Synthesis, binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Article abstract of DOI:10.1016/j.bmc.2010.06.020

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or 'attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.

Full text of DOI:10.1016/j.bmc.2010.06.020

Bioorganic & Medicinal Chemistry 18 (2010) 5647–5660
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, binding and bioactivity of c-methylene c-lactam ecdysone
receptor ligands: Advantages of QSAR models for flexible receptors
Woldeamanuel Birru a, Ross T. Fernley b, Lloyd D. Graham c, Julian Grusovin b, Ronald J. Hill c,
Albert Hofmann a, Linda Howell a, Peter J. James d, Karen E. Jarvis a, Wynona M. Johnson a, Dionne A. Jones a,
Christa Leitner a, Andris J. Liepa a, George O. Lovrecz b, Louis Lu b, Roland H. Nearn a, Brian J. O’Driscoll a,
a,
Tram Phan b, Matthew Pollard c,1, Kathleen A. Turner a, , David A. Winkler
*
*
a CSIRO Molecular and Health Technologies, Ian Wark Laboratory, Bag 10, Clayton South, Vic. 3169, Australia
b CSIRO Molecular and Health Technologies, Parkville Laboratory, 343 Royal Parade, Parkville, Vic. 3052, Australia
c CSIRO Food and Nutritional Sciences, Sydney Laboratory, PO Box 52, North Ryde, NSW 1670, Australia
d Animal Research Institute, Department of Primary Industries and Fisheries, 665 Fairfield Rd., Yeerongpilly, Qld 4105, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to
ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding,
making modelling methods such as docking extremely challenging. It is, however, possible to generate
excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively
restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding
Received 16 April 2010
Revised 2 June 2010
Accepted 7 June 2010
Available online 15 June 2010
and selected in vivo toxicity data for c-methylene c-lactams, a new class of high-affinity ligands for ecdy-
This paper is dedicated to the memory of
Dr. Dionne Jones, deceased 5/10/2000
sone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR
study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this
class of ligands is indeed recognised by a single conformation of the EcR binding pocket.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords:
Biological activity
Structure–activity relationships
Synthesis
Ecdysone receptor
1. Introduction
oids. As the ecdysone receptor is absent from other organisms, it
is an attractive target for the generation of safe insecticides. One
Nuclear hormone receptors are a large family of ligand-acti-
vated proteins which bind to DNA sequences and regulate the
expression of genes within the cell nucleus. These regulatory pro-
teins control homeostasis, development and differentiation of tis-
sues within an organism.
class of ecdysone receptor ligands, the diacylhydrazines, has been
commercialised for use as insecticides.
Due to its absence from most taxa, EcR and its functional do-
mains are also used as components of ecdysone switches for the
control of reporter and therapeutic genes in mammalian cells,
and for control of transgenes more generally in agriculturally
important plant and animal species. In this role, gene constructs
whose expression can be controlled by the level of ecdysteroid
can be prepared relatively easily. As ecdysone receptors are natu-
rally absent from mammalian cells, they provide an orthogonal
gene switch that offers great promise for the safe control of trans-
gene expression in medical and veterinary applications.2,3
The ecdysone receptor protein (EcR)1 is a nuclear hormone
receptor that occurs almost exclusively in the phylum Arthropoda
(e.g., insects, arachnids and crustaceans). The functional receptor
for ecdysteroid hormones is a non-covalent heterodimer of EcR
with a second nuclear receptor, the ultraspiracle protein (USP),
the latter being a homologue of the retinoid X receptor, RXR. The
functional ecdysone receptor is activated by binding to ecdyster-
The ligand binding domains (LBDs) of nuclear hormone recep-
tors frequently display structural plasticity and can bind to struc-
turally diverse ligands by adapting the shape of their ligand
binding pockets.4,5 In solution, the unliganded receptor LBD will
have a number of different conformations in dynamic equilibrium.6
A ligand will sample these conformations and bind to the one that
is complementary to its shape and other properties. This shifts the
* Corresponding authors. Tel.: +613 9545 2586; fax: +613 9545 2589 (K.A.T.); tel.:
+613 9545 2477; fax: +613 9545 2446 (D.A.W.).
E-mail addresses: kathleen.turner@csiro.au (K.A. Turner), david.winkler@csiro.au
(D.A. Winkler).
1
Present address: Arana Therapeutics Ltd, Level 2, 37 Epping Rd., Macquarie Park,
NSW 2113, Australia.
0968-0896/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.020

5648
W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
equilibrium towards the conformation that binds this ligand.7
It has been demonstrated that the ecdysone receptor displays such
behaviour.8,9
Potentially, the binding site of the ecdysone receptor might
change smoothly and continuously in size and shape when differ-
ent types of ligands bind. However, 3D quantitative structure–
activity relationships (QSAR) studies have suggested that, for a
given chemotype, the shape of the binding pocket does not change
much in response to variation in substitution on the ligand.10 This
suggests that the binding pocket may be capable of adopting only a
finite number of relatively stable shapes. The existence of robust
chemotype-specific binding pocket conformations would provide
greater confidence in binding predictions for ligand analogue
optimisation programs. In addition, if all of these can be identified
by structural biology, the task of predicting the activity of novel
ligands by docking would be greatly facilitated.
In EcR, as in other nuclear receptors, the ligand binding pocket
is likely to be open and accessible in the apo-LBD. Upon ligand
binding, the energetically preferred conformation then changes
to one where helix 12 is positioned over the mouth of the pocket,
acting as a lid. X-ray crystallography (Fig. 1) has shown that the
EcR LBD adopts a ‘sock’ shaped pocket when binding the natural
steroid hormone, 20-hydroxyecdysone (Fig. 2). Such studies have
also shown that the binding pocket for the diacylhydrazines,
although utilising some of the same amino acid residues, is very
different in shape and occupies a somewhat different position in
the LBD, as Figure 1 illustrates.8,11–13 Previous attempts to use
docking to the ecdysteroid-bound LBD conformation to understand
the mode of binding of the diacyhydrazines to the ecdysone recep-
tor and to predict binding affinities resulted in models14,15 which
were ultimately proved incorrect by crystallography.11
Variability in ecdysone receptor structure between insect
orders can be looked upon as providing a resource for tailoring
the breadth of control spectra, potentially giving an advantage over
broad-spectrum insecticides. The diacylhydrazines display remark-
able selectivity for insect ecdysone receptors from different taxo-
nomic orders. For example, Tebufenozide (Fig. 2) is two to three
orders of magnitude more effective against Lepidoptera (moths/
butterflies) than it is against Diptera (flies).16 The selectivity for
certain insect orders limits the environmental impact of this type
of insecticide. The effectiveness of diacylhydrazines as insecticides
Figure 2. Examples from known ecdysone receptor ligand classes.
generally correlates with their affinities for the LBDs of the corre-
sponding ecdysone receptors.17,18 While all insect ecdysone recep-
tors bind the physiological ligand (20-hydroxyecdysone) using
highly conserved amino acid residues, non-ecdysteroid ligands
employ interactions with other residues in the binding pocket.19
Since these residues are less conserved in EcRs from different tax-
onomic orders, the binding affinity of each non-ecdysteroid chem-
otype can also vary greatly across orders.
Despite considerable research in this area, no class of ecdysone
receptor ligand with selectivity outside the Lepidoptera and Cole-
optera has yet been developed for commercial use. One hurdle in
this respect is the plasticity of the EcR LBD, which makes develop-
ment of a good model for binding of ligand to the receptor more
Figure 1. EcR-LBD binding pocket (a) from Bemisia tabaci bound to an ecdysteroid or (b) Heliothis virescens bound to a diacylhydrazine. Reproduced with permission from
Ref. 11.

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5649
difficult. A few other classes of ecdysone receptor ligands have
been discovered that have not survived the pathway to commercial
insecticides, but have potential applications in ecdysone based
gene switches, for example, tetrahydroquinolines9,20,21 and
oxadiazolines22 (Fig. 2).
High-throughput screening can provide a large volume of data
from which to develop QSAR to guide synthesis and optimisation
of activity against a selected target. This is particularly important
for a structurally adaptive receptor as it may provide a model that
cannot reliably be obtained by docking procedures. Others have re-
cently used high-throughput screens based on transgenic reporter
cell lines to identify new classes of ecdysone receptor ligand.23 We
have developed an in vitro fluorescence polarisation assay for the
ecdysone receptor.24 This can be automated as a high-throughput
screen to efficiently and reliably assess the binding activity of the
ecdysone receptor against thousands of compounds from a chem-
ical library.18
L. cuprina (sheep blowfly) was assayed by high-throughput screen-
ing in vitro. The IC50 of a selection of these compounds was then
determined. Many of the methylene lactams were good ligands
for the recombinant ecdysone receptor LBDs, binding with compa-
rable affinity to the natural ligand 20-hydroxyecdysone. Some (e.g.,
11e) appeared to be almost as effective as the high-affinity phy-
toecdysteroid ponasterone A.
None of the hydroxylactams 3 demonstrated any binding to the
ecdysone receptor, indicating that the methylene at position 5 is
important. For B. ovis, replacement of a hydrogen on this methy-
lene results in a decrease in binding as the size of the group in-
creases, leading to complete loss of activity when R8 is n-propyl.
For B. ovis, a variety of substituents are tolerated around the
aromatic ring in 4–14. Polysubstitution is preferred over monosub-
stitution and 2,5-disubstitution and 2,4,5-trisubstitution were
most consistently favoured. There is clearly some room for steric
bulk in the 2 position. t-Butyl substituents in the 2-position are
favoured, but only when there is no substituent on the 6-position.
Larger groups such as benzyl and phenoxy in the 2-position are
also tolerated. Chlorination generally leads to the strongest
binders.
Using high-throughput screening we have discovered a new
class of compounds, the
c-methylene c-lactams, which bind to
recombinant insect ecdysone receptor LBDs with high affinity.25,26
We report the synthesis and an extensive 3D QSAR study of
c
-methylene
c
-lactams as ligands for the ecdysone receptor from
There is no clear indication of an effect of the size of the substit-
uents R7 on the activity. Methyl compounds 4, phenyl compounds
12 and n-hexyl compound 13a all showed similar propensity to
bind. No heteroatoms were investigated at this position.
Replacement of the aromatic ring on the lactam nitrogen with
an alicyclic ring generally leads to decreased binding. One signifi-
cant exception to this is 15d, where R7 is phenyl. It is possible that
this binds in the receptor in the opposite orientation. Substitution
of the aromatic ring for a heterocyclic ring, benzyl moiety or an
amide also generally resulted in decreased binding.
an insect of the order Phthiraptera (lice) and another from the
order Diptera. We also demonstrate that these methylene lactams
can disrupt the nymphal development of Bovicola ovis (sheep body
louse).
2. Results and discussion
2.1. Chemistry
In order to probe the volume around R1, compounds 16 with
acyl and aminoacyl groups at this position were synthesised. Their
binding affinity as compared to the nitriles was inconsistent, being
sometimes greater, sometimes less. These were not stable for ex-
tended periods and in some cases this may have affected the
results.
Only twenty-two IC50 values were determined for binding to
the L. cuprina ecdysone receptor, insufficient to carry out a detailed
SAR analysis. However, it can be seen that methylene lactams
clearly exhibit binding affinity for the L. cuprina receptor. In many
cases the IC50 values are similar to those for binding to the B. ovis
protein, although some differ significantly, no doubt reflecting
local differences in the ligand binding pockets. Our data add weight
to the notion that new ligand chemistries can be discovered
for ecdysone receptors outside the orders Lepidoptera and
Coleopterea.
Acetamides 1 were generally prepared from the corresponding
amines by literature procedures. Procedures requiring heating
were improved by use of microwave irradiation. Condensation of
acetamides 1 with vicinal diones 2 provided hydroxylactams 3
which were dehydrated without further purification (Scheme 1).
This procedure sometimes required protracted reaction times,
but attempts to decrease these by raising the temperature resulted
in generation of significantly more by-products. Dehydration of 3
provided the desired lactams 4–16. Again the reaction time can
be decreased by employing microwave irradiation.
2.2. Structure–activity relationships from ecdysone receptor
binding assays
The binding of three hundred and fifty compounds of types
4–16 to recombinant ecdysone receptor LBDs from B. ovis and/or
Given that the conjugated cyano-substituted diene motif pres-
ent in the methylene lactams 4–16 represents a potential Michael
acceptor, we were concerned that these compounds might inhibit
the receptor by forming covalent adducts rather than by reversible
binding. To investigate the likelihood of this possibility, a number
of examples were treated with a variety of nucleophiles under
acidic, basic and neutral conditions. Under basic conditions reac-
tion did occur to provide complex intractable products, however
under neutral and acidic conditions no reaction occurred. There
was just one exception: on treatment with a considerable excess
of sodium 4-toluene sulfinate in acetic acid, an adduct was isolated
which corresponded to reaction at the exocyclic methylene. How-
ever, attempts to purify this material by recrystallisation resulted
in loss of the sulfinyl moiety and recovery of the pure methylene
lactam. As physiological pH is close to neutral, on the basis of this
evidence, it is unlikely that the methylene lactams undergo a
Michael reaction with the receptor. This is supported by the obser-
vation that there is no particular correlation between Michael
acceptor ability and binding affinity.
Scheme 1. Reagents and conditions: (a) piperidine, morpholine, DABCO or DBU,
DMF, 1-43 days (method D); (b) formic acid, TFA or MeSO3H 18–80 °C, 4–18 h
(method E); TFA or TfOH, DCM, rt, 10 min–24 h (method F); formic acid,
lW, 120 °C,
10 min (method G).

