Synthesis and methemoglobinemia-inducing properties of analogues of para-aminopropiophenone designed as humane rodenticides

Article abstract of DOI:10.1016/j.bmcl.2013.10.046

A number of structural analogues of the known toxicant para- aminopropiophenone (PAPP) have been 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 for alkyl analogues of PAPP (aminophenones 1-20; compound 6 metHb% = 74.1 ± 2). Besides lipophilicity, this structural sub-class suggested there were certain structural requirements for activity, with both branched (10-16) and cyclic (17-20) alkyl analogues exhibiting inferior in vitro metHb induction. Of the four candidates (compounds 4, 6, 13 and 23) evaluated in vivo, 4 exhibited the greatest toxicity. In parallel, aminophenone bioisosteres, including oximes 30-32, sulfoxide 33, sulfone 34 and sulfonamides 35-36, were found to be inferior metHb inducers to lead ketone 4. Closer examination of Hammett substituent constants suggests that a particular combination of the field and resonance parameters may be significant with respect to the redox mechanisms behind PAPPs metHb toxicity.

Full text of DOI:10.1016/j.bmcl.2013.10.046

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6629–6635
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and methemoglobinemia-inducing properties of analogues
of para-aminopropiophenone designed as humane rodenticides
a
b
b
c,d
David Rennison a, , Daniel Conole , Malcolm D. Tingle , Junpeng Yang , Charles T. Eason
,
⇑
Margaret A. Brimble 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 structural analogues of the known toxicant para-aminopropiophenone (PAPP) have been
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 for alkyl analogues of PAPP (aminophenones 1–20; com-
pound 6 metHb% = 74.1 2). Besides lipophilicity, this structural sub-class suggested there were certain
structural requirements for activity, with both branched (10–16) and cyclic (17–20) alkyl analogues
exhibiting inferior in vitro metHb induction. Of the four candidates (compounds 4, 6, 13 and 23) evalu-
ated in vivo, 4 exhibited the greatest toxicity. In parallel, aminophenone bioisosteres, including oximes
30–32, sulfoxide 33, sulfone 34 and sulfonamides 35–36, were found to be inferior metHb inducers to
lead ketone 4. Closer examination of Hammett substituent constants suggests that a particular combina-
tion of the field and resonance parameters may be significant with respect to the redox mechanisms
behind PAPPs metHb toxicity.
Received 30 August 2013
Revised 22 October 2013
Accepted 23 October 2013
Available online 31 October 2013
Keywords:
para-Aminopropiophenone (PAPP)
Methemoglobin
Methemoglobinemia
Rodenticide
Rat toxicant
Ó 2013 Elsevier Ltd. All rights reserved.
Rats can be counted amongst man’s most serious competitors
causing substantial damage to agricultural interests worldwide.
The World Health Organization estimates that 20% of all human
food is destroyed or contaminated by rodents each year,1 while
one US government report claims that each rat damages up to
$10 worth of food and stored grains per annum, and contaminates
5–10 times that amount.2 More recently, the Food and Agriculture
Organization of the United Nations reported that, globally, rats
ruined more than 42 million tons of food, worth an estimated
US$30 billion p.a.3 On a domestic level, property damage can also
be estimated in millions of dollars annually, often the product of
building fires attributed to rodents chewing through lead gas pipes
or stripping insulation from electrical wires.3 In addition to their
substantial contribution to economic loss, rats constitute a major
threat to health, primarily as a consequence of the diseases they
harbour. Acting as vectors for both viral and bacterial diseases, rats
are responsible for transmitting more than 35 types of disease to
humans including cholera, dysentery, salmonella and the bubonic
plague.2 In the past century alone, more than 10 million people
have died from rodent-borne diseases.3 Furthermore, as an inva-
sive species second only to humans, rats are responsible for a loss
of biodiversity through predation and habitat destruction.4
In New Zealand alone, almost NZ$200 million is spent on verte-
brate pest control products annually.5 Most of these, however,
share common disadvantages such as secondary non-target poi-
soning, risks of environmental contamination, and accumulation/
persistence of residues in the food chain. Moreover, most of these
agents lack humaneness. In the 1980s, the United States Fish and
Wildlife Services investigated para-aminopropiophenone 2 (PAPP)
(Fig. 1) for the control of coyotes (Canis latrans), demonstrating it to
be highly toxic to canids per se.6 Subsequently, PAPP has been eval-
uated in both New Zealand and Australia for the humane control of
introduced predators such as feral cats and stoats.7–9 In 2011 PAPP
was successfully registered for the control of these pests in New
Zealand, and has since been marketed under the trade name Preda-
STOPÒ.10,11 In addition to its intrinsic toxicity against such problem
species, the use of PAPP as a pest control agent offers a number of
complimentary benefits—one being its comparatively humane
mode of action.7 PAPP induces its lethal effect by reducing oxygen
supply to the brain, leading to symptoms such as progressive leth-
argy, drowsiness, stupor and ultimately unconsciousness prior to
death. From a welfare perspective, this mechanism of toxicity
⇑
Corresponding authors. Tel.: +64 9 373 7599x83881 (D. Rennison); tel.: +64 9
373 7599x88259; fax: +64 9 373 7422 (M.A. Brimble).
