Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors

Article abstract of DOI:10.3109/14756366.2011.651466

This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA cysteine protease inhibitors. The structure of the inhibitors consists of a R1-Phe-R2-AS-Ph scaffold (AS=allyl sulfone). R1 was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R2 was varied with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine proteases cathepsin B and calpain I with a kobs/[I] of 6080±1390M^-1s^-1.

Full text of DOI:10.3109/14756366.2011.651466

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 468–478
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.651466
RESEARCH ARTICLE
Optimization of peptidyl allyl sulfones as clan CA cysteine
protease inhibitors
Brandon D. Fennell1, Julia M. Warren1, Kevin K. Chung1, Hannah L. Main1, Andrew B. Arend1,
Anna Tochowicz2, and Marion G. Götz1
1Department of Chemistry, Whitman College, WA, USA and 2Sandler Center for Drug Discovery, Department of
Pathology, University of California San Francisco, San Francisco, CA, USA
Abstract
This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA
cysteine protease inhibitors. The structure of the inhibitors consists of a R1-Phe-R2-AS-Ph scaffold (AS=allyl sulfone). R1
was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R2 was varied
with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization
to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all
factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with
three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The
most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine
proteases cathepsin B and calpain I with a kobs/[I] of 6080 1390M−1s−1.
Keywords: Cruzain, Chagas disease, peptidyl vinyl sulfone
Introduction
Cathepsins are members of family C1. Eleven cathep-
Proteases are a group of enzymes that are responsible for
a variety of metabolic functions based on their ability to
cleave peptide bonds1–4. ey are grouped by catalytic
mechanism into five major classes: metallo, aspartate,
threonine, serine and cysteine proteases5. Cysteine pro-
teases catalyze the cleavage of peptide bonds using a
catalytic dyad of Cys and His residues6. Each of the five
major protease classes is further divided into clans and
families7. Clan CA (C indicates cysteine) is the largest
of the cysteine protease clans and contains a variety of
enzymes including calpains, cathepsins and cruzain.
Calpains are members of family C2, requiring Ca2+ for
activation8. ey have been linked to a number of differ-
ent cellular functions including signal transduction, cell
fusion, mitosis and protein turnover. Mutations and other
defects in calpains have been implicated in hypertension
and muscular dystrophy as well as neurodegeneration9.
To date, no highly selective and potent inhibitors of cal-
pains have been synthesized.
sins are currently known including cathepsin B, C, F, H,
K (O2), L, O, S, V (L2), W and X (parentheses indicate
secondary names). Cathepsin B is the most abundant
of the cathepsins, serving a variety of cell degradation
processes10 in addition to potentially functioning as a
defence mechanism against amyloid plaque aggrega-
tion11. Cathepsin B has been associated with tumour
progression6,12,13. More generally, cathepsins have been
linked to rheumatoid arthritis, osteoarthritis, neurologi-
cal disorders and lysosomal storage diseases14,15. Because
of their role in these diseases, cathepsins have been tar-
gets for drug development16–22. Continued investigation
of new cathepsin inhibitors provides a route for expand-
ing treatment of these diseases.
Cruzain is another member of family C1. It is a key
enzyme in the life cycle of Trypanosoma cruzi, the para-
sitic vector of Chagas disease23–25. Chagas disease is an
increasing problem in Latin America with 10 million
people suffering from infection and millions more at
Address for Correspondence: Dr. Marion G. Götz, Department of Chemistry, Whitman College, 345 Boyer Ave, Walla Walla, WA 99362, USA.
Tel: +1-509-527-5957. Fax: +1-509-237-5904. E-mail: gotzmg@whitman.edu
(Received 24 November 2011; revised 15 December 2011; accepted 16 December 2011)
468

