Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.05.048

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC₅₀ values of 290 and 25 nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

Full text of DOI:10.1016/j.bmc.2008.05.048

Bioorganic & Medicinal Chemistry 16 (2008) 6911–6923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modelling of N-heterocyclic
dipeptide aldehydes as selective calpain inhibitors
a
a
b
Matthew A. Jones a, James D. Morton b, , James M. Coxon , Stephen B. McNabb , Hannah Y.-Y. Lee ,
*
Steven G. Aitken a, Janna M. Mehrtens a, Lucinda J. G. Robertson b, Axel T. Neffe a, , Shigeru Miyamoto a,
Roy Bickerstaffe b, Karl Gately b, Jacqueline M. Wood b, Andrew D. Abell a, ,à
*
a Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
b Agriculture and Life Sciences Division, Post Office Box 84, Lincoln University, Canterbury, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-heterocyclic dipeptide aldehydes 4–13 have been synthesised and evaluated as inhibitors of
ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC50 values of 290 and
25 nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor,
displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been deter-
mined. Because of the lack of available structural information on the ovine calpains, in silico homology
models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-
CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed
SAR for compounds 4–13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9
(CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens
from calcium-induced opacification.
Received 28 March 2008
Revised 21 May 2008
Accepted 22 May 2008
Available online 27 May 2008
Keywords:
Calpain
Isoform selectivity
Ovine calpain 1 and 2
In silico calpain homology model
Substituted heterocyclic dipeptides
CAT0059
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
opment of a potent and selective calpain 2 inhibitor would be a po-
tential means of treating cortical cataract.
Calpains are calcium activated neutral proteases belonging to
the papain superfamily of cysteine proteases. The structure, mech-
anism of activation and pharmacological actions of calpain have
been recently reviewed.1 Over-activation of calpain is central to a
number of medical conditions associated with cellular damage.
These include traumatic brain injury, muscular dystrophy and cat-
aract.2 In the case of cortical cataract, increased lens calcium3 and
subsequent over-activation of calpain 2 have been linked to the
breakdown of lens proteins, resulting in clouding of the lens and
ultimately blindness.4,5 Therefore, it has been proposed that devel-
In this paper, we present SAR data on a series of N-heterocyclic
peptidyl aldehydes 4–13 (see Schemes 1–3) that were designed to
interact with the S3 binding pocket of calpains. Due to sequence
differences between calpain 1 and 2 it was anticipated that the
use of N-terminal heterocyclic moieties that interact differentially
with either o-CAPN1 or o-CAPN2 would result in isoform selective
calpain inhibitors.
A variety of N-terminal heterocyclic constituents of protease
inhibitors have been shown to interact with key residues in the
S3 hydrophobic pocket of cysteine proteases (Fig. 1). The quinoline
derivative 1 was designed based on the expectation that the forma-
tion of additional hydrogen bonds might be possible via an interac-
tion between the quinoline NH and calpain residues6; the P3
morpholine urea 2 exhibits a favourable electrostatic interaction
with the sidechain ammonium group of Lys64 in the S3 pocket of
Cathepsin S7; and the extended P3 methylpiperazine-thiazoleben-
zamide 3 is capable of forming an ionic interaction to Asp61 in the
S3 pocket of Cathepsin K.8
Abbreviations: DIPEA, N,N-diisopropylethylamine; HATU, O-(7-azabenzotri-
azole-1-yl)-N,N,N0N0-tetramethyluronium hexafluorophosphate; pyr, pyridine;
o-CAPN1, ovine calpain 1; o-CAPN2, ovine calpain 2; h-CAPN1, human calpain 1;
h-CAPN2, human calpain 2; p-CAPN1, porcine calpain 1; p-CAPN2, porcine
calpain 2; r-CAPN1, rat calpain 1; r-CAPN2, rat calpain 2.
* Corresponding authors. Tel.: +64 3 3253803x8169; fax: +64 3 3253851 (J.D.
Morton); tel.: +61 8 8303 5652; fax: +61 8 8303 4358 (A.D. Abell).
E-mail addresses: mortonj@lincoln.ac.nz (J.D. Morton), andrew.abell@adelaide.
edu.au (A.D. Abell).
In this paper, we extend this methodology by designing prote-
ase inhibitors with N-terminal heterocyclic moieties that are capa-
ble of preferentially binding o-CAPN2 over o-CAPN1, resulting in
isoform selective calpain inhibitors.
Present address: GKSS Research Centre, Institute of Polymer Research, Kantst-
rasse 55, 14513 Teltow, Germany.
à
Present address: School of Chemistry & Physics, The University of Adelaide, North
Terrace, Adelaide, SA 5005, Australia.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.048

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M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
the efficacy of the inhibitors using ovine calpains and an ovine lens
O
culture assay.
O
H
N
NH2
2. Results and discussion
N
H
O
O
2.1. Preparation of N-heterocyclic dipeptide aldehydes
1
The target compounds 4–8 were prepared as detailed in Scheme
1. N-Boc-valine was reacted with S-leucinol to give the Boc
protected dipeptide 14. Treatment with HCl/dioxane gave the
hydrochloride salt 15 in a quantitative yield.16 This key intermedi-
ate was separately reacted with five heterocyclic acids or acid
chlorides to give the dipeptide alcohols 16–20.17 Subsequent
oxidation of the alcohols 16–20 with SO3-pyridine (SO3-pyr) and
DSMO (the Parikh–Doering18 oxidation reaction) gave the alde-
hydes 4–8 in good yields (Scheme 1).
O
O
O
CF3
H
N
N
N
N
H
H
O
SO2CH2Ph
2
Aldehydes 9 and 10 were prepared as described in Scheme 2.
Formylation of 2-pyrrole carboxylic acid ethyl ester gave a mixture
of 5- and 4-formyl-pyrrole ethyl ester isomers 21 and 22 (2.4:1)
that were separable by chromatography.19 Separate ester hydroly-
sis of ester isomers 21 and 22 resulted in the formation of 5-for-
myl-2-pyrrole carboxylic acid 23 and 4-formyl-2-pyrrole
carboxylic acid 24, respectively. The acids 23 and 24 were reacted
with 15 to give the alcohols 25 and 26. Oxidation gave the corre-
sponding aldehydes 9 and 10 (Scheme 2).
O
H
N
CN
N
H
N
N
O
N
S
3
Figure 1. Protease inhibitors 1–3 designed to form hydrogen-bonding interactions
in the S3 pockets of calpain, cathepsin S and cathepsin K, respectively.6–8
Aldehydes 11–13 were prepared as described in Scheme 3. The
substituted 2-pyrrole carboxylic acid precursors 27–29 were syn-
thesised using standard literature procedures.20,21 5-Formyl-N-
methyl-pyrrole-2-carboxylic acid 27 was synthesised via alkylation
of 2-pyrrole carboxylic acid using methyl iodide and NaOH followed
by base-mediated ester hydrolysis.20 5-Methyl-pyrrole-2-carboxyl-
ate 28 was synthesised from the reaction of diethyl acetamidomalo-
nate and 1,4-dichloro-2-butyne using NaOEt as a base in refluxing
EtOH according to the method developed by Curran and Keaney21
followed by ester hydrolysis. 5-Methoxycarbonyl-2-pyrrole carbox-
ylic acid 29 was synthesised via KMnO4 oxidation of the correspond-
ing 5-formyl ester in methanol.20 The pyrrole acids 27–29 were
Rodent models are most commonly used to assess the effect of
anti-cataract agents.9–12 However, the rodent is a poor model for
human cataract as its lenses are smaller, spherical, and have a dif-
ferent depth focus mechanism compared to human lenses.13 The
ovine lens is a better model as it has a similar size, shape and
accommodative index to the human lens.14 In addition, the ovine
lens proteins are more similar to human lens proteins than those
of the rat lens as shown by the close homology between sequences
of ovine and human crystallins.15 For these reasons we have tested
BocHN
CO2H
O
H
O
H
N
N
O
H
O
N
H
N
H
X
X
O
H
(i)
O
O
H
O
4 X = O
5 X = S
6 X = NH
7 X = O
8 X = S
BocHN
OH
N
H
14
(iv)
(vi)
(ii)
O
H
O
H
O
Cl
H3N
(iii)
(v)
N
OH
H
N
OH
OH
N
N
X
N
H
X
H
H
O
O
O
16 X = O
17 X = S
18 X = NH
19 X = O
20 X = S
15
Scheme 1. Synthesis of inhibitors 4–8. Reagents: (i) S-leucinol, HATU, DIPEA, DMF (68%); (ii) 4 M HCl/dioxane (99%); (iii) DIPEA, DMF, 2-furan-carbonyl chloride or
2-thiophene-carbonyl chloride or (2-pyrrole-carboxylic acid and HATU) (48%, 56% and 84%, respectively); (iv) SO3-Pyr, DMSO, DCM, DIPEA (58%, 51% and 71%, respectively);
(v) DIPEA, DMF, HATU 5-formyl-2-furan-carboxylic acid or 5-formyl-2-thiophene-carboxylic acid (30% and 88%, respectively); (vi) SO3-Pyr, DMSO, DCM, DIPEA (80% and 89%,
respectively).

M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
6913
reacted with 15 to give alcohols 30–32 that were oxidised to give the
corresponding aldehydes 11–13 (Scheme 3).
OEt
N
H
O
2.2. Ovine calpain sequence
(i)
O
The amino acid sequences of the catalytic subunits of o-CAPN1
and o-CAPN2 were deduced from their cDNA sequences (GenBank
Accession Nos. EU623071 and EU161096, respectively). o-CAPN2
has two alleles with a G replaced by a T at 741 and a C replaced
by a T at 1539. However, neither of these alter the amino acid se-
quence. The human calpain sequences, h-CAPN122 and h-CAPN2,23
are more homologous to the ovine calpains, o-CAPN1 and o-CAPN2,
than to the rat calpains, r-CAPN124 and r-CAPN2.25 The o-CAPN1
amino acid sequence is 95% homologous with the h-CAPN1, con-
siderably closer than the 89% homology of r-CAPN1 to h-CAPN1.
The large subunits of o-CAPN2 and h-CAPN2 are 95% homologous.
This compares with 94% for rat with human. Of the 35 amino acids
which differ between o-CAPN2 and h-CAPN2, 18 are conservative
substitutions (similar amino acids) and the changes are clustered
in non-essential regions of the protein. The homology between o-
CAPN2 and o-CAPN1 is 62%, which is similar to that found in other
species. o-CAPN2 is also 62% homologous to h-CAPN1.
Alignment of selected residues in the catalytic domain of ovine,
human and rat calpain highlights the greater homology between
ovine calpains and h-CAPN1 compared to r-CAPN1 (Fig. 2). Within
these sections there are 11 amino acids different between h-CAPN1
and r-CAPN1 and only two between h-CAPN1 and o-CAPN1. There
are nine differences between h-CAPN1 and o-CAPN2. However,
only two of these differences occur in proximity to the active site,
namely Ser209 and Met260 in h-CAPN1, which correspond to Ala199
and Ser250 in o-CAPN2, respectively.