E-mail address: d.rennison@auckland.ac.nz (D. Rennison).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.046

6630
D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
O
X
NADH-cytochrome b r eductase
(MtHb reductase)
Aminophenones 1-29
R
R
2
R = CH2CH3 (PAPP)
H2
N
HbFe3+ (metHb)
Compounds 30-36
HbFe2+
H2
N
Figure 1. Aminophenones 1–29, including PAPP 2, and compounds 30–36 (amin-
ophenone bioisosteres); see Tables 1 and 3.
RED BLOOD CELL
O2
H2O2
OH- + OH
could be viewed as advantageous in minimizing animal distress.7
Moreover, the time to death is relatively short, usually within
1–2 h following ingestion.12,13 Having induced its toxic effect, PAPP
is readily converted to comparatively innocuous metabolites, and
as a consequence is unlikely to be present in carcasses at levels that
will cause secondary poisoning to non-target species, nor should it
persist in the food chain.6 Indeed, should the accidental poisoning
of non-target species such as pets or farm dogs occur, a simple and
relatively inexpensive antidote, methylene blue, is available.14 Fur-
ther to this, PAPP is relatively non-toxic to most bird species (LD50
>100 mg/kg),6,7,13,15 so from an avian conservation perspective this
agent is particularly attractive.
O
O
HO
N
H
O=N
PNPP 2b
PHAPP 2a
d iaphorase
Mechanism of action—The toxic effects of PAPP can be attributed
to its capacity to induce the formation of methemoglobin
(metHb),16 leading to a physiological phenomenon known as
methemoglobinemia, an impairment of the red blood cells’ (eryth-
rocytes) capacity to transport oxygen—resulting in cerebral hypox-
ia, central nervous system depression and ultimately death.15 Upon
systemic absorption, PAPP is metabolized by hepatic mixed func-
tion oxidases to para-hydroxylaminopropiophenone 2a (PHAPP).17
Once taken up into the erythrocyte, and in the presence of oxygen,
PHAPP undergoes a coupled, or two-stage, redox reaction with
hemoglobin (HbFe2+) to form methemoglobin (HbFe3+, metHb),
the toxic endpoint (Fig. 2). The first stage involves the oxidation
of PHAPP to para-nitrosopropiophenone 2b (PNPP), and the com-
plementary reduction of molecular oxygen to hydrogen peroxide.
Consequently, the hydrogen peroxide reacts in situ to oxidize
hemoglobin to methemoglobin.18 Catalytic methemoglobin induc-
tion is an NADPH-dependent process involving reductive enzymes
present in erythrocytes such as diaphorase and NADP flavin reduc-
tase, as well as the antioxidant glutathione. These enzymes work to
drive the catalytic cycle of metHb formation via the back-reduction
of PNPP to PHAPP, the net result being a small quantity of PAPP
having the capacity to turnover a large number of hemoglobin
equivalents to methemoglobin.17,19 To regulate this process
NADH-cytochrome b reductase oxidase, also referred to as methe-
moglobin reductase, is known to reduce methemoglobin back to
hemoglobin.20,21 In addition to PAPP’s well documented properties
as a toxicant, Pan et al.22 demonstrated that for a homologous
series of related aminophenones, acute oral toxicity in rats was ele-
vated for p-aminobutyrophenone 3 and p-aminovalerophenone 4,
when directly compared to p-aminoacetophenone 1, p-aminoca-
prophenone 5 and PAPP itself (Table 3). Although investigated no
further, Pan et al attributed such observations to be the result of
high levels of methemoglobineamia. On the basis of such findings
it was now our intention to prepare a series of PAPP-related ana-
logues to further investigate the structural features and physico-
chemical properties responsible for, and the in vitro biological
markers indicative of, the in vivo toxicity of aminophenones in
rats.
NADPH flavin reductase
glutathione
PASSIVE DIFFUSION
LIVER
O
O
HEPATIC OXIDATION
HO
N
H
H2N
PHAPP 2a
PA
PP 2
Figure 2. Postulated mechanism for the in vivo formation of metHb by PAPP.
oxide, afforded the corresponding aminophenones (Scheme 1).
Aminoacetophenone 1 was subject to a one-step Ru(II)-catalyzed
a
-alkylation25 with the appropriate alcohol or aldehyde, in the
presence of dichlorotris(triphenylphosphine)ruthenium(II) and
potassium hydroxide at elevated temperature, to afford aminophe-
nones 6–9, 23 and 25–29 (Scheme 2). As a consequence of our
failure to access pivaloyl aminophenone 13 using the above Fri-
edel–Crafts acylation route,23 13 was synthesized through a
Cu(I)-promoted acylation26 of p-bromobenzoyl chloride (38) with
tert-butyllithium in the presence of copper(I) bromide dimethyl
sulfide complex, again progressing via a bromophenone intermedi-
ate. Subsequent amination afforded pivaloyl aminophenone 13
over two steps. Aminophenone 21 was prepared from p-bromo-
benzoyl chloride using Friedel–Crafts chemistry (Scheme 3).