Optimization of peptidyl allyl sulfones 469
Hfe, homo-phenylalanine residue
Abbreviations
AMC, 7-aminomethyl coumarin
AS, allyl sulfone
Cbz, benzyloxycarbonyl
DTT, dithiothreitol
HOBT, hydroxybenzotriazole
iBCF, isobutyl chloroformate
Mp, N-methylpiperazinocarbonyl
Mu, morpholinocarbonyl
NMM, N-methylmorpholine
VS, vinyl sulfone
EDCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
Elim, eliminated sulfone (-C(O)-N=C(R)-CH=CH2)
risk26. Current treatment utilizes benznidazole (a nitro-
imidazole derivative)27 and nifurtimox (a 5-nitrofuran
derivative)28, which are effective in the acute phase of
the disease; however, these treatments are plagued with
severe side effects and increasing resistance29,30. As a
result, demand for developing new treatments for this
disease remains prevalent.
of flexibility of the phenyl group after the introduction of
the planar allyl sulfone moiety.
We have previously synthesized allyl sulfones from
vinyl sulfone precursors and determined that stereo-
chemistry of the P1 residue of the vinyl sulfone influ-
enced the stereochemistry of the allyl sulfone product,
although the contributing effect was not determined.
To further investigate this, both enantiomers of the P1
amino acid residue were used during the synthesis of the
dipeptidyl allyl sulfones.
Todate,anumberofcysteineproteaseinhibitorshavebeen
developed using a variety of reactive functional groups31, also
referred to as “warheads,” including aldehydes20, fluorom-
ethyl ketones21, nitriles32, α-ketoesters, amides, and acids33,
tetrafluorophenoxymethyl ketones34, epoxysuccinates35,
thiosemicarbazones36, hydrazone derivatives37,38, vinyl sul-
fones39 and allyl sulfones40. ese warheads are attached to
Materials and methods
Materials
either a peptidyl or peptidomimetic scaffold16,41
.
Starting materials and reagents were purchased from
VWR International (Radnor, Pennsylvania) and used
without further purification. Amino acids were purchased
from Advanced ChemTech (Louisville, Kentucky) and
enzymes and substrates from Calbiochem (San Diego,
California)andEnzoLifeSciences(AnnArbor, Michigan).
Compound purity was confirmed by MS electron spray
ionization (ESI) and NMR (13C and 1H with chemical
shifts reported in ppm relative tetramethyl silane). MS
analysis was performed at the University of Arkansas
High Performance Mass Spectrometry Laboratory using
a Bruker Esquire-LC Ion Trap LC/MS. NMR experiments
were conducted using a BrukerAvance III 400MHz
UltrashieldPlus Spectrometer. Compounds were purified
via gravity column chromatography using Sigma-Aldrich
Fluka analytical silica (60 Å, 70–230 mesh). Reaction
progress was monitored using EMD 60 F254 TLC plates.
Fluorogenic enzyme assays were performed on a BioTek
Flx800 Plate Reader. Molecular modelling data was
acquired using chemical computing group’s Molecular
Operating Environment (MOE) software package.
We selected cruzain as our primary target due to the
high demand for the development of new treatments
for Chagas disease. Currently, the most effective known
inhibitor of cruzain, referred to as K11777, is a dipeptidyl
vinyl sulfone (Mp-Phe-Hfe-VS-Ph)42. Both metabolic43
and animal studies44,45 have confirmed that K11777 is a
highly selective and potent inhibitor of cruzain in vivo
and is currently entering clinical trials46. We have demon-
strated previously that use of an allyl sulfone in place of
the vinyl sulfone moiety showed enhancements in inhibi-
tory potency for clan CA cysteine proteases (Figure 1)40.
In order to optimize the lead allyl sulfones for inhi-
bition of cruzain, we chose to modify the P3 and P1
positions of the scaffold. e peptidyl scaffold was main-
tained because no peptidomimetic scaffolds have been
able to achieve in vivo potency equivalent to K11777.
e goal of the P3 modification was to vary solubility
and polarity inside the active site using Cbz, Mp and Mu
groups. Based on previous kinetic data, the P2 and P1′
positions were kept constant, as Phe and SO2Ph, respec-
tively, in all analogues39. e P1 residue was varied, using
either Phe or Hfe residues, in order to examine the effect
General synthesis of inhibitors
Trans-vinyl sulfones 1–4 were prepared as previously
described via Wadsworth-Emmons chemistry using chi-
ral amino acid aldehydes and a sulfonylphosphonate40,47
.
Phe-OMe was reacted with morpholinocarbonyl chlo-
ride and saponified to carboxylic acid 5 (Mu-Phe-OH) in
71% overall yield (Scheme 1). Phe-OBz was reacted with
N-methylpiperazinocarbonyl chloride and hydrogenated
to give carboxylic acid 6 (Mp-Phe-OH) in 89% yield. Tri-
fluoroacetic acid (TFA) salts 1 and 2 (TFA·L/D-Hfe-VS-Ph)
were coupled to 5 using 1-ethyl-3-(3-dimethylaminopropyl)
Figure 1. Basic scaffold of dipeptidyl allyl sulfone inhibitors with
enzyme subsite designations P3, P2, P1 and P1′.
© 2013 Informa UK, Ltd.

