The preparation of macrocyclic calpain inhibitors by ring closing metathesis and cross metathesis

Article abstract of DOI:10.1071/CH14121

Ring closing metathesis and cross metathesis approaches to a new macrocyclic peptidomimetic aldehyde 2 have been developed, with the former route being the most convenient. Aldehyde 2 is a potent inhibitor of calpain II (IC50 of 45nM) with comparable activity to the benchmark acyclic inhibitor SJA6017 4. Both compounds contain an N-terminal 4-fluorophenylsulfonyl group. The P2 Ile analogue of 2 (16) is significantly less active (IC50 of 2000nM) which reflects an unusually subtle importance of the P2 residue for active site binding.

Full text of DOI:10.1071/CH14121

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2014, 67, 1257–1263
Full Paper
http://dx.doi.org/10.1071/CH14121
The Preparation of Macrocyclic Calpain Inhibitors
by Ring Closing Metathesis and Cross Metathesis*
A
A
B
Seth A. Jones, Joanna Duncan, Steven G. Aitken,
B
A C
,
James M. Coxon, and Andrew D. Abell
A
School of Chemistry and Physics, The University of Adelaide,
North Terrace, Adelaide, SA 5005, Australia.
B
Department of Chemistry, University of Canterbury, Private Bag 4800,
Christchurch 8140, New Zealand.
C
Corresponding author. Email: andrew.abell@adelaide.edu.au
Ring closing metathesis and cross metathesis approaches to a new macrocyclic peptidomimetic aldehyde 2 have been
developed, with the former route being the most convenient. Aldehyde 2 is a potent inhibitor of calpain II (IC50 of 45 nM)
with comparable activity to the benchmark acyclic inhibitor SJA6017 4. Both compounds contain an N-terminal
4-fluorophenylsulfonyl group. The P2 Ile analogue of 2 (16) is significantly less active (IC50 of 2000 nM) which reflects an
unusually subtle importance of the P2 residue for active site binding.
Manuscript received: 7 March 2014.
Manuscript accepted: 16 April 2014.
Published online: 15 May 2014.
Results and Discussion
Introduction
The key precursor 6 to the macrocycle 3 was prepared by
reaction of the tyrosine derivative 5 with 4-fluorophenysulfonyl
chloride, followed by methyl ester hydrolysis, and (1-[bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate) (HATU) mediated coupling of
the resulting acid with leucine tert-butyl ester. The first route to
the macrocycle involved hydrolysis of the C-terminal tert-butyl
ester of 6, on treatment with triflouroacetic acid (TFA), followed
by coupling with allyl-glycine methyl ester, in the presence of
HATU, to give the diene 7 in 84 % over both steps. RCM of the
diene 7 under thermal conditions with one portion of Grubbs’
Second Generation Catalyst (3 mol-%) gave an inseparable
mixture (25 : 2) of macrocyclic alkenes 8. Catalytic hydro-
genation of the mixture gave the desired macrocyclic methyl
ester 3 in 89 %.
With few exceptions, inhibitors and substrates of proteases bind
to the active site of the enzyme in a b-strand conformation.[1]
On this basis we[2,3] and others,[4] have reported the synthesis
of macrocyclic templates that typically link the P1 and P3
residues in order to constrain the peptide backbone in this
geometry. The component macrocycle effectively reduces the
entropy loss associated with ligand–receptor binding, while also
enhancing biostability. These templates can be elaborated into
inhibitors of several proteases, e.g. CAT0811 1 (Fig. 1) is a
potent inhibitor of calpain II (half maximal inhibitory concen-
tration (IC50) of 30 nM)[3] that shows promise in slowing the
progression of calpain-induced cataracts.[5]
Structure–activity relationship (SAR) studies on these
macrocyclic protease inhibitors reveal that the nature of the
N-terminal group is important for potency, presumably by
forming key hydrogen bonds with the active site according
to docking studies.[6] As part of an ongoing project to improve
the versatility of such macrocyclic inhibitors, we now
report the preparation and assay of an analogue of CAT811 1
that contains an N-terminal 4-fluorophenylsulfonyl group
(see structure 2). Such a sulfonyl group is known to provide
potent acyclic calpain inhibitors, e.g. SJA6067 4.[7] We
present a comparative study on the preparation of 2 from a
common precursor 6 using ring closing metathesis (RCM)[3,8]
and a new sequence involving cross metathesis (CM) followed
by lactamisation, see Scheme 1. The assay of this new macro-
cyclic aldehyde and a P2 Ile analogue (see 16), against calpain
II, is also reported.
The key step in the second route to 3 involved CM of two
mole equivalents of the dipeptide alkene 6 with the allyl glycine
dimer 9a,[9] in the presence of Grubbs Second Generation
Catalyst, to give 11 as a mixture of isomers that could not be
1
separated (92 : 8 by H NMR spectroscopy) in modest yield
(33 %). This mixture was hydrogenated over 10 % Pd on C to
give 12. Treatment with TFA removes both the tert-butyl ester
and N-Boc protecting groups. Lactamisation with HATU then
gave the desired lactam 3 in 42 % yield over two steps.
Interestingly the use of N-Boc allylglycine methyl ester 9b,
instead of the dimer 9a, in a CM reaction with 6 resulted in
complex mixtures, while reaction of the corresponding dimer 10
and 9b gave 11 in a somewhat improved yield of 51 %.
*Dedicated to the late Professor Roger F. C. Brown.
Journal compilation Ó CSIRO 2014
www.publish.csiro.au/journals/ajc

1258
S. A. Jones et al.
with that of CAT811 1 and the benchmark calpain inhibitor
SJA6017 4. This acyclic inhibitor contains the same N-sulfonyl
group and exhibits an IC50 of 40 nM in our assay system.[14]
Thus an N-terminal 4-fluorophenylsulfonyl group appears to
be favoured for both acyclic and macrocyclic calpain inhibi-
tors, where the nature of the N-terminal group is empirically
known[13] to significantly influence potency.