Treatment of ketone 2 with O-methyl hydroxylamine hydro-
chloride and sodium carbonate at elevated temperature27 directly
afforded O-methyl ketoxime 30 in 70% yield, exclusively as the E
isomer (Scheme 4). The synthesis of O-methyl
a-chloroaldoxime
31 commenced with the condensation28 of p-nitrobenzaldehyde
(39) and hydroxylamine hydrochloride to afford aldoxime 40 in
90% yield. Alkylation29 of 40 with methyl iodide led to O-methyl
aldoxime 41 in 59% yield. Treatment of 41 with N-chlorosuccini-
mide28 afforded O-methyl
a
-chloroaldoxime 42 in 39% yield, with
subsequent reduction30 using iron and hydrochloric acid furnish-
ing the desired O-methyl -chloroaldoxime 31 in 35% yield,
exclusively as the Z isomer (Scheme 5). The preparation of
O-methyl -cyanoaldoxime 32 began with the chlorination of
aldoxime 40, as previously described,28 to give 43. The cyano group
was installed through treatment of -chloroaldoxime 43 with
Synthesis—Aminophenones 3, 5, 10–12, 14–20 and 22 were pre-
pared over two steps, via their corresponding bromophenones, by
Friedel–Crafts acylation23 of bromobenzene (37) with the appro-
priate acid chloride, employing anhydrous aluminum chloride
under solvent-free conditions. Subsequent copper(I)-catalyzed
amination,24 using aqueous ammonia in the presence of copper(I)
a
a
a

D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
6631
O
Table 1
a, b
Physicochemical properties and in vitro methemoglobin formation for compounds
R
1–29 (see Fig. 1)
H2
N
Br
37
d
13C–NH2
% metHbc
a
b
No.
R
logPexp.
3
R = (CH2
)
2CH3
4CH3
(16%)
(31%)
(15%)
(2%)
5
R = (CH2
)
1
CH3
1.34
1.65
2.06
2.48
3.01
3.49
3.91
4.58
5.15
2.02
2.44
2.46
2.46
2.84
2.85
2.95
1.82
2.29
2.71
3.07
2.42
2.58
3.07
3.20
1.84
2.58
2.92
3.81
3.37
151.1
151.0
150.9
151.0
150.8
150.8
150.8
150.8
150.9
150.9
150.8
151.0
149.8
151.1
151.2
150.8
150.9
150.8
150.7
150.8
150.9
151.2
151.3
151.1
151.3
151.1
151.1
150.9
150.9
4.5 0.9
15.2 3.1
40.8 2.2
58.1 3.8
70.9 0.7
74.1 2.8
54.2 1.0
29.0 1.6
13.3 1.0
19.7 2.8
31.1 1.2
36.9 1.7
54.2 1.6
49.0 1.0
49.5 0.7
52.7 1.6
23.7 2.1
29.3 3.3
33.5 1.1
40.5 1.6
38.0 3.6
47.0 1.9
57.3 1.5
29.6 1.4
31.0 1.1
45.5 1.1
52.3 1.3
51.1 0.3
62.4 0.4
10
R = CH(CH3
)
2
2
CH2CH3
11 R = CH(CH3)CH2CH3
(49%)
(6%)
12
14
R = CH2CH(CH3
)
R = CH(CH3)CH2C2 H2CH3
3
(CH2)2CH3
4
(CH2)3CH3
(8%)
15 R = CH2CH(CH3)CH2CH3
(33%)
(91%)
(7%)
16
17
R = CH2CH2CH(CH3
)
2
5
(CH2)4CH3
R = CHc(CH2
)
2
3
4
5
6
(CH2)5CH3
18 R = CHc(CH2
)
(67%)
(38%)
(21%)
19
20
R = CHc(CH2
R = CHc(CH2
)
)
7
(CH2)6CH3
8
(CH2)8CH3
22 R = CH2C6H5
9
(CH2)10CH3
CH(CH3)2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Scheme 1. Reagents and conditions: (a) RCOCl, AlCl3, rt, 3–6 h; (b) aq NH3, CuO,
DMSO, 90 °C (sealed-tube), 16 h.
CH(CH3)CH2CH3
CH2CH(CH3)2
C(CH3)3
CH(CH3)CH2CH2CH3
CH2CH(CH3)CH2CH3
CH2CH2CH(CH3)2
CHc(CH2)2
CHc(CH2)3
CHc(CH2)4
CHc(CH2)5
Ph
CH2Ph
CH2CH2Ph
CH@CHPh
CH2CH2-(2-pyridyl)
CH2CH2-(2-furanyl)
CH2CH2-(2-thienyl)
CH2CH2CHc(CH2)5
CH2CH2CH2Ph
O
O
a
R
H2N
H2
N
1
6
R = (CH2
)
5CH3
(17%)
(9%)
7
R = (CH2
)
6CH3
8CH3
10CH3
2Ph
8
R = (CH2
)
(11%)
(9%)
9
R = (CH2)
(35%)
(4%)
23
R = (CH2
)
25 R = (CH2
)
2-(2-pyridyl)
2-(2-furanyl)
2-(2-thienyl)
(15%)
(40%)
(19%)
(24%)
26
27
R = (CH2
R = (CH2
)
)
28 R = (CH2
)
2CHc(CH2
)
5
29
R = (CH2)3Ph
Scheme 2. Reagents and conditions: (a) RCH2OH (for 6–9, 29) or RCHO (for 23, 25–
29), RuCl2(PPh3)3, KOH, 1,4-dioxane, 90 °C (sealed-tube), 42 h.
