Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides

Article abstract of DOI:10.1016/j.bmc.2014.02.013

A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared and evaluated for their capacity to induce methemoglobinemia - with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb% = 61.0 ± 3.6) and 1,3,4-oxadiazole 10 (metHb% = 52.4 ± 0.9) demonstrating the greatest activity. Of the 5 candidates (compounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120 mg/kg was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as the CYPs, was postulated as one reason for their failure.

Full text of DOI:10.1016/j.bmc.2014.02.013

Bioorganic & Medicinal Chemistry 22 (2014) 2220–2235
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and methemoglobinemia-inducing properties
of benzocaine isosteres designed as humane rodenticides
Daniel Conole a, Thorsten M. Beck a, Morgan Jay-Smith a, Malcolm D. Tingle b, Charles T. Eason c,d
,
Margaret A. Brimble a, , David Rennison a,
⇑
⇑
a School of Chemical Sciences, University of Auckland, Auckland, New Zealand
b Department of Pharmacology & Clinical Pharmacology, University of Auckland, Auckland, New Zealand
c Centre for Wildlife Management and Conservation, Lincoln University, Lincoln, New Zealand
d Cawthron Institute, Nelson, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared
and evaluated for their capacity to induce methemoglobinemia—with a view to their possible application
as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemo-
globin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb% = 61.0 3.6) and
1,3,4-oxadiazole 10 (metHb% = 52.4 0.9) demonstrating the greatest activity. Of the 5 candidates (com-
pounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120 mg/kg
was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as
the CYPs, was postulated as one reason for their failure.
Received 18 December 2013
Revised 3 February 2014
Accepted 11 February 2014
Available online 20 February 2014
Keywords:
Benzocaine
Isostere
Methemoglobin
Methemoglobinemia
Rodenticide
Rat toxicant
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
para-aminopropiophenone (PAPP) (Fig. 1) has more recently been
evaluated in both New Zealand and Australia for the humane con-
In New Zealand alone, almost NZ$200 million is spent on ver-
tebrate pest control products annually.1 Most of these, however,
share common disadvantages such as secondary non-target poi-
soning, risks of environmental contamination, and accumula-
tion/persistence of residues in the food chain. Moreover, most
of these agents lack humaneness. In 1901 benzocaine (Fig. 1)
was found to possess anaesthetic properties,2 and was subse-
quently introduced to the market under the trade name Anesthe-
trol of introduced predators such as feral cats and stoats.12–14
Interestingly, the toxic effects of PAPP have likewise been attrib-
uted to its capacity to induce the formation of methemoglobin
(metHb),15 leading to cerebral hypoxia, central nervous system
depression and ultimately death.16 In a drug comparison study,
Guertler et al.17 reported that sheep, dosed either intranasally with
benzocaine or intravenously with PAPP, developed similar levels of
methemoglobinemia. Further to this research, Coleman and
Kuhns,18 demonstrated that in vitro benzocaine was a more potent
inducer of methemoglobin than PAPP. Considering their structural
and electronic similarities, in combination with their apparent
dependency on NADPH as a co-factor in metHb induction,18 it is
generally accepted that benzocaine and PAPP induce their toxic ef-
fects by analogous biochemical pathways (Fig. 2).17
In addition to their intrinsic toxicity, the use of methemoglobi-
nemia-inducing agents in pest control offers a number of comple-
mentary benefits—principally their comparatively humane mode
of action.12 For example, PAPP is known to induce its lethal effect
by reducing oxygen supply to the brain, leading to symptoms such
as progressive lethargy, drowsiness, stupor and ultimately uncon-
sciousness prior to death. From a welfare perspective, particularly
in terms of minimizing animal distress, this mechanism of toxicity
sinÒ.3 Still widely used today in the clinic as
a topical
anaesthetic,4 benzocaine has been observed to induce methemo-
globinemia in some patients,5–7 that is an impairment of the
red blood cells capacity to transport oxygen, in some instances
leading to potentially dangerous levels.8–10 While such a side
effect naturally poses a concern to medicine, benzocaine-induced
methemoglobinemia, and its potential application to pest man-
agement, could be considered for further exploration.
Originally investigated by the United States Fish and Wildlife
Services in the 1980s for the control of coyotes (Canis latrans),11
⇑
Corresponding authors. Tel.: +64
7599x83881 (D.R.); fax: +64 9 373 7422 (school).
9 373 7599x88259 (M.A.B.), +64 9 373
E-mail address: d.rennison@auckland.ac.nz (D. Rennison).
http://dx.doi.org/10.1016/j.bmc.2014.02.013
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2221
could be viewed as advantageous.12 Furthermore, time to death is
NADH-cytochrome b reductase
(metHb red uctase)
relatively short, usually within 1–2 h following ingestion.19,20 Hav-
ing induced their toxic effects, such toxicants are readily converted
to comparatively innocuous metabolites, and as a consequence are
unlikely to be present in carcasses at levels that will cause second-
ary poisoning to non-target species, nor should they persist in the
food chain.11 Indeed, should the accidental poisoning of non-target
species occur, a simple and relatively inexpensive antidote, meth-
ylene blue, is available.21
HbFe3+ (metHb)
HbFe2+
RED BLOOD CELL
O2
H2O2
OH- + OH
2. Results and discussion
2.1. Synthesis
O
O
5-(40-Aminophenyl)-1,2,4-oxadiazoles 1–6 were prepared over
three steps via amidoximes 36–41,22 themselves the dehydration
product of the reaction between the corresponding nitrile
(30–35) and hydroxylamine. A sequential one-pot base-catalyzed
acylation of 36–41 using 4-nitrobenzoyl chloride (42), with
concomitant dehydration,22 led to nitro-substituted oxadiazoles
43–48 (2–18%, over two steps). Compounds 43–48 were then
carefully reduced using sodium sulfide23 to afford target oxadiaz-
oles 1–6 (47–87%) (Scheme 1). The key step towards the synthesis
of 5-(40-aminophenyl)-1,2,4-thiadiazole 7 involved the union of thi-
obenzamide 50 and dimethylacetal 5124 (92%);25 compound 50 was
accessed in two steps from commercially available 4-nitrobenzoyl
chloride via benzamide 49. Subsequent cyclization of carbothioa-
mide 52 in the presence of hydroxylamine-O-sulfonic acid (HSA)
gave nitro-substituted thiadiazole 53 (70%).25 Ultimately, reduction
furnished target thiadiazole 7 (69%) (Scheme 2).23
O
O
HO
N
H
O=N
Ethyl par a-nitrosobenzoate
d iaphorase
NADPH flavin reductase
glutathione
PASSIVE DIFFUSION
LIVER
O
O
HEPATIC
O
O
OXIDATION
HO
N
H2N
Ethyl
H
The synthesis of 2-(40-aminophenyl)-1,3,4-oxadiazoles 8–15
proceeded via common hydrazide intermediate 55, which in turn
was acylated26 with the corresponding acid chlorides to afford
N-acylhydrazides 56–63 (10-87%). Subsequent cyclization26 in
the presence of phosphorus oxychloride at elevated temperature
afforded nitro-substituted oxadiazoles 64–71 (12–60%). Finally,
sodium sulfide reduction, as described previously,23 afforded target
oxadiazoles 8–15 (9–98%) (Scheme 3). 2-(40-Aminophenyl)-1,3,
4-thiadiazole 16 was conveniently accessed in two steps from pre-
viously described26 N-acylhydrazide 58 via a one-pot thiolation
and condensation,27 using Lawesson’s reagent at elevated temper-
ature, to afford nitro-substituted thiadiazole 72 (56%). Ultimately,
reduction23 afforded target thiadiazole 16 (46%) (Scheme 4).
Preparation of 3-(40-aminophenyl)-1,2,4-oxadiazoles 17–19
centred around the synthesis of key benzamidoxime intermediate
75. Treatment of 4-nitrobenzaldehyde (73) with hydroxylamine,
and concomitant dehydration, gave 4-nitrobenzonitrile (74).28 Fur-
ther treatment of nitrile 74 with hydroxylamine, as previously
described,22 led to benzamidoxime intermediate 75 (11%). A one-
pot base-catalyzed acylation and dehydration22 in the presence
of the corresponding acid chlorides afforded nitro-substituted
oxadiazoles 76–78 (21–56%). Reduction23 of compounds 76–78
revealed target oxadiazoles 17–19 (42-70%) (Scheme 5).
inobenzo
para-am
ate
Ethyl
-hydroxylaminobenzoate
(B
enzocain
para
e)
Figure 2. Postulated mechanism for the formation of metHb by aminobenzenes,
taking benzocaine as an example.68 Upon systemic absorption certain aminobenz-
enes are metabolized by hepatic mixed function oxidases to the corresponding N-
phenylhydroxylamines.69 Once taken up into the erythrocyte, and in the presence of
oxygen, N-phenylhydroxylamines undergo a coupled, or two-stage, redox reaction
with hemoglobin (HbFe2+) to form methemoglobin (HbFe3+, metHb), the toxic
endpoint. The first stage involves the oxidation of the N-phenylhydroxylamine to a
nitrosobenzene, and the complimentary reduction of molecular oxygen to hydrogen
peroxide. Consequently, the hydrogen peroxide reacts in situ to oxidize hemoglobin
to methemoglobin.70 Catalytic methemoglobin induction is an NADPH-dependent
process involving reductive enzymes present in erythrocytes such as diaphorase
and NADP flavin reductase, as well as the antioxidant glutathione. These enzymes
work to drive the catalytic cycle of metHb formation via the back-reduction of
nitrosobenzene to N-phenylhydroxylamine, the net result being a small quantity of
aminobenzene having the capacity to turnover a large number of hemoglobin
equivalents to methemoglobin.68,69 To regulate this process NADH-cytochrome b
reductase oxidase, also referred to as methemoglobin reductase, is known to reduce
methemoglobin back to hemoglobin.71,72
hyde (73) and propyltriphenylphosphonium iodide to give nitro-
styrene 79 (54%),29 as a mixture of E and Z isomers. Styrene 79
was subsequently converted over two steps to oxadiazole N-oxide
80, using sodium nitrite and acetic acid,30 followed by chlorosul-
fonic acid in dimethylformamide at elevated temperature. Finally,
concomitant reduction of both the N-oxide and the nitrophenyl
groups of 80, using zinc and an excess of ammonium formate,31
furnished target oxadiazole 20 in a single step (16%) (Scheme 6).
Synthesis of 4-(40-aminophenyl)-1,2,5-oxadiazole 21 proceeded
via intermediate 81, which was prepared in three steps from
4-nitrobenzaldehyde (73).32 Treatment33 of 81 with hydroxyl-
The first step towards the synthesis of 3-(40-aminophenyl)-
1,2,5-oxadiazole 20 was the Wittig reaction of 4-nitrobenzalde-
O
O
R
OEt
H2N
H2N
amine afforded a-oximidoacetoxime 82 (49%). Reaction of 82 with
Benzocaine
PAPP (R = Et)
PAVP (R = nBu)
sodium acetate at elevated temperature for five days eventually
afforded amino-oxadiazole 83 (44%). Diazotization33 of compound
83 to access chloro-oxadiazole 84 at first proved problematic.
However, having determined that the order of addition was crucial
Figure 1. Benzocaine, para-aminopropiophenone (PAPP) and para-aminovaler-
ophenone (PAVP).