O
O
H
N
H
N
O
N
H
H
O
O
O
1 CAT0811
Conclusion
IC50 Calpain II ϭ 30 nM
The synthesis of a new macrocyclic peptidomimetic aldehyde
2, containing an N-terminal 4-fluorophenylsulfonyl group, is
described. The macrocycle is prepared from a common pre-
cursor dipeptide 6 in either of two ways. The preferred route
involves elaboration of 6 to diene 7, which then undergoes RCM
to give the desired macrocycle. Alternatively, the components of
the macrocycle are introduced first by CM of 6 with an allyl
glycine derivative. Simultaneous C- and N-terminal deprotec-
tion then allows formation of the macrocycle by lactamisation.
The macrocycle 2 was shown to be a potent inhibitor of calpain
II, with comparable activity to that of SJA6067 4. As noted
above, the nature of the P2 amino acid appears to be critical for
activity, with the P2 Ile analogue 16 being significantly less
active. The macrocycle 2, with a comparable IC50 and greater
ease of synthesis, is an attractive alternative to the benchmark
macrocycle CAT0811 1.
F
O
O
H
N
H
N
N
R
S
H
O
O
O
O
2 R ϭ H
3 R ϭ OMe
F
O
H
N
H
N
H
S
O
O
O
Experimental
4 SJA6017
General Remarks
IC50 Calpain II ϭ 40 nM
Thin-layer chromatography (TLC) was performed with Merck
aluminium sheets coated with silica gel 60 F254 or plastic backed
Merck Kieselgel KG60F254 silica plates. Flash column chro-
matography was performed using Merck or Scharlau silica gel
60 (230 – 400 mesh). Proton NMR spectra were acquired on a
Varian Inova 500 spectrometer (operating at 500 MHz), Varian
Inova 600 spectrometer (operating at 600 MHz), or a Varian
Gemini 2000 spectrometer (operating at 300 MHz). Carbon
NMR spectra were acquired on a Varian Gemini 2000 spec-
trometer (operating at 75 MHz) or on a Varian Inova 600
spectrometer (operating at 150 MHz) both with a delay (D1) of
1 s. All spectra were obtained at 238C and chemical shifts are
reported in parts per million (ppm) and are referenced relative to
residual solvent (e.g. CHCl3 at dH 7.26 for CDCl3, DMSO at dH
2.50 for DMSO-d6, dH 3.31 for CD3OD). Electrospray ionisa-
tion (ESI) high resolution mass spectra (HRMS) were recorded
at the University of Canterbury, New Zealand on a Micromass
LCT spectrometer using a probe voltage of 3200 V, an operating
temperature of 1508C, and a source temperature of 808C. Direct
ionisation used 10 mL of a 10 mg mLꢀ1 solution, using a carrier
Fig. 1. Calpain inhibitors CAT0811 1 and SJA6017 4 and target macro-
cycles (half maximal inhibitory concentration ¼ IC50).
However, this last reaction suffers from the disadvantage that it
requires preparation of 10 by CM dimerisation of
(see Scheme 1) and as such it was not pursued further.
6
Our previously reported b-strand template 13[10] also pro-
vides the macrocyclic ester 3 on reaction with 4-fluoropheny-
sulfonyl chloride, albeit in low yield (7 %) (see Scheme 2). The
Ile P2 analogue of 3 (see 15) was similarly prepared from 14, but
also in a low yield of 10 %. In contrast, the equivalent reaction of
13 with benzyl chloroformate allows for the efficient introduc-
tion of the N-Cbz group of CAT811 1.[3] While this route does
provide valuable N-protected macrocycles such as CAT811 1,
its usefulness is limited by low yields in N-4-fluoropheny-
sulfonyl protection and by the multistep synthesis of the key
starting materials, 13 and 14.[10]
solvent of 50 % acetonitrile/water at a flow rate of 20 mL minꢀ1
.
The esters of 3 and 15 were separately reduced with LiBH4 in
THF and the resulting primary alcohols oxidised with Dess–
Martin periodinane and a sulfur trioxide pyridine complex,
respectively, to give the desired aldehydes 2 and 16 (see
Schemes 1 and 2). The aldehydes 2 and 16 were then assayed
against calpain II using an in vitro fluorescence-based assay[12]
and resulted in IC50 values of 45 and 2000 nM, respectively.
The observed difference in activity of 2 and 16 is consider-
able and reflects the importance of the P2 amino acid in defin-
ing potency against calpain II.[10] While Leu and Ile are both
hydrophobic amino acids, the subtle difference between the two
appears to have a significant effect on potency. This highlights
the importance of the P2 residue for binding to the active site of
calpain.[13] Importantly, the activity of 2, the most potent
macrocycle in the study reported here, compares favourably
Ionisation was assisted by the addition of 0.5 % formic acid. ESI
mass spectra were determined using a Finnigan LCQ Ion Trap
mass spectrometer at the University of Adelaide. Electrospray
conditions were as follows: needle potential, 4500 V; tube lens,
60 V; heated capillary, 2008C, 30 V; sheath gas flow, 30 psi.
Microwave irradiation refers to heating in a microwave at
1200 W, under an inert atmosphere, and with a QuickFit reflux
condenser attached.