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).
O
O
a or b, c
Percentage methemoglobin formation in vitro at a compound concentration of
Cl
R
10
l
M (37 °C, 1 h, n = 3). Data represented as mean SD.50
Br
H2
R = C(CH3
N
38
13
21 R = Ph
)
(6%, 2 steps)
(22%, 2 steps)
3
Table 2
In vitro metabolism and in vitro erythrocytic uptake for compounds 1–6 (see Fig. 1)
Scheme 3. Reagents and conditions: (a) tBuLi, CuBrÁCH3SCH3, THF, À78 °C, 4 h (for
13); (b) AlCl3, C6H6, reflux, 36 h (for 21); (c) aq NH3, CuO, DMSO, 90 °C (sealed-
tube), 16 h.
No.
% Microsomal metabolisma
% Erythrocytic uptakeb
1
2
3
4
5
6
7.63 1.1
11.0 1.8
16.7 2.8
15.3 2.8
14.9 1.3
17.8 2.5
46.6 3.0
59.8 2.2
70.4 1.3
78.9 0.8
88.5 2.3
94.0 0.5
O
O
N
a
H2N
H2N
a
Percentage loss of parent compound post incubation with rat liver microsomes
30
2
(70%)
and NADPH (37 °C, 0.5 h, n = 4). Data represented as mean SE.44
b
Percentage compound extracted from rat erythrocytes following incubation
Scheme 4. Reagents and conditions: (a) NH2OMeÁHCl, Na2CO3, EtOH, H2O, reflux,
(37 °C, 0.5 h, n = 4). Data represented as mean SE.44
36 h.
sodium hydroxide at elevated temperature, afforded O-methyl
Table 3
a
-cyanoaldoximes 45 in 66% yield. Ultimately, 45 was reduced as
Physicochemical properties and in vitro methemoglobin formation for compounds
30–36 (see Fig. 1)
described previously30 to access the desired O-methyl
a-cya-
a
b
noaldoxime 32 in 32% yield, exclusively as the Z isomer (Scheme 5).
Note that the geometrical assignment of (Z)-oximes 31, 32, 41–45
was made through comparison of their 1H NMR data to that of
related literature examples.30
No.
X
R
logPexp.
d
13C–NH2
% metHbc
30
31
32
33
34
35
36
N@OCH3
N@OCH3
N@OCH3
S@O
SO2
SO2NH
SO2NCH3
CH2CH3
Cl
CN
(CH2)4CH3
(CH2)4CH3
(CH2)3CH3
(CH2)3CH3
2.30
2.42
2.44
2.26
2.53
2.05
2.73
147.3
148.7
149.3
149.4
151.3
150.5
150.4
6.4 0.8
8.2 0.9
8.4 1.9
13.2 0.8
33.5 2.9
5.1 0.4
22.0 0.5
Aminophenyl alkyl sulfoxide 33 and aminophenyl alkyl sulfone
34 were prepared from the common nitrophenyl alkyl sulfide
intermediate 47, which in turn was obtained by thiolation31 of
p-chloronitrobenzene (46), using n-pentanethiol in the presence
of potassium hydroxide at elevated temperature. Reduction32 of
nitrophenyl alkyl sulfide 47 with tin(II) chloride afforded an ami-
nophenyl alkyl sulfide intermediate, which was subsequently sub-
jected to mild oxidation conditions33 using hydrogen peroxide to
afford the desired aminophenyl alkyl sulfoxide 33 in low yield over
two steps. Oxidation31 of nitrophenyl alkyl sulfide 47 using
m-chloroperoxybenzoic acid yielded a nitrophenyl alkyl sulfone
intermediate. Selective nitro group reduction31 using tin(II)
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 SD.50
sodium cyanide in the presence of triethylamine,30 affording
-cyanoaldoxime 44 in 19% yield. Alkylation30 of 44 using methyl
iodide, in the presence of tetra-n-butylammonium bromide and
a

6632
D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
OR
X
O
O
OR
N
N
O
S
NH2
a, b
c
N
R
a
g
H
X
O2N
O2N
O2N
O2N
H2N
48
39
49
R = H
(68% over 2 steps)
(82%)
50 R = Me
31
32
40 X = H, R = H
41
(90%)
(59%)
R = Me, X = Cl (35%)
(32%)
R = Me, X = CN
X = H, R = Me
via steps b-f
42 X = Cl, R = Me (39%)
(90%)
(19%)
(66%)
43 X = Cl, R = H
44
O
O
d
S
X = CN, R = H
45 X = CN R = Me
35 R = H
(38%)
N
R
36
R = Me (86%)
H2N
Scheme 5. Reagents and conditions: (a) NH2OHÁHCl, NaOH, H2O, EtOH, rt, 3 h (to
give 40); (b) (i) 40, NaH, DMF, rt, 0.25 h, (ii) MeI, DMF, rt, 0.5 h (41); (c) 41, NCS,
DMF, 0 °C ? rt, 48 h (42); (d) 40, NCS, DMF, 0 °C ? rt, 24 h (43); (e) 43, NaCN, NEt3,
iPrOH, DCE, H2O, 0 °C, 5 h (44); (f) 44, MeI, TBAB, NaOH, THF, H2O, 70 °C, 3 h (45);
(g) 42, Fe, aq 10 M HCl, EtOH, H2O, 70 °C, 3 h (31) or 45, Fe, aq 10 M HCl, EtOH, H2O,
70 °C, 3 h (32).
