infected with P. cynomolgi sporozoites at a dose of
10 mg/kg ꢁ 3 days by im dosing. Nevertheless, the IZ derivatives
showed very weak or no in vitro cell growth inhibition against
Received: January 31, 2011
Published: May 31, 2011
r
2011 American Chemical Society
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ARTICLE
Figure 1. Chemical structures of imidazolinediones and s-triazine derivatives.
yield by coupling of 1-alkyl-2-methylsulfanylimidazolin-4-one
(13aꢀc) with 3,4-dichlorophenylguanidine.
Carbamates 8dꢀg was prepared by treatment of 8a, 8b, or 8c
with 1 equiv of di-tert-butyl dicarbonate or alkyl chloroformate
under basic conditions. Similarly, treatment of compound 8b
with 1 equiv of trimethylacetyl chloride or 2-ethylbutyryl chlor-
ide resulted in the formation of carboxamide 8h or 8i, respec-
tively. However, it is interesting to note that with treatment of 8b
with more than 4 equiv of acyl chloride, carboxylation of the
4-oxo and the guanidine amino group took place to form
compound 14. For example, treatment of 8b with 4 equiv of
trimethylacetyl chloride at room temperature for 2 h in the
presence of Et3N and DMAP gave 14 in 95% yield. The chemical
Figure 2. New 4-oxoimidazoline derivatives.
1
structure of the product 14 was determined by H NMR, 13C
of chemically more stable 2-guanidino-4-oxoimidazoline deriva-
tives. The unstable imidazolinedione rings of the IZ molecules
(1ꢀ6) were replaced with a chemically more stable 4-oxo-
imidazoline ring (8, 9), as shown in Figure 2.
NMR, and LC/MS. In the 1H NMR spectra of 8h, nine signals
were observed as follows: two NH protons at 14.01 and 11.59 ppm
(N-H), three aromatic protons at 8.10, 7.40, and 7.24 ppm -
(aromatic protons), and aliphatic protons at 4.54 (-CH-), 3.81
(CO-CH2-), 1.36 (3 ꢁ CH3), and 1.26 ppm (2 ꢁ CH3). In the
1H NMR spectra of 14, the methylene (-COCH2-) proton
signals at 3.81 ppm observed in 8h disappeared; a new olefinic
proton signal at 6.65 ppm and a new tert-butyl group signal at
1.33 ppm were observed. Likewise, in the 13C NMR spectra of
14, the methylene carbon signal of 8h at 44.90 ppm vanished and
a new olefinic signal at 97.6 ppm appeared.
’ CHEMISTRY
The new compounds 8 and 9 are deoxo analogues of the
imidazolinedione derivatives (IZ) which exhibited potent causal
prophylactic activity and curative activity in Rhesus models by
intramuscular injection. Though oral activity was observed in
mice at high dose (>160 mg/kg ꢁ 3), IZ compounds 1ꢀ6
showed no oral activity in the Rhesus model. The lack of oral
activity was at least in part due to the short plasma half-life which
in turn is due to the chemical instability of the five-membered
imidazolinedione ring of the IZ compounds. Since the 4-oxoi-
midazolines (deoxo-IZs) are chemically more stable than 4,5-
imidazolinediones (IZs), deoxo-IZ analogues 8 and 9 may
possess better oral activity than the corresponding IZ derivatives
(3ꢀ6). Deoxo-IZ (8aꢀc), N-carbamates (8d,g), and N-carbox-
amides (8h,i) were prepared according to Scheme 1. The starting
materials N-alkylaminoacetic acid (10aꢀc) and 1-alkyl-2-thio-
hydantoin (11aꢀc) were prepared according to the reported
procedures.21,22 The starting materials 10aꢀc were obtained in
over 90% yield by treatment of hydroxyacetonitrile with primary
amine at room temperature for 1 h, followed by NaOH hydro-
lysis. Fusion of N-alkylaminoacetic acids 10a,b with NH4SCN at
140 °C gave compounds 11a,b in 65% yield. Although treatment
of chloroacetic acid with tert-butylamine gave a decent yield of
N-tert-butylaminoacetic acid (10c), fusion of 10c with NH4SCN
gave poor yields of compound 11c. Regioselective S-alkylation of
11aꢀc with 1.5 equiv of CH3I gave 12aꢀc HI salt in good
yield.23 The salts 12aꢀc were converted to free base compounds
13aꢀc in 95% yield by basification with K2CO3. The desired
guanidine derivatives (8aꢀc) were readily prepared in good
2-Amino-1-isopropylimidazolin-4-one (15b) was prepared
either by treatment of 2-methylsulfanylimidazolin-4-one (13b)
with ammonia in methanol23 or by treatment of sodium iso-
propylcyanamide with chloroacetamide in CH3CN24 The latter
3
method provided the product in one step from the commercially
available materials in 80ꢀ85% yield, while the former method
required multiple-step synthesis from hydroxyacetonitrile. Both
methods were used to prepare 15aꢀc in good yield.
The isopropyl group was considered an essential substituent at
the N1-position of IZ antimalarials 1ꢀ6 in previous studies. To
further examine the importance of the isopropyl group in the
deoxo-IZ analogues, a number of N-CH3 and N-C(CH3)3
analogues 8a and 8c and their carbamates 8d and 8f were
designed and prepared as shown in Scheme 1.
2-Amino-1-methylimidazolin-4-one (15a) and 2-amino-1-tert-
butylimidazolin-4-one (15c) were prepared using the same
method as for the synthesis of 2-amino-1-isopropylimidazolin-
4-one (15b). Compounds 8a and 8c were also prepared in
moderate yields (50ꢀ60%) by heating 15a or 15c with 3,4-
dichlorophenylcyanamide in n-propanol. Since fusion of 10c
with NH4SCN gave poor yield of 11c, compound 8c was mainly
prepared by treatment of 15c with 3,4-dichlorophenylcyanamide
in n-propanol. (Scheme 1).
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Scheme 1. Synthesis of 2-Guanidino-4-oxoimidazoline Derivatives 8aꢀia
a Reagents and conditions: (i) NaOH/H2O, 90%; (ii) 140 °C, fusion, 4 h, 70%; (iii) CH3I, DMF, 10 h, 25 °C, 90%; (iv) K2CO3, H2O; (v) EtOH, 82 °C,
24 h, 75%; (vi) RCOCl or (RO)2CdO, Et3N, DMF, DMAP, 25 °C, 50ꢀ65%; (vii) 7.0 M NH3 in MeOH, 70 °C, 4 h, 85%; (viii) CH3CN,
70 °C, 4 h, 80%.
While carbamates 8dꢀg and carboxamides 8h,i of the deoxo-
IZs were prepared by derivatization of biguanides 8aꢀc as shown
in Scheme 1, the synthesis of the N-alkyl analogues (8jꢀn) were
prepared by coupling of S-methyl-N-alkyl-N0-(3,4-dichlorophe-
nyl)thioureas 18aꢀd and 15aꢀc in one step as shown in
Scheme 2. The free base, 2-methylisothioureas 18aꢀd were
prepared via the reaction of 3,4-dichlorophenyl isothiocyanate
(16) with a variety of primary amines to form thiourea derivatives
(17aꢀd) followed by methylation with CH3I.25 The desired
N-alkyl guanidine derivatives 8jꢀn were readily prepared by
coupling of the free base (18aꢀd) with 2-aminoimidazolines
15aꢀc. After the samples were heated at 100 °C for 24 h, the
substituted guanidine derivatives (8jꢀn) were obtained in mod-
erate to good yield (50ꢀ75%). It was noted that the coupling
reaction required base as catalyst to give good yields. Likewise,
preparation of the cyclic amine analogues was achieved by
treatment of isothiocyanate with pyrrolidine and piperidine to
give the corresponding thiourea derivatives 19a and 19b, respec-
tively. Reaction of 19a,b with CH3I afforded S-methylthiourea
HI salts 20a,b which were converted to free base prior to
coupling with 15b to afford the desired derivatives 21a,b in
moderate yields (41ꢀ50%) (Scheme 2).
corresponding derivatives of 2. However, the latter compounds
are more active than the former in exoerythrocytic mouse
tests.17,18 On the contrary, derivatives of compound 1 are more
active than the corresponding less stable derivatives of 2 in the
Rhesus monkey test. Since replacement of one carbonyl group in
the imidazolidin-4,5-dione derivatives (1ꢀ6) with a methylene
group will result in chemically more stable imidazolidin-4-one
analogues, a series of deoxo analogues of compound 2 deriv-
atives, 9aꢀe and 25aꢀc, were also prepared and tested in this study.
N-[1-(3,4-Dichlorophenyl)-4-oxo-4,5-dihydro-1H-imidazol-
2-yl]-N0-isopropylguanidine (9a) and its carbamate (9b,c) and
carboxamide (9d,e) derivatives were prepared as illustrated in
Scheme 3. 3,4-Dichlorophenylcyanamide (22) was prepared by a
known methods26 and was converted to sodium salt 23 using
sodium metal in absolute ethanol before reacting with chloroa-
cetamide in CH3CN to give 2-amino-1-(3,4-dichlorophenyl)-
1,5-dihydroimidazolin-4-one (24). Treatment of compound 24
with isopropylcyanamide in 1-propanol resulted in formation of
guanidine derivative 9a which was converted to carbamates
9bꢀc and carboxamides 9dꢀe in moderate yields (40ꢀ50%).
N-Alkyl-N0-(3,4-dichlorophenyl)biguanidine analogues 25aꢀc
were prepared by coupling of 2-amino-3-(3,4-dichlorophe-
nyl)imidazolin-4-one (24) with compounds 18aꢀd in the pre-
sence of K2CO3.
Our previous structureꢀactivity relationship (SAR) studies
indicated that derivatives of 1 are chemically more stable than the
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Scheme 2. Synthesis of N-(3,4-Dichlorophenyl)-N0-subistituted-N00-(1-isopropyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidines
8jꢀn and 21a,ba
a Reagents and conditions: (i) RNH2, CH2Cl2, 25 °C, 2 h; (ii) CH3I, acetone, reflux, 2 h; (iii) Na2CO3, H2O, 25 °C; (iv) 15aꢀc, K2CO3,
CH3CH2CH2OH, reflux, 24 h; (v) RRNH, CH2Cl2, 25 °C, 2 h.