470 B.D. Fennell et al.
Scheme 1. (a) EDCl, HOBT, Et3N, DCM, overnight; (b) iBCF, NMM, THF/DMF, overnight; (c) 1 eq.n-BuLi, 4.5 eq. t-BuOOH, −20°C for
70min, 9h or 18h.
carbodiimide (EDCl) and hydroxybenzotriazole (HOBT) to
produce dipeptidyl vinyl sulfones 7and 8(Mu-Phe-L/D-Hfe-
VS-Ph).TFAsalts 3and4(TFA·L/D-Phe-VS-Ph)werecoupled
to Cbz-Phe-OH using EDCl and HOBT to yield dipeptidyl
vinyl sulfones 9 and 10 (Cbz-Phe-L/D-Phe-VS-Ph). TFA salts
1–3 were coupled to 6 using iBCF and N-methylmorpholine
(NMM) to give dipeptidyl vinyl sulfones 11–13 (Mp-Phe-
L/D-Hfe-VS-Ph, Mp-Phe-L-Phe-VS-Ph). Dipeptidyl vinyl sul-
fones 7–13 were then isomerized to allyl sulfone analogues
14–20 using t-BuOOH and n-BuLi.
under reduced pressure to give 5 as a white powder in a
76.5% yield. 1H NMR (400 MHz, DMSO-d6) δ 2.90 (dd, 1H,
CH2Ph J= 13 Hz, 10 Hz), 3.03 (dd, 1H, CH2Ph, J= 15 Hz, 5
Hz), 3.23 (m, 4H, CH2NCH2), 3.49 (m, 4H, CH2OCH2), 4.23
(m, 1H, α-H), 6.73 (d, 1H, NH), 7.17–7.30 (m, 5H, Ph),
12.53 (s, broad, 1H, COOH).
Synthesis of (2S)-2-(N-methylpiperazine-4-carboxamido)-3-
phenylpropanoic acid (6, Mp-Phe-OH) e hydrochloride
salt of N-methylpiperazinecarbonyl chloride (1.50g, 7.54
mmol) was dissolved in THF (50mL) under argon at −15°C.
Et3N (0.839g, 8.29 mmol) was added and the reaction was
stirredfor15min. ehydrochloridesaltofPhe-OBz(2.00g,
6.84 mmol) was dissolved in THF (50mL) under argon
at −15°C. Et3N (0.839g, 8.29 mmol) was added and stirred
for 15min. e two reactions were combined and kept
at −15°C for 15min, then allowed to warm to room tem-
perature and stirred overnight. e reaction was quenched
with water (80mL) and the product was extracted with
EtOAc (3×50mL). e organic layer was washed with water
(3×50mL) and brine (2×50mL) and dried (MgSO4). e
solvent was removed under reduced pressure. e crude
product was purified via column chromatography (silica,
5% MeOH in DCM, 0.5% Et3N). e pure product (Mp-Phe-
OBz) was isolated as a clear colorless syrup with a yield of
88.5%. 1H NMR (400MHz, CDCl3) δ 2.31 (s, 3H, CH3N), 2.37
(t, 4H, CH2N(CH3)CH2), 3.14 (m, 2H, CH2Ph), 3.36 (m, 4H,
CH2NCH2), 3.88 (broad, 2H, α-H and NH), 7.00–7.03 (m, 2H,
Ph), 7.21–7.41 (m, 8H, Ph). Mp-Phe-OBz was deprotected
using a procedure adapted from Maring et al.48. Mp-Phe-
OBz (0.600g, 1.52 mmol) was dissolved in ethanol (100mL)
and degassed. Ammonium formate (0.957g, 15.171 mmol)
and Pd/C (120mg) were added. e reaction was refluxed
for 40min. e resulting solution was filtered over Celite
and the solvent was removed under reduced pressure.
e product was isolated as a white powder with a yield
of 85.3%. 1H NMR (400MHz, CDCl3) δ 2.37 (s, 3H, CH3N),
2.54 (broad, 4H, CH2N(CH3)CH2), 3.01 (m, 1H, CH2Ph), 3.18
(m, 1H, CH2Ph), 3.34–3.45 (m, 4H, CH2NCH2), 4.44 (m, 1H,
α-H), 5.73 (d, 1H, NH), 7.12–7.26 (m, 5H, Ph). Acid peak not
observed.
Detailed synthesis
Preparation of Amino Acid Vinyl Sulfone TFA Salts
TFA salts 1–4 were synthesized using previously reported
methods40.
Synthesis
of
(2S)-2-(morpholine-4-carboxamido)-3-
phenylpropanoic acid (5, Mu-Phe-OH) e hydrochloride
salt of Phe-OMe (7.00 g, 32.5 mmol) was dissolved in
THF (150 mL) under argon at −15°C. NMM (7.25g, 71.4
mmol) was added followed by the dropwise addition of
morpholinecarbonylchloride (5.34 g, 35.7 mmol). e
reaction was allowed to warm to room temperature
and stirred overnight. e reaction was poured into aq.
HCl (1 N, 100 mL) and extracted with EtOAc (3 × 60 mL).
e organic extract was then washed with aq. HCl (1
N, 3 × 60 mL) and brine (3 × 60 mL) and dried (MgSO4).
e solvent was removed under reduced pressure. e
product was initially isolated as a syrup that spontane-
ously crystallized into white crystals with a 92% yield. 1H
NMR (400 MHz, CDCl3) δ 3.14 (t, 2H, CHCH2Ph), 3.33 (m,
4H, CH2NCH2), 3.75 (t, 4H, CH2OCH2), 4.81 (m, 1H, α-H),
4.87 (d, 1H, NH), 7.11–7.12 (m, 2H, Ph), 7.28–7.32 (m, 3H,
Ph). Mu-Phe-OMe (9.47 g, 32.5 mmol) was dissolved in
methanol (150 mL). Sodium hydroxide (2 N, 2.62 g, 48.5
mmol) was added. After 4 h, the solvent was removed
under reduced pressure and the mixture was suspended
in water (100 mL). e solution was acidified to pH 2 by
addition of aq. HCl (3 N, approx. 20 mL). e product
was extracted with EtOAc (3 × 30 mL), washed with brine
(2 × 25 mL) and dried (MgSO4). e solvent was removed
Journal of Enzyme Inhibition and Medicinal Chemistry