Scheme 7. Reagents and conditions: (a) (i) NaNO2, aq 10 M HCl, H2O, 0 °C, 0.25 h,
(ii) SOCl2, CuCl, H2O, 0 °C ? rt, 1.25 h; (b) CH3(CH2)3NH2, THF, rt, 0.5 h; (c) NaH,
MeI, 0 °C ? reflux, 18 h; (d) 49, Fe, NH4Cl, MeOH, H2O, rt ? 70 °C, 2.5 h (to give 35)
or 50, Fe, NH4Cl, MeOH, H2O, rt ? 70 °C, 2.5 h (36).
determine the effect of alkyl chain branching on in vitro metHb
induction. Despite possessing similar partition coefficients,
branched aminophenones 11 (metHb 31.1 1.2%) and 12 (metHb
36.9 1.7%) were demonstrated to induce lower levels of metHb
chloride furnished aminophenyl alkyl sulfone 34, again in low
yield, over two steps (Scheme 6).
The first step towards the synthesis of aminophenyl alkyl sul-
fonamide 35 involved diazotization34 of 4-nitroaniline (48), with
subsequent treatment of the resulting diazonium salt with thionyl
chloride yielding a sulfonyl chloride intermediate. Subsequent
amination35 with butylamine afforded nitrophenyl alkyl sulfon-
amide 49 in 68% yield over two steps. Reduction36 of the nitro
group of 49 using iron and ammonium chloride at elevated
temperature furnished the desired aminophenyl alkyl sulfonamide
35 in 38% yield. Methylation37 of nitrophenyl alkyl sulfonamide 49
using sodium hydride and methyl iodide afforded N-methyl
nitrophenyl alkyl sulfonamide 50 in 82% yield, which underwent
reduction of the nitro group as described previously36 to afford
aminophenyl alkyl N-methyl sulfonamide 36 in 86% yield
(Scheme 7). Aminophenones 1, 2, and 4 were obtained from com-
mercial sources.38,39 All new compounds were fully characterized
spectroscopically (see Supplementary data).
when compared to straight chain aminophenone
4 (metHb
58.1 1.5%). This pattern was further reinforced on comparing
aminophenones 14 (metHb 49.0 1.0%), 15 (metHb 49.5 0.7%)
and 16 (metHb 52.7 1.6%) to aminophenone
5 (metHb
70.9 0.7%) (Table 1, Figs. 1 and 3).
Contrary to the trend, pivaloyl aminophenone 13 (metHb
54.2 1.6%), constituting the greatest degree of alkyl chain branch-
ing, induced the highest levels of metHb formation within the
branched chain series. This anomalous result may have been influ-
enced by the slightly lower electron-withdrawing nature of the
pivaloyl substituent, relative to other alkyl aminophenones within
the series; as evidenced by the upfield chemical shift of its 13C–NH2
signal (d 149.8 ppm), taken as an approximate measure of electron-
richness of the aryl amine ring, when compared to the same carbon
in PAPP (d 151.0 ppm). Such observations were largely in agree-
ment with the literature, with the reported Hammett substituent
constant for the pivaloyl substituent (rpara (COtBu) +0.32) being
appreciably lower than that of the propionyl group (rpara (COEt)
+0.48).41 One plausible explanation for this anomaly has been pro-
posed by Bowden et al.,42 who suggested that the electronic contri-
bution of the pivaloyl group is less than that of its branched
counterparts due to its steric bulk interacting with the aromatic
ring, resulting in significant twisting and subsequent deconjuga-
Biological activity—Structural and electronic considerations
aside, the aforementioned trend in acute oral toxicity reported by
Pan et al.,22 presumed to be a consequence of methemoglobinemia
in vivo, suggests that there is a potential correlation between the
lipophilic parameter (p) and the metHb inducing properties of
these compounds. Alkyl aminophenones—In accordance with gen-
eral partition coefficient theory,40 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. Evaluation of the partition
coefficient of aminophenones 1–20 using RP-HPLC methods con-
firmed that their lipophilic properties increased in a stepwise fash-
ion, by approximately 0.4 log P units per carbon atom, as the length
of the alkyl chain increased (Table 1, Fig. 1). Straight chain amin-
ophenones 1–9 exhibited a steady increase in their capacity to in-
duce the formation of metHb in vitro until the alkyl chain reached
6 carbons in length (compound 6 metHb 74.1 2.8%), after which
further extension caused the level of metHb induction to decline.
Building on this established relationship, further structural
exploration through aminophenones 10–16 was undertaken to
tion of the carbonyl group relative to the plane of the
p-system.
To complete the series a selection of cyclic alkyl chain aminophe-
nones, namely 17–20, were prepared. In vitro evaluation of amin-
ophenones 17 (metHb 23.7 2.1%), 18 (metHb 29.3 3.3%), 19
(metHb 33.5 1.6%) and 20 (metHb 40.5 1.1%) suggested that
metHb induction was again strongly influenced by the lipophilic
parameter, though once again the levels of metHb formation
in vitro were found to be lower when compared to those of the
unbranched alkyl aminophenone series (Table 1, Figs. 1 and 3).
