Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

Article abstract of DOI:10.1016/j.bmc.2004.03.054

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1, 2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5days at 50mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.

Full text of DOI:10.1016/j.bmc.2004.03.054

Bioorganic & Medicinal Chemistry 12 (2004) 2815–2824
Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-
1,2,4-oxadiazoles
Denise M. Cottrell,a Jeffrey Capers,a Manar M. Salem,a Kate DeLuca-Fradley,b
Simon L. Croftb and Karl A. Werbovetza,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
500 West 12th Avenue, Columbus, OH 43210, USA
bDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
Received 24 November 2003; revised 19 March 2004; accepted 24 March 2004
Available online 30 April 2004
Abstract—A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity
against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with
acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium
thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypan-
osoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3
prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanato-
methyl)-1,2,4-oxadiazole (compound 4h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a
murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five
daily i.v. doses at 5 mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thio-
cyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
maintained by the World Health Organization (see
http://www.who.int/inf-fs/en/fact116.html and http://
www.who.int/inf-fs/en/fact259.html). Unfortunately, the
drugs that are commonly used against leishmaniasis and
African trypanosomiasis are administered by injection,
are moderately to extremely toxic, and have been com-
promised by the development of resistance in some areas
(see Refs. 1 and 2 for recent reviews). The registration of
miltefosine in India as the first oral treatment for vis-
ceral leishmaniasis3 and ongoing clinical trials for the
orally available pentamidine analog DB289 as a candi-
date for the treatment of early stage African trypano-
somiasis4 provide hope that improved drugs for these
diseases may soon be within reach. The widespread
utility of these agents is not yet established, however,
and the chemotherapeutic armamentarium against
kinetoplastid parasites remains limited. New drug can-
didates are clearly needed in the fight against these
pathogens.
Leishmaniasis and African trypanosomiasis continue to
place a heavy burden on millions of people in developing
nations. Both are vector-borne diseases caused by pro-
tozoan parasites of the phylogenetic order Kinetoplast-
ida. Depending on the causative Leishmania species,
leishmaniasis presents as a spectrum of disease ranging
from self-resolving cutaneous infections to life-threat-
ening visceral manifestations. Trypanosoma brucei rho-
desiense and Trypanosoma brucei gambiense parasites
give rise to acute and chronic central nervous system
infections, respectively, that are ultimately fatal in the
absence of treatment. Further information regarding the
transmission, symptoms, geographical distribution, and
prevalence of these diseases can be found on websites
Keywords: Leishmania; African trypanosomes; Chemotherapy;
Thiocyanate; 1,2,4-Oxadiazole.
Recent work in our laboratory has focused on the
investigation of tubulin as an antikinetoplastid drug
* Corresponding author. Tel.: +1-614-292-5499; fax: +1-614-292-2435;
e-mail: werbovetz.1@osu.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.054

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D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
target.5;6 In the context of these studies, we found that
an oxadiazole-containing aromatic thiocyanate from the
Walter Reed Chemical Inventory, 3-(4-chlorophenyl)-5-
(thiocyanatomethyl)-1,2,4-oxadiazole (WR85915), pos-
sessed intriguing antileishmanial activity.7 This com-
pound blocked the assembly of leishmanial tubulin in
vitro, presumably by binding covalently to one or more
sulfhydryl residues of the protein,8 and inhibited the
growth of L. donovani axenic amastigote-like parasites
by 50% at a concentration of 4.3 lM.7 Cellular studies
with WR85915 suggested that microtubule disruption
was not the primary mechanism of action of this com-
pound in Leishmania, however, indicating that one or
more other targets in the parasite are affected.7 An
independent report appearing at about the same time as
our work also demonstrated that three simple aromatic
thiocyanates possess micromolar activity against the
related kinetoplastid parasite Trypanosoma cruzi, the
causative agent of Chagas’ disease.9 Antimonial and
arsenical drugs, which have long been the mainstays of
antikinetoplastid chemotherapy, react readily with
sulfhydryl residues in their +3 oxidation states. Wild-
type L. donovani amastigotes reduce Sb(V) to the thiol-
reactive Sb(III) species, but organisms resistant to the
Sb(V)-containing antileishmanial drug Pentostam do
not perform this reduction.10 Given the known activity
of sulfhydryl-reactive compounds against kinetoplastid
parasites and the promising in vitro activity of aromatic
thiocyanates against these organisms, we chose to pre-
pare analogs of the oxadiazole-containing thiocyanate
WR85915 and examine their activity against Leishmania
and African trypanosomes.
Inclusion of 8-hydroxyquinoline in these reactions
decreased the fraction of the unwanted benzamide side
product, presumably by chelating trace metal ions as
suggested by Yamanaka et al.11 Benzamidoximes 2a–h
were converted to 3-aryl-5-halomethyl oxadiazoles 3a–h
in a two-step reaction sequence. Treatment of 2a–h with
either chloroacetyl chloride, bromoacetyl bromide, or
bromoacetyl chloride in the presence of equimolar
potassium carbonate in acetone solvent afforded O-acyl
adducts that were subsequently converted to the
halomethyl oxadiazoles in good to excellent overall
yields by heating at reflux in toluene.12 Procedures
employing chloroacetyl chloride or bromoacetyl bro-
mide were preferable to those using bromoacetyl chlo-
ride because exchange of chlorine for bromine led to the
formation of a mixture of bromomethyl and chloro-
methyl oxadiazoles. Halomethyl oxadazoles 3a–h were
transformed into the corresponding methyl thiocyanates
4a–h in fair to good yield by treatment of 3a–h with
ammonium thiocyanate in DMF. This protocol was
based on previous procedures where thiocyanatomethyl
oxadiazoles were prepared from halomethyl oxadiazoles
using potassium thiocyanate or ammonium thiocyanate
in DMSO.13;14 5-Alkyl oxadiazoles were synthesized as
shown in Scheme 2. Transformation of benzamidoximes
2a–c into the 3-aryl-5-alkyl-1,2,4-oxadiazoles 5a–f was
readily accomplished by treatment of 2a–c with acetyl or
isobutyryl chloride in pyridine.15
2.2. Antiparasitic activity
We first investigated the antiparasitic activities of the
5-thiocyanotomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiaz-
oles against Leishmania donovani axenic amastigote-like
parasites in vitro. In the series of 5-thiocyanotomethyl-
3-aryl-1,2,4-oxadiazoles, different substituents were
introduced on the 3-aryl ring in order to assess the
effects of lipophilicity, steric bulk, and electronic factors
on antiparasitic activity at that site. The thiocyanate-
containing compounds 4a–h displayed antileishmanial
IC50 values at concentrations ranging from 2.3 to 12 lM.