2222
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
R
N
R
N
N
OH
N
N
c
a
b
O
O
R
CN
R
NH2
O2N
H2N
36
37
1
2
30
R = Me
R = Et
43
44
R = Me
R = Et
(87%)
(81%)
(69%)
(63%)
(83%)
(47%)
R = Me
R = Et
R = Me (7%)
31
(7%)
R = Et
38 R = nPr
(11%)
(4%)
3 R = nPr
32 R = nPr
45 R = nPr
39
40
41 R = Bn
4
5
33
34
R = iPr
R = Ph
46
47
48 R = Bn
R = iPr
R = Ph
R = iPr
R = Ph
R = iPr
(18%)
(2%)
R = Ph
6 R = Bn
35 R = Bn
Scheme 1. Reagents and conditions: (a) NH2OHꢀHCl, NaOH, EtOH, H2O, reflux, 36 h; (b) 4-nitrobenzoyl chloride (42), pyridine, reflux, 0.5 h (yield over two steps); (c)
Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
O
O
S
a
b
Cl
NH2
NH2
O2N
O2N
O2N
42
49 (99%)
50 (58%)
Et
N
Et
N
O
O
S
Et
N
N
Et
N
51
d
e
S
S
N
N
c
H2N
O2N
O2N
52
53
7
(69%)
(92%)
(70%)
Scheme 2. Reagents and conditions: (a) aq NH3, rt, 0.5 h;73 (b) Lawesson’s reagent, THF, reflux, 5 h;74 (c) 51, rt, 1 h; (d) hydroxylamine-O-sulfonic acid (HSA), pyridine, EtOH,
MeOH, rt, 1 h; (e) Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
O
O
O
H
N
b
R
NH2
a
Cl
N
H
N
H
O
O2N
O2N
O2N
42
55
56
57
(55%)
R = Me (31%)
(29%)
R = Et
58 R = nPr (10%)
N
N
N
N
59
60
R = iPr (36%)
(83%)
R = nBu
R
R
c
d
O
O
61 R = tBu (50%)
62
63
R = Ph (76%)
(87%)
H2N
O2N
R = Bn
8
9
64
R = Me
R = Et
(55%)
(70%)
R = Me (44%)
(20%)
65
R = Et
10 R = nPr (98%)
66 R = nPr (57%)
11
12
67
68
R = iPr (83%)
(31%)
R = iPr (12%)
(25%)
R = nBu
R = nBu
13 R = tBu (79%)
69 R = tBu (50%)
14
15
70
71
R = Ph (65%)
(9%)
R = Ph (35%)
(60%)
R = Bn
R = Bn
Scheme 3. Reagents and conditions: (a) (i) MeOH, rt, 0.5 h (to give methyl para-nitrobenzoate 54); (ii) 54, N2H4ꢀH2O, MeOH, reflux, 0.5 h;26 (b) RCOCl, DMA, 0 °C, 6 h; (c)
POCl3, 80 °C, 20–48 h; (d) Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
in the formation of the diazonium salt, gratifyingly, a modified
diazotization procedure34 afforded compound 84 (22%). Subse-
quent treatment33 of 84 with freshly prepared sodium ethoxide
afforded afford nitro-substituted ethoxy-oxadiazole 85, which,
due to its instability towards chromatographic conditions, was
reduced23 without further delay to furnish target oxadiazole 21
(55%, over two steps) (Scheme 7).
5-(40-Aminophenyl)tetrazole 23 was likewise prepared in three
steps beginning with the click reaction36 of 4-nitrobenzonitrile
(74) and sodium azide, which proceeded smoothly to afford
1H-tetrazole 89 (93%). In turn, 1H-tetrazole 89 was alkylated37 using
propyl iodide to give nitro-substituted 2H-tetrazole 90 (45%). Final-
ly, reduction23 of 90 furnished target tetrazole 23 (93%) (Scheme 9).
Heterocycles 24–29 were prepared via a Suzuki-cross coupling
strategy, employing commercially available 4-acetamidophenylbo-
ronic acid pinacol ester (91) as the boronate coupling partner.
Treatment38 of 91 with the corresponding aryl halides 92–97 in
the presence of bis(triphenylphosphine)palladium(II) chloride
and sodium bicarbonate under microwave irradiation afforded
1-(40-Aminophenyl)tetrazole 22 was prepared from 4-nitroani-
line (86) over three steps via acetanilide 87. Subsequent treatment
with sodium azide and phosphorus oxychloride,35 at elevated
temperature, afforded nitro-substituted tetrazole 88 (17%), with
ensuing reduction23 affording target tetrazole 22 (52%) (Scheme 8).

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2223
N
of chloropyridazine 102 with ammonium formate in the presence
of palladium-on-carbon afforded pyridazine 105.39 Subsequent
acid-hydrolysis40 of acetamides 98–100 and 103–105 proceeded
smoothly to furnish target heterocycles 24–29 (78-96%)
(Scheme 10).
O
N
H
N
nPr
nPr
a
S
N
H
O
O2N
O2N
58
72
(56%)
N
N
2.2. Physicochemical properties and in vitro methemoglobin
formation studies
nPr
b
S
H2N
In-house evaluation of benzocaine (metHb 0.3 0.1%) and PAPP
(metHb 24.8 0.6%) for their capacity to induce the formation of
methemoglobin in vitro revealed benzocaine to be devoid of all
activity under our assay conditions. Such observations were
hypothesized to be a consequence of benzocaine’s inherent hydro-
lytic instability, a process presumed to be under the catalysis of
residual plasma esterases present among erythrocyte isolates.17,41
To provide evidence that hydrolysis was occurring during the
metHb experiment, bis(4-nitrophenyl) phosphate (BNPP), a com-
mercially available reversible esterase inhibitor, was employed to
16
(46%)
Scheme 4. Reagents and conditions: (a) Lawesson’s reagent, PhMe, reflux, 5 h; (b)
Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
heterocycles 98–103 (47–80%). Chloropyrimidine 101 was subse-
quently reduced using zinc and aqueous ammonia at elevated tem-
perature to afford pyrimidine 104 (15%, over two steps). Treatment
OH
N
O
CN
a
b
H
NH2
O2N
O2N
74
O2N
73
75
(11%)
(94%)
O2N
O
O
N
N
N
R
R
c
d
N
H2N
17
76
R = Me (21%)
(60%)
78 R = nPr (78%)
R = Me (42%)
(70%)
19 R = nPr (54%)
77
18
R = Et
R = Et
Scheme 5. Reagents and conditions: (a) NH2OHꢀHCl, DMSO, rt ? 100 °C, 3 h; (b) NH2OHꢀHCl, NaOH, EtOH, H2O, reflux, 36 h; (c) RCOCl, pyridine, reflux, 0.5 h; (d) Na2Sꢀ9H2O,
H2O, 1,4-dioxane, 80 °C, 1 h.
O
N
O
N
N
N
O
O
Et
a
d
b, c
H
Et
Et
O2N
O2N
O2N
H2N
79
73
80
20
(16%)
(45% over 3 steps)
Scheme 6. Reagents and conditions: (a) (i) CH3CH2CH2PPh3+Iꢁ, nBuLi, THF, ꢁ20 °C, 0.75 h; (ii) then 73, THF, ꢁ20 °C ? rt, 2 h; (b) NaNO2, AcOH, H2O, 5 °C ? rt, 18 h; (c)
ClSO3H, DMF, 100 °C, 0.5 h; (d) Zn, NH4Cl, THF, H2O, reflux, 5 h.
OH
CN
OH
O
N
N
O
N
N
NH2
a, b, c
d
e
H
NH2
N
O2N
O2N
O2N
O2N
OH
73
81
82
83
(44%)
(15% over 3 steps)
(49%)
O
N
O
N
O
N
N
N
N
h
g
f
Cl
OEt
OEt
O2N
O2N
H2N
21
84
85
(22%)
(55% over 2 steps)
Scheme 7. Reagents and conditions: (a) NH2OHꢀHCl, NaOH, H2O, EtOH, rt, 3 h; (b) NCS, DMF, 0 °C ? rt, 24 h; (c) NaCN, iPrOH, DCE, H2O, 0 °C, 5 h; (d) NH2OHꢀHCl, NaOAc,
EtOH, reflux, 6 h; (e) NaOAc, EtOH, reflux, 120 h; (f) (i) NaNO2/H2O, AcOH/aq 10 M HCl/H2O, 0 °C, 0. 5 h; (ii) CuCl, aq 10 M HCl, 0 °C ? rt, 0.5 h; (g) (i) Na, EtOH, rt, 0.5 h (ii)
then 84, NaOEt, EtOH, rt, 2 h; (h) Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.

2224
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
N
N
N
N
N
N
H
N
N
N
nPr
NH2
a
c
b
nPr
nPr
O
H2N
O2N
O2N
O2N
86
87 (80%)
88 (17%)
22 (52%)
Scheme 8. Reagents and conditions: (a) (CH3CH2CH2CO)2O, NEt3, 1,4-dioxane, rt, 1 h; (b) NaN3, POCl3, MeCN, reflux 10 h; (c) Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
N
N
N
N
N
N
N
N
N
nPr
nPr
CN
a
b
c
N
N
N
H
O2N
O2N
H2N
O2N
74
89 (93%)
90 (45%)
23 (93%)
Scheme 9. Reagents and conditions: (a) NaN3, NH4Cl, DMF, reflux, 12 h; (b) NaH, nPrI, DMF, 0 °C ? 5 °C, 1.5 h; (c) Na2Sꢀ9H2O, H2O, 1,4-dioxane, 80 °C, 1 h.
N
99
R = Ac (80%)
c
25 R = H (88%)
N
N
N
93
N
92
RHN
RHN
N
Br
Br
98
R = Ac (74%)
c
a
a
24 R = H (89%)
N
N
N
N
N
O
B
94
97
N
N
Cl
N
Cl
O
O
RHN
c
91
a
RHN
100
N
H
a
103
R = Ac (47%)
R = Ac (56%)
a
29 R = H (78%)
26 R = H (96%)
a
a
Cl
N
N
Cl
N
95
N
X
N
N
N
N
X
96
Cl
Cl
102
R = Ac, X = Cl (25%)
RHN
d
c
RHN
101 R = Ac, X = Cl
104
105 R = Ac, X = H
28
R = H, X = H (58% over two steps)
b
R = Ac, X = H (15% over two steps)
c
27 R = H, X = H (93%)
Scheme 10. Reagents and conditions: (a) PdCl2(PPh3)2, NaHCO3, PhMe, EtOH, H2O, 120 °C, MW, 50 W, 1 h; (b) Zn, aq NH3, THF, reflux, 5 h; (c) aq 1 M HCl, MeOH, reflux, 1 h;
(d) NH4Ac, 10 mol % Pd/C, MeOH, reflux, 18 h.
Table 1
block the postulated hydrolytic action of these enzymes. Gratify-
ingly, an immediate recovery in activity was noted (benzocaine
in the presence of BNPP, metHb 45.0 0.8%), supporting the postu-
late that metabolism was indeed likely accountable for the earlier
failure of benzocaine to induce metHb; note that neither BNPP
alone, nor p-aminobenzoic acid, the cleavage product of benzo-
caine hydrolysis, led to the formation of metHb (Table 1).
With such evidence in hand, it was now our intention to pre-
pare a series of benzocaine isosteres of improved metabolic stabil-
ity, while retaining the structural and physicochemical properties
responsible for methemoglobin induction in the parent compound
(Fig. 3). Of the non-classical isosteres developed for the ester func-
tionality, the 1,2,4-oxadiazole moiety is the most frequently em-
ployed surrogate in the literature.42–44 In light of this a series of
1,2,4-oxadiazoles (1–6), incorporating either alkyl or aryl substitu-
ents at the 3-position, were synthesized and subsequently
Physicochemical properties and in vitro methemoglobin formation for benzocaine,
PAPP and PAVP
d
13C–NH2
%metHbc
a
b
Compd
LogPexp.
Benzocaine
PAPP
PAVP
2.04
1.65
2.48
150.7
151.0
151.0
0.3 0.1, 45.0 0.8d
24.8 0.6
61.9 1.9
a
b
c
LogPexp. determined by RP-HPLC methods using a standard curve.
Chemical shift for 13C–NH2 as measured by 13C NMR (in ppm).
Percentage methemoglobin formation in vitro at a compound concentration of
10
l
M (37 °C, 1 h, n = 3). Data represented as mean S.D.67
d
In the presence of an esterase inhibitor [bis-(4-nitrophenyl) phosphate, 1 mM].
evaluated for their capacity to induce the formation of methemo-
globin in vitro. Recently we reported a series of PAPP-related ana-
logues, consequently revealing a strong correlation between the