Aryl aminophenones—In a parallel study, attention focused on the
preparation and evaluation of a series of aryl aminophenones. De-
spite sharing similar lipophilic profiles, aminobenzophenone 21
(metHb 39.1 2.1%) displayed markedly lower levels of in vitro
metHb induction when compared to alkyl aminophenone 4 (metHb
58.1 3.8). The introduction of a methylene spacer between the
carbonyl group and the aryl ring of 21 afforded aminophenone 22
(metHb 47.0 1.9%), and a moderate gain in activity, while further
extending the length of the spacer led to a stepwise increase in
metHb formation, as demonstrated in aminophenones 23 (metHb
57.3 1.5%) and 29 (metHb 62.4 0.4%). Conversely, replacement
of the saturated ethylene spacer of dihydrochalcone 23 with an
O
S
b, c
H2N
33
(2% over 2
steps)
Cl
S
a
O
O
S
O2N
O2N
46
47 (91%)
d, e
H2N
34 (7% over 2
steps)
olefin bond, generating an
a,b-unsaturated ketone, resulted in a
notable decrease in metHb induction, as demonstrated by aminoch-
alcone 24 (metHb 29.6 1.4%) (Table 1, Fig. 1). Given that the
observed 13C–NH2 chemical shifts of aryl aminophenones 21–24
Scheme 6. Reagents and conditions: (a) CH3(CH2)4SH, KOH, DMF, 100 °C, 6 h; (b)
SnCl2, EtOH, 70 °C, 5 h; (c) H2O2, H2O, 70 °C, 1 h; (d) m-CPBA, CH2Cl2, 0 °C ? rt, 4 h;
(e) SnCl2, EtOH, 70 °C, 4 h.

D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
6633
80
70
60
50
40
30
20
10
0
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Log
P
1
2
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
7
9
11
Number of carbons in the alkyl chain (R); compound number above bar
Straight chain
Branched chain
Cyclic chain
Figure 3. Percentage methemoglobin formation in vitro for aminophenones 1–20 (37 °C, 1 h, n = 3). Data represented as mean SD.50
and 29 were closely related to that of PAPP (d 151.0 ppm) this again
suggests that the electronic contribution of the side chain to the
25
aryl amine ring has a minimal effect on the observed activity, even
in the case of aminobenzophenone 21 (d 150.9 ppm) and aminoch-
20
alcone 24 (d 151.1 ppm), where conjugation was anticipated to be a
factor. Such observations were again generally in agreement with
15
the literature, for example, the experimentally determined Ham-
mett substituent constants for the propionyl (rpara (COEt) +0.48)
and benzoyl (rpara COPh +0.43) groups, as featuring in PAPP and
aminobenzophenone 21, respectively, were likewise found to be
comparable.41 Given that both the partition coefficients and elec-
tronic properties of aminochalcone 24 and aminodihydrochalcone
23 (d 151.3 ppm) were largely analogous, it is hypothesized that a
loss in conformational flexibility may be responsible for the
observed fall in metHb induction in the former. In summary, aryl
aminophenone homologues 21–23 and 29 exhibited a similar trend
to that observed within the parallel alkyl aminophenone series,
again predominantly influenced by the partition coefficient.
Finally, substitution of the phenyl group of compound 23 with a
selection of heteroaromatic ring equivalents completed our aryl
aminophenone series. As anticipated, the partition coefficients of
aminophenones 25–27 were largely influenced by the contribution
of the heterocycle. All heterocyclic bioisosteres 25 (metHb
34.5 1.1%), 26 (metHb 50.6 1.1%), 27 (metHb 58.2 1.3%)
explored exhibited decreased in vitro metHb induction compared
to aminodihydrochalcone 23 (metHb 63.9 0.6%). This was partic-
ularly evident in the case of the more polar examples, whereas
non-aromatic aminophenone 28 (metHb 56.9 0.3%) appeared to
be of comparable efficacy; once again such observations largely
correlated to the lipophilic parameter.
In vitro summary (aminophenones)—From these preliminary
studies, it became apparent that metHb induction in vitro, as ob-
served in vivo, was strongly influenced by the lipophilic parameter
of the analogue. One hypothesis for the aforementioned observa-
tions is that in vitro, lead aminophenones 5 and 6 are more readily
metabolized to their corresponding N-hydroxyl metabolites.43
However, when measuring the loss of parent compound following
incubation in the presence of rat liver microsomes and NADPH, col-
laborative research by Yang44 demonstrated that while compounds
1 and 2 appeared less susceptible to metabolism, no significant
difference was observed between aminophenones 3–6 (Table 2,
Fig. 4). A second explanation could relate to the uptake of these
N-hydroxyl metabolites into the erythrocyte. Uncharged molecules
10
5
0
1
2
3
4
5
6
Compound
Figure 4. Effect of increasing alkyl chain length on the metabolism of aminophe-
nones 1–6 following incubation in the presence of rat liver microsomes and NADPH
(37 °C, 0.5 h, n = 4). Data represented as mean SE.44
are predominantly transported into erythrocytes via passive diffu-
sion down a concentration gradient, thus the greater levels of
in vitro metHb induction observed for aminophenones 5 and 6
may be a result of the enhanced penetration of their corresponding
N-hydroxyl metabolites across the red blood cell membrane.45
Indeed, further studies by Yang44 would suggest that there is a
relationship between the lipophilicity of the aminophenone and
its uptake into erythrocytes (Table 2, Fig. 5); note that these exper-
iments were conducted using the parent aminophenones and not
their N-hydroxyl metabolites—in silico generated partition coeffi-
cients would suggest the log P values of N-hydroxyaminophenones
to be 0.5–1logP units below that of the corresponding aminophe-
nones. A third rationalization for the observed tail off in metHb
induction at log P values greater than 3.5 may be that these com-
pounds simply begin to lose their solubility under assay conditions,
leading to precipitation and consequently reduced metabolism/
uptake.