Although this range of IC50 values is rather narrow, the
2. Results and discussion
2.1. Chemistry
The desired thiocyanates were prepared as outlined in
Scheme 1. Conversion of 1a–h into benzamidoximes 2a–
h was accomplished by treatment of the benzonitriles
with hydroxylamine hydrochloride in ethanol in the
presence of the chelating agent 8-hydroxyquinoline.11
OH
O
N
N
N
CN
i
X
NH2
ii, iii
R1
R3
R1
R3
R1
R3
R2
3a-h
1 = R2 = R3 = H, X = Br
b: R1 = Cl, R2 = R3 = H, X = Br
c: R1 = OCH3, R2 = R3 = H, X = Cl
d: R1 = CH3, R2 = R3 = H, X = Cl
e: R1 = F, R2 = R3 = H, X = Cl
f: R1 = R3 = H, R2 = Cl, X = Cl
g: R1 = R2 = H, R3 = Cl, X = Cl
h: R1 = R2 = Cl, R3 = H, X = Br
R2
2a-h
R2
1a-h
O
a:
R
N
N
SCN
iv
R1
R3
R2
4a-h
Scheme 1. Reagents and conditions: (i) hydroxylamine hydrochloride, 8-hydroxyquinoline, EtOH; (ii) haloacetyl halide, K2CO3, acetone; (iii)
toluene, reflux; (iv) ammonium thiocyanate, DMF.

D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
2817
OH
O
N
5a: R1 = H, R2 = CH3
N
N
R2
5b: R1 = Cl, R2 = CH3
NH2
5c: R1 = OCH3, R2 = CH3
5d: R1 = H, R2 = CH(CH3)2
5e: R1 = Cl, R2 = CH(CH3)2
5f: R1 = OCH3, R2 = CH(CH3)2
i
R1
R1
2a-c
5a-f
Scheme 2. Reagents and conditions: (i) acetyl or isobutyryl chloride, pyridine.
order of antileishmanial activity in the series 4h (3,4
di-Cl) > 4b (4-Cl) > 4d (4-CH3) > 4c (4-OCH3) > 4a (4-H)
suggests that greater lipophilicity in the 3-aryl ring
augments antileishmanial potency in this group of
thiocyanate-containing oxadiazoles.16 The in vitro
antileishmanial activities of 4b, 4e, and 4h approach that
of the clinically useful antileishmanial drug pentamidine,
indicating that these aromatic thiocyanates were worthy
of further investigation. Oxadiazoles bearing 5-alkyl
substitutions (5a–f) were ineffective against Leishmania,
strongly suggesting that the 5-thiocyanatomethyl group
is essential for antikinetoplastid activity in this group of
molecules. Aromatic thiocyanates 4a–h were also tested
against bloodstream-form Trypanosoma brucei. Little
difference in antitrypanosomal potency was observed
among the eight compounds, with IC50 values ranging
from 5.2 to 7.4 lM. As with L. donovani, compounds
5a–f exhibited weaker activity against T. brucei. Al-
though 4a–h affect the growth of both African trypano-
somes and Leishmania at comparable concentrations
in vitro, clinically effective antitrypanosomal drugs such
as pentamidine and suramin display in vitro activity at
nanomolar concentrations (see Table 1). Subsequent
studies thus focused on the antileishmanial potential of
the 5-thiocyanatomethyl-3-aryl-1,2,4-oxadiazoles.
macrophage cell line, respectively. While compounds 4b
and 4d displayed modest selectivity against PC3 prostate
cancer cells, being 3.8-fold and 2.9-fold more potent
against L. donovani, compounds 4f and 4h displayed less
than 2-fold selectivity compared to this cancer cell line.
When the two most active agents against Leishmania
were examined against African Green Monkey kidney
(Vero) cells, greater selectivity was observed (9.1-fold for
4b and 30-fold for 4h). Compound 4b, which displayed
modest but consistent selectivity across the three cell
lines in vitro, was chosen for further antileishmanial
evaluation in an intracellular model employing L. mexi-
cana-infected J774 macrophages (Table 2). Although the
IC50 value of 4b against J774 macrophages is reported as
29 lM in Table 1, 50 lM concentrations of 4b could be
employed in this intracellular antileishmanial assay
without obvious toxicity to the mammalian cells because
a 50-fold higher starting concentration of macrophages
is used in the intracellular assay. Compared to controls,
parasite burdens were decreased by about half at 50 lM
4b and by approximately one-third at 25 lM 4b, while
12.5 lM concentrations of this compound had essen-
tially no effect on intracellular L. mexicana. Our mea-
sured activity of amphotericin B, the most consistent
and effective standard compound in this assay, was
comparable to the 40 nM ED50 for the reduction of
amastigotes in peritoneal macrophages reported by Neal
and Croft.17
The antileishmanial selectivity of 4a–h was examined by
evaluating the effect of these compounds on the growth
of mammalian cell lines. Compounds 4b, 4d, 4f, and 4h
showed modest in vitro selectivity against amastigote-
like L. donovani compared to J774 murine macrophages,
with these agents being 6.4-fold, 3.4-fold, and 4.3-fold,
and 7.4-fold more active against Leishmania than the
While amphotericin B is clearly superior to 4b in the
Leishmania-infected macrophage assay, other clinically
useful antileishmanial compounds such as pentamidine
and Pentostam are much less active than amphotericin B
Table 1. IC50 values (lM) of 3-aryl-5-substituted-1,2,4-oxadiazoles against axenic L. donovani, T. brucei, and mammalian cell lines
Compound
L. donovani axenic amastigotes
T. b. brucei variant 221
J774 macrophages
PC3 prostate
Vero cells
4a
4b
9.5 2.5
4.5 1.8
7.3 3.3
6.2 0.6
4.9 1.8
6.5 0.6
7.4 1.4
5.2 1.7
6.9 0.3
6.7 0.3
6.4 0.5
5.2 1.7
6.7 0.2
5.2 2.0
>25
7.2 0.4
6.2 0.7
NDa
41 14
ND
29
8.7 1.1
21
9.5 0.6
7
17
8.4 4.2
18
6
4c
4d
6
8
ND
ND
4e
7.5 3.2
8.1 1.1
8.4 3.9
3.9 2.2
ND
4f
4g
28
11
8
1
2
ND
ND
12
0
4h
5a
2.3 1.0
>200
>200
17
70 22
ND
ND
ND
ND
ND
ND
ND
ND
23
5b
5c
ND
>25
ND
ND
>200
>200
ND
ND
5d
5e
>25
>25
ND
ND
>200
>100
ND
ND
5f
>25
0.008 0.006
0.20 0.05
ND
32 14
ND
Pentamidine
Suramin
1.1 0.1
ND
6
>100
ND
ND
a ND––not determined.