Scheme 3. Synthesis of N-[1-(3,4-Dichlorophenyl)-4-oxo-4,5-dihydro-1H-imidazol-2-yl]-N0-isopropylguanidine
Derivatives (9aꢀe)a
a Reagents and conditions: (i) BrCN/AcOH, 4 h, 25 °C, 85%; (ii) Na, EtOH, 25 °C; (iii) ClCH2CONH2/CH3CN, reflux, 4 h, 80%; (iv) 18, K2CO3,
1-propanol/DMF, 100 °C, 24 h; (v) (CH3)2CHNHCN/CH3CH2CH2OH, reflux, 24 h, 65%; (vi) (RO)2CdO or RCOCl, Et3N, DMF, DMAP,
25 °C, 45ꢀ55%.
’ EXPERIMENTAL SECTION
was performed with UV absorbance. Flash chromatography was con-
ducted with silica gel 60 Å (200ꢀ400 mesh) from Sigma-Aldrich Co.
Solvents and reagents obtained from commercial sources were used
without purification, unless otherwise noted. Reactions were carried out
under an inert atmosphere of nitrogen. Elemental analysis was per-
formed 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 analysis of purity and chromophore properties. The
system consisted of an Agilent 1100 series LC/UVꢀvis system online
with a ThermoFinnigan (now Thermo Scientific; Waltham, MA) LCQ
A. Chemistry. Melting points were determined in open capillary
tubes on an OptiMelt melting point apparatus (Standard Research
Systems, U.S.) and are uncorrected. H NMR and 13C NMR spectra
1
were recorded using Bruker Avance 300 and Bruker Avance 600
spectrometers (Bruker Instruments, 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
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MS instrument equipped with an electrospray ionization (ESI) source.
Samples were analyzed using shallow CH3CN/1% HCOOH/H2O gra-
dients at low flow rate. The purity of all final products was g95%.
General Procedure for the Preparation of 11aꢀc. A mixture of
2-alkylaminoacetic acid (0.1 mol) and NH4SCN (22.8 g, 0.3 mol) was
heated at 140 °C for 6 h. The dark red solution was cooled. The solid
cake formed was crushed with a spatula, washed with 40 mL of H2O,
and collected. The crystals were then washed successively with H2O,
95% ethanol, and hexane to give 7.5 g (65%) of light tan crystals after
drying. The products were determined to be at least 93ꢀ95% pure by
LC/MS analytical method described in Experimental Section above
and were used without further purification.
added sodium metal (0.38 g, 16.5 mmol) in small portions. The mixture
was stirred at room temperature until the sodium metal was consumed.
The solvent was evaporated to dryness under reduced pressure. The
solid was suspended in 20 mL of CH3CN. To the suspension chlor-
oacetamide (1.7 g, 18.1 mmol) was added and the mixture was refluxed
for 5 h. The white precipitates were collected after cooling, washed
successively with H2O and acetone, and dried to give the product in
80ꢀ85% yield. The products were used without further purification.
2-Amino-1-methyl-1,5-dihydroimidazol-4-one (15a). 15a was pre-
1
pared by method A. Mp 295ꢀ296 °C. White solid. H NMR (D2O):
δ 4.70 (s, 2H), 3.20 (s, 3H). 13C NMR (D2O): δ 186.3, 184.0, 57.7,
34.8. MS (EI): m/z 113.0 [M]þ.
2-Amino-1-isopropyl-1,5-dihydroimidazol-4-one (15b). 15b was
prepared by methods A and B. Mp 225ꢀ226 °C. White solid. 1H NMR
(MeOD): δ 4.07 (q, J = 6.72 Hz, 1H), 3.90 (s, 1H), 1.24 (s, 3H), 1.23
(s, 3H). 13C NMR (CD3OD): 187.0, 168.9, 45.0, 21.2, 19.0. IR (neat):
3308, 1658, and 1497 cmꢀ1. MS (EI): m/z 141.0 [M]þ.
1-Methyl-2-thioxoimidazolidin-4-one (11a). Mp 222ꢀ224 °C.
Yield: 45%. Light tan crystals. 1H NMR (DMSO-d6): δ 11.67 (s, 1H),
4.27 (s, 2H), 3.23 (s, 3H). 13C NMR (DMSO-d6): δ 181.5, 172.1, 55.1,
and 32.7. MS (EI): m/z 130.0 [M]þ.
1-Isopropyl-2-thioxoimidazolidin-4-one (11b). Mp 158 °C. Yield:
1
2-Amino-1-tert-butyl-1,5-dihydroimidazol-4-one (15c). 15c was pre-
pared by method B. Mp 230ꢀ231 °C. Yield: 50%. White solid. 1H NMR
(DMSO-d6): δ 7.02 (s, 2H), 3.87 (s, 2H), 1.34 (s, 9H). 13C NMR
(DMSO-d6): δ 184.3, 169.6, 53.9, 53.3, 28.4. IR (neat): 3300, 1665, 1470,
and 1415 cmꢀ1. MS (ESI): m/z 156.0 [M þ 1]þ.
55%. H NMR (CDCl3): δ 9.26 (s, 1H), 4.95 (m, 1H), 4.01 (s, 2H),
1.26 (d, 6H). 13C NMR (CDCl3): δ 179.8, 171.0, 48.3, 47.3, 19.6. MS
(EI): m/z 158.0 [M]þ
1-tert-Butyl-2-thioxoimidazolidin-4-one (11c). Mp 119ꢀ120 °C.
1
Yield: 40%. H NMR (CDCl3): δ 9.20 (s, 1H), 3.80 (s, 2H), 1.45 (s,
9H). 13C NMR (CDCl3): δ 180.9, 170.7, 47.1, 29.4. MS (ESI): m/z
General Procedure for the Preparation of 8a,b. To the solution of
free base 2-methylthio-imidazol-4-one (13, 7.0 mmol) in 10 mL of
EtOH was added 3,4-dichlorophenylguanidine (1.7 g, 8.3 mmol). The
reaction mixture was heated at 80 °C for 48 h. The pale solid precipitates
were collected, washed with CH3OH, and dried to afford the product as
a pale white solid.
172.2 [M]þ.
General Procedure for the Preparation of 12a,b. A solution of
2-thiohydantoin 11 (5 mmol) in 5 mL of DMF at 0 °C was treated with
CH3I (0.8 mL, 10.6 mmol). The solution was warmed slowly to 35 °C
over 30 min and stirred for an additional 5 h. The precipitated
compound was collected and washed with ethanol followed by hexanes.
The products were determined to be at least 93ꢀ95% pure by LC/MS
described in Experimental Section above and were used without further
purification.
N-(3,4-Dichlorophenyl)-N0-(1-methyl-4-oxo-4,5-dihydro-1H-imida-
zol-2-yl)guanidine (8a). Yield 70%. Mp 256.6ꢀ258.5 °C. White solid.
1H NMR (DMSO-d6): δ 9.50 (s, 1H), 8.04 (d, J = 2.45 Hz, 1H), 7.58 (d,
J = 8.76 Hz, 1H), 7.35 (dd, J = 8.74 Hz, J = 2.45 Hz, 1H), 3.75 (s, 2H),
3.02 (s, 3H). 13C NMR (DMSO-d6): δ 185.1, 174.2, 157.7, 139.1, 131.2,
130.9, 125.2, 123.1, 121.6, 54.2, 30.6. IR (neat): 3148, 1878, 1550,
and 1470 cmꢀ1. MS (EI): m/z 299.0 [M]þ. Anal. (C11H11Cl2N5O) C,
H, N, Cl.
1-Methyl-2-methylsulfanyl-1,5-dihydroimidazol-4-one HI Salt (12a).
1
Yield: 88%. Mp 226ꢀ228 °C. White solid. H NMR (D2O): δ 4.70
(s, 2H), 3.25 (s, 3H), 2.73 (s, 3H). 13C NMR (D2O): δ 176.6, 174.0,
57.7, 34.8, 15.2. MS (EI): m/z 144.0 [M]þ.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-4-oxo-4,5-dihydro-1H-imi-
dazol-2-yl)guanidine (8b). Yield 75%. Mp 231ꢀ232 °C. White solid.
1H NMR (DMSO-d6): δ 9.45 (s, 1H), 8.06 (d, J = 2.36 Hz, 1H), 7.54 (d,
J = 8.75 Hz, 1H), 7.29 (dd, J = 8.75 Hz, J = 2.36 Hz, 1H), 4.39 (m, 1H),
3.70 (s, 2H), 1.16 (s, 3H), 1.14 (s, 3H). 13C NMR (DMSO-d6): δ 185.1,
173.1, 157.6, 139.2, 131.2, 130.8, 125.1, 123.1, 121.4, 47.4, 44.2, 20.7. IR
(neat): 3150, 1680, 1600, and 1475 cmꢀ1. MS (EI): m/z 327.0 [M]þ.
Anal. (C13H15Cl2N5O) C, H, N, Cl.
1-Isopropyl-2-methylsulfanyl-1,5-dihydroimidazol-4-one HI Salt (12b).
Yield: 90%. Mp 198 °C. White solid. 1H NMR (D2O): δ 4.69 (s, 2H),
4.19 (m, 1H), 2.66 (s, 3H), 1.21 (s, 3H), 1.19 (s, 3H). 13C NMR (D2O):
δ 175.1, 173.6, 51.4, 18.9, 14.1. MS (EI): m/z 172.0 [M]þ.
General Procedure for the Preparation of 13a,b. To the water
solution of 12a,b salt (1.3 g) was added saturated Na2CO3 solution at
ice-cold temperature until the pH reached ∼9ꢀ10. The reaction mixture
was extracted with CH2Cl2 three times. The organic solution was dried
over Na2SO4 and evaporated to dryness under reduced pressure. The
residue was purified by a silica gel column using hexane/EtOAc (4:1 v/v)
to give product 13.