4-Fluoro-N-((7S,10S,13S)-7-formyl-10-isobutyl-9,
12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]-noadeca-
1(18),15(19),16-trien-13-yl)-benzenesulfonamide (2)
To a solution of 3 (50 mg, 0.09 mmol) in dry THF (1 mL) was
added LiBH4 (0.31 mL, 1 M in THF) dropwise. The reaction

Macrocyclic Calpain Inhibitors
1259
O
F
O
ϪClϩH3N
H
N
OMe
OtBu
S
N
H
O2
O
O
5
O
10
2
a
g
f
F
F
O
O
H
N
H
N
OtBu
OtBu
S
N
H
S
N
H
e
O2
O2
O
Boc
O
O
NH
O
O
6
11
OMe
h
b
F
F
O
O
O
H
H
H
N
N
OtBu
N
S
N
H
N
H
OMe
S
O2
O2
O
O
Boc
O
O
NH
O
7
12
OMe
c
i
F
F
O
O
R
O
O
H
N
H
N
H
H
N
N
S
S
N
H
N
H
OMe
O2
O2
O
O
O
O
d
3 R ϭ OMe
2 R ϭ H
8
j
O
BocNH
BocNH
OMe
9a
OMe
BocNH
OMe
O
9b
O
Scheme 1. Reagents and conditions: a: 4-FPS-Cl, DIPEA, DMF; followed by NaOH, MeOH/H2O/THF; and then
Leu tert-Bu ester, EDCI, DIPEA; b: TFA, DCM; followed by allyl-Gly methyl ester, HATU, DIPEA, DMP;
c: Grubbs Second Generation Catalyst, DCM; d: H2, Pd on C, EtOAc; e: 9a, Grubbs Second Generation Catalyst,
DCM, reflux; f: Grubbs Second Generation Catalyst, BCl2(Cy)2, DCM, microwave; g: 9b, Grubbs Second
Generation Catalyst, DCM, reflux; h: H2, Pd on C, DCM/MeOH; i: TFA, DCM; followed by HATU, HOAt,
DIPEA; j: LiBH4, THF; followed by Dess–Martin periodinane, DCM. 4-FPS-Cl ¼ 4-fluorobenzene sulfonyl
chloride; DIPEA ¼ N,N-diisopropylethylamine; EDCI ¼ 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-
1-amine; HATU ¼ 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoropho-
sphate; HOAt ¼ 1-hydroxy-7-azabenzotriazole.
mixture was stirred at room temperature for 16 h and the
solvent was removed under vacuum. The resultant residue was
dissolved in ethyl acetate (5.0 mL) and washed with water
(5.0 mL). The organic phase was dried over MgSO4 and the
solvent was removed under vacuum to give the intermediate
alcohol (42 mg) that was not purified further. dH (600 MHz,
CDCl3) 7.89–7.86 (2H, m), 7.15–7.12 (2H, m), 6.96 (1H, d,
J 6.0), 6.77–6.75 (2H, m), 5.81 (1H, d, J 6.0), 5.25 (1H, d, J 9.6),
4.31–4.28 (1H, m), 4.11–4.07 (1H, m), 3.97–3.93 (1H, m),
3.86–3.83 (1H, m), 3.62 (1H, dd, J 6.0, 13.8), 3.54 (1H, dd, J 4.2,
10.8), 3.46 (1H, dd, J 4.2, 10.8), 3.12 (1H, dd, 6.0, 13.2), 2.65
(1H, t, J 13.2), 2.22–2.19 (1H, m), 1.85–1.79 (1H, m), 1.60–1.45
(2H, m), 1.35–1.21 (2H, m), 1.06–1.03 (1H, m), 0.973–0.91
(1H, m), 0.87 (3H, d, J 6.6), 0.78 (3H, d, J 6.6).
To a solution of this alcohol (42 mg) in dichloromethane
(DCM) (5.0 mL) under inert atmosphere was added Dess–
Martin periodinane (97 mg) and the mixture stirred vigorously
for 1.5 h at room temperature. The reaction was quenched
with saturated aqueous NaHCO3 (5.0 mL) containing Na2S2O5
(2 mmol) and the aqueous layer extracted with DCM
(2 ꢁ 10 mL). The combined organic layers were washed with
water (5.0 mL), the organic phase dried over MgSO4, and the
solvents removed under vacuum. The residue was purified by
flash chromatography on silica gel (elution with 2 : 1 ethyl

1260
S. A. Jones et al.
P2
O
O
H
N
a
HlCl H2N
P3
O
N
H
3
O
O
P1
13
O
O
H
N
F
HlCl H2N
O
O
N
H
O
H
N
H
N
O
O
N
H
a
S
O2
R
O
O
14
15 R ϭ OMe
16 R ϭ H
b
Scheme 2. Reagents and conditions: a: 4-FPS-Cl, DIPEA, DMF; b: (i) LiBH4, THF; (ii) SO3Py, IPA, DMSO,
DIPEA. P1-P3 nomenclature based on that of Schechter and Berger.[11] 4-FPS-Cl ¼ 4-fluorobenzene sulfonyl
chloride; DIPEA ¼ N,N-diisopropylethylamine; IPA ¼ isopropyl alcohol.
acetate/petroleum ether) to give 2 (26 mg, 53 % over two steps)
as a white solid.
A solution of the alkenes 8 in ethyl acetate was stirred under a
hydrogen atmosphere, with 10 % Pd on C, at room temperature,
for 1 h. The mixture was filtered through celite and concentrated
under vacuum to give 3 as a white solid (26 mg, 60 %). Data as
recorded below.
dH (600 MHz, CDCl3) 9.48 (1H, s), 7.88–7.86 (2H, m), 7.14–
7.11 (2H, m), 6.97 (2H, d, J 7.2), 6.75 (2H, d, J 7.2), 5.77–5.75
(2H, m), 5.52 (1H, d, J 9.6), 4.52 (1H, ddd, J 4.8, 9.0, 16.2),
4.24–4.20 (1H, m), 4.14–4.10 (1H, m), 3.86–3.82 (1H, m), 3.74
(1H, dd, J 6.6, 13.8), 3.11 (1H, dd, J 6.0, 12.0), 2.66 (1H, t, J
12.0), 2.04–1.96 (1H, m), 1.82–1.74 (1H, m), 1.60–1.44 (2H, m),
1.38–1.20 (2H, m), 1.16–1.10 (1H, m), 1.00–0.94 (1H, m), 0.88–
0.83 (1H, m), 0.81 (3H, d, J 6.0), 0.79 (3H, d, J 6.0). dC (75 MHz,
CDCl3) 197.7, 170.8, 168.7, 165.9, 164.2, 157.5, 136.6, 129.8,
129.7, 116.4, 116.3, 115.9, 66.5, 58.8, 58.2, 51.8, 43.2, 40.4,
29.7, 27.9, 24.5, 22.9, 22.0, 20.1. m/z 548.2226. HRMS (ESþ)
Anal. Calc. for C27H34FN3O6S [M þ H]þ: 548.2230.