H
H
OR
N
H
O
O
OR
N
H
O
21 R = Et
22 R = Et
(ii)
(v)
23 R = H
(iii)
24 R = H
O
H
N
(vi)
H
OH
N
H
N
H
O
O
O
25
H
O
H
N
OH
N
H
N
H
(iv)
O
26
O
H
(vii)
H
N
O
N
H
N
H
O
O
H
O
H
9
O
H
N
2.3. Biological activity
O
N
H
N
H
O
H
The compounds 4–13 were assayed against o-CAPN1 and
o-CAPN2, purified from ovine lung tissue, using a fluorescence-
based assay to determine in vitro potency and selectivity,26 and
the results summarised in Table 1. All compounds inhibited both
o-CAPN1 and o-CAPN2 with IC50 values below 1 lM. 5-Formyl-
pyrrole 9 (IC50 values of 290 and 25 nM against o-CAPN1 and
10
Scheme 2. Synthesis of inhibitors 9 and 10. Reagents: (i) POCl3, DMF (21, 59%; 22
25%); (ii) NaOH, MeOH, H2O (93%); (iii) HATU, DIPEA, DMF, 15 (39%); (iv) SO3-Pyr,
DMSO, DCM, DIPEA (71%); (v) NaOH, THF, H2O (85%); (vi) HATU, DIPEA, DMF, 15
(36%); (vii) SO3-Pyr, DMSO, DCM, DIPEA (90%).
O
O
H
H
N
(i)
(iv)
H
N
O
H
OH
H
OH
N
H
N
H
N
N
N
O
O
H
O
O
O
O
Me
Me
Me
27
30
11
O
O
H
H
(v)
(ii)
N
O
N
OH
OH
N
N
N
N
N
H
H
H
H
O
H
H
O
O
12
28
31
O
O
H
N
H
N
(iii)
(vi)
O
O
O
OH
O
OH
N
N
N
H
N
H
N
H
H
H
O
O
H
O
O
O
O
13
29
32
Scheme 3. Synthesis of inhibitors 11–13. Reagents: (i) 15, HATU, DIPEA, DMF (56%); (ii) 15, HATU, DIPEA, DMF, (29%); (iii) 15, HATU, DIPEA, DMF (64%); (vi) SO3-Pyr, DMSO,
DCM, DIPEA (75%); (v) SO3-Pyr, DMSO, DCM, DIPEA (63%); (vi) SO3-Pyr, DMSO, DCM, DIPEA (69%).

6914
M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
Figure 2. Sequence alignment of r, h and o-CAPN1, and o-CAPN2. Amino acid residues forming part of the active site cleft and binding pockets are in bold type. Amino acid
substitutions compared to h-CAPN1 are shaded.
o-CAPN2, respectively) was the most potent and selective o-CAPN2
inhibitor, displaying >11-fold selectivity. In contrast, other well-
documented dipeptidyl aldehyde calpain inhibitors such as
Table 1
In vitro inhibition data
a
SJA601727 (IC50 values of 7.5 and 78 nM against porcine calpain 1
(p-CAPN1) and porcine calpain 2 (p-CAPN2), respectively) and
MDL2817028 (IC50 values of 56 and 350 nM against h-CAPN1 and
h-CAPN2, respectively) display selectivity for calpain 1 over calpain
2. Formyl-furan 7 (IC50 values of 960 and 100 nM against o-CAPN1
and o-CAPN2, respectively) was also a highly potent and selective
o-CAPN2 inhibitor, displaying >9-fold selectivity for o-CAPN2 com-
pared to o-CAPN1. The inhibitors 4–6, 8, 10, 12 and 13 displayed
moderate selectivity (between 2- and 7-fold) for o-CAPN2, while
N-methyl-pyrrole 11 was non-selective.
Compound
IC50 (nM)
Selectivity o-CAPN2
over o-CAPN1
o-CAPN1
o-CAPN2
4
5
6
7
8
790
680
650
960
440
290
530
150
340
290
135
100
315
100
85
5.85
6.80
2.06
9.60
5.18
11.6
5.30
1.00
3.09
2.07
9
25
10
11
12
13
100
150
110
140
Compounds 4–10, 12 and 13 are all more potent against
o-CAPN2 than o-CAPN1 (Table 1). The assay results for o-CAPN1
a
Values are means of three experiments. Variation between experiments is < 10%.

M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
6915
(Table 1) show that the pyrrole containing derivatives, 6 and 9–13,
are generally more potent than the furan or thiophene analogues 4,
5, 7 and 8. A 5-carbonyl substituent on pyrrole (9, 11, 13) appears
to be important for optimum activity against o-CAPN1, as pyrrole
analogues lacking this group (6, 10 and 12) show reduced potency.
It is also noteworthy that the 5-formyl and 5-methoxycarbonyl
pyrrole derivatives 9 and 13 are equally potent against o-CAPN1.
In addition, the N-methyl-pyrrole derivative 11 is more potent
against o-CAPN1 than its unsubstituted analogue 9.
The assay results show the 5 formyl thiophene 8 and 5-formyl-
pyrrole 9 to be the most potent analogues against o-CAPN2. The
importance of a 5-carbonyl substituent parallels the o-CAPN1 as-
say results. The N-methyl-pyrrole 11 is less potent against o-
CAPN2 than the unsubstituted analogue 9. This order of potency
is the reverse of that observed for o-CAPN1.
tal structures have been published29,32; however, one is in an inac-
tive form.32 The sequence alignment of selected residues of h-
CAPN1,22 r-CAPN1,24 o-CAPN1 and o-CAPN2 clearly shows that a
high degree of homology exists within the residues that form the ac-
tive site cleft of ovine calpains and h-CAPN1 (Fig. 2). Therefore, h-
CAPN1 X-ray crystal structure (1ZCM) was used as the structural ba-
sis for the o-CAPN1 homology model.
A similar strategy was employed to build the o-CAPN2 homol-
ogy model. Despite a number of differences in the amino acid se-
quence of h-CAPN1 and o-CAPN2 (Fig. 2), it was decided to build
the in silico o-CAPN2 homology model based on the h-CAPN1 X-
ray crystal structure (1ZCM)29 as this is the most appropriate X-
ray crystal structure available. Therefore, the model based on this
X-ray structure is likely to be the best available to rationalise the
observed SAR of compounds 4–13.
2.4. Construction of in silico ovine calpain homology models
2.5. Molecular modelling and SAR discussion
Molecular modelling was used to rationalise the observed SAR
of compounds 4–13. Novel in silico ovine calpain homology models
were constructed using the o-CAPN1 and o-CAPN2 sequences and
h-CAPN1 X-ray crystal structure data available from the Protein
Data Bank (PDB code 1ZCM).29
The h-CAPN1X-ray crystal structure, 1ZCM, was mutated in silico
around the active site cleft to mimic the amino acid sequence of
either o-CAPN1 or o-CAPN2 (see Section 4.5). These two ovine cal-
pain homology models were then used to dock compounds 4–
13.33,34
The most appropriate calpain X-ray crystal structure information
is required to build accurate in silico ovine calpain models. Ten X-ray
crystal structures of the active catalytic domains of calpains 1 and 2
have been published.30 Eight of these crystal structures have been
obtained by Davies using a rat calpain 1 (r-CAPN1) construct. Unfor-
tunately, no useful structural information is available for the rat cal-
Aldehyde inhibitors of cysteine proteases are considered to act
by reversible formation of a thio-hemiacetal. Docking studies with
Glide34 do not allow for covalent bond formation between the cys-
teine thiol and the aldehyde. The potential inhibitor and enzyme
necessarily remain as separate molecular entities. Such studies al-
low the potential of a compound to position itself to act as an
inhibitor to be evaluated. They show whether or not the aldehyde
carbonyl carbon is positioned appropriately for attack by the
pain 2 (r-CAPN2) construct due to an instability in helix a7, which
results in intrinsic inactivation.31 Only two h-CAPN1 construct crys-
Table 2
Docking results and parameter for compounds 4–13
Compound
Grid
No. starting conf.
No. poses generated
No. poses with WHD < 4.5a
Most representative pose
H bondsb,c
WHDa
Emodel
Einternal
4
5
OvineI
OvineII
4
3
4
4
5
4
4
3
3
4
39
34
36
34
A, B, Ser251
A, C
3.38
3.31
ꢀ56.04
ꢀ47.95
4.48
5.84
OvineI
OvineII
27
22
20
17
B, C, Ser251
B, C, Gly261
3.67
3.47
ꢀ52.03
ꢀ53.65
1.78
7.87
6
OvineI
OvineII
40
40
17
26
B, C
B, C, Ser261
3.72
3.46
ꢀ50.55
ꢀ50.44
4.26
4.91
7
OvineI
OvineII
40
32
19
7
B, C
A, B, C, Ile244
4.02
3.81
ꢀ54.27
ꢀ50.65
6.42
6.84
8
OvineI
OvineII
35
49
28
44
A, B, C
A, B, C
3.97
3.69
ꢀ54.54
ꢀ49.40
5.58
5.23
9
OvineI
OvineII
40
32
27
6
A, B, C, Ile254
Gln99, Gly261, 2ꢁ Gly198
4.15
3.54
ꢀ53.66
ꢀ50.49
1.15
3.69
10
11
12
OvineI
OvineII
37
32
16
23
B, C
B, C
4.17
3.46
ꢀ50.86
ꢀ55.13
3.72
3.83
OvineI
OvineII
30
17
23
13
A, B, C, Ile254
A, B, C, Ile244
3.72
3.74
ꢀ54.05
ꢀ52.95
5.84
6.96
OvineI
OvineII
17
27
8
8
B, C, Thr210
C, Gly271
3.54
3.48
ꢀ56.14
ꢀ53.94
4.86
3.66
13
OvineI
OvineII
34
36
9
15
A, B, C, Ile254
B, C, Ile244
4.40
3.47
ꢀ54.76
ꢀ53.39
3.81
2.74
a
War head distance (WHD) is the distance between the carbonyl carbon of the aldehyde and the active site cysteine sulfur in Å.
b
Hydrogen bonds from the carbonyl group of Gly208, the NH group of Gly208 and the carbonyl group of Gly271 of the o-CAPN1 homology model are labelled A, B and C,
respectively.
c
The analogous hydrogen bonds from the carbonyl group of Gly198, the NH group of Gly198, and the carbonyl group of Gly261 of the o-CAPN2 homology model are also
labelled A, B and C, respectively.

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M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
nucleophilic sulfur of the cysteine as required for the compound to
act as a reversible covalent inhibitor.
of the o-CAPN1 and o-CAPN2 models. For example, the representa-
tive poses of 9 in the o-CAPN1 model show the inhibitor in a
b-strand conformation. The most representative pose is shown in
Figure 3c. The representative poses38 of 9 in o-CAPN2 show the
inhibitor is in a unique shunted arrangement and conformation.39
The most representative pose is shown in Figure 3d. The two poses
(Fig. 3c and d) have similar Glide Emodel40 scores (ꢀ53.66 and
ꢀ50.49, respectively) and Glide Einternal41 scores (1.15 and
3.69 kJ, respectively). This suggests that the shunted arrangement
of 9 (Fig. 3d) is of similar energy to the more common b-strand
binding mode (Fig. 3c). The most representative shunted arrange-
ment of 9 (Fig. 3d) has the carbonyl carbon of the C-terminal alde-
hyde in proximity to the active site cysteine (3.54 Å) with an
additional hydrogen bond between the aldehyde oxygen and
Gln99. A hydrogen bond is also formed between the pyrrole –NH
and Gly198 (Table 2, see Section 4).
For the non-selective inhibitor 11, the most representative
poses in the o-CAPN1 and o-CAPN2 models are almost identical.
This is consistent with its equal potency against o-CAPN1 and o-
CAPN2 (Table 1).
The similar potency of 5-formyl-pyrrole 9 and its N-methyl
derivative 11 against o-CAPN1 (Table 1) is consistent with a com-
mon mode of binding. However, the uniquely shunted arrange-
ment and conformation of 9 bound to o-CAPN2 (as discussed
above) contrasts the more common b-strand inhibitor conforma-
tion observed for 11. This may explain the enhanced potency of 9
against o-CAPN2.