5650
W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
Table 1
Structures and IC50 for binding to the Bovicola ovis ecdysone receptor
Compound
Clog P
R
R1
R2
R3
R4
R5
R6
R7
R8
IC50 (B. ovis)
M)
IC50 (L. cuprina)
(lM)
(
l
4a
4b
4c
4d
4e
4f
4g
4h
4i
2.67
2.76
2.45
3.38
3.78
4.24
3.24
2.80
3.38
2.80
4.45
4.02
3.07
4.45
2.91
4.24
2.52
1.37
1.32
3.26
2.47
2.36
5.35
5.35
4.12
2.70
3.13
2.32
3.09
4.41
3.88
4.41
2.20
3.66
1.94
1.47
3.75
3.78
3.85
3.97
5.21
3.48
3.27
4.19
3.05
4.23
4.31
3.91
3.79
3.70
5.02
3.61
4.01
4.50
4.22
4.44
4.14
4.54
4.71
5.42
4.41
4.41
4.19
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
Cl
Br
Me
iPr
tBu
Ph
CF3
MeO
CF3O
EtO
PhO
PhCH2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
55
37.5
80
22.5
17.5
25
27.5
11
45
350
25
22.5
19
18
48
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
Cl
PhO
MeS
CF3
25
22
35
Ph
NMe2
NHCOMe
N(Me)COMe
150
575
400
22.5
90
150
12.5
5
4.5
92.5
650
55
Cl
F
Cl
F
MeO
Cl
tBu
CF3
OH
MeO
MeO
EtCOO
iPr
tBu
tBu
NO2
MeO
Cl
tBu
CF3
Cl
Cl
NO2
Cl
6.5
55
iPr
Me
Et
20.8
160
150
160
15
23.5
Me
Cl
NO2
–OCH2O–
Cl
Cl
Cl
F
20
67.5
9.8
15
7
11.3
55
48
20
68
160
40
26
17.5
15
15
80
Cl
27.5
8.5
Cl
Cl
Cl
Cl
Cl
Cl
tBu
Cl
Cl
Cl
Me
Me
Cl
Cl
Me
Me
Me
Me
Me
Me
iPr
Me
CF(CF3)2
tBu
CF3O
Cl
Me
tBu
Et
iPr
Cl
Cl
Cl
Et
Et
Et
Et
Et
Et
Et
Et
Et
nPr
nPr
nPr
iPr
iPr
iPr
Cl
Cl
Cl
11
Me
Me
7g
7h
7i
45
Cl
Cl
Cl
Cl
5.5
160
8.5
48
10.5
9.5
6.5
27.5
62.5
11
7.25
Cl
8a
8b
8c
9a
9b
9c
9d
9e
Me
Me
iPr
tBu
tBu
iPr
Cl
15
42
47
480
Cl
Me
Me
Me
Cl
Cl
iPr
iPr
Me

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5651
Table 1 (continued)
Compound
Clog P
R
R1
R2
R3
Cl
R4
R5
R6
R7
R8
IC50 (B. ovis)
M)
IC50 (L. cuprina)
(lM)
(
l
9f
3.98
4.97
4.75
4.75
6.08
4.75
4.14
4.67
5.07
3.78
5.78
4.64
3.24
3.91
4.84
5.24
5.58
5.43
4.51
5.74
4.05
3.50
4.15
6.76
4.22
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
Me
CF3O
Me
Me
tBu
Cl
Me
Et
iPr
Me
Me
iPr
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
55
15
4.3
6
4
6
10a
10b
10c
10d
10e
10f
10g
10h
10i
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
8.3
12.5
5.5
56
Cl
Cl
Me
Me
Me
Me
NO2 nBu
Me
Me
35
46
20.3
71
17.5
11
21
11
6.8
5.5
6.3
2.7
30
45
8.5
12.5
43
22
85
33
Me
iPr
Me
10j
10k
10l
Cl
Me
nBu
nBu
nBu
iBu
iBu
iBu
iBu
iBu
iBu
iBu
Ph
MeO MeO
MeO
11a
11b
11c
11d
11e
11f
11g
12a
12b
12c
13a
14a
Me
Cl
Cl
Cl
CF3
Cl
Cl
CF3
Cl
Me
Cl
Cl
Me
Cl
Cl
F
iPr
Me
Cl
5.5
175
Me
Cl
Cl
Cl
Me
Ph
Ph
nOct
Me
Me
Me
Cl
55
Me
Cl
78:22
E:Z-Me
E-nPr
Z-iPr
H
H
H
H
H
14b
14c
15a
15b
15c
15d
15e
15f
6.18
2.36
1.67
2.23
3.82
3.62
3.27
2.51
CN
CN
CN
CN
CN
CN
CN
CN
tBu
Cl
MeO
Me
Me
Me
Me
Me
nPr
Ph
2300
65
85
MeO
Cyclopentyl
Cyclohexyl
Cyclohexyl
50
27.5
7.5
250
48
Cyclohexyl
12.5
2,3-Me2cyclohexyl
Bicyclo[2.2.1]hept-
2-yl
Me
Me
H
15g
3.93
Tetrahydro-
naphthalen-1-yl
CN
Me
H
26
15h
15i
15j
15k
15l
15m
15n
15o
15p
16a
16b
16c
16d
16e
16f
4.24
2.57
3.00
2.71
2.15
1.26
2.00
2.24
0.70
3.87
4.70
5.01
5.17
6.54
5.42
6.34
5.91
4.99
5.24
4-Chloronaphth-1-yl CN
Me
Me
Et
H
H
H
(CH2)3
H
H
H
H
H
H
H
H
H
H
H
H
H
15
2-Chlorobenzyl
4-ClPhCONH
CN
CN
37.5
72.5
88
125
95
45
26
90
45
16
11
12.5
18
58
2250
9
44
Cyclohexylcon(Me)
Piperidin-1-yl
5-Clpyrid-2-yl
3,5-Cl2pyrid-2-yl
Quinolin-3-yl
Thiazol-2-yl
CN
CN
CN
CN
CN
CN
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
MeCO
i-PrCO
EtOCO
PhCO
PhCO
PhCO
PhCO
PhCO
PhCO
3-
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CF3
17.5
250
Cl
CF3
Cl
iPr
iPr
Cl
Me
iPr
16g
16h
16i
Cl
Me
Me
Cl
Me
H
H
16j
12.5
ClPhNHCO
Ponasterone A
20-Hydroxy-
ecdysone
1.1
24
0.9
5.5
2.2.1. 3D QSAR models
stricted rotation around the N-Ph bond needing to be considered
(Fig. 3).
Both CoMFA and COMSIA models were derived from the data.
The CoMFA models using steric and electrostatic fields alone, or
augmented by calculated log P, were of higher statistical signifi-
cance than the COMSIA models using a combination of steric, elec-
trostatic, lipophilic, donor and acceptor fields. Consequently only
the CoMFA results are reported here. Table 2 shows that the statis-
tical quality of the CoMFA models was similar. The compounds in
the data set were methylene lactams, with one of the most active
members providing the template for 3D QSAR alignment. This
compound (6b) was also conformationally simple, with only re-
The model using steric, and electrostatic CoMFA fields had the
best statistical quality. Figure 4 shows the predicted values for
B. ovis pI50 values compared to the measured values. Compounds
in the training set were predicted within a factor of 1.5 on average.
The cross-validation predictions were within a factor of 2.7 on
average. Lipophilicity appears to be important for activity, as a plot
of pI50 against calculated log P (Fig. 5) clearly shows. Only com-
pounds with n-butyl substituents on the methylene moiety of
the methylene lactam and very large substituents on the ortho

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W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
Table 2
Results of the 3D QSAR modelling studies for B. ovis
Parameter
CoMFA
CoMFA + log P
CoMFA refined
SEP
0.435
7
0.48
0.196
0.89
127
0.440
8
0.47
0.186
0.91
125
0.424
8
0.51
0.183
0.91
129
# Components
q2 (LOO)
SEE
r2
F
Contribution %
Steric
Electrostatic
Lipophilic/log P
Number of compounds
63
37
63
33
4
62
38
113
113
113
Figure 5. Relationship between B. ovis pI50 and calculated log P. The template
molecule 6b, and four other compounds mentioned in the text, have been omitted.
Figure 3. Methylene lactam 6b, used for CoMFA alignments.
Figure 4. Predicted pI50 value for B. ovis from CoMFA model using steric and
electrostatic fields.
position of the phenyl ring, deviate markedly from this relation-
ship. However, as log P (lipophilicity) and calculated molar refrac-
tivity MR (size and polarisability) also correlated (r2 = 0.74) with
one another, the apparent effect of lipophilicity could be due to
molecular size.
Figure 6. CoMFA steric map with an active template lactam 11e. Green regions
favour additional steric bulk, yellow regions disfavour additional steric bulk.
gand then there would be poor correlation in the model. The high
degree of correlation in this model supports the conclusion from
previous QSAR studies on ecdysteroids27,28 and diacylhydra-
zines29,30 that the LBD does not greatly alter its conformation on
binding variously substituted ligands within each chemotype. This
is consistent with the idea that the EcR LBD does indeed adopt a
limited number of stable binding pocket configurations.
Given the tendency of leave-one-out cross-validation to overes-
timate the predictivity of models, we also carried out a four group
cross-validation study for the best QSAR model, CoMFA refined.
This omits 29 compounds from the training set and predicts their
activities using the QSAR model derived from the remaining com-
pounds. We obtained an SEP of 0.46 and a cross-validated r2 of 0.42
for six components. This is slightly inferior to the LOO model re-
ported in Table 2, but is still statistically significant and may be a
more accurate reflection of the predictivity of the model.
These structure–activity relationships are consistent with the
fact that nuclear hormone receptors bind endogenous ligands that
are relatively rigid and quite lipophilic. If the ecdysone receptor
structure deformed continuously in response to changes in the li-
2.3. Molecular field maps
The molecular field maps in Figures 6 and 7 summarise the con-
tributions that molecular size and/or lipophilicity and partial
charge make to the binding of the analogues in the data set. The
steric map in Figure 6 shows that there is a sterically favoured