Method B
TFA (2 mL) was added to a solution of 12 (19 mg,
0.03 mmol) in DCM (2.0 mL) under a N2 atmosphere and the
mixture stirred at room temperature for 3 h. The solvent was
removed under vacuum and the residue dissolved in toluene
before removal of all volatiles under vacuum to give a colourless
oil (14 mg). The oil was dissolved in DMF (2.1 mL) and treated
with HATU (9 mg, 0.02 mmol), 1-hydroxy-7-azabenzotriazole
(HOAt, 3 mg, 0.02 mmol), and N,N-diisopropylethylamine
(DIPEA, 0.014 mL, 0.09 mmol). The solution was stirred at
room temperature for 18 h, diluted with ethyl acetate, and
washed successively with 1 M aqueous HCl, saturated aqueous
NaHCO3, and brine. The combined organic phase was dried
over MgSO4 and the solvent removed under vacuum. The
residue was absorbed onto silica gel and elution with ethyl
acetate/petroleum ether (2 : 1) gave 3 (6 mg, 42 %) as an amor-
phous white solid. Data as recorded below.
(7S,10S,13S)-13-(4-Fluoro-benzenesulfonylamino)-
10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]
nonadeca-1(18),15(19),16-triene-7-carboxylic
Acid Methyl Ester (3)
Method A
To a solution of diene 7 (44 mg, 0.07 mmol) and DCM
(1.5 mL) under refluxing conditions was added Grubbs’ Second
Generation Catalyst (2 mg, 3 mol-%) and the mixture stirred
for 45 min. After removal of solvent under vacuum the crude
residue was recrystallised from ethyl acetate to give 8 as mixture
Method C
1
of E and Z-isomers that could not be separated (25 : 2 by H
To a solution of 13 (232 mg, 0.51 mmol) in DMF (5.1 mL)
was added 4-fluorobenzene sulfonyl chloride (89 mg,
0.56 mmol) and DIPEA (0.18 mL, 1.00 mmol). The solution
was stirred at room temperature for 18 h, diluted with ethyl
acetate (5 mL), and washed successively with 1 M aqueous HCl
(5.0 mL) and brine (5.0 mL). The organic layer was separated
and dried over MgSO4 and the volatiles removed under vacuum.
The residue was absorbed onto silica gel and elution with ethyl
acetate and petroleum ether (2 : 1) gave 3 as a white amorphous
solid (20 mg, 7 %).
NMR spectroscopy) as an amorphous white solid. Selected data
from mixture, major isomer: dH (600 MHz, CDCl3) 7.87–7.84
(2H, m), 7.14–7.11 (2H, m), 7.01–6.91 (2H, m), 6.81 (1H, d,
J 6.6), 6.76–6.64 (2H, m), 5.62 (1H, d, J 9), 5.56–5.51 (1H, m),
5.50 (1H, d, J 6.6), 5.48–5.43 (1H, m), 4.68–4.60 (2H, m), 4.54
(1H, dd, J 4.2, 15.6), 3.84–3.75 (2H, m), 3.72 (3H, s), 3.08 (1H,
dd, J 5.4, 13.2), 2.73–2.68 (2H, m), 2.24–2.18 (1H, m), 1.26–
1.19 (2H, m), 0.99–0.95 (1H, m), 0.79 (3H, d, J 6.6), 0.77 (3H, d,
J 6.6). Selected data from mixture, minor isomer: dH (600 MHz,
CDCl3) 6.85–6.82 (2H, m), 5.02–4.99 (1H, m), 4.25–4.14 (2H,
m), 3.65 (3H, s), 5.53–3.49 (1H, m), 3.33–3.29 (1H, m).
dH (600 MHz, CDCl3) 7.92–7.88 (2H, m), 7.16–7.12 (2H, m),
6.98 (2H, d, J 8.4), 6.77 (2H, d, J 8.4), 5.83 (1H, d, J 6.6), 5.77

Macrocyclic Calpain Inhibitors
1261
(1H, d, J 8.4), 5.59 (1H, d, J 10.2), 4.51 (1H, ddd, J 3.6, 8.6,
16.8), 4.27–4.21 (1H, m), 4.14–4.07 (1H, m), 3.90–3.84 (1H, m),
3.74–3.68 (1H, m), 3.73 (3H, s), 3.12 (1H, dd, J 6.0, 12.8), 2.68
(1H, t, J 12.6), 1.94–1.86 (1H, m), 1.75–1.70 (1H, m), 1.57–1.51
(1H, m), 1.37–1.31 (1H, m), 1.27–1.21 (2H, m), 1.14–1.09 (1H,
m), 0.97–0.85 (2H, m), 0.82 (3H, d, J 6.6), 0.80 (3H, d, J 6.6). dC
(75 MHz, CDCl3) 172.4, 170.4, 168.9, 165.9, 164.2, 157.4,
136.6, 130.1, 127.5, 116.5, 116.3, 66.6, 58.8, 52.7, 51.8, 51.4,
43.1, 40.4, 31.3, 27.8, 24.4, 22.9, 21.9, 21.0. m/z 578.2337.