Optimization of peptidyl allyl sulfones 471
pressure. e product was purified via column chroma-
tography (silica, 1:1 Hex: EtOAc) as a white powder with
a 36.4% yield. H NMR (400 MHz, CDCl3) δ 2.81 (d, 2H,
CH2Ph), 2.95–3.05 (m, 2H, CH2Ph), 4.29 (q, 1H, α-H), 4.93
(q, 1H, α-H), 5.07 (s, 2H, OCH2Ph), 5.14 (m, 1H, NH),
5.73 (d, 1H, NH), 5.95 (d, 1H, CH=CH-SO2), 6.80 (dd, 1H,
CH=CH-SO2, J= 15 Hz, 5 Hz), 6.99–7.46 (m, 15H, 3× Ph),
7.52–7.83 (m, 5H, Ph-SO2).
General procedure for peptide coupling reaction using
Mu-Phe-OH. Phenyl (3S)-3-(N-morpholine-4-carboxamido
phenylalanyl)amino-5-phenylpent-1-enylsulfone(7,Mu-Phe-
L-Hfe-VS-Ph) TFA salt 1 (TFA·D-Hfe-VS-Ph) (2.89 g, 6.95
mmol) was dissolved in DCM (120 mL) and Et3N (1.06 g,
10.4 mmol) was added. Compound 5 (Mu-Phe-OH)
(1.99 g, 6.95 mmol) was dissolved in DCM (80mL) and
added to the reaction followed by HOBT (1.03g, 7.64
mmol) and EDCl (1.19 g, 7.64 mmol). Additional Et3N was
added to adjust the pH to between 9 and 10. e reaction
was stirred overnight. e mixture was washed with aq.
HCl (3N, 3 × 50 mL), aq. NaHCO3 (10%, 3 × 50 mL), brine
(3 × 50 mL) and dried (MgSO4). e solvent was