Design and synthesis of prodrugs of the rat selective toxicant norbormide

Article abstract of DOI:10.1016/j.bmc.2012.05.014

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2- pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, one major drawback of NRB as a viable rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due to either its unpleasant 'taste' or rapid onset of effects. A series of NRB prodrugs were prepared in an effort to 'mask' this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Compound 19 displayed the most promising profile with respect to a delay in the onset of symptoms and was subsequently demonstrated to be significantly more palatable to rats.

Full text of DOI:10.1016/j.bmc.2012.05.014

Bioorganic & Medicinal Chemistry 20 (2012) 3997–4011
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of prodrugs of the rat selective toxicant norbormide
David Rennison a, , Olivia Laita a, Sergio Bova b, Maurizio Cavalli b, Brian Hopkins c, Darwin S. Linthicum c,
⇑
Margaret A. Brimble a,
⇑
a Department of Chemistry, University of Auckland, 23 Symonds Street, Auckland, New Zealand
b Department of Pharmacology and Anesthesiology, Pharmacology Division, University of Padova, Padova, Italy
c Landcare Research, PO Box 40, Canterbury Agriculture and Science Centre, Gerald Street, Lincoln, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Norbormide [5-(a-hydroxy-a-2-pyridylbenzyl)-7-(a-2-pyridylbenzylidene)-5-norbornene-2,3-dicarbox-
Received 3 April 2012
Revised 30 April 2012
Accepted 8 May 2012
Available online 15 May 2012
imide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but rel-
atively harmless to other rodents and mammals. However, one major drawback of NRB as a viable
rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due
to either its unpleasant ‘taste’ or rapid onset of effects. A series of NRB prodrugs were prepared in an
effort to ‘mask’ this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory
activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Com-
pound 19 displayed the most promising profile with respect to a delay in the onset of symptoms and was
subsequently demonstrated to be significantly more palatable to rats.
Keywords:
Norbormide
Prodrug
Rodenticide
Rat toxicant
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
difacoum, bromadiolone, bromethalin, difethialone, difenacoum)
with varying degrees of success.5 Annually, more than US$500 mil-
Rats cause substantial damage each year to agricultural inter-
ests 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 annually, and
contaminates 5 to 10 times that amount.2 More recently, the Food
and Agriculture Organization of the United Nations reported that,
worldwide, rats ruined more than 42 million tons of food, worth
an estimated US$30 billion.3 In addition to this vast economic loss,
rats are responsible for a number of health problems, acting as vec-
tors for both viral and bacterial diseases, transmitting more than
35 types of disease to humans such as leptospirosis, cholera, sal-
monella and the bubonic plague.2 Furthermore, rats are known to
be one of the most invasive species responsible for loss of biodiver-
sity and native habitats, second only to humans, threatening both
plant and animal survival through predation and habitat destruc-
tion.4 Currently, there are a number of toxicants on the market that
are effective in controlling rats, almost all being non-specific
broad-spectrum rodenticides. To date, rodent control has been
achieved through the use of sub-chronic poisons (e.g., cholecalcif-
erol, bromethalin), acute poisons (e.g., zinc phosphide), first-gener-
ation anticoagulants (e.g., warfarin, coumatetralyl, diphacinone,
chlorophacinone) and second-generation anticoagulants (e.g., bro-
lion is spent on rodent control products, with second-generation
anticoagulants being the most preferred products. Most of these,
however, have one common disadvantage in that they have sec-
ondary non-target risks associated with them and are dangerous
not only to children, but also to domestic pets, wildlife, and live-
stock. Rodenticides rank second in the number of pesticide related
poisonings recorded each year, with a recent study revealing just
under 15,000 people were exposed to such toxicants in the US in
2008 alone, 86% being children under the age of six.6 Additionally,
these poisons also pose increasing risks through environmental
contamination, accumulation of residues in the food chain and a
general lack of humaneness.
Norbormide (NRB), a vasoactive also known under the trade
names ShoxinÒ and Raticate,Ò was first introduced to the market
as a rodenticide over forty years ago (Fig. 1). Discovered in the
N
O
OH
NR
NRB R = H; 1 R = Me
O
N
⇑
Corresponding author. Tel.: +64 9 373 7599x83881; fax: +64 9 373 7422.
E-mail addresses: d.rennison@auckland.ac.nz (D. Rennison), d.rennison
@auckland.ac.nz (M.A. Brimble).
Figure 1. Norbormide (NRB) and N-methyl norbormide 1.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.014

3998
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
1960s NRB was found to be uniquely toxic to rats but relatively
harmless to other rodents and mammals.7,8 NRB displays unique
species-specific constrictor activity that is restricted to the periph-
eral arteries of the rat. In arteries from all other species tested, as
well as in rat aorta and extravascular smooth muscle tissue, NRB
exhibits vasorelaxant properties at concentrations that induce
vasoconstriction in the rat peripheral arteries.9 Furthermore, de-
tailed studies conducted upon the individual stereoisomers of
NRB, isolated from the endo rich stereoisomeric mixture, found
the parent compound’s physiological effects to be strongly stereo-
specific. In rat peripheral arteries only the endo isomers of NRB re-
tained the contractile activity elicited by the stereoisomeric
mixture, with both the endo and exo isomers exhibiting vasodilato-
ry activity in rat aorta.10 In vivo evaluation established that only
the endo isomers of NRB were toxic in rats.11 The mechanisms in-
volved in these divergent effects of NRB have yet to be clarified.
Available evidence suggests that the vasoconstrictor effect may
be mediated by the stimulation of a number of signal transduction
pathways12 that lead to modulation of calcium influx, presumably
mediated by phospholipase C (PLC)-coupled receptors expressed in
rat peripheral artery myocytes,12 whereas the relaxant effect may
be the result of a reduction of Ca2+ entry through L-type Ca2+
channels.13
To date, efforts to establish NRB as a viable rodenticide have
been largely unsuccessful. Over time, rats as a species have devel-
oped an evolutionary trait relating to how they sample food, par-
ticularly novel food, such that they do not ingest a potentially
toxic dose at the first encounter. This survival strategy is most
likely linked to their lack of an emetic centre, and thus their inca-
pacity, as a species, to vomit. As an acute poison, NRB has a rapid
onset of action with toxic symptoms being recorded almost imme-
diately, and evidence suggests rats develop a learnt aversion to this
poison following the consumption of sub-lethal doses during sam-
pling, a phenomenon referred to as bait-shyness.14,15 NRB is also
known to be relatively unpalatable to rats.16–18
to be a consequence of NRB-induced vasoconstriction of the blood
vessels of the buccal cavity, a primary affect leading to bait-shy-
ness. Although efforts to address this palatability problem using
microencapsulation technologies have had varying degrees of suc-
cess, the rapid release of the toxicant in vivo has not been suffi-
ciently delayed and bait shyness has remained
a major
problem.19,20 In an endeavour to circumnavigate these detrimental
qualities we herein report the synthesis of a select series of NRB
prodrugs, designed to both mask and delay such unfavourable
‘side-effects’ prior to systemic breakdown, whereby the active tox-
icant would then subsequently be released leading to death.
2. Results and discussion
2.1. Synthesis and in vitro evaluation for vasoconstrictory
activity pre-cleavage
The synthesis of NRB has been reported previously.7,10,21 Earlier
studies have demonstrated that it is exclusively the endo-isomers
of NRB which are responsible for both the unique vasoconstrictory
activity and species-selective lethality observed in rats.11 Purifica-
tion by recrystallisation allowed the exclusive isolation of mixed
endo-NRB, with all NRB prodrugs herein prepared as endo-specific
stereoisomeric mixtures.22
The first series of prodrugs to be explored were based on N-(a-
acyloxyalkyl)esters in the form of pyridinium salts of type 2–4,
using established methods similar to those reported by Davidsen
et al. (Fig. 2, Table 1).23 With the knowledge that N-methyl norbor-
Table 1
In vitro evaluation for vasoconstrictory activity of 2–4
Compd
R
Yield (%)
Vasoconstrictiona
2
3
4
t-Bu
Ph
CHPh2
76
37
51
P132b
P132b
P132b
While a pre-requisite for lethality in rats, it is this aforemen-
tioned intrinsic vasoconstrictory activity which is believed to be
the most significant shortcoming of NRB as a viable rodenticide.
Sub-lethal dosing due to the toxicants unpleasant ‘taste’ is believed
a
Maximum contractile effect as a % of 90 mM KCl contraction (rat caudal artery);
stereoisomeric mixture of NRB = 132%.
b
lag time ca. 5–10 min.
R
O
O
H-6'
I
N
N
N
N
C-8
O
O
O
O
OH
OH
OH
OH
N
N
N
O
X
N
O
O
C-5
R
O
O
O
O
N
N
O
N
O
N
O
8
21
X = (CH2)2
2
R = t-Bu
R = t-Bu
3 R = Ph
4 R = CHPh2
9 R = (CH)2CH3
22 X = (CH2)4
10
11
23
24
R = (CH)6CH3
R = (CH)10CH3
X = (CH2)6
X = (CH2)8
12 R = Ph
25 X = (CH2)10
13
14
26
27
R = o-OMePh
R = m-OMePh
X = p-(C6H4)
X = (CH2)2C(O)O(CH2)2OC(O)(CH2)2
15 R = p-OMePh
16
17
R = CH2Ph
R = CHPh2
18 R = CH2CH2Ph
19
20
R = CH=CHPh
R = 2-Naph
Figure 2. N-(a-Acyloxymethyl)pyridinium iodide prodrugs 2–4, N-(a-acyloxymethyl)dicarboximide prodrugs 8–20 and N-(a-acyloxymethyl)dicarboximide dimer prodrugs
21–27 (Tables 1–5).

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
3999
R
N
O
O
I
N
N
O
c
O
b
O
OH
OH
OH
NR
N
N
O
R
O
O
O
O
O
N
N
N
O
41, 45, 50
41-53
R
O
Cl
R
O
Cl
2
8
R = t-Bu
3 R = Ph
4 R = CHPh2
NRB R = H
R = t-Bu
1 R = Me
9 R = (CH)2CH3
10 R = (CH)6CH3
11
12
R = (CH)10CH3
R = Ph
13 R = o-OMePh
14 R = m-OMePh
15
R = p-OMePh
16 R = CH2Ph
17 R = CHPh2
18
19
R = CH2CH2Ph
R = CH=CHPh
20 R = 2-Naph
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 16 h, 75% (to give 1); (b) (i) 41, 45 or 50, NaI, acetone, rt, 3 h, (ii) then 1, MeCN, 80 °C (see Table 1); (c) 41–53,
K2CO3, DMF, rt, 16 h (see Table 2).
mide 1 shares a similar pharmacological profile to NRB,11 albeit of
O
O
a, b
marginally lower potency, 1 was selected over NRB for this partic-
ular class of prodrug on synthetic grounds, on the basis that the
dicarboximide nitrogen would not require protection within this
series (Fig. 1). Methylation of the imide nitrogen24 using iodometh-
ane afforded 1 in 75% yield (Scheme 1). Initial attention focused on
R
OH
R
O
Cl
28
41
R = t-Bu
R = t-Bu
(59%)
(100%)*
29 R = (CH)2CH3
30 R = (CH)6CH3
42 R = (CH)2CH3
43 R = (CH)6CH3
(43%)
31
44
R = (CH)10CH3 (14%)
R = (CH)10CH3
the synthesis of N-(a-acyloxymethyl)pyridinium iodide prodrugs
32 R = Ph
45 R = Ph
(49%)
(42%)
33 R = o-OMePh
46 R = o-OMePh
2–4, our design rationale throughout being based on the concept
that the introduction of such steric bulk into the toxicant by way
of such a pro-moiety would impair its intrinsic capacity to elicit
vasoconstriction pre-cleavage.
Chloromethyl esters 41, 45 and 50 were prepared from the cor-
responding acid chlorides 28, 32 and 37, respectively, through treat-
ment with paraformaldehyde in the presence of zinc chloride under
heating (Scheme 2).25 Conversion to the corresponding iodides
in situ using sodium iodide,26 and subsequent alkylation23 of 1 at
34
47
R = m-OMePh
R = m-OMePh (26%)
35 R = p-OMePh
36 R = CH2Ph
48 R = p-OMePh
(12%)
(35%)
(37%)
49 R = CH2Ph
50
37
R = CHPh2
R = CHPh2
38 R = CH2CH2Ph
39 R = CH=CHPh
51 R = CH2CH2Ph (42%)
52 R = CH=CHPh
53
(5%)
(6%)
40
R = 2-Naph
R = 2-Naph
O
O
O
O
a, b
HO
X
OH
Cl
60
O
X
O
Cl
elevated temperature afforded N-(a-acyloxymethyl)pyridinium io-
dide salts 2, 3 and 4 in 76%, 37% and 51% yields, respectively
(Scheme 1, Table 1). Although the formation of N-pyridinium dial-
kylation products of 1 were initially anticipated, given that NRB
contains two pyridine rings, the 1H NMR spectra of the products
formed indicated the formation of only a single monoalkylation
product. A significant 1H NMR resonance assigned to the more
deshielded H-60 proton on the C-8 pyridyl ring was observed in all
three pyridinium salt prodrugs 2–4, resonating in the range d
9.00–9.50 ppm compared to d 8.43–8.64 ppm in 1 (Fig. 2). It is
known, from X-ray crystallographic data,27 that a hydrogen bond
exists between the hydroxyl group of the carbinol carbon and the
nitrogen of the pyridyl ring at C-5. This is put forward as one poten-
tial hypothesis for the exclusive recovery of monoalkylation prod-
ucts only, due to the inability to effect alkylation at the C-5
pyridyl ring as a consequence of this hydrogen bonding.
54
X = (CH2)2
X = (CH2)2
(26%)
(15%)
(17%)
(40%)
(21%)
(21%)
55 X = (CH2)4
56 X = (CH2)6
61 X = (CH2)4
62 X = (CH2)6
57
63
X = (CH2)8
X = (CH2)8
58 X = (CH2)10
59 X = p-(C6H4)
64 X = (CH2)10
65 X = p-(C6H4)
O
O
O
O
O
c
O
O
OH
O
HO
O
66
67
O
O
a, b
O
O
Cl
Cl
O
O
O
O
(9% over 3 steps)
68
In vitro evaluation of N-(a-acyloxymethyl)pyridinium iodides
Scheme 2. Reagents and conditions: (a) SOCl2, reflux, 1 h; (b) paraformaldehyde,
ZnCl2, 80 °C, 2 h or ⁄ClSO3CH2Cl, NaHCO3, TBABr, DCM, H2O, rt, 2 h; (c) ethylene
glycol, pyridine, reflux, 16 h, 95%.
2–4 for pre-cleavage vasoconstrictory activity revealed all three
prodrugs to exhibit NRB-like vasoconstriction in rat peripheral
arteries at levels comparable to the parent compound, albeit with
a lag time of around 5–10 min (Fig. 2, Table 1). Subsequent hydro-
lytic stability studies later revealed compounds 2–4 to be suscep-
tible to cleavage under bioassay conditions (Table 4), providing
one possible explanation for the observed delayed vasoconstrictory
activity in vitro. As a result N-(
a-acyloxyalkyl)pyridinium salt
prodrugs of NRB were abandoned. It should be stated however,