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2225
R3
N
O1
N
4 Et
1
O
N4
2
N
4 N
3 N
2 N
R5
R5
2
5
5
X
2
X
3
3
N
1
1
4
H2N
H2N
H2N
H2N
20
X = O
X = O
17 R5 = Me
18 R5 = Et
1 R3 = Me
2 R3 = Et
8 R5 = Me
9 R5 = Et
19 R5 = nPr
O1
N
3OEt
5
N
3 R3 = nPr
10 R5 = nPr
2
3
5
4
5
6
11
12
13
14
R
= iPr
= Ph
= Bn
R = iPr
4
3
R
R
R
R
5 = nBu
5 = tBu
5 = Ph
3
H2N
R
21
X = S
15 R5 = Bn
7 R3 = Et
X = S
16 R5 = nPr
1
N
N3
N3
2
3
2
4
N
1
6
5
2
N
N2
N
N
4
5
1 N
N 3
4
nPr
6
N
5 nPr
4
5
1
H2N
H2N
H2N
H2N
22
23
24
25
1
N
6
6
6
3
2
5
4
1
6
5
5
5
1 N
N
1 N
N
3
2
2
N
4
2
4
N
N
4
3
3
H2N
H2N
H2N
H2N
26
27
28
29
Figure 3. Compounds 1–29.
Table 2
lipophilic parameter (p) and their in vitro metHb inducing proper-
Physicochemical properties and in vitro methemoglobin formation for compounds 1–
19
ties.45 In accordance with general partition coefficient theory,46 for
each extension of the alkyl chain by a single carbon unit within
such a series, it is generally accepted that there is a corresponding
stepwise increase in the overall lipophilicity of the molecule. Eval-
uation of the partition coefficients of compounds 1–6 using RP-
HPLC methods revealed that oxadiazoles 1–3 increased in a step-
wise fashion, by approximately 0.3 log P units per carbon atom,
as the length of the alkyl chain increased. Contrary to expectation,
benzyl-substituted oxadiazole 6 was revealed to be less lipophilic
than phenyl-substituted oxadiazole 5, despite possessing an addi-
tional methylene unit. However, upon further investigation, such
an observation appears to be in agreement with Hansch’s hydro-
phobic constants for phenyl and benzyl substituents.47 Taken as
an approximate measure of the electron-richness of the aryl amine
ring, NMR experiments revealed the 13C–NH2 chemical shift for
oxadiazoles 1–6 (150.5–150.6 ppm) to closely resemble that of
benzocaine (150.7 ppm), suggesting that the 1,2,4-oxadiazole moi-
ety provides an electronic contribution similar to that of the ester
substituent present in benzocaine (Table 2).
a
b
Compd
X
R3
R5
LogPexp.
d
13C–NH2
%metHbc
1
2
3
4
5
6
7
8
O
O
O
O
O
O
S
O
O
O
O
O
O
O
O
S
Me
Et
—
—
—
—
—
—
—
Me
Et
nPr
iPr
nBu
tBu
Ph
Bn
nPr
—
1.89
2.22
2.61
2.57
3.48
3.24
2.39
1.63
1.92
2.27
2.23
2.66
2.53
2.91
2.82
2.53
1.71
2.13
2.54
150.5
150.6
150.5
150.5
150.6
150.6
149.8
149.5
149.5
149.5
149.5
149.5
149.8
149.7
149.6
149.1
149.2
149.1
149.1
49.2 1.3
54.3 0.6
61.0 3.6
56.5 1.8
8.8 1.2
nPr
iPr
Ph
Bn
Et
—
—
—
—
—
—
—
—
—
Me
Et
45.1 1.1
52.0 1.3
37.1 0.9
46.6 0.6
52.4 0.9
45.6 1.3
52.8 0.7
42.0 2.5
20.8 2.7
42.7 3.4
38.0 1.2
42.4 2.7
50.0 2.7
58.2 1.7
9
10
11
12
13
14
15
16
17
18
19
O
O
O
—
—
nPr
a
b
c
LogPexp. determined by RP-HPLC methods using a standard curve.
Owing to previously disclosed concerns over the stability of
benzocaine during in vitro metHb experiments, known metHb
inducers PAPP (metHb 24.8 0.6%) and PAVP (metHb 61.9 1.9%)
were employed as alternative positive controls (Fig. 4 and Table 1).
Note that in a previous study,45 PAVP was revealed to exhibit the
greatest toxicity in rats, hence, from a pharmacokinetic perspec-
tive, in terms of optimal lipophilicity, all compounds were subse-
quently modeled on PAVP. Oxadiazoles 1 (metHb 49.2 1.3%), 2
(metHb 54.3 0.6%) and 3 (metHb 61.0 3.6%) demonstrated a
steady increase in their capacity to induce the formation of metHb
Chemical shift for 13C–NH2 as measured by 13C NMR (in ppm).
Percentage methemoglobin formation in vitro at a compound concentration of
10
l
M (37 °C, 1 h, n = 3). Data represented as mean S.D.67
in vitro with increasing length of the alkyl chain substituent.
Branched chain oxadiazole 4 (metHb 56.5 1.8%) was found to
be marginally less active than its straight-chain homologue 3. A
considerable fall in metHb induction was recorded for phenyl oxa-
diazole 5 (metHb 8.8 1.2%), with a recovery in activity being

2226
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
70
60
50
40
30
20
10
0
3.5
3
19
4
2
12
10
7
18
1
3.0
9
11
6
15
17
16
20
8
2.5 Log P
2.0
21
14
5
1.5
Alkyl chain substituent (R); compound number above bar
2-Subsꢀtuted-1,3,4-oxadiazoles
2-Subsꢀtuted-1,3,4-thiadiazoles
5-Subsꢀtuted-1,2,4-oxadiazoles
5-Subsꢀtuted-1,2,4-thiadiazoles
3-Subsꢀtuted-1,2,4-oxadiazoles
3- and 5-Subsꢀtuted-1,2,5-oxadiazoles
Figure 4. Percentage methemoglobin formation in vitro for oxadiazoles 1–6, 8–15 and 17–21 and thiadiazoles 7 and 16 (37 °C, 1 h, n = 3). Data represented as mean S.D.
observed for benzyl oxadiazole 6 (metHb 45.1 1.1), possibly
relating to its increased conformational flexibility (Fig. 4 and
Table 2). Overall, the 1,2,4-oxadiazole moiety would appear to con-
stitute a suitable ester isostere with regard to its influence on the
methemoglobin-inducing properties of these molecules. With
these promising results in hand, the strategy of developing isoster-
ic analogues of benzocaine was further pursued.
elaboration of our oxadiazole series, it was postulated that the iso-
electronic replacement of the oxygen atom in our oxadiazole ring
system with sulfur would allow a subtle manipulation of the global
electronic contribution towards the aryl amine ring. With guidance
from physicochemical and in vitro metHb induction data derived
from previously prepared oxadiazoles, attention focused on the
preparation and evaluation of thiadiazoles 7 and 16. As expected,
thiadiazoles 7 and 16 were found to be marginally more lipophlilic
Although traditionally employed as an amide isostere,44 the
1,3,4-oxadiazole moiety was considered for investigation on the
grounds that it may possess comparable global electronics to that
of the 1,2,4-oxadiazole ring. As expected, the lipophilicity of oxadi-
azoles 8–10 increased in a stepwise trend, largely influenced by the
hydrophobic nature of the substituent at the 5-position. Compari-
son of the partition coefficients of oxadiazoles 8–10 to their 1,2,4-
oxadiazole counterparts (1–3) suggested that the former structural
subclass was inherently more polar; hence the inclusion of n-bu-
tyl-substituted oxadiazole 12, in an effort to attain a level of lipo-
philicity comparable to that of lead oxadiazole 3. Providing further
support of the counterintuitive trend in lipophilicity observed for
1,2,4-oxadiazoles 5 and 6, benzyl-substituted oxadiazole 15 was
again found to be marginally more polar than phenyl-substituted
oxadiazole 14. Oxadiazoles 8–12 (149.5 ppm) exhibited 13C–NH2
chemical shifts slightly upfield from those of both the correspond-
ing 1,2,4-oxadiazoles 1–4 (150.5–150.6 ppm) and benzocaine
(150.7 ppm) itself; such observations would suggest that the
1,3,4-oxadiazole moiety is marginally less electron-withdrawing
than both its 1,2,4-oxadiazole counterpart and the parent ester
group of benzocaine. Similar trends to those observed within the
1,2,4-oxadiazole series were noted, again suggesting that metHb
induction was strongly influenced by the lipophilic parameter,
with a general intolerance of side chain branching. Oxadiazoles
10 (metHb 52.4 0.9%) and 12 (metHb 52.8 0.7%) induced the
highest levels of metHb formation within the series, likely a
reflection of their favourable lipophilic profiles. However, when
compared to lead 1,2,4-oxadiazole 3 (metHb 61.0 3.6%), the
1,3,4-oxadiazole family were revealed to be slightly poorer induc-
ers of methemoglobin formation in vitro, one explanation for this
observation being that the weaker electron-withdrawing nature
of the 1,3,4-oxadiazole substituent provided a sub-optimal elec-
tronic contribution (Fig. 4 and Table 2).
than their corresponding oxadiazoles, compounds
2 and 10,
respectively. Likewise, the 13C–NH2 chemical shift of thiadiazole
7 (149.8 ppm) was revealed to be upfield from that of oxadiazole
2 (150.6 ppm), as was thiadiazole 16 (149.1 ppm) when compared
to oxadiazole 10 (149.5 ppm), indicating a subtle decrease in the
electron-withdrawing nature of the heterocyclic ring. In terms of
their metHb inducing properties, thiadiazole
7
(metHb
52.0 1.3%) and oxadiazole 2 (metHb 54.3 0.6%) were found to
be similar. Comparison of thiadiazole 16 (metHb 38.0 1.2%) with
its alkyl chain length equivalent oxadiazole 10 (metHb
52.4 0.9%), suggested that exchanging sulfur for oxygen within
the heterocyclic ring was detrimental in terms of its potential to in-
duce the formation of metHb in vitro. While the observed decrease
in activity for thiadiazoles 7 and 16 may be postulated as a conse-
quence of the marginally reduced electron-withdrawing potential
of the thiadiazole moiety, a second explanation may be a slight
structural intolerance of the larger sulfur atom within the hetero-
cyclic system (Fig. 4 and Table 2).
Based on the promising activity demonstrated by 5-(40-amino-
phenyl)-1,2,4-oxadiazoles, a select series of regioisomeric ana-
logues, namely 3-(40-aminophenyl)-1,2,4-oxadiazoles 17–19,
were prepared. As a result of the inferior in vitro metHb induction
displayed by oxadiazoles 4, 5, 6, 11, 14 and 15, the corresponding
regioisomeric 1,2,4-oxadiazoles were not pursued. Likewise, thia-
diazole analogues were discounted on similar grounds. As ob-
served previously, the lipophilicity of oxadiazoles 17–19
increased in a stepwise fashion as the length of the alkyl chain in-
creased (at the 5 position). Oxadiazoles 17–19 exhibited 13C–NH2
chemical shifts (149.1–149.2 ppm) significantly upfield of those
of regioisomeric oxadiazoles 1–3 (150.5–150.6 ppm), suggesting
that substitution at the 3-position of the 1,2,4-oxadiazole resulted
in a more electron-rich aryl amine ring when directly compared to
connection through the 5-position. Once again, familiar trends in
activity were observed within the series, whereby an increase in al-
kyl chain length, through oxadiazoles 17 (metHb 42.4 2.7%), 18
In studies involving oxadiazole-containing compounds it has
been demonstrated that an increase in potency can be achieved
through the corresponding thiadiazole.33,43,48 Thus, in a further