1-Methyl-2-methylsulfanyl-1,5-dihydroimidazol-4-one (13a). Yield:
98%. Mp 118.0ꢀ120 °C. White solid. 1H NMR (CDCl3): δ 3.99 (s, 2H),
3.15 (s, 3H), 2.66 (s, 3H). 13C NMR (CDCl3): δ 186.1, 184.9, 57.5, 32.8,
15.0. MS (EI): m/z 144.0 [M]þ.
1-Isopropyl-2-methylsulfanyl-1,5-dihydroimidazol-4-one (13b). Yield:
98%. Colorless oil. 1H NMR (CDCl3): δ 4.05 (m, 1H), 3.91 (s, 2H), 2.67
(s, 3H), 1.28 (s, 3H), 1.26 (s, 3H). 13C NMR (CDCl3): δ 184.9, 184.5,
50.3, 20.7, 14.8. MS (EI): m/z 172.0 [M]þ.
General Procedure for the Preparation of Compounds 15aꢀc.
Method A. 2-Methylthioimidazolin-4-one 13b (0.86 g, 5 mmol) in
MeOH was added to 8 mL of 7.0 M ammonia in MeOH. The mixture
was refluxed at 65 °C for 5 h. The MeOH was removed under reduced
pressure. The crude product was purified by first suspending in 20 mL of
CH2Cl2. The product was collected by filtration, washed twice with
CH2Cl2, and dried to give the white product in 80ꢀ88% yield.
Method B. To alkylcyanamide (RNHCN, where R = ꢀCH3, ꢀCH-
(CH3)2, or ꢀC(CH3)3, 16.5 mmol) in 15 mL of absolute EtOH was
Synthesis of N-(3,4-Dichlorophenyl)-N0-(1-tert-butyl-4-oxo-4,5-di-
hydro-1H-imidazol-2-yl)guanidine (8c). A mixture consisting of 1-tert-
butyl-2-aminoimidazolin-4-one (15c) (3.0 g, 19.3 mmol) and 3,4-di-
chlorophenylcyanamide (3.7 g, 20 mmol) in 1-propanol (30 mL) was
heated at 100 °C for 24 h. After cooling, the solution was filtered and the
solvent was removed under reduced pressure. The crude product was
purified by a silica gel column using CH2Cl2/MeOH (10:1 v/v) as
eluent and recrystallized from MeOH to give 3.9 g of white crystals. Mp
214ꢀ216 °C. 1H NMR (DMSO-d6): δ 9.40 (s, 1H), 7.88 (s, 1H), 7.54
(d, J = 8.65 Hz, 1H), 7.20 (dd, J = 8.65 Hz, J = 2.40 Hz, 1H), 3.87 (s, 2H),
1.36 (s, 9H). 13C NMR (DMSO-d6): δ 184.3, 173.5, 157.3, 138.6, 131.3,
130.8, 126.0, 125.0, 124.8, 122.9, 122.8, 54.4, 51.6, 28.7. IR (neat):
3367.4, 1660.9, 1466.3, and 407.2 cmꢀ1. MS (ESI): m/z 341.97
[M þ 1]þ. Anal. (C14H17Cl2N5O) C, H, N, Cl.
General Procedure for the Preparation of Compounds 8dꢀi. To a
mixture of 8a, 8b, or 8c (5.40 mmol) and DMF (10 mL) were added
DMAP (0.3 g, 2.67 mmol) and Et3N (0.5 mL), followed by addition of
dicarbonate (5.61 mmol) or acyl chloride (5.61 mmol). The reaction
mixture was stirred at room temperature for 12 h and evaporated to
dryness. The solid was dissolved in CH2Cl2 (30 mL), filtered, and the
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filtrate was washed with water, dried over Na2SO4, and evaporated to
dryness to give the crude product. The crude product was purified by a
silica gel column and eluted with EtOAc/hexane (4:1 v/v) to give the
desired product.
25.0, 20.8, 11.6. MS (ESI): 426 m/z [M þ 1]þ. Anal. (C19H25Cl2N5O2)
C, H, N, Cl.
General Procedure for the Preparation of Compounds 8jꢀn.
A mixture of 2-aminoimidazolin-4-one (15, 14.2 mmol), 2-methyli-
sothiourea 18 (20.0 mmol), and K2CO3 (2.0 g, 14.2 mmol) in
1-propanol (20 mL) was heated at 110 °C for 48 h and filtered to
remove the K2CO3. The solvent was evaporated to dryness, and the
residue was suspended in 10 mL of CH2Cl2. The solid K2CO3 was
removed by filtration, and the filtrate was evaporated to dryness. The
crude product was purified with a silica gel column using CH2Cl2/
MeOH (10:1 v/v) as eluent to give the final product which was further
purified by recrystallization from Et2O and EtOAc.
N-(tert-Butoxycarbonyl)-N0-(3,4-dichlorophenyl)-N00-(1-methyl-4-
oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (8d). The title com-
pound was prepared according to the general procedure using 8a and
di-tert-butyl dicarbonate as acylating agent to give 45% yield of the
product as white crystals. Mp 234ꢀ235 °C. 1H NMR (CDCl3): δ 12.80
(s, 1H), 10.81 (s, 1H), 8.00 (d, J = 2.46 Hz, 1H), 7.43 (d, J = 8.70 Hz,
1H), 7.39 (dd, J = 8.70 Hz, J = 2.46 Hz, 1H), 3.86 (s, 2H), 3.14 (s, 3H),
1.54 (s, 9H). 13C NMR (CDCl3): δ 184.3, 172.4, 153.8, 152.9, 136.0,
132.5, 130.2, 128.6, 124.5, 121.6, 84.6, 54.2, 30.8, 28.0. IR (neat):
3105, 1670, 1555, and 1470 cmꢀ1. MS (EI): m/z 399 [M]þ. Anal.
(C16H19Cl2N5O3) C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-isopropyl-N00-(1-isopropyl-4-oxo-4,5-di-
hydro-1H-imidazol-2-yl)guanidine (8j). The title compound was pre-
pared from 15b and 18a. Yield: 75%. Mp 195ꢀ196 °C. White crystals.
1H NMR (CDCl3): δ 11.83 (br, 1H), 7.47 (d, J = 8.50 Hz, 1H), 7.36 (d,
J = 2.36 Hz, 1H), 7.04 (dd, J = 8.50 Hz, J = 2.36 Hz, 1H), 4.57 (q, J = 6.90
Hz, 1H), 4.11 (br, 1H), 3.70 (s, 2H), 1.24 (s, 3H), 1.23 (s, 3H), 1.21 (s,
3H), 1.19 (s, 3H). 13C NMR (CDCl3): δ 188.1, 172.3, 155.5, 135.0,
142.1, 130.5, 128.4, 123.3, 46.9, 43.8, 22.7, 20.6. IR (neat): 3300,
1680, 1660, and 1470 cmꢀ1. MS (EI): m/z 369.0 [M]þ. Anal.
(C16H21Cl2N5O) C, H, N, Cl.
N-(tert-Butoxycarbonyl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-
4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (8e). Compound 8e
was prepared by reaction of 8b with di-tert-butyl dicarbonate. Yield
50%. White crystals. Mp 152ꢀ153 °C. 1H NMR (CDCl3): δ 12.73 (s,
1H), 10.81 (s, 1H), 8.05 (d, J = 2.37 Hz, 1H), 7.43 (d, J = 8.67 Hz, 1H),
7.24 (dd, J = 8.67 Hz, J = 2.37 Hz, 1H), 4.51 (m, 1H), 3.80 (s, 2H), 1.54
(s, 9H), 1.24 (s, 3H), 1.22 (s, 3H). 13C NMR (CDCl3): δ 184.68,
171.30, 153.88, 152.76, 136.11, 132.50, 130.22, 128.50, 124.50, 121.45,
84.58, 47.26, 44.83, 28.09, 20.79. IR (neat): 3100, 1658, 1574,
and 1450 cmꢀ1. MS (EI): m/z 427.0 [M]þ. Anal. (C18H23Cl2N5O3)
C, H, N, Cl.
N-(tert-Butyl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4-oxo-4,5-
dihydro-1H-imidazol-2-yl)guanidine (8k). Compound 8k was pre-
pared from 15b and 18b. Yield: 75%. Mp 178ꢀ179 °C. White crystals.
1H NMR (CDCl3): δ 11.95 (s, 1H), 7.48 (d, J = 8.53 Hz, 1H), 7.33 (d,
J = 2.1 Hz, 1H), 7.04 (dd, J = 8.53 Hz, J = 2.1 Hz, 1H), 4.70 (br, 1H), 4.61
(q, J = 6.70 Hz, 1H), 3.74 (s, 2H), 1.43 (s, 9H), 1.23 (s, 3H), 1.21 (s,
3H). 13C NMR (CDCl3): δ 185.5, 171.7, 155.7, 136.0, 133.7 131.5,
130.5, 127.4, 124.7, 52.6, 46.8, 44.1, 29.5, 20.7. IR (neat): 3300,
1692, 1564, and 1506 cmꢀ1. MS (EI]: m/z 383.0 [Mþ]. Anal.
(C17H23Cl2N5O) C, H, N, Cl.
N-(tert-Butoxycarbonyl)-N0-(3,4-dichlorophenyl)-N00-(1-tert-butyl-
4-oxo-4,5-dihydro-1H-imidazol-2-yl)guanidine (8f). Compound 8f
was prepared by treatment of 8c with di-tert-butyl dicarbonate. Yield
55%. White crystals. Mp 168ꢀ169 °C. 1H NMR (CDCl3): δ 13.02 (s,
1H), 10.64 (s, 1H), 7.76 (d, J = 2.37 Hz, 1H), 7.43 (d, J = 8.61 Hz, 1H),
7.14 (dd, J = 8.61 Hz, J = 2.37 Hz, 1H), 3.91 (s, 2H), 1.50 (s, 9H), 1.35 (s,
9H). 13C NMR (CDCl3): δ 183.27, 171.59, 154.35, 153.25, 135.77,
132.58, 130.27, 129.63, 126.90, 123.67, 84.21, 55.30, 51.47, 28.84, 28.10.
IR (neat): 3100, 1650, 1575, and 1460 cmꢀ1. MS (ESI): m/z 442.3
[M þ 1]þ. Anal. (C19H25Cl2N5O3) C, H, N, Cl.
N-(Ethyl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4-oxo-4,5-dihy-
dro-1H-imidazol-2-yl)guanidine (8l). Compound 8l was prepared from
1
15b and 18c. Yield: 70%. Mp 178ꢀ179 °C. White crystals. H NMR
(CDCl3): δ 11.78 (s, 1H), 7.31ꢀ7.41 (m, 2H), 7.06 (s, 1H), 5.13 (s,
1H), 4.57 (s, 1H), 3.69 (s, 2H), 3.43 (q, J = 6.63 Hz, 2H), 1.18 (m, 9H).