4000
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
that although LC–MS was strongly supportive of N-(
a
-acyloxym-
currently, with full vasoconstrictory activity observed in the
unsubstituted aromatic N-( -acyloxymethyl)dicarboximide series,
ethyl)pyridinium iodide formation, the failure to obtain full NMR
characterization, due to solubility problems, means such data
should be treated with a degree of caution. Attention now shifted
to utilizing the hydroxyl group at C-5 as a potential ‘handle’ for
prodrug formation, through a series of O-(a-acyloxymethyl)ethers
of 1 (Fig. 3). Disappointingly, all efforts to prepare prodrugs of type
5–7 using standard literature procedures failed, with the recovery
of starting materials in all instances. Consequently, O-(a-acy-
loxyalkyl)ether prodrugs of NRB were likewise abandoned.
a
efforts were made to further increase the size of the benzoate
derived pro-moiety through the insertion of a ‘spacer’ between
the carbonyl carbon of the ester and the phenyl group, revealing
alkylene chain homologues phenylacetate 16 and dihydrocinna-
mate 18. Additional steric bulk was introduced into phenylacetate
16 through the incorporation of a second phenyl ring to give diph-
enylacetate 17. Conformationally-restricted cinnamate 19, along
with naphthoate 20, were also prepared to further expand the ser-
ies. In vitro evaluation revealed diphenylacetate 17, cinnamate 19
and naphthoate 20 to be devoid of all vasoconstrictory activity pre-
cleavage. By contrast, phenylacetate 16 and dihydrocinnamate 18
remained partially active as vasoconstrictors, albeit displaying
markedly reduced levels of efficacy, 6% and 13%, respectively. It
is hypothesized that the marginal activity observed in phenylace-
tate 16 is a consequence of insufficient steric bulk, while conform-
ationally flexible dihydrocinnamate 18, versus conformationally
restricted non-vasoconstricting cinnamate 19, permits the pro-
moiety to orientate itself so as to allow some degree of weak bind-
ing, thus permitting low level efficacy (Fig. 2, Table 2).
Difficulties encountered in the preparation of both N-(
a-acyl-
oxymethyl)pyridinium salts and O-( -acyloxymethyl)ether pro-
a
drugs of NRB led us to utilizing the dicarboximide nitrogen as a
third and final potential alkylation site for the formation of pro-
drugs of NRB (Fig. 2, Table 2). Treatment of NRB with chloromethyl
esters 41–53, prepared analogously to those described previously
(Scheme 2), in the presence of potassium carbonate at room tem-
perature, afforded N-(
a-acyloxymethyl)dicarboximide prodrugs
8–20 (Scheme 1, Table 2).
In vitro evaluation of prodrugs 8 and 12 for pre-cleavage vaso-
constrictory activity in rat blood vessels revealed both the pivalate
and benzoate pro-moieties, respectively, to be of insufficient steric
bulk to hinder vasoconstriction at all; in both cases eliciting levels
of efficacy similar to that observed for the parent compound. With-
We now elected to explore a series of N-(a-acyloxyalkyl)dicarb-
oximide dimer prodrugs of type 21–27 (Fig. 2, Table 3), making use
of the considerable steric bulk of a second NRB molecule, intro-
duced through a linker, to act as the pro-moiety; with the concept
that two equivalents of NRB would now be released upon cleavage.
in the aliphatic N-(a-acyloxymethyl)dicarboximide series, n-but-
anoate 9 was found to display partial vasoconstriction, while
efforts to further increase the size of the pro-moiety, through alkyl
chain homologues n-octanoate 10 and n-dodecanoate 11, proved
successful, in both examples being devoid of all vasoconstrictory
activity pre-cleavage. A strategy to incorporate further steric bulk
into benzoate 12, through the introduction of ring substituents,
proceeded via methoxybenzoate analogues 13 (ortho), 14 (meta)
and 15 (para). In vitro evaluation revealed o-methoxybenzoate
13 to be a partial vasoconstrictor, while structural regioisomers
14 and 15 both failed to elicit any vasoconstrictory response. Con-
As a more elaborate example of a NRB dimer prodrug, N-[a-(ethyl-
ene bis(hydrogensuccinoyloxy))methyl]dicarboximide tetraester
27 was proposed to provide a molecule incorporating a second
independent site of cleavage, positioned further away from the
sterically demanding NRB molecule, with a view to making it more
susceptible to enzymatic breakdown. Commercially available
diacid chlorides 54–59 were converted to the corresponding
dichloromethyl esters 60–65 using conditions similar to those de-
tailed previously.28 Treatment of dichloromethyl esters 60–65 with
two equivalents of NRB, in the presence of potassium carbonate,
afforded dimers 21–26, respectively, in yields ranging from 12%
to 81%.24 Ethylene bis(hydrogen succinate)29 (67) was prepared
in 95% yield via the addition of ethylene glycol to a solution of
succinic anhydride (66) in pyridine, under heating. Diacid 67 was
then sequentially taken through to the corresponding diacid chlo-
ride,30 dichloromethyl ester 68,28 and finally, via the subsequent
reaction of in situ generated iodide with two equivalents of NRB,
to the desired dimer 27, in 26% yield overall (Schemes 2 and 3, Ta-
ble 3).24,26 All dicarboximide dimers evaluated within this study
were found to be devoid of all pre-cleavage vasoconstrictory activ-
ity (Table 3).
N
O
R
O
5 R = t-Bu; 6 R = Ph; 7 R = Ac
O
O
N
C-5
O
N
Figure 3. O-(a-Acyloxymethyl)ether prodrug candidates 5–7.
2.2. In vitro cleavage studies and in vivo evaluation
Table 2
In vitro evaluation for vasoconstrictory activity of 8–20
To evaluate the hydrolytic stability of lead N-(a-acyloxymeth-
yl)dicarboximide prodrug candidates 10–11, 14–17, 19 and 20
Compd
R
Yield (%)
Vasoconstrictiona
8
9
t-Bu
48
62
56
32
71
65
34
65
19
15
6
P132
86
0
0
P132
90
0
0
6
(CH2)2CH3
(CH2)6CH3
(CH2)10CH3
Ph
o-OMePh
m-OMePh
p-OMePh
CH2Ph
Table 3
10
11
12
13
14
15
16
17
18
19
20
In vitro evaluation for vasoconstrictory activity of 21–27
Compd
X
Yield (%)
Vasoconstrictiona
21
22
23
24
25
26
27
(CH2)2
(CH2)4
(CH2)6
(CH2)8
17
16
6
81
12
21
26
0
0
0
0
0
0
0
CHPh2
0
13
0
CH2CH2Ph
CH@CHPh
2-Naph
(CH2)10
p-(C6H4)
(CH2)2C(O)O(CH2)2OC(O)(CH2)2
12
32
0
a
a
Maximum contractile effect as a% of 90 mM KCl contraction (rat caudal artery);
stereoisomeric mixture of NRB = 132%.
Maximum contractile effect as a% of 90 mM KCl contraction (rat caudal artery);
stereoisomeric mixture of NRB = 132%.

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4001
N
N
N
O
O
a
O
OH
OH
OH
N
N
NH
O
O
X
O
O
O
O
O
N
N
O
N
O
Cl
O
X
O
Cl
60-65, 68
21
X = (CH2)2
X = (CH2)4
22
23 X = (CH2)6
24
25
X = (CH2)8
X = (CH2)10
26 X = p-(C6H4)
27
X = (CH2)2C(O)O(CH2)2OC(O)(CH2)2
Scheme 3. Reagents and conditions: (a) (i) 60–65, 68, NaI, acetone, rt, 3 h, (ii) then NRB, K2CO3, DMF, rt, 48 h (see Table 3).
120
100
80
60
40
20
0
16
10
11
19
15
14
20
21
17
27
23
22
24
Figure 4. In vitro enzymatic cleavage studies on selected prodrugs using rat serum (37 °C, 3 h, n = 3).
(pre-cleavage vasoconstrictory activity <10%) in rat blood, in vitro
enzymatic cleavage studies using rat serum were conducted in-
house (Fig. 4, Table 4). Results showed full cleavage of phenylace-
tate 16 following the standard adopted incubation time of 3 h,
revealing our most labile prodrug under such conditions within
this study. Given that compound 16 incorporated the smallest
pro-moiety of those prodrugs under evaluation this data was lar-
gely expected. Next most susceptible were alkyl chain homologues
n-octanoate 10 and n-dodecanoate 11 (ca. 89% and 83% NRB re-
lease, respectively) while methoxybenzoate analogues 14 (meta)
and 15 (para) both cleaved in the region of 65%. Cinnamate 19
was found to release around 77% NRB post exposure to rat serum,
with related naphthoate 20 being considerably less prone to cleav-
age, displaying similar rates of enzymatic breakdown to dipheny-
lacetate 17, with toxicant release levels both in the region of 30%.
Surprisingly, and contrary to our design rationale with respect
to the dimer concept, we discovered that it was in fact the diester
prodrugs of increased chain length which were the least suscepti-
ble to enzymatic degradation. Within this series dodecanedioate
25 (n = 10, <5% NRB release) was observed to be practically inert
towards cleavage while sebacate 24 (n = 8) was found to release
around 10% toxicant following the 3 h exposure period. By con-
trast, succinate 21 (n = 2, ca. 30% NRB release) was observed to
cleave the most readily in the presence of rat serum. The remaining
alkyl chain diesters, adipate 22 and suberate 23 (n = 4, 6, respec-
tively), cleaved in the region of 15%. Efforts to further increase
the susceptibility of the dicarboximide diesters towards enzymatic
degradation, through the incorporation of a second independent
site of cleavage within the linker, were found to be moderately suc-
cessful, with compound 27 (ca. 21% NRB release) revealing a higher
degree of cleavage (vs un-functionalized chain length equivalent,
dodecanedioate 25, <5% NRB release). With regards to aromatic es-
ter derived prodrug 26, incorporating a terephthalate linker, rela-
tively low levels of NRB release were observed (ca. 5%). As a
consequence of their common relative resistance to hydrolytic
cleavage (<30% NRB release, 3 h), and on synthetic grounds, dicarb-
oximide dimer prodrugs 21–27 were taken no further; given that
the rate of NRB clearance31 in rats is known to be relatively high
the aforementioned candidates were viewed to be of inadequate
lability, if the goal of lethal systemic levels of toxicant were ulti-
mately to be attained.
Having now established a relative order of hydrolytic stability
against rat blood enzymes within our non-vasoconstricting pro-
drug series, six candidates (10, 14, 16, 17, 19 and 20; ranging from
ca. 100% to 29% toxicant release, 3 h) were nominated for further
evaluation (Fig. 4, Table 4). As an alternative source of carboxylest-
erase, and with the knowledge that the liver has the highest levels
of carboxylesterase activity in the biological system of many

4002
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
Table 4
Pre-cleavage vasoconstrictory activity, in vitro hydrolytic stability (low pH, rat blood and liver enzymes) and in vivo lethality of NRB and prodrugs 2–4, 8–27 in rats
Compd
Vasoconstrictiona
% NRB released
Rat serumd
In vivo lethality (iv)
In vivo lethality (oral)
Hydrolytic stability
Rat liver S9e
endo-NRB
2
3
4
8
132
132
132
132
132
86
0
—
—
—
—
—
—
—
—
—
—
—
—
—
Yesf,g
—
—
—
—
—
—
—
—
Yesh,i
—
—
—
—
—
—
—
—
100b
100b
100b
0b
9
0b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
20c
20c
0b
89.1 1.2
83.2 1.8
—
73.0 5.2
—
—
—
53.1 3.3
—
69.9 0.8
12.4 0.3
0
132
90
0
0
6
0
13
0
0
0
0
0
0
0
0
0
0b
—
—
—
—
—
—
—
0c
64.2 3.0
67.7 3.1
100.0 0.1
29.1 1.0
—
76.9 0.5
31.3 2.7
29.6 5.4
13.4 0.2
14.9 5.0
8.6 0.5
<5
0c
0b, <5c
0c
Yesf,g
ntj
—
—
Noh
—
0b
—
0c
55.4 2.4
20.2 0.8
Yesf,g
Yesf,g
—
Yesh,i
Noh
—
0c
0c
—
—
—
—
—
—
—
0c
—
—
—
—
—
—
—
—
—
—
—
—
0c
0c
0c
0c
<5
20.8 0.7
0c
a
b
c
d
e
f
Maximum contractile effect as a% of 90 mM KCl contraction (rat caudal artery, n = 3), stereoisomeric mixture of NRB = 132%.
For vasoconstricting prodrugs pre-cleavage, Tyrode solution (37 °C, 1 h, n = 3).
For non-vasoconstricting prodrugs pre-cleavage, simulated gastric fluid without pepsin (pH 1.2, 37 °C, 1 h, n = 3).
80% rat serum (37 °C, 3 h, n = 3).
1 mg/mL rat liver S9 fraction (37 °C, 6 h, n = 3);
20 mg/Kg (rat, iv, n = 3).
10 mg/Kg (rat, iv, n = 3).
40 mg/Kg (rat, oral, n = 3).
20 mg/Kg (rat, oral, n = 3).
Solubility problems encountered preventing injection by iv nt = not tested.
g
h
i
j
mammals, including rats,32 further information was sought on the
probable routes of cleavage of the aforementioned prodrugs
through their incubation in the presence of rat liver S9 fraction.
Compounds 10 and 16 were once again shown to cleave most read-
ily (73% and 69%, respectively, 6 h), as revealed earlier in our serum
based experiments, while prodrugs 19, 14, 20 and 17 (ca. 55%, 53%,
20% and 12% NRB release, 6 h, respectively) also appeared to
roughly fit a similar trend to previous observations (Fig. 5, Table 4).
All prodrug candidates considered for further evaluation were
now subject to stability appraisal under simulated gastric fluid
(SGF) conditions (without pepsin)33 prior to in vivo evaluation, to
reveal any potential chemical instabilities that may occur in the
acidic environment of the stomach prior to uptake (Table 4).
Prodrugs 14, 17, 19 and 20 were found to be stable at low pH
following the standard adopted incubation period of 1 h, with
low levels of cleavage (<5%) observed for phenylacetate 16. Com-
pound 10 was found to be unstable (ca. 20% cleavage) under the
aforementioned conditions and were thus taken no further.
As part of our preliminary in vivo assessment, and in an endeav-
our to ascertain proof-in-principle, prodrugs 16, 19 and 20 were
successfully administered intravenously to euthanized rats; for-
mulation problems prevented the delivery of compound 17 by iv
(Table 4). Doses of both 20 and 10 mg/Kg resulted in instant death,
or death within minutes, in all cases, illustrating the rapid cleavage
of the aforementioned prodrugs in vivo. Oral gavage experiments
at an initial dose of 40 mg/Kg were next undertaken for prodrugs
14, 17, 19 and 20, leading to a lethal end-point in the case of com-
pounds 14 and 19, while compounds 17 and 20 both failed to in-
duce death; potentially a consequence of the cleavage (too slow)
versus clearance phenomenon, thus leading to sub-lethal dosing
(Table 4). At a reduced dose of 20 mg/Kg compound 19 was again
found to be lethal in rats (mean time to death 90 min), with an
all important lag time in the onset of symptoms of around
30 min. At this same dose, NRB (see positive control, endo-NRB)
would on average result in death within 35 min, and more signifi-
cantly with symptoms being evident within approximately 5 min
of administration (Tables 4 and 5).
80
70
60
50
40
30
20
10
0
In order to provide further experimental evidence that NRB’s
unpleasant ‘taste’, a probable primary affect leading to bait-shy-
ness, is indeed a consequence of the parent compound’s inherent
capacity to elicit constriction of the blood vessels of the buccal cav-
ity, a palatability trial was commissioned to further evaluate pro-
drug 19 (Fig. 6, Table 5). Following 4 days ‘pre-baiting’ with a
peanut butter formulation free of toxicant rats were fed with a bait
containing 1% w/w prodrug in peanut butter. Upon exchanging the
‘placebo’ bait for one containing compound 19 consumption levels
10
16
19
14
20
17
24
Figure 5. In vitro enzymatic cleavage studies on selected prodrugs using rat liver S9
fraction (37 °C, 6 h, n = 3).