2818
D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
Table 2. Activity of 4b and amphotericin B against intracellular
L. mexicana
3. Experimental
3.1. General methods
Compound
Concentration
(lM)
% Reduction in amastigotes/
macrophage
All reagents were obtained from commercial vendors
and were used without further purification unless
otherwise indicated. Nuclear magnetic resonance spectra
4b
50
25
13
48 17
36 14
2
14
1
were obtained at 250, 400, or 600 MHz for H and 62.5,
Amphotericin B
0.50
0.13
0.031
99
0
100, or 150 MHz for 13C using instruments from Bruker.
Elemental analyses were performed by Atlantic
Microlabs (Norcross, GA) and were within 0.4% of the
calculated values.
77 25
29 16
in this intracellular model.17 Considering the in vitro
potency and selectivity of 4b, we examined the activity
of this compound in a rodent model of visceral leish-
maniasis (Table 3). The activity of Pentostam observed
here was comparable to the roughly 50% inhibition of
liver parasitemia reported previously for an identical
dose of this antimonial drug given by the same route.18
When administered i.v. at a dose of 5 mg/kg, compound
4b was slightly less active than a 15 mg/kg s.c. dose of
Pentostam but more active than the highest tolerable
dose of i.v.-administered pentamidine. In a separate
experiment, 4b suppressed liver parasitemia by 61%
when given at 50 mg/kg in five daily oral doses. No
toxicity was observed when 4b was given at the 5 mg/kg
i.v. dose, while a slight weight loss was observed in the
group treated with the 50 mg/kg oral dose of this com-
pound.
3.2. Benzonitriles 1a–h
With the exception of the costly 3,4-dichlorobenzonitrile
(1h), which was prepared by the reaction of 3,4-dichloro-
benzaldehyde with hydroxylamine and phthalic anhy-
dride,19 benzonitrile precursors were obtained from
commercial sources.
3.3. Preparation of benzamidoximes 2a–h
Benzamidoximes were synthesized by the reaction of 1a–
h with hydroxylamine hydrochloride in the presence of
sodium carbonate and 8-hydroxyquinoline. Representa-
tive procedure for the preparation of 4-methyl-
benzamidoxime 2d. To a solution of 1d (25.0 g, 0.21 mol)
in ethanol (200 mL) was added 8-hydroxyquinoline
(0.077 g, 0.53 mmol). Hydroxylamine hydrochloride
(31.3 g, 0.45 mol) and sodium carbonate (35.8 g,
0.34 mol), each dissolved in water (100 mL), were
sequentially added over a period of 20 min, then the
reaction mixture was heated to reflux for 4.5 h. After
cooling the mixture, solvent was removed in vacuo to
give a light olive green solid. Chromatography on silica
gel using ethyl acetate as eluent gave 31.6 g of a slightly
yellow crystalline solid. This crude material was further
purified by recrystallization from ethanol to give 17.8 g
(56%) of the title compound as a white crystalline solid,
mp 145–147 ꢁC, lit. mp 146 ꢁC.20 Concentration of the
mother liquors via aspirator afforded an additional 5.3 g
(16%) of 2d, mp 140–146 ꢁC. Melting points for com-
pounds 2a–c,20 2e,21 and 2f21 were also consistent with
those reported in the literature.
2.3. Conclusions
Compound 4b (WR85915) displays good in vitro and in
vivo activity against Leishmania. While the antiparasitic
selectivity of such aromatic thiocyanates is clearly a
concern, electrophilic molecules have a long history of
clinical success in treating diseases caused by kineto-
plastid parasites. The synthesis of aromatic thiocyanates
such as 4b is also relatively straightforward, suggesting
that these types of molecules could be produced inex-
pensively for treating diseases that are prevalent in the
developing world. Future studies seeking to identify the
major antiparasitic target(s) of 4b and aiming to increase
the potency and specificity of analogs of this compound
against these target(s) could prove to be a fruitful
approach in the search for critically-needed antileish-
manial agents.
Table 3. Activity of 4b, Pentostam, and pentamidine against L. donovani in BALB/c mice
Compound
Dosing regimen
Actual total dose received
% Inhibition S.E.M.
Experiment 1
4b
5 mg/kg i.v. · 5
2.5 mg
7.5 mg Sb(V)
1.5 mga
48
60
31
8
5
5
Pentostam
Pentamidine
15 mg Sb(V)/kg s.c. · 5
5 mg/kg i.v. · 5a
Experiment 2
4b
Pentostam
50 mg/kg p.o. · 5b
15 mg Sb(V)/kg s.c. · 5
25 mgb
7.5 mg Sb(V)
61
47
8
5
a Pentamidine produced irritation on administration of full dose on day 1. The dose was reduced by 50% for all mice for the following 4 days.
b A slight loss of weight in this group of mice was observed.

D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
2819
3.3.1. 2-Chlorobenzamidoxime 2g. Melting point 119–
121 ꢁC, lit. 116–117 ꢁC.21 1H NMR (400 MHz, CDCl3): d
8.76 (br s, 1H), 7.52 (dd, J1 ¼ 7:5 Hz, J2 ¼ 1:4 Hz, 1H),
7.42 (m, 1H), 7.37–7.26 (m, 2H), 4.95 (s, 2H).
(83%) of the O-acyl adduct as a slightly yellow solid, mp
126–130 ꢁC. A solution of this O-acyl adduct (24.0 g,
0.082 mol) in 500 mL of toluene was refluxed and stirred
for 5 h. Solvent was removed under vacuum from the
hot solution to give the crude product as a yellow solid.
The product was dissolved in a minimum volume of
ethyl acetate and chromatographed on silica gel with
ethyl acetate/hexane (1:9). Elution of the column affor-
ded 20.5 g (92%) of the title compound as a slightly
yellow solid, mp 67–70 ꢁC. 1H NMR (400 MHz, CDCl3):
d 8.01 (d, J ¼ 8:7 Hz, 2H), 7.46 (d, J ¼ 8:7 Hz, 2H), 4.55
(s, 2H). 13C NMR (150 MHz, CDCl3): d 174.8, 168.2,
137.7, 129.3, 128.8, 124.7, 16.3. Note: Integration of the
1H NMR spectrum indicated that 4% of the chloro-
methyl compound (singlet at 4.74 d) had been formed by
exchange of chlorine for bromine, which apparently
took place under the reaction conditions used in the
preparation of the precursor O-acyl adduct.
3.3.2. 3,4-Dichlorobenzamidoxime 2h. Melting point
129–135 ꢁC, lit. 138.5–142 ꢁC.21 1H NMR (400 MHz,
CDCl3): d 7.85 (br s, 1H), 7.74 (m, 1H), 7.48 (m, 2H),
4.85 (s, 2H).