13C NMR (CDCl3): δ 185.7, 171.8, 156.2, 135.2, 134.0, 131.4, 130.4,
128.1, 126.5, 46.8, 43.9, 36.6, 20.6, 14.9. IR (neat): 3200, 1686, 1507, and
1439 cmꢀ1. MS (ESI): m/z 356.12 [M þ 1]þ. Anal. (C15H19Cl2N5O)
C, H, N, Cl.
N-(Ethoxycarbonyl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4-oxo-
4,5-dihydro-1H-imidazol-2-yl)guanidine (8g). The title compound was
prepared by treatment of 8b with ethyl chloroformate. Yield: 48%. Mp
279.3ꢀ280.0 °C. White crystals. 1H NMR (CDCl3): δ 13.1 (br s, 1H),
10.7 (s, 1H), 8.03 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.20 (dd,
J = 8.60 Hz, J = 2.3 Hz, 1H), 4.53 (m, 1H), 4.26 (q, J = 7.0 Hz, 2H), 3.80
(s, 2H), 1.37 (t, J = 7.0 Hz, 3H), 1.25 (d, J = 6.5 Hz, 6H). 13C NMR
(CDCl3): δ 184.8, 169.9, 155.6, 153.5, 136.1, 132.5, 130.2, 128.6, 124.6,
121.4, 62.9, 47.1, 44.9, 20.7, 14.2. MS (ESI): m/z 400 [M þ 1]þ. Anal.
(C16H19Cl2N5O3) C, H, N, Cl.
N-(Adamantyl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4-oxo-
4,5-dihydro-1H-imidazol-2-yl)guanidine (8m). Compound 8m was
prepared from 15b and 18d. Yield: 75%. Mp 197ꢀ198 °C. White
crystals. 1H NMR (CDCl3): δ 11.95 (br, 1H), 7.47 (d, J = 8.53 Hz, 1H),
7.34 (d, J = 2.37 Hz, 1H), 7.05 (dd, J = 8.53 Hz, J = 2.37 Hz, 1H), 4.57
(m, 2H), 3.74 (s, 2H), 2.08 (m, 9H), 1.66 (m, 6H), 1.26 (s, 3H), 1.22 (s,
3H). 13C NMR (CDCl3): δ 185.5, 171.5, 155.4, 136.0, 133.7, 131.4,
130.5, 127.4, 124.7, 53.2, 46.9, 44.2, 42.2, 36.3, 29.4, 20.8. IR (neat):
3200, 1702, 1565, and 1503 cmꢀ1. MS (EI): m/z 461.0 [M]þ. Anal.
(C23H29Cl2N5O) C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-4-oxo-4,5-dihydro-1H-imi-
dazol-2-yl)-N00-(trimethylacetyl)guanidine (8h). Compound 8h was
prepared by treatment of 8b with trimethylacetyl chloride. Yield: 45%.
Mp 189ꢀ190 °C. White crystals. 1H NMR (CDCl3): δ 14.01 (s, 1H),
11.59 (s, 1H), 8.10 (d, J = 2.40 Hz, 1H), 7.40 (d, J = 8.70 Hz, 1H), 7.24
(dd, J = 8.67 Hz, J = 2.40 Hz, 1H), 4.54 (q, J = 7.50 Hz, 1H), 3.81 (s, 2H),
1.36 (s, 9H), 1.26 (s, 3H), 1.24 (s, 3H). 13C NMR (CDCl3): δ 184.0,
182.6, 171.6, 153.0, 136.0, 132.5, 130.3, 128.7, 124.5, 121.4, 47.4, 44.9,
41.0, 27.0, 20.7. IR (neat): 3100, 1670, 1558, and 1468 cmꢀ1. MS (ESI):
m/z 412.22 [M þ 1]þ. Anal. (C18H23Cl2N5O2) C, H, N, Cl
N-(Isopropyl)-N0-(3,4-dichlorophenyl)-N00-(1-methyl-4-oxo-4,5-di-
hydro-1H-imidazol-2-yl)guanidine (8n). The title compound was pre-
pared from 15a and 18a. Yield: 70%. Mp 208ꢀ210 °C. White crystals.
1H NMR (DMSO-d6): δ 10.11 (br, 1H), 8.86 (s, 1H), 7.88 (s, 1H), 7.59
(d, J = 8.64 Hz, 1H), 7.46 (m, 1H), 4.09 (br, 1H), 3.68 (s, 2H), 2.93 (s,
3H), 1.22 (s, 3H), 1.20 (s, 3H). 13C NMR (DMSO-d6): δ 184.5, 173.0,
155.2, 141.0, 130.5, 125.8, 124.1, 53.9, 43.4, 30.4, 23.1. IR (neat): 1690,
1514, 1460, and 302 cmꢀ1. MS (ESI): m/z 342.06 [M þ 1]þ. Anal.
(C14H17Cl2N5O) C, H, N, Cl.
N-(2-Ethylbutyryl)-N0-(3,4-dichlorophenyl)-N00-(1-isopropyl-4-oxo-
4,5-dihydro-1H-imidazol-2-yl)guanidine (8i). Compound 8i was pre-
pared by treatment of 8b with 2-ethylbutyryl chloride. Yield: 32%. White
solid. Mp 142.6ꢀ143.8 °C. 1H NMR (CDCl3): δ 13.9 (br s, 1H), 11.5
(s, 1H), 8.12 (br s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H),
4.58 (m, 1H), 3.82 (s, 2H), 2.35 (m, 1H), 1.68 (m, 4H), 1.26 (d, J = 6.7
Hz, 6H), 0.97 (t, J = 7.3 Hz, 6H). 13C NMR (CDCl3): δ 184.2, 179.8,
171.6, 152.6, 135.9, 132.6, 130.3, 128.7, 124.5, 121.4, 52.3, 47.4, 44.9,
Synthesis of 2,2-Dimethylpropionic Acid 2-[N0-(3,4-Dichlorophenyl)-
N00-(2,2-dimethylpropionyl)guanidino]-1-isopropyl-1H-imidazol-4-
yl Ester (14). To a mixture of starting material 1-isopropyl-4-oxoimida-
zolinoguanidine 8b (0.55 g, 1.68 mmol) in CHCl3 (10 mL) were added
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DMAP (0.41 g, 3.36 mmol) and Et3N (0.5 g, 5.0 mmol), followed by
addition of trimethylacetyl chloride (0.42 g, 3.36 mmol) at 0 °C. The
reaction mixture was stirred at room temperature for 2 h. The solvent
was evaporated to dryness and the product was purified by silica gel
column using hexane/EtOAc (10:1 v/v) to give 0.80 g (95%) of
compound 14 as white crystals. Mp 128ꢀ129 °C. 1H NMR (CDCl3):
δ 13.28 (s, 1H), 10.93 (s, 1H), 8.28 (d, J = 2.43 Hz, 1H), 7.33 (d, J = 8.70
Hz, 1H), 7.23 (dd, J = 8.70 Hz, J = 2.43 Hz, 1H), 6.65 (s, 1H), 4.76 (q, J =
6.01 Hz, 1H), 1.44 (s, 3H), 1.42 (s, 3H), 1.35 (s, 9H), 1.33 (s, 9H). 13C
NMR (CDCl3): δ 181.3, 175.6, 145.3, 142.7, 142.6, 137.9, 132.3, 130.0,
125.9, 122.3, 119.6, 97.6, 46.2, 40.8, 39.0, 27.0, 26.5, 22.6. MS (ESI): m/z
496.25 [M þ 1]þ. Anal. (C23H31Cl2N5O3) C, H, N, Cl.
N-[1-(3,4-Dichlorophenyl)-4-oxo-4,5-dihydro-1H-imidazol-2-yl]-N0-
isopropyl-N00-(2-ethtylbutyryl)guanidine (9e). Compound 9e was pre-
pared from 9a and 2-ethylbutyryl chloride. Yield: 45%, white crystals.
1
Mp 149.3ꢀ150.2 °C. H NMR (CDCl3): δ 13.5 (s, 1H), 9.59 (d,
J = 6.4 Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.20 (dd,
J = 8.70 Hz, J = 2.3 Hz, 1H), 4.53 (m, 1H), 4.27 (m, 3H), 2.30 (m, 1H),
1.67 (m, 4H), 1.33 (d, J = 6.5 Hz, 6H), 0.96 (t, J = 7.4 Hz, 6H). 13C
NMR: (CDCl3) δ 181.9, 179.4, 170.6, 154.5, 137.5, 132.6, 130.2, 127.8,
123.6, 119.6, 52.5, 52.2, 44.1, 25.0, 22.4, 11.7. MS (ESI): m/z 426.0
[M þ 1]þ. Anal. (C19H25Cl2N5O2) C, H, N, Cl.
General Procedure for the Preparation of 17aꢀd. 3,4-Dichlorophe-
nyl isothiocyanate (16, 5.0 g, 24.5 mmol) was added dropwise to an ap-
propriate amine (27 mmol) in CH2Cl2 (15 mL). The mixture was stirred
for 18 h at room temperature, and the solvent was then removed under
reduced pressure. The solid product was suspended in Et2O (20 mL),
collected, washed several times with fresh Et2O, and dried.