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4003
Table 5
In vivo lethality and palatability trial observations for NRB and prodrug 19 in rats
Compd
In vivo lethalitya
Onset of Symptomsb (min)
Palatability triald
Average Bait Consumed (g) (of 5 g)
Time to Death (min)
No of deaths observed
endo-NRB
19
PB
5c
35c
90c
—
1.9 0.5
4.3 0.2
5.0 0.2
3/6
6/6
0/6
30c
—
a
b
c
20 mg/Kg (rat, oral, n = 3).
Visual signs of distress (laboured/irregular breathing, lethargy, tail twitching), consistent with NRB-like symptoms.
Mean time.
Rats fed with 1% w/w toxicant (n = 6) in a peanut butter bait following 4 days ‘pre-baiting’ with a peanut butter formulation free of toxicant. PB = Peanut butter only.
d
was carried out using a Büchi GKR-51 Kugelrohr apparatus. Melting
6
5
4
3
2
1
0
points in degrees Celsius (°C) were measured on an ElectrothermalÒ
melting point apparatus and are uncorrected. Nuclear Magnetic
Resonance (NMR) spectra were recorded on a Bruker AVANCE
DRX400 (1H, 400 MHz; 13C, 100 MHz) or a Bruker AVANCE 300
(1H, 300 MHz; 13C, 75 MHz) spectrometer at 298 K. For 1H NMR
data, chemical shifts are described in parts per million (ppm) rela-
tive to tetramethylsilane (d 0.00) and are reported consecutively
as position (dH), relative integral, multiplicity (s = singlet, bs = broad
singlet, d = doublet, bd = broad doublet, t = triplet, q = quartet,
dd = doublet of doublets, dt = doublet of triplets, qd = quartet of
doublets, m = multiplet, bm = broad multiplet), coupling constant
(J/Hz) and assignment. For 13C NMR data, chemical shifts (ppm)
are referenced internally to CDCl3 (d 77.0) and are reported consec-
utively as position (dC) and degree of hybridization. Assignments
were aided by DEPT135 and HSQC experiments. Infrared spectra
were recorded on a Perkin–Elmer Spectrum One Fourier Transform
NRB
19
PB
Figure 6. Palatability trial observations for NRB and prodrug 19; rats fed with 1%
w/w toxicant (n = 6) in a peanut butter bait following 4 days ‘pre-baiting’ with a
peanut butter formulation free of toxicant. PB = Peanut butter only.
Absorption peaks are reported in wavenumbers (m
, cmꢀ1), with the
remained high (4.3 0.2 g of 5.0 g, n = 6), versus the control bait
(peanut butter only; complete consumption observed, 5 g, n = 6);
and enhanced with respect to the average consumption of norbor-
mide-containing baits (1.9 0.5 g of 5.0 g, n = 6). Strikingly, a kill
rate of six from six rats was observed for compound 19, versus 3/
6 for the parent toxicant in non-prodrug form.
major peaks assigned to the appropriate functional groups. Mass
spectra were recorded on a VG-70SE mass spectrometer (EI, CI
and FAB). High-resolution mass spectra were recorded at a nominal
resolution of 5000. The purity of all target compounds was assigned
using reverse-phase HPLC [Dionex P680 system using a Phenome-
nex Gemini C18-Si column (50 mm ꢁ 2 mm, 5
lm)]—eluted using
a gradient of 100:0% A/B to 5:95% A/B over 15 min at 0.2 mL/min;
where solvent A was water (0.1% formic acid) and solvent B was
CH3CN (0.1% formic acid); with detection at 254 and 280 nm.
3. Conclusion
In summary, in an endeavour to circumnavigate the well docu-
mented problem of NRB-associated bait shyness in rats, a select ser-
ies of prodrugs in the form of N-(a-acyloxymethyl)dicarboximides
4.1.1. 5-(a-Hydroxy-a-2-pyridylbenzyl)-N-methyl-7-(a-2-
pyridylbenzylidene)-5-norbornene-2,3-dicarboximide11 (1)
Compound 1 was prepared by a procedure similar to that of
Hursthouse et al.24 To a solution of NRB (0.5 g, 0.98 mmol) in
dimethylformamide (5 mL) was added dropwise a solution of iodo-
methane (0.15 mL, 2.44 mmol) in dimethylformamide (1 mL), fol-
lowed by potassium carbonate (0.34 g, 2.44 mmol), and the
reaction stirred at room temperature for 16 h. The mixture was ta-
ken up in chloroform/water (1:1) (30 mL) and the organic phase
separated. The aqueous layer was further extracted with chloroform
(3 ꢁ 10 mL) and the combined organic phases washed with water
(2 ꢁ 20 mL), dried over anhydrous sodium sulfate and the solvent
removed in vacuo to give crude 1 as a yellow residue. Purification
by flash chromatography (hexane/ethyl acetate 1:2) afforded 1 as
a colourless solid (150 mg, 0.28 mmol, 30%). 1H NMR (300 MHz,
CDCl3) d 2.93 and 2.96 (3H, s, NMe), 3.29–3.40 (0.7H, m, 0.2H W/
H-3 and 0.5H Y/H-3), 3.43–3.54 (0.4H, m, 0.2H U/H-2 and U/H-3,
0.2H V/H-2), 3.58–3.66 (1.1H, m, 0.2H V/H-3, 0.4H W/H-2 and W/
H-4, 0.5H Y/H-2), 3.85–3.90 (0.3H, m, 0.1H U/H-1 and 0.2H V/H-
1), 3.94 (0.5H, dt, J = 4.5 and 1.4 Hz, Y/H-4), 4.13–4.16 (0.1H, m, U/
H-4), 4.28 (0.2H, dt, J = 4.5 and 1.4 Hz, V/H-4), 4.44–4.48 (0.7H, m,
0.2H W/H-1 and 0.5H Y/H-1), 5.48 (0.2H, dd, J = 3.3 and 1.3 Hz, V/
H-6), 5.50–5.52 (0.7H, m, 0.5H Y/H-6 and 0.2H OH), 5.54 (0.5H, s,
OH), 5.59 (0.3H, s, OH), 5.96–5.99 (0.3H, m, 0.1H U/H-6 and 0.2H
were prepared. Having successfully demonstrated a number of
these analogues to be devoid of any undesirable vasoconstrictory
activity pre-cleavage, subsequent in vitro cleavage studies revealed
a broad range of hydrolytic stabilities in the presence of both rat
blood and liver carboxylesterases. Of the six candidates evaluated
in vivo NRB prodrug 19 displayed the most promising profile with
respect to a delay in the onset of symptoms and was subsequently
demonstrated to be significantly more palatable to rats. On the basis
of the findings of this study compound 19 has since been selected to
undergo advanced appraisal in a two-choice bait trial, an undertak-
ing which sits outside the scope of this manuscript.
4. Experimental (Chemistry)
4.1. General experimental methods
All reagents were used as supplied unless otherwise stated. Sol-
vents were purified by standard methods.34 Analytical thin layer
chromatography (TLC) was carried out on pre-coated silica gel
plates (Merck/UV254) and products were visualized by UV fluores-
cence and/or staining. Potassium permanganate solution was the
stain of choice. Flash chromatography was performed using silica
gel (Riedel-de Haën, particle size 0.032–0.063 mm). Distillation
W/H-6), 6.63–7.67 (16H, m, Ar), 8.43–8.45 (0.2H, m, 2U/aPyr),