Modelling studies show the inhibitors 4–8 and 10–13 are docked
in the o-CAPN1 and o-CAPN2 models in a similar conformation, ori-
entation and hydrogen-bonding arrangement (Table 2, see Section
4) to that observed in the crystal structures of leupeptin and
SNJ1715 each co-crystallised in r-CAPN1.35,36 The inhibitors 4–13
can be divided into three groups based on the observed selectivity
for o-CAPN2 over o-CAPN1: 4–6, 8, 10, 12 and 13 display moderate
isoform selectivity (2- to 7-fold); 7 and 9 are the most selective
(9.60- and 11.6-fold, respectively); while 11 is non-selective.
The inhibitor conformations, orientations and hydrogen-bond-
ing arrangements for each of 4–6, 8, 10, 12 and 13 are similar for
the two calpain models. This similarity could explain why the po-
tency of each inhibitor is comparable in o-CAPN1 and o-CAPN2 and
why the inhibitors only display moderate isoform selectivity (Table
1). For example, the representative poses37 of 8 in the o-CAPN1 and
o-CAPN2 models show that the inhibitor is in a b-strand conforma-
tion. The most representative poses of 8, with o-CAPN1 and with o-
CAPN2, are shown in Figure 3a and b, respectively. The carbonyl
carbon of the C-terminal aldehyde, in the representative poses
for 8 with o-CAPN1 and o-CAPN2, is positioned to allow for attack
by the nucleophilic sulfur of the active site cysteine. Here the dis-
tance between the carbonyl carbon and the active site cysteine sul-
fur is 3.97 Å and 3.44 Å, respectively.
Representative poses of the most selective inhibitors 7 and 9
(Table 1) appear to have different inhibitor conformations in each
Figure 3. Most representative poses of 8 docked to (a) o-CAPN1 homology model in a b-strand conformation and (b) o-CAPN2 homology model in a b-strand conformation
and 9 docked to (c) o-CAPN1 homology model in a b-strand conformation and (d) o-CAPN2 homology model in a unique shunted arrangement. The parameters for the
representative poses of 4–13 docked into the o-CAPN1 and o-CAPN2 homology models are shown in Table 2 in Section 4.

M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
4. Experimental
6917
Lens 1
Lens 2
4.1. Chemistry
Proton NMR spectra were acquired on a Varian Inova 500
spectrometer recorded at 500 MHz unless otherwise stated. Car-
bon NMR spectra were obtained on a Varian Unity XL 300 MHz
Fourier Transform spectrometer recorded at 75 MHz with a delay
(D1) of 1 s. Unless otherwise stated, all spectra were obtained at
23 °C. Chemical shifts are reported in parts per million (ppm, d)
and are referenced relative to residual solvent, for example,
CDCl3 (CHCl3 at dH 7.26 ppm). Electrospray ionisation (ESI) mass
spectra were detected on a micromass LCT TOF mass spectrome-
ter with a probe voltage of 3200 V and a source temperature of
80 °C. Infrared spectra were obtained using a Shimadzu 9201PC
series FTIR interfaced with an Intel 486 PC operating Shimadzu
HyperIR software. Diffuse reflectance spectra were obtained in
a solid KBr matrix. Melting points were obtained on an electro-
thermal melting point apparatus and are uncalibrated. Microanal-
ysis was performed at the University of Otago Microanalytical
Laboratory. All reported values are within 0.4% of the calculated
value. Thin layer chromatography (TLC) was performed on alu-
minium-backed Merck Kieselgel KG60F silica plates and flash col-
umn chromatography was carried out under a positive pressure
of dry nitrogen using Merck silica gel 60 (230–400 mesh). All
chemicals were purchased from Aldrich in the highest possible
purity.
Mean opacification grading
score ( sd)
45.1 2.9
27.9 5.5
Figure 4. Calcium-induced cataract in ovine lenses. The scores represent the ave-
rage result using three lens pairs. Opacification scores of 100 = full opacity, whereas
a score of 1 = clear and transparent. The loss of transparency was significantly re-
duced by o-CAPN2 selective inhibitor 9.
2.6. In vitro lens culture assay
Overactivation of calpain 2 has been linked to the develop-
ment of cortical cataract hence the o-CAPN2 selective inhibitor
9 was assayed in an in vitro lens culture system. The ability of
9 to retard calpain-induced cell damage in ovine lenses was
studied. A detailed method for this assay has been previously re-
ported.16 Inhibitor 9 (10
lM) was added to one lens from each
pair of sheep lenses in culture media. After 2 h incubation, cal-
cium was added to all lenses to activate the constituent calpains
and induce cataract formation. After 44 h all lenses were photo-
graphed and the opacity graded; see Figure 4 for a representa-
tive pair of lenses. Lenses treated with calcium only (e.g., lens
1 in Fig. 4) clearly showed the opacity associated with cataract
formation; however, lenses treated with calcium in the presence
of 9 (e.g., lens 2) remained essentially transparent, as revealed
by the reference grid placed behind each lens. The loss of trans-
parency was significantly reduced by 9 (p < 0.005 in a paired t-
test).
4.1.1. General procedure A: amide bond formation from an acid
chloride
To a stirred solution of the acid chloride (1.0 equiv) in dry N,N-
dimethylformamide were added the amine (1.0 equiv) and DIPEA
(2.0 equiv), and the resulting solution was stirred at rt overnight.
The reaction mixture was diluted with ethyl acetate and washed
successively with aqueous 1 M HCl, saturated aqueous sodium
bicarbonate and brine. The organic phase was separated, then
dried over MgSO4 and concentrated in vacuo.
3. Conclusion
4.1.1.1. Furan-2-carboxylic acid [(S)-1-((S)-1-hydroxymethyl-3-
methyl-butylcarbamoyl)-2-methyl-propyl]-amide (16). To
a
A range of N-terminal hetrocyclic dipeptide aldehydes 4–13,
designed to interact with the S3 binding pocket of calpain, have been
prepared and assayed in vitro against o-CAPN1 and o-CAPN2. Com-
pounds 4–13 are potent calpain inhibitors with IC50 values below
stirred solution of 14 was added 4 M HCl in dioxane (10.0 equiv),
and the resulting solution was stirred at rt overnight. Concentra-
tion in vacuo gave the hydrochloride salt 15, which was used
subsequently without further purification. Reaction of 15 with
2-furan-carbonyl chloride according to general procedure A gave
1 lM and show differing degrees of isoform selectivity. The 5-for-
myl-pyrrole 9 was highly active against o-CAPN2 (IC50 25 nM) and
exhibited >11-fold selectivity for o-CAPN2 over o-CAPN1. In con-
trast, other documented dipeptidyl aldehyde calpain inhibitors such
as SJA6017 and MDL28170 display selectivity for calpain 1 over cal-
pain 2.
The amino acid sequences of catalytic subunits of o-CAPN1 and o-
CAPN2 were deduced from their cDNA sequences and have 95%
homology to the analogous human isoforms. In silico homology
models were generated from the o-CAPN1 and o-CAPN2 sequences
and h-CAPN1 X-ray crystal structure data. The models have been
used to rationalise the observed SAR of compounds 4–13. The obser-
vation that the representative poses of 9 in o-CAPN2 show the inhib-
itor in a unique shunted arrangement and different from all other
inhibitor poses in both calpain models could explain the high iso-
form selectivity (>11-fold) of this inhibitor for o-CAPN2 over o-
CAPN1.
16 as
C
a
colourless oil (0.15 g, 48%). HRMS (ES+) calcd for
16H27N2O4 (M+H+) 311.1971 found 311.1973; 1H NMR
(500 MHz, CD3OD) 0.85 (3H, d, J = 6.4 Hz, CH3), 0.87 (3H, d,
J = 6.4 Hz, CH3), 0.98 (3H, d, J = 6.8 Hz, CH3), 1.00 (3H, d,
J = 6.8 Hz, CH3), 1.32–1.44 (2H, m, CH2CH(CH3)2), 1.58–68 (1H, m,
CH2CH(CH3)2), 2.10–2.20 (1H, m, CH(CH3)2), 3.44–3.55 (2H, m,
CH2OH), 3.96–4.06 (1H, m, CHCH2CH(CH3)2), 4.34–4.42 (1H, m,
CHCH(CH3)2), 6.54–6.58 (1H, m, CHCHCH), 7.16 (1H, d, J = 3.4 Hz,
CHCHCH), 7.65 (1H, m, CHCHCH), 7.95 (1H, d, J = 8.8 Hz, NH),
7.99 (1H, d, J = 8.3 Hz, NH); 13C NMR (75 MHz, CD3OD) 17.8
(CH3), 18.6 (CH3), 21.1 (CH3), 22.5 (CH3), 24.6 (CHCH2CH(CH3)2),
30.8 (CHCH(CH3)2), 39.7 (CHCH2CH(CH3)2), 49.5 (CHCH2CH(CH3)2),
59.0 (CH2OH), 64.3 (CHCH(CH3)2), 111.8 (CHCHCH), 114.4,
(CHCHCH), 145.1 (CHCHCH), 147.2 (CO), 159.0 (CONH), 172.0
(CONH); m/z (ES) 311 [M+(H)]+, 100%; 333 [M+(Na)]+, 20%.
The o-CAPN2 selective inhibitor 9 was assessed in an in vitro
lens culture assay and shown to successfully protect the lens from
calcium-induced opacification. Development of 9 (CAT0059) is
ongoing42 and its suitability for use as an in vivo anti-cataract
agent will be reported in a future publication.
4.1.1.2. Thiophene-2-carboxylic acid [(S)-1-((S)-1-hydroxyme-
thyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (17).
Reaction of 15 (as prepared above) with 2-thiophene-carbonyl
chloride according to general procedure A gave 17 as a colourless