Synthesis of N-[1-(3,4-Dichlorophenyl)-4-oxo-4,5-dihydro-1H-imi-
dazol-2-yl]-N0-isopropylguanidine (9a). A mixture of 2-amino-3-(3,4-
dichlorophenyl)imidazolin-4-one (24, 3.0 g, 12.3 mmol) and isopro-
pylcyanamide (2.1 g, 24.6 mmol) in 1-propylanol (30 mL) was heated at
110 °C for 48 h, filtered, and the solvent was removed under reduced
pressure. The product was purified by a silica gel column to give 1.7 g
(45%) of the product which was recrystallized from MeOH. Mp
1-(3,4-Dichlorophenyl)-3-isopropylthiourea (17a). The title com-
pound was prepared by treatment of 3,4-dichlorophenyl isothiocy-
anate 16 with isopropylamine as described above. Yield: 86%. Mp
1
1
260ꢀ262 °C. H NMR (DMSO-d6): δ 8.68 (br, 1H), 8.37 (br, 1H),
145ꢀ146 °C. White solid. H NMR (CDCl3): δ 8.44 (s, 1H), 7.47
7.95 (m, 1H), 7.52 (m, 2H), 7.14 (s, 1H), 4.03 (s, 2H), 4.01 (q, J = 6.0
Hz, 1H), 1.19 (s, 3H), 1.17 (s, 3H). 13C NMR (DMSO-d6): δ 182.8,
171.1, 159.2, 139.4, 131.1, 130.4, 124.5, 122.3, 121.7, 120.8, 120.1, 52.3,
43.1, 11.5. IR (neat): 3414, 1703, 1654, and 1476 cmꢀ1. MS (ESI): m/z
328.06 [M þ 1]þ. Anal. (C13H15Cl2N5O) C, H, N, Cl.
(d, J = 8.55 Hz, 1H), 7.34 (d, J = 2.65 Hz, 1H), 7.08 (dd, J = 8.55 Hz, J =
2.65 Hz, 1H), 5.86 (s, 1H), 4.53 (q, J = 6.28 Hz, 1H), 1.26 (s, 3H), 1.24
(s, 3H). 13C NMR (CDCl3): δ 179.0, 135.8, 133.9, 131.6, 130.8, 126.5,
124.0, 47.5, 22.3. IR (neat): 3308, 1583, 1538, and 1474 cmꢀ1. MS (EI):
m/z 264.0 [M]þ.
General Procedure for the Preparation of 9bꢀe. To a mixture of
compound 9a (1.80 g, 5.40 mmol) and DMF (10 mL) was added DMAP
(0.3 g, 2.67 mmol) and Et3N (0.5 mL), followed by addition of
dicarbonate (5.61 mmol) or acyl chloride (5.61 mmol). The reaction
mixture was stirred at 30 °C for 12 h, and the solvent was evaporated to
dryness. The residue was suspended in CH2Cl2 (30 mL) and filtered to
remove the unreacted starting material. The filtrate was evaporated to
dryness and purified by a silica gel column, eluting with hexane/ethyl
acetate (1:1 v/v) mixed solvent. The product was recrystallized from
hexane/EtOAc.
1-tert-Butyl-3-(3,4-dichlorophenyl)thiourea (17b). Title compound
was prepared by treatment of 16 with tert-butylamine. Yield: 80%. Mp
157ꢀ158 °C. White solid. 1H NMR (CDCl3): δ 8.07 (s, 1H), 7.48 (d,
J = 8.54 Hz, 1H), 7.35 (s, 1H), 7.09 (d, J = 8.39 Hz, 1H), 6.07 (s, 1H),
1.52 (s, 9H). 13C NMR (CDCl3): δ 179.1, 136.4, 133.7, 131.4, 130.3,
126.3, 123.9, 54.3, 28.9. IR (neat): 3205, 1558, 1532, and 1473 cmꢀ1
.
MS (EI): m/z 276.0 [M]þ.
1-(3,4-Dichlorophenyl)-3-ethylthiourea (17c). The title compound
was prepared from 16 and ethylamine according to the general
procedure described above. Yield: 87%. Mp 113ꢀ114 °C. White solid.
1H NMR (CDCl3): δ 8.54 (s, 1H), 7.46 (d, J = 8.54 Hz, 1H), 7.28 (d, J =
2.1 Hz, 1H), 7.12 (dd, J = 8.54 Hz, J = 2.1 Hz, 1H), 6.09 (br, 1H), 3.65
(q, J = 6.56 Hz, 2H), 1.21 (t, J = 2.75 Hz, 3H). 13C NMR (CDCl3): δ
180.1, 135.9, 133.7, 131.5, 130.8, 126.6, 124.2, 40.3, 14.2. IR (neat):
3119, 1631, 1535, and 1473 cmꢀ1. MS (EI): m/z 248.0 [M]þ.
1-(Adamantan-1-yl)-3-(3,4-dichlorophenyl)thiourea (17d). The ti-
tle compound was prepared from 16 and adamantylamine. Yield: 87%.
Mp 179ꢀ180 °C. White solid. 1H NMR (CDCl3): δ 9.44 (s, 1H), 7.97
(s, 1H), 7.47 (d, J = 7.76 Hz, 1H), 7.34 (s, 1H), 7.31 (d, J = 8.73 Hz, 1H),
2.20 (m, 6H), 2.02 (m, 3H), 1.61 (m, 6H). 13C NMR (CDCl3): δ 178.8,
140.4, 130.7, 130.3, 125.2, 124.1, 122.9, 54.0, 41.0, 40.5, 40.2, 39.9, 39.6,
39.4, 36.4, 29.4. IR (neat): 3119, 1540, 1500, and 1460 cmꢀ1. MS (EI):
m/z 354.0 [M]þ.
General Procedure for the Preparation of 18aꢀd. Methyl iodide
(3.2 g, 22.5 mmol) was added dropwise to thiourea 17 (15.0 mmol) in
acetone (50 mL) solution. The mixture was stirred at 60 °C for 4 h, and the
solvent was evaporated to dryness under the reduced pressure. The product
was suspended in Et2O, collected, and washed with fresh ether. The white
solid was dissolved in 50 mL of H2O and THF (1:1 v/v) mixed solvent. The
solution was basified with 1.0 M K2CO3 solution to pH 10 and extracted 3
times with 50 mL of CH2Cl2. The extracts were combined, dried over
Na2SO4, and evaporated to dryness to give the product 18 in over 96%
yield. The products are pure enough to be used without further purification.
1-(3,4-Dichlorophenyl)-3-isopropyl-2-methylisothiourea (18a). Yield:
98%. Pale yellow oil. 1H NMR (CDCl3): δ 7.29 (d, J = 8.45 Hz, 1H), 7.00
(d, J = 2.37 Hz, 1H), 6.72 (dd, J = 8.45 Hz, J = 2.39 Hz, 1H), 4.29 (m, 1H),
4.02(m, 1H), 2.06 (s, 3H), 1.22 (s, 3H), 1.20 (s, 3H). 13C NMR (CDCl3):
δ 149.5, 132.3, 130.4, 125.4, 124.1, 121.9, 44.7, 22.9, 14.1. MS (EI): m/z
278 [M]þ.
N-(tert-Butoxycarbonyl)-N0-[1-(3,4-dichlorophenyl)-4-oxo-4,5-di-
hydro-1H-imidazol-2-yl]-N00-isopropylguanidine (9b). Compound 9b
was prepared from 9a and di-tert-butyl dicarbonate. Yield: 48%. Mp
246ꢀ248 °C. White crystals. 1HNMR (CDCl3): δ 12.41 (s, 1H), 8.83
(d, J = 6.27 Hz, 1H), 8.09 (d, J = 2.58 Hz, 1H), 7.39 (d, J = 8.61 Hz, 1H),
7.17 (dd, J = 8.61 Hz, J = 2.58 Hz, 1H), 4.24ꢀ4.26 (m, 1H), 4.20 (s, 2H),
1.52 (s, 9H), 1.31 (s, 3H), 1.30 (s, 3H). 13C NMR (CDCl3): δ 182.4,
170.5, 154.6, 153.4, 137.6, 132.5, 130.1, 127.5, 123.5, 119.5, 84.1, 52.4,
44.0, 28.0, 22.4. IR (neat): 1723, 1606, 1505, and 1152 cmꢀ1. MS (ESI):
m/z 430.0 [M þ 1]þ. Anal. (C18H23Cl2N5O3) C, H, N, Cl.
N-(Ethoxycarbonyl)-N0-[1-(3,4-dichlorophenyl)-4-oxo-4,5-dihydro-
1H-imidazol-2-yl]-N00-isopropylguanidine (9c). Compound 9c was pre-
pared from 9a and ethyl chloroformate. Yield: 50%. Mp 193.5ꢀ195 °C.
White crystals. 1H NMR (CDCl3): 1H NMR (CDCl3): δ 12.9 (br, 1H),
8.76 (br, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.20 (dd,
J = 2.5 Hz, J = 8.8 Hz, 1H), 4.26ꢀ4.25 (m, 5H), 1.35 (m, 9H). 13C NMR
(CDCl3): δ 182.2, 170.6, 154.4, 154.3, 137.6, 132.5, 130.2, 127.7, 123.6,
119.6, 62.9, 52.5, 44.2, 22.4, 14.2. MS (ESI): m/z 400.05 [M þ 1]þ. Anal.
(C16H19Cl2N5O3) C, H, N, Cl.
N-[1-(3,4-Dichlorophenyl)-4-oxo-4,5-dihydro-1H-imidazol-2-yl]-
N0-isopropyl-N00-(trimethylacetyl)guanidine (9d). Compound 9d was
prepared from compound 9a and trimethylacetyl chloride. Yield: 50%.
White crystals. Mp 182ꢀ183 °C. 1H NMR (CDCl3): 13.68 (s, 1H), 9.63
(s, 1H), 8.12 (d, J = 2.37 Hz, 1H), 7.42ꢀ7.39 (d, J = 8.76 Hz, 1H),
7.21ꢀ7.18 (dd, J = 8.76 Hz, J = 2.37 Hz, 1H), 4.29 (m, 1H), 4.21 (s, 2H),
1.34ꢀ1.12 (m, 15H). 13C NMR (CDCl3): 182.1, 181.7, 170.5, 158.1,
154.9, 137.5, 132.5, 130.2, 127.7, 123.5, 119.5, 118.2, 52.5, 44.1, 26.9. IR
(neat): 1731, 1612, 1571, and 1484 cmꢀ1. MS (ESI): m/z 412.40
[M þ 1]þ. Anal. (C18H23Cl2N5O2) C, H, N, Cl.