4004
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
8.46–8.50 (1.1H, m, 0.4H 2V/
a
Pyr, 0.2H W/
a
Pyr and 0.5H Y/
Pyr).
a
Pyr),
phy (hexane/ethyl acetate 1:1) afforded 8 as a colourless solid
(118 mg, 0.19 mmol, 48%). mp 95–98 °C; 1H NMR (400 MHz,
CDCl3) d 1.18–1.19 (8.4H, m, tBu), 1.25–1.26 (0.6H, m, tBu), 3.38
(0.2H, dd, J = 7.9 and 4.5 Hz, W/H-3), 3.45 (0.5H, dd, J = 7.9 and
4.5 Hz, Y/H-3), 3.51–3.59 (0.4H, m, 0.2H U/H-2 and U/H-3, 0.2H
V/H-2), 3.63–3.64 (0.2H, m, W/H-4), 3.67–3.76 (0.9H, m, 0.2H V/
H-3, 0.2H W/H-2 and 0.5H Y/H-2), 3.88–3.92 (0.3H, m, 0.1H U/H-
1 and 0.2H V/H-1), 4.00–4.01 (0.5H, dt, J = 4.5 and 1.3 Hz, Y/H-4),
4.20–4.21 (0.1H, m, U/H-4), 4.35–4.36 (0.2H, dt, J = 4.5 and
1.3 Hz, V/H-4), 4.49–4.52 (0.7H, m, 0.2H W/H-1 and 0.5H Y/H-1),
5.26–5.29 (0.7H, m, CH2), 5.31–5.62 (3H, m, 1.3H CH2, 0.2H V/H-
6, 0.5H Y/H-6 and 1H OH), 6.04–6.06 (0.3H, m, 0.1H U/H-6 and
0.2H W/H-6), 6.71–7.57 (16H, m, Ar), 8.42–8.50 (0.6H, m, 0.2H
8.61–8.64 (0.7H, m, 0.2H W/
a
Pyr and 0.5H Y/a
4.1.2. 5-( -Hydroxy-a-2-pyridylbenzyl)-N-methyl-7-[a-2-pyri
a
dyl-N-(pivaloyloxymethyl)benzylidene]-5-norbornene-2,3-
dicarboximide iodide (2)
Compound 2 was prepared by a procedure similar to that of
Davidsen and co-workers,23 and Bodor and co-workers.26 To a solu-
tion of chloromethyl pivalate (41) (345 mg, 2.28 mmol) in acetone
(3 mL) was added a solution of sodium iodide (342 mg, 2.28 mmol)
in acetone (2 mL), and the mixture stirred at room temperature for
3 h. The solvent was removed in vacuo and the crude residue taken
up in acetonitrile (2 mL). A solution of 1 (300 mg, 0.60 mmol) in
acetonitrile (1.5 mL) was added and the mixture stirred at 80 °C
for 16 h. The solvent was removed in vacuo with purification by
flash chromatography (dichloromethane/methanol 20:1) affording
2 as a yellow solid (348 mg, 0.45 mmol, 76%). mp 111–120, 150–
2U/
and 0.5H Y/
Pyr); 13C NMR (100 MHz, CDCl3) d 26.9 (CH3), 44.0–46.9 (CH),
a
Pyr and 0.4H 2V/
a
Pyr), 8.54–8.55 (0.7H, m, 0.2H W/
aPyr
aPyr), 8.61–8.63 (0.7H, m, 0.2H W/aPyr and 0.5H Y/
a
49.0–49.6 (CH), 62.0–62.2 (CH2), 77.7–77.8 (C), 121.7–121.8 (CH),
122.4–122.8 (CH), 123.7–124.3 (CH), 124.9 (CH), 126.5–130.2
(CH), 133.2 (CH), 133.9 (CH), 135.1 (CH), 135.8–135.9 (CH),
136.4–136.6 (CH), 138.3–138.5 (C), 141.8 (C), 142.2 (C), 142.8 (C),
143.1 (C), 147.7–148.1 (CH), 148.9–149.3 (CH), 152.6 (C), 154.0–
155.5 (C), 157.9–158.3 (C), 160.6–161.0 (C), 174.6 (C), 175.0–
168 °C; m
max(NaCl)/cmꢀ1 1104 and 1277 (C–O ester), 1691 (C@O
imide), 1751 (C@O ester); m/z (FAB+) 640 (M+, 100%); (Found: M+
640.2803, C40H38N3O5 requires 640.2812).
4.1.3. 5-(a-Hydroxy-a-2-pyridylbenzyl)-N-methyl-7-[a-2-pyri
dyl-N-(benzoyloxymethyl)benzylidene]-5-norbornene-2,3-
175.5 (C), 177.3–177.4 (C); m
max(nujol)/cmꢀ1 1140 and 1212 (C–O
dicarboximide iodide (3)
ester), 1586 (C@O imide), 1715 (C@O ester); m/z (FAB+) 626
(MH+, 67%), 120 (100); (Found: MH+ 626.2644, C39H36N3O5 re-
quires 626.2655).
A similar procedure23,26 to that previously described for the
preparation of 2 was followed using chloromethyl benzoate (45)
(106 mg, 0.76 mmol) in acetone (0.2 mL), sodium iodide (339 mg,
2.28 mmol) in acetone (2.5 mL), and the mixture stirred at room
temperature for 30 min. The solvent was removed in vacuo and
the residue taken up in acetonitrile (1.5 mL). A solution of 1
(200 mg, 0.38 mmol) in acetonitrile (2 mL) was added and the mix-
ture stirred at 80 °C for 16 h. The solvent was removed in vacuo
with purification by flash chromatography (dichloromethane/
methanol 20:1) affording 3 as a yellow solid (110 mg, 0.14 mmol,
4.1.6. N-Butanoyloxymethyl-5-(
a
-hydroxy-a-2-pyridylbenzyl)-
7-(a-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide
(9)
Compound 9 was prepared by a procedure similar to that of
Hursthouse and co-workers,24 and Binderup and co-workers.35 To
a mixture of butyric acid (426 L, 4.6 mmol), water (5 mL), dichlo-
l
romethane (5 mL), sodium hydrogen carbonate (1.46 g, 17.5 mmol)
and tetrabutylammonium bromide (148 mg, 0.46 mmol) was
added dropwise chloromethyl chlorosulfate (534 lL, 5.28 mmol)
37%). mp 165–169 °C; m
max(NaCl)/cmꢀ1 1087 and 1261 (C–O ester),
1697 (C@O imide), 1733 (C@O ester); m/z (FAB+) 660 (M+, 100%);
(Found: M+ 660.2490, C42H34N3O5 requires 660.2498).
in dichloromethane (1.5 mL). The reaction was stirred at room
temperature for 16 h and the organic layer separated, dried over
anhydrous sodium sulfate and the solvent removed in vacuo. The
crude residue was taken up in dimethylformamide (1 mL) and
added to a solution of NRB (500 mg, 0.98 mmol) in dimethylform-
amide (2 mL), followed by potassium carbonate (135 mg,
0.98 mmol). The mixture was stirred at room temperature for
16 h, taken up in dichloromethane/water (1:1) (30 mL), washed
with water (2 ꢁ 20 mL), dried over anhydrous magnesium sulfate
and the solvent removed in vacuo. Purification by flash chromatog-
raphy (hexane/ethyl acetate 1:1) afforded 9 as a colourless solid
(375 mg, 0.61 mmol, 62%). mp 68–79 °C; 1H NMR (300 MHz,
CDCl3) d 0.89–0.96 (3H, m, Me), 1.55–1.71 (2H, m, OCOCH2CH2),
2.25–2.35 (2H, m, OCOCH2CH2), 3.37–3.74 (2.2H, m, 0.2H U/H-2
and U/H-3, 0.6H V/H-2 and V/H-3, 0.6H W/H-2, W/H-3 and W/H-
4, 0.8H Y/H-2 and Y/H-3), 3.86–3.93 (0.4H, m, 0.1H U/H-1 and
0.3H V/H-1), 3.96–4.02 (0.4H, m, Y/H-4), 4.19–4.24 (0.1H, m, U/
H-1), 4.37–4.43 (0.3H, m, V/H-4), 4.50–4.59 (0.6H, m, 0.2H W/H-
1 and 0.4H Y/H-1), 5.30–5.35 (0.7H, m, 0.3H V/H-6 and 0.4H Y/H-
6), 5.44–5.64 (3H, m, 2H NCH2O and 1H OH), 6.03–6.08 (0.3H, m,
0.1H U/H-6 and 0.2H W/H-6), 6.75–7.58 (16H, m, Ar), 8.37–8.44
4.1.4. 5-(a-Hydroxy-a-2-pyridylbenzyl)-N-methyl-7-[a-2-pyri
dyl-N-(diphenylacetoyloxymethyl)benzylidene]-5-norbornene-
2,3-dicarboximide iodide (4)
A similar procedure23,26 to that previously described for the
preparation of 2 was followed using chloromethyl diphenylacetate
(50) (197 mg, 0.76 mmol) in acetone (1 mL), sodium iodide
(113 mg, 0.76 mmol) in acetone (0.5 mL), and the mixture stirred
at room temperature for 3 h. The solvent was removed in vacuo
and the crude residue taken up in acetonitrile (0.5 mL). A solution
of 1 (199 mg, 0.38 mmol) in acetonitrile (1 mL) was added and the
mixture stirred at 80 °C for 16 h. The solvent was removed in vacuo
with purification by flash chromatography (dichloromethane/
methanol 20:1) affording 4 as a yellow solid (171 mg, 0.20 mmol,
51%). mp 145–155 °C; m
max(nujol)/cmꢀ1 1112 and 1277 (C–O es-
ter), 1618 (C@O imide), 1693 (C@O ester); m/z (FAB+) 750 (M+,
84%), 167 (100); (Found: M+ 750.2963, C49H40N3O5 requires
750.2968).
4.1.5. 5-(
-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (8)
A similar procedure24 to that previously described for the prep-
a-Hydroxy-a-2-pyridylbenzyl)-N-pivaloyloxymethyl-7-
(a
(0.8H, m, 0.2H 2U/
0.2H W/ Pyr and 0.4H Y/
and 0.4H Y/
Pyr); 13C NMR (75 MHz, CDCl3) d 13.2 (CH3), 17.7
a
Pyr and 0.6H 2V/
a
Pyr), 8.49–8.51 (0.6H, m,
a
aPyr), 8.58–8.63 (0.6H, m, 0.2H W/
a
Pyr
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (2 mL), chloromethyl pivalate (41) (59 mg,
0.39 mmol) in dimethylformamide (0.5 mL) and potassium carbon-
ate (54 mg, 0.39 mmol). The mixture was stirred at room temper-
ature for 16 h, taken up in chloroform (15 mL), washed with
water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
a
(CH2), 35.2 (CH2), 43.8–46.7 (CH), 48.8–49.3 (CH), 61.2–61.4
(CH2), 77.2 (C), 121.3–122.4 (CH), 123.6–124.0 (CH), 126.4–130.0
(CH), 132.8–133.5 (CH), 135.6–136.4 (CH), 138.0–138.3 (C),
141.7–142.9 (C), 147.4–147.8 (CH), 148.7–148.9 (CH), 152.4 (C),
153.7 (C), 155.2 (C), 157.7–158.0 (C), 160.3–160.7 (C), 172.0–
172.1 (C), 174.3–175.1 (C); m
max/cmꢀ1 1041 and 1208 (C–O ester),

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4005
1585 (C@O imide), 1713 (C@O ester); m/z (ESI) 612 (MH+, 100%);
0.1H U/
0.2H W/
a
a
Pyr, 0.2H W/
Pyr and 0.4H Y/a
a
Pyr and 0.4H Y/
a
Pyr), 8.60–8.61 (0.6H, m,
(Found: MH+ 612.2489, C38H34N3O5 requires 612.2493).
Pyr); 13C NMR (75 MHz, CDCl3) d 13.9
(CH3), 22.4 (CH2), 24.4 (CH2), 28.8–29.2 (CH2), 31.6 (CH2), 33.5
(CH2), 43.9–46.8 (CH), 48.9–49.4 (CH), 61.3–61.6 (CH2), 77.1–77.6
(CH), 121.4–122.5 (CH), 123.7–124.1 (CH), 126.5–130.1 (CH),
132.9 (CH), 133.7 (CH), 135.6–136.4 (CH), 138.1–138.4 (C),
141.8–143.0 (C), 147.6–147.9 (CH), 148.9–149.1 (CH), 152.5 (C),
153.8–155.4 (C), 157.8–158.1 (C), 160.5–160.9 (C), 172.3–172.5
4.1.7. 5-(
7-( -2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide
(10)
a-Hydroxy-a-2-pyridylbenzyl)-N-octanoyloxymethyl-
a
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (500 mg, 0.98 mmol) in
dimethylformamide (2 mL), chloromethyl octanoate (43)
(377 mg, 1.96 mmol) in dimethylformamide (0.5 mL) and potas-
sium carbonate (135 mg, 0.98 mmol). The mixture was stirred at
room temperature for 16 h, taken up in dichloromethane/water
(1:1) (30 mL), washed with water (2 ꢁ 20 mL), dried over anhy-
drous magnesium sulfate and the solvent removed in vacuo. Puri-
fication by flash chromatography (hexane/ethyl acetate 2:1)
afforded 10 as an oily residue (367 mg, 0.55 mmol, 56%). 1H NMR
(300 MHz, CDCl3) d 0.85–0.89 (3H, m, Me), 1.21–1.38 (8H, m,
OCO(CH2)2(CH2)4), 1.55–1.69 (2H, m, OCOCH2CH2), 2.29–2.37
(2H, m, OCOCH2), 3.36 (0.2H, dd, J = 7.5 and 4.5 Hz, W/H-3), 3.42
(0.4H, dd, J = 7.5 and 4.5 Hz, Y/H-3), 3.48–3.74 (1.6H, m, 0.2H U/
H-2 and U/H-3, 0.6H V/H-2 and V/H-3, 0.4H W/H-2 and W/H-4,
0.4H Y/H-2), 3.86–3.91 (0.4H, m, 0.1H U/H-1 and 0.3H V/H-1),
3.97–4.00 (0.4H, m, Y/H-4), 4.20–4.22 (0.1H, m, U/H-4), 4.36–
4.39 (0.3H, m, V/H-4), 4.50–4.54 (0.6H, m, 0.2H W/H-1 and 0.4H
Y/H-1), 5.28–5.32 (0.7H, m, NCH2O), 5.46–5.60 (3H, m, 1.3H
NCH2O, 0.3H V/H-6 and 0.4H Y/H-6), 6.03–6.07 (0.3H, m, 0.1H U/
H-6 and 0.2H W/H-6), 6.74–7.58 (16H, m, Ar), 8.40–8.48 (0.7H,
(C), 174.4–175.2 (C); m
max/cmꢀ1 1041 and 1209 (C–O ester), 1585
(C@O imide), 1715 (C@O ester); m/z (ESI) 724 (MH+, 1%), 706
(MH+-H2O, 100); (Found: MH+ 724.3747, C46H50N3O5 requires
724.3745).
4.1.9. N-Benzoyloxymethyl-5-(
-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (12)
A similar procedure24 to that previously described for the prep-
a-hydroxy-a-2-pyridylbenzyl)-7-
(a
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (2 mL), chloromethyl benzoate (45) (66 mg,
0.39 mmol) in dimethylformamide (0.5 mL) and potassium carbon-
ate (54 mg, 0.39 mmol). The mixture was stirred at room temper-
ature for 16 h, taken up in chloroform (15 mL), washed with
water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (hexane/ethyl acetate 1:1) afforded 12 as a colourless solid
(180 mg, 0.28 mmol, 71%). mp 94–96, 105–108 °C; 1H NMR
(400 MHz, CDCl3) d 3.41–3.44 (0.2H, dd, J = 8.0 and 4.4 Hz, W/H-
3), 3.48–3.52 (0.4H, dd, J = 7.9 and 4.6 Hz, Y/H-3), 3.54–3.63
(0.5H, m, 0.2H U/H-2 and U/H-3 and 0.3H V/H-2), 3.65–3.67
(0.2H, m, W/H-2), 3.70–3.79 (0.9H, m, 0.3H V/H-3, 0.3H W/H-2
and 0.4H Y/H-2), 3.91–3.95 (0.4H, m, 0.1H U/H-1 and 0.3H V/H-
1), 4.02–4.03 (0.4H, m, Y/H-4), 4.23–4.24 (0.1H, m, U/H-4), 4.39–
4.40 (0.3H, m, V/H-4), 4.54–4.56 (0.6H, m, 0.2H W/H-1 and 0.4H
Y/H-1), 5.53–5.58 (1.9H, m, 0.3H V/H-6, 0.4H Y/H-6 and 1.2H
CH2), 5.64 (0.3H, s, OH), 5.65 (0.2H, s, OH), 5.68–5.75 (0.6H, m,
CH2 and OH), 5.82–5.86 (0.7H, m, CH2 and OH), 6.11–6.12 (0.3H,
m, 0.1H U/H-6 and 0.2H W/H-6), 6.74–7.59 (19.2H, m, Ar), 8.01–
m, 0.1H U/
a
a
Pyr and 0.6H 2V/
Pyr, 0.2H W/ Pyr and 0.4H Y/
Pyr and 0.4H Y/
Pyr); 13C NMR (75 MHz, CDCl3) d 13.9
a
Pyr), 8.51–8.53 (0.7H, m, 0.1H U/
a
aPyr), 8.60–8.63 (0.6H, m, 0.2H
W/a
a
(CH3), 22.4 (CH2), 24.4 (CH2), 28.7–28.8 (CH2), 31.4 (CH2), 33.6
(CH2), 43.9–46.9 (CH), 49.0–49.5 (CH), 61.4–61.7 (CH2), 77.2 (C),
77.6 (C), 77.7 (C), 121.5–122.6 (CH), 123.8–124.2 (CH), 126.6–
130.2 (CH), 133.0–133.8 (CH), 135.7–136.5 (CH), 138.2–138.5 (C),
141.8–143.0 (C), 147.6–148.0 (CH), 149.0–149.2 (CH), 152.5 (C),
153.9–155.4 (C), 157.9–158.2 (C), 160.5–160.9 (C), 172.4–172.5
(C), 174.5–175.3 (C);
m
max/cmꢀ1 1040 and 1214 (C–O ester), 1585
8.03 (1.8H, m, OCOPh), 8.40–8.50 (1.4H, m, 0.2H 2U/
2V/ Pyr, 0.2H W/ Pyr and 0.4H Y/ Pyr), 8.61–8.62 (0.6H, m,
0.2H W/ Pyr and 0.4H Y/
Pyr); 13C NMR (100 MHz, CDCl3) d
aPyr, 0.6H
(C@O imide), 1712 (C@O ester); m/z (ESI) 668 (MH+, 1%), 650
(MH+-H2O, 100); (Found: MH+ 668.3123, C42H42N3O5 requires
668.3119).
a
a
a
a
a
44.0–46.9 (CH), 49.0–49.6 (CH), 62.0–62.2 (CH2), 77.7–77.8 (C),
121.7–122.6 (CH), 123.9–124.2 (CH), 126.5–130.2 (CH), 133.2
(CH), 133.9 (CH), 135.8–136.6 (CH), 138.2–138.5 (C), 141.8 (C),
142.2 (C), 142.8 (C), 147.8–148.0 (CH), 149.0–149.2 (CH), 152.6
(C), 154.0–154.2 (C), 154.7–154.9 (C), 155.5 (C), 157.8–158.2 (C),
4.1.8. N-Dodecanoyloxymethyl-5-(
a
-hydroxy-a-2-pyridylbenzyl
)-7-(a-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide
(11)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (500 mg, 0.98 mmol) in
dimethylformamide (2 mL), chloromethyl dodecanoate (44)
(488 mg, 1.96 mmol) in dimethylformamide (0.5 mL) and potas-
sium carbonate (135 mg, 0.98 mmol). The mixture was stirred at
room temperature for 16 h, taken up in dichloromethane/water
(1:1) (30 mL), washed with water (2 ꢁ 20 mL), dried over anhy-
drous magnesium sulfate and the solvent removed in vacuo. Puri-
fication by flash chromatography (hexane/ethyl acetate 2:1)
afforded 11 as a colourless oil (231 mg, 0.32 mmol, 32%). 1H NMR
(300 MHz, CDCl3) d 0.85–0.89 (3H, m, Me), 1.20–1.36 (16H, m,
OCO(CH2)2(CH2)8), 1.55–1.68 (2H, m, OCOCH2CH2), 2.29–2.36
(2H, m, OCOCH2), 3.36 (0.2H, dd, J = 9.0 and 3.0 Hz, W/H-3), 3.42
(0.4H, dd, J = 9.0 and 3.0 Hz, Y/H-3), 3.48–3.74 (1.6H, m, 0.2H U/
H-2 and U/H-3, 0.6H V/H-2 and V/H-3, 0.4H W/H-2 and W/H-4,
0.4H Y/H-2), 3.86–3.92 (0.4H, m, 0.1H U/H-1 and 0.3H V/H-1),
3.98–3.99 (0.4H, m, Y/H-4), 4.20–4.21 (0.1H, m, U/H-4), 4.37–
4.38 (0.3H, m, V/H-4), 4.51–4.54 (0.6H, m, 0.2H W/H-1 and 0.4H
Y/H-1), 5.29–5.33 (0.7H, m, NCH2O), 5.43–5.60 (3H, m, 1.3H
NCH2O, 0.3H V/H-6, 0.4H Y/H-6 and 1H OH), 6.03–6.07 (0.3H, m,
0.1H U/H-6 and 0.2H W/H-6), 6.75–7.58 (16H, m, Ar), 8.39–8.46
160.5–160.9 (C), 165.2–165.4 (C), 174.6–175.4 (C);
m
max(NaCl)/cm
ꢀ1
1092 and 1265 (C–O ester), 1585 (C@O imide), 1720 (C@O es-
ter); m/z (FAB+) 646 (MH+, 8%), 120 (100); (Found: MH+
646.2349, C41H32N3O5 requires 646.2342).
4.1.10. 5-(
a-Hydroxy-a-2-pyridylbenzyl)-N-o-methoxybenzoyl
oxymethyl-7-(
a-2-pyridylbenzylidene)-5-norbornene-2,3-
dicarboximide (13)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (500 mg, 0.98 mmol) in
dimethylformamide (2 mL), chloromethyl o-methoxybenzoate
(46) (393 mg, 1.96 mmol) in dimethylformamide (1 mL) and potas-
sium carbonate (135 mg, 0.98 mmol). The mixture was stirred at
room temperature for 16 h, taken up in dichloromethane/water
(1:1) (30 mL), washed with water (2 ꢁ 20 mL), dried over anhy-
drous sodium sulfate and the solvent removed in vacuo. Purifica-
tion by flash chromatography (hexane/ethyl acetate 1:1) afforded
13 as a colourless solid (432 mg, 0.64 mmol, 65%). mp 83–88 °C;
1H NMR (300 MHz, CDCl3) d 3.39–3.43 (0.2H, m, W/H-3), 3.45–
3.49 (0.5H, m, Y/H-3), 3.53 (0.3H, dd, J = 9.0 and 6.0 Hz, V/H-2),
3.64–3.77 (1H, m, 0.3H V/H-3, 0.2H W/H-2 and 0.5H Y/H-2), 3.80
and 3.83 (3H, s, OMe), 3.90–3.94 (0.3H, m, V/H-1), 4.00–4.05
(0.7H, m, 0.1H U/aPyr and 0.6H 2V/aPyr), 8.51–8.52 (0.7H, m,