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2227
Table 4
(metHb 50.0 2.7%) and 19 (metHb 58.2 1.7%), correlated to
Hammett substituent constants51
greater in vitro metHb induction. Overall, the in vitro metHb
induction exhibited by oxadiazoles 17–19 was marginally inferior
to that of lead oxadiazole 3 (metHb 61.0 3.6%). Akin to 2-substi-
tuted-1,3,4-oxadiazoles 8–10, the lower levels of in vitro metHb
induction displayed by oxadiazoles 17–19 is most likely attributed
to the sub-optimal electronic contribution of the heterocycle (Fig. 4
and Table 2).
R
rpara
R
rpara
O
O
N
+0.45
+0.48
+0.50
+0.44
+0.48
+0.53
O
N
N
N
To complete our study on oxadiazoles as isosteres for the ester
substituent, the 1,2,5-oxadiazole43 moiety was investigated
through 3-(40-aminophenyl)-1,2,5-oxadiazole 20 (R4 = Et). Addi-
tionally, with the knowledge that 3/4-substituted 1,2,5-oxadiaz-
oles were anticipated to have the lowest electron-withdrawing
potential within the oxadiazole series, 4-(40-aminophenyl)-1,2,5-
oxadiazole 21 (R3 = OEt), possessing an inductively electron-with-
drawing heterocyclic ring substituent, was also evaluated. While
physicochemical evaluation demonstrated oxadiazole 20 to be
lipophilically comparable to lead oxadiazole 3, contrary to expecta-
tion, oxadiazole 21 was observed to have a higher log P than antic-
ipated (Table 3). The 13C–NH2 chemical shifts of oxadiazoles 20
(148.4 ppm) and 21 (148.6 ppm) were again revealed to be consid-
erably further upfield than those of benzocaine and lead oxadiazole
3. This confirmed the 1,2,5-oxadiazole ring to be less electron-
withdrawing than its 1,2,4-counterpart. Disappointingly, the
introduction of an ethoxy group at the 3-position of the 1,2,
5-oxadiazole ring resulted in only a subtle decrease in the elec-
tron-richness of the aryl amine ring. Interestingly, oxadiazole 21
(metHb 28.0 1.9%) exhibited significantly poorer in vitro metHb
induction compared to oxadiazole 20 (metHb 42.0 4.3%).
Considering that the incorporation of an alkoxy substituent into
the 1,2,5-oxadiazole ring did not significantly alter its electronic
profile, this result suggests there may be a structural intolerance
to such a modification (Fig. 4 and Table 3).
N
N
N
N
N
N
N
N
N
N
N
+0.56
+0.39
+0.63
n/a
N
H
N
N
N
alkylation of the 5-(1H)-tetrazole ring nitrogen, leading to
5-(2H)-tetrazoles, would be potentially analogous to the alkylation
of a carboxylic acid in the formation of an ester; thus permitting
the removal of the tetrazole’s acidic character with minimal impact
on the electronic properties of the heterocycle. With guidance from
in silico logP calculations, attention focused on the preparation and
evaluation of (1H)-tetrazole 22 and (2H)-tetrazole 23, each incor-
porating an alkyl substituent tailored to replicate those found in
the preceding oxadiazole series. While tetrazole 23 (metHb
42.4.0 3.3%) was demonstrated to be moderately active, albeit
inferior to the oxadiazoles per se, tetrazole 22 (metHb 8.4 0.5%)
was found to be a very poor inducer of methemoglobin in vitro. Gi-
ven the similar electronic contributions from their respective het-
erocyclic substituents, one explanation for the marked differences
in activity of tetrazoles 22 (148.5 ppm) and 23 (148.4 ppm) may
again be structurally founded (Table 3).
Traditionally employed as a heterocyclic isostere for the car-
boxyl group,49 the tetrazole moiety has received attention in the
literature as a viable alternative to the oxadiazole moiety.50 As evi-
denced by their literature Hammett substituent constants,51 the
Further structurally diverse heterocycles reported in the litera-
ture as potential surrogates for the oxadiazole moiety include the
six-membered diazine family.53 Upon closer examination of their
Hammett substituent constants, the pyrimidines and 3-substituted
pyridazines, together with para-substituted pyridines, were con-
firmed as having electronic properties similar to that of the ester
electronic profiles of 1-(1H)-tetrazole (
tetrazole ( para = +0.56) were demonstrated to be largely compara-
ble to that of the parent ethyl ester ( para = +0.45) of benzocaine
rpara = +0.50) and 5-(1H)-
r
r
(Table 4). Furthermore, tetrazoles have been reported to show
good resistance towards oxidative metabolism, a pathway reported
to be of possible concern in the deactivation of oxadiazoles.52 Inter-
group (Table 4).51 Despite no experimentally measured
rpara values
being available, pyrazines were also considered for further consid-
eration on the basis that their electrostatic mapping has been re-
ported to be similar to that of the 1,2,4-oxadiazole ring.53 In light
of these findings pyridine 24, pyrimidines 25–27, pyridazine 28
and pyrazine 29 were prepared for physicochemical and in vitro
evaluation. In general, heterocycles 24–29 were shown to be sig-
nificantly more polar than those compounds studied previously;
which in terms of their lipophilic profile had purposely been tai-
lored to resemble lead in vivo metHb inducer PAVP. Succinctly,
pyridine 24 (147.8 ppm), pyrimidines 25 (147.4 ppm), 26
(149.1 ppm) and 27 (149.5 ppm), pyridazine 28 (148.5 ppm) and
pyrazine 29 (148.3 ppm) exhibited a broad range of 13C–NH2
chemical shifts, and thus a probing array of electronic properties.
Gratifyingly, the trend in chemical shift of heterocycles 24–28
was in general agreement with their Hammett substituent con-
stants. However, when directly comparing the 13C–NH2 chemical
shifts of compounds 24–28 to benzocaine, these values appeared
to be significantly upfield from that suggested by their Hammett
constants;51 this would imply that their electron-withdrawing po-
tential was lower than that of ester derivatives. Pyrimidine 26
(metHb 14.0 3.5%) exhibited the highest level of in vitro metHb
induction within the series, followed by pyridine 24 (metHb
4.9 0.7%), pyrazine 29 (metHb 4.2 4.0%) and pyrimidine 27
estingly, the parent carboxylic acid substituent (rpara = +0.45) was
likewise revealed to be electronically similar, suggesting that ester-
ification has a minimal impact on the overall electronic contribu-
tion of the group. In light of this, it was postulated that
Table 3
Physicochemical properties and in vitro methemoglobin formation for compounds
20–29
a
b
Compound
LogPexp.
d
13C–NH2
%metHbc
20
21
22
23
24
25
26
27
28
29
2.46
2.79
1.84
2.29
1.47
1.58
1.67
1.70
1.54
1.68
148.4
148.6
148.5
148.4
147.8
147.4
149.1
149.5
148.5
148.3
42.0 4.3
28.0 1.9
8.4 0.5
42.4 3.3
4.9 0.7
0
14.0 3.5
3.5 0.5
0
4.2 4.0
a
b
c
LogPexp. determined by RP-HPLC methods using a standard curve.
Chemical shift for 13C–NH2 as measured by 13C NMR (in ppm).
Percentage methemoglobin formation in vitro at a compound concentration of
10
l
M (37 °C, 1 h, n = 3). Data represented as mean S.D.67

2228
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
(metHb 3.5 0.5%). Unexpectedly, pyrimidine 25 (metHb 0%) and
pyridazine 28 (metHb 0%) were found to be devoid of all activity.
Disappointingly no relationship could be identified between the
activity of heterocycles 24–29 and any one physicochemical
parameter (Table 3).
to possess considerable levels of hydrolytic activity, with each bio-
logical medium effecting ca. 50% and 100% loss of parent com-
pound, respectively (Table 6).
As a consequence of our failure to identify a pathway for the
putative metabolism of oxadiazoles 3 and 10, attention next turned
to the microsomal fraction of the rat liver as the potential source
for metabolic deactivation. A literature search revealed a number
of examples of microsomal CYP-catalyzed 1,2,4-oxadiazole ring
opening, primarily by means of a reductive57–59 or oxidative60–62
cleavage process, with the former being more prominent.59,60 Lan
et al.58 reported that 1,2,4-oxadiazoles undergo reductive ring
opening through an NADPH-dependent reductive NAO bond cleav-
age, forming amidine, amide and carboxylic acid metabolites. On
the other hand, cleavage of the 3-methyl-1,2,4-oxadiazole moiety
has been reported by Yabuki et al.,61 whereby hydroxylation of
the methyl group attached at the 3-position of the oxadiazole ring
resulted in a spontaneous rearrangement and formation of a novel
2.3. In vivo evaluation and stability studies
Having evaluated a number of isosteres of benzocaine for
metHb induction in vitro, attention next turned to their appraisal
in vivo—in an effort to determine whether such compounds
possessed sufficient toxicity to induce death in rats. Initially two
candidates were nominated, namely 1,2,4-oxadiazole 3 and 1,3,4-
oxadiazole 10; selected on the basis that they exhibited the great-
est in vitro metHb induction within the isostere series, combined
with the fact that they could potentially possess difference meta-
bolic stability profiles. PAVP, a proven methemoglobinemia-induc-
ing toxicant in rats, was to act as
a
positive control.
nitrile metabolite in rats. A similar oxidation/ring opening
Disappointingly, at doses of 120 mg/Kg, oxadiazoles 3 and 10 failed
to elicit a lethal endpoint in all instances (n = 4). Shortly after dos-
ing with compounds 3 and 10, rats exhibited symptoms character-
istic of sub-lethal methemoglobinemia (blue paws, tail and nose),
however these symptoms were short lived (ca. 45 min), with all
rats making a full recovery. Conversely, the symptoms observed
(blue paws, tail and nose, lethargy, ataxia, prone) for rats dosed
with PAVP continued to become more pronounced, leading to
death in all cases (4:4) (Table 5).
Having earlier demonstrated compounds 3 and 10 to be equipo-
tent to PAVP in vitro, attention next focused on determining possi-
ble reasons for their low toxicity in rats. One conceivable
hypothesis is that oxadiazoles 3 and 10 may have been the subject
of metabolism, thus rendering them inactive shortly after adminis-
tration. Through a series of stability experiments, employing vari-
ous biological mediums, the metabolic sensitivity of oxadiazoles 3
and 10 was evaluated. Firstly, the potential for chemical degrada-
tion of compounds 3 and 10 in the acidic environment of the stom-
ach following oral gavage was investigated. However, post-
incubation with simulated gastric fluid (SGF), analysis revealed a
negligible loss of parent compound in both examples, demonstrat-
ing these compounds to be stable at low pH. Next, given that the
serum is known to possess a large expression of metabolic
enzymes,54,55 attention turned to examining the stability of oxadi-
azoles 3 and 10 in a rat blood model. Subsequent incubation in the
presence of rat serum again revealed a minimal loss of parent com-
pound. Having successfully demonstrated stability against rat
blood enzymes, and with the knowledge that the liver possesses
the highest enzyme activity in the biological system of many mam-
mals, including rats, the metabolic breakdown pathway of com-
sequence involving 1,2,4-oxadiazoles has also been reported by
Bateman et al.62 Conversely, reported biotransformations of the
1,3,4-oxadiazole ring are rare, suggesting this regioisomer to be
generally more metabolically stable than its 1,2,4-counterpart.52,63
Maciolek et al.,64 reported that the 1,3,4-oxadiazole moiety, how-
ever, exhibits some susceptibility to oxidative ring opening, cata-
lyzed by the CYP1A2 isozyme expressed largely in rat liver
microsomes. In our hands, attempts to identify various routes for
the metabolic breakdown of oxadiazoles 3 and 10 through incuba-
tion with microsomes proved problematic. Identification of new
metabolites was further complicated as the microsomes likewise
facilitated the formation of the corresponding N-phenylhydroxyl-
amines, themselves unstable and prone to undergoing redox-
mediated side reactions with their nitrosobenzene oxidation
products, to generate a number of dimeric (azoxy and azo) metab-
olites.65 As a result of such complications, further efforts towards
the identification of the metabolic pathway responsible for the
detoxicification of oxadiazoles 3 and 10 were abandoned.
Instead, attention now turned to revealing oxadiazoles with im-
proved resistance to the metabolic pathways which compounds 3
and 10 were presumed vulnerable. One established literature
method for blocking the aforementioned CYP-induced side chain
a-hydroxylation is through the incorporation of a fluorine
substituent.66 Given that such a modification would likely have
an adverse effect on the global electronic properties of our hetero-
cycles, particularly the redox potential of the aryl amine, this strat-
egy was deemed unsuitable and not considered further. Instead, a
second recognized strategy to impede metabolism was imple-
mented through the introduction of further steric bulk around
the presumed site of metabolism, in an effort to make such hetero-
cycles poorer substrates for CYP enzymes. To test this approach,
iso-propyl- and tert-butyl-substituted 1,3,4-oxadiazoles, com-
pounds 11 and 13, respectively, were put forward for appraisal
in vivo; having earlier being demonstrated to be only marginally
inferior metHb inducers in vitro to related 1,3,4-oxadiazole 10.
On the grounds that tetrazoles52 have received attention for
pounds
3 and 10 was further probed in a hepatic model.
Incubation in the presence of rat liver S9 fraction56 again revealed
oxadiazoles 3 and 10 to be stable under assay conditions, suggest-
ing these compounds were likewise stable to hepatic hydrolases.
Note; using benzocaine as a positive control, both the rat serum
and S9 fraction employed in these experiments were demonstrated
Table 5
In vivo evaluation of PAVP and compounds 3, 10, 11, 13 and 23 in rats (po)
Compd
Dose (mg/Kg)
No of deaths observed
Symptoms recorded
PAVP
3
10
11
13
23
120
120
120
120
120
120
4/4
0/4
0/4
0/2
0/2
0/2
Blue paws, tail and nose; lethargy; ataxia; pronea
Faint blue paws, tail and nosea
Faint blue paws, tail and nosea
Faint blue paws, tail and earsa
Faint blue paws, tail and earsa
Faint blue paws, tail and earsa
a
Visual signs consistent with methemoglobinemia.