4529
dx.doi.org/10.1021/jm200111g |J. Med. Chem. 2011, 54, 4523–4535

Journal of Medicinal Chemistry
ARTICLE
1-tert-Butyl-3-(3,4-dichlorophenyl)-2-methylisothiourea (18b). Yield:
99%. Colorless oil. 1H NMR(CDCl3):δ 7.29 (d, J =8.51 Hz, 1H), 7.00 (d,
J = 2.4 Hz, 1H), 6.73 (dd, J = 8.50 Hz, J = 2.4 Hz, 1H), 4.40 (s, 1H), 2.14 (s,
3H), 1.41 (s, 9H). 13C NMR (CDCl3): δ 150.3, 149.3, 132.0, 130.1, 125.0,
123.8, 121.7, 53.3, 28.7. IR (neat): 2995, 1584, 1475, and 1292 cmꢀ1. MS
(EI): m/z 290.0 [M]þ.
1-(3,4-Dichlorophenyl)-3-ethyl-2-methylisothiourea (18c). Yield: 98%.
Colorless oil. 1H NMR (CDCl3): δ 7.29 (d, J = 8.49 Hz, 1H), 7.00 (d, J =
2.40 Hz, 1H), 6.73 (dd, J=8.49Hz, J=2.39Hz, 1H), 4.45(br, 1H), 3.63(q,
J = 6.56 Hz, 2H), 2.28 (s, 3H), 1.21 (t, J = 7.16 Hz, 3H). 13C NMR
(CDCl3): δ 149.4, 132.3, 130.4, 125.6, 124.1, 122.0, 38.0, 14.7, 14.0. IR:
3115, 1605, 1519, and 1468 cmꢀ1. MS (EI): m/z 262.0 [M]þ.
1-(Adamantan-1-yl)-3-(3,4-dichlorophenyl)-2-methylisothiourea
(18d). Yield: 99%. Mp 111ꢀ112 °C. White solid. 1H NMR (CDCl3): δ
7.29 (d, J = 8.49 Hz, 1H), 6.98 (d, J = 2.34 Hz, 1H), 6.74 (dd, J = 8.49 Hz,
J = 2.34 Hz, 1H), 4.30 (s, 1H), 2.14 (s, 3H), 2.07 (m, 9H), 1.66 (m, 6H).
13C NMR (CDCl3): δ 149.3, 132.1, 130.1, 124.9, 123.8, 121.7, 53.9,
41.7, 36.3, 29.5, 15.0. IR (neat): 2910, 1583, 1528, and 1473 cmꢀ1. MS
(EI): m/z 368.0 [M]þ.
General Procedure for the Preparation of Compounds 21a,b. A
reaction mixture consisting of 2-aminoimidazolin-4-one (15b, 1.0 g,
7.08 mmol), 2-methylisourea 20 (10.6 mmol), K2CO3 (1.0 g, 7.08 mmol),
and 1-propanol (50 mL) was refluxed for 12 h and, after cooling to room
temperature, was filtered to remove the K2CO3. The filtrate was
evaporated to dryness under reduced pressure. The crude product was
purified with a silica gel column using CH2Cl2/MeOH (10:1 v/v) as
eluent. The product was further purified by recrystallization from ethyl
acetate/MeOH mixed solvent.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-4-oxo-4,5-dihydro-1H-imi-
dazol-2-yl)pyrrolidine-1-carboxamidine (21a). The title compound
was prepared from 20a and 15b in 50% yield as white crystals. Mp
177ꢀ178 °C. 1H NMR (CDCl3): δ 7.33 (d, J = 8.57 Hz, 1H), 7.14 (d,
J = 2.40 Hz, 1H), 6.89 (dd, J = 8.57 Hz, J = 2.40 Hz, 1H), 4.57 (q, J = 6.70
Hz, 1H), 3.72 (s, 2H), 3.34 (m, 4H), 1.85 (m, 4H), 1.21 (s, 3H), 1.19 (s,
3H). 13C NMR (CDCl3): δ 185.2, 171.0, 156.8, 138.6, 132.8, 130.5,
128.1, 124.9, 122.7, 49.5, 47.1, 44.0, 25.2, 20.5. IR (neat): 3000, 1681,
1555, and 1425 cmꢀ1. MS (ESI): m/z 382.01 [M þ 1]þ. Anal.
(C17H21Cl2N5O) C, H, N, Cl.
General Procedure for the Preparation of Compounds 19aꢀb. 3,4-
Dichlorophenyl isothiocyanate (16) (4.0 g, 19.6 mmol) in 10.0 mL of
CH2Cl2 was added dropwise to cyclic amine (29.4 mmol) in CH2Cl2
(20 mL). The mixture was stirred at room temperature until the starting
material isothiocyanate 16 was consumed. The crude product was
suspended in Et2O (50 mL), and the solid product was collected and
dried. The compound was pure enough for the next step synthesis
without further purification.
N-(3,4-Dichlorophenyl)-N0-(1-isopropyl-4-oxo-4,5-dihydro-1H-imi-
dazol-2-yl)piperidine-1-carboxamidine (21b). The title compound
was prepared from 20b and 15b in 48% yield as white crystals. Mp
195ꢀ197 °C. 1H NMR (CDCl3): δ 7.33 (d, J = 8.65 Hz, 1H), 7.14 (d,
J = 2.55 Hz, 1H), 6.91 (dd, J = 8.65 Hz, J = 2.55 Hz, 1H), 4.57 (q, J = 6.74
Hz, 1H), 3.73 (s, 2H), 3.41 (m, 4H), 1.60 (m, 6H), 1.22 (s, 3H), 1.19 (s,
3H). 13C NMR (CDCl3): δ 185.1, 171.6, 157.8, 139.5, 133.1, 130.8,
127.3, 122.4, 120.3, 48.3, 47.1, 44.2, 25.5, 24.2, 20.6. IR (neat): 1711,
1632, 1502, and 1438 cmꢀ1. MS (ESI): m/z 396.1 [M þ 1]þ. Anal.
(C18H23Cl2N5O) C, H, N, Cl.
Pyrrolidine-1-carbothioic Acid (3,4-Dichlorophenyl)amide (19a).
1
Yield: 92%. Mp 187ꢀ188 °C. White solid. H NMR (DMSO-d6): δ
9.12 (s, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.48 (m, 2H), 3.64 (m, 4H), 1.82
(m, 4H). 13C NMR (DMSO-d6): δ 177.4, 141.3, 130.2, 129.9, 126.8,
126.1, 125.5, 65.3, 44.9, 24.1, 15.6. IR (neat): 3400, 2917, 1579, 1531,
1422, and 1306 cmꢀ1. MS (EI): m/z 274.0 [M]þ.
Synthesis of 2-Amino-1-(3,4-dichlorophenyl)-1,5-dihydroimidazol-
4-one (24). 3,4-Dichlorophenylcyanamide (22, 3.1 g, 16.5 mmol) in
20 mL of EtOH was added sodium metal (0.38 g, 16.5 mmol). The
mixture was stirred at room temperature until sodium was consumed.
The solvent was evaporated to dryness, and the residue was dissolved in
50 mL of CH3CN. Chloroacetamide (1.7 g, 18.1 mmol) was added to
the solution, and the mixture was refluxed for 5 h. When the mixture was
cooled, the white solid precipitates were collected, washed successively
with H2O and acetone, and dried over Na2SO4 to give 2.7 g (67%) of
Piperidine-1-carbothioic Acid (3,4-Dichlorophenyl)amide (19b).
1
Yield 98%. Mp 199ꢀ200 °C. White solid. H NMR (DMSO-d6): δ
9.31 (s, 1H), 7.59 (d, J = 2.19 Hz, 1H), 7.47 (d, J = 8.69 Hz, 1H), 7.29
(dd, J = 8.69 Hz, J = 2.19 Hz, 1H), 3.85 (m, 4H), 1.56 (m, 6H). 13C
NMR (DMSO-d6): δ 180.6, 142.0, 130.3, 130.0, 126.4, 125.9, 125.1,
49.8, 25.9, 24.2. IR (neat): 3400, 2917, 1579, 1531, 1422, and
1306 cmꢀ1. MS (EI): m/z 288.8 [M]þ.
1
compound 24. Mp 285ꢀ287 °C. H NMR (DMSO-d6): δ 7.66 (m,
4H), 7.35 (m, 1H), 4.21(s, 2H). 13C NMR (DMSO-d6): δ 184.1, 169.5,
137.9, 132.1, 131.6, 127.0, 126.7, 125.0, 56.9. IR (neat): 3400, 1570,
1531, and 1420 cmꢀ1. MS (ESI): m/z 244.1 [M þ 1]þ.
General Procedure for the Preparation of 20a,b. Methyl iodide (3.9 g,
27.30 mmol) was added dropwise to thiourea 19 (18.2 mmol) and
acetone (50 mL) solution. The mixture was stirred at 60 °C for 4 h, and
the solvent was evaporated to dryness. The product was suspended in
Et2O, collected, and washed with fresh Et2O. The white solid was
dissolved in 50 mL of H2O and THF (1:1 v/v) mixed solvent. The
solution was basified with 1.0 M K2CO3 solution to pH 10 and extracted
3 times with 50 mL of CH2Cl2. The extracts were combined, dried over
Na2SO4, and evaporated to dryness to give the product 20 in over 96%
yield and was used without further purification.
N-(3,4-Dichlorophenyl)pyrrolidine-1-carboximidothioic Acid Methyl
Ester (20a). The title compound was prepared from 19a and CH3I in
90% yield as a colorless oil. 1H NMR (CDCl3): δ 7.27 (d, J = 8.56 Hz,
1H), 7.05 (d, J = 2.44 Hz, 1H), 6.79 (dd, J = 8.56 Hz, J = 2.44 Hz, 1H),
3.52 (m, 4H), 2.05 (s, 3H), 1.92 (m, 4H). 13C NMR (CDCl3): δ 154.2,
149.8, 132.0, 130.0, 123.8, 123.2, 121.4, 49.3, 25.3, 14.0. IR (neat): 3015,
1585, 1458, and 1387 cmꢀ1. MS (EI): m/z 288.0 [M]þ.
N-(3,4-Dichlorophenyl)piperidine-1-carboximidothioic Acid Methyl
Ester (20b). Compound 20b was prepared from 19b and CH3I in 93%
yield as a colorless oil. 1H NMR (DMSO-d6): δ 7.70 (m, 2H), 7.31 (m,
1H), 3.75 (m, 4H), 2.42 (s, 3H), 1.68 (m, 6H). 13C NMR (DMSO-
d6): δ 168.7, 139.5, 132.0, 131.6, 128.8, 125.4, 123.9, 53.1, 25.8, 23.3,
17.0. IR (neat): 3015, 1583, 1448, and 1238 cmꢀ1. MS (EI): m/z
302.0 [M]þ.