6918
M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
crystalline solid (0.7 g, 56%). Mp 181–183 °C; HRMS (ES+) calcd for
16H27N2O3S (M+H+) 327.1742 found 327.1758; 1H NMR
4.1.2.3. 5-Formyl-thiophene-2-carboxylic acid
[(S)-1-((S)-1-
C
hydroxymethyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-
amide (20). Reaction of 15 (as prepared above) with (5-formyl)-
thiophene-2-carboxylic acid according to general procedure B gave
20 as a yellow solid (1.2 g, 88%). Mp 133–135 °C; HRMS (ES+) calcd
for C17H27N2O4S (M+H+) 355.1692 found 355.1695; 1H NMR
(500 MHz, CDCl3) 0.88 (6H, m, 2ꢁ CH3), 0.88 (3H, d, J = 6.3 Hz,
CH3), 1.03 (3H, d, J = 5.1 Hz, CH3), 1.04 (3H, d, J = 6.1 Hz, CH3),
1.32–1.46 (2H, m, CH2CH(CH3)2), 1.56–1.66 (1H, m, CH2CH(CH3)2),
2.16–2.26 (1H, m, CH(CH3)2), 2.94–3.14 (1H, m, OH), 3.60 (1H, dd,
J = 11.0 Hz, J = 5.7 Hz, CH2OH), 3.70 (1H, dd, J = 11.0 Hz, J = 3.7 Hz,
CH2OH), 4.02–4.11 (1H, m, CHCH2OH), 4.41 (1H, app t, J = 8.4 Hz,
CHCH(CH3)2), 6.4 (1H, d, J = 8.1 Hz, NH), 7.16 (1H, d, J = 8.4 Hz,
NH), 7.60 (1H, d, J = 4.0 Hz, CHCH), 7.73 (1H, d, J = 4.0 Hz, CHCH),
9.95 (1H, s, CHO); 13C NMR (75 MHz, CDCl3) 18.8 (CH3), 19.2
(CH3), 22.2 (CH3), 22.8 (CH3), 24.8 (CHCH2CH(CH3)2), 31.0
(CHCH(CH3)2), 39.7 (CHCH2CH(CH3)2), 50.5 (CH2OH), 59.9
(CHCH2CH(CH3)2), 65.8 (CHCH(CH3)2) 128.7 (CHCH), 128.7
(CHCCHO), 135.5 (CCONH), 146.4 (CS), 161.2 (CONH), 171.8
(CONH), 183.2 (CCHO); m/z (ES) 355 [M+(H)]+, 100%; 377
[M+(Na)]+, 50%. Anal. Calcd for C17H26N2O4S: C, 57.60; H, 7.39; N,
7.90. Found C, 57.34; H, 7.69; N, 8.10.
(500 MHz, CDCl3) 0.86 (6H, d, J = 7.0 Hz, 2ꢁ CH3), 1.03 (3H, d,
J = 7.0 Hz, CH3), 1.04 (3H, d, J = 7.0 Hz, CH3), 1.30–1.44 (2H, m,
CH2CH(CH3)2), 1.54–1.64 (1H, m, CH2CH(CH3)2), 2.21 (1H, sextet,
J = 7.0 Hz, CH(CH3)2), 2.80 (1H, br s, OH), 3.54–3.62 (1H, m, CH2OH),
3.66–3.74 (1H, m, CH2OH), 4.00–4.10 (1H, m, CHCH2CH(CH3)2),
4.45 (1H, app t, J = 8.0 Hz, CHCH(CH3)2), 6.68 (1H, d, J = 8.0 Hz, NH),
6.91 (1H, d, J = 8.0 Hz, NH), 7.08 (1H, t, J = 4.0 Hz, CHCHCH), 7.50
(1H, d, J = 5.0 Hz, SCHCHCH), 7.61 (1H, d, J = 3.0 Hz, CHCHCH); 13C
NMR (75 MHz, CDCl3) 18.8 (CH3), 19.2 (CH3), 22.2 (CH3), 22.7
(CH3), 24.7 (CHCH2CH(CH3)2), 31.1 (CHCH(CH3)2), 39.6
(CHCH2CH(CH3)2), 50.5 (CHCH2CH(CH3)2), 59.6 (CH2OH), 66.0
(CHCH(CH3)2), 127.5 (CHCHCH), 128.4, (CHCHCH), 130.4 (CHCHCH),
138.5 (CS), 162.1 (CONH), 172.3 (CONH); m/z (ES) 327 [M+(H)]+,
100%; 349 [M+(Na)]+, 90%. Anal. Calcd for C16H26N2O3S: C, 58.87;
H, 8.03; N, 8.58. Found C, 58.74; H, 8.05; N, 8.56.
4.1.2. General procedure B: amide bond formation
To a solution of N,N-dimethylformamide were added the amine
(1.2 equiv), the carboxylic acid (1.0 equiv), HATU (1.0 equiv)
and DIPEA (2.5 equiv). The solution was stirred at rt overnight then
diluted with ethyl acetate and washed with water and brine. The
organic phase was separated, dried over MgSO4 and concentrated
in vacuo.
4.1.2.4. 5-Formyl-pyrrole-2-carboxylic acid [(S)-1-((S)-1-hydr-
oxymethyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide
(25). Reaction of 15 (as prepared above) with (5-formyl)-1H-pyr-
role-2-carboxylic acid 23 according to general procedure B gave
25 as a yellow solid (0.5 g, 39%). Mp 102–104 °C; HRMS (ES+) calcd
4.1.2.1. 1H-Pyrrole-2-carboxylic acid [(S)-1-((S)-1-hydroxyme-
thyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (18).
Reaction of 15 (as prepared above) with 1H-pyrrole-2-carboxylic
acid according to general procedure B gave 18 as an orange solid
(0.26 g, 84%). Mp 110–112 °C; HRMS (ES+) calcd for C16H28N3O3
(M+H+) 310.2131 found 310.2134; 1H NMR (500 MHz, CD3OD)
0.86 (3H, d, J = 7.0 Hz, CH3), 0.89 (3H, d, J = 7.0 Hz, CH3), 0.97–
1.00 (6H, m, 2ꢁ CH3), 1.35–1.40 (2H, m, CH2CH(CH3)2), 1.55–1.59
(1H, m, CH2CH(CH3)2), 2.11–2.12 (1H, m, CH(CH3)2), 3.43–3.48
(2H, m, CH2OH), 3.98–4.02 (1H, m, CHCH2CH(CH3)2), 4.26 (1H,
dd, J = 9.0 Hz, J = 2.0 Hz, CHCH(CH3)2), 6.16–6.17 (1H, m, CHCHCH),
6.86–6.87 (1H, m, CHCHCH), 6.91–6.92 (1H, m, CHCHCH), 7.60 (1H,
d, J = 9.0 Hz, NH), 7.85 (1H, d, J = 9.0 Hz, NH); 13C NMR (75 MHz,
CD3OD) 19.4 (CH3), 20.2 (CH3), 22.6 (CH3), 24.0 (CH3), 26.1
(CHCH2CH(CH3)2), 31.9 (CHCH(CH3)2), 41.3 (CHCH2CH(CH3)2),
51.0 (CHCH2CH(CH3)2), 60.6 (CH2OH), 65.8 (CHCH(CH3)2), 110.6
(CHCHCH), 112.8 (CHCHCH), 123.5 (CHCHCH), 126.7 (CN), 163.6
(CONH), 174.2 (CONH); m/z (ES) 310 [M+(H)]+, 100%; 332
[M+(Na)]+, 30%. Anal. Calcd for C16H27N3O3: C, 62.11; H, 8.80; N,
13.58. Found C, 61.89; H, 8.44; N, 13.70.
for
C
17H27N3O4 (M+H+) 338.2080 found 338.2086; 1H NMR
(500 MHz, CDCl3) 0.80 (3H, d, J = 6.3 Hz, CH3), 0.82 (3H, d,
J = 6.3 Hz, CH3), 1.02 (3H, d, J = 6.8 Hz, CH3), 1.05 (3H, d,
J = 6.8 Hz, CH3), 1.35–1.37 (2H, m, CH2CH(CH3)2), 1.51–1.58 (1H,
m, CH2CH(CH3)2), 2.09–2.16 (1H, m, CH(CH3)2), 3.80–3.89 (2H, m,
CH2OH) 4.17–4.20 (1H, m, CHCH2CH(CH3)2), 5.01 (1H, app t,
J = 15.0 Hz, CHCH(CH3)2), 6.78–6.80 (1H, m, CHCH), 6.97–6.99
(1H, m, CHCCHO), 7.23 (1H, d, J = 16.0 Hz, NH), 8.17 (1H, d,
J = 16.0 Hz, NH), 9.53 (1H, s, CHO), 11.78 (1H, br s, NH); 13C NMR
(75 MHz, CDCl3) 18.9 (CH3), 19.1 (CH3), 22.4 (CH3), 22.8 (CH3),
24.9 (CHCH2CH(CH3)2), 32.2 (CHCH(CH3)2), 40.0 (CHCH2CH(CH3)2),
48.6 (CHCH2CH(CH3)2), 58.4 (CH2OH), 64.8 (CHCH(CH3)2), 110.7
(CHCH), 122.0, (CHCCHO), 132.3 (CCONH), 133.6 (CN), 159.5
(CONH), 171.0 (CONH), 181.0 (CCHO); m/z (ES) 338 [M+(H)]+,
30%; 360 [M+(Na)]+, 100%. Anal. Calcd for C17H27N3O4: C, 60.51;
H, 8.07; N, 12.45. Found C, 60.57; H, 8.01; N, 12.57.
4.1.2.5. 4-Formyl-1H-pyrrole-2-carboxylic acid
[(S)-1-((S)-1-
hydroxymethyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-
amide (26). Reaction of 15 (as prepared above) with
(4-formyl)-1H-pyrrole-2-carboxylic acid 24 according to general
procedure B gave 26 as a pale yellow glassy solid (0.1 g, 36%).
Mp 112–115 °C; HRMS (ES+) calcd for C17H27N3O4 (M+H+)
338.2080 found 338.2066; 1H NMR (500 MHz, CD3OD) 0.86 (3H,
d, J = 6.8 Hz, CH3), 0.89 (3H, d, J = 6.8 Hz, CH3), 0.98–1.02 (6H, m,
2ꢁ CH3), 1.00 (3H, d, J = 5.0 Hz, CH3), 1.32–1.44 (2H, m,
CH2CH(CH3)2),1.60–1.68 (1H, m, CH2CH(CH3)2), 2.08–2.13 (1H, m,
CH(CH3)2), 3.42–3.52 (2H, m, CH2OH), 3.96–4.02 (1H, m,
CHCH2CH(CH3)2), 4.22–4.28 (1H, m, CHCH(CH3)2), 6.84–6.88 (1H,
m, CHC(CHO)), 6.92–6.96 (1H, m, CHNH), 7.64–7.68 (1H, m, NH),
7.82–8.00 (1H, m, NH) 9.73 (1H, s, CHO); 13C NMR (75 MHz,
(CD3)2SO) 19.5 (CH3), 20.0 (CH3), 22.5 (CH3), 24.1 (CH3), 24.8
(CHCH2CH(CH3)2), 32.2 (CHCH(CH3)2), 41.1 (CHCH2CH(CH3)2),
49.4 (CHCH2CH(CH3)2), 59.3 (CH2OH), 64.6 (CHCH(CH3)2), 109.5
(CHCH), 127.1, (CHCCHO), 129.2 (CCONH), 130.9 (CN), 160.6
(CONH), 171.3 (CONH), 186.6 (CCHO); m/z (ES) 338 [M+(H)]+,
10%; 360 [M+(Na)]+, 100%. Anal. Calcd for C17H27N3O4: C, 60.51;
H, 8.07; N, 12.45. Found C, 60.12; H, 8.01; N, 12.44.
4.1.2.2. 5-Formyl-furan-2-carboxylic acid [(S)-1-((S)-1-hydroxy-
methyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (19).
Reaction of 15 (as prepared above) with (5-formyl)-furan-2-car-
boxylic acid according to general procedure B gave 19 as white so-
lid (180 mg, 30%). Mp 46–48 °C; HRMS (ES+) calcd for C17H26N2O5
(M+H+) 339.1906 found 339.1920; 1H NMR (500 MHz, CDCl3)
0.89 (6H, m, 2ꢁ CH3), 1.04 (6H, m, 2ꢁ CH3), 1.37–1.43 (2H, m,
CH2CH(CH3)2), 1.62 (1H, m, CH2CH(CH3)2), 2.17–2.26 (1H, m,
CH(CH3)2), 3.60 (1H, dd, J = 5.5 Hz, J = 11.0 Hz, CH2OH), 3.71 (1H,
dd, J = 3.4 Hz, J = 11.0 Hz, CH2OH), 4.05–4.10 (1H, m, CHCH2OH),
4.38–4.41 (1H, m, CHCH(CH3)2), 6.21 (1H, d, J = 8.0 Hz, NH), 7.28
(2H, m, CHCH and CHCH), 9.75 (1H, s, CHO); 13C NMR (75 MHz,
CDCl3); 18.5 (CH3), 19.1 (CH3), 22.2 (CH3), 22.7 (CH3), 24.7
(CHCH2CH(CH3)2), 31.4 (CHCH(CH3)2), 39.7 (CHCH2CH(CH3)2),
49.8 (CHCH2CH(CH3)2), 58.7 (CH2OH), 65.0 (CHCH(CH3)2), 115.9
(CHCH), 121.4 (CHCCHO), 150.6 (CCONH), 152.5 (CO), 157.3
(CONH), 170.9 (CONH), 178.5 (CCHO); m/z (ES) 339 [M+(H)]+,
100%. Anal. Calcd for C17H26N2O5: C, 60.34; H, 7.74; N, 8.28. Found
C, 60.58; H, 7.57; N, 8.09.