4006
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
(0.5H, m, Y/H-4), 4.37–4.42 (0.3H, m, V/H-4), 4.50–4.57 (0.7H, m,
0.2H W/H-1 and 0.5H Y/H-1), 5.49–5.57 (2.3H, m, 2H CH2, 0.3H
V/H-6 or OH), 5.65–5.68, 5.79–5.84 (1.5H, m, 0.3H V/H-6 or OH,
0.5H Y/H-6 and 0.7H OH), 6.08–6.09 (0.2H, m, W/H-6), 6.72–7.78
94–112 °C; 1H NMR (400 MHz, CDCl3) d 3.40 (0.2H, dd, J = 8.0 and
4.4 Hz, W/H-3), 3.47 (0.4H, dd, J = 8.0 and 4.8 Hz, Y/H-3), 3.53–
3.62 (0.5H, m, 0.2H U/H-2 and U/H-3, 0.3H V/H-2), 3.65–3.67
(0.2H, m, W/H-4), 3.68–3.78 (0.9H, m, 0.3H V/H-3, 0.2H W/H-2
and 0.4H Y/H-2), 3.84 and 3.85 (3H, s, OMe), 3.91–3.95 (0.4H, m,
0.1H U/H-1 and 0.3H V/H-1), 4.00–4.04 (0.4H, m, Y/H-4), 4.22–
4.24 (0.1H, m, U/H-4), 4.34–4.40 (0.3H, m, V/H-4), 4.51–4.56
(0.6H, m, 0.2H W/H-1 and 0.4H Y/H-1), 5.51–5.54, 5.59–5.60,
5.64–5.70 and 5.78–5.81 (3.7H, m, 2H CH2, 0.3H V/H-6, 0.4H Y/
H-6 and 1 OH), 6.10–6.12 (0.3H, m, 0.1H U/H-6 and 0.2H W/H-6),
6.71–7.61 and 7.95–8.02 (20H, m, Ar), 8.41–8.54 (1.4H, m, 0.2H
(20H, m, Ar), 8.37–8.43 (1.3H, m, 0.6H 2V/
a
Pyr, 0.2H W/aPyr and
0.5H Y/aaPyr), 8.58–8.60 (0.7H, m, 0.2H W/
a
Pyr and 0.5H Y/
a
Pyr);
13C NMR (75 MHz, CDCl3) d 44.2–46.3 (CH), 48.8–49.3 (CH), 55.6
(CH3), 61.8–62.1 (CH2), 77.1 (C), 77.2 (CH), 77.4–77.5 (C), 111.7
(CH), 118.5–118.6 (CH), 119.7 (CH), 121.4–122.4 (CH), 123.8–
124.0 (CH), 126.3 (CH), 126.9–128.0 (CH), 128.9–129.2 (CH),
129.9 (CH), 131.4 (CH), 133.5–133.7 (CH), 135.7–136.4 (CH),
138.0–138.3 (C), 141.6 (C), 142.1 (C), 142.6 (C), 147.5–147.8 (CH),
148.8–148.9 (CH), 152.4 (C), 153.7–155.3 (C), 157.6–158.1 (C),
2U/
a
Pyr, 0.6H 2V/
a
a
Pyr, 0.2H W/
Pyr and 0.4H Y/
a
Pyr and 0.4H Y/
aPyr), 8.61–8.65
(0.6H, m, 0.2H W/
a
Pyr); 13C NMR (100 MHz,
159.1 (C), 160.3–160.6 (C), 164.4 (C), 174.4–175.1 (C);
m
max/cmꢀ1
CDCl3) d 44.2–47.1 (CH), 49.2–49.8 (CH), 55.5 (CH3), 62.0–62.3
(CH2), 77.4 (CH), 77.9–78.0 (C), 113.7 (CH), 121.5–122.8 (CH),
124.0–124.4 (CH), 126.8–130.4 (CH), 132.0 (CH), 133.2 (CH),
134.0 (CH), 136.0–136.9 (CH), 138.4–138.7 (C), 142.0–143.3 (C),
148.0–148.2 (CH), 149.2–149.4 (CH), 152.8 (C), 154.2–155.7 (C),
158.1–158.4 (C), 160.8–161.2 (C), 163.7 (C), 165.1–165.2 (C),
1124 and 1235 (C–O ester), 1584 (C@O imide), 1713 (C@O ester);
m/z (ESI) 676 (MH+, 10%), 658 (MH+-H2O, 100); (Found: MH+
676.2448, C42H34N3O6 requires 676.2442).
4.1.11. 5-(
a-Hydroxy-a-2-pyridylbenzyl)-N-m-methoxybenzoyl
oxymethyl-7-(
a-2-pyridylbenzylidene)-5-norbornene-2,3-dicar
174.8–175.6 (C); m
max/cmꢀ1 1079 and 1251 (C–O ester), 1584
boximide (14)
(C@O imide), 1712 (C@O ester); m/z (ESI) 676 (MH+, 1%), 658
(MH+-H2O, 100); (Found: MH+ 676.2434, C42H34N3O6 requires
676.2442).
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (500 mg, 0.98 mmol) in
dimethylformamide (2 mL), chloromethyl m-methoxybenzoate
(47) (393 mg, 1.96 mmol) in dimethylformamide (1 mL) and potas-
sium carbonate (135 mg, 0.98 mmol). The mixture was stirred at
room temperature for 16 h, taken up in dichloromethane/water
(1:1) (30 mL), washed with water (2 ꢁ 20 mL), dried over anhy-
drous magnesium sulfate and the solvent removed in vacuo. Puri-
fication by flash chromatography (hexane/ethyl acetate 1:1)
afforded 14 as a colourless solid (224 mg, 0.33 mmol, 34%). mp
171–180 °C; 1H NMR (300 MHz, CDCl3) d 3.40 (0.2H, dd, J = 7.5
and 4.5 Hz, W/H-3), 3.47 (0.5H, dd, J = 9.0 and 6.0 Hz, Y/H-3),
3.56 (0.3H, dd, J = 7.5 and 4.5 Hz, V/H-2), 3.65–3.66 (0.2H, m, W/
H-4), 3.70–3.79 (4H, m, 3H OMe, 0.3H V/H-3, 0.2H W/H-2 and
0.5H Y/H-2), 3.91–3.94 (0.3H, m, V/H-1), 4.00–4.03 (0.5H, m, Y/
H-4), 4.41–4.42 (0.3H, m, V/H-4), 4.54–4.56 (0.7H, m, 0.2H W/H-
1 and 0.5H Y/H-1), 5.54–5.76 and 5.82–5.87 (3.8H, m, 2H CH2,
0.3H V/H-6, 0.5H Y/H-6 and 1H OH), 6.11–6.13 (0.2H, m, W/H-6),
4.1.13. 5-(
ethyl-7-(
mide (16)
a
-Hydroxy-
a-2-pyridylbenzyl)-N-phenylacetyloxym
a
-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboxi
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (2 mL), chloromethyl phenylacetate (49)
(144 mg, 0.78 mmol) in dimethylformamide (0.4 mL) and potas-
sium carbonate (54 mg, 0.39 mmol). The mixture was stirred at
room temperature for 16 h, taken up in chloroform (15 mL),
washed with water (2 ꢁ 10 mL), dried over anhydrous magnesium
sulfate and the solvent removed in vacuo. Purification by flash
chromatography (hexane/ethyl acetate 1:2) afforded 16 as a col-
ourless solid (50 mg, 0.08 mmol, 19%). mp 71–78 °C; 1H NMR
(400 MHz, CDCl3) d 3.35 (0.2H, dd, J = 7.9 and 4.5 Hz, W/H-3),
3.40 (0.4H, dd, J = 7.8 and 4.5 Hz, Y/H-3), 3.45 (0.3H, dd, J = 7.9
and 5.0 Hz, V/H-2), 3.62–3.71 (3.3H, m, 2H CH2Ph, 0.2H U/H-2
and U/H-3, 0.3H V/H-3, 0.4H W/H-2 and W/H-4, 0.4H Y/H-2),
3.87–3.94 (0.4H, m, 0.1H U/H-1 and 0.3H V/H-1), 3.99–4.00
(0.4H, m, Y/H-4), 4.10–4.11 (0.1H, m, U/H-4), 4.27–4.29 (0.3H, m,
V/H-4), 4.43–4.46 (0.6H, m, 0.2H W/H-1 and 0.4H Y/H-1), 5.30
(0.45H, s, Ha/NCH2O), 5.31 (0.45H, s, Hb/NCH2O), 5.48–5.52 (1.5H,
m, 0.3H V/H-6, 0.4H Y/H-6, 0.8H NCH2O and OH), 5.56–5.62
(1.3H, m, NCH2O and OH), 6.06–6.07 (0.3H, m, 0.1H U/H-6 and
6.75–7.62 (20H, m, Ar), 8.41–8.48 (1.3H, m, 0.6H 2V/
W/ Pyr and 0.5H Y/ Pyr), 8.59–8.61 (0.7H, m, 0.2H W/
0.5H Y/
Pyr); 13C NMR (75 MHz, CDCl3) d 44.3–46.4 (CH), 48.9–
a
Pyr, 0.2H
a
a
aPyr and
a
49.4 (CH), 55.1 (CH3), 61.8–62.2 (CH2), 77.0 (C), 77.2 (CH), 77.5–
77.6 (C), 114.0 (CH), 119.5 (CH), 121.5–122.5 (CH), 123.8–124.0
(CH), 126.4–130.2 (CH), 133.7 (CH), 135.7–136.4 (CH), 138.1–
138.4 (C), 141.6–142.6 (C), 147.5–147.8 (CH), 148.8–149.0 (CH),
152.4 (C), 153.8–155.3 (C), 157.7–158.0 (C), 159.2 (C), 160.3–
160.8 (C), 164.9–165.0 (C), 174.4–175.3 (C);
m
max/cmꢀ1 1040 and
0.2H W/H-6), 6.74–7.59 (21H, m, Ar), 8.46–8.47 (0.2H, m, 2U/
8.50–8.51 (1.2H, m, 0.6H 2V/ Pyr, 0.2H W/ Pyr and 0.4H Y/
8.63–8.65 (0.6H, m, 0.2H W/ Pyr and 0.4H Y/
a
a
Pyr),
Pyr),
1218 (C–O ester), 1586 (C@O imide), 1712 (C@O ester); m/z (ESI)
a
a
676 (MH+, 3%), 658 (MH+-H2O, 100); (Found: MH+ 676.2452,
a
a
Pyr); 13C NMR
C
42H34N3O6 requires 676.2442).
(100 MHz, CDCl3) d 40.6 (CH2), 41.4 (CH2), 44.4–46.6 (CH), 49.1–
49.6 (CH), 62.0–62.3 (CH2), 77.7–77.8 (C), 121.7–122.7 (CH),
124.3–124.4 (CH), 126.7–129.5 (CH), 130.3 (CH), 133.1 (C), 133.9
(C), 136.3–136.7 (CH), 138.2–138.3 (C), 141.9–142.2 (CH), 147.5–
148.2 (C), 149.0 (C), 152.6–155.6 (C), 157.8–158.2 (C), 161.0 (C),
4.1.12. 5-(
oxymethyl-7-(
dicarboximide (15)
a
-Hydroxy-
a-2-pyridylbenzyl)-N-p-methoxybenzoyl
a-2-pyridylbenzylidene)-5-norbornene-2,3-
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (1 mL), chloromethyl p-methoxybenzoate
(48) (157 mg, 0.78 mmol) in dimethylformamide (0.5 mL) and
potassium carbonate (54 mg, 0.39 mmol). The mixture was stirred
at room temperature for 16 h, taken up in dichloromethane/water
(1:1) (15 mL), washed with water (2 ꢁ 10 mL), dried over anhy-
drous magnesium sulfate and the solvent removed in vacuo. Puri-
fication by flash chromatography (hexane/ethyl acetate 1:1)
afforded 15 as a colourless solid (172 mg, 0.25 mmol, 65%). mp
170.6 (C), 174.4–175.4 (C); m
max/cmꢀ1 1138 and 1211 (C–O ester),
1585 (C@O imide), 1714 (C@O ester); m/z (FAB+) 660 (100%);
(Found: MH+ 660.2501, C42H34N3O5 requires 660.2498).
4.1.14. N-Diphenylacetyloxymethyl-5-(
a-hydroxy-a-2-pyridylb
enzyl)-7-(
mide (17)
a-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboxi
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (2 mL), chloromethyl diphenylacetate (50)