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5653
Table 3
LC50 for test compounds against B. ovis
Compound
LC50
(
lg/mL)
95% Confidence limits
5o
7e
8a
10b
11c
11d
15d
15l
167
190
66
175
119
26
29–462
46–343
10–168
66–888
62–182
6–74
130
244
72–205
85–458
in vivo using a B. ovis nymphal development assay. LC50 values
for compounds tested in feeding assays are shown in Table 3.
The values ranged from 26 to 244 lg/mL. The correlation coeffi-
cient for the association between IC50 (Table 1) and LC50 in the
louse nymphal assay was r = 0.64 (p < 0.1). This correlation was
influenced by a high leverage value (compound 15l) which had
markedly higher IC50 and LC50 than other values in the data set.
Omitting this point still gave a correlation of r = 0.45, suggesting
a relationship between the binding values measured in the com-
petitive inhibition assay and toxicity measured in the louse nym-
phal assays.
3. Conclusion
We have discovered a new class of ligands for the ecdysone
receptor in the c-methylene c-lactams, some of which bind with
an affinity comparable to that observed for ecdysteroids. The
degree of correlation in the QSAR studies on this series of mole-
cules supports the argument that they all bind to one particular
conformation of the receptor binding pocket. This in turn adds to
the weight of evidence that the ecdysone receptor ligand binding
pocket does not deform continuously in response to different li-
gand chemistries, but rather has a discrete binding conformation
for each ligand chemotype.
We have demonstrated that methylene lactams bind to
recombinant ecdysone receptor LBDs from more than one order
of insects and, in the case of B. ovis, that this binding translates
to developmental toxicity in vivo. Insecticidal activity towards
non-lepidopteran species provides hope that new classes of ecdy-
sone receptor modulators can be developed into commercially via-
ble insecticides with activity spectra different from that of the
diacylhydrazines.
Figure 7. CoMFA electrostatic map with an active template lactam 11e. Blue
regions indicate where partial positive charge increases bioactivity, red regions
where partial negative charge favours bioactivity.
(green) region above and on one face of the lactam ring. Alkyl sub-
stituents on the R7 position interact with this region, which results
in increased binding. There is another sterically favoured region
below and on one side of the phenyl or other ring moiety that
is attached to the lactam nitrogen atom. Sterically unfavourable
(yellow) regions exist largely on the region of space on the oppo-
site side to the sterically favoured region (which is beside and
below the phenyl ring or equivalent). There is also a sterically dis-
favoured region in line with the methylene moiety, consistent with
the poor activities of analogues that have longer alkyl substituents
on the methylene group.
The electrostatic map in Figure 7 is more difficult to interpret in
terms of structure–activity relationships. There are essentially two
main regions, one that favours partial positive charge (blue field
map) behind the lactam ring, and one that favours partial negative
charge below the phenyl ring or equivalent (red field map). The lat-
ter region may reflect the favourable presence of halogens at or
near the 4-position of the phenyl ring. These are electronegative
atoms and so tend to adopt partial negative charges. The large blue
region, favouring partial positive charge, may not be indicating
that partial positive charge is beneficial, but rather, it may reflect
the relatively strong binding of alkyl substituents in the 6-position
and the weaker binding exhibited by analogues with partially neg-
atively charged substituents (such as nitro, hydroxyl, or ether) in
this region.
4. Experimental section
4.1. Chemistry
4.1.1. General notes
Microwave reactions were conducted in a Biotage Initiator
microwave reactor. Melting points were determined on a Bausch
& Lomb hot-stage melting point apparatus and are uncorrected.
1H and 13C spectra were recorded on a Bruker AV400 at 400 and
100.6 MHz, respectively, unless otherwise stated. Chemical shifts
were measured in ppm relative to CDCl3 then related to tetrameth-
ylsilane (d = 0.00). Multiplicity is reported as s = singlet, d = dou-
blet, t = triplet, q = quartet, m = multiplet and br = broad. Infrared
spectra were recorded on a Perkin Elmer 842 spectrophotometer.
Positive ion electron impact mass spectra (EIMS) and high resolu-
tion electron impact mass spectra (HR-EIMS) were recorded on a
Thermoquest MAT 95XL, using an ionisation energy of 70 eV.
Accurate mass measurements were obtained with a resolution of
2.4. Whole insect B. ovis toxicity
A number of the compounds which displayed binding to the re-
combinant B. ovis ecdysone receptor LBD in vitro were tested

5654
W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5000–10,000 using perfluorokerosene (PFK) as the reference com-
pound. Known compounds were prepared by the routes previously
described in the literature31 or by the methods outlined below and
had physical and spectral characteristics in accordance with those
reported. Cyanoacetic acid was dried at 50 °C under vacuum over
CaCl2 before use. Dry N,N-dimethylformamide (DMF) was obtained
from a solvent dispensing system built by J. C. Meyer based on a
design developed by Pangborn et al.32 Petroleum spirit refers to
the fraction boiling between 40 and 60 °C unless otherwise stated.
Chromatographic purification was conducted by radial chromatog-
raphy on Merck Silica Gel 60PF254 on a chromatotron (Harrison
Research) unless otherwise stated. Dry vacuum column chroma-
3H, CH3), 5.12 (d, Jgem = 2.7 Hz, 1H, @CH), 5.28 (d, Jgem = 2.7 Hz,
1H, @CH), 7.16–7.20 (m, 1H, ArH), 7.28–7.30 (m, 1H, ArH), 7.38–
7.46 (m, 2H, ArH); 13C NMR (CDCl3, 50 MHz): d 12.5, 100.6,
107.9, 111.5, 125.9, 127.9, 128.8, 130.6, 134.5, 135.2, 145.2,
157.7, 163.0; IR (KBr):
m 3403, 3084, 2228, 1705, 1611, 1595,
1389, 1153, 784, 685 cmꢀ1; HRMS (EI) m/z: calcd for C13H9Cl2N2O:
244.0398 [M]+Å; found: 244.0405.
4.1.6. 1-[3-(Trifluoromethyl)phenyl]-4-methyl-5-methylene-2-
oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (4p)
Prepared by method D using morpholine as the base, overnight,
followed by method E, trifluoroacetic acid, 18 °C, overnight. The
cooled reaction solution was diluted with aqueous isopropanol
(1:1, 20 mL) to precipitate 5r (0.71 g, 85%) as colourless needles:
tography was conducted on Merck Silica Gel 60 15–40
are not optimised.
l
m.33 Yields
mp 158 °C; 1H NMR (CDCl3):
d 2.46 (s, 3H, CH3), 5.11 (d,
4.1.2. 1-(2-Ethoxyphenyl)-4,5-dimethyl-5-hydroxy-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile (3a) (method D)
Jgem = 2.7 Hz, 1H, @CH), 5.33 (d, Jgem = 2.7 Hz, 1H, @CH), 7.48 (d,
J = Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H2), 7.60–7.68 (m, 2H, Ar-H);
13C NMR (CDCl3): d 12.4, 101.0, 107.7, 111.5, 124.5 (q, JCF = 4 Hz),
123.4 (q, JCF = 272 Hz), 125.3 (q, JCF = 4 Hz), 130.4, 131.0, 132.1 (q,
Morpholine (five drops) was added to a solution of N-[2-ethoxy-
phenyl]cyanoacetamide (2.04 g, 10 mmol) and butane-2,3-dione
(0.94 g, 6 mmol) in dry DMF (4 mL). The resulting mixture was stir-
red at room temperature for 6 h then diluted slowly with water
(20 mL). After standing overnight the precipitate was filtered off
to give crude 3a as a solid which was used in the next step without
further purification.
JCF = 33 Hz), 134.0, 144.9, 158.2, 163.1; IR (KBr):
m 3413, 2227,
1709, 1631, 1610, 1329, 1188, 1157, 1123, 808, 698 cmꢀ1
;
HRMS (EI) m/z: calcd for C14H9F3N2O: 278.0661 [M]+Å; found:
278.0655.
4.1.7. 1-(2,4-Dimethoxyphenyl)-4-methyl-5-methylene-2-oxo-
2,5-dihydro-1H-pyrrole-3-carbonitrile (5c)
4.1.3. 1-(2-Ethoxyphenyl)-4-methyl-5-methylene-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile (4j) (method E)
Prepared by method D using morpholine as the base, 28 days,
followed by method G, chromatography eluent 1:1 dichlorometh-
ane/petroleum spirit followed by recrystallisation (methanol) to
give 5c (24%) as yellow crystals: mp 176 °C; 1H NMR (CDCl3): d
2.42 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.78 (d,
Jgem = 2.0 Hz, 1H, @CH), 5.14 (d, Jgem = 2.0 Hz, 1H, @CH), 6.51–
6.57 (m, 2H, Ar-H5, Ar-H6), 7.06 (d, J3,5 = 8.4 Hz, 1H, Ar-H3); 13C
NMR (CDCl3): d 12.5, 55.6, 55.8, 99.8, 99.9, 105.0, 108.1, 112.1,
Crude 1-(2-ethoxyphenyl)-4,5-dimethyl-5-hydroxy-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile from the previous step was ta-
ken up in formic acid (6 mL) and heated at 60 °C for 4 h. The cooled
solution was diluted with water and extracted with ether (30 mL).
The ether phase was dried (MgSO4), filtered and concentrated un-
der reduced pressure. Purification by radial chromatography
(dichloromethane elution) then recrystallisation from isopropanol
gave 4j (1.49 g, 59%) as colourless needles mp 110 °C; 1H NMR
(CDCl3): d 1.27 (t, J = 6.8 Hz, 3H, OCH2CH3), 2.42 (s, 3H, CH3), 4.01
(s, J = 7.0 Hz, 2H, OCH2CH3), 4.78 (s, 1H, @CH), 5.17 (s, 1H, @CH),
6.99 (t, J = 6.4 Hz, 1H, Ar-H4), 7.00 (d, J = 7.7 Hz, 1H, Ar-H3), 7.15
(d, J = 7.3 Hz, 1H, Ar-H6), 7.36 (t, J = 7.6 Hz, 1H, Ar-H5); 13C NMR
(CDCl3): d 12.4, 14.6, 64.2, 100.2, 108.0, 112.1, 113.5, 120.9,
114.5, 130.8, 145.7, 156.7, 157.6, 161.6, 163.6; IR (KBr):
m 3419,
2938, 2228, 1714, 1632, 1514, 1315, 1295, 1213, 1030 831 cmꢀ1
;
HRMS (EI) m/z: calcd for C15H14N2O3 270.0999 [M]+Å; found
270.1000.
4.1.8. N-[2,5-Bis(trifluoromethyl)phenyl]cyanoacetamide (1a)
(method A)
122.0, 130.3, 130.6, 145.3, 155.0, 157.9, 163.4; IR (KBr):
m 3420,
2985, 2224, 1715, 1633, 1506, 1391, 1290, 1170, 1048, 893,
758 cmꢀ1; HRMS (EI) m/z: calcd for C15H14N2O2: 254.1050 [M]+Å;
found: 254.1046.
1,3-Diisopropylcarbodiimide (DIC) (1.95 mL, 12.6 mmol) was
added dropwise to a stirred, chilled (0–5 °C) suspension of 2,5-
bis(trifluoromethyl)aniline (2.86 g, 12.5 mmol) and cyanoacetic
acid (1.15 g, 13.5 mmol) in dry DMF (25 mL) maintained under
nitrogen. The resulting mixture was stirred at room temperature
for 2 days, filtered then poured into water (150 mL). The precipi-
tated solid was filtered off, washed with water and dried under
vacuum to give 1a (2.77 g, 75%) as a white solid: mp 158 °C sub;
1H NMR (CDCl3): d 3.64 (s, 2H, CH2), 7.60 (d, J3,4 = 8.2 Hz, 1H,
Ar-H4), 7.82 (d, J3,4 = 8.2 Hz, 1H, Ar-H3), 8.13 (br s, 1H, NH), 8.49
(s, 1H, Ar-H6); 13C NMR (DMSO-d6): d 26.4, 116.0, 123.1 (q,
JCF = 274 Hz), 123.5 (q, JCF = 273 Hz), 124.3 (m), 127.1 (q, JCF = 3 Hz),
128.5 (q, JCF = 5 Hz), 128.6 (q, JCF = 29 Hz), 133.6 (q, JCF = 34 Hz),
4.1.4. 1-[3-Chlorophenyl]-4, 5-dimethyl-5-hydroxy-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile (3b)
1,8-Diazabicyclo[5.4.0]undec-7-ene (four drops) was added to a
solution of N-[3-chlorophenyl)-2-cyanoacetamide (2.00, 10.0
mmol) and butane-2,3-dione (0.93 g, 10.5 mmol) in dry DMF
(4 mL) (exotherm). The resulting mixture was stirred at room tem-
perature for 30 min then water (20 mL) and diethyl ether (30 mL)
were added. The aqueous layer was acidified to pH 3 with concen-
trated hydrochloric acid. The layers were separated and the organic
layer was washed with water (20 mL). The organic layer was con-
centrated to give crude 3b which was used in the next step without
further purification
136.1 (q, JCF = 2 Hz); IR (KBr)
m 3279, 1684, 1595, 1545, 1438,
1393, 1314, 1267, 1172, 1124, 1083, 1044, 897, 837, 657 cmꢀ1
;
HRMS (EI) m/z: calcd for C11H6F6N2O: 296.0379 [M]+Å; found:
296.0375.
4.1.5. 1-[3-Chlorophenyl]-4-methyl-5-methylene-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile (4m)
4.1.9. 1-[2,5-Bis(trifluoromethyl)phenyl]-4,5-dimethyl-5-
hydroxy-2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (3c)
Morpholine (seven drops) was added to a solution of N-[2,5-
bis(trifluoromethyl)phenyl]cyanoacetamide (1.40 g, 4.7 mmol)
and butane-2,3-dione (0.41 mL, 4.7 mmol) in dry DMF (1.5 mL).
The resulting mixture was stood at room temperature for 4 weeks
then poured into water (50 mL) and extracted with ethyl acetate
Prepared by method E, 90 °C, 90% formic acid, 1.5 h. After cool-
ing, the reaction mixture was diluted with methanol (8 mL), stir-
red, then filtered and washed with methanol to give 4m as a
yellow solid (1.81 g, 74%). A sample was recrystallised from dichlo-
romethane and methanol with carbon filtration to give fine, flat,
pale yellow needles: mp 203 °C dec. 1H NMR (CDCl3): d 2.46 (s,