3.4. Preparation of halomethyl oxadiazoles
3.4.1. 3-Phenyl-5-(bromomethyl)-1,2,4-oxadiazole 3a. To
a solution of 2a (13.6 g, 0.10 mol) in chloroform
(300 mL) was added sodium carbonate (10.6 g,
0.10 mol). Bromoacetyl chloride (21.3 g, 0.135 mol) in
chloroform (9 mL) was added to the reaction mixture
over a period of 75 min. A white solid suspended in the
solution formed upon addition of the bromoacetyl
chloride. The reaction mixture was stirred an additional
60 min, then solvent was removed in vacuo to give the
crude product. The solid was dissolved in ethyl acetate
(400 mL), then the organic layer was washed with water
(3 · 100 mL), brine (3 · 100 mL), dried (Na2SO4), and
filtered. Removal of the solvent by evaporation under
vacuum gave 23.9 g (93%) of the O-acyl adduct as a
white powder. This crude adduct (22.9 g, 0.089 mol) was
suspended in 500 mL of o-xylene and refluxed for 3 h.
Solvent was removed under vacuum from the hot reac-
tion mixture to give the product as a yellow oil, which
crystallized on standing. The crude product was dis-
solved in ethyl acetate (300 mL), then the organic layer
was washed with water (3 · 100 mL), brine (3 · 100 mL),
and dried (Na2SO4). The solution was filtered and sol-
vent was evaporated to give 21.1 g of a slightly yellow
solid. Chromatography on silica gel using ethyl acetate/
hexane (1:4) gave 11.4 g (54%) of the title compound
as a white crystalline solid, mp 47–52 ꢁC. 1H NMR
(400 MHz, CDCl3): d 8.11–8.08 (m, 2H), 7.56–7.48 (m,
3H), 4.57 (s, 2H). 13C NMR (150 MHz, CDCl3): d 174.5,
168.9, 131.5, 128.9, 127.5, 126.2, 16.4. Note: Integration
of the 1H NMR spectrum indicated that 6% of the
chloromethyl compound (singlet at 4.77 d) had been
formed by exchange of chlorine for bromine, which
apparently took place under the reaction conditions
used in the preparation of the precursor O-acyl adduct.
3.4.3. 3-(4-Methoxyphenyl)-5-(chloromethyl)-1,2,4-oxa-
diazole 3c. To a solution of 2c (14.0 g, 0.084 mol) in
dry acetone (225 mL) was added potassium carbonate
(11.6 g, 0.084 mol). Chloroacetyl chloride (8.07 mL,
11.3 g, 0.10 mol) was added dropwise over a period of
1.5 h to the stirred reaction mixture, and the reaction
was allowed to stir overnight at room temperature.
Solvent was then removed under reduced pressure to
give the product as a white powder. This solid was dis-
solved in ethyl acetate (400 mL) and was partitioned
once with water (100 mL). The organic layer was then
washed with water (3 · 50 mL), brine (3 · 50 mL), dried
over Na2SO4, and concentrated in vacuo to give 8.71 g
(43%) of the product as a light yellow gummy solid. The
O-acyl adduct (8.4 g, 0.035 mol) was added to 150 mL of
toluene and was heated to reflux for 5 h. Removal of the
solvent in vacuo gave 7.4 g of the product as a brown oil.
The oil was chromatographed on silica gel using ethyl
acetate/hexane (1: 9) as an eluent to afford 5.75 g of the
product as an off-white solid. Recrystallization of the
product from hexane gave 4.71 g (60%) of the title
compound as a white crystalline solid, mp 53–56 ꢁC
(Note: this compound was previously listed as a
liquid12). 1H NMR (400 MHz, CDCl3): d 8.02 (d,
J ¼ 8:9 Hz, 2H), 6.99 (d, J ¼ 8:9 Hz, 2H), 4.73 (s, 2H),
3.87 (s, 3H). 13C NMR (150 MHz, CDCl3): d 174.0,
168.6, 162.2, 129.2, 118.6, 114.4, 55.4, 33.4.
3.4.4. 3-(4-Methylphenyl)-5-(chloromethyl)-1,2,4-oxadiaz-
ole 3d. To a solution of 2d (17.10 g, 0.110 mol) in 250 mL
of dry acetone was added potassium carbonate (15.2 g,
0.11 mol). Chloroacetyl chloride (9.61 mL, 13.6 g,
0.12 mol) was added dropwise over a period of 1 h to the
stirred reaction mixture, and the reaction was allowed to
stir overnight at room temperature. Solvent was then
removed under reduced pressure to give the product as a
white solid. This solid was dissolved in ethyl acetate
(500 mL), the organic layer was washed with water
(4 · 200 mL), brine (1 · 200 mL), dried over Na2SO4, and
filtered. Solvent was removed in vacuo to give a white
crystalline solid, mp 116–119 ꢁC. The product was
recrystallized from ethanol/chloroform/hexane (1:1:1) to
3.4.2. 3-(4-Chlorophenyl)-5-(bromomethyl)-1,2,4,-oxadiaz-
ole 3b. To a solution of 2b (17.6 g, 0.10 mol) in methy-
lene chloride (300 mL) was added potassium carbonate
(13.8 g, 0.10 mol). Bromoacetyl chloride (21.1 g,
0.13 mol) was added dropwise over a period of 3 h.
Solvent was evaporated to give the crude product as a
white powder. The adduct was dissolved in ethyl acetate
(500 mL), the organic layer was washed with water
(3 · 300 mL) and brine (3 · 300 mL), then the ethyl ace-
tate solution was dried over sodium sulfate and filtered.
Evaporation of the solvent gave the crude product as a
yellow solid. The material was chromatographed on
silica gel with ethyl acetate/hexane (3:7) to give 24.0 g

2820
D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
give 9.85 g (40%) of the O-acyl adduct as a white crys-
talline solid. The O-acyl adduct (8.50 g, 0.038 mol) was
suspended in 200 mL of toluene, then the reaction mix-
ture was heated to reflux and stirred for 5 h. Removal of
the solvent in vacuo gave 7.81 g of the crude product as
a light yellow oil, which crystallized on standing. This
solid was chromatographed on silica gel with ethyl
acetate/hexane (1:9) to afford 7.45 g of a white crystalline
solid. The product was recrystallized from hexane to
give 5.40 g (68%) of the title compound as a white
crystalline solid, mp 46–48 ꢁC, lit. 40–41 ꢁC.22 1H NMR
(400 MHz, CDCl3): d 7.96 (d, J ¼ 8:2 Hz, 2H), 7.29 (d,
J ¼ 7:9 Hz, 2H), 4.73 (s, 2H), 2.41 (s, 3H). 13C NMR
(150 MHz, CDCl3): d 174.1, 168.9, 142.0, 129.7, 127.4,
123.4, 33.4, 21.6.
under vacuum to give a yellow oil. This oil was chro-
matographed on silica gel with ethyl acetate/hexane (1:9)
to afford 7.47 g of a colorless oil, which crystallized on
standing. The product was recrystallized from hexane to
give the title compound as a white crystalline solid
(6.47 g, 69%), mp 44–46 ꢁC, lit. mp 41–42 ꢁC.12 1H NMR
(400 MHz, CDCl3): d 8.08 (m, 1H), 7.97 (d, J ¼ 7:7 Hz,
1H), 7.50 (m, 1H), 7.43 (m, 1H), 4.75 (s, 2H). 13C NMR
(150 MHz, CDCl3): d 174.6, 167.9, 135.1, 131.6, 130.3,
127.9, 127.6, 125.6, 33.3.