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2229
3.1.1. 5-(40-Aminophenyl)-3-methyl-1,2,4-oxadiazole (1)
Table 6
In vitro stability (low pH, rat blood and liver enzyme models) for benzocaine and
Compound 1 was prepared by a procedure similar to that of Lin
and Lang,23 To a stirring solution of 3-methyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (43) (50 mg, 0.24 mmol) in 1,4-dioxane (1 mL)
at 80 °C was added a hot solution (75 °C) of sodium sodium sulfide
nonahydrate (0.14 g, 0.58 mmol) in water (1 mL), and the reaction
mixture was stirred at 80 °C for 45 min. The solution was allowed
to cool and the solvent removed in vacuo. The residue was diluted
with ethyl acetate (50 mL), washed with water (50 mL), the sepa-
rated aqueous layer further extracted with ethyl acetate
(3 ꢂ 50 mL) and the combined organic phases dried over anhy-
drous sodium sulfate, filtered and the solvent removed in vacuo.
Purification by flash chromatography (hexane/ethyl acetate 9:1)
compounds 3 and 10
% Parent compound remaininga
Compd
SGFb
Rat serumc
Rat liver S9d
Benzocaine
3
10
>97
>97
>97
52 2.0e
>97
>97
0e
>97
>97
a
b
c
As determined by RP-HPLC.
Simulated gastric fluid without pepsin (pH 1.2, 37 °C, 1 h, n = 3).
80% rat serum in phosphate buffer (pH 7.4, 37 °C, 3 h, n = 3).
4 mg/mL rat liver S9 fraction in phosphate buffer (pH 7.4, 37 °C, 6 h, n = 3).
Major metabolite identified as para-aminobenzoic acid.
d
e
afforded
1 as a pale brown solid (37 mg, 0.21 mmol, 87%).
Rf = 0.45 (hexane/ethyl acetate, 1:1); mp 185–190 °C [lit.23 mp
186–188 °C]; 1H NMR (300 MHz; CDCl3) dH 2.42 (3H, s, CH3), 4.13
(2H, s, NH2), 6.72 (2H, d, J = 8.7 Hz, H-30, H-50) and 7.90 (2H, d,
J = 8.7 Hz, H-20, H-60); 13C NMR (75 MHz, CDCl3) dC 11.7 (CH3),
113.8 (C), 114.5 (CH), 129.8 (CH), 150.5 (C), 167.4 (C) and 175.7
(C); MS (ESI, 70 eV) m/z 176 (M+, 100%). Found (M+, 176.0822),
C9H10N3O requires 176.0818.
demonstrating superior resistance towards oxidative metabolism
than their oxadiazole counterparts, compound 23 was also selected
for further appraisal. Regrettably, at doses of 120 mg/Kg, oxadiaz-
oles 11 and 13, and tetrazole 23, failed to elicit death in all in-
stances (n = 2). Similar to observations recorded for compounds 3
and 10, all rats exhibited symptoms characteristic of sub-lethal
methemoglobinemia (blue paws, tail and ears), again without
attaining a lethal endpoint. Based on such observations, the order
of severity of methemoglobinemia was reported as (from most se-
vere to least severe case)—compound 23 > 13 > 11 (Table 5).
In summary, in an endeavor to further develop the known met-
hemoglobinemia-inducing agent benzocaine as a pest control
agent, a series of heterocyclic isosteres were prepared and evalu-
ated in vitro. Of these compounds, oxadiazoles 3 and 10 were
found to induce the formation of methemoglobin to the greatest
extent. Disappointingly, such promising findings in vitro failed to
translate into toxicity in rats in vivo, potentially a consequence
of the suboptimal metabolic stability of these compounds in the
test species.
3.1.2. 5-(40-Aminophenyl)-3-ethyl-1,2,4-oxadiazole (2)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-ethyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (44) (0.58 g, 2.64 mmol) in 1,4-dioxane (10 mL)
and sodium sulfide nonahydrate (1.52 g, 6.34 mmol) in water
(10 mL). Purification by flash chromatography (hexane/ethyl ace-
tate 4:1) afforded 2 as a pale brown solid (0.40 g, 2.13 mmol, 81%).
Rf = 0.30 (hexane/ethyl acetate, 2:1); mp 100–105 °C; 1H NMR
(400 MHz; CDCl3) dH 1.37 (3H, t, J = 8.0 Hz, CH2CH3), 2.79 (2H, q,
J = 8.0 Hz, CH2CH3), 4.22 (2H, s, NH2), 6.71 (2H, d, J = 8.8 Hz, H-30,
H-50) and 7.90 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz,
CDCl3) dC 11.4 (CH3), 19.8 (CH2), 113.7 (C), 114.3 (CH), 129.8 (CH),
150.6 (C), 171.8 (C) and 175.6 (C); IR (m
max/cmꢁ1) 683, 751, 825,
3. Experimental (chemistry)
1254 (C–N), 1322, 1484, 1581 (N–H), 1645, 2926, 2980, 3228,
3334 (N–H) and 3446; MS (ESI, 70 eV) m/z 190 (M+, 100%). Found
(M+, 190.0976), C10H12N3O requires 190.0975.
3.1. General experimental methods
Reactions were carried out using oven-dried glassware under
dry nitrogen unless stated otherwise. Anhydrous tetrahydrofuran
was distilled from sodium wire and dichloromethane was dried
over calcium hydride. Flash chromatography was carried out using
3.1.3. 5-(40-Aminophenyl)-3-propyl-1,2,4-oxadiazole (3)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-propyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (45) (0.77 g, 3.30 mmol) in 1,4-dioxane (10 mL)
and sodium sulfide nonahydrate (1.90 g, 7.92 mmol) in water
(10 mL). Purification by flash chromatography (hexane/ethyl ace-
tate 4:1) afforded 3 as a pale brown solid (0.46 g, 2.26 mmol,
69%). Rf = 0.32 (hexane/ethyl acetate, 2:1); mp 96-100 °C; 1H
NMR (400 MHz; CDCl3) dH 1.02 (3H, t, J = 7.3 Hz, CH2CH2CH3),
1.82 (2H, m, CH2CH2CH3), 2.74 (2H, t, J = 7.3 Hz, CH2CH2CH3),
4.13 (2H, s, NH2), 6.71 (2H, d, J = 8.4 Hz, H-30, H-50) and 7.91 (2H,
d, J = 8.4 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 13.7 (CH3),
20.5 (CH2), 28.0 (CH2), 113.8 (C), 114.4 (CH), 129.8 (CH), 150.5
silica gel (Riedel-de Haën, particle size 40-63 lm). Thin layer chro-
matography was performed using silica coated aluminium plates
(60, F254; Merck); compound identification was achieved through
staining with ninhydrin, potassium permanganate or iodine on
silica.
Melting point determinations were performed on an Electrom-
thermalÒ and X-4 Melting Point Apparatus and are reported,
uncorrected, in degrees Celsius (°C). Infrared spectra were recorded
using a Perkin–Elmer Spectrum 1000 series Fourier Transform IR
spectrometer as a thin film between sodium chloride plates; the
absorption maxima are expressed in wavenumbers (m
, cmꢁ1).
(C), 170.7 (C) and 175.5 (C); IR (m
max/cmꢁ1) 709, 759, 839, 1179,
NMR spectra were recorded on either a Bruker DRX300 spectrom-
eter (300 MHz for 1H nuclei and at 75 MHz for 13C nuclei); or a Bru-
ker DRX400 spectrometer (400 MHz for 1H nuclei and at 100 MHz
for 13C nuclei). All chemical shifts are reported in parts per million
(ppm) relative to CDCl3, d6-DMSO or MeOD. The 1H NMR data is re-
ported in the following order: chemical shift (d, ppm), relative inte-
gral, multiplicity (s, singlet; d, doublet; dd, doublet of doublets; dt,
doublet of triplets; t, triplet; q, quartet; m, multiplet), coupling
constant(s) (J in Hz), signal assignment. The 13C NMR data is re-
ported in the following format: chemical shift (d, ppm), degree of
hybridization. High resolution mass spectra were recorded on a
VG-70SE spectrometer using electrospray ionization (ESI) or atmo-
spheric pressure chemical ionization (APCI) methods.
1230 (C–N), 1326, 1491, 1578 (N–H), 1628, 2872, 2961, 3205,
3324 (N–H) and 3473; MS (ESI, 70 eV) m/z 204 (M+, 100%); Found
(M+, 204.1134), C11H14N3O requires 204.1131.
3.1.4. 5-(40-Aminophenyl)-3-isopropyl-1,2,4-oxadiazole (4)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 3-isopropyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (46) (0.24 g, 1.01 mmol) in 1,4-dioxane (5 mL)
and sodium sulfide nonahydrate (0.58 g, 2.42 mmol) in water
(5 mL). Purification by flash chromatography (hexane/ethyl acetate
2:1) afforded 4 as a pale brown solid (0.13 g, 0.64 mmol, 63%).
Rf = 0.30 (hexane/ethyl acetate, 2:1); mp 90-95 °C; 1H NMR
(400 MHz; CDCl3) dH 1.38 (6H, d, J = 7.2 Hz, CH(CH3)2), 3.12 (1H,