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4007
(102 mg, 0.39 mmol) in dimethylformamide (0.5 mL) and potas-
4.1.16. N-Cinnamoyloxymethyl-5-(
zyl)-7-( -2-pyridylbenzylidene)-5-norbornene-2,3-dicarboxi
mide (19)
A similar procedure24 to that previously described for the prep-
a-hydroxy-a-2-pyridylben
sium carbonate (54 mg, 0.39 mmol). The mixture was stirred at
room temperature for 16 h, taken up in chloroform (15 mL),
washed with water (2 ꢁ 10 mL), dried over anhydrous magnesium
sulfate and the solvent removed in vacuo. Purification by flash
chromatography (hexane/ethyl acetate 2:1) afforded 17 as a col-
ourless solid (42 mg, 0.06 mmol, 15%). mp 113–115 °C; 1H NMR
(400 MHz, CDCl3) d 3.35 (0.7H, dd, J = 7.9 and 4.6 Hz, Y/H-3), 3.41
(0.3H, dd, J = 7.9 and 4.9 Hz, V/H-2), 3.58–3.67 (1H, m, V/H-3 and
Y/H-2), 3.86–3.89 (0.3H, m, V/H-1), 3.96 (0.7H, dt, J = 4.6 and
1.4 Hz, Y/H-4), 4.29 (0.3H, dt, J = 4.5 and 1.5 Hz, V/H-4), 4.45–
4.48 (0.7H, m, Y/H-1), 5.05 (0.7H, s, Y/CHPh2), 5.06 (0.3H, s, V/
CHPh2), 5.36 (0.5H, s, Ha/CH2), 5.39 (0.5H, s, Hb/CH2), 5.46–5.49
(2H, m, 0.6H V/H-6 and V/OH, 1.4H Y/H-6 and Y/OH), 5.64–5.66
(1H, m, CH2), 6.70–7.58 (26H, m, Ar), 8.47–8.50 (1.3H, m, 0.6H
a
aration of 1 was followed using NRB (174 mg, 0.34 mmol) in
dimethylformamide (1.5 mL), chloromethyl cinnamate (52)
(68 mg, 0.34 mmol) in dimethylformamide (0.5 mL) and potassium
carbonate (47 mg, 0.34 mmol). The mixture was stirred at room
temperature for 16 h, taken up in chloroform (15 mL), washed with
water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (hexane/ethyl acetate 4:1, then 1:1) afforded 19 as a colourless
solid (28 mg, 0.04 mmol, 12%). mp 108–113 °C; 1H NMR (400 MHz,
CDCl3) d 3.46 (0.8H, dd, J = 8.0 and 4.6 Hz, Y/H-3), 3.55 (0.2H, dd,
J = 8.0 and 4.6 Hz, V/H-2), 3.69–3.77 (1H, m, 0.2 V/H-3 and 0.8 Y/
H-2), 3.90–3.94 (0.2H, m, V/H-1), 4.00 (0.8H, dt, J = 4.5 and
1.4 Hz, Y/H-4), 4.37 (0.2H, dt, J = 4.5 and 1.4 Hz, V/H-4), 4.51–
4.54 (0.8H, m, Y/H-1), 5.43 (0.5H, s, Ha/CH2), 5.46 (0.5H, s, Hb/
CH2), 5.53–5.56 (1.8H, m, 0.2H V/H-6, 0.8H Y/H-6 and 0.8H OH),
5.64 (0.2H, bs, OH), 5.69 (0.5H, s, Ha/CH2), 5.70 (0.5H, s, Hb/CH2),
6.43 (0.8H, d, J = 16.1 Hz, Y/OCOCH), 6.45 (0.2H, d, J = 16.1 Hz, V/
OCOCH), 6.75–7.59 (21H, m, Ar), 7.70 (1H, d, J = 16.1 Hz, 0.2H V/
2V/aPyr and 0.7H Y/aPyr), 8.62–8.63 (0.7H, m, Y/a
Pyr); 13C NMR
(100 MHz, CDCl3) d 44.5 (CH), 45.1 (CH), 45.9–46.5 (CH), 49.1–
49.4 (CH), 56.5 (CH), 62.2–62.3 (CH2), 77.8–77.9 (C), 121.8–121.9
(CH), 122.6–122.7 (CH), 124.1–124.3 (CH), 127.1–129.5 (CH),
130.2 (CH), 136.0–136.1 (CH), 136.5 (CH), 138.1–138.5 (C), 141.8
(C), 142.2 (C), 147.9–148.0 (CH), 149.1 (CH), 149.3 (CH), 154.1–
154.3 (C), 154.8 (C), 155.6 (C), 157.9 (C), 158.3 (C), 161.2 (C),
171.6 (C), 175.0–175.4 (C);
m
max(NaCl)/cmꢀ1 1139 and 1215 (C–O
CHPh and 0.8H Y/CHPh), 8.48–8.50 (0.2H, m, V/
a
Pyr), 8.52–8.55
Pyr);
ester), 1585 (C@O imide), 1715 (C@O ester); m/z (ESI+) 736
(MH+, 27%), 120 (100); (Found: MH+ 736.2814, C48H38N3O5 re-
quires 736.2811).
(1H, m, 0.2H V/ Pyr and 0.8H Y/ Pyr), 8.62–8.64 (0.8H, m, Y/a
a
a
13C NMR (75 MHz, CDCl3) d 44.5 (CH), 45.2 (CH), 46.0–46.2 (CH),
46.8 (CH), 49.2–49.5 (CH), 62.0 (CH2), 77.8–77.9 (C), 116.9 (CH),
121.8–122.0 (CH), 122.6–122.7 (CH), 124.1–124.4 (CH), 127.4
(CH), 127.4–129.7 (CH), 130.4–130.5 (CH), 134.2 (C), 136.0 (CH),
136.5 (CH), 138.4–138.6 (C), 142.0–142.4 (C), 146.1 (CH), 148.0
(CH), 149.2–149.4 (C), 154.1 (C), 154.9 (C), 158.4 (C), 161.1 (C),
4.1.15. N-Dihydrocinnamoyloxymethyl-5-(
a-hydroxy-a-2-pyr
idylbenzyl)-7-( -2-pyridylbenzylidene)-5-norbornene-2,3-
a
dicarboximide (18)
Compound 18 was prepared by a procedure similar to that of
Hursthouse and co-workers,24 and Bodor and co-workers.26 To a
solution of chloromethyl dihydrocinnamate (51) (155 mg,
0.78 mmol) in acetone (1.5 mL) was added sodium iodide
(117 mg, 0.78 mmol), and the mixture stirred at room temperature
for 3 h. The solvent was removed in vacuo and the crude iodo-
methyl dihydrocinnamate was taken through to the next step with-
out further purification. A solution of NRB (200 mg, 0.39 mmol),
iodomethyl dihydrocinnamate and potassium carbonate (54 mg,
0.39 mmol) in dimethylformamide (1.5 mL) was stirred at room
temperature for 16 h. The mixture was then taken up in chloroform
(15 mL), washed with water (2 ꢁ 10 mL), dried over anhydrous
magnesium sulfate and the solvent removed in vacuo. Purification
by flash chromatography (hexane/ethyl acetate 1:2) afforded 18
as a colourless solid (15 mg, 0.02 mmol, 6%). mp 89–94 °C; 1H
NMR (300 MHz, CDCl3) d 2.66-2.71 (2H, m, OCOCH2), 2.94-2.99
(2H, m, CH2Ph), 3.36–3.40 (0.2H, m, W/H-3), 3.42–3.47 (0.3H, m,
Y/H-3), 3.52–3.56 (0.7H, m, 0.4H U/H-2 and U/H-3, 0.3H V/H-2),
3.62–3.74 (1H, m, 0.3H V/H-3, 0.4H W/H-2 and W/H-4, 0.3H Y/H-
2), 3.85-3.94 (0.5H, m, 0.2H U/H-1 and 0.3H V/H-1), 3.98-4.00
(0.3H, m, Y/H-4), 4.18–4.24 (0.2H, m, U/H-4), 4.35–4.40 (0.3H, m,
V/H-4), 4.48–4.49 (0.5H, m, 0.2H W/H-1 and 0.3H Y/H-1), 5.29–
5.33 (0.6H, m, 0.3H V/H-6 and 0.3H Y/H-6), 5.43–5.60 (3H, m, 2H
NCH2O and 1H OH), 6.03-6.07 (0.4H, m, 0.2H U/H-6 and 0.2H W/
165.7 (C), 175.5 (C); m
max/cmꢀ1 1144 and 1202 (C–O ester), 1584
(C@O imide), 1712 (C@O ester); m/z (FAB+) 672 (MH+, 100%);
(Found: MH+ 672.2486, C43H34N3O5 requires 672.2498).
4.1.17. 5-(
yl-7-( -2-pyridylbenzylidene)-5-norbornene-2,3-dicarboxi
mide (20)
A similar procedure24 to that previously described for the prep-
a-Hydroxy-a
-2-pyridylbenzyl)-20-naphthoyloxymeth
a
aration of 1 was followed using NRB (184 mg, 0.36 mmol) in
dimethylformamide (1.5 mL), chloromethyl 2-naphthoate (53)
(80 mg, 0.36 mmol) in dimethylformamide (0.5 mL) and potassium
carbonate (47 mg, 0.34 mmol). The mixture was stirred at room
temperature for 16 h, taken up in chloroform (15 mL), washed with
water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (hexane/ethyl acetate 4:1, then 1:1) afforded 20 as a colourless
solid (80 mg, 0.11 mmol, 32%). mp 108–112 °C; 1H NMR (400 MHz,
CDCl3) d 3.51 (0.8H, dd, J = 7.9 and 4.5 Hz, Y/H-3), 3.60 (0.2H, dd,
J = 7.9 and 5.0 Hz, V/H-2), 3.74–3.81 (1H, m, 0.2H V/H-3 and 0.8H
Y/H-2), 3.94–3.96 (0.2H, m, V/H-1), 4.03–4.04 (0.8H, m, Y/H-4),
4.39–4.40 (0.2H, m, V/H-4), 4.54–4.57 (0.8H, m, Y/H-1), 5.55–
5.57 (1.8H, m, 0.2H V/H-6, 0.8H Y/H-6 and 0.8H OH), 5.61–5.63
(1.2H, m, CH2 and OH), 5.87 (0.1H, s, Ha/CH2), 5.88 (0.4H, s, Ha/
CH2), 5.89 (0.1H, s, Hb/CH2), 5.90 (0.4H, s, Hb/CH2), 6.73–7.61
(18H, m, Ar), 7.86–7.95 (3H, m, Ar), 8.02–8.05 (1H, m, Ar), 8.48–
H-6), 6.74–7.63 (21H, m, Ar), 8.42–8.52 (1.5H, m, 0.4H 2U/
0.4H 2V/ Pyr, 0.2H W/ Pyr and 0.3H Y/ Pyr), 8.63-8.65 (0.5H, m,
0.2H W/ Pyr and 0.3H Y/
Pyr); 13C NMR (75 MHz, CDCl3) d 30.6
aPyr,
a
a
a
8.52 (1.2H, m, 0.4H 2V/
aPyr and 0.8H Y/aPyr), 8.59–8.64 (1.8H,
a
a
m, 0.8H Y/
a
Pyr and 1H Ar); 13C NMR (75 MHz, CDCl3) d 45.2
(CH2), 35.4 (CH2), 44.5–46.7 (CH), 49.2–49.7 (CH), 61.7–61.9
(CH2), 79.2 (C), 121.8–122.7 (CH), 124.0–124.8 (CH), 126.3–130.3
(CH), 133.8–134.0 (CH), 136.0–136.8 (CH), 138.4–138.6 (C), 140.2
(C), 142.0 (C), 147.9–148.4 (CH), 149.2–149.4 (CH), 152.7 (C),
(CH), 46.1 (CH), 46.7 (CH), 49.5 (CH), 62.5 (CH2), 77.8 (C), 121.7–
121.8 (CH), 122.7 (CH), 124.3 (CH), 125.2 (CH), 126.4–128.4 (CH),
129.4 (CH), 130.3 (CH), 131.6 (CH), 132.3 (C), 135.7 (C), 136.0
(CH), 136.5 (CH), 138.4 (C), 141.9 (C), 148.0 (CH), 149.2 (CH),
154.1 (C), 154.8 (C), 158.4 (C), 161.1 (C), 165.7 (C), 175.6 (C);
155.6 (C), 158.4 (C), 161.1 (C), 168.4 (C), 171.8 (C), 175.5 (C);mmax(-
NaCl)/cmꢀ1 1141 and 1214 (C–O ester), 1582 (C@O imide), 1717
(C@O ester); m/z (ESI+) 674 (MH+, 100%), 656 (MH+-H2O, 15);
(Found: MH+ 674.2649, C43H36N3O5 requires 674.2665).
m
max/cmꢀ1 1079 and 1263 (C–O ester), 1585 (C@O imide), 1712
(C@O ester); m/z (FAB+) 696 (MH+, 9%), 120 (100); (Found: MH+
696.2504, C45H34N3O5 requires 696.2498).