3.4.7. 3-(2-Chlorophenyl)-5-(chloromethyl)-1,2,4-oxadi-
azole 3g. To a solution of 2g (16.5 g, 0.096 mol) in
anhydrous acetone (250 mL) was added potassium car-
bonate (13.8 g, 0.100 mol), then chloroacetyl chloride
(8.07 mL, 11.3 g, 0.100 mol) was added to the stirred
reaction mixture over a period of 1 h. The reaction
mixture was stirred overnight at room temperature, then
solvent was removed under reduced pressure to give the
product as a white solid. This solid was dissolved in
ethyl acetate (400 mL), the organic layer was washed
with water (4 · 100 mL), brine (4 · 100 mL), dried over
Na2SO4, and filtered. Solvent was removed in vacuo to
give 20.4 g (85%) of the O-acyl adduct as a white solid.
This O-acyl adduct (14.1 g, 0.057 mol) was suspended in
200 mL of toluene, then the reaction mixture was heated
to reflux and stirred for 6 h. Solvent was removed in
vacuo from the hot reaction mixture to give the crude
product as a light yellow oil. This oil was chromato-
graphed on silica gel with ethyl acetate/hexane (1:9) to
give 12.1 g of the product as a colorless oil, which
crystallized on standing, mp 39–40 ꢁC. The solid was
recrystallized from hexane/chloroform to give 10.2 g
(78%) of the title compound as a white crystalline solid,
3.4.5. 3-(4-Fluorophenyl)-5-(chloromethyl)-1,2,4-oxadi-
azole 3e. To a stirred solution of 2e (16.2 g, 0.105 mol) in
500 mL of dry acetone was added potassium carbonate
(14.5 g, 0.105 mol). Over a period of 3 h, a solution of
chloroacetyl chloride (8.3 mL, 11.8 g, 0.10 mol) in 10 mL
of dry acetone was added dropwise to the reaction
mixture. Stirring was continued for an additional 3 h at
room temperature, then solvent was removed in vacuo
from the reaction mixture to give an off-white solid. The
solid was washed with water (4 · 100 mL), then was
dissolved in ethyl acetate (400 mL). This organic layer
was washed with water (4 · 100 mL), brine (4 · 100 mL),
then dried over Na2SO4. Evaporation of the solvent
gave 19.6 g (81%) of the O-acyl adduct. This O-acyl-
adduct (16.3 g, 0.07 mol) was added to 250 mL of tolu-
ene, then the solution was heated to reflux with stirring
for 6 h. Solvent was evaporated from the reaction mix-
ture to give 15.8 g of the crude product as an oil. This oil
was chromatographed on silica gel with ether/hexane
(1:4) to afford the oxadiazole as a colorless oil, which
solidified upon refrigeration. Recrystallization of the
product from hexane afforded 13.1 g (88%) of the title
1
mp 40–42 ꢁC. H NMR (400 MHz, CDCl3): d 7.92 (dd,
J1 ¼ 7:6 Hz, J2 ¼ 1:8 Hz, 1H), 7.55 (dd, J1 ¼ 7:9 Hz,
J2 ¼ 1:3 Hz, 1H), 7.47–7.37 (m, 2H), 4.78 (s, 2H). Note:
these data are consistent with the 1H NMR spectral data
provided earlier for this compound.23 13C NMR
(100 MHz, CDCl3): d 173.9, 167.7, 133.5, 132.0, 131.8,
131.0, 127.0, 125.5, 33.3.
1
compound as a white crystalline solid, mp 31–32 ꢁC. H
NMR (400 MHz, CDCl3): d 8.09 (m, 2H), 7.18 (m, 2H),
4.75 (s, 2H). 13C NMR (150 MHz, CDCl3): d 174.5,
168.1, 164.8 (d, JCF ¼ 251:8 Hz), 129.7 (d, JCF ¼ 9:0 Hz),
122.4 (d, JCF ¼ 3:2 Hz), 116.2 (d, JCF ¼ 21:9 Hz), 33.3.
3.4.8.
3-(3,4-Dichlorophenyl)-5-(bromomethyl)-1,2,4-
oxadiazole 3h. To a solution of 2h (16.1 g, 0.078 mol)
dissolved in 500 mL of dry acetone was added potassium
carbonate (12.3 g, 0.078 mol). Bromoacetyl bromide
(15.8 g, 0.078 mol) dissolved in 20 mL of dry acetone was
added dropwise to the stirred reaction mixture over a
period of 3 h, then the reaction mixture was allowed to
stir for an additional 2 h. Solvent was removed in vacuo
to give the crude product as a slightly yellow solid. The
solid was dissolved in ethyl acetate (500 mL), the organic
layer was washed with water (4 · 100 mL), dried with
Na2SO4, then filtered. Solvent was evaporated to give
21.5 g of the crude product. Two recrystallizations of the
adduct from ethanol afforded 8.89 g (35%) of the O-acyl
adduct as a white crystalline solid, mp 129–130 ꢁC. This
O-acyl adduct (7.87 g, 0.024 mol) was added to 300 mL
of toluene, then the reaction mixture was heated to
reflux and stirred for 5 h. Removal of the solvent in
vacuo gave a light yellow oil, which solidified on
3.4.6. 3-(3-Chlorophenyl)-5-(chloromethyl)-1,2,4-oxadi-
azole 3f. To a solution of 2f (16.4 g, 0.096 mol) in
anhydrous acetone (300 mL) was added potassium car-
bonate (13.8 g, 0.100 mol), then chloroacetyl chloride
(8.07 mL, 11.3 g, 0.100 mol) was added to the stirred
reaction mixture over a period of 3 h. The reaction
mixture was stirred an additional 3 h at room tempera-
ture, then solvent was evaporated under vacuum to give
the product as a white solid. The solid was dissolved in
ethyl acetate (500 mL), the organic layer was washed
with water (4 · 100 mL), brine (4 · 100 mL), dried over
Na2SO4, and filtered. Solvent was removed in vacuo to
give 10.3 g (43%) of the O-acyl adduct as a white crys-
talline solid, mp 105–108 ꢁC. This O-acyl adduct (10.2 g,
0.041 mol) was added to 150 mL of toluene, then the
reaction mixture was heated to reflux and stirred for 6 h.