HRMS (ESþ) Anal. Calc. for C28H36FN3O7S [M þ H]þ:
578.2336.
methyl ester (1.1 equiv.), HATU (1.1 equiv.), and DIPEA
(4 equiv.) were added and the mixture stirred at room tem-
perature overnight. The reaction mixture was diluted with
ethyl acetate (2.0 mL) and washed sequentially with 1 M aque-
ous HCl (2 ꢁ 2.0 mL), NaHCO3 (2.0 mL), and brine (2.0 mL),
and then dried over MgSO4. After removal of solvent under
vacuum, the residue was absorbed onto silica gel. Elution with
ethyl acetate and hexane (1 : 1) gave 7 as a white amorphous
solid (45 mg, 84 %).
dH (600 MHz, CDCl3) 7.64–7.58 (2H, m), 7.12–7.02 (2H, m),
6.82 (2H, d, J 9.2), 6.72 (2H, d, J 9.2), 6.68 (1H, d, J 8.0), 6.56
(1H, d, J 7.8), 6.10–6.02 (1H, m), 5.76–5.62 (1H, m), 5.44–5.40
(1H, m), 5.34–5.30 (1H, m), 5.15–5.11 (2H, m), 4.83 (1H, d, J
5.0), 4.66–4.60 (1H, m), 4.51–4.42 (3H, m), 3.75 (3H, s), 3.70–
3.74 (1H, m), 3.08–3.02 (1H, m), 2.77–2.70 (1H, m), 2.61–2.57
(1H, m), 2.58–2.47 (1H, m), 1.78–1.74 (1H, m), 1.44–1.56 (2H,
m), 0.94 (3H, d, J 6.6) 0.92 (3H, J 6.6). dC (75 MHz, CDCl3)
171.8, 171.1, 169.8, 166.2, 164.5, 158.1, 133.5, 133.0, 132.5,
129.4, 126.7, 119.0, 117.9, 116.6, 115.1, 68.8, 58.2, 52.5, 51.7,
40.9, 37.8, 36.9, 29.6, 24.5, 22.9, 21.9. m/z 604.2478. HRMS
(ESþ) Anal. Calc. for C30H38FN3O7S [M þ H]þ: 604.2487.
(S)-2-[(S)-3-(4-Allyloxy-phenyl)-2-(4-fluoro-
benzenesulfonylamino)-propionylamino]-4-methyl-
pentanoic Acid tert-Butyl Ester (6)
To a solution of 5 (4.10 g, 15 mmol) in DMF (150 mL) was
added 4-fluorobenzene sulfonyl chloride (3.2 g, 17 mmol) and
DIPEA (7.5 mL, 45 mmol) and the solution stirred at room
temperature for 18 h. The mixture was diluted with ethyl acetate,
washed successively with 1 M aqueous HCl and brine, and dried
over MgSO4. The volatiles were removed under vacuum and the
residue absorbed onto silica gel. Elution with ethyl acetate/
petroleum ether (1 : 2) gave 4-fluorobenzene sulfonyl-protected
tyrosine methyl ester as a white amorphous solid that was used
directly in the next step. dH (300 MHz, CDCl3) 7.75–7.68 (2H,
m), 7.12–7.05 (2H, m), 6.95 (2H, d, J 8.6), 6.76 (2H, d, J 8.6),
6.03 (1H, tdd, J 17.2, 10.6, 5.3), 5.42–5.37 (2H, m), 5.28 (1H, qd,
J 10.5, 1.3), 4.48 (2H, td, J 5.3, 1.5), 4.13 (1H, td, J 9.2, 6.3), 3.53
(3H, s), 2.99 (1H, dd, J 13.9, 5.7), 2.91 (1H, dd, J 13.9, 6.7).
A solution of this N-protected methyl ester (2.3 g, 5.9 mmol)
in THF (17 mL) was added 1.6 M aqueous NaOH (5.9 mL)
and MeOH (10 mL) and the solution stirred at room temperature
for 16 h. The solvent was removed under vacuum to give a
colourless oil that was dissolved in DMF (95 mL). Leucine
tert-butyl ester (1.4 g, 6.4 mmol), 3-(ethyliminomethylenea-
mino)-N,N-dimethyl-propan-1-amine (EDCI, 1.2 g, 6.4 mmol),
1-hydroxybenzotriazole (0.81 g, 6.4 mmol), and DIPEA
(3.8 mL, 22 mmol) were added and the solution was stirred at
room temperature for 18 h. The resulting mixture was diluted
with ethyl acetate, and washed successively with 1 M aqueous
HCl, saturated aqueous NaHCO3, and brine. The organic phase
was dried over MgSO4 and the solvent removed under vacuum.
The crude residue was absorbed onto silica gel and elution with
ethyl acetate and petroleum ether (1 : 1) gave 6 (2.96 g, 92 %) as
a foamy solid.
(R)-tert-Butyl 6-(4-((S)-3-((S)-1-tert-butoxy-4-methyl-
1-oxopentan-2-ylamino)-2-(4-fluorophenylsulfonamido)-
3-oxopropyl)phenoxy)-2-(tert-butoxycarbonylamino)
hexanoate (12)
Method A for Preparation of Precursor 11
A mixture of olefins 9a[8] (47 mg, 0.09 mmol) and 6 (19 mg,
0.04 mmol) were dissolved in freshly distilled DCM (1 mL)
and Grubbs’ Second Generation Catalyst (4 mg, 0.005 mmol)
was added. The solution was heated under reflux for 18 h,
concentrated under vacuum, and the residue absorbed onto
silica gel. Elution with a gradient of ethyl acetate and petroleum
ether gave 11 (22 mg, 33 %) as a mixture of E and Z-isomers
1
that could not be separated (92 : 8 by H NMR spectroscopy).