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5655
(3 ꢁ 50 mL). The combined organic phases were washed with
water (3 ꢁ 50 mL), dried (MgSO4), filtered and concentrated
under reduced pressure to give crude 3c (1.65 g) as a violet foam
which was used in the subsequent step without further puri-
fication.
J5,6 = 9.0, JHF = 10.1 Hz, 1H, Ar-H5), 7.62 (ddd, J2,6 = 2.7, JHF = 3.8,
J5,6 = 9.0 Hz, 1H, Ar-H6), 8.03 (dd, J2,6 = 2.7, JHF = 6.5 Hz, 1H, Ar-H2);
13C NMR (CDCl3): d 12.5, 100.7, 107.9, 111.1, 119.9 (d, JCF = 22 Hz),
125.2, 129.7 (d, JCF = 4 Hz), 134.7 (d, JCF = 9 Hz), 137.7 (d, JCF = 4 Hz),
144.6, 154.8 (d, JCF = 268 Hz), 158.2, 162.9; IR (KBr)
m 3413, 3082,
2236, 1710, 1633, 1538, 1350, 1259, 1160 cmꢀ1; HRMS (EI) m/z:
4.1.10. 1-[2,5-Bis(trifluoromethyl)phenyl]-4-methyl-5-
calcd for C13H8FN3O3: 273.0544 [M]+Å; found: 273.0545.
methylene-2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (5f)
(method G)
4.1.14. 1-(2,4,5-Trichlorophenyl)-4-methyl-5-methylene-2-oxo-
2,5-dihydro-1H-pyrrole-3-carbonitrile (6b)
A solution of crude 1-[2,5-bis(trifluoromethyl)phenyl]-4,5-di-
methyl-5-hydroxy-2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile
(0.53 g, 1.5 mmol) in formic acid (2 mL) was heated by microwave
to 120 °C for 10 min then cooled, poured into water (50 mL) and
extracted with ethyl acetate (3 ꢁ 50 mL). The combined organic
phases were washed with water (3 ꢁ 50 mL), dried (MgSO4) and
concentrated under reduced pressure. The crude material was sub-
jected to radial chromatography (10:1 dichloromethane/petroleum
spirit) and recrystallised from ethyl acetate/petroleum spirit to
provide 5f (67 mg, 13% over 2 steps) as cream crystals: mp
150 °C; 1H NMR (CDCl3): d 2.48 (s, 3H, CH3), 4.69 (d, Jgem = 3.1 Hz,
1H, @CH), 5.28 (d, Jgem = 3.1 Hz, 1H, @CH), 7.59 (s, 1H, Ar-H6),
7.90 (d, J3,4 = 8.2 Hz, 1H, Ar-H4), 8.00 (d, J3,4 = 8.2 Hz, 1H, Ar-H3);
13C NMR (CDCl3): d 12.5, 100.9, 107.9, 111.2, 122.0 (q, JCF = 274 Hz),
122.5 (q, JCF = 274 Hz), 127.3 (q, JCF = 4 Hz), 128.9 (q, JCF = 5 Hz),
129.2 (q, JCF = 4 Hz), 132.5 (q, JCF = 1 Hz), 133.8 (q, JCF = 32 Hz),
Prepared by method D using morpholine as the base, 2 days, fol-
lowed by method E, 50 °C, 16 h. 17%, purified by recrystallisation
[diethyl ether/petroleum spirit (bp 80–100 °C)] to give 6b as a
fawn solid: mp 178 °C; 1H NMR (CDCl3): d 2.45 (s, 3H, CH3), 4.82
(d, Jgem = 2.6 Hz, 1H, @CH), 5.31 (d, Jgem = 2.6 Hz, 1H, @CH), 7.41
(s, 1H, Ar-H6), 7.66 (s, 1H, Ar-H3); 13C NMR (CDCl3): d 12.6,
110.2, 107.8, 111.4, 130.4, 131.8, 132.1, 132.3, 132.7, 134.9,
143.9, 158.6, 162.7; IR (KBr):
m 3429, 2225, 1722, 1633, 1469,
901, 880 cmꢀ1; HRMS (EI) m/z: calcd for C13H7Cl3N2O: 311.9618
[M]+Å; found: 311.9615.
4.1.15. N-[4-Chloro-2-isopropyl-6-methylphenyl]
cyanoacetamide (1c)
Prepared by method A, 2 h, and recrystallised from chloroform
to give 1c (27%) as a white solid: mp 168 °C; 1H NMR (CDCl3): d
1.20 (d, J = 6.8 Hz, 6H, CH(CH3)2), 2.20 (s, 3H, CH3), 3.00 (sept,
J = 6.8 Hz, 1H, CH(CH3)2), 3.57 (s, 2H, CH2), 7.10 (d, J = 1.8 Hz, 1H,
Ar-H), 7.15 (d, J = 1.8 Hz, 1H, Ar-H), 7.38 (br s, 1H, NH); 13C NMR
(CDCl3): d 18.3, 23.2, 25.8, 28.9, 114.7, 124.2, 128.2, 129.6, 134.4,
135.9 (q, JCF = 34 Hz), 145.8, 158.5, 163.2; IR (KBr):
m 3072, 2231,
1726, 1637, 1442, 1333, 1319, 1187, 1125, 1085 cmꢀ1; HRMS (EI)
m/z: calcd for C15H8F6N2O: 346.0535 [M]+Å; found: 346.0538.
4.1.11. 1-(2-Methoxy-5-chlorophenyl)-4-methyl-5-methylene-
2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (5h)
137.8, 147.7, 159.9; IR (KBr): m 3251, 2965, 2929, 2258, 1652,
1533, 1386, 1346, 1237, 1222, 892, 864 cmꢀ1; HRMS (EI) m/z: calcd
Prepared by method D using morpholine as the base, 28 days,
followed by method G, chromatography eluent 3:1 dichlorometh-
ane/petroleum spirit to give 5h (21%) as a yellow solid: mp
190 °C; 1H NMR (CDCl3): d 2.44 (s, 3H, CH3), 3.76 (s, 3H, OCH3),
4.81 (d, J = 2.6 Hz, 1H, @CH), 5.19 (d, J = 2.6 Hz, 1H, @CH), 6.96 (d,
J = 9.0 Hz, 1H, H3), 7.18 (d, J = 2.6 Hz, 1H, H6), 7.37 (dd, J = 2.6,
9.0 Hz, 1H, H4); 13C NMR (CDCl3): d 12.5, 56.1, 100.2, 108.1,
111.8, 113.5, 122.7, 125.5, 130.3, 130.6, 144.9, 154.5, 158.0,
for C13H15ClN2O: 250.0867 [M]+Å; found: 250.0861
4.1.16. 1-(4-Chloro-2-isopropyl-6-methylphenyl)-4-methyl-5-
methylene-2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (6f)
Prepared by method D using morpholine as the base, 7 days, fol-
lowed by method E, 70 °C, 2 h, chromatography eluent 1:1 dichlo-
romethane/petroleum spirit. Purified by recrystallisation (diethyl
ether/petroleum spirit) to give 6f (20%) as amber prisms: mp
153 °C; 1H NMR (CDCl3): d 1.13 (t, J = 7.1 Hz, 6H, (CH3)2CH), 2.02
(s, 3H, Ar-CH3), 2.48 (s, 3H, CH3), 2.59 (sept, J = 7.0 Hz, 1H,
(CH3)2CH), 4.66 (d, J = 2.2 Hz, 1H, @CH), 5.18 (d, J = 2.2 Hz, 1H,
@CH), 7.16 (d, J = 1.8 Hz, 1H, Ar-H), 7.23 (d, J = 1.8 Hz, 1H, Ar-H);
13C NMR (CDCl3, 50 MHz): d 12.5, 17.8, 23.5, 24.1, 28.9, 99.7,
108.7, 111.6, 124.9, 128.2, 128.6, 135.8, 139.2, 145.2, 150.2,
163.1; IR (KBr):
m 3420, 3070, 2228, 1717, 1633, 1502, 1165,
816 cmꢀ1; HRMS (EI) m/z: calcd for C14H11ClN2O2: 274.0509
[M]+Å; found: 274.0497.
4.1.12. N-(3-Nitro-4-fluorophenyl)cyanoacetamide (1b)
Diphenyl phosphate (4.85 g, 24 mmol) was added to a stirred
solution of 4-fluoronitroaniline (2.8 g, 20 mmol) and cyanoacetic
acid (2.04 g, 24 mmol) in DMF (6 mL) followed by pyridine
(1.92 g, 24 mmol). The resulting mixture was stirred overnight
then slowly diluted with water (60 mL) and the precipitated solid
filtered off to give 1b as light brown crystals (2.53 g, 61%): mp
157.5, 163.0; IR (KBr):
m 3415, 3125, 2965, 1713, 1633, 1387,
1170, 901, 882, 859 cmꢀ1; HRMS (EI) m/z: calcd for C17H17ClN2O:
300.1024 [M]+Å; found: 300.1015.
4.1.17. N-{4,6-Dimethyl-2-[fluorobis(trifluoromethyl)methyl]
phenyl}-cyanoacetamide (1d)
167 °C; 1H NMR (CDCl3):
d 3.94 (s, 2H, CH2CN), 7.56 (dd,
JHH = 9.1, JHF = 11.1 Hz, 1 H, Ar-H5), 7.81 (ddd, JHH = 3.8, 9.1 Hz,
JHF = 2.9 Hz, 1H, Ar-H6), 8.42 (dd, JHH = 2.7 Hz, JHF = 6.8 Hz, 1H, Ar-
H2), 10.75 (br s, 1H, NH); 13C NMR (CDCl3): d 27.2, 115.9, 116.1
(d, JCF = 3 Hz), 119.3 (d, JCF = 22 Hz), 126.9 (d, JCF = 8 Hz), 135.4 (d,
JCF = 3 Hz), 136.7 (d, JCF = 8 Hz), 151.1 (d, JCF = 260 Hz), 162.1; IR
A mixture of 4,6-dimethyl-2-[fluorobis(trifluoromethyl)methyl]
aniline (1.45 g, 5 mmol) and cyanoacetic acid (1.28 g, 15 mmol) in
diethylcyanamide (8 mL) was heated at 60 °C for 1 h. Additional
cyanoacetic acid (0.86 g, 10 mmol) was added and heating contin-
ued for 1 h. The cooled mixture was stirred into water (50 mL) and
extracted with ether (30 mL). The organic phase was removed and
washed with water (3 ꢁ 30 mL), dried (MgSO4), filtered and con-
centrated under reduced pressure. The residue was subjected to ra-
dial chromatography (dichloromethane elution) to give the crude
product as a pale brown oil which slowly solidified. Purification
by column chromatography (eluent:gradient of ethyl acetate in
60–80 °C bp petroleum spirit) gave 1d (0.79 g, 39%) as colourless
fluffy needles: mp 141 °C; 1H NMR (CDCl3, 200 MHz): d 2.26 (s,
3H, Ar-6-CH3), 2.38 (s, 3H, Ar-4-CH3), 3.51 (s, 2H, CH2CN), 7.25
(br s, 1H, Ar-H), 7.30 (br s, 1H, Ar-H), 7.57 (br s, 1H, NH); 13C
(KBr):
m
3311, 2264, 1678, 1562, 1347, 1229, 839 cmꢀ1; HRMS
(EI) m/z: calcd for C9H6FN3O3: 223.0388 [M]+Å; found: 223.0381.
4.1.13. 1-(3-Nitro-4-fluorophenyl)-4-methyl-5-methylene-2-
oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile (5p)
Prepared by method D using morpholine as the base, overnight,
followed by method E, 18 °C, 3 days. The reaction solution was stir-
red into ice/water and the precipitate filtered off to give 5o (49%) as a
solid: mp 198 °C; 1H NMR (CDCl3): d 2.49 (s, 3H, CH3), 5.15 (d,
J = 3.1 Hz, 1H, @CH), 5.37 (d, J = 3.1 Hz, 1H, @CH), 7.46 (dd,