2230
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
m, CH(CH3)2), 4.15 (2H, s, NH2), 6.70 (2H, d, J = 8.6 Hz, H-30, H-50)
and 7.91 (2H, d, J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3)
dC 20.5 (CH3), 26.8 (CH), 113.8 (C), 114.3 (CH), 129.8 (CH), 150.5
Rf = 0.15 (hexane/ethyl acetate, 2:1); mp 190–194 °C; 1H NMR
(400 MHz; CDCl3) dH 2.57 (3H, s, CH3), 4.05 (2H, s, NH2), 6.72 (2H,
d, J = 8.6 Hz, H-30, H-50) and 7.81 (2H, d, J = 8.6 Hz, H-20, H-60); 13C
NMR (75 MHz, CDCl3) dC 11.0 (CH3), 113.7 (C), 114.6 (CH), 129.4
(C), 175.3 (C) and 175.5 (C); IR (m
max/cmꢁ1) 661, 766, 837, 1162,
1252 (C–N), 1387, 1490, 1583 (N–H), 1626, 2873, 2927, 3192,
3301 (N–H) and 3470; MS (ESI, 70 eV) m/z 204 (M+, 100%). Found
(M+, 204.1127), C11H14N3O requires 204.1131.
(CH), 149.5 (C), 162.6 (C) and 165.2 (C); IR (m
max/cmꢁ1) 701, 827,
1170 (C–O), 1349 (C–N), 1494, 1557 (N–H), 1600, 2927, 3214,
3335 (N–H) and 3399; MS (ESI, 70 eV) m/z 176 (M+, 100%); Found
(M+, 176.0822), C9H10N3O requires 176.0818.
3.1.5. 5-(40-Aminophenyl)-3-phenyl-1,2,4-oxadiazole (5)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-phenyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (47) (0.35 g, 1.32 mmol) in 1,4-dioxane (3 mL)
and sodium sulfide nonahydrate (0.76 g, 3.17 mmol) in water
(3 mL). Purification by flash chromatography (hexane/ethyl acetate
2:1) afforded 5 as a pale brown solid (0.26 g, 1.10 mmol, 83%).
Rf = 0.40 (hexane/ethyl acetate, 1:1); mp 165–170 °C; 1H NMR
(400 MHz; CDCl3) dH 4.15 (2H, s, NH2), 6.76 (2H, d, J = 8.8 Hz, H-
30, H-50), 7.49–7.51 (3H, m, ArH), 8.02 (2H, d, J = 8.8 Hz, H-20, H-
60) and 8.15 (2H, m, ArH); 13C NMR (100 MHz, CDCl3): dC = 114.0
(C), 114.5 (CH), 127.3 (C), 127.5 (CH), 128.8 (CH), 130.0 (CH),
3.1.9. 2-(40-Aminophenyl)-5-ethyl-1,3,4-oxadiazole (9)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-ethyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (65) (50 mg, 0.23 mmol) in 1,4-dioxane (1 mL)
and sodium sulfide nonahydrate (0.13 g, 0.55 mmol) in water
(1 mL). Purification by flash chromatography (hexane/ethyl acetate
1:1) afforded 9 as a pale brown solid (30 mg, 0.16 mmol, 70%).
Rf = 0.15 (hexane/ethyl acetate, 2:1); mp 138–142 °C; 1H NMR
(400 MHz; CDCl3) dH 1.41 (3H, t, J = 7.6 Hz, CH2CH3), 2.91 (2H, q,
J = 7.6 Hz, CH2CH3), 4.05 (2H, s, NH2), 6.72 (2H, d, J = 8.8 Hz, H-30,
H-50) and 7.82 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz,
CDCl3) dC 10.9 (CH3), 19.1 (CH2), 113.8 (C), 114.6 (CH), 128.8
130.9 (CH), 150.6 (C), 168.6 (C) and 176.0 (C); IR (m )
max/cmꢁ1
689, 748, 1217 (C–N), 1366, 1500, 1604 (N–H), 1739, 2970, 3221,
3324 (N–H) and 3392; MS (ESI, 70 eV) m/z 238 (M+, 100%). Found
(M+, 238.0970), C14H12N3O requires 238.0975.
(CH), 149.5 (C), 165.0 (C) and 166.8 (C); IR (m
max/cmꢁ1) 834, 1175
(C–O), 1366 (C–N), 1497, 1574 (N–H), 1608, 1738, 2924, 3216,
3332 (N–H) and 3446; MS (ESI, 70 eV) m/z 190 (M+, 100%); Found
(M+, 190.0975), C10H12N3O requires 190.0975.
3.1.6. 5-(40-Aminophenyl)-3-benzyl-1,2,4-oxadiazole (6)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-benzyl-5-(40-nitrophenyl)-
1,2,4-oxadiazole (48) (60 mg, 0.21 mmol) in 1,4-dioxane (5 mL)
and sodium sulfide nonahydrate (0.12 g, 0.51 mmol) in water
(5 mL). Purification by flash chromatography (hexane/ethyl acetate
4:1) afforded 6 as a pale brown solid (25 mg, 0.10 mmol, 47%).
Rf = 0.38 (hexane/ethyl acetate, 2:1); mp 89–92 °C; 1H NMR
(400 MHz; CDCl3) dH 4.10 (4H, s, CH2, NH2), 6.67 (2H, d,
J = 8.8 Hz, H-30, H-50), 7.25–7.38 (5H, m, ArH) and 7.88 (2H, d,
J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 32.4 (CH2),
113.8 (C), 114.4 (CH), 126.9 (CH), 128.6 (CH), 129.0 (CH), 129.9
3.1.10. 2-(40-Aminophenyl)-5-propyl-1,3,4-oxadiazole (10)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-propyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (66) (27 mg, 0.12 mmol) in 1,4-dioxane (1 mL)
and sodium sulfide nonahydrate (67 mg, 0.28 mmol) in water
(1 mL). Purification by flash chromatography (hexane/ethyl acetate
1:1) afforded 10 as a pale brown solid (23 mg, 0.11 mmol, 98%).
Rf = 0.15 (hexane/ethyl acetate, 1:1); mp 127–132 °C; 1H NMR
(400 MHz; CDCl3) dH 1.05 (3H, t, J = 7.5 Hz, CH2CH2CH3), 1.86 (2H,
m, CH2CH2CH3), 2.86 (2H, t, J = 7.5 Hz, CH2CH2CH3), 4.09 (2H, s,
NH2), 6.72 (2H, d, J = 8.4 Hz, H-30, H-50) and 7.82 (2H, d,
J = 8.4 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 13.6 (CH3),
20.1 (CH2), 27.2 (CH2), 113.7 (C), 114.6 (CH), 128.4 (CH), 149.5
(CH), 135.8 (C), 150.6 (C), 169.7 (C) and 176.0 (C); IR (m )
max/cmꢁ1
706, 754, 1181, 1289 (C–N), 1364, 1488, 1607 (N–H), 2924, 3029,
3217, 3345 (N–H) and 3427; MS (ESI, 70 eV) m/z 273 (MNa+,
100%). Found (MNa+, 274.0950), C15H13N3NaO requires 274.0951.
(C), 165.0 (C) and 165.8 (C); IR (m
max/cmꢁ1) 826, 1172 (C–O),
1315 (C–N), 1498, 1569 (N–H), 1607, 1739, 2970, 3207, 3324 (N–
H) and 3425; MS (ESI, 70 eV) m/z 204 (M+, 100%); Found (M+,
204.1131), C11H14N3O requires 204.1131.
3.1.7. 5-(40-Aminophenyl)-3-ethyl-1,2,4-thiadiazole (7)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-ethyl-5-(40-nitrophenyl)-
1,2,4-thiadiazole (53) (50 mg, 0.21 mmol) in 1,4-dioxane (2 mL)
and sodium sulfide nonahydrate (0.12 g, 0.51 mmol) in water
(2 mL). Purification by flash chromatography (hexane/ethyl acetate,
2:1) afforded 7 as a light brown solid (30 mg, 0.15 mmol, 69%).
Rf = 0.25 (hexane/ethyl acetate, 2:1); mp 138–143 °C; 1H NMR
(400 MHz; CDCl3) dH 1.42 (3H, t, J = 7.6 Hz, CH2CH3), 3.01 (2H, q,
J = 7.6 Hz, CH2CH3), 4.06 (2H, s, NH2), 6.70 (2H, d, J = 8.6 Hz, H-30,
H-50) and 7.75 (2H, d, J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz,
CDCl3) dC 12.3 (CH3), 26.6 (CH2), 114.3 (CH), 121.0 (C), 129.1 (CH),
3.1.11. 2-(40-Aminophenyl)-5-isopropyl-1,3,4-oxadiazole (11)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 3-isopropyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (67) (25 mg, 0.11 mmol) in 1,4-dioxane (1 mL)
and sodium sulfide nonahydrate (62 mg, 0.26 mmol) in water
(1 mL). Purification by flash chromatography (hexane/ethyl acetate
2:1) afforded 11 as a pale brown solid (18 mg, 0.09 mmol, 83%).
Rf = 0.15 (hexane/ethyl acetate, 1:1); mp 110–114 °C; 1H NMR
(400 MHz; CDCl3) dH 1.42 (6H, d, J = 7.2 Hz, CH(CH3)2), 3.23 (1H,
m, CH(CH3)2), 4.15 (2H, s, NH2), 6.73 (2H, d, J = 8.8 Hz, H-30, H-50)
and 7.82 (2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3)
dC 20.1 (CH3), 26.3 (CH), 113.8 (C), 114.6 (CH), 128.4 (CH), 149.5
149.8 (C), 178.6 (C) and 187.9 (C); IR (m
max/cmꢁ1) 819, 1174, 1302
(C–N), 1419, 1477, 1601 (N–H), 1650, 2972, 3328 (N–H) and
3386; MS (ESI, 70 eV) m/z 206 (M+, 100%); Found (M+, 206.0743),
(C), 164.9 (C) and 169.8 (C); IR (m
max/cmꢁ1) 834, 1176 (C–O),
C
10H12N3S requires 206.0746.
1365 (C–N), 1498, 1566 (N–H), 1608, 1738, 2971, 3220, 3328 (N–
H) and 3407; MS (ESI, 70 eV) m/z 204 (M+, 100%); Found (M+,
204.1133), C11H14N3O requires 204.1131.
3.1.8. 2-(40-Aminophenyl)-5-methyl-1,3,4-oxadiazole (8)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 2-methyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (64) (47 mg, 0.23 mmol) in 1,4-dioxane (1 mL)
and sodium sulfide nonahydrate (0.13 g, 0.55 mmol) in water
(1 mL). Purification by flash chromatography (hexane/ethyl acetate
2:1) afforded 8 as a pale brown solid (22 mg, 0.13 mmol, 55%).
3.1.12. 2-(40-Aminophenyl)-5-butyl-1,3,4-oxadiazole (12)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 3-butyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (68) (0.23 g, 0.93 mmol) in 1,4-dioxane (5 mL)
and sodium sulfide nonahydrate (0.54 g, 2.23 mmol) in water

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2231
(5 mL). Purification by flash chromatography (hexane/ethyl acetate
1:1) afforded 12 as a pale brown solid (69 mg, 0.30 mmol, 31%).
Rf = 0.40 (hexane/ethyl acetate, 1:1); mp 119–122 °C; 1H NMR
(400 MHz; CDCl3) dH 0.97 (3H, t, J = 7.5 Hz, CH2CH2CH2CH3), 1.45
(2H, m, CH2CH2CH2CH3), 1.81 (2H, m, CH2CH2CH2CH3), 2.88 (2H,
t, J = 7.5 Hz, CH2CH2CH2CH3), 4.05 (2H, s, NH2), 6.72 (2H, d,
J = 8.8 Hz, H-30, H-50) and 7.81 (2H, d, J = 8.8 Hz, H-20, H-60); 13C
NMR (100 MHz, CDCl3) dC 13.6 (CH3), 22.1 (CH2), 25.1 (CH2), 28.7
(CH2), 113.9 (C), 114.6 (CH), 128.4 (CH), 149.5 (C), 165.0 (C) and
thiadiazole (72) (50 mg, 0.20 mmol) in 1,4-dioxane (2.5 mL) and
sodium sulfide nonahydrate (0.12 g, 0.48 mmol) in water
(2.5 mL). Purification by flash chromatography (hexane/ethyl ace-
tate 2:1) afforded 16 as a light brown solid (20 mg, 0.09 mmol,
46%). Rf = 0.15 (hexane/ethyl acetate, 2:1); mp 102–107 °C; 1H
NMR (400 MHz; CDCl3) dH 1.04 (3H, t, J = 7.5 Hz, CH2CH2CH3),
1.84 (2H, m, CH2CH2CH3), 3.06 (2H, t, J = 7.5 Hz, CH2CH2CH3),
4.04 (2H, s, NH2), 6.70 (2H, d, J = 8.8 Hz, H-30, H-50) and 7.72 (2H,
d, J = 8.8 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 13.5 (CH3),
23.4 (CH2), 32.0 (CH2), 114.8 (CH), 120.4 (C), 129.3 (CH), 149.1
166.0 (C); IR (m
max/cmꢁ1) 828, 1173 (C–O), 1314 (C–N), 1498,
1572 (N–H), 1606, 1738, 2863, 2962, 3212, 3323 (N–H) and
(C), 168.5 (C) and 168.7 (C); IR (m
max/cmꢁ1) 842, 1174 (C–O),
3428; MS (ESI, 70 eV) m/z 218 (M+, 100%). Found (M+, 218.1279),
1306 (C–N), 1416, 1450, 1602 (N–H), 1623, 2852, 2961, 3211,
3322 (N–H) and 3414; MS (ESI, 70 eV) m/z 220 (M+, 100%); Found
(M+, 220.0906), C11H14N3S requires 220.0903.
C12H16N3O requires 218.1288.
3.1.13. 5-(40-Aminophenyl)-5-tert-butyl-1,3,4-oxadiazole (13)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 2-tert-butyl-5-(40-nitrophenyl)-
1,3,4-oxadiazole (69) (0.16 g, 0.63 mmol) in 1,4-dioxane (4 mL)
and sodium sulfide nonahydrate (0.36 g, 1.51 mmol) in water
(4 mL). Purification by flash chromatography (hexane/ethyl acetate
1:1) afforded 13 as a pale brown solid (0.11 g, 0.50 mmol, 79%).
Rf = 0.20 (hexane/ethyl acetate, 1:1); mp 140–145 °C; 1H NMR
(400 MHz; CDCl3) dH 1.46 (9H, s, C(CH3)3), 4.31 (2H, s, NH2), 6.72
(2H, d, J = 8.8 Hz, H-30, H-50) and 7.80 (2H, d, J = 8.8 Hz, H-20, H-
60); 13C NMR (100 MHz, CDCl3): dC 28.1 (CH3), 32.2 (C), 113.3 (C),
3.1.17. 3-(40-Aminophenyl)-5-methyl-1,2,4-oxadiazole (17)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 5-methyl-3-(40-nitrophenyl)-
1,2,4-oxadiazole (76) (36 mg, 0.18 mmol) in 1,4-dioxane (1 mL)
and sodium sulfide nonahydrate (0.10 g, 0.42 mmol) in water
(1 mL). Purification by flash chromatography (hexane/ethyl acetate
2:1) afforded 17 as a yellow-brown solid (13 mg, 0.07 mmol, 42%).
Rf = 0.20 (hexane/ethyl acetate, 2:1); 1H NMR (400 MHz; CDCl3) dH
2.62 (3H, s, CH3), 3.96 (2H, s, NH2), 6.72 (2H, d, J = 8.4 Hz, H-30, H-
50) and 7.85 (2H, d, J = 8.4 Hz, H-20, H-60); 13C NMR (100 MHz,
CDCl3): dC 12.4 (CH3), 114.7 (CH), 116.6 (C), 128.8 (CH), 149.1 (C),
168.3 (C) and 175.9 (C); MS (ESI, 70 eV) m/z 176 (M+, 100%); Found
(M+, 176.0823), C9H10N3O requires 176.0818.
114.4 (CH), 128.2 (CH), 149.8 (C), 164.9 (C) and 172.0 (C); IR (mmax
/
cmꢁ1) 831, 1161 (C–O), 1313 (C–N), 1500, 1557 (N–H), 1609, 2977,
3241, 3342 (N–H) and 3462; MS (ESI, 70 eV) m/z 218 (M+, 100%);
Found (M+, 218.1297), C12H16N3O requires 218.1288.
3.1.18. 3-(40-Aminophenyl)-5-ethyl-1,2,4-oxadiazole (18)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 5-ethyl-3-(4-nitrophenyl)-1,2,4-
oxadiazole (77) (0.25 g, 1.12 mmol) in dioxane (7 mL) and sodium
sulfide (0.28 g, 3.51 mmol) in water (7 mL). Purification by flash
chromatography (hexane/ethyl acetate 3:2) afforded 18 as a yellow
solid (0.15 g, 0.79 mmol, 70%). Rf = 0.50 (hexane/ethyl acetate, 3:2).
mp 94–99 °C/[lit.75 mp 96 °C]; 1H NMR (400 MHz; CDCl3): dH 1.43
(3H, t, J = 7.6 Hz, CH2CH3), 2.93 (2H, q, J = 7.6 Hz, CH2CH3), 3.95 (2H,
s, NH2), 6.72 (2H, d, J = 8.7 Hz, H-30, H-50) and 7.86 (2H, d, J = 8.7 Hz,
H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 10.9 (CH3), 20.4 (CH2),
114.8 (CH), 116.8 (C), 128.9 (CH), 149.1 (C), 168.2 (C) and 180.2
(C); MS (ESI, 70 eV) m/z 190 (M+, 100%); Found (M+, 190.0974),
C10H12N3O requires 190.0975.
3.1.14. 2-(40-Aminophenyl)-5-phenyl-1,3,4-oxadiazole (14)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 3-phenyl-5-(40-nitrophenyl)-1,3,4-
oxadiazole (70) (52 mg, 0.19 mmol) in 1,4-dioxane (3 mL) and so-
dium sulfide nonahydrate (0.11 g, 0.47 mmol) in water (3 mL).
Purification by flash chromatography (hexane/ethyl acetate 2:1)
afforded 14 as a pale brown solid (30 mg, 0.13 mmol, 65%).
Rf = 0.25 (hexane/ethyl acetate, 2:1); 1H NMR (400 MHz; CDCl3)
dH 4.11 (2H, s, NH2), 6.76 (2H, d, J = 8.8 Hz, H-30, H-50), 7.51–7.53
(3H, m, ArH), 7.93 (2H, d, J = 8.8 Hz, H-20, H-60) and 8.12 (2H, m,
ArH); 13C NMR (100 MHz, CDCl3) dC 113.7 (C), 114.7 (CH), 124.3
(C), 126.7 (CH), 128.7 (CH), 129.0 (CH), 131.3 (CH), 149.7 (C),
163.7 (C) and 165.0 (C); MS (ESI, 70 eV) m/z 238 (M+, 100%); Found
(M+, 238.0973), C14H12N3O requires 238.0975.
3.1.19. 3-(40-Aminophenyl)-5-propyl-1,2,4-oxadiazole (19)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 5-propyl-3-(4-nitrophenyl)-1,2,4-
oxadiazole (78) (0.34 g, 1.37 mmol) in dioxane (10 mL) and sodium
sulfide (0.28 g, 3.55 mmol) in water (10 mL). Purification by flash
chromatography (hexane/ethyl acetate 3:2) afforded 19 as a yellow
solid (0.16 g, 0.79 mmol, 54%). Rf = 0.80 (hexane/ethyl acetate, 1:1).
Mp 87–91 °C/[lit.75 mp 88 °C]; 1H NMR (400 MHz; CDCl3) dH 1.04
(3H, t, J = 7.5 Hz, CH2CH2CH3), 1.89 (2H, m, CH2CH2CH3), 2.88
(2H, t, J = 7.5 Hz, CH2CH2CH3), 3.95 (2H, s, NH2), 6.72 (2H, d,
J = 8.6 Hz, H-30, H-50) and 7.86 (2H, d, J = 8.6 Hz, H-20, H-60); 13C
NMR (100 MHz, CDCl3) dC 13.7 (CH3), 20.3 (CH2), 28.5 (CH2),
114.7 (CH), 116.8 (C), 128.9 (CH), 149.1 (C), 168.2 (C) and 179.3
(C); MS (ESI, 70 eV) m/z 204 (M+, 100%); Found (M+, 204.1133),
3.1.15. 2-(40-Aminophenyl)-5-benzyl-1,3,4-oxadiazole (15)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 3-benzyl-5-(40-nitrophenyl)-1,3,4-
oxadiazole (71) (0.25 g, 0.89 mmol) in 1,4-dioxane (5 mL) and so-
dium sulfide nonahydrate (0.51 g, 2.13 mmol) in water (5 mL).
Purification by flash chromatography (hexane/ethyl acetate 1:1)
afforded 15 as
a pale brown solid (20 mg, 0.08 mmol, 9%).
Rf = 0.30 (hexane/ethyl acetate, 1:1); mp 131–135 °C; 1H NMR
(400 MHz; CDCl3) dH 4.03 (2H, s, NH2), 4.24 (2H, s, CH2), 6.69
(2H, d, J = 8.4 Hz, H-30, H-50), 7.28–7.35 (5H, m, ArH) and 7.78
(2H, d, J = 8.4 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 31.9
(CH2), 113.6 (C), 114.6 (CH), 127.4 (CH), 128.5 (CH), 128.8 (CH),
128.8 (CH), 134.2 (C), 149.6 (C), 164.2 (C) and 165.5 (C); IR (mmax
/
cmꢁ1) 723, 841, 1179 (C–O), 1371 (C–N), 1498, 1571 (N–H),
1608, 1702, 2924, 3216, 3354 (N–H) and 3455; MS (ESI, 70 eV)
m/z 274 (MNa+, 100%); Found (MNa+, 274.0942), C15H13N3NaO re-
quires 274.0951.
C11H14N3O requires 204.1131.
3.1.20. 4-(40-Aminophenyl)-4-ethyl-1,2,5-oxadiazole (20)
Compound 20 was prepared by a procedure similar to that of
Boiani et al.31 To a stirring solution of 3-ethyl-4-(40-nitrophenyl)-
2-oxide-1,2,5-oxadiazole (80) (0.13 g, 0.59 mmol) and zinc dust
(0.19 g, 2.97 mmol) in tetrahydrofuran (5 mL) at room temperature
was added ammonium chloride (1.57 g, 59.36 mmol) in water
3.1.16. 2-(40-Aminophenyl)-5-propyl-1,3,4-thiadiazole (16)
A similar procedure23 to that previously described for the prep-
aration of 1 was followed using 2-propyl-5-(40-nitrophenyl)-1,3,4-