4008
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4.1.18. 5-(
a
-Hydroxy-
a
-2-pyridylbenzyl)-7-(
a
-2-pyridylbenzylid
4.1.20. 5-(a-Hydroxy-a-2-pyridylbenzyl)-7-(a-2-pyridylbenzy
ene)-N-succinoyloxymethyl-5-norbornene-2,3-dicarboximide
dimer (21)
lidene)-N-suberoyloxymethyl-5-norbornene-2,3-dicarboximide
dimer (23)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (2 mL), dichloromethyl succinate (60)
(42 mg, 0.20 mmol) in dimethylformamide (0.5 mL) and potassium
carbonate (54 mg, 0.40 mmol). The mixture was stirred at room
temperature for 16 h, taken up in chloroform (20 mL), washed with
water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (chloroform/methanol 100:1) afforded 21 as a colourless solid
(40 mg, 0.03 mmol, 17%). mp 129–148 °C; 1H NMR (300 MHz,
CDCl3) d 2.65–2.71 (4H, m, 2x OCOCH2), 3.38–3.76 (4.3H, m, H-1,
H-2, H-3 and H-4), 3.87–3.93 (0.4H, m, H-1, H-2, H-3 and H-4),
3.98–3.99 (1.1H, m, H-1, H-2, H-3 and H-4), 4.17–4.19 (0.4H, m,
H-1, H-2, H-3 and H-4), 4.34–4.35 (0.4H, m, H-1, H-2, H-3 and H-
4), 4.49–4.51 (1.4H, m, H-1, H-2, H-3 and H-4), 5.30–5.34 (1.5H,
m, NCH2O and OH), 5.48–5.64 (5.1H, m, NCH2O, H-6 and OH),
5.79–5.80 (0.6H, m, NCH2O, H-6 and OH), 6.04–6.07 (0.8H, m, H-
A similar procedure24,26 to that previously described for the
preparation of 18 was followed using dichloromethyl suberate
(62) (44 mg, 0.16 mmol) in acetone (0.2 mL), and sodium iodide
(49 mg, 0.32 mmol) in acetone (0.2 mL), at room temperature for
3 h. The solvent was removed in vacuo and the crude diiodomethyl
suberoate was taken through to the next step without further puri-
fication. A solution of NRB (164 mg, 0.32 mmol), diiodomethyl sub-
eroate and potassium carbonate (50 mg, 0.36 mmol) in
dimethylformamide (1.5 mL) was stirred at room temperature for
48 hours. The mixture was taken up in chloroform (15 mL), washed
with water (2 ꢁ 10 mL), dried over anhydrous magnesium sulfate
and the solvent removed in vacuo. Purification by flash chromatog-
raphy (hexane/ethyl acetate 1:2) afforded 23 as a colourless solid
(15 mg, 0.01 mmol, 6%). mp 112–116 °C; 1H NMR (300 MHz, CDCl3)
d 1.20–1.41 (4H, m, 2ꢁ OCOCH2CH2CH2), 1.49–1.76 (4H, m, 2ꢁ
OCOCH2CH2), 2.21–2.44 (4H, m, 2ꢁ OCOCH2), 3.36 (0.6H, dd,
J = 8.1 and 4.5 Hz, W/H-3), 3.43 (0.8H, dd, J = 7.8 and 4.5 Hz, Y/H-
3), 3.52–3.74 (3.2H, m, 1.2H V/H-2 and V/H-3, 1.2H W/H-2 and
W/H-4 and 0.8H Y/H-2), 3.86–3.92 (0.6H, m, V/H-1), 3.95–4.03
(0.8H, m, Y/H-4), 4.34–4.39 (0.6H, m, V/H-4), 4.48–4.59 (1.4H, m,
0.6H W/H-1 and 0.8H Y/H-1), 5.27 (0.85H, s, Ha/NCH2O), 5.30
(0.85H, s, Hb/NCH2O), 5.45–5.58 (5.3H, m, 2.3H NCH2O, 0.6H V/H-
6, 0.8H Y/H-6 and 1.6H OH), 5.74 (0.4H, s, OH), 6.05–6.06 (0.6H,
m, W/H-6), 6.73–7.60 (32H, m, Ar), 8.48–8.54 (2.6H, m, 1.2H 2V/
6 and OH), 6.74–7.61 (32H, m, Ar), 8.42–8.47 (1.2H, m,
aPyr),
8.55–8.56 (1.3H, m, Pyr), 8.61–8.62 (1.5H, m,
a
a
Pyr); 13C NMR
(75 MHz, CDCl3) d 28.6 (CH2), 44.7–46.6 (CH), 49.5 (CH), 49.7
(CH), 61.7–62.0 (CH2), 77.7 (C), 121.7–122.7 (CH), 124.3 (CH),
126.6–130.3 (CH), 133.9 (CH), 136.0–136.6 (CH), 138.4–138.6 (C),
142.0 (C), 142.8 (C), 148.0–148.1 (CH), 149.2 (CH), 152.7 (C),
154.0–155.0 (C), 158.0–158.3 (C), 160.7–161.0 (C), 170.8–170.9
(C), 174.6 (C), 175.2–175.4 (C);
m
max(NaCl)/cmꢀ1 1144 and 1265
a
Pyr, 0.6H W/
aPyr, 0.8H Y/aPyr), 8.62–8.64 (1.4H, m, 0.6H W/aPyr
(C–O ester), 1585 (C@O imide), 1716 (C@O ester); m/z (FAB+)
and 0.8H Y/
a
Pyr); 13C NMR (100 MHz, CDCl3) d 24.3–24.4 (CH2),
1165 (MH+, 14%), 397 (100); (Found: MH+ 1165.4106,
28.6 (CH2), 29.7 (CH2), 33.7–33.8 (CH2), 44.5–46.7 (CH), 49.2–
49.7 (CH), 61.9 (CH2), 77.8 (C), 121.7–121.9 (CH), 122.6–122.7
(CH), 124.3 (CH), 126.7–129.6 (CH), 130.3 (CH), 136.0–136.6
(CH), 138.4 (C), 142.0 (C), 148.0 (CH), 149.2–149.3 (CH), 154.1
(C), 154.9 (C), 158.4 (C), 161.1 (C), 172.6 (C), 175.4–175.5 (C);
C72H57N6O10 requires 1165.4136).
4.1.19. N-Adipoyloxymethyl-5-(
7-( -2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide
dimer (22)
a-hydroxy-a-2-pyridylbenzyl)-
a
m
max/cmꢀ1 1118 and 1211 (C–O ester), 1584 (C@O imide), 1715
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (100 mg, 0.20 mmol) in
dimethylformamide (1 mL), dichloromethyl adipate (61) (24 mg,
0.10 mmol) in dimethylformamide (0.5 mL) and potassium carbon-
ate (27 mg, 0.20 mmol). The mixture was stirred at room temper-
ature for 16 h, taken up in chloroform (10 mL), washed with
water (2 ꢁ 5 mL), dried over anhydrous magnesium sulfate and
the solvent removed in vacuo. Purification by flash chromatogra-
phy (chloroform/methanol 100:1) afforded 22 as a colourless solid
(40 mg, 0.03 mmol, 16%). mp 117–124 °C; 1H NMR (300 MHz,
CDCl3) d 1.56–1.80 (4H, m, 2ꢁ OCOCH2CH2), 2.26–2.48 (4H, m,
2ꢁ OCOCH2), 3.37–3.78 (4.5H, m, H-1, H-2, H-3 and H-4), 3.84–
3.92 (0.7H, m, H-1, H-2, H-3 and H-4), 3.97–3.99 (0.9H, bm, H-1,
H-2, H-3 and H-4), 4.16–4.21 (0.2H, bm, H-1, H-2, H-3 and H-4),
4.32–4.39 (0.5H, bm, H-1, H-2, H-3 and H-4), 4.45–4.55 (1.2H,
bm, H-1, H-2, H-3 and H-4), 5.28–5.36 (1.4H, m, NCH2O and OH),
5.42–5.61 (5.4H, m, NCH2O, H-6 and OH), 5.74, 5.75 (0.5H, bs,
OH), 5.82 (0.1H, bs, OH), 6.03–6.06 (0.6H, m, H-6 and OH), 6.74–
(C@O ester); m/z (FAB+) 1221 (MH+, 28%), 397 (100); (Found:
MH+ 1221.4771, C76H65N6O10 requires 1221.4762).
4.1.21. 5-(a-Hydroxy-a-2-pyridylbenzyl)-7-(a-2-pyridylbenzy
lidene)-N-sebacoyloxymethyl-5-norbornene-2,3-dicarboximide
dimer (24)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (100 mg, 0.20 mmol) in
dimethylformamide (1 mL), dichloromethyl sebacate (63)
(29 mg, 0.10 mmol) in dimethylformamide (0.2 mL) and potas-
sium carbonate (27 mg, 0.20 mmol). The mixture was stirred at
room temperature for 48 h, taken up in chloroform (10 mL),
washed with water (2 ꢁ 5 mL), dried over anhydrous magnesium
sulfate and the solvent removed in vacuo. Purification by flash
chromatography (chloroform/methanol 100:1) afforded 24 as a
colourless solid (101 mg, 0.08 mmol, 81%). mp 113–121 °C; 1H
NMR (400 MHz, CDCl3) d 1.23–1.37 (8H, bm, 2x OCO(CH2)2(CH2)2),
1.55–1.67 (4H, bm, 2ꢁ OCOCH2CH2), 2.26–2.38 (4H, m, 2x
OCOCH2), 3.37 (0.4H, dd, J = 7.9 and 4.5 Hz, W/H-3), 3.43 (0.9H,
dd, J = 7.9 and 4.5 Hz, Y/H-3), 3.50–3.59 (0.9H, m, 0.4H U/H-2
and U/H-3 and 0.5H V/H-2), 3.62–3.74 (2.2H, m, 0.5H V/H-3,
0.8H W/H-2 and W/H-4 and 0.9H Y/H-2), 3.88–3.92 (0.7H, m,
0.2H U/H-1 and 0.5H V/H-1), 3.98 (0.9H, dt, J = 4.4 and 1.3 Hz,
Y/H-4), 4.19–4.20 (0.2H, m, U/H-4), 4.36–4.37 (0.5H, m, V/H-4),
4.50–4.53 (1.3H, m, 0.4H W/H-1 and 0.9H Y/H-1), 5.28–5.31
(1.4H, m, NCH2O), 5.42–5.60 (4H, m, 2.6H NCH2O, 0.5H V/H-6,
0.9H Y/H-6), 5.74 (2H, s, OH), 6.03 (0.2H, dd, J = 3.3 and 1.1 Hz,
U/H-6), 6.05 (0.4H, dd, J = 3.3 and 1.1 Hz, W/H-6), 6.74–7.59
7.58 (32H, m, Ar), 8.43–8.54 (2.8H, m,
aPyr), 8.61–8.62 (1.2H, m,
aPyr); 13C NMR (75 MHz, CDCl3) d 23.8–23.9 (CH2), 33.3–33.4
(CH2), 44.1–47.0 (CH), 49.2–49.7 (CH), 61.6–61.8 (CH2), 77.8 (C),
78.7 (C) 79.1 (C), 121.7–122.8 (CH), 123.9–124.3 (CH), 126.7–
130.3 (CH), 133.9 (CH), 135.8–136.0 (CH), 136.5–136.6 (CH),
138.4–138.6 (C), 142.0 (C), 142.4 (C), 142.9 (C), 147.8–148.2 (C),
149.2–149.4 (C), 152.7 (C), 154.0–155.6 (C), 158.1–158.4 (C),
160.7–161.1 (C), 171.9–172.1 (C), 174.7–175.5 (C);
m
max(NaCl)/cm
ꢀ1
1139 and 1216 (C–O ester), 1585 (C@O imide), 1717 (C@O es-
ter); m/z (FAB+) 1193 (MH+ 33%), 397 (100); (Found: MH+
1193.4463, C74H61N6O10 requires 1193.4449).
(32H, m, Ar), 8.41–8.48 (1.4H, m, 0.4H 2U/aPyr and 1H 2V/aPyr),