5656
W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
NMR (DMSO): d 18.3, 20.9, 25.7, 93.3 (dsept, JCF = 33, 207 Hz),
116.2, 120.7 (dq, JCF = 28, 288 Hz), 122.8 (m), 125.3 (m), 132.4
tals: mp 112 °C; 1H NMR (CDCl3, 200 MHz): d 1.08–1.80 (m, 10H,
cyclohexyl-H), 2.35 (tt, J = 3.1, 11.1 Hz, 0.5H, cyclohexyl-H1),
3.06, 3.14 (2s, 3H, NCH3), 4.06 (br s, 2H, NH2); 13C NMR (CDCl3,
50 MHz): d 25.6, 25.7, 25.9, 28.9, 38.0, 38.4, 39.2, 40.3, 174.1,
(m), 135.1, 138.2 (m), 140.0, 162.1; IR (KBr):
1673, 1520, 1268, 1216, 981, 737 cmꢀ1; HRMS (EI) m/z: calcd for
14H11F7N2O: 356.0754 [M]+Å; found: 356.0748.
m 3255, 2939, 2263,
C
178.5; IR (KBr): m 3222, 3219, 2936, 2855, 1661, 1621, 1446,
1392, 1217, 1102, 995 cmꢀ1; HRMS (EI) m/z: calcd for C8H16N2O:
4.1.18. 1-{4,6-Dimethyl-2-[fluorobis(trifluoromethyl)methyl]
phenyl}-4-methyl-5-methylene-2-oxo-2,5-dihydro-1H-pyrrole-
3-carbonitrile (6h)
156.1257 [M]+Å; found: 156.1255.
4.1.21. E- and Z-N0-(2-Cyanoacetyl)-N-methylcyclohex-
Prepared by method D using morpholine as the base, 6 h, fol-
lowed by method E, formic acid, 80 °C, 1 h. The solid which precip-
itated on cooling the reaction mixture was filtered off and washed
lightly with aqueous isopropanol to give 6h (0.85 g, 73%) as colour-
less plates: mp 188 °C (sublimes); 1H NMR (CDCl3): d 2.04 (s, 3H,
CH3), 2.42 (s, 3H, CH3), 2.45 (s, 3H, CH3), 4.59 (br d, 1H, @CH),
5.18 (d, J = 2.2 Hz, 1H, @CH), 7.32 (s, 1H, Ar-H), 7.35 (s, 1H, Ar-H);
13C NMR (CDCl3): d 12.4, 17.7, 21.3, 93.0 (dsept, JCF = 33, 209 Hz),
99.9, 108.2, 111.6, 120.2 (dq, JCF = 28, 288 Hz), 120.6 (dq, JCF = 28,
286 Hz), 124.8 (d, JCF = 17 Hz), 126.1 (br m, JCF = 2 Hz), 128.3,
anecarbohydrazide (1e)
A mixture of E- and Z-N-methylcyclohexanecarbohydrazide
(1.00 g, 6.4 mmol), cyanoacetic acid (0.55 g, 6.4 mmol) and phos-
phorus oxychloride (1.96 g, 12.8 mmol) in dichloroethane (10 mL)
was heated at 90 °C (oil bath) for 2.5 h. It was then cooled to room
temperature, diluted with ice (10 g) and stirred for 30 min. The lay-
ers were separated then the aqueous layer extracted with dichloro-
methane (3 ꢁ 20 mL). The combined organic extracts were
extracted with 10% sodium hydroxide (2 ꢁ 20 mL). The sodium
hydroxide extracts were acidified with concentrated hydrochloric
acid and then extracted with dichloromethane (2 x20 mL). The
dichloromethane layers were dried (MgSO4), filtered and concen-
trated under reduced pressure to give a yellow oil (1.15 g, 86%).
Purification by dry vacuum column chromatography, eluting with
a gradient of methanol in chloroform gave 1e as a pale yellow oil
(0.81 g, 57%). 1H NMR (CDCl3): d 1.04–1.39 (m, 5H, CH2), 1.57–
1.79 (m, 5H, CH2), 2.46 (tm, J = 11.2 Hz, 1H, CH), 3.06 (s, 2H,
CH2CN), 3.27, 3.53 (2s, 3H, N-CH3), 9.44, 9.92 (2s, 1H, NH); 13C
NMR (CDCl3): d 24.4, 25.4, 25.6, 28.9, 29.3, 35.5, 38.5, 39.9, 40.7,
134.9, 140.2 (d, JCF = 1 Hz), 140.8, 145.1, 157.9, 162.8; IR (KBr):
m
3424, 2231, 1717, 1291, 1267, 1243, 1229, 1214, 975 cmꢀ1; HRMS
(EI) m/z: calcd for C18H13F7N2O: 406.0911 [M]+Å; found: 406.0907.
4.1.19. (Z) and (E)-1-(3,5-Dimethoxyphenyl)-4-methyl-5-(2-
methylpropylidene)-2-oxo-2,5-dihydro-1H-pyrrole-3-
carbonitrile (14c) and (14e)
Prepared by method D using morpholine as the base, 6 days, fol-
lowed by method E using formic acid, 60 °C, 24 h, chromatography
eluent dichloromethane to give a 3:1 mixture of two isomers
(23%). These were separated by repeated radial chromatography
(dichloromethane elution) then recrystallised from methanol to
give cream crystals. Compound 14c (major isomer): mp 142 °C; 1H
NMR (CDCl3): d 0.87 (d, J = 6.4 Hz, 6H, CH(CH3)2), 2.09–2.23 (m,
1H, CH(CH3)2), 2.39 (s, 3H, CH3), 3.78 (s, 6H, OCH3), 5.40 (d,
J = 11.0 Hz, 1H, @CH), 6.39 (d, J = 2.4 Hz, 2H, Ar-H2, Ar-H6) , 6.52
(t, J = 2.4 Hz, 1H, Ar-H4); 13C NMR (CDCl3): d 12.5, 22.5, 25.9, 55.5,
101.4, 105.6, 106.6, 112.3, 129.1, 136.4, 137.5, 159.4, 161.2, 164.6;
113.9, 114.4, 161.4, 161.8, 175.9, 179.0; IR (KBr):
m 3312. 3015,
2304, 1800, 1710, 1504, 1441, 1393, 1319, 1128 cmꢀ1; HRMS (EI)
m/z: calcd for C11H17N3O2: 223.1315 [M]+Å; found: 223.1313.
4.1.22. E- and Z-N-(3-Cyano-2-oxo-2,4,5,6-tetrahydro-1H-indol-
1-yl)-N-methylcyclohexanecarboxamide (15k)
Prepared by method D using morpholine as the base, 3 days, fol-
lowed by method E, 90 °C, 4 h, chromatography eluent 1:1:2 ethyl
acetate: dichloromethane: petroleum spirit to give 15k (45%) as a
pale yellow gum which was an inseparable 6:1 mixture of isomers:
1H NMR (CDCl3): d 1.03–1.92 (m, 10H, cyclohexyl-H), 1.97 (quintet,
J = 6.0 Hz, 2H, H5), 2.03 (m, 1H), 2.42, 2.51 (2q, J = 5.1 Hz, 2H, H6),
2.83, 2.91 (2t, J = 6.4 Hz, 2H, H4), 3.08, 3.36 (2s, 3H, NCH3), 5.80,
6.00 (2t, J = 4.6 Hz, 1H, H7); 13C NMR (CDCl3): d 22.3, 24.1, 24.2,
24.3, 25.3, 25.4, 25.5, 28.4, 29.0, 29.3, 29.7, 34.0, 38.1, 39.7, 40.6,
100.5, 111.3, 118.1, 119.6, 135.1, 135.2, 157.1, 157.8, 161.9,
IR (KBr):
m ;
3416, 2968, 2228, 1712, 1614, 1195, 1153, 847 cmꢀ1
HRMS (EI) m/z: calcd for C18H20N2O3: 312.1468 [M]+Å; found:
312.1467. Compound 14e (minor isomer): mp 167 °C; 1H NMR
(CDCl3): d 1.08 (d, J = 6.6 Hz, 6H, CH(CH3)2), 2.62 (s, 3H, CH3), 3.04–
3.17 (m, 1H, CH(CH3)2), 3.79 (s, 6H, OCH3), 5.47 (d, J = 11.0 Hz, 1H,
@CH), 6.32 (d, J = 2.2 Hz, 2H, Ar-H2, Ar-H6) , 6.51 (t, J = 2.2 Hz, 1H,
Ar-H4); 13C NMR (CDCl3): d 16.5, 23.2, 27.2, 55.5, 100.9, 107.0,
109.3, 112.2, 132.2, 135.1, 137.2, 156.1, 161.3, 162.4; IR (KBr):
m
175.2, 178.8; IR (KBr): m 3480, 2934, 2859, 2230, 1723, 1680,
3419, 2969, 2225, 1713, 1613, 1194, 1155, 1059, 847 cmꢀ1; HRMS
1602, 1452, 1377, 1342, 1148, 735 cmꢀ1; HRMS (EI) m/z: calcd
(EI) m/z: calcd for C18H20N2O3: 312.1468 [M]+Å; found: 312.1464.
for C17H21N3O2: 299.1628 [M]+Å; found: 299.1621.
4.1.20. E- and Z-N-Methylcyclohexanecarbohydrazide
4.1.23. 1-(5-Chloropyrid-2-yl)-4,5-dimethyl-5-hydroxy-2-oxo-
2,5-dihydro-1H-pyrrole-3-carbonitrile (3d)
Prepared by method D using morpholine as the base, 16 h, to
give the crude product which was triturated with isopropanol, fil-
tered off and used in the next step without further purification.
A solution of cyclohexanecarboxylic chloride (14.4 g, 98 mmol)
in dry dichloromethane (28 mL) was added dropwise to a stirred,
chilled (-35 °C) solution of methylhydrazine (15.5 mL, 294 mmol)
in dry dichloromethane (28 mL). The addition rate and cooling bath
(acetone/CO2) temperature were controlled so as to maintain the
internal reaction temperature between ꢀ35 °C and ꢀ40 °C. The
reaction mixture was then allowed to warm to 10 °C and ice
(50 g) added. The reaction mixture was stirred for 30 min then
the organic layer was removed and the aqueous layer extracted
with 2 M hydrochloric acid (3 ꢁ 100 mL). The combined acidic ex-
tracts were washed with dichloromethane (100 mL), basified with
10% aqueous sodium hydroxide solution and extracted with
dichloromethane (3 ꢁ 100 mL). These combined dichloromethane
extracts were dried (MgSO4), filtered and concentrated under re-
duced pressure to give a pale yellow solid, which was recrystallised
from diethyl ether to give an inseparable mixture of E- and Z-N-
Methylcyclohexanecarbohydrazide (4.51 g, 29%) as colourless crys-
4.1.24. 1-(5-Chloropyrid-2-yl)-4-methyl-5-methylene-2-oxo-
2,5-dihydro-1H-pyrrole-3-carbonitrile (15m) (method F)
Trifluoromethanesulfonic acid (1.5 mL, 17 mmol) was added to a
stirred, chilled (0–5 °C) solution of 1-(5-chloropyrid-2-yl)-4,5-
dimethyl-5-hydroxy-2-oxo-2,5-dihydro-1H-pyrrole-3-carbonitrile
(460 mg, 1.7 mmol) in dichloromethane (5 mL). The resulting solu-
tion was stirred at 0–5 °C for 2 h then diluted with ice-water
(25 mL). The organic layer was removed and washed with water
(2 ꢁ 25 mL), dried (MgSO4), filtered and concentrated under re-
duced pressure to a mustard solid. Recrystallisation from methanol
gave
1-(5-chloropyrid-2-yl)-4-methyl-5-methylene-2-oxo-2,5-
dihydro-1H-pyrrole-3-carbonitrile (0.25 g, 60%) as beige needles:

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5657
mp 173 °C; 1H NMR (CDCl3): d 2.44 (s, 3H, CH3), 5.50 (d, Jgem = 2.5 Hz,
1H, @CH), 6.04 (d, Jgem = 2.0 Hz, 1H, @CH), 7.66 (d, J3,4 = 8.6 Hz, 1H,
Py-H3), 7.80 (dd, J4,6 = 2.4 Hz, J3,4 = 8.6 Hz, 1H, Py-H4), 8.44 (d,
J4,6 = 2.4 Hz, 1H, Py-H6); 13C NMR (CDCl3): d 12.4, 105.2, 107.1,
111.5, 120.9, 130.1, 138.2, 142.6, 146.6, 147.2, 159.2, 163.2; IR
4.1.29. 3-Ethoxycarbonyl-1-(2,4,5-trichlorophenyl)-4-methyl-5-
methylene-2-oxo-2,5-dihydro-1H-pyrrole (16c)
Prepared by method D using morpholine as the base, 38 days, fol-
lowed by method G, chromatography eluent 1:1 dichloromethane/
petroleum spirit then recrystallised from methanol to give 16c
(34%) as a pale yellow solid: mp 121 °C; 1H NMR (CDCl3): d 1.36 (t,
J = 7.1 Hz, 3H, OCH2CH3), 2.48 (s, 3H, CH3), 4.35 (q, J = 7.1 Hz, 2H,
OCH2CH3), 4.67 (d, Jgem = 1.6 Hz, 1H, @CH), 5.19 (d, Jgem = 1.6 Hz,
1H, @CH), 7.39 (s, 1H, Ar-H6), 7.63 (s, 1H, Ar-H3); 13C NMR (CDCl3):
d 11.7, 14.2, 61.2, 98.3, 122.1, 131.4, 131.6, 131.9, 132.5, 132.8, 134.2,
(KBr):
m ;
3426, 2231, 1723, 1473, 1389, 1112, 891, 762 cmꢀ1
HRMS (EI) m/z: calcd for C12H8ClN3O: 245.0350 [M]+Å; found:
245.0351.
4.1.25. 3-Acetyl-1-(2,4,5-trichlorophenyl)-4,5-dimethyl-5-
hydroxy-2-oxo-2,5-dihydro-1H-pyrrole (3e) (method D)
DABCO (1,4-diazabicyclo[2.2.2]octane) (20 mg, 0.18 mmol) was
added to a mixture of 3-oxo-N-(2,4,5-trichlorophenyl)butanamide
(1.40 g, 5 mmol) and butanedione (0.44 mL, 5.0 mmol) in dry DMF
(1.5 mL). The resulting solution was stirred at room temperature
for 39 days then poured into water (25 mL) and extracted with ethyl
acetate (3 ꢁ 25 mL). The combined organic phases were washed
with water (2 ꢁ 25 mL), dried (MgSO4), filtered and concentrated
under reduced pressure to give crude 3e (2.16 g) as a brown gum
which was used in the next step without further purification.
144.6, 154.3, 162.3, 164.5; IR (KBr): m 3128, 2981, 1736, 1629, 1473,
1403, 1357, 1266, 1156, 1131, 1101, 1082, 1034, 912 cmꢀ1; HRMS
(EI) m/z: calcd for C15H12Cl3NO3: 358.9877 [M]+Å; found: 358.9862.
4.1.30. N-[2-Chloro-5-(trifluoromethyl)phenyl]-3-oxo-3-phenyl
propanamide (1g) (method C)
3-Amino-4-chlorobenzotrifluoromethane (1.00 g, 5.1 mmol)
and ethyl benzoylacetate (1.20 g, 6.2 mmol) were subjected to
microwave irradiation at 200 °C for 2 h. This reaction was carried
out five times. Solid from the combined reaction mixtures was pre-
cipitated out with 75% ethanol in water to give 1g (1.36 g, 16%). A
sample recrystallised from diethyl ether and light petroleum gave
colourless, fluffy needles: mp 135 °C. 1H NMR (CDCl3): d 4.19 (s,
2H, CH2), 7.30 (br d, J = 8.1 Hz, 1H, NHAr-H4), 7.47–7.56 (m, 3H,
NHAr-H3, Ph-H3, -H5), 7.65 (t, J = 7.3 Hz, 1H, Ph-H4), 8.03 (d,
J = 7.9 Hz, 2H, Ph-H2, -H6), 8.78 (1H, NHAr-H6), 10.17 (s, 1H,
NH); 13C NMR (CDCl3): d 45.0, 118.6 (q, JCF = 4 Hz), 121.3 (q,
JCF = 4 Hz), 123.6 (q, JCF = 272 Hz), 126.6, 128.5, 129.0, 129.6,
4.1.26. 3-Acetyl-1-(2,4,5-trichlorophenyl)-4-methyl-
5-methylene-2-oxo-2,5-dihydro-1H-pyrrole (16a)
Prepared by Method G, chromatography eluent 1:1 dichloro-
methane: petroleum spirit to provide 16a (6% over 2 steps) as a
pale yellow gum: 1H NMR (CDCl3, 200 MHz): d 2.51 (s, 3H, CH3),
2.62 (s, 3H, COCH3), 4.76 (d, Jgem = 2.4 Hz, 1H, @CH), 5.26 (d,
Jgem = 2.4 Hz, 1H, @CH), 7.43 (s, 1H, Ar-H6), 7.68 (s, 1H, Ar-H3);
13C NMR (CDCl3, 50 MHz): d 10.4, 29.4, 99.6, 127.0, 131.4, 131.5,
130.0 (q, JCF = 33 Hz), 134.5, 135.3, 135.8, 164.3, 196.1; IR (KBr):
m
3191, 1693, 1662, 1587, 1532, 1428, 1332, 1219, 1167, 1123,
1083 cmꢀ1; HRMS (EI) m/z: calcd for C16H11ClF3NO2: 341.0425
[M]+Å; found: 341.0427.
131.7, 132.6, 133.0, 133.9, 145.0, 154.6, 166.7, 195.8; IR (neat):
m
2932, 1719, 1630, 1466, 1134, 1081 cmꢀ1; HRMS (EI) m/z: calcd
for C14H10Cl3NO2: 328.9772 [M]+Å; found: 328.9773.
4.1.31. 3-Benzoyl-1-[2-chloro-5-(trifluoromethyl)phenyl]-4-
methyl-5-methylene-2-oxo-2,5-dihydro-1H-pyrrole (16e)
Prepared by method D using 1,8-diazabicyclo[5.4.0]undec-7-
ene as the base, 7 days, followed by method E, formic acid, 45 °C,
4 h, dry column vacuum chromatography eluent 1:9 ethyl ace-
tate/bp 60–80 °C petroleum spirit to give 17f (44%) as a pale yellow
semicrystalline mass: 1H NMR (CDCl3): d 2.32 (s, 3H, CH3), 4.71 (d,
Jgem = 2.7 Hz, 1H, @CH), 5.19 (d, Jgem = 2.7 Hz, 1H, @CH), 7.49 (tm,
J = 7.7 Hz, 2H, COPh-H3, -H5), 7.61 (tm, J = 7.4 Hz, 1H, COPh-H4),
7.65 (br s, 1H, N-Ar-H6), 7.67 (t, 1H, N-Ar-H4), 7.69 (t, 1H, N-Ar-
H3), 7.69 (t, J = 8.2/8.8 Hz, 2H, Ar-H), 7.92 (dm, J = 7.7 Hz, 2H,
4.1.27. 4-Methyl-3-oxo-N-(2,4,5-trichlorophenyl) pentanamide
(1f)
Pyridine (0.1 mL, 1.2 mmol) was added to a solution of 2,4,5-tri-
chloroaniline (3.92 g, 20 mmol) and ethyl isobutyryl acetate
(5.2 mL, 32 mmol) in xylene (30 mL). The resulting solution was stir-
red and heated to reflux for 1 h then additional pyridine (0.1 mL)
added and the mixture heated to reflux for 1 h. A portion of the xy-
lene (25 mL) was distilled off then the mixture allowed to cool to
room temperature. The precipitated solid was filtered off and dried
under vacuum over CaCl2 to give 1f (2.49 g, 40%) as pale grey nee-
dles: mp 96 °C; 1H NMR (CDCl3): d 1.19 (d, J = 6.4 Hz, 6H, CH3),
2.73 (sept, J = 6.4 Hz, 1H, CH(CH3)2), 3.66 (s, 2H, CH2), 7.48 (s, 1H,
Ar-H6), 8.60 (s, 1H, Ar-H3); 13C NMR (CDCl3): d 17.7, 42.3, 46.5,
COPh-H2, -H6); 13C NMR (CDCl3):
d 11.4, 97.5, 123.1 (q,
JCF = 272 Hz), 127.1 (q, JCF = 4 Hz), 128.47 (q, JCF = 4 Hz), 128.54,
129.7, 130.5 (q, JCF = 34 Hz), 130.6, 131.4, 132.8, 133.9, 136.8,
138.0 (q, J = 1 Hz), 145.3, 150.3, 165.7, 190.8; IR (KBr):
m 3391,
121.8, 122.5, 127.4, 129.9, 131.5, 134.1, 164.0, 211.1; IR (KBr):
m
3057, 1698, 1652, 1619, 1492, 1443, 1326, 1262, 1215, 1187,
3201, 2979, 1707, 1686, 1570, 1496, 1360, 1074, 886 cmꢀ1; HRMS
1119, 1086, 1026, 951, 836, 687 cmꢀ1; HRMS (EI) m/z: calcd for
(EI) m/z: calcd for C12H12Cl3NO2: 306.9928 [M]+Å; found: 306.9943.
C
20H13ClF3NO2: 391.0581 [M]+Å; found: 391.0567.
4.1.28. 3-Isobutyryl-1-(2,4,5-trichlorophenyl)-4-methyl-5-
methylene-2-oxo-2,5-dihydro-1H-pyrrole (16b)
4.1.32. N-(2-Isopropyl-6-methylphenyl)-3-oxo-3-phenylpro-
panamide (1h) (method B)
Prepared by method D using DABCO as the base, 39 days, fol-
lowed by method G, chromatography eluent 1:3 dichlorometh-
ane/petroleum spirit to give 16b (51%) as a colourless solid; mp
103 °C; 1H NMR (CDCl3): d 1.13 (d, J = 6.8 Hz, 3H, CH(CH3)2), 1.14
(d, J = 6.8 Hz, 3H, CH(CH3)2), 2.46 (s, 3H, CH3), 3.67 (sept,
J = 6.8 Hz, 1H, CH(CH3)2), 4.74 (d, Jgem = 2.7 Hz,1H, @CH), 5.22 (d,
Jgem = 2.7 Hz, 1H, CH), 7.44 (s, 1H, Ar-H6), 7.67 (s, 1H, Ar-H3); 13C
NMR (CDCl3): d 11.7, 17.8, 17.9, 39.1, 98.5, 127.4, 131.4, 131.7,
A solution of 2-isopropyl-6-methylaniline (14.92 g, 100 mmol)
and ethyl benzoylacetate (21.14 g, 110 mmol) in DMF (60 mL) was
stirred and heated at 150 °C for 4 h then cooled to 80 °C. Water
was added until the mixture was turbid. It was stirred for 30 min,
water and ethanol added and then stirred for a further 1 h. The pre-
cipitated solid was filtered off to give 1h(20.4 g, 72%). A sample was
recrystallised from methanol to give a colourless solid: mp 289 °C
sub; 1H NMR (CDCl3, 40 °C, 500 MHz): d 1.14 (d, J = 6.9 Hz, 6H,
CH(CH3)2), 2.18 (s, 3H, CH3), 3.03 (sept, J = 6.9 Hz, 1H, CH(CH3)2),
4.15 (s, 2H, CH2), 7.05 (br d, J = 7.6 Hz, 1H, NHAr-H3), 7.13 (br d,
J = 7.6 Hz, 1H, NHAr-H5), 7.17 (t, J = 7.6 Hz, 1H, NHAr-H4), 7.50 (t,
J = 7.6 Hz, 2H, COPh-H3, H5), 7.62 (t, J = 7.6 Hz, 1H, COPh-H4), 8.05
131.9, 132.5, 132.9, 134.3, 145.1, 154.2, 166.0, 202.6; IR (KBr):
m
3092, 2976, 2936, 1679, 1631, 1591, 1474, 1407, 1389, 1354,
1167, 1128, 1082, 888, 871, 828 cmꢀ1; HRMS (EI) m/z: calcd for
C16H14Cl3NO2: 357.0085 [M]+Å; found: 357.0075.