General Procedure for the Preparation of 25aꢀc. 2-Amino-1-(3,4-
dichlorophenyl)-1,5-dihydroimidazol-4-one (24, 4.0 g, 16.39 mmol),
K2CO3 (1.3 g, 17.6 mmol), and 2-methylisothiourea 18 (25 mmol) were
suspended in 1-propanol (25 mL) and DMF (5 mL) mixed solvent and
was refluxed at 110 °C for 48 h. The solvent was removed under the
reduced pressure. The crude product was dissolved in 20 mL of CH2Cl2
and washed with 15 mL of water. The organic layer was dried over
Na2SO4 and evaporated to dryness. The product was purified with a
silica gel column, followed by recrystallization from MeOH.
N-(3,4-Dichlorophenyl)-N0-[1-(3,4-dichlorophenyl)-4-oxo-4,5-dihy-
dro-1H-imidazol-2-yl]-N00-isopropylguanidine (25a). The title com-
pound was prepared from 24 and 18a in 70% yield as white crystals. Mp
1
208ꢀ210 °C. H NMR CDCl3): δ 11.98 (br s, 1H), 8.19 (br s, 1H),
7.35ꢀ7.51 (m, 3H), 7.08ꢀ7.22 (m, 3H), 4.86 (s, 1H), 4.27 (m, 1H),
4.18 (s, 2H), 1.26 (s, 3H), 1.24 (s, 3H). 13C NMR (CDCl3): δ 182.6,
170.7, 156.2, 137.8, 135.0, 134.8, 132.4, 131.7, 127.8, 127.2, 125.0, 123.1,
119.2, 52.2, 44.3, 22.8. IR (neat): 1700, 1574, 1470, 1384. MS (EI): m/z
473.0 [M]þ. Anal. (C19H17Cl4N5O) C, H, N, Cl.
N-tert-Butyl-N0-(3,4-dichlorophenyl)-N00-[1-(3,4-dichlorophenyl)-4-
oxo-4,5-dihydro-1H-imidazol-2-yl]guanidine (25b). The title com-
pound was prepared from 24 and 18b in 65% yield as white crystals.
Mp 167ꢀ168 °C. 1H NMR (CDCl3): δ 12.01 (br s, 1H), 7.88 (s, 1H),
4530
dx.doi.org/10.1021/jm200111g |J. Med. Chem. 2011, 54, 4523–4535

Journal of Medicinal Chemistry
ARTICLE
Table 1. Causal Prophylactic Activity, Metabolic Stability, and in Vitro Potencya
metab stability t1/2 (min)
P. falciparum IC50 (μg/mL)
compd
oral doseb (mg/kg)
human
mouse
EE P. berghei
inactive
D6
TM
W2
cLogP
8a
8b
160
160
40
>60
>60
>60
40
3.42
3.42
3.31
3.31
2.57
2.57
1.35
2.23
inactive
DP: 6d (1/5)
CP: (2/5)
8c
160
>60
<10
>60
<10
2.75
1.26
>5.00
2.54
1.01
1.72
2.58
DP: 2-3d (1/5)
CP: (5/5)
320
160
8d
CP: (4/5)
DP: 1d (1/5)
DP: 2-3d (2/5)
CP: (5/5)
40
8e
8f
160
160
320
12.77
<10
15.76
<10
1.60
2.90
2.86
>5
0.74
>5.0
2.80
4.35
4.70
3.66
CP: (5/5)
>5.0
8g
34.3
>60
CP: (2/5)
DP: 2-3d (2/5)
DP: 2-11d (2/5)
inactive: (3/5)
CP: (3/5)
160
160
8h
8i
>60
14
>60
5
3.19
0.06
>1.28
0.16
4.90
0.09
3.90
4.61
DP: 3d (1/5)
inactive
40
320
CP: (4/5)
DP: 3d (1/5)
DP: 2-5d (4/5)
inactive: (1/5)
CP: (5/5)
160
8j
320
160
53
17
1.65
>5.0
0.37
3.94
CP: (4/5)
DP: 1d (1/5)
inactive
14
8k
8l
160
160
>60
>60
>12
3.0
>12
5.2
>12
1.49
2.27
2.51
>5.0
5.83
4.29
3.59
5.94
3.06
39.72
>60
1.72
>60
inactive
3.38
1.55
>5.0
3.01
2.60
>5.0
8m
8n
160
320
160
20.90
>60
22.33
49.82
inactive
CP: (4/5)
CP: (1/5)
DP: 2d (4/5)
9a
9b
9c
9d
320
320
320
320
53.74
>60
13.51
>60
1.1
2.19
0.90
0.70
0.63
0.25
4.87
1.4
0.56
0.15
4.65
3.52
2.83
3.07
>60
>60
inactive
40.45
13.50
CP: (4/5)
DP: 2d (1/5)
CP: (3/5)
DP: 2-4d (2/5)
CP: (3/5)
DP: 2d (2/5)
CP: (1/5)
DP: 2d (4/5)
inactive
160
320
160
9e
59.2
>60
>60
>60
0.78
>5.0
1.55
2.2
3.78
2.76
21a
>5.0
2.83
21b
25a
160
160
320
28
18
>5.0
1.66
1.98
2.86
3.32
5.87
39.21
27.25
inactive
DP: 1-2d (4/5)
inactive: (1/5)
inactive
25b
25c
160
160
>60
>60
>60
>60
2.45
2.6
2.58
>5.0
6.22
5.53
inactive
>5.0
a TM = TM91C235; EE P. berghei = sporozoites infected mouse model; DP = delayed in patency; CP = causal prophylaxis. b The dose unit is
(mg/kg)/day, and mice were treated for 3 consecutive days on days ꢀ1, 0, 1.
4531
dx.doi.org/10.1021/jm200111g |J. Med. Chem. 2011, 54, 4523–4535

Journal of Medicinal Chemistry
ARTICLE
Table 2. Causal Prophylactive Activity of Deoxo-IZ
Analoguesa
po. Those given sc were suspended in peanut oil. Each compound was
prepared at different dose levels. Compounds were administered either
po or sc to mice once a day for 3 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 biweekly
for 31 days. Mice alive on day 31 with no parasites found in any blood
films were considered protected. Table 1 summarizes the test results of
the new compounds.
(iv) Assessment of Causal Prophylactic/Radical Curative Activities
in Rhesus Monkeys. The causal prophylactic and radical curative
antimalarial activity of the new derivatives 8e, 8h, and 8j were assessed
in P. cynomolgi sporozoites challenged Rhesus monkey model. Detailed
procedures of sporozoite harvest and drug tests are described in the
previous reports.16ꢀ20 The results are shown in Table 2. Assessment of
radical curative activity of the test compounds was carried out using the
monkeys' developed parasitemia during the causal prophylactic
experiments when the test compounds showed no or weak activity.
Monkeys were treated with chloroquine (10 (mg/kg)/day) orally
for 7 consecutive days and the test compounds by im dosing for
3 consecutive days after the parasitemia level reached 5000 parasites/
mm3. Chloroquine at 10 (mg/kg)/day ꢁ 7 days eliminates the blood
stage parasites but not the liver stage hypnozoites. Compounds with
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, 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 is expected in the control group. Relapse in the treated group
indicates failure of the test compounds. Monkeys showing no relapse
after 100 days are considered radically cured. Relapses of the control
monkeys are treated with chloroquine once daily for 7 days and observed
for second relapse. Relapse in experimental animals and the second
relapse of the control monkeys are treated with the standard 7-day oral
CQ and PQ (1.78 mg of base/kg). After standard treatment, blood
smears were monitored daily for 4 consecutive days and 2 times weekly
for 2 weeks. The results are shown in Table 3.
results
dose
(mg/kg/ days
first day
delayed
drug
day)
treated
vehicle route patency patency (day)
vehicle
N/A
ꢀ1, 0, 1 HECT po
8
8
valid control
valid control
PQ
TQ
1.78
ꢀ1, 0, 1 MC
po
po
13
13
11
10
11
12
12
10
13
12
8
5
5
3
2
3
4
4
2
5
4
0
0
0.316 ꢀ1, 0, 1 MC
Malarone 14
ꢀ1, 0, 1 HECT po
ꢀ1, 0, 1 HECT po
ꢀ1, 0, 1 HECT po
ꢀ1, 0, 1 HECT po
8e
8j
50
50
50
8h
8
a PQ = primaquine; TQ = tafenoquine; DP = delayed parasitemia
patency; Malarone = atovaquone (10 mg/kg) þ proguanil (4 mg/kg).
7.51ꢀ7.48 (d, J = 8.50 Hz, 1H), 7.42ꢀ7.35 (m, 2H), 7.19 (d, J = 2.41 Hz,
1H), 7.09ꢀ7.06 (dd, J = 8.50 Hz, J = 2.41 Hz, 1H), 4.96 (br s, 1H), 4.20
(s, 2H), 1.37 (s, 9H). 13C NMR (CDCl3): δ 183.0, 170.8, 156.4, 137.6,
135.4, 134.0, 132.5, 131.6, 131.3, 130.2, 128.4, 127.6, 125.3, 124.8, 121.5,
53.1, 53.0, 29.5. IR (neat): 1698, 1567, 1470, and 1390 cmꢀ1. MS (ESI):
m/z 487.93 [M þ 1]þ. Anal. (C20H19Cl4N5O) C, H, N, Cl.
N-(3,4-Dichlorophenyl)-N0-[1-(3,4-dichlorophenyl)-4-oxo-4,5-dihy-
dro-1H-imidazol-2-yl]-N00-ethylguanidine (25c). The title compound
was prepared from 24 and 18c in 66% yield as white crystals. Mp
229ꢀ231 °C 1H NMR (DMSO-d6): δ 10.30 (s, 1H), 9.21 (s, 1H), 7.87
(d, J = 8.50 Hz, 1H), 7.58ꢀ7.64 (m, 3H), 7.29ꢀ7.32 (m, 3H), 4.21 (s,
2H), 3.36 (q, J = 6.70 Hz, 2H), 1.14 (t, J = 6.65 Hz, 3H). 13C NMR
(DMSO-d6): δ 182.0, 170.1, 156.7, 138.4, 137.9, 130.7, 130.2, 139.7,
127.3, 125.5, 124.6, 122.0, 120.0, 52.0, 36.4, 14.3. IR (neat): 1699, 1558,
1474, and 1395 cmꢀ1. MS (ESI): m/z 460.0 [M þ 1]þ. Anal.