M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
6919
4.1.2.6. 5-Formyl-1-methyl-1H-pyrrole-2-carboxylic acid [(S)-1-
((S)-1-hydroxymethyl-3-methylbutylcarbamoyl)-2-methyl-pro-
pyl]-amide (30). Reaction of 15 (as prepared above) with
(5-formyl)-1-methyl-1H-pyrrole-2-carboxylic acid 27 according to
general procedure B gave 30 as an orange solid (0.32 g, 56%). Mp
178–180 °C; HRMS (ES+) calcd for C18H30N3O4 (M+H+) 352.2236
found 352.2245; 1H NMR (500 MHz, CD3OD) 0.88–0.93 (6H, m, 2ꢁ
CH3), 1.00–1.05 (6H, m, 2ꢁ CH3), 1.32–1.46 (2H, m, CH2CH(CH3)2),
1.58–1.66 (1H, m, CH2CH(CH3)2), 2.14–2.24 (1H, m, CH(CH3)2),
3.54–3.62 (1H, m, CH2OH), 3.67–3.74 (1H, m, CH2OH), 4.03–4.11
(1H, m, CHCH2CH(CH3)2), 4.21 (3H, s, NCH3), 4.32 (1H, dd,
J = 7.2 Hz, J = 8.4 Hz, CHCH(CH3)2), 6.09 (1H, d, J = 7.9 Hz, NH), 6.63
(1H, d, J = 4.4 Hz, CHCH), 6.76 (1H, d, J = 8.4 Hz, NH), 6.88 (1H, d,
J = 4.4 Hz, CHCH), 9.69 (1H, s, CHO); 13C NMR (75 MHz, CDCl3) 18.7
(CH3), 19.5 (CH3), 21.4 (CH3), 23.2 (CH3), 25.1 (CHCH2CH(CH3)2),
31.5 (CHCH(CH3)2), 34.8 (CHCH2CH(CH3)2), 40.2 (CH2OH) 50.5
(CHCH2CH(CH3)2), 59.3 (CHCH(CH3)2), 66.0 (NCH3), 112.2 (CHCH),
122.6 (CHCCHO), 133.6 (CCONH), 135.0 (CNCH3), 161.5 (CONH),
171.6 (CONH), 181.3 (CCHO); m/z (ES) 352 [M+(H)]+, 100%. Anal.
Calcd for C18H29N3O4: C, 60.52; H, 8.32; N, 11.96. Found C, 60.46;
H, 8.21; N, 11.69.
4.1.3. General procedure C: oxidation
A solution of the alcohol (1.0 equiv) in a 1:3 mixture of dimeth-
ylsulfoxide and dichloromethane was cooled over an ice bath, fol-
lowed by the addition of DIPEA (4.3 equiv). To this ice-cold
reaction mixture, a solution of SO3ꢂPyr complex (4.5 equiv) dis-
solved in dimethylsulfoxide was added. The reaction mixture was
maintained at a low temperature for a further 2 h (or until TLC
indicated complete consumption of the starting alcohol). The reac-
tion mixture was diluted with ethyl acetate and partitioned be-
tween ethyl acetate and 1 M HCl. The organic phase was washed
with saturated aqueous sodium bicarbonate and brine, then dried
over MgSO4 and concentrated in vacuo.
4.1.3.1. Furan-2-carboxylic acid [(S)-1-((S)-1-formyl-3-methyl-
butylcarbamoyl)-2-methyl-propyl]-amide (4). Oxidation of16
according to general procedure C gave 4 as a colourless oil
(0.10 g, 58%). HRMS (ES+) calcd for C16H25N2O4 (M+H+) 309.1820
found 309.1814; m
max (KBr) 1738 (CHO); 1H NMR (500 MHz, CDCl3)
0.80 (3H, d, J = 5.6 Hz, CH3), 0.83 (3H, d, J = 5.6 Hz, CH3), 1.00 (3H, d,
J = 6.4 Hz, CH3), 1.04 (3H, d, J = 6.4 Hz, CH3), 1.34–1.44 (1H, m,
CH2CH(CH3)2), 1.56–1.66 (2H, m, CH2CH(CH3)2), 2.15–2.24 (1H,
m, CH(CH3)2), 4.40–4.48 (1H, m, CHCH2CH(CH3)2), 4.71 (1H, dd,
J = 9.1 Hz, J = 7.1 Hz, CHCH(CH3)2), 6.46 (1H, m, CHCHCH), 7.07
(1H, d, J = 3.6 Hz, CHCHCH), 7.19 (1H, d, J = 9.1 Hz, NH), 7.43 (1H,
s, CHCHCH), 7.63 (1H, d, J = 7.4 Hz, NH), 9.56 (1H, s, CHO); 13C
NMR (75 MHz, CDCl3) 18.2 (CH3), 19.2 (CH3), 21.7 (CH3), 22.8
(CH3), 24.6 (CHCH2CH(CH3)2), 31.6 (CHCH(CH3)2), 37.1
(CHCH2CH(CH3)2), 57.2 (CHCH2CH(CH3)2), 57.7 (CHCH(CH3)2),
112.0 (CHCHCH), 114.7, (CHCHCH), 144.4 (CHCHCH), 147.2 (CO),
158.2 (CONH), 171.6 (CONH), 199.6 (CHO); m/z (ES) 310
[M+(H)]+, 100%; 332 [M+(Na)]+, 20%.
4.1.2.7. 5-Methyl-1H-pyrrole-2-carboxylic acid [(S)-1-((S)-1-
hydroxymethyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-
amide (31). Reaction of 15 (as prepared above) with (5-methyl)-
1H-pyrrole-2-carboxylic acid 28 according to general procedure
B gave 31 as a white solid (0.27 g, 29%). Mp 173–175 °C; HRMS
(ES+) calcd for C17H30N3O3 (M+H+) 324.2287 found 352.2286; 1H
NMR (500 MHz, CD3OD) 0.86 (3H, d, J = 6.5 Hz, CH3), 0.88 (3H, d,
J = 6.5 Hz CH3), 01.03 (3H, d, J = 6.5 Hz, CH3), 1.04 (3H, d,
J = 6.5 Hz CH3), 1.26–1.42 (2H, m, CH2CH(CH3)2), 1.50–1.62 (1H,
m, CH2CH(CH3)2), 2.12–2.22 (1H, m, CH(CH3)2), 2.28 (3H, s,
CCH3), 3.61 (1H, dd, J = 11.2 Hz, J = 4.8 Hz, CH2OH), 3.74 (1H,
dd, J = 11.2 Hz, J = 2.9 Hz, CH2OH), 4.04–4.12 (1H, m,
CHCH2CH(CH3)2), 4.50 (1H, app t, J = 8.4 Hz, CHCH(CH3)2), 5.92
(1H, br s, CHCH), 6.65 (1H, br s, CHCH), 6.71 (1H, br s, NH),
7.35 (1H, br s, NH), 9.92 (1H, s, CHO); 13C NMR (75 MHz, CD3OD)
16.8 (CH3), 18.0 (CH3), 20.2 (CH3), 22.4 (CH3), 23.6
(CHCH2CH(CH3)2), 30.0 (CHCH(CH3)2), 36.8 (CHCH2CH(CH3)2),
49.2 (CHCH2CH(CH3)2), 60.5 (CH2OH), 65.8 (CHCH(CH3)2), 105.3
(CH3), 126.9 (CHCH), 127.5 (CHCH), 135.9 (CCH3), 156.3 (CONH),
171.6 (CONH); m/z (ES) 324 [M+(H)]+, 100%; 346 [M+(Na)]+, 50%.
Anal. Calcd for C17H29N3O3: C, 63.13; H, 9.04; N, 12.99. Found C,
62.85; H, 9.16; N, 12.89.
4.1.3.2. Thiophene-2-carboxylic acid
[(S)-1-((S)-1-formyl-3-
methyl-butylcarbamoyl)-2-methyl-propyl]-amide (5). Oxidation
of 17 according to general procedure C gave 5 as a colourless crys-
talline solid (0.13 g, 51%). Mp 178–180 °C; HRMS (ES+) calcd for
C
16H25N2O3S (M+H+) 325.1586 found 325.1578; mmax (KBr) 1736
(CHO); 1H NMR (500 MHz, CD3OD) 0.87 (3H, d, J = 4.5 Hz, CH3),
0.89 (3H, d, J = 4.0 Hz, CH3), 1.02 (3H, d, J = 11.5 Hz, CH3), 1.06
(3H, d, J = 7.0 Hz, CH3), 1.43–1.50 (2H, m, CH2CH(CH3)2), 1.65–
1.72 (1H, m, CH2CH(CH3)2), 2.20–2.26 (1H, m, CH(CH3)2), 4.45–
4.49 (1H, m, CHCH(CH3)2), 4.62 (1H, dd, J = 7.5 Hz, J = 8.0 Hz,
CHCH2CH(CH3)2), 6.97 (1H, d, J = 9.0 Hz, CHCHCH), 7.07 (1H, dd,
J = 4.5 Hz, J = 3.5 Hz, CHCHCH), 7.12–7.16 (1H, m, CHCHCH), 7.50
(1H, d, J = 6.0 Hz, NH), 7.62 (1H, d, J = 5.0 Hz, NH), 9.57 (CHO); 13C
NMR (75 MHz, CDCl3) 18.5 (CH3), 19.3 (CH3), 21.8 (CH3), 22.9
(CH3), 24.7 (CHCH2CH(CH3)2), 31.4 (CHCH(CH3)2), 37.4
(CHCH2CH(CH3)2), 57.5 (CHCH2CH(CH3)2), 58.7 (CHCH(CH3)2),
127.8 (CHCHCH), 128.5, (CHCHCH), 130.5 (CHCHCH), 138.3 (CS),
162.0 (CONH), 171.8 (CONH), 200.20 (CHO); m/z (ES) 325
[M+(H)]+, 100%; 345 [M+(Na)]+, 20%. Anal. Calcd for C16H24N2O3S:
C, 59.23; H, 7.46; N, 8.63. Found C, 59.32; H, 7.59; N; 8.61.