Solvent was removed from the hot reaction mixture

D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
2821
standing. The crude product was dissolved in a mini-
mum volume of ethyl acetate and chromatographed on
silica gel with ether/hexane (1:9). Elution of the column
gave 5.75 g (78%) of the product as a white crystalline
solid, mp 54–55 ꢁC. 1H NMR (400 MHz, CDCl3): d 8.16
(d, J ¼ 1:9 Hz, 1H), 7.90 (dd, J1 ¼ 8:4 Hz, J2 ¼ 2:0 Hz,
1H), 7.55 (d, J ¼ 8:4 Hz, 1H), 4.55 (s, 2H). 13C NMR
(150 MHz, CDCl3): d 175.5, 167.7, 136.3, 133.9, 131.5,
129.7, 126.9, 126.5, 16.7.
30 mL of dry DMF was added ammonium thiocyanate
(2.6 g, 0.030 mol). The solution was heated and stirred at
60–90 ꢁC for 30 min. Ice/water (80 mL) was added to the
cooled reaction mixture followed by stirring for 20 min
until precipitation of the product was complete. The
yellow precipitate was filtered, washed with water
(3 · 80 mL), then chromatographed on silica gel
using ethyl acetate/hexane (1:3) as eluent to give 1.8 g
(56%) of the title compound as a light yellow crystalline
solid, mp 90–92 ꢁC. An analytical sample was obtained
by sublimation of the product at 80–86 ꢁC (0.5–
0.75 Torr) to give a white crystalline solid, mp un-
changed. 1H NMR (400 MHz, CDCl3): d 8.02 (d,
J ¼ 8:9 Hz, 2H), 7.00 (d, J ¼ 8:9 Hz, 2H), 4.37 (s, 2H),
3.87 (s, 3H). 13C NMR (150 MHz, CDCl3): d 172.5,
168.8, 162.3, 129.2, 118.3, 114.4, 109.5, 55.4, 27.4. Anal.
(C11H9N3O2S) C, H, N.
3.5. Preparation of 3-aryl-5-thio-cyanatomethyl-1,2,4-
oxadiazoles
3.5.1. 3-Phenyl-5-(thiocyanatomethyl)-1,2,4-oxadiazole
4a. To a solution of 3a (3.0 g, 0.013 mol) in 40 mL of
dry DMF was added ammonium thiocyanate (2.2 g,
0.03 mol). The reaction mixture was heated and stirred
at 70–90 ꢁC for 1 h, then the solution was cooled and ice/
water (40 mL) was added with stirring to produce a
yellow precipitate. Filtration of the solid followed by
washing with water (200 mL) afforded 1.9 g of a yellow
powder. The product was chromatographed on silica gel
with ethyl acetate/hexane (1:4) as eluent to give the
product as a white solid. This solid was recrystallized
from hexane/chloroform to give 1.06 g (39%) of the title
compound as a white crystalline solid, mp 77–79 ꢁC, lit.
75–77 ꢁC.13 The analytical sample was obtained by
sublimation of the recrystallized solid at 65–70 ꢁC (0.50–
3.5.4. 3-(4-Methylphenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4d. To a solution of 3d (3.00 g, 0.014 mol)
in 25 mL of dry DMF was added ammonium thiocya-
nate (2.26 g, 0.030 mol). The solution was heated and
stirred at 70–95 ꢁC for 30 min. Ice/water (60 mL) was
added to the cooled reaction mixture followed by stir-
ring for 30 min until precipitation of the product was
complete. The yellow solid that was formed was filtered,
washed with water (3 · 50 mL), and dissolved in ethyl
acetate (200 mL). This organic layer was washed with
water (1 · 50 mL), brine (3 · 50 mL), dried over Na2SO4,
then solvent was evaporated to give 3.08 g of a light
yellow oil, which crystallized on standing. Chromatog-
raphy on silica gel using ethyl acetate/hexane (1:3) as
eluent gave 3.00 g of product as a light yellow crystalline
solid. Recrystallization of the product from hexane/ethyl
acetate afforded 1.82 g (56%) of the compound as a
white crystalline solid, mp 96–98 ꢁC. The analytical
sample was obtained by sublimation of the product at
80–85 ꢁC (0.75 Torr). 1H NMR (400 MHz, CDCl3): d
7.98 (d, J ¼ 7:9 Hz, 2H), 7.31 (d, J ¼ 7:9 Hz, 2H), 4.39
(s, 2H), 2.43 (s, 3H). 13C NMR (150 MHz, CDCl3): d
172.7, 169.1, 142.2, 129.7, 127.5, 123.1, 109.4, 27.5, 21.6.
Anal. (C11H9N3OS) C, H, N.
1
1.0 Torr). H NMR (400 MHz, CDCl3): d 8.10–8.08 (m,
2H), 7.56–7.47 (m, 3H), 4.40 (s, 2H); 13C NMR
(150 MHz, CDCl3): d 172.8, 169.0, 131.7, 129.0, 127.5,
125.8, 109.4, 27.4. Anal. (C10H7N3OS) C, H, N.
3.5.2. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxa-
diazole 4b. To a solution of 3b (7.04 g, 0.026 mol) dis-
solved in 30 mL of dry DMF was added ammonium
thiocyanate (4.57 g, 0.06 mol) in 20 mL of dry DMF.
The reaction mixture was heated and stirred at 70–90 ꢁC
for 75 min, then ice/water (50 mL) was added to the
cooled reaction mixture. A yellow precipitate formed,
which was filtered and washed with water (100 mL). This
yellow precipitate was dissolved in ethyl acetate
(500 mL), the organic layer was extracted with distilled
water (2 · 200 mL) and brine (2 · 200 mL), dried over
Na2SO4, then filtered. Removal of the solvent in vacuo
gave 5.86 g of crude oxadiazole as a yellow crystalline
solid. The product was dissolved in a minimum amount
of ethyl acetate and chromatographed on silica gel with
ethyl acetate/hexane (1:9) to give 5.4 g (83%) of the title
compound as light yellow crystals, mp 100–102 ꢁC. The
analytical sample was obtained by sublimation of the
light yellow solid at 85–90 ꢁC (1 Torr) to give the com-
3.5.5.