In vivo evaluation (aminophenones)—Interestingly, the degree of
lipophilicity required for maximum in vitro metHb induction did
not appear to correlate with the aforementioned observations of
Pan et al.,22 with respect to in vivo toxicity, in rats. To validate this
discrepancy, four candidates were nominated for appraisal in vivo,
namely aminophenones 4, 6, 13 and aminodihydrochalcone 23.
Aminophenone 4 was found to be the most toxic compound
in vivo, again, presumably a consequence of methemoglobinemia,

6634
D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
100
80
60
40
20
0
In a study employing oxime ethers as ester surrogates, Bro-
midge et al.48 demonstrated that the pKa of azabicyclic muscarinic
agonists could be manipulated through the introduction of an
electron-withdrawing group into the oxime function. In light of
this O-methyl
a-chloroaldoxime 31 and O-methyl a-cya-
noaldoxime 32 were prepared and subsequently demonstrated to
be of comparable lipophilicity to both ketoxime 30 and ketone
4.47 As anticipated, the electronic trend within this series was of
increasing 13C–NH2 chemical shift relative to the increasing elec-
tron-withdrawing potential of the oxime derivative, as evidenced
by compounds 30 (d 147.3 ppm), 31 (d 148.7 ppm) and 32 (d
149.3 ppm). Disappointingly, ketoxime 30 (metHb 6.4 0.8%) in-
duced only low levels of metHb in vitro, which was postulated to
be a consequence of the relatively weak electron-withdrawing
nature of the oxime ether moiety compared with that of the parent
1
2
3
4
5
6
Compound
ketone. However, contrary to expectation,
a-chloroaldoxime 31
Figure 5. Effect of increasing alkyl chain length on the uptake of aminophenones 1–
6 into rat erythrocytes post incubation (37 °C, 0.5 h, n = 4). Data represented as
mean SE.44
(metHb 8.4 0.9%) and -cyanoaldoxime 32 (metHb 8.2 1.9%)
a
were likewise found to induce similarly low levels of metHb, thus
providing no clear relationship between the electronic parameter
and metHb induction within this series. In summary, these results
would suggest that the derivatized oxime moieties were insuffi-
ciently electron-withdrawing to provide an optimal electronic
contribution to the aryl amine ring. Structural requirements should
also be considered with respect to in vitro metHb activity, as deriv-
atized ketoxime moieties within this series may not be as well tol-
erated as the ketone (Table 3, Fig. 1).
while compounds 6 and 23, both potent metHb inducers in vitro,
were shown to be considerably less toxic. In both cases, rats exhib-
ited symptoms characteristic of sub-lethal methemoglobinemia
(blue paws, tail and nose, lethargy and ataxia), and in one example
in each treatment group death eventuated. Surprisingly, compound
13 was found to be largely devoid of toxicity at the administered
dose (Table 4). One possible explanation for these in vitro/in vivo
discrepancies could relate to aminophenones 3 and 4 exhibiting
greater bioavailability than aminophenones 5 and 6. With these
additional complicating factors in mind, synthesis of metHb induc-
ers from this point forward focused on the design of bioisosteric
analogues possessing a log P value similar to that of ketone 4, an
established potent in vivo metHb inducer,22 despite aminophenone
6 exhibiting superior in vitro metHb induction.
In an effort to further extend our study on the role of electronics
in metHb toxicity, an emphasis was now placed on carbonyl isos-
teric replacements possessing stronger electron-withdrawing
properties; as evidenced by their literature Hammett substituent
constants41—including bioisosteres based on the sulfoxide (rpara
(SOMe) +0.49) and sulfone (
tionally a bioisostere for the carboxyl moiety,49 the sulfonamide
rpara (SO2NMe2) +0.65) group was also considered, due to its
r
para (SO2Me) +0.72) groups.41,46 Tradi-
(
Carbonyl isosteres—Attention next turned to investigating the
role of the ketone itself. Taking into consideration the aforemen-
tioned biochemical mechanism behind metHb induction, it was
postulated that the electronic contribution of the ketone to the
aryl amine ring was fundamental to PAPP’s toxicity. To further
probe this theory a number of carbonyl bioisosteres were ex-
plored, starting with the commonly employed oxime moiety.46
With guidance from in silico partition coefficient calculations,
O-methyl ketoxime 30 was proposed, incorporating an O-alkyl
chain tailored to achieve a lipophilic profile comparable to that
of lead methemoglobinemia inducing agent aminophenone 4
(our most potent in vivo metHb inducer).47 As predicted, the
experimental partition coefficient of ketoxime 30 was comparable
to that of 4. The 13C–NH2 chemical shift of compound 30
(d 147.3 ppm), however, was found to be appreciably upfield of
that of PAPP (d 151.0 ppm), suggesting the O-alkyl ketoxime
moiety to be less electron-withdrawing than the parent ketone
(Table 3, Fig. 1).