2232
D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
(5 mL), and the mixture heated under reflux for 5 h. The solution
was filtered through celite and the solvent removed in vacuo.
The resulting residue was taken up in ethyl acetate (50 mL),
washed with water (50 mL), the separated aqueous layer further
extracted with ethyl acetate (2 ꢂ 50 mL) and the combined organic
phases dried over anhydrous magnesium sulfate, filtered and the
solvent removed in vacuo. Purification by flash chromatography
(dichloromethane/methanol 199:1) afforded 20 as a yellow solid
(18 mg, 0.10 mmol, 16%). Rf = 0.30 (dichloromethane/methanol
199:1); mp 51–54 °C; 1H NMR (400 MHz; CDCl3): dH 1.38 (3H, t,
J = 7.4 Hz, CH2CH3), 2.91 (2H, q, J = 7.4 Hz, CH2CH3), 3.94 (2H, s,
NH2), 6.76 (2H, d, J = 8.4 Hz, H-30, H-50) and 7.51 (2H, d,
J = 8.4 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 11.9 (CH3),
18.2 (CH2), 115.0 (CH), 115.8 (C), 129.3 (CH), 148.4 (C), 153.3 (C)
3443; MS (ESI, 70 eV) m/z 204 (M+, 100%). Found (M+, 204.1238),
C10H14N5 requires 204.1244.
3.1.23. 5-(40-Aminophenyl)-2-propyl-2H-tetrazole (23)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 5-(40-nitrophenyl)-2-propyl-
2H-tetrazole (90) (0.11 g, 0.47 mmol) in 1,4-dioxane (2.5 mL) and
sodium sulfide nonahydrate (0.26 g, 1.08 mmol) in water
(2.5 mL). Purification by flash chromatography (hexane/ethyl ace-
tate 2:1) afforded 23 as a pale brown solid (89 mg, 0.44 mmol,
93%). Rf = 0.25 (hexane/ethyl acetate, 2:1); mp 74–78 °C; 1H NMR
(400 MHz; CDCl3) dH 0.98 (3H, t, J = 7.4 Hz, CH2CH2CH3), 2.06 (2H,
m, CH2CH2CH3), 3.97 (2H, s, NH2), 4.56 (2H, t, J = 7.4 Hz, CH2CH2-
CH3), 6.74 (2H, d, J = 8.4 Hz, H-30, H-50) and 7.93 (2H, d, J = 8.4 Hz,
H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 10.9 (CH3), 22.8 (CH2),
54.4 (CH2), 114.8 (CH), 117.4 (C), 128.0 (CH), 148.4 (C) and 165.2
and 154.4 (C); IR (m
max/cmꢁ1) 829, 1046, 1074, 1186, 1294, 1444,
1530 (N–O), 1608 (N–H), 1631, 2852, 2925, 3230, 3355 (N–H)
and 3447; MS (ESI, 70 eV) m/z 190 (M+, 100%). Found (M+,
190.0975), C10H12N3O requires 190.0975.
(C); IR (m
max/cmꢁ1) 763, 838, 1173, 1303 (C–N), 1442, 1460, 1610
(N–H), 2878, 2967, 3225, 3331 (N–H), 3372, 3431 and 3475; MS
(ESI, 70 eV) m/z 204 (M+, 100%). Found (M+, 204.1239), C10H14N5
requires 204.1244.
3.1.21. 4-(40-Aminophenyl)-3-ethoxy-1,2,5-oxadiazole (21)
Compound 21 was prepared by a two-step procedure similar
to that of Sauerberg and co-workers33 and a similar procedure23
to that previously described for the preparation of 1. In the first
step, to a stirring solution of sodium metal (10 mg, 0.26 mmol) in
ethanol (2 mL) at room temperature was added 3-chloro-4-
(40-nitrophenyl)-1,2,5-oxadiazole (84) (20 mg, 0.09 mmol), and
the reaction mixture stirred at room temperature for a further
2 h. The solvent was removed in vacuo and the resulting residue
was taken up in ethyl acetate (25 mL), washed with water
(25 mL), the separated aqueous layer further extracted with ethyl
acetate (2 ꢂ 25 mL) and the combined organic phases dried over
anhydrous magnesium sulfate, filtered and the solvent removed
in vacuo to afford crude 3-ethoxy-4-(40-nitrophenyl)-1,2,5-oxadi-
azole (85) as a light brown solid (12 mg), which was used without
further purification. In the second step, a similar procedure23 to that
previously described for the preparation of 1 was followed using
crude 3-ethoxy-4-(40-aminophenyl)-1,2,5-oxadiazole (85) (12 mg)
in 1,4-dioxane (1 mL) and sodium sulfide nonahydrate (25 mg,
0.10 mmol) in water (1 mL). Purification by flash chromatography
(hexane/ethyl acetate 4:1) afforded 21 as a brown solid (10 mg,
0.05 mmol, 55% over two steps); Rf = 0.35 (hexane/ethyl acetate,
1:1); mp 80–85 °C; 1H NMR (400 MHz; CDCl3): dH 1.53 (3H, t,
J = 7.2 Hz, CH2CH3), 4.50 (2H, q, J = 7.2 Hz, CH2CH3), 6.73 (2H, d,
J = 8.6 Hz, H-30, H-50) and 7.81 (2H, d, J = 8.6 Hz, H-20, H-60); 13C
NMR (100 MHz, CDCl3) dC 14.6 (CH3), 68.5 (CH2), 114.8 (CH),
115.1 (C), 128.9 (CH), 145.1 (C), 148.6 (C) and 163.4 (C); IR
3.1.24. 4-(40-Aminophenyl)pyridine (24)
Compound 24 was prepared by a procedure similar to that of
Detsi et al.40 A mixture of 4-(40-acetamidophenyl)pyridine (98)
(55 mg, 0.26 mmol) in methanol (2.5 mL) and hydrochloric acid
(2.5 mL, 2 M) was heated under reflux for 1 h. The reaction was
quenched with an aqueous solution of sodium hydroxide (20 mL,
1 M), extracted with ethyl acetate (20 mL ꢂ 2), dried over anhy-
drous sodium sulfate, filtered and the solvent removed in vacuo.
Purification by flash chromatography (hexane/ethyl acetate 1:1)
afforded 24 as a pale brown solid (39 mg, 0.23 mmol, 89%).
Rf = 0.25 (hexane/ethyl acetate, 1:1); 1H NMR (400 MHz; CDCl3)
dH 6.78 (2H, d, J = 8.4 Hz, H-30, H-50), 7.48–7.51 (4H, m, H-3, H-5,
H-20, H-60) and 8.51 (2H, d, J = 4.0 Hz, H-2, H-6); 13C NMR
(100 MHz, CDCl3) dC 115.3 (CH), 120.7 (CH), 127.2 (C), 127.9
(CH), 147.8 (C), 148.7 (C) and 149.2 (CH); MS (ESI, 70 eV) m/z
171 (M+, 100%); Found (M+, 171.0923), C11H11N2 requires
171.0917.
3.1.25. 5-(40-Aminophenyl)pyrimidine (25)
A similar procedure40 to that previously described for the prep-
aration of 24 was followed using 5-(40-acetamidophenyl)pyrimi-
dine (99) (65 mg, 0.30 mmol) in methanol (2.5 mL) and
hydrochloric acid (2 M, 2.5 mL). Purification by flash chromatogra-
phy (hexane/ethyl acetate 1:1) afforded 25 as a pale brown solid
(46 mg, 0.27 mmol, 88%). Rf = 0.30 (hexane/ethyl acetate, 1:1); 1H
NMR (400 MHz; CDCl3) dH 3.89 (2H, s, NH2), 6.80 (2H, d,
J = 8.8 Hz, H-30, H-50), 7.40 (2H, d, J = 8.8 Hz, H-20, H-60), 8.89 (2H,
s, H-4, H-6) and 9.11 (1H, s, H-2); 13C NMR (100 MHz, CDCl3) dC
115.6 (CH), 124.0 (C), 127.9 (CH), 134.2 (C), 147.4 (C), 154.1 (CH)
and 156.4 (CH); MS (ESI, 70 eV) m/z 172 (M+, 100%); Found (M+,
172.0869), C10H10N3 requires 172.0869.
(m
max/cmꢁ1) 573, 829, 988, 1031, 1189 (C–O), 1285 (C–N), 1380,
1490 (N-O), 1564, 1606, 1723, 2924, 2997, 3214, 3320 (N–H) and
3435; MS (ESI, 70 eV) m/z 206 (M+, 100%). Found (M+, 206.0928),
C10H12N3O2 requires 206.0924.
3.1.22. 1-(40-Aminophenyl)-5-propyl-1H-tetrazole (22)
A similar procedure23 to that previously described for the
preparation of 1 was followed using 1-(40-nitrophenyl)-5-propyl-
1H-tetrazole (88) (0.11 g, 0.47 mmol) in 1,4-dioxane (3 mL) and so-
dium sulfide nonahydrate (0.27 g, 1.13 mmol) in water (3 mL).
Purification by flash chromatography (hexane/ethyl acetate 2:1)
afforded 22 as a pale brown solid (50 mg, 0.25 mmol, 52%).
Rf = 0.15 (hexane/ethyl acetate, 2:1); mp 77–80 °C; 1H NMR
(400 MHz; CDCl3) dH 0.96 (3H, t, J = 7.6 Hz, CH2CH2CH3), 1.78 (2H,
m, CH2CH2CH3), 2.81 (2H, t, J = 7.6 Hz, CH2CH2CH3), 4.18 (2H, s,
NH2), 6.80 (2H, d, J = 8.6 Hz, H-30, H-50) and 7.15 (2H, d,
J = 8.6 Hz, H-20, H-60); 13C NMR (100 MHz, CDCl3) dC 13.6 (CH3),
20.6 (CH2), 25.3 (CH2), 115.0 (CH), 123.7 (C), 126.2 (CH), 148.5
3.1.26. 2-(40-Aminophenyl)pyrimidine (26)
A similar procedure40 to that previously described for the prep-
aration of 24 was followed using 2-(40-acetamidophenyl)pyrimi-
dine (100) (40 mg, 0.19 mmol) in methanol (2.5 mL) and
hydrochloric acid (2.5 mL, 2 M). Purification by flash chromatogra-
phy (hexane/ethyl acetate 2:1) afforded 26 as a pale brown solid
(31 mg, 0.18 mmol, 96%). Rf = 0.35 (hexane/ethyl acetate, 2:1); 1H
NMR (400 MHz; CDCl3) dH 3.95 (2H, s, NH2), 6.75 (2H, d,
J = 8.8 Hz, H-30, H-50), 7.05 (1H, t, J = 4.6 Hz, H-5), 8.26 (2H, d,
J = 8.8 Hz, H-20, H-60) and 8.70 (2H, d, J = 4.8 Hz, H-4, H-6); 13C
NMR (100 MHz, CDCl3) dC 114.6 (CH), 117.8 (CH), 127.8 (C),
129.7 (CH), 149.1 (C), 157.0 (CH) and 164.8 (C); MS (ESI, 70 eV)
(C) and 155.2 (C); IR (
1504, 1520, 1607 (N–H), 2873, 2960, 3235, 3348 (N–H) and
m
max/cmꢁ1) 811, 830, 1172, 1304 (C–N),