4.1.2.8. 5-[(S)-1-((S)-1-Hydroxymethyl-3-methyl-butylcar-bamoyl)-
2-methyl-propylcarbamoyl]-1H-pyrrole-2-carboxylic acid methyl
ester (32). Reaction of 15 (as prepared above) with 1H-Pyrrole-
2,5-dicarboxylic acid monomethyl ester 29 according to general
procedure B gave 32 as a white solid (0.42 g, 64%). Mp 115–
117 °C; HRMS (ES+) calcd for C18H30N3O5 (M+H+) 368.2185 found
368.2185; 1H NMR (500 MHz, CDCl3) 0.85 (3H, d, J = 7.0 Hz, CH3),
0.89 (3H, d, J = 7.0 Hz, CH3), 0.95 (3H, d, J = 7.0 Hz, CH3), 1.00 (3H,
d, J = 7.0 Hz, CH3), 1.38–1.40 (1H, m, CH2CH(CH3)2), 1.41–1.58
(2H, m, CH2CH(CH3)2), 2.16–2.17 (1H, m, CH(CH3)2), 3.80–3.84
(2H, m, CH2OH), 3.87 (1H, s, OCH3), 4.17–4.19 (1H, m,
CHCH2CH(CH3)2), 4.94 (1H, app t, J = 9.0 Hz, CHCH(CH3)2), 6.73
(1H, s, CHCH), 6.86–6.88 (1H, m, CHCCOMe), 7.17 (1H, d,
J = 9.0 Hz, NH), 8.24 (1H, d, J = 8.5 Hz, NH); 13C NMR (75 MHz,
CDCl3) 18.6 (CH3), 19.5 (CH3), 22.9 (CH3), 23.0 (CH3), 25.2
(CHCH2CH(CH3)2), 31.8 (CHCH(CH3)2), 34.8 (CHCH2CH(CH3)2),
49.2 (CH2OH) 52.1 (CHCH2CH(CH3)2), 59.2 (CHCH(CH3)2), 65.4
(OCH3), 111.2 (CHCH), 116.3 (CHCCHO), 125.0 (CCONH), 130.4
(CCO2Me), 160.2 (CONH), 162.3 (CONH), 172.2 (CCO2Me); m/z
(ES) 368 [M+(H)]+, 100%. Anal. Calcd for C18H29N3O5: C, 58.84; H,
7.96; N, 11.44. Found C, 58.62; H, 7.98; N, 11.43.
4.1.3.3. 1H-Pyrrole-2-carboxylic acid
[(S)-1-((S)-1-formyl-3-
methyl-butylcarbamoyl)-2-methyl-propyl]-amide (6). Oxidation
of 18 according to general procedure C gave 6 as a colourless crys-
talline solid (0.20 g, 71%). Mp 165–167 °C; HRMS (ES+) calcd for
C
16H26N3O3 (M+H+) 308.1974 found 308.1961; mmax (KBr) 1741
(CHO); 1H NMR (500 MHz, CD3OD) 0.79 (3H, d, J = 10.5 Hz, CH3),
0.81 (3H, d, J = 11.5 Hz, CH3), 0.87 (3H, d, J = 12.0 Hz, CH3), 0.89
(3H, d, J = 12.0 Hz, CH3), 1.33–1.38 (2H, m, CH2CH(CH3)2), 1.53–
1.56 (1H, m, CH2CH(CH3)2), 2.10–2.15 (1H, m, CH(CH3)2), 4.02–
4.07 (1H, m, CHCH2CH(CH3)2), 4.30 (1H, m, CHCH(CH3)2), 6.03–
6.05 (1H, m, CHCHCH), 6.83–6.85 (1H, m, CHCHCH), 6.85–6.87
(1H, m, CHCHCH) 7.92 (1H, d, J = 9.0 Hz, NH), 8.40 (1H, d,
J = 9.0 Hz, NH), 9.36 (1H, s, CHO), 11.45 (1H, s, NH); 13C NMR

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M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
(75 MHz, (CD3)2SO) 18.7 (CH3), 18.8 (CH3), 19.4 (CH3), 21.4 (CH3),
23.2 (CHCH2CH(CH3)2), 30.3 (CHCH(CH3)2), 36.4 (CHCH2CH(CH3)2),
56.8 (CHCH2CH(CH3)2), 57.9 (CHCH(CH3)2), 108.7 (CHCHCH), 111.2,
(CHCHCH), 121.6 (CHCHCH), 125.9 (CN), 160.5 (CONH), 172.0
(CONH), 201.3 (CHO); m/z (ES) 308 [M+(H)]+, 100%. Anal. Calcd
for C16H25N3O3: C, 62.52; H, 8.20; N, 13.67. Found C, 62.66; H,
8.36; N, 13.59.
4.1.3.7. 4-Formyl-1H-pyrrole-2-carboxylic acid [(S)-1-((S)-1-
formyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (10).
Oxidation of 26 according to general procedure C gave 10 as a col-
ourless glassy solid (48 mg, 90%). Mp 101–103 °C; HRMS (ES+)
calcd for C17H25N3O4 (M+H+) 336.1923 found 336.1922; mmax
(KBr) 1736 (CHO); 1H NMR (500 MHz, (CD3)2SO) 0.82 (3H, d, J =
6.5 Hz, CH3), 0.86 (3H, d, J = 6.0 Hz, CH3), 0.90 (3H, d, J = 7.0 Hz,
CH3), 1.06 (3H, d, J = 7.5 Hz, CH3), 1.41–1.44 (1H, m, CH2CH(CH3)2),
1.50–1.56 (1H, m, CH2CH(CH3)2), 1.61–1.65 (1H, m, CH2CH(CH3)2),
2.05–2.11 (1H, m, CH(CH3)2), 4.10–4.14 (1H, m, CHCH2CH(CH3)2),
4.30 (1H, app t, J = 8.5 Hz, CHCH(CH3)2), 7.41 (1H, s, CHCH), 7.69
(1H, s, CHCCHO), 8.17 (1H, d, J = 9.0 Hz, NH), 8.42 (1H, d, J =
7.5 Hz, NH), 9.38 (1H, s, CHCHO), 9.72 (1H, s, CCHO), 12.30 (1H,
br s, NH); 13C NMR (75 MHz, (CD3)2SO) 18.7 (CH3), 19.4 (CH3),
21.6 (CH3), 23.8 (CH3), 24.1 (CHCH2CH(CH3)2), 30.0 (CHCH(CH3)2),
36.2 (CHCH2CH(CH3)2), 58.3 (CHCH2CH(CH3)2), 59.8 (CHCH(CH3)2),
119.0 (CHCH), 126.5, (CHCCHO), 128.3 (CCONH), 130.3 (CN), 160.0
(CONH), 171.7 (CONH), 185.9 (CCHO) 201.2 (CHCHO); m/z (ES) 336
[M+(H)]+, 100%. Anal. Calcd for C17H25N3O4: C, 60.88; H, 7.51; N,
12.53. Found C, 60.67; H, 7.46; N, 12.55.
4.1.3.4. 5-Formyl-furan-2-carboxylic acid [(S)-1-((S)-1-formyl-
3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (7). Oxidation
of 19 according to general procedure C gave 7 as a colourless oil
(100 mg, 80%). HRMS (ES+) calcd for C17H25N2O5 (M+H+)
337.1740 found 337.1763; mmax (KBr) 1734 (CHO); 1H NMR
(500 MHz, CDCl3); 0.87–0.94 (6H, m, 2ꢁ CH3), 1.03 (3H, d,
J = 6.7 Hz, CH3), 1.04 (3H, d, J = 6.7 Hz, CH3), 1.40–1.49 (1H, m,
CH2CH(CH3)2), 1.64–1.74 (1H, m, CH2CH(CH3)2), 2.18–2.28 (1H,
m, CH(CH3)2), 4.54–4.62 (2H, m, CHCH(CH3)2 and CHCHO), 6.85
(1H, d, J = 7.1 Hz, NH), 7.20–7.32 (2H, m, CHCH and CHCH), 7.36
(1H, d, J = 9.1 Hz, NH), 9.59 (1H, s, CHCHO), 9.73 (1H, s, CCHO);
13C NMR (75 MHz, CD3OD) 18.4 (CH3), 19.3 (CH3), 21.8 (CH3),
22.9 (CH3), 24.7 (CHCH2CH(CH3)2), 31.4 (CHCH(CH3)2), 37.3
(CHCH2CH(CH3)2), 57.4 (CHCH2CH(CH3)2), 58.4 (CHCH(CH3)2),
116.0 (CHCH), 121.1 (CHCCHO), 150.5 (CCONH), 152.6 (CO), 157.4
(CONH), 171.2 (CONH), 178.5 (CCHO), 199.6 (CHCHO); m/z (ES)
337 [M+H]+, 100%.
4.1.3.8. 5-Formyl-1-methyl-1H-pyrrole-2-carboxylic acid [(S)-
1-((S)-1-formyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-
amide (11). Oxidation of 30 according to general procedure C gave
11 as an orange solid (150 mg, 75%). Mp 134–136 °C; HRMS (ES+)
calcd for C18H28N3O4 (M+H+) 350.2080 found 350.2086; mmax
(KBr) 1741 (CHO); 1H NMR (500 MHz, CDCl3) 0.87 (3H, d,
J = 7.0 Hz, CH3), 0.92 (3H, d, J = 7.0 Hz, CH3), 0.96 (3H, d,
J = 7.0 Hz, CH3), 1.00 (3H, d, J = 7.0 Hz, CH3), 1.43–1.48 (1H, m,
CH2CH(CH3)2), 1.70–1.77 (2H, m, CH2CH(CH3)2), 2.14–2.24 (1H,
m, CH(CH3)2), 4.21 (3H, s, NCH3), 4.41–4.44 (1H, m,
CHCH2CH(CH3)2), 4.62 (1H, app t, J = 8.0 Hz, CHCH(CH3)2), 6.19
(1H, d, J = 8.0 Hz, NH), 6.61–6.66 (1H, m, CHCH), 6.88 (1H, d,
J = 4.0 Hz, NH), 7.25–7.27 (1H, m, CHCCHO), 9.69 (1H, s, CHO),
9.70 (1H, s, CHO); 13C NMR (75 MHz, CDCl3); 18.5 (CH3), 19.5
(CH3), 22.1 (CH3), 23.3 (CH3), 25.1 (CHCH2CH(CH3)2), 31.7
(CHCH(CH3)2), 38.1 (CHCH2CH(CH3)2), 57.8 (CHCH2CH(CH3)2),
58.7 (CHCH(CH3)2), 64.0 (NCH3) 112.1 (CHCH), 122.5, (CHCCHO),
128.6 (CCONH), 130.1 (CNCH3), 181.2 (CONH), 185.7 (CONH),
199.6 (CCHO), 199.0 (CHCHO); m/z (ES) 350 [M+(H)]+, 100%. Anal.
Calcd for C18H27N3O4: C, 61.87; H, 7.79; N, 12.03. Found C, 62.11;
H, 8.11; N, 11.98.
4.1.3.5. 5-Formyl-thiophene-2-carboxylic acid [(S)-1-((S)-1-for-
myl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (8).
Oxidation of 20 according to general procedure C gave 8 as a yel-
low solid (0.47 g, 89%). Mp 162–164 °C; HRMS (ES+) calcd for
C
17H25N2O4S (M+H+) 353.1535 found 353.1521; mmax (KBr) 1738
(CHO); 1H NMR (500 MHz, CDCl3) 0.88 (6H, m, 2ꢁ CH3), 1.03 (3H,
d, J = 6.6 Hz, CH3), 1.08 (3H, d, J = 6.6 Hz, CH3), 1.46–1.75 (2H, m,
CH2CH(CH3)2), 1.63–1.73 (1H, m, CH2CH(CH3)2), 2.16–2.26 (1H,
m, CH(CH3)2), 4.35–4.41 (1H, m, CHCHO), 4.66 (1H, app t,
J = 8.7 Hz, CHCH(CH3)2), 7.71 (1H, d, J = 4.0 Hz, CHCH), 7.78 (1H,
d, J = 4.0 Hz, CHCH), 7.82–7.90 (1H, m, NH), 8.19 (1H, d, J = 8.4 Hz,
NH), 9.57 (1H, s, CHCHO), 9.95 (1H, s, CCHO); 13C NMR (75 MHz,
CDCl3) 19.4 (CH3), 19.5 (CH3), 21.9 (CH3), 23.2 (CH3), 25.0
(CHCH2CH(CH3)2), 31.0 (CHCH(CH3)2), 37.3 (CHCH2CH(CH3)2),
60.1 (CHCH2CH(CH3)2), 129.2 (CHCH), 135.9 (CHCCHO), 146.8
(CCONH), 146.8 (CS), 161.6 (CONH), 172.6 (CONH), 183.6 (CCHO),
199.4 (CHO); m/z (ES) 353 [M+(H)]+ 100%. Anal. Calcd for
C17H24N2O4S: C, 57.93; H, 6.86; N, 7.95. Found C, 57.55; H, 6.80;
4.1.3.9. 5-Methyl-1H-pyrrole-2-carboxylic acid
[(S)-1-((S)-1-
N, 7.60.
formyl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (12).