Selected data from mixture, major isomer: dH (300 MHz,
CDCl3) 7.70 (2H, app dd, J 7.6, 5.3), 7.13–7.04 (2H, m), 6.92
(2H, d, J 8.0), 6.75–6.65 (2H, m), 6.46 (1H, d, J 8.3), 5.80–5.67
(2H, m), 5.18 (1H, d, J 7.2), 5.06 (1H, d, J 8.4), 4.60–4.33 10
(4H, m), 3.97–3.87 (1H, m), 3.74–3.72 (3H, m), 2.92 (2H, d, J
5.5), 2.63–2.49 (2H, m), 1.63–1.40 (21H, m), 0.89–0.79 (6H, m).
m/z 748.3. LRMS (ES) Anal. Calc. for C37H51FN3O10S
[M ꢀ H]ꢀ: 748.3. Selected data from mixture, minor isomer:
dH (300 MHz, CDCl3) 7.86 (2H, br s), 5.60–5.54 (2H, m), 3.72
(3H, s), 3.07–3.02 (2H, m), 0.79 (6H, br s).
dH (300 MHz, CDCl3) 7.71 (2H, app dd, J 8.9, 5.0), 7.12–7.05
(2H, m), 6.91 (2H, app d, J 8.6), 6.73 (2H, d, J 8.6), 6.43 (1H, d, J
8.1), 6.05 (1H, tdd, J 17.3, 10.6, 5.3), 5.42 (1H, ddd, J 17.3, 2.9,
1.3), 5.30 (1H, ddd, J 10.5, 3.1, 1.2), 5.10 (1H, d, J 7.7), 4.49 (2H,
ddd, J 5.3, 1.5, 1.5), 4.41–4.29 (1H, m), 3.87 (1H, td, J 12.7, 6.4),
3.01–2.85 (2H, m), 1.53–1.34 (12H, m), 0.88 (6H, dd, J 6.1, 3.2).
dC (75 MHz, CDCl3) 171.7, 170.1, 158.1, 135.7, 133.5, 130.7,
130.3, 130.2, 127.6, 118.1, 116.7, 116.4, 115.2, 82.5, 69.0, 58.3,
52.0, 42.1, 38.2, 28.3, 25.1, 23.0, 22.4. m/z 547.3. LRMS (ES)
Anal. Calc. for C28H36FN2O6S [M ꢀ H]ꢀ: 547.2.
Method B for Preparation of Precursor 11
A mixture of 6 (660 mg, 1.2 mmol), Grubbs’ Second Gene-
ration Catalyst (41 mg, 0.05 mmol), and 1 M BCl2(Cy)2
(0.19 mL, 0.19 mmol) in DCM (3.0 mL) was irradiated in a
microwave for 2 min. Three more batches of catalyst
(3 ꢁ 41 mg) were added, with microwave irradiation for 2 min
after each addition. The solvent was removed under reduced
pressure, the residue was absorbed onto silica gel, and elution
with ethyl acetate and petroleum ether (1 : 1) gave 10 (560 mg,
87 %, an inseparable mixture of E and Z-isomers that could not
be separated, 81 : 19 by 1H NMR spectroscopy) as a colourless
oil. dH (300 MHz, CDCl3) 7.73–7.68 (4H, m), 7.11–7.04
(4H, m), 6.92 (4H, app d, J 8.6), 6.73 (4H, app d, J 8.5), 6.46
(2H, d, J 8.2), 6.08 (2H, app t, J 2.3), 5.20–5.03 (2H, m), 4.54
(4H, app d, J 3.2), 4.39–4.32 (2H, m), 3.94–3.84 (2H, m), 3.02–
2.89 (4H, m), 1.56–1.30 (24H, m), 0.88 (12H, dd, J 5.9, 3.4).
(S)-2-{(S)-2-[(S)-3-(-4-Allyloxy-phenyl)-2-(4-fluoro-
benzenesulfonylamino)-propionylamino]-4-methyl-
pentanoylamino}-pent-4-enoic Acid Methyl Ester (7)
To a solution of dipeptide 6 (50 mg, 0.09 mmol) in DCM (1 mL)
was added TFA (4 equiv.) and the reaction mixture was stirred at
room temperature for 18 h and then concentrated under vacuum.
The product was dissolved in DMF (1 mL) to which allyl glycine

1262
S. A. Jones et al.
To a solution of 10 (29 mg, 0.03 mmol) and 9b (39 mg,
extracted with ethyl acetate, and the combined organic phase
washed with brine. After drying over MgSO4, the solvent was
removed under vacuum to give the alcohol (32 mg, 78 %) as a
white solid that was not purified further. dH (600 MHz, DMSO-
d6) 7.95 (2H, app dd, J 8.9, 5.3), 7.35 (2H, app t, J 8.9), 7.31 (1H,
d, J 9.2), 7.18 (1H, d, J 7.8), 6.94 (2H, d, J 7.4), 6.70 (2H, d, J
8.5), 4.30–4.28 (2H, m), 4.02–3.99 (1H, m), 3.67–3.63 (2H, m),
3.34 (1H, br s), 3.15–3.11 (1H, m), 3.04–3.00 (1H, m), 2.71 (1H,
dd, J 12.9, 5.9), 2.58 (1H, app t, J 12.2), 1.74–1.70 (1H, m),
1.47–1.44 (1H, m), 1.27–1.08 (6H, m), 0.74–0.65 (4H, m), 0.48
(3H, d, J 6.8). dC (150 MHz, DMSO-d6) 168.6, 168.3, 156.0,
138.1, 130.1, 129.6, 129.5, 127.6, 115.9, 115.8, 115.3, 66.3,
64.3, 56.6, 55.9, 49.0, 38.5, 29.8, 27.6, 24.4, 21.9, 14.4, 11.6.
m/z 550.2381. HRMS (ESþ) Anal. Calc. for C27H37N3O6SF
[M þ H]þ: 550.2387.