D. Conole et al. / Bioorg. Med. Chem. 22 (2014) 2220–2235
2233
m/z 172 (M+, 100%); Found (M+, 172.0873), C10H10N3 requires
172.0869.
(C–N), 1473, 1512, 1603 (N–H), 1655, 2851, 2923, 3076, 3216,
3340 (N–H) and 3400; MS (ESI, 70 eV) m/z 172 (M+, 100%); Found
(M+, 172.0870), C10H10N3 requires 172.0869.
3.1.27. 4-(40-Aminophenyl)pyrimidine (27)
A similar procedure40 to that previously described for the prep-
aration of 24 was followed using 4-(40-acetamidophenyl)pyrimi-
dine (104) (11 mg, 0.06 mmol) in methanol (1.25 mL) and
hydrochloric acid (1.25 mL, 2 M). Purification by flash chromatog-
raphy (hexane/ethyl acetate 1:1) afforded 27 as a pale brown solid
(9 mg, 0.06 mmol, 93%). Rf = 0.25 (hexane/ethyl acetate, 1:1); mp
195–200 °C [lit.76 mp 202–203 °C]; 1H NMR (300 MHz; CDCl3) dH
3.99 (2H, s, NH2), 6.76 (2H, d, J = 8.7 Hz, H-30, H-50), 7.59 (1H, dd,
J = 5.4, 2.0 Hz, H-5), 7.95 (2H, d, J = 8.7 Hz, H-20, H-60), 8.64 (1H,
d, J = 5.4 Hz, H-6) and 9.15 (1H, d, J = 1.6 Hz, H-2); 13C NMR
(75 MHz, CDCl3) dC 114.9 (CH), 115.5 (CH), 126.3 (C), 128.7 (CH),
149.5 (C), 156.9 (CH), 158.9 (CH) and 163.6 (C); MS (ESI, 70 eV)
m/z 172 (M+, 100%); Found (M+, 172.0871), C10H10N3 requires
172.0869.
4. Experimental (pharmacology)
4.1. RP-HPLC assay for purity, log P and stability determination
Purity and experimental log P values for all pharmacologically
evaluated compounds were assigned using RP-HPLC [Dionex Ulti-
mate 3000 System using a Phenomenex Gemini C18-Si column
(100 Å, 50 mm ꢂ 2.0 mm;
5 lm)]—eluted using a gradient of
99:1% A/B to 0:100% A/B over 60 min at 0.5 mL/min; where eluent
A was water and eluent B was acetonitrile—with detection at
254 nm. Solvents for RP-HPLC were purchased as HPLC grade and
used without further purification.
4.2. In vitro methemoglobin formation assay
3.1.28. 3-(40-Aminophenyl)pyridazine (28)
Microsomes pooled from male and female Sprague-Dawley rats
(250–350 g) were prepared using the classical differential sedi-
mentation method described by Gill et al.78 Microsomal protein
concentration was determined by the method of Lowry et al.79
CYP1A, CYP2B and CYP2E1 microsomal activity was determined
by the methods of Koop et al.80 and Pohl et al.81 Microsomes were
Compound 28 was prepared by a two-step procedure similar
to that of Guzzo et al.39 and a similar procedure40 to that previ-
ously described for the preparation of 24. In the first step, a mix-
ture of 3-chloro-6-(40-acetamidophenyl)pyridazine (102) (24 mg,
0.10 mmol), ammonium formate (32 mg, 0.50 mmol) and palla-
dium-on-carbon (6 mg, 0.02 mmol, 10% w/w) in methanol
(5 mL) was heated under reflux for 18 h. The reaction was
allowed to cool, filtered through celite and the solvent removed
in vacuo. The residue was diluted with ethyl acetate (50 mL),
washed with an aqueous solution of sodium hydroxide (50 mL,
1 M), the separated aqueous layer further extracted with ethyl
acetate (2 ꢂ 50 mL) and the combined organic phases dried over
anhydrous magnesium sulfate, filtered and the solvent removed
in vacuo to afford crude 3-(40-acetamidophenyl)pyridazine (105)
as a light brown solid (12 mg), which was used without further
purification. In the second step, a mixture of crude 3-(40-acet-
amidophenyl)pyridazine (105) (12 mg) in methanol (2.5 mL) and
hydrochloric acid (2 M, 2.5 mL) was heated under reflux for 1 h.
The reaction was quenched with an aqueous solution of sodium
hydroxide (20 mL, 1 M), extracted with ethyl acetate
(2 ꢂ 20 mL), dried over anhydrous sodium sulfate, filtered and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (hexane/ethyl acetate 1:3) afforded 28 as an off white solid
(10 mg, 0.06 mmol, 58% over two steps). Rf = 0.25 (hexane/ethyl
acetate, 1:3); mp 168–173 °C [lit.77 mp 173–174 °C]; 1H NMR
(400 MHz; CDCl3) dH 3.94 (2H, s, NH2), 6.80 (2H, d, J = 8.8 Hz,
H-30, H-50), 7.44 (1H, m, H-5), 7.75 (1H, dd, J = 8.8, 1.8 Hz, H-4),
7.94 (2H, d, J = 8.8 Hz, H-20, H-60) and 9.05 (1H, dd, J = 8.8 Hz,
J = 1.4 Hz, H-6); 13C NMR (100 MHz, CDCl3) dC 115.1 (CH), 122.7
(CH), 126.3 (C), 126.5 (CH), 128.3 (CH), 148.5 (C), 149.1 (CH)
and 159.2 (C); MS (ESI, 70 eV) m/z 172 (M+, 100%); Found (M+,
172.0871), C10H10N3 requires 172.0869.
diluted to 28 mg/ml, split into 300 lL aliquots and stored at
ꢁ80 °C; to ensure that each batch was only subjected to a single
freeze-thaw cycle prior to use. Erythrocytes, obtained from male
Sprague-Dawley rats, were centrifuged at 4000 rpm for 5 min at
4 °C using an IEC Centra CL3RÒ refrigerated centrifuge, then
washed with phosphate buffered saline (PBS) (0.10 M, pH 7.4),
re-centrifuged and re-suspended in PBS to approximately 50%
hematocrit. Standard metHb assay conditions similar to those of
Coleman et al.82 were followed: washed erythrocytes (100
microsomes (1 mg/ml), NADPH (1 mM) and compound (10
l
L),
lM,
1% v/v dimethyl sulfoxide), diluted with PBS (0.1 M, pH 7.4) to a fi-
nal volume of 200 lL, were incubated (37 °C, 1 h, n = 3) in an
Eppendorf Thermomixer CompactÒ. Dimethyl sulfoxide, PAPP and
PAVP were used as assay calibration standards. Subsequent to
incubation, samples were immediately put on ice and a 35 lL ali-
quot assayed for methemoglobin formation using an ABL700 series
blood-gas analyzer, 100–240 V, 50–60 Hz, 90 W.
4.3. Simulated gastric fluid assay
All in vivo candidates were appraised for hydrolytic stability in
the presence of SGF, without pepsin:83 [200
lL total volume, so-
dium chloride solution (0.03 M) acidified to pH 1.2 using concen-
trated hydrochloric acid; at a final compound concentration of
200 lM, 2.5% v/v dimethyl sulfoxide overall, 37 °C, n = 3]. Analysis
was performed by RP-HPLC at an injection volume of 5 lL.
4.4. Rat serum assay
3.1.29. 2-(40-Aminophenyl)pyrazine (29)
A similar procedure40 to that previously described for the prep-
aration of 24 was followed using 40-acetamidophenylpyrazine
(103) (35 mg, 0.19 mmol) in methanol (2.5 mL) and hydrochloric
acid (2 M, 2.5 mL). Purification by flash chromatography (hexane/
ethyl acetate 1:1) afforded 29 as a pale brown solid (22 mg,
0.13 mmol, 78%). Rf = 0.30 (hexane/ethyl acetate, 1:1); mp 125–
130 °C; 1H NMR (400 MHz; CDCl3) dH 3.94 (2H, s, NH2), 6.78 (2H,
d, J = 8.6 Hz, H-30, H-50), 7.85 (2H, d, J = 8.6 Hz, H-20, H-60), 8.47
(1H, d, J = 2.8 Hz, PyrH), 8.53 (1H, dd, J = 2.8, 1.2 Hz, PyrH) and
8.93 (1H, d, J = 1.6 Hz, PyrH); 13C NMR (100 MHz, CDCl3) dC 115.1
(CH), 126.3 (C), 128.1 (CH), 141.2 (CH), 141.5 (CH), 143.8 (CH),
Rat serum was obtained from Sigma–Aldrich and stored at
ꢁ78 °C. Similar assay conditions to those reported by Di et al.84 were
followed: [200 lL total volume, 80% rat serum (diluting with phos-
phate buffer (0.1 M, pH 7.4)), at a final compound concentration of
200 M, 2.5% v/v dimethyl sulfoxide overall, 37 °C, 3 h, n = 3] using
an Eppendorf Thermomixer Compact. Reactions were quenched by
transferring 150 L of the incubation mixture to 450 L of ice-cold
acetonitrile, affording a final compound concentration of 50 M.
l
l
l
l
Samples were centrifuged at 14,500ꢂg for 15 min at ambient tem-
perature using an Eppendorf Mini Spin PlusÒ centrifuge. Analysis
148.3 (C) and 152.8 (C); IR (m
max/cmꢁ1) 812, 1081, 1185, 1369
was performed by RP-HPLC at an injection volume of 50 lL.

2234
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Acknowledgments
The authors would like to acknowledge the Ministry of Science
and Innovation, New Zealand and Connovation Ltd, New Zealand
for their financial support and assistance (D.C.). Thanks also go to
Lee Shapiro (Connovation Ltd) and Elaine Murphy (Department
of Conservation/Lincoln University, New Zealand) for their invalu-
able help during the animal experiments, and to Rose-Marie Daniel
(Radiometer) for providing the blood-gas analyzer (in vitro methe-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.013.
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