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W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
(d, J = 7.6 Hz, 2H, COPh-H2, H6), 8.46 (br s, 1H, NH); 13C NMR (CDCl3,
40 °C, 125 MHz): d 18.6, 23.4, 28.7, 45.6, 123.5, 127.9, 128.1, 128.7,
COPh-H3, -H5), 7.48 (s, 1H, NAr-H6), 7.58 (t, J = 7.1 Hz, 1H, COPh-
H4), 7.66 (s, 1H, N-Ar-H3), 7.89 (d, J = 7.7 Hz, 2H, COPh-H2, -H6);
13C NMR (CDCl3): d 11.3, 97.6, 128.5, 129.6, 130.5, 131.4, 131.7,
131.9, 132.5, 132.8, 133.9, 134.2, 136.7, 145.1, 150.2, 165.6,
129.0, 132.4, 134.3, 135.9, 136.3, 145.6, 164.4, 196.5; IR (KBr):
m
3258, 2963, 1625, 1550, 1469, 1257, 778 cmꢀ1; HRMS (EI) m/z: calcd
for C19H21NO2: 295.1567 [M]+Å; found: 295.1567.
190.7; IR (KBr):
1360, 889, 845, 708, 693 cmꢀ1
C
19H11Cl3NO2 [M-H]+: 389.9850; found: 389.9848.
m
3416, 3082, 3061, 1713, 1659, 1469, 1394,
;
HRMS (EI) m/z: calcd for
4.1.33. 3-Benzoyl-1-[2-isopropyl-6-methylphenyl]-4-methyl-5-
methylene-2-oxo-2,5-dihydro-1H-pyrrole (16f)
Prepared by method D using DABCO as the base, 21 days, fol-
lowed by method E, 18 °C, 1 h to give 16f (55%) as a yellow solid:
mp 125 °C; 1H NMR (CDCl3): d 1.17 (d, J = 6.8 Hz, 3H, CH(CH3)2),
1.18 (d, J = 6.8 Hz, 3H, CH(CH3)2), 2.13 (s, 3H, Ar-CH3), 2.30 (s, 3H,
CH3), 2.78 (sept, J = 6.8 Hz, 1H, CH(CH3)2), 4.57 (d, J = 1.3 Hz, 1H,
@CH), 5.07 (d, J = 1.3 Hz, 1H, @CH), 7.14 (d, J = 7.3 Hz, 1H, Ar-H3),
7.25 (d, J = 7.3 Hz, 1H, Ar-H5), 7.31 (t, J = 7.7 Hz, 1H, Ar-H4), 7.45
(t, J = 7.3 Hz, 2H, 7.7 Hz, COPh-H3, -H5), 7.57 (t, J = 7.3 Hz, 1H,
COPh-H4), 7.90 (d, J = 7.3 Hz, 2H, COPh-H2, -H6); 13C NMR (CDCl3):
d 11.3, 18.1, 23.6, 24.3, 28.6, 97.0, 124.2, 128.3, 128.4, 129.4, 129.7,
130.4, 131.0, 133.8, 137.0, 137.3, 146.3, 148.3, 149.4, 166.3, 191.4;
4.1.37. N,1-Bis(3-chlorophenyl)-4-methyl-5-methylene-2-oxo-
N-phenyl-2,5-dihydro-1H-pyrrole-3-carboxamide (16j)
Prepared by method D using morpholine as the base, overnight,
followed by method E, 18 °C, overnight. The reaction solution was
diluted with aqueous isopropanol (1:1) to precipitate the product
which was filtered off and dried to give 16j (75%) as pale yellow
needles: mp 164 °C; 1H NMR (CDCl3): d 2.70 (s, 3H, CH3), 5.15 (d,
J = 2.6 Hz, 1H, @CH), 5.39 (d, J = 2.6 Hz, 1H, @CH), 7.07 (dm,
J = 7.9 Hz, 1H, Ar-H), 7.20–7.25 (m, 2H, Ar-H), 7.33 (t, J = 1.8 Hz,
1H, Ar-H), 7.38–7.48 (m, 3H, Ar-H), 7.88 (t, J = 2.0 Hz, 1H, Ar-H),
10.57 (s, 1H, NH); 13C NMR (CDCl3): d 11.5, 100.8, 117.9, 120.1,
120.5, 124.4, 125.9, 128.0, 128.8, 129.9, 130.5, 134.61, 134.63,
IR (KBr):
m ;
3423, 2963, 1692, 1654, 1628, 1472, 1449, 1164 cmꢀ1
HRMS (EI) m/z: calcd for C23H23NO2: 345.1723 [M]+Å; found:
135.1, 139.0, 145.9, 154.6, 160.0, 168.3; IR (KBr):
1660, 1631, 1588, 1543, 875, 780 cmꢀ1; HRMS (EI) m/z: calcd for
19H14Cl2N2O2: 372.0427 [M]+Å; found: 372.0417.
m 3072, 1701,
345.1715.
C
4.1.34. 3-Benzoyl-1-[2,6-diisopropylphenyl]-4-methyl-5-methy
lene-2-oxo-2,5-dihydro-1H-pyrrole (16g)
4.2. Receptor screening
Prepared by method D using DABCO as the base, 21 days, fol-
lowed by method E, formic acid, 18 °C, 1 h, chromatography eluent
1:19 ethyl acetate/petroleum spirit. Purified by recrystallisation
(ethanol/water) to give 16g (23%) as pale yellow needles: mp
164 °C; 1H NMR (CDCl3): d 1.19 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.20
(d, J = 6.8 Hz, 6H, CH(CH3)2), 2.33 (s, 3H, CH3), 2.73 (quint,
J = 6.8 Hz, 2H, CH(CH3)2), 4.58 (d, Jgem = 1.5 Hz, 1H, @CH), 5.08 (d,
Jgem = 1.5 Hz, 1H, @CH), 7.26 (d, J = 7.7 Hz, 2H, Ar-H3, -H5), 7.41
(t, J = 7.7 Hz, 1H, Ar-H4), 7.45 (t, J = 7.7 Hz, 2H, COPh-H3, -H5),
7.57 (t, J = 7.3 Hz, 1H, COPh-H4), 7.91 (d, J = 7.3 Hz, 2H, COPh-H2,
-H6); 13C NMR (CDCl3): d 11.2, 23.8, 24.3, 28.9, 97.1, 124.0, 128.3,
129.0, 129.8, 130.9, 133.7, 137.0, 147.5, 148.0, 149.5, 166.8,
The ligand-binding regions from the EcR and USP proteins of the
insect pests, B. ovis34 (BoEcRDEF/BoUSPDEF) and Lucilia cuprina,
(LcEcRDEF/LcUSPDEF) were co-expressed using a baculovirus vector,
purified as a recombinant heterodimer by immobilised metal affin-
ity chromatography,35 and used in fluorescence polarisation
assays.24
The standard fluorescence polarisation (FP) assay buffer used
was 50 mM sodium phosphate, 100 mM NaCl, pH 7.424 with a final
volume of 200 lL for all assays. The assays were set up in opaque
black flat-bottomed 96-well plates and were incubated at 4 °C
overnight, then re-equilibrated at room temperature for 3 h before
reading the polarisation (mP) values. The plate reader was a PHER-
Astar instrument (BMG Labtech, Offenburg, Germany) with the
fluorescence polarisation optic module FP 485 520A 520B (in-
stalled according to manufacture’s instructions) and operated by
the PHERAstar software. The standard instrument setup used the
default FP basic parameters for a 96-well plate and involved a
3 s, 3 mm linear shake before each cycle.
191.5; IR (KBr):
m 3385, 2963, 2869, 1693, 1660, 1629, 1610,
1386, 1345, 1157, 860, 822, 712, 689 cmꢀ1; HRMS (EI) m/z: calcd
for C25H27NO2: 373.2036 [M]+Å; found: 373.2027.
4.1.35. N-(2,4,5-Trichlorophenyl)-3-oxo-3-phenylpropanamide
(1i)
Prepared by method B, to give 1i (28%) as near colourless solid
which was a mixture of the ketone and enol forms in solution: mp
168 °C; 1H NMR (DMSO-d6): d 4.31 (s, 2H, COCH2), 6.32 (s, 1H,
COCH=C), 7.46–7.59 (m, 5H, COPh-H3, -H4, -H5 enol form), 7.55
(t, J = 7.5 Hz, 2H, COPh-H3, H5 ketone form), 7.67 (t, J = 7.3 Hz,
1H, COPh-H4 ketone form), 7.76 (d, J = 5.9 Hz, 2H, COPh-H2, H6
enol form), 7.89 (s, NHAr-H6 ketone form), 7.91 (NHAr-H6 enol
form), 7.99 (d, J = 7.3 Hz, 2H, COPh-H2, H6 ketone form), 8.21 (s,
1H, NHAr-H3 enol form), 8.22 (s, 1H, NHAr-H3 ketone form); 13C
NMR (DMSO-d6): d 48.0, 89.9, 124.5‘125.3, 126.0, 127.3, 128.8,
129.2, 129.3, 130.3, 131.0, 131.8, 133.8, 134.1, 134.9, 135.4,
4.2.1. High-throughput screening
All preparative pipetting steps for the high-throughput polari-
sation assays were carried out by the TECAN Genesis 200 robot
run by Gemini software V.4.2.0.0 and using TECAN 200 lL dispos-
able conductive tips (TECAN Cat. No. 10612510). The plate reader
was normalised to the polarisation value of 100mP for the un-
bound fluorescein–inokosterone A conjugate (MB4628)24 and the
gain and focal height were adjusted against a blank (lacking re-
combinant heterodimer) at the start of each test run for each plate.
Each plate contained 6 lL of 60 different compounds in dimethyl
136.5, 167.0, 170.1, 171.4, 194.9; IR (KBr):
m 3446, 3186, 2355,
1691, 1577, 1517, 1360, 1075, 756 cmꢀ1; HRMS (EI) m/z: calcd
sulfoxide (DMSO) randomly chosen from the CSIRO compound li-
brary36 that had been dispensed into the centre wells of the 96-well
plate for the high-throughput screening of a total of three thousand
compounds. The plates were stored at ꢀ20 °C until ready for use
and were allowed to thaw at room temperature for three hours
prior to commencement of the assay. A script was written for the
for C15H10Cl3NO2: 340.9772 [M]+Å; found: 340.9786.
4.1.36. 3-Benzoyl-1-(2,4,5-trichlorophenyl)-4-methyl-5-methy
lene-2-oxo-2,5-dihydro-1H-pyrrole (16h)
Prepared by method D using DABCO as the base, 43 days, fol-
lowed by method G, chromatography eluent 1:1 dichlorometh-
ane/petroleum spirit to give 16h (61%) as a yellow solid: mp
166 °C; 1H NMR (CDCl3): d 2.27 (s, 3H, CH3), 4.72 (d, J = 2.6 Hz,
1H, @CH), 5.17 (d, J = 2.6 Hz, 1H, @CH), 7.46 (t, J = 7.7 Hz, 2H,
TECAN robot to pipette 194 lL of a buffer mixture into the wells
of the plates containing the library compounds. The buffer mixture
contained 2.51 mg/mL of purified BoEcRDEF/BoUSPDEF heterodi-
mer or 1.14 mg/mL LcEcRDEF/LcUSPDEF heterodimer, 0.5 mg/mL of