(C18H15Cl4N5O) C, H, N, Cl.
’ RESULTS AND DISCUSSION
B. Biological Studies. (i) Assessment of Metabolic Stability. The
metabolic stability assay was performed in a 96-well plate on a TECAN
Genesis robotic sample processor following WRAIR SOP SP 01-02.
Samples wereanalyzedbyLCꢀMS/MS using fast LCgradient orisocratic
methods. The parent drug was quantified using external calibration and
plots of parent drug response vs amount. The results are shownin Table 1.
(ii) In Vitro Antimalarial Studies. The in vitro assays were conducted
by using a modification of the semiautomated microdilution techniques
of Desjardins et al. and Chulay et al.27,28 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.29 The results are shown in
Table 1.
(iii) Assessment of Causal Prophylactic Activity in Mice. New
compounds were assessed for their causal prophylactic activity in
exoerythrocytic (EE) mouse model using sporozoites of P. berghei.
The procedures have been previously described.18ꢀ20 Briefly, each
compound was ground with a mortar and pestle and suspended in
hydroxyethylcellulose and Tween 80 for compounds to be administered
The test results of metabolic stability, 50% inhibitory con-
centration (IC50) in three clones of P. falciparum (D-6, W-2, and
TM91C235), and antimalarial activity in P. berghei sporozoites
infected mice of the new compounds are summarized in Table 1.
Calculated cLogP values were also included in the table to
facilitate the examination of the relationship between lipophyli-
city and antimalarial activity of the new compounds. The results
indicated that the cLogP values of the new compounds with good
in vitro and in vivo activity are well within 3ꢀ4.5, a value shared
by the majority of the exiting antimalarial drugs. As was observed
with imidazolinedione (IZ) derivatives (1ꢀ6), the deoxo-IZ
analogues (8, 9) prepared in this study exhibited only weak in
vitro activity against blood stage malaria, P. falciparum, except 8j,
8h, 9b, and 9c, which showed moderate activity with IC50 less
than 1 μg/mL.
The results also indicated that the deoxo-IZ compounds (8, 9)
are in general metabolically unstable, especially analogues with
good prophylactic activity, such as 8d, 8e, 8f, 8i, and 8j. To
identify the active metabolites, extensive metabolic studies of the
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Table 3. Radical Curative Activity of Deoxo-IZ Analogues
drug 2
drug 1
dose ((mg/kg)/day)
route
no. days delayed in relapse after treatment
11
chloroquine
(10 mg/kg/day)
po for 7 days
none
11
8e
30 ꢁ 3 days
50 ꢁ 7 days
30 ꢁ 3 days
30 ꢁ 3 days
1.78 ꢁ 7 days
im
po
im
im
po
radical cure
radical cure
28
radical cure
8j
42
38
8h
PQ
16
15
radical cure
Scheme 4. Major Metabolic Pathway of Imidazolin-4-one Derivatives
most active compounds 8e and 8f in mice were conducted.
Preliminary results indicated that these compounds were meta-
bolized extensively to yield a host of metabolites in mice. The
major metabolites,31 however, were identified as the correspond-
ing hydroxyimidazolinones (II) and imidazolinediones (III) as
shown in Scheme 4. For example, compounds 8e and 8j were
metabolized to the corresponding IZ derivatives 8e0 and 8j0,
respectively. The LC/MS results of compounds 8e0 and 8j0 are
identical to the results of the known compound 3 and the N,N0-
diisopropyl-IZ analogue,32 respectively, in LC retention time and
mass spectrum. Since deoxo-IZ compounds 8e and 8j are
metabolically vulnerable and their major metabolites (8e0
and 8j0) are potent antimalarials, most likely the new class of
deoxo-IZ (I) may act as prodrugs of the IZ compounds (III) as
shown in Scheme 4.
Rhesus monkeys infected with sporozoites of P. cynomolgi to
assess their casual prophylactic and radical curative activities.
Three clinical drugs, PQ, TQ, and Malarone, were also tested in
the same experiment to determine their benchmark dosage as
prophylactic drugs and to serve as positive control of the
experiment. The primaquine dose of 1.78 mg/kg ꢁ 3 was used,
as it cured 100% of the relapsed monkeys when in combination
use with 10 mg/kg chloroquine for 7 days by oral route. The
tafenoquine dose of 0.316 mg/kg was based on the report of
Puri.30 The Malarone dose (14 mg/kg) was derived from the
recommended human dose. The results showed that both 8e and
8j showed comparable activity as that of PQ, TQ, and Malarone
and delayed patency of the treated monkeys 2ꢀ5 days longer
than the untreated control by oral administration (Table 2). As
was reported earlier, the IZ class of compounds (1, 3, and 5)
showed good casual prophylactic activity in Rhesus only by im
but not by oral dosing. Although 2ꢀ3 days of delay in patency is
far from impressive, this is the first time significant oral activity of
this class of compounds was observed in Rhesus monkeys.
Furthermore, the most commonly used clinical drugs, PQ, TQ
and Malarone, also did not show much activity in the same test,
suggesting the need to amend the current protocol used to assess
the casual prophylactic activity in Rhesus. Compound 8h is a
carboxamide derivative that exhibited good casual prophylactic
activity in mice, but no activity was observed in the same Rhesus
test by im at 30 mg/kg ꢁ 3. The result further confirmed our
previous observations that carboxamide-IZ derivatives, such as 5
and 6, are generally less active than the corresponding N-alkyl or
N-carbamate derivatives in the Rhesus test.17,18,32
The SAR of the new compounds indicated that isopropyl is
a preferred substituent at R1 and R2 of deoxo compounds
8 and 9 over the other groups. Among the nine compounds
(8b, 8e, 8g, 8h, 8i, 8j, 8k, 8l, and 8m) with R1 being isopropyl,
six (8e, 8g, 8h, 8i, 8j, and 8l) showed curative activity in the
prophylactic test in mice. Compound 8j, with both R1 and R2
being the isopropyl group, showed the best activity among the
compounds tested, with 5/5 and 4/5 mice protected at doses of
320 and 160 mg/kg ꢁ 3, respectively. Besides the isopropyl group,
t-Boc also appears to be a favorable substituent at R2, as 8d, 8e,
and 8f all provided good protection to the infected mice at
160 mg/kg ꢁ 3 dosing.
On the basis of promising efficacy results in the mouse model,
compounds 8e, 8h, and 8j were selected for further testing in
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Journal of Medicinal Chemistry
ARTICLE
Monkeys failed in the causal tests were used for assessment of
radical curative activity of 8e, 8h, and 8j according to the method
described above. The results are shown in Table 3. Compound
8e at 30 mg/kg ꢁ 3 by im, combined with 10 mg/kg of CQ by
oral, showed complete cure of both treated monkeys. No cure
was observed for monkeys treated with 8j and 8h, but the relapse
time was delayed from 11 days for untreated control to 40 days
and 15.5 days for monkeys treated with 8j and 8h, respectively.
Compound 8e, the most active among the three tested, was
further evaluated for oral radical curative activity at a dose of 50
(mg/kg)/day for 7 consecutive days. One of the treated monkeys
was cured and the other was protected for 28 days. PQ at
1.78 mg/kg/day in combination with 10 mg/kg of CQ for 7
consecutive days by oral administration results in a complete
radical cure.
The Rhesus PK data revealed that t1/2 of compound 3 is much
longer when the drug was administered by im (∼70.5 h) than by
oral (8.9 h) dosing, likely because of the depot effect of the drug at
the injection sites.31 Although absorption factors remain unknown
via im route, a compound with a half-life of 70.5 h would take
roughly 14 days to clear 97% of the absorbed dose. Our previous
studies also indicated that compound 3 showed good protective
activity in Rhesus by im, but not by oral dosing.17ꢀ19 Further PK
study showed the poor oral activity of 3 was not due to poor oral
absorption. In fact, compound 3 was well absorbed by oral route;
peak absorption of a 60 mg/kg dose given orally was nearly 10-fold
higher than 30 mg/kg dose by im route. The Rhesus PK and the
efficacy data suggested that long plasma half-life of the drug is critical
for its activity against liver stage malaria. Furthermore, analysis of the
relative presence of metabolites indicated that compound 3 is the
major species, comprising 86% and 96% of measurable analytes at
24 h via the oral and im routes, respectively. A small amount
(4ꢀ10%) of the metabolites is the original lead compound 1 and a
very small amount (<0.5%) is metabolized to s-triazenes (7) at 24 h,
suggesting that the parent compound 3 is the active species.31 As the
major metabolite of the deoxo-IZ (I) is its corresponding IZ
derivative III (Scheme 4) and the latter is highly active in mouse
and monkey models, the deoxo-IZ (I) analogues prepared in this
study may act as prodrugs of the corresponding IZ derivative (III).
Notes
This manuscript has been reviewed by Walter Reed Army
Institute of Research; Walter Reed Army Institute of Research
has no objection to its presentation and/or publication. The
opinions or assertions contained herein are the private views of
the authors and are not to be construed as official or as reflecting
the views of the Department of the Army or the Department of
Defense.
’ ACKNOWLEDGMENT
This research was conducted in compliance with the Animal
Welfare Act and other federal statutes and regulations relating to
animals and to 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 (Grant A40191_09_WR), U.S. Army Medical
Research and Material Command, Department of Defense, U.S.,
Peer Reviewed Medical Research Program (PRMRP) (Grant
PR054609), and Medicines for Malaria Venture (MMV 04/
0013), Geneva, Switzerland.
’ ABBREVIATIONS USED
PQ, primaquine; TQ, tafenoquine; DP, delayed parasitemia pa-
tency; Malarone, atovaquone (10 mg/kg) þ proguanil (4 mg/kg);
IZ, guanidylimidazolinedione; G6PD, glucose 6-phosphate
dehydrogenase
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’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis data. This
b
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 301-319-9084. Fax: 301-319-9449. E-mail: ai.lin@
us.army.mil.
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