D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
4009
8.52–8.53 (1.3H, m, 0.4H W/
a
Pyr and 0.9H Y/
a
Pyr) 8.61–8.63
H-1), 5.53–5.57 (3.4H, m, NCH2O and OH), 5.64–5.74 (2.3H, m,
1H NCH2O and OH, 0.5H V/H-6, 0.8H Y/H-6), 5.82–5.86 (1.3H, m,
NCH2O and OH), 6.09–6.16 (0.7H, m, 0.2H U/H-6 and 0.5H W/H-
6), 6.26 (0.3H, s, OH), 6.74–7.60 (32H, m, Ar), 8.04–8.16 (4H, m,
(1.3H, m, 0.4H W/ Pyr and 0.9H Y/
a
a
Pyr); 13C NMR (100 MHz,
CDCl3) d 24.5 (CH2), 28.8–28.9 (CH2), 33.7–33.8 (CH2), 44.0–46.9
(CH), 49.1–49.6 (CH), 61.5–61.8 (CH2), 77.5–77.7 (C), 79.0 (C),
121.6–122.2 (CH), 122.4–122.7 (CH), 123.9–124.2 (CH), 126.6–
130.2 (CH), 133.1 (CH), 133.9 (CH), 135.8–136.6 (CH), 138.3–
138.5 (C), 141.9–143.1 (C), 147.7–148.1 (CH), 149.1–149.3 (CH),
152.6 (C), 153.9–155.5 (C), 158.0–158.3 (C), 160.6–161.0 (C),
Ar), 8.41–8.50 (2.7H, m, 0.4H 2U/
and 0.8H Y/ Pyr), 8.62–8.63 (1.3H, m, 0.5H W/
Pyr); 13C NMR (100 MHz, CDCl3) d 44.1–47.0 (CH), 49.2–51.1
aPyr, 1H 2U/
aPyr, 0.5H W/aPyr
a
aPyr and 0.8H/
a
(CH), 62.3–62.6 (CH2), 77.8 (C), 121.7–122.8 (CH), 124.0–124.3
(CH), 126.6–130.9 (CH), 133.2–134.0 (CH), 135.9–136.7 (CH),
138.3–138.5 (C), 141.8–143.1 (C), 147.8–148.0 (CH), 149.1–149.3
(CH), 152.6 (C), 154.0–155.6 (C), 157.9–158.3 (C), 160.6–161.0
172.3–172.6 (C), 174.6–175.4 (C);
m
max(NaCl)/cmꢀ1 1152 and
1211 (C–O ester), 1585 (C@O imide), 1718 (C@O ester); m/z
(FAB+) 1249 (MH+, 19%), 397 (100); (Found: MH+ 1249.5065,
C78H69N6O10 requires 1249.5075).
(C), 164.3–164.6 (C), 174.6–175.5 (C); m
max/cmꢀ1 1079 and 1241
(C–O ester), 1585 (C@O imide), 1713 (C@O ester); m/z (FAB+)
1213 (MH+, 4%), 120 (100); (Found: MH+ 1213.4124, C76H57N6O10
requires 1213.4136).
4.1.22. N-Dodecanedioyloxymethyl-5-(
a-hydroxy-a-2-pyridylb
enzyl)-7-( -2-pyridylbenzylidene)-5-norbornene-2,3-dicarboxi
a
mide dimer (25)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (1 mL), dichloromethyl dodecanedioate (64)
(66 mg, 0.20 mmol) in dimethylformamide (0.2 mL) and potassium
carbonate (54 mg, 0.39 mmol). The mixture was stirred at room
temperature for 48 h, taken up in chloroform (10 mL), washed with
water (2 ꢁ 5 mL), dried over anhydrous magnesium sulfate and the
solvent removed in vacuo. Purification by flash chromatography
(hexane/ethyl acetate 1:2) afforded 25 as a colourless solid
(60 mg, 0.05 mmol, 12%). mp 105–110 °C; 1H NMR (300 MHz,
CDCl3) d 1.20–1.39 (12H, bm, 2ꢁ OCO(CH2)2(CH2)3), 1.54–1.73
(4H, m, 2ꢁ OCOCH2CH2), 2.28–2.40 (4H, m, 2ꢁ OCOCH2), 3.43
(1.5H, dd, J = 7.8 and 4.5 Hz, Y/H-3), 3.52 (0.5H, dd, J = 8.1 and
5.0 Hz, V/H-2), 3.66–3.74 (2H, m, 0.5H V/H-3, 1.5H Y/H-2), 3.88–
3.92 (0.5H, m, V/H-1), 3.98–4.00 (1.5H, m, Y/H-4), 4.35–4.37
(0.5H, m, V/H-4), 4.50–4.53 (1.5H, m, Y/H-1), 5.28–5.32 (2H, m,
NCH2O), 5.46–5.59 (5.5H, m, 2H NCH2O, 0.5H V/H-6, 1.5H Y/H-6
and 1.6H OH), 5.74 (0.4H, s, OH), 6.74–7.59 (32H, m, Ar), 8.48–
4.1.24. N-[
hydroxy-
bornene-2,3-dicarboximide dimer (27)
a
-(Ethylenebis(hydrogensuccinoyloxy))methyl]-5-(
a-
a-2-pyridylbenzyl)-7-( -2-pyridylbenzylidene)-5-nor
a
A similar procedure24,26 to that previously described for the
preparation of 18 was followed using dichloromethyl ethylene
bis(hydrogen succinate) (68) (56 mg, 0.2 mmol) in acetone
(0.5 mL), and sodium iodide (30 mg, 0.2 mmol) in acetone
(0.5 mL), at room temperature for 3 h. The solvent was removed
in vacuo and the crude iodomethyl ethylene bis(hydrogen succi-
nate) was taken through to the next step without further purifica-
tion.
A solution of NRB (200 mg, 0.39 mmol), diiodomethyl
ethylene bis(hydrogen succinate) and potassium carbonate
(54.0 mg, 0.40 mmol) in dimethylformamide (2 mL) was then stir-
red at room temperature for 16 h. The mixture was taken up in
chloroform (20 mL), washed with water (2 ꢁ 10 mL), dried over
anhydrous magnesium sulfate and the solvent removed in vacuo.
Purification by flash chromatography (chloroform/methanol
100:1, then hexane/ethyl acetate 4:1–3:2) afforded 27 as an oily
colourless solid (68 mg, 0.05 mmol, 26%). mp 49 °C; 1H NMR
(400 MHz, CDCl3) d 2.61–2.73 (8H, m, 2ꢁ NCH2OCO(CH2)2), 3.37
(0.6H, dd, J = 7.9 and 4.4 Hz, W/H-3), 3.44 (1H, dd, J = 7.9 and
4.6 Hz, Y/H-3), 3.50–3.74 (3H, m, 0.4H U/H-2 and U/H-3, 0.4H V/
H-2 and V/H-3, 1.2H W/H-2 and W/H-4 and 1H Y/H-2), 3.87–
3.91 (0.4H, m, 0.2H U/H-1 and 0.2H V/H-1), 3.97 (1H, dt, J = 4.4
and 1.2 Hz, Y/H-4), 4.18–4.19 (0.2H, m, U/H-4), 4.26–4.36 (4.2H,
m, 0.2H V/H-4, 4H COO(CH2)2OCO), 4.50–4.51 (1.6H, m, 0.6H W/
H-1 and 1H Y/H-1), 5.30–5.34 (1.3H, m, NCH2O and OH), 5.47–
5.60 (5.9H, m, 4.7H NCH2O and OH, 0.2H V/H-6, 1H Y/H-6), 6.04
(0.2H, dd, J = 3.3 and 1.2 Hz, U/H-6), 6.06 (0.6H, dd, J = 3.2 and
1.0 Hz, W/H-6), 6.75–7.59 (32H, m, Ar), 8.42–8.54 (2.4H, m, 0.4H
8.54 (2.5H, m, 1H 2V/
aPyr and 1.5H Y/aPyr), 8.62–8.64 (1.5H, m,
1.5H Y/
a
Pyr); 13C NMR (100 MHz, CDCl3) d 24.6 (CH2), 29.0–29.6
(CH2), 33.8–33.9 (CH2), 44.5–46.7 (CH), 49.2–49.4 (CH), 61.8
(CH2), 77.8 (C), 79.0 (C), 121.7–122.0 (CH), 122.6–122.8 (CH),
124.1–124.3 (CH), 126.7–130.3 (CH), 136.0–136.5 (CH), 138.4–
138.6 (C), 142.0–142.4 (C), 147.8–147.9 (CH), 149.2–149.4 (CH),
154.0 (C), 154.9 (C), 155.6 (C), 158.0–158.4 (C), 161.1 (C), 172.5–
172.7 (C), 175.2–175.5 (C); m
max/cmꢀ1 1149 and 1209 (C–O ester),
1585 (C@O imide), 1713 (C@O ester); m/z (ESI+) 1277 (MH+, 28%),
627 (100); (Found: MH+ 1277.5441, C76H65N6O10 requires
1277.5383).
2U/
a
Pyr, 0.4H 2V/
a
Pyr, 0.6H W/
a
a
Pyr and 1H Y/aaPyr), 8.62–8.63
4.1.23. 5-(a-Hydroxy-a-2-pyridylbenzyl)-7-(a-2-pyridylbenzylid
ene)-N-terephthaloyloxymethyl-5-norbornene-2,3-dicarboxi
(1.6H, m, 0.6H W/
a
Pyr and 1H Y/
Pyr); 13C NMR (100 MHz, CDCl3)
d 28.7 (CH2), 29.6 (CH2), 44.0–46.6 (CH), 49.1–49.6 (CH), 61.7–62.4
(CH2), 77.7 (CH), 121.7–122.7 (CH), 123.9–124.3 (CH), 126.6–130.3
(CH), 133.2–134.0 (CH), 135.8–136.6 (CH), 138.3–138.6 (C), 141.9–
142.8 (C), 147.8–148.1 (CH), 149.2–149.4 (CH), 152.6–155.5 (C),
158.0–158.3 (C), 160.6–161.0 (C), 171.0–171.7 (C), 174.6–175.4
mide dimer (26)
A similar procedure24 to that previously described for the prep-
aration of 1 was followed using NRB (200 mg, 0.39 mmol) in
dimethylformamide (1 mL), dichloromethyl terephthalate (65)
(53 mg, 0.20 mmol) in dimethylformamide (0.2 mL) and potassium
carbonate (54 mg, 0.39 mmol). The mixture was stirred at room
temperature for 48 h, taken up in chloroform (10 mL), washed with
water (2 ꢁ 5 mL), dried over anhydrous magnesium sulfate and the
solvent removed in vacuo. Purification by flash chromatography
(hexane/ethyl acetate 1:2) afforded 26 as a colourless solid
(49 mg, 0.04 mmol, 21%). mp 150–157 °C; 1H NMR (400 MHz,
CDCl3) d 3.42 (0.5H, dd, J = 8.0 and 4.4 Hz, W/H-3), 3.49 (0.8H, dd,
J = 8.0 and 4.4 Hz, Y/H-3), 3.55–3.65 and 3.70–3.80 (3.2H, 0.4H U/
H-2 and U/H-3, 1H V/H-2 and V/H-3, 1H W/H-1 and W/H-2, 0.8H
Y/H-2), 3.90–3.95 (0.7H, m, 0.2H U/H-1 and 0.5H V/H-1), 3.99–
4.05 (0.8H, m, Y/H-4), 4.19–4.23 (0.2H, m, U/H-4), 4.35–4.40
(0.5H, m, V/H-4), 4.48–4.56 (1.3H, bm, 0.5H W/H-1 and 0.8H Y/
(C);
m
max(NaCl)/cmꢀ1 1147 and 1214 (C–O ester), 1586 (C@O
imide), 1714 (C@O ester); m/z (FAB+) 1309 (MH+, 8%), 120 (100);
(Found: MH+ 1309.4546, C78H65N6O14 requires 1309.4559).
5. Experimental (Pharmacology)
5.1. Rat caudal artery and aortic ring isolation and recording of
contractile force
Male Wistar rats (150–250 g) were obtained from Charles River
Italia (Milano, Italy) and killed by decapitation. Ventral caudal ar-
tery was isolated, placed in Tyrode solution at room temperature

4010
D. Rennison et al. / Bioorg. Med. Chem. 20 (2012) 3997–4011
and cleaned of extraneous fatty and connective tissue under a dis-
section microscope. All vessels were cut into rings 2 mm long,
mounted on a custom-built plexiglass support by means of two
intraluminal tungsten wires and placed in 20 mL double-jacketed
organ baths filled with Tyrode solution of the following composi-
tion (mM): NaCl 125, KCl 5, CaCl2 2.7, MgSO4 1, KH2PO4 1.2, NaH-
CO3 25 and glucose 11, maintained at 37 °C, pH 7.35, bubbled with
95% O2 and CO2. The endothelium was removed by gently rubbing
the lumen of the rings with a very thin rough-surfaced tungsten
wire (caudal artery). The mechanical activity of the rings was de-
tected by means of an isometric force transducer (Ugo Basile,
Comerio, Italy) coupled to a pen recorder (Ugo Basile, Comerio,
Italy). Rings were passively stretched to impose a resting tension
(2 g) and were allowed to equilibrate for 60 min. After equilibra-
tion, each ring was repeatedly simulated with both KCl (90 mM)
concentration of 200 lM, 2.5% DMSO overall, 37 °C, 6 h, n = 3]. The
assay protocol followed is as that described above for the rat serum
assay, with an adjusted end-point of 6 h.
5.5. Simulated gastric fluid (SGF) assay
All in vivo prodrug candidates were subject to a 1 h hydrolytic
stability appraisal in the presence of SGF, without pepsin33
[200
to pH 1.2 with concentrated hydrochloric acid, at a final prodrug
concentration of 200 M, 2.5% DMSO overall, 37 °C, n = 3). The
lL total volume, sodium chloride solution (0.03 M) acidified
l
RP-LCMS analysis protocol followed is as that described above for
the hydrolytic stability assay.
5.6. In vivo experiments and palatability trial
and phenylephrine (10
tained. To verify the absence of the endothelium, rings contracted
with 1 M phenylephrine were exposed to 2 m carbamylcholine.
The absence of the endothelium was revealed by the lack of car-
bamylcholine-induced relaxation. Each compound was tested for
lM) until reproducible responses were ob-
Wistar rats (150–200 g) were used to appraise the rodenticidal
activity of the compounds put forward for in vivo evaluation.
Briefly, prior to administration the prodrug candidates were dis-
solved in 0.2 M hydrochloric acid (5% DMSO overall), to the desired
dose, and without delay either injected intravenously, via the tail,
or orally gavaged. All treated animals were housed in a quiet room
and monitored every minute (iv) or every 15 min (oral) to verify
early signs of toxicity. Wistar rats (ca. 250 g) were presented with
1% w/w toxicant (n = 6) in a peanut butter bait, following 4 days
‘pre-baiting’ with a peanut butter formulation free of toxicant.
l
l
the vasoconstrictor activity at a concentration of 50 lM, which
has been previously reported to be the one evoking the maximal
response to norbormide.36 The contractile responses to the com-
pounds were expressed as percent of the 90 mM KCl response.
5.2. Hydrolytic stability assay
All prodrugs were subject to a 1 h hydrolytic stability appraisal
[200 lL total volume, at a final prodrug concentration of 200 lM,
Acknowledgment
2.5% DMSO overall, 37 °C, n = 3] using Tyrode solution for those
candidates which displayed vasoconstrictory activity in the rat
caudal artery contractile experiment, and phosphate buffer
(0.1 M, pH 7.4) for those revealed to be non-vasoconstricting pre-
cleavage. Analysis was by RP-LCMS at an injection volume of
The authors wish to thank Landcare Research for their financial
support and assistance.
Supplementary data
5
l
L. Each prodrug and corresponding drug were subject to exter-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.014.
nal calibration curves at 50, 100 and 200 M to allow conversion of
absorbance unit area into nmol, from which the percentage of drug
l
released from each prodrug was calculated.
References and notes
5.3. Rat serum assay
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ꢀ78 °C. Similar assay conditions to those reported by Li Di and
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lL total volume, 80% rat serum
(diluting with phosphate buffer (0.1 M, pH 7.4)), at a final prodrug
concentration of 200 M, 2.5% DMSO 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 prodrug/drug con-
centration of 50
M. Samples were centrifuged at 14,500ꢁg for
15 min using an Eppendorf Mini Spin Plus centrifuge, at ambient
temperature. 400 L of the supernatant was removed and trans-
ferred to clean tubes. Analysis was by RP-LCMS at an injection vol-
ume of 20 L. Each prodrug and corresponding drug were subject
to external calibration curves at 50 M, 100 and 200 M to allow
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l
l
l
l
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l
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22. As a mixture of cis-threo/trans-threo/cis-erythro/trans-eyrthro stereoisomers;10
of comparable isomeric ratio to that of the pre-cursor endo-NRB mixture.
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lL total volume, 1 mg/mL rat liver S9 fraction
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