evidently suitable electronic properties. With guidance from in sil-
ico log P calculations, aminophenyl sulfoxide 33, sulfone 34, and
sulfonamides 35 and 36 were proposed, again each incorporating
an alkyl chain tailored to achieve a lipophilic profile comparable
to that of lead aminophenone 4.47
Sulfone 34 (metHb 33.5 2.9%), bearing the largest positive
Hammett substituent constant within the series, demonstrated
the greatest level of activity, while sulfoxide 33 (metHb
13.2 0.8%), possessing a rpara value virtually analogous to that
of the ketone group, was found to be a poor inducer of metHb
in vitro. Again, despite its perceived favorable electronics, sulfon-
amide 35 (metHb 5.1 0.4%) exhibited the weakest in vitro metHb
induction within the study. The low level of activity observed for
compound 35 was loosely attributed to the presence of the N–H
hydrogen bond donor function of the sulfonamide, and its pre-
sumed detrimental impact on the mechanisms behind metHb
induction. Gratifyingly, N-alkylation of sulfonamide 35 to reveal
N-methyl sulfonamide 36 (metHb 22.0 0.5%) led to a significant
Table 4
In vivo evaluation of compounds 1–6, 13 and 23, in rats (p.o.)
No.
LD50 (mg/Kg)22
Dose (mg/Kg)
No. of deaths observed
Symptoms recorded
1
2
3
4
5
6
13
23
381 22
221 26
85 31
85 31
216 43
—
—
—
—
60
—
120
120
120
—
—
—
5/7
—
1/4
0/2
1/4
—
—
—
Blue paws, tail and nose; lethargy; ataxia; pronea
—
Blue paws, tail and nose; lethargy; ataxiaa
Blue paws; lethargya
—
—
Blue paws, tail and nose; lethargy; ataxiaa
a
Visual signs consistent with methemoglobinemia.

D. Rennison et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6629–6635
6635
Table 5
References and notes
Hammett substituent constants41
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Mouse; World Health Organization, 1971.
R
rpara
F
R
COEt
COtBu
SOMe
SO2NMe2
SO2Me
+0.48
+0.32
+0.49
+0.65
+0.72
+0.34
+0.26
+0.52
+0.44
+0.53
+0.14
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À0.03
+0.21
+0.19
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recovery in activity. Largely in disagreement with their literature
Hammett substituent constants, the 13C–NH2 chemical shift of sul-
fone 34 (d 151.3 ppm) was found to closely resemble that of ketone
2 (d 151.0 ppm), while that of sulfoxide 33 (d 149.4 ppm), and to a
lesser extent sulfonamide 36 (d 150.4 ppm), were revealed to be
less comparable (Table 3, Fig. 1).
One explanation for such anomalies, or at least in the example
of sulfoxide 33, may relate to the ‘component specific’ nature of
each substituent’s electronic contribution. The Hammett substitu-
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and a resonance (R) component—the former being dictated by the
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derived from the delocalization of the electrons in a
p-system.
On the surface, evaluation of the Hammett substituent constants
for the propionyl and sulfoxide groups, as featured in PAPP and
sulfoxide 33, respectively, would suggest these substituents to be
relatively similar in terms of their electronic nature. Upon closer
inspection of each substituents corresponding F and R components,
however, the resonance (R) parameter is seen to afford a larger
contribution to the overall rpara constant of that found in PAPP,
compared with that found for sulfoxide 33 (Table 5). It could be
postulated that such variations in F/R composition influence the
critical electronic factors behind metHb toxicity, namely
N-hydroxylation and the accompanying coupled redox cycle.
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methemoglobinemia-inducing agent PAPP, a series of related
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Acknowledgements
46. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.
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-
moglobin experiments).
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48. Bromidge, S. M.; Brown, F.; Cassidy, F.; Clark, M. S. G.; Dabbs, S.; Hadley, M. S.;
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1997, 40, 4265.
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50. Standard metHb assay conditions similar to those of Coleman et al. (Pharm.
Pharmacol. 1991, 43, 779–784) were adopted: washed erthyrocytes (100
were incubated (37 °C, 1 h, n = 3) with compound (10 mM) in DMSO (1% v/v),
microsomes (1 mg/mL) and NADPH (1 mM), with a final volume of 200 L,
diluted with PBS (0.1 M, pH 7.4), in an Eppendorf Thermomixer Compact. PAPP
was used as a standard to calibrate the assay. Subsequent to incubation,
lL)
l
samples were immediately put on ice and a 35 lL aliquot of sample assayed for
CO-oximetry parameters using an ABL700 series blood-gas analyzer, 100–
240 V, 50–60 Hz, 90 W.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10.046.