Oxidation of 31 according to general procedure C gave 12 as a col-
ourless solid (90 mg, 63%). Mp 159–161 °C; HRMS (ES+) calcd for
4.1.3.6. 5-Formyl-pyrrole-2-carboxylic acid
[(S)-1-((S)-1-for
myl-3-methyl-butylcarbamoyl)-2-methyl-propyl]-amide (9).
Oxidation of 25 according to general procedure C gave9 as a yellow
solid (0.2 g, 71%). Mp 70–72 °C; HRMS (ES+) calcd for C17H25N3O4
(M+H+) 336.1923 found 336.1925; mmax (KBr) 1738 (CHO); 1H
NMR (500 MHz, CDCl3) 0.79 (3H, d, J = 7.0 Hz, CH3), 0.82 (3H, d,
J = 7.0 Hz, CH3), 1.01 (3H, d, J = 7.0 Hz, CH3), 1.06 (3H, d,
J = 7.0 Hz, CH3), 1.23–1.26 (1H, m, CH2CH(CH3)2), 1.63–1.653 (2H,
m, CH2CH(CH3)2), 2.01–2.03 (1H, m, CH(CH3)2), 4.02–4.04 (1H, m,
CHCH2CH(CH3)2), 4.80 (1H, app t, J = 8.0 Hz, CHCH(CH3)2), 6.84–
6.88 (1H, m, CHCH), 6.93–6.95 (1H, m, CHCCHO), 7.75 (1H, d,
J = 7.0 Hz, NH), 7.82 (1H, d, J = 7.0 Hz, NH), 9.58 (1H, s, CHCHO),
9.60 (1H, s, CCHO), 11.82 (1H, br s, NH); 13C NMR (75 MHz, CDCl3)
18.9 (CH3), 19.2 (CH3), 21.63 (CH3), 22.8 (CH3), 24.6
(CHCH2CH(CH3)2), 30.8 (CHCH(CH3)2), 37.2 (CHCH2CH(CH3)2),
57.5 (CHCH2CH(CH3)2), 59.1 (CHCH(CH3)2), 111.9 (CHCH), 121.1,
(CHCCHO), 132.2 (CCONH), 134.2 (CN), 160.1 (CONH), 172.5
(CONH), 180.6 (CCHO), 199.5 (CHCHO); m/z (ES) 336 [M+(H)]+,
100%; 358 [M+(Na)]+, 10%. Anal. Calcd for C17H25N3O4: C, 60.88;
H, 7.51; N, 12.53. Found C, 60.99; H, 7.28; N, 12.57.
C
17H29N3O3 (M+H+) 322.2131 found 322.2141; mmax (KBr) 1736
(CHO); 1H NMR (500 MHz, CDCl3) 0.85 (3H, d, J = 7.0 Hz, CH3),
0.90 (3H, d, J = 7.0 Hz, CH3), 0.99 (3H, d, J = 7.0 Hz, CH3), 1.01 (3H,
d, J = 7.0 Hz, CH3), 1.24–1.27 (1H, m, CH2CH(CH3)2), 1.63–1.69
(2H, m, CH2CH(CH3)2), 2.18–2.20 (1H, m, CH(CH3)2), 2.30 (3H, s,
CCH3) 3.66–3.68 (1H, m, CHCH2CH(CH3)2), 4.57–4.62 (1H, m,
CHCH(CH3)2), 5.94 (1H, br s, CHCH), 6.45 (1H, br s, NH), 7.25–
7.27 (1H, m, CHCCH3), 7.32 (1H, br s, NH), 9.59 (1H, s, CHO); 13C
NMR (75 MHz, CDCl3) 16.5 (CH3), 18.6 (CH3), 19.0 (CH3), 22.9
(CH3), 23.8 (CHCH2CH(CH3)2), 30.8 (CHCH(CH3)2), 36.3
(CHCH2CH(CH3)2), 60.6 (CHCH2CH(CH3)2), 66.8 (CHCH(CH3)2),
105.5 (CH3), 126.3 (CHCH), 128.0 (CHCH), 135.0 (CCH3), 158.0
(CONH), 169.3 (CONH); 199.6 (CHO); m/z (ES) 322 [M+(H)]+,
100%. Anal. Calcd for C17H27N3O3: C, 63.53; H, 8.47; N, 13.07. Found
C, 63.90; H, 8.22; N, 12.70.
4.1.3.10.
5-[(S)-1-((S)-1-Formyl-3-methyl-butylcarbamoyl)-2-
methyl-propylcarbamoyl]-1H-pyrrole-2-carboxylic acid methyl
ester (13). Oxidation of 32 according to general procedure C gave

M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
6921
13 as a colourless solid (0.33 g, 69%). Mp 93–95 °C; HRMS (ES+)
calcd for C18H28N3O5 (M+H+) 366.2029 found 366.2033; mmax
(KBr) 1736 (CHO); 1H NMR (500 MHz, CDCl3) 0.84 (3H, d,
J = 6.1 Hz, CH3), 0.86 (3H, d, J = 6.1 Hz, CH3), 0.99 (3H, d,
J = 6.6 Hz, CH3), 1.02 (3H, d, J = 6.6 Hz, CH3), 1.38–1.45 (1H, m,
CH2CH(CH3)2), 1.60–1.72 (2H, m, CH2CH(CH3)2), 2.12–2.22 (1H,
m, CH(CH3)2), 3.86 (1H, s, OCH3), 4.48–4.55 (1H, m,
CHCH2CH(CH3)2), 4.64 (1H, app t, J = 8.5 Hz, CHCH(CH3)2), 6.72
(1H, s, CHCH), 6.84–6.87 (1H, m, CHCCOMe), 7.15 (1H, d,
J = 7.0 Hz, NH), 7.60 (1H, br s, NH), 9.54 (1H, br s, CHO), 10.85
(1H, br s, NH); 13C NMR (75 MHz, CDCl3); 18.6 (CH3), 19.6 (CH3),
22.0 (CH3), 23.4 (CH3), 24.9 (CHCH2CH(CH3)2), 31.6 (CHCH(CH3)2),
38.0 (CHCH2CH(CH3)2), 57.7 (CHCH2CH(CH3)2), 58.5 (CHCH(CH3)2),
63.9 (OCH3), 111.8 (CHCH), 125.4, (CHCCHO), 129.9 (CCONH),
130.0 (CN), 161.5 (CONH), 171.7 (CONH),199.9 (CHCHO); m/z (ES)
366 [M+(H)]+, 100%. Anal. Calcd for C18H27N3O5: C, 59.16; H,
7.45; N, 11.50. Found C, 59.41; H, 7.87; N, 11.82.
Fidelity (Invitrogen, California, USA), 1
l
L gene specific cDNA and
water added up to volume. The samples were amplified using the
following parameters: initial denaturation at 94 °C for 10 min, fol-
lowed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for
3 min followed by a final extension at 72 °C for 10 min.
PCR amplicons were sequenced several times in the forward
and reverse directions using the previously mentioned primers
(along with other internal primers designed from the developing
ovine sequence) at the Waikato University DNA Sequencing Facil-
ity (University of Waikato, Waikato, New Zealand). The sequences
were compiled using the GeneDoc Multiple Sequence Alignment
Editor & Shading Utility (version, 2.6.002, Pittsburgh Supercomput-
ing Centre, Pittsburgh, PA, USA).
The consensus coding sequences for ovine calpain 1 (2151 bp)
and calpain 2 (2103 bp) were constructed from sequence data de-
rived from PCRs performed on several non-related animals and the
calpain
2 sequence submitted to GenBank (Accession No.
EU161096).
4.2. Sequencing of ovine calpains
4.3. Enzyme assays
RNA was isolated from ovine muscle tissue using an RNeasyÒ
Mini Kit (Qiagen GmbH, Hilden, Germany) as per the manufac-
turer’s instructions.
Calpain 1 cDNA was synthesised following the manufacturer’s
protocol using oligo(dt)12–18 primers and Superscript III Reverse
Transcriptase (Invitrogen, California, USA). The calpain I coding se-
quence was amplified in two fragments using gene specific primers
designed on the bovine sequence (GenBank Accession No.
NM174259) using DNAMANTM (version 4.0, Lynnon Biosoft, Quebec,
Canada). The 50 amplicon PCR was carried out in a total reaction
o-CAPN1 and o-CAPN2, partially purified from sheep lung by
hydrophobic and ion-exchange chromatography, were diluted in
a mixture containing 20 mM MOPS, 2 mM EGTA, 2 mM EDTA and
0.5% b-mercapto-ethanol (pH 7.5) to give a linear response over
the course of the assay. The substrate solution (0.0005% BODIPY-
FL casein in 10 mM CaCl2, 0.1 mM NaN3 and 0.1% b-mercapto-eth-
anol) was prepared fresh each day. Calpain inhibition assays were
performed in 96-well black WhatmanÓ plates at 25 °C. Calpain
control assays contained 50
lL of enzyme and 50
lL of sample buf-
volume of 20
USA), 4
l
L consisting of 1ꢁ PCR buffer (Invitrogen, California,
fer. The reaction was initiated by the addition of 100 l
L of sub-
l
L Q solution (Invitrogen, California, USA), 2.5 mM MgCl2,
strate solution. The progress of the reaction was followed for
10 min in a (BMG) Fluorostar with excitation at 485 nm and emis-
sion at 530 nm. For inhibitor assays the sample buffer was replaced
250 nm forward primer (50-ACCGTGAATTAGAGAGATCGTC-30),
250 nm reverse primer (50-ACAGGGTGGTGTTCCAGTTG-30),
125
lM dNTPs, 1 U Taq DNA Polymerase (Qiagen Gmbh, Germany),
with 50 lL of inhibitor dissolved in DMSO (2% total concentration)
1
l
L gene specific cDNA and water added up to volume. The ther-
and diluted with water. The percentage inhibition was determined
as 100 times the activity with inhibitor present divided by the
activity of the control assay. The reported IC50 values are the aver-
age of triplicate determinations.
mo-cycling profile consisted of initial denaturation at 94 °C for
10 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and
72 °C for 1 min and one final extension step of 72 °C for 10 min.
The 30 amplicon PCR was carried out in a total reaction volume
of 20
4 lL Q
l
L consisting of 1ꢁ PCR buffer (Invitrogen, California, USA),
solution (Invitrogen, California, USA), 2.5 mM MgCl2,
250 nm forward primer (50-TCCGAGACTTCATGCGTG-30), 250 nm
reverse primer (50-AGGCACTTGCAGCTGGTG-30), 125
M dNTPs,
1 U Taq DNA Polymerase (Qiagen Gmbh, Germany), 1 L gene spe-
4.4. Lens culture
The EMEM culture medium for normal culture (EMEM, pH 7.4)
was prepared from minimum essential medium powder (or MEM,
purchased from Sigma, product number M0643, 2003), 26 mM
NaHCO3, 0.02 mg/mL gentamycin (antibiotic, from Sigma) and
l
l
cific cDNA and water added up to volume. The thermo-cycling pro-
file consisted of: initial denaturation at 94 °C for 10 min, followed
by 35 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min and
one final extension step of 72 °C for 10 min.