3-(4-Fluorophenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4e. To a solution of 3e (7.04 g, 0.033 mol)
in 30 mL of dry DMF was added ammonium thiocya-
nate (6.85 g, 0.09 mol). The reaction mixture was stirred
and heated with a water bath at 60–95 ꢁC for 30 min. Ice/
water (60 mL) was added to the cooled reaction mixture
followed by stirring until precipitation of the product
was complete. The precipitate was filtered, washed
with water (2 · 100 mL), and the light yellow solid was
dissolved in ethyl acetate (500 mL). This organic layer
was extracted with water (3 · 150 mL) and brine
(3 · 150 mL), dried over Na2SO4, and filtered. Removal
of the solvent afforded 6.50 g of the crude product as a
light yellow oil, which crystallized on standing. Gradient
elution from a silica gel column (ether/hexane (1:4) to
ether/hexane (1:1)) gave 6.38 g of white crystalline solid,
which was recrystallized from hexane/chloroform to
afford 3.9 g (50%) of the title compound, mp 74–76 ꢁC.
1
pound as a white crystalline solid, mp unchanged. H
NMR (400 MHz, CDCl3): d 8.03 (d, J ¼ 8:6 Hz, 2H),
7.48 (d, J ¼ 8:6 Hz, 2H), 4.39 (s, 2H); 13C NMR
(150 MHz, CDCl3): d 173.1, 168.3, 138.0, 129.4, 128.9,
124.4, 109.4, 27.3. Anal. (C10H6ClN3OS) C, H, N.
3.5.3. 3-(4-Methoxyphenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4c. To a solution of 3c (3.0 g, 0.013 mol) in

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D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
The analytical sample was obtained by sublimation of
the recrystallized solid at 60–70 ꢁC (0.75–1 Torr). 1H
NMR (400 MHz, CDCl3): d 8.09 (m, 2H), 7.18 (m, 2H),
4.40 (s, 2H); 13C NMR (150 MHz, CDCl3): d 173.0,
168.3, 164.9 (d, JCF ¼ 252:5 Hz), 129.8 (d, JCF ¼ 8:8 Hz),
122.1 (d, JCF ¼ 2:3 Hz), 116.3 (d, JCF ¼ 22:1 Hz), 109.4,
27.4. Anal. (C10H6FN3OS) C, H, N.
50 mL of dry DMF was added ammonium thiocyanate
(2.6 g, 0.03 mol). The reaction mixture was heated and
stirred at 75–95 ꢁC for 1 h, then cooled. Ice/water
(50 mL) was added with stirring to the reaction mixture
to precipitate the product, then the yellow solid was
filtered, washed with water, and dissolved in ethyl
acetate (500 mL). The organic layer was washed with
water (3 · 100 mL), brine (2 · 100 mL), and dried
(Na2SO4). Solvent was removed in vacuo to give the
crude product as an orange-yellow solid. Gradient elu-
tion of the product from a silica gel column using
chloroform/hexane (1:4 to 1:1) afforded 1.87 g of prod-
uct. Recrystallization from chloroform-hexane gave
1.44 g (50%) of the title compound as a light yellow
solid, mp 106–108 ꢁC. Sublimation of the solid at 90–
95 ꢁC (0.50 Torr) gave the analytical sample as a white
3.5.6.
3-(3-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4f. To a solution of 3f (4.0 g, 0.017 mol) in
40 mL of dry DMF was added ammonium thiocyanate
(3.88 g, 0.051 mol). This solution was heated and stirred
at 60–90 ꢁC for 30 min. To the cooled solution was
added ice/water (80 mL), followed by stirring for 15 min to
give a light yellow precipitate. The solid was filtered and
washed with water (2 · 100 mL). This solid was dissolved
in ethyl acetate (200 mL), then the organic layer was
washed with water (3 · 100 mL), brine (3 · 100 mL),
dried over Na2SO4, and the solvent removed in vacuo to
give a yellow solid. Chromatography on silica gel
employing ethyl acetate/hexane (1:4) as the eluent gave
2.5 g of the product as a pale yellow solid. The product
was recrystallized from chloroform to afford 2.35 g
(54%) of the title compound as an off-white crystalline
solid, mp 113.5–115 ꢁC. The analytical sample was
obtained by sublimation of the solid at 90 ꢁC (1 Torr) to
give the compound as a white crystalline solid. 1H NMR
(600 MHz, CDCl3): d 8.09 (m, 1H), 7.98 (d, J ¼ 7:6 Hz,
1H), 7.52 (m, 1H), 7.45 (dd, J1 ¼ J2 ¼ 7:8 Hz, 1H), 4.41
(s, 2H); 13C NMR (150 MHz, CDCl3): d 173.3, 168.1,
135.1, 131.8, 130.4, 127.7, 127.6, 125.7, 109.3, 27.4.
Anal. (C10H6ClN3OS) C, H, N.
1
crystalline solid, mp 108–109 ꢁC. H NMR (400 MHz,
CDCl3):
d
8.20 (d, J ¼ 2:0 Hz, 1H), 7.94 (dd,
J1 ¼ 8:4 Hz, J2 ¼ 2:0 Hz, 1H), 7.59 (d, J ¼ 8:4 Hz, 1H),
4.40 (s, 2H). 13C NMR (150 MHz, CDCl3): d 173.4,
167.5, 136.2, 133.6, 131.2, 129.4, 126.6, 125.8, 109.2,
27.3. Anal. (C10H5Cl2N3OS) C, H, N.
3.6. Preparation of 3-aryl-5-alkyl-1,2,4-oxadiazoles 5a–f
These compounds were prepared from benzamidoximes
2a–c and acetyl or isobutyryl chloride in pyridine
according to the general procedure of Chiou and
Shine.15 Representative procedure for the preparation of
3-(4-methoxyphenyl)-5-methyl-1,2,4-oxadiazole 5c. To a
three-necked flask equipped with an addition funnel and
a condenser was added 2c (3.04 g, 0.018 mol) and pyr-
idine (4 mL). Acetyl chloride (2.75 g, 0.035 mol) was then
added dropwise to this suspension over a period of
15 min, then the reaction mixture was heated to reflux
for 45 min. Upon cooling, the mixture solidified. Water
(30 mL) was added, at which point the product precip-
itated. The precipitate was filtered, washed with water,
then chromatographed on silica gel using a methylene
chloride/hexane (1:4 to 2:3) step gradient as eluent to
give 2.90 g (85%) of the desired product as a white
crystalline solid, mp 61–63 ꢁC, lit. 58–59 ꢁC.24 1H NMR
(250 MHz, CDCl3): d 8.00 (d, J ¼ 8:9 Hz, 2H), 6.98 (d,
J ¼ 8:9 Hz, 2H), 3.87 (s, 3H), 2.64 (s, 3H).