0.17 mmol) in DCM (2.0 mL) was added Grubbs’ Second
Generation Catalyst (1 mg). The mixture was heated under
reflux for 18 h, further catalyst (1 mg) was added, and reflux
continued for a further 2 h. A third batch of catalyst (1 mg) was
added, and the mixture refluxed for an additional 3 h. The
solvent was removed under reduced pressure, the residue
absorbed onto silica gel, and elution with a gradient of ethyl
acetate and petroleum ether gave 11 (20 mg, 51 %) as a mixture
1
of E and Z-isomers that could not be separated (87 : 13 by H
NMR spectroscopy). NMR data for 11 as reported above.
A mixture of alkenes 11 (25 mg, 0.03 mmol) was dissolved
in DCM and methanol (1 : 1, 1.0 mL), 10 % Pd on C (7 mg)
was added, and the mixture stirred under a H2 atmosphere for
16 h. The mixture was filtered through celite, and the solvent
removed under vacuum to give 12 (25 mg, quantitative) as a
colourless oil.
dH (300 MHz, CDCl3) 7.72–7.67 (2H, m), 7.10–7.02 (2H, m),
6.93–6.87 (2H, m), 6.69–6.65 (2H, m), 6.47 (1H, d, J 7.2),
5.23–5.15 (1H, m), 5.05 (1H, d, J 8.4), 4.45–4.30 (2H, m),
3.91–3.84 (3H, m), 3.74 (3H, s), 2.92 (2H, d, J 6.4), 1.91–1.60
(4H, m), 1.60–1.40 (23H, m), 0.89–0.86 (6H, m). dC NMR
(75 MHz, CDCl3) 171.5, 170.7, 169.7, 163.6, 158.5, 130.6 (br),
130.2, 130.1, 127.1, 116.7, 116.4, 114.9, 82.5, 67.7, 58.1, 53.6,
52.6, 51.9, 42.1, 38.2, 32.8, 29.1, 28.6, 28.2, 25.0, 22.9, 22.3. m/z
750.4. LRMS (ES) Anal. Calc. for C37H53FN3O10S [M ꢀ H]ꢀ
750.3.
A solution of the preceding alcohol (23 mg, 0.04 mmol)
and propan-2-ol (0.025 mL, 0.33 mmol) in DMSO (4.0 mL)
and DCM (2.0 mL) was cooled in an ice bath with stirring under
a nitrogen atmosphere. DIPEA (0.26 mL, 1.49 mmol) was
added, followed by a solution of SO3ꢂPy complex (237 mg,
1.49 mmol) in DMSO (1.5 mL) dropwise over 5 min. The ice
bath was removed and the mixture left stirring for 3 h. The
mixture was diluted with ethyl acetate, washed with 1 M
aqueous HCl, saturated aqueous NaHCO3, and brine. The
organic phase was dried over MgSO4 and the solvent removed
under vacuum. The residue was absorbed onto silica gel and
eluted with ethyl acetate to give 16 (7 mg, 30 %) as a white solid.
dH (600 MHz, DMSO-d6) 9.49 (1H, s), 7.90–7.87 (2H, m),
7.15–7.13 (2H, m), 6.98 (2H, br s), 6.76 (2H, d, J 7.1), 5.92 (1H,
d, J 6.2), 5.62 (1H, d, J 7.6), 5.56 (1H, d, J 9.5), 4.59–4.54 (1H,
m), 4.26 (1H, ddd, J 11.8, 6.3, 3.8), 4.13 (1H, ddd, J 11.7, 8.1,
3.6), 3.91–3.87 (1H, m), 3.70 (1H, dd, J 6.2, 3.4), 3.15 (1H, dd, J
13.0, 5.9), 2.68 (1H, dd, J 13.0, 11.6), 2.10–1.94 (1H, m), 1.86–
1.72 (1H, m), 1.63–1.25 (6H, m), 1.09–0.97 (1H, m), 0.95–0.82
(3H, m), 0.46 (3H, d, J 6.8). dC (150 MHz, DMSO-d6) 197.8,
168.9, 168.7, 157.3, 136.5, 129.8, 129.7, 127.4, 116.4, 116.3,
66.5, 58.8, 58.1, 57.4, 40.4, 38.6, 29.7, 27.9, 27.8, 25.8, 21.1,
14.0, 12.0. m/z 548.2239. HRMS (ESþ) Anal. Calc. for
C27H35N3O6SF [M þ H]þ: 548.2231.
(7S,10S,13S)-10-s-Butyl-13-(4-fluoro-
benzenesulfonylamino)-9,12-dioxo-2-oxa-8,
11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),
16-triene-7-carboxylic Acid Methyl Ester (15)
To a solution of 14 (71 mg, 0.16 mmol) in DMF (1 mL) was
added 4-fluorobenzene sulfonyl chloride (30 mg, 0.16 mmol)
and DIPEA (2 equiv.). The solution was stirred at room tem-
perature for 16 h, diluted with ethyl acetate, and washed with
1 M aqueous HCl (ꢁ 2) and brine. The organic layer was sepa-
rated, dried over MgSO4, and the volatiles removed under
vacuum. The residue was absorbed onto silica gel and elution
with a gradient of ethyl acetate and petroleum ether gave 15 as a
white amorphous solid (20 mg, 7 %).