2.5
culture medium was immediately sterilised by filtering with
0.2
m pore size filters into autoclaved bottles. 2 M Ca2+ solution
lg/mL amphotericin B (anti-fungal, from Sigma) in dH2O. The
l
The 50 and 30 amplicons were sequenced several times from sev-
eral animals in the forward and reverse directions using the above-
mentioned primers at the Allan Wilson Centre (Massey University,
Palmerston North, New Zealand).
in sterilised dH2O was added to 10 mL culture medium to give a fi-
nal concentration of 5 mM Ca2+. Calpain inhibitors were solubilised
in DMSO to give a final concentration of DMSO in EMEM of less
than 0.05%. Adequate amounts of DMSO were added to the med-
ium containing 5 mM Ca2+ so there was no effect attributed by
DMSO. Groups of ovine eye globes from 9 to 12 month old lambs
were collected from a local slaughterhouse immediately after
slaughter and delivered to the laboratory for lens dissection. Pairs
of lenses from the eyes of each animal were kept together. Three
pairs of lenses were dissected within 2 h of death of the animal
using the posterior approach. The intact lenses were immediately
placed in a sterile culture dish containing 10 mL of culture medium
per lens. The entire lens was submerged with its anterior epithe-
lium facing upward in the medium, which had been pre-incubated
at 37 °C with 5% CO2 for 48 h in a sterilised chamber. One of each of
Calpain 2 cDNA was synthesised as per the manufacturer’s pro-
tocol using Superscript III Reverse Transcriptase (Invitrogen, Cali-
fornia, USA) and
a calpain 2
gene specific 30 primer (50-
AAAAGTTTCTCCGTGGAGGCT-30). Calpain 2 was then amplified by
PCR using primers flanking the coding region designed from the
rat sequence (GenBank Accession No. NM017116) using DNA-
MANTM (version 4.0, Lynnon Biosoft, Quebec, Canada). PCRs were
carried out in a total reaction volume of 25
l
L consisting of 1ꢁ
PCRꢁ Enhancer Buffer (Invitrogen, California, USA), 200
l
M dNTPs,
1.5 mM MgCl2, 200 nM forward primer (50-GACGACACCATGGC
GGGCATCGCGGCC-30), 200 nM reverse primer (50-ATTAACAAGCTT
TCAAAGTACTGAAAAACAC-30), 5
l
L PCRꢁ Enhancer Solution (Invit-
the paired lenses was pre-incubated with 9 (10
2.5 h, whilst the other was cultured in EMEM only. Both lenses
lM) in EMEM for
rogen, California, USA), 1 U PlatinumÒ Taq DNA Polymerase High

6922
M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
were then exposed to a final concentration of 5 mM Ca2+ in EMEM,
by the addition of 16
L of 2 M Ca2+ stock solution in dH2O. All
parameters of the most representative poses of compounds 4–13
docked to the o-CAPN1 and o-CAPN2 homology models are shown
in Table 2.
l
lenses were incubated at 37 °C under 5% CO2 for 44 h. All the lenses
were then photographed using a digital camera (Sony Cybershot
DSCF505V) fitted to a stand. A transparent flat bottom culture dish
containing a lens and medium was placed on black grid lines
(1 ꢁ 1 mm) with a white background light. Images of the anterior
epithelium of the lens were taken with an image resolution of
1856 ꢁ 1392 pixels as a RGB true colour JPEG image. The lens opac-
ity grade was scored by an image analysis system that was pro-
grammed to grade the extent of opacification captured by a
digital photo image. The software used to grade the opacification
captured was Image Pro-Plus v 4.1. A macro-script was created
to automatically analyse the digital images of lenses (pixel size
1856 ꢁ 1392) placed on a 1 mm ꢁ 1 mm black grid. The grading
system was developed on the basis of selecting predefined pixel
RGB values, after area defining and sharpening of the image. Lenses
were graded on a scale of 1 to 100, with fully opaque lens scoring
100 and transparent lens 1.
Acknowledgments
We thank Matthew Muir for the supply of calpain and Dr. Quen-
tin MacDonald (QBit NZ) for helpful discussions. The research was
supported by a University of Canterbury Fellowship (A.T.N., S.G.A.),
grants from the New Zealand Public Good Science and Technology
Fund LINX205 (including support for M.A.J., J.M.M.), a Tertiary Edu-
cation Commission enterprise scholarship (HYYL), the Foundation
for Research Science and Technology (SBM) and Douglas Pharma-
ceuticals Limited.
References and notes
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All molecular modelling experiments were conducted with the
Schrödinger suite, 2005. Conformational searches on 4–13 were
carried out with MacroModel 9.1,33 generating an ensemble of
low energy conformers to establish suitable starting conformations
of each compound for the docking studies. The searches were con-
ducted with the MCMM method using a GB/SA water model and
the OPLS2001 force field, with 3000 steps for the conformational
search and up to 5000 iterations for the minimisation of each gen-
erated structure. The minimisation was stopped with the default
gradient convergence threshold of d = 0.05 kJ/(mol Å). The default
Polak-Ribiere Conjugate gradient method was used for all minimi-
sations. The crystal structure of human mini calpain 1 (PDB code
1ZCM)29 was prepared for use in the modelling studies using the
protein preparation facility in GLIDE 4.034 followed by deprotona-
tion of Cys115 and protonation of His272. The in silico ovine homol-
ogy models were created by the virtual mutation of the
appropriate residues around the active site cleft. These structures
were minimised using the OPLS2005 force field with a GB/SA water
model over 500 iterations. All residues within a 5 Å distance to the
calcium ions, the calcium ions and the key residues Gly208 Gly271
and Cys115 (o-CAPN1) or Gly198, Gly261 and Cys105 (o-CAPN2) of
the structures were kept frozen during this minimisation. The cen-
tre of the docking grid was defined as the centroid of the residues
Cys115, Gly208, and Gly271 (o-CAPN1) or Gly198, Gly261 and Cys105 (o-
CAPN2) and was generated with GLIDE 4.0 using default settings.
The centre of the docked ligands was defined within a 12 Å box.
The docking of flexible ligands to the rigid calpain model with
GLIDE 4.0 was performed with the following parameters:
OPLS2001 force field, extra precision mode, 90,000 poses per ligand
for the initial docking, keep best 1000 poses per ligand for energy
minimisation, energy minimisation with a distance dependent
dielectric constant of 2 and a maximum of 5000 conjugate gradient
steps.
24. Sorimachi, H.; Amano, S.; Ishiura, S.; Suzuki, K. Biochim. Biophys. Acta 1996,
1309, 37–41.
For the docking studies of 4–13 a conformational search was
conducted for each compound and X-cluster used to choose repre-
sentative conformers from the ensemble within a 12 kJ/mole of the
global minimum. These, usually three to five, were used as starting
conformers in the docking studies. From each docking study up to
ten poses as defined by the program were collected. Representative
poses were chosen such that (i) the distance between the carbonyl
carbon of the aldehyde group and the active site cysteine sulfur is
less than 4.5 Å; (ii) trans Val amide bonds were preferred36 to cis
(iii) appropriate hydrogen bonds in the active site are present
and (iv) poses have a low energy Glide Emodel score. The associated
25. Deluca, C. I.; Davies, P. L.; Samis, J. A.; Elce, J. S. Biochim. Biophys. Acta 1993,
1216, 81–93.
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178.
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Commun. 1988, 157.
29. Li, Qingshan. Q.; Hanzlik, R. P.; Weaver, R. F.; Schönbrunn, E. Biochemistry 2006,
45, 701–708.
30. PBD file numbers: 1MDW, 1TL9, 1TLO, 1KXR, 2G8J, 2G8E, 2NQG, 2NQI, 2ARY,
1ZCM.
31. Moldoveanu, T.; Hosfield, C. M.; Lim, D.; Jia, Z.; Davies, P. L. Nat. Struct. Biol.
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M. A. Jones et al. / Bioorg. Med. Chem. 16 (2008) 6911–6923
6923
32. Davis, T. L.; Walker, J. R.; Finerty, P. J.; Mackenzie, F.; Newman, E. M.; Dhe-
Paganon, S. J. Mol. Biol. 2007, 366, 216–229.
33. MacroModel, version 9.1, Schrödinger, LLC, New York, NY, 2005.
34. Glide, version 4.0, Schrödinger, LLC, New York, NY, 2005.
38. Of the 32 poses generated for inhibitor 9 docked in o-CAPN2 model only six
gave the carbonyl carbon of the aldehyde in proximity to the active site
cysteine (<4.5 Å). Of these six poses five including the lowest energy poses
were in the shunted arrangement with only a single pose with the inhibitor in
a more common binding mode as defined by hydrogen bonds A, B and C.
39. The 5-formyl-pyrrole 9 possesses two aldehyde groups. Only two of the 72
poses collected in the docking studies with o-CANP1 and o-CANP2 show the
pyrrole aldehyde in proximity to the active site cysteine sulfur (<4.5 Å). Thirty-
three poses have the Leu aldehyde in proximity to the active site cysteine
sulfur. Furthermore, the Leu aldehyde is the more reactive to nucleophilic
attack since the nitrogen of the pyrrole aldehyde reduces the electrophilic
character of the carbonyl. The dialdehyde 9 reacts with hydroxylamine only at
the Leu aldehyde. This supports the Leu aldehyde as being responsible for the
potency of 9.
35. Calpain inhibitors typically adopt
a b-strand conformation that spans ca.
15 Å of the active site cleft of calpain. An X-ray structure of leupeptin
co-crystallised with r-CAPN1 construct (PDB code 1TL9) shows two key
hydrogen bonds between the NH and carbonyl groups of Leu (P2) and
Gly208 and also an additional hydrogen bond between the NH of the
P1-P2 amide bond and Gly271. Recent X-ray crystal structures, for
example, SNJ1715 co-crystallised with r-CAPN1 construct (PDB code
2G8E), confirm the importance of these hydrogen bonds. These hydrogen
bonds equate to the hydrogen bonds labelled A, B and C as reported in
Table 2.
36. The Leu amide in all of the docked compounds 4–13 is trans in all poses. We did
not limit the programme to collecting only poses where the amide(s) is trans
and for some poses the Val amine is in a cis configuration.
40. Glide Emodel combines the energy grid score, the binding affinity predicted by
the GlideScore and the internal strain energy of the ligand for the model
potential used to direct the conformational-search algorithm.
37. Of the 84 poses generated for inhibitor 8 docked in the o-CAPN1 and o-
CAPN2 models, 72 gave the carbonyl carbon of the aldehyde in proximity to
the active site cysteine (<4.5 Å). Of these 72 poses 42 exhibited a b-strand
arrangement of the inhibitor and 20 of these had the Val amide in a trans
configuration.
41. Glide Einternal score is the internal strain energy of the ligand.
42. Abell, A. D.; Coxon, J. M.; Miyamoto, S.; Jones, M. A.; Neffe, A. T.; Aitken, S. G.;
Stuart, B. G.; Nikkel, J. M.; Morton, J. D.; Bickerstaffe, R.; Robertson, L. J.;
Lee, H. Y.; Muir. M. S. N.Z. Patent. 2007, 547303; U.S. Patent Appl., 2007,
20070293560.