3.5.7. 3-(2-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4g. To a solution of 3g (4.0 g, 0.017 mol) in
30 mL of dry DMF was added ammonium thiocyanate
(3.88 g, 0.051 mol). The reaction mixture was heated and
stirred at 60–90 ꢁC for 30 min. Ice/water (80 mL) was
added to the cooled reaction mixture followed by stir-
ring for 20 min until precipitation of the product was
complete. The yellow solid that was formed was filtered,
washed with water (3 · 50 mL), and dissolved in ethyl
acetate (300 mL). This organic layer was washed with
water (1 · 50 mL), brine (3 · 50 mL), dried over Na2SO4,
then solvent was evaporated to give the product as a
yellow solid. Chromatography on silica gel using ethyl
acetate/hexane (1.5:8.5) as eluent gave 3.72 g of product,
which was recrystallized from chloroform/hexane to give
3.01 g (70%) of the title compound as a light yellow
crystalline solid, mp 86–90 ꢁC. The analytical sample
was obtained by sublimation of the light yellow recrys-
tallized compound at 60–80 ꢁC (0.6–0.7 Torr) to give the
title compound as a white crystalline compound, mp
3.6.1. 3-Phenyl-5-methyl-1,2,4-oxadiazole 5a. Melting
point 40–41 ꢁC, lit. 39–41 ꢁC.15 1H NMR (250 MHz,
CDCl3): d 8.06 (m, 2H), 7.48 (m, 3H), 2.66 (s, 3H).
3.6.2. 3-(4-Chlorophenyl)-5-methyl-1,2,4-oxadiazole 5b.
Melting point 103–105 ꢁC, lit. 90–92 ꢁC.25 1H NMR
(250 MHz, CDCl3): d 8.01 (d, J ¼ 8:4 Hz, 2H), 7.46 (d,
J ¼ 8:4 Hz, 2H), 2.66 (s, 3H).
1
unchanged. H NMR (400 MHz, CDCl3): d 7.96 (dd,
J1 ¼ 7:6 Hz, J2 ¼ 1:8 Hz, 1H), 7.56 (dd, J1 ¼ 7:9 Hz,
J2 ¼ 1:3 Hz, 1H), 7.49–7.39 (m, 2H), 4.44 (s, 2H). 13C
NMR (150 MHz, CDCl3): d 172.5, 167.9, 133.5, 132.2,
131.8, 131.1, 127.0, 125.2, 109.4, 27.3. Anal.
(C10H6ClN3OS) C, H, N.
3.6.3. 3-Phenyl-5-isopropyl-1,2,4-oxadiazole 5d. Color-
less oil,26 1H NMR (250 MHz, CDCl3): d 8.09 (m, 2H),
7.48 (m, 3H), 3.30 (sep, J ¼ 7:0 Hz, 1H), 1.47 (d,
J ¼ 7:0 Hz, 6H).
3.5.8. 3-(3,4-Dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-
oxadiazole 4h. To a solution of 3h (3.17 g, 0.01 mol) in

D. M. Cottrell et al. / Bioorg. Med. Chem. 12 (2004) 2815–2824
2823
3.6.4.
3-(4-Chlorophenyl)-5-isopropyl-1,2,4-oxadiazole
ough washing in flowing tap water, the slides were al-
lowed to air dry before being viewed by oil immersion
microscopy to determine the percentage of infected cells.
For each in vitro determination, experiments were per-
formed at least twice, with the mean standard devia-
tion given in each case.
5e. Colorless oil,26 1H NMR (250 MHz, CDCl3): d
8.02 (d, J ¼ 8:4 Hz, 2H), 7.45 (d, J ¼ 8:4 Hz, 2H), 3.28
(sep, J ¼ 7:0 Hz, 1H), 1.46 (d, J ¼ 7:0 Hz, 6H).
3.6.5. 3-(4-Methoxyphenyl)-5-isopropyl-1,2,4-oxadiazole
5f. Colorless oil,26 1H NMR (250 MHz, CDCl3): d 8.01
(d, J ¼ 8:4 Hz, 2H), 6.98 (d, J ¼ 8:4 Hz, 2H), 3.87 (s,
3H), 3.27 (sep, J ¼ 7:1 Hz, 1H), 1.45 (d, J ¼ 7:1 Hz,
6H).
3.8. In vivo antileishmanial assays
The in vivo efficacy of compounds against L. donovani
(strain MHOM/ET/67/82/L82) was measured according
to general procedures outlined previously.18 In these
experiments, female BALB/c mice were infected intra-
venously, via the lateral tail vein, with 1.5 · 107
L. donovani amastigotes freshly isolated from the spleen
of an infected hamster. Infected mice were then ran-
domly sorted into groups of five. Treatment groups
received the test or standard compound subcutaneously
(s.c.), intravenously (i.v.), or orally (p.o.) once per day
for 5 days on days 7–11 of infection. In addition, one
group of five mice was given the drug vehicle only. Three
days after the completion of treatment, the mice were
killed and their livers were removed and weighed.
Smears were then prepared, methanol fixed, and Giemsa
stained. The activity of the compounds was determined
by comparing the number of amastigotes per 500 liver
cells times the tissue weight in animals from treated and
untreated groups.
3.7. In vitro assays
The activity of the test compounds against L. donovani
axenic amastigotes (WHO designation MHOM/SD/62/
1S-CL2D) was measured as described previously.5;27
Assessment of the antiproliferative activity of com-
pounds against bloodstream-form Trypanosoma brucei
brucei (MITat 1.2, variant 221) and J774 murine mac-
rophages was also carried out as outlined earlier.5 The
toxicity of compounds to PC3 cells and Vero cells was
measured in the same manner as for the macrophages,
except that the medium differed in each case. For the
PC3 cells, the medium was F12K (Kaighn’s modifica-
tion, from Gibco) supplemented with 50 units/mL pen-
icillin, 50 lg/mL streptomycin, and 10% fetal bovine
serum, while Vero cells were grown in Eagle’s Minimal
Essential Medium with Earle’s Balanced Salt Solution
(from ATCC), which includes 2 mM glutamine, non-
essential amino acids and is supplemented with 50 units/
mL penicillin, 50 lg/mL streptomycin, and 10% fetal
bovine serum. Selected compounds were tested for their
activity against Leishmania-infected J774 macrophages
essentially as described earlier28 with some modifica-
tions. Briefly, macrophages were mixed with a late log
phase culture of L. mexicana (WHO designation:
MNYC/BCZ/62/M379) promastigotes to yield a solu-
tion containing 5 · 105 macrophages/mL and 37.5 ·
105 promastigotes/mL in DMEM supplemented with
Acknowledgements
We would like to thank the American Association of
Colleges of Pharmacy New Investigator Program and
the College of Pharmacy at the Ohio State University
for financial support.
References and notes
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