Enzyme Inhibition Assay[12]
dH (500 MHz, CDCl3) 7.87–7.92 (2H, m), 7.14 (2H, app t, J
8.1), 6.98 (2H, br d, J 7.3), 6.77 (2H, br d, J 7.4), 5.98–6.04 (1H,
m), 5.69–5.75 (2H, m), 4.51–4.56 (1H, m), 4.24–4.29 (1H, m),
4.08–4.13 (1H, m), 3.91–3.96 (1H, m), 3.73 (3H, s), 3.67–3.71
(1H, m), 3.14 (1H, dd, J 5.5, 13.1), 2.67 (1H, app t, J 11.9), 1.89–
1.94 (1H, m), 1.76–1.82 (1H, m), 1.59–1.63 (1H, m), 1.50–1.56
(2H, m), 1.26–1.40 (3H, m), 0.99–1.05 (1H, m), 0.85 (3H, br t, J
6.6), 0.47 (3H, app br s). dC NMR (75 MHz, CDCl3) 172.8,
168.9, 168.8, 157.5, 130.3, 130.1, 130.0, 127.6, 116.7, 116.4,
116.2, 66.9, 58.9, 57.6, 52.9, 51.4, 40.6, 39.0, 31.8, 27.8, 26.0,
21.5, 14.2, 12.3. m/z 578.2317. HRMS (ESþ) Anal. Calc. for
C28H36FN3O7S [M þ H]þ: 578.2336.
Ovine calpain 2 (o-CAPN2) was partially purified from sheep
lung by hydrophobic and ion-exchange chromatography.
The calpain was diluted in 0.5 % mercaptoethanol, 2 mM ethy-
lene glycol tetraacetic acid (EGTA), 2 mM ethylenediamine-
tetraacetic acid (EDTA), and 20 mM 3-(N-morpholino)
propanesulfonic acid (MOPS), pH 7.5. A substrate solution
(0.0005 % BODIPY-FL casein in 10 mM 2-amino-2-hydro-
xymethyl-propane-1,3-diol (TRIS) HCl, pH 7.5, containing
10 mM CaCl2, 0.1 mM NaN3, and 0.1 % mercaptoethanol) was
prepared fresh for the assay. These solutions of enzyme and
substrate were prepared at concentrations such as to provide a
linear increase in fluorescence over the course of the assay
(10 min). The calpain inhibition assays were performed in
96-well black Whatman plates at 258C. Calpain control assays
contained 50 mL of enzyme and 50 mL of sample buffer. Each
assay was initiated by the addition of 100 mL of substrate solu-
tion and the progress of the reactions were followed for 10 min in
a (BMG) Fluostar with an excitation wavelength of 485 nm and
by measuring emission at a wavelength of 530 nm. For inhibitor
assays the sample buffer was replaced with 50 mL of inhibitor
dissolved in DMSO (2 % total concentration) and diluted with
water. The percentage inhibition was determined as 100 times
N-((7S,10S,13S)-10-s-Butyl-7-formyl-9,12-dioxo-2-oxa-
8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-
trien-13-yl)-4-fluoro-benzenesulfonamide (16)
To an ice cooled solution of 15 (44 mg, 0.08 mmol) in THF
(2.0 mL) was added LiBH4 in THF (2 M, 0.19 mL) dropwise,
and the solution allowed to warm to room temperature with
stirring, for 16 h. Water (1.0 mL) was added, the volatiles
removed under vacuum, and the residue partitioned between
1 M aqueous HCl and ethyl acetate. The aqueous phase was

Macrocyclic Calpain Inhibitors
1263
the activity with inhibitor present divided by the activity of the
control assay. The reported IC50 values are the average of trip-
licate determinations, with standard errors ,50 %.
(b) D. R. March, G. Abbenante, D. A. Bergman, R. I. Brinkworth,
W. Wickramasinghe, J. Begun, J. L. Martin, D. P. Fairlie, J. Am. Chem.
Soc. 1996, 118, 3375. doi:10.1021/JA953790Z
(c) A. K. Ghosh, S. Kulkarni, D. D. Anderson, L. Hong, A. Baldridge,
Y.-F. Wang, A. A. Chumanevich, A. Y. Kovalevsky, Y. Tojo,
M. Amano, Y. Koh, J. Tang, I. T. Weber, H. J. Mitsuya, J. Med. Chem.
2009, 52, 7689. doi:10.1021/JM900695W
Supplementary Material
1H NMR and 13C NMR spectra for compounds 2, 3, 6, 7, 12, 15,
and 16 are available on the Journal’s website
[5] (a) J. D. Morton, H. Y. Y. Lee, J. D. McDermott, L. J. G. Robertson,
R. Bickerstaffe, M. A. Jones, J. M. Coxon, A. D. Abell, Invest. Oph-
thalmol. Vis. Sci. 2013, 54, 389. doi:10.1167/IOVS.12-11088
(b) H. Y. Y. Lee, J. D. Morton, L. J. G. Robertson, J. McDermott,
R. Bickerstaffe, A. D. Abell, M. A. Jones, J. M. Mertons, J. M. Coxon,
Clin. Experiment. Ophthalmol. 2008, 36, 852. doi:10.1111/J.1442-
9071.2009.01925.X
[6] (a) B. G. Stuart, J. M. Coxon, J. D. Morton, A. D. Abell,
D. Q. McDonald, S. G. Aitken, M. A. Jones, R. Bickerstaffe, J. Med.
Chem. 2011, 54, 7503. doi:10.1021/JM200471R
Acknowledgements
Financial support from the Australian Research Council, the New Zealand
Public Good Science and Technology Fund, Foundation for Research
Science and Technology, and Douglas Pharmaceuticals is gratefully
acknowledged. The authors also thank Dr Janna Mehrtens and Dr Matthew
Jones for conducting the calpain inhibition assay and Matthew Muir for the
supply of calpain.
(b) S. A. Jones, M. A. Jones, S. B. McNabb, S. G. Aitken, J. M. Coxon,
A. D. Abell, Protein Pept. Lett. 2009, 16, 1466. doi:10.2174/
092986609789839296
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