W. Birru et al. / Bioorg. Med. Chem. 18 (2010) 5647–5660
5659
bovine serum albumin (BSA) and 36 nM fluorescein–inokosterone
A conjugate in the standard FP assay buffer.
(2002).38 LC50 values were corrected for the mortality observed in
solvent-only control experiments.
Ten wells on each plate were manually set up to contain the na-
tive hormone (20-hydroxyecdysone) as positive controls at three
different concentrations, in triplicate, and one well was used for
the heterodimer-free blank. Buffer mixture was manually added
4.4. Computational methods
Molecular modelling was carried out using Sybyl 8.1 on Silicon
Graphics Fuel and Red Hat Linux PC workstations. Structures were
constructed using modelling and molecular mechanics minimisa-
tion in Sybyl using the Tripos forcefield and default values. Gastei-
ger–Huckel charges were used in the minimisation calculations.
Structures were energy minimized using the default Sybyl energy
minimizer and Gasteiger–Huckel charges.
to the controls and contained 2.51 mg/mL of purified BoEcRDEF
BoUSPDEF or 1.14 g/mL LcEcRDEF/LcUSPDEF receptor, 0.5 mg/mL of
bovine serum albumin (BSA), 36 nM fluorescein–inokosterone A
conjugate and 6 L of DMSO in the standard FP assay buffer. The
blank contained 0.5 mg/mL of bovine serum albumin (BSA),
/
l
l
36 nM fluorescein–inokosterone A conjugate and 6
in the standard FP assay buffer.
lL of DMSO
The compounds were screened at a rate of 10 plates (600 com-
pounds) per day. The plates were set up in the afternoon, stored at
4 °C overnight and were then equilibrated at room temperature for
three hours before reading the mP values. Data were plotted in Ex-
cel and used to calculate the IC50 for the binding activity of each
compound.
4.4.1. Alignment and choice of conformation
The highly active and relatively rigid molecule, lactam 6b, was
used as a template molecule. Molecules were set to a conformation
that most closely matched that of the template, and the Sybyl de-
fault minimizer was used to relieve any strain in the molecules.
The molecules were aligned on the template to maximize the over-
lap between the methylene lactam ring and the aromatic ring at-
tached to the lactam nitrogen atom. Where two conformations of
similar energy existed for rotation around the lactam-aromatic
ring bond, molecules were placed in a consistent conformation
with placement of any ortho substituents done consistently so that
moieties with similar properties were given maximum opportunity
to overlap. Where it was not clear from energetic or conforma-
tional considerations, which of two possible conformers is the cor-
rect one, both conformations were used to develop an initial
CoMFA model, which was then used to eliminate the conformation
whose activity was predicted least accurately. The stereochemistry
4.2.2. IC50 determinations
A 30 mM stock solution of the test compound (i.e., the putative
ligand) in DMSO was used to prepare a dilution series covering the
assay range of 3 nM to 3 mM (final concentration). The diluent for
the test compound was DMSO. These preparative pipetting steps
were performed in 0.6 mL microfuge tubes. Each 200
cluded 6 L of test compound in DMSO (3% v/v final concentration
DMSO in each assay), 0.5 mg/mL bovine serum albumin (BSA),
36 nM fluorescein–inokosterone conjugate and 2.51 mg/mL
lL assay in-
l
A
BoEcRDEF/BoUSPDEF receptor or 1.14 mg/mL LcEcRDEF/LcUSPDEF
receptor in standard FP assay buffer.
of the c-methylene substituent in compounds with non-hydrogen
The plate reader was normalised to 100 mP using a 200
l
L sam-
substituents was fixed to E. A final CoMFA model was then devel-
oped using the conformation that was most consistent with the
activities of other members of the data set. The conformations of
the alkyl substituents on the methylene lactam ring were set to
near orthogonality to the lactam ring, to minimize the energies
of the ecdysone agonists. The conformations of other substituents
on the lactam ring were set to be extended, then energy mini-
mized. The alignment of all molecules on the template is illustrated
in Figure 8.
ple containing 36 nM fluorescent conjugate, 0.5 mg/mL BSA and 3%
v/v DMSO in standard FP assay buffer. Data were plotted in Excel
(Microsoft Corporation). All samples were assayed in triplicate on
a single plate and IC50 values were determined from the mean.
Random samples of 10% of the IC50 determinations on the BoEcR-
DEF/BoUSPDEF receptor and the LcEcRDEF/LcUSPDEF receptor dis-
played a coefficient of variation of 1–6% and 1–4%, respectively,
with the exception of one sample which displayed a coefficient
of variation of 18% in the IC50 for binding to the BoEcRDEF/BoUSPDEF
receptor. Another compound was assayed on two separate occa-
sions and displayed a coefficient of variation of 9% in the IC50 for
binding to the BoEcRDEF/BoUSPDEF receptor.
4.4.2. 3D QSAR modelling
3D QSAR models were built using both the comparative molec-
ular field analysis method (CoMFA)39 and the comparative molec-
ular similarity analysis method (COMSIA).40 COMSIA dissects
molecular interactions into five properties, and uses a Gaussian
function to probe the similarity of each molecule with respect to
the probe within the lattice. In contrast, CoMFA used only steric
and electrostatic fields, which may explain why CoMFA models
that included log P were generally also significant. Sybyl 8.1 was
used to generate the CoMFA fields using Gasteiger–Huckel charges,
with an sp3 hybridized carbon probe with a charge of +1.0 for the
CoMFA fields, and a grid spacing of 2 Å. The default values for the
field cut offs were used. The log P (octanol–water) values calcu-
lated using the Pomona College Clog P method.41 The COMSIA
fields were also generated using Sybyl 8.0 and Gasteiger–Huckel
charges. The standard COMSIA probe with a unit radius, +1.0
charge, and unit hydrophobicity were used. Steric, electrostatic,
lipophilic, donor, and acceptor fields were generated.
The QSAR models were derived using the partial least squares
method. The sampls method was used to generate a cross-vali-
dated the leave-one-out (LOO) predictivity estimate of the models,
and to determine the correct number of principal components for
the final non-cross-validated models. Region focusing was also
used to refine the CoMFA models, which resulted in a small
improvement in statistical quality.
4.3. B. ovis toxicity assay
Wool snippets 1–1.5 cm in length were weighed to 20 mg
amounts, added to flat bottom vials and treated with 200
tonic solutions of test compounds at concentrations ranging from
0.01 to 1000
g/mL. Louse diet37 was weighed to 10 mg quantities,
added to 75 mm ꢁ 12 mm borosilicate culture tubes and treated
with 50 L of similar concentrations of test compound. Diet and
lL of ace-
l
l
wool were allowed to dry overnight at 5 °C. Lice were collected
from sheep penned adjacent to the laboratory, provided with un-
treated louse diet and held overnight in an incubator at 36.5 °C
and 68% relative humidity. The next morning the treated wool
snippets were added to the culture tubes containing diet treated
with corresponding concentrations of test compound together
with ten third instar B. ovis nymphs. Four replicate tubes were used
for each concentration and diet and wool in the control tubes were
treated with acetone. All tubes were held in an incubator at 36.5 °C
and 68% relative humidity for nine days to allow time for the
nymphs to moult. On day nine lice were removed from the tubes
and the numbers of dead lice recorded. LC50 values and 95%
confidence limits were calculated using PoloPlusÓ LeOra Software

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Acknowledgements
The authors would like to acknowledge Luci Mokbel for some
preliminary chemical synthesis. We are grateful to Australian Wool
Innovation Ltd for financial support.
Supplementary data
33. Pedersen, S. J.; Rosenbohm, C. Synthesis 2001, 16, 2431.
34. Pollard, M.; Hannan, G. N.; Graham, L. D.; Hill, R. J. International Patent WO-
2008092212, 2008; Chem. Abstr. 2008, 149, 240285.
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N.; Pawlak-Skrzecz, A.; Noyce, L.; Lovrecz, G. O.; Lu, L.; Hill, R. J. Prot. Exp. Purif.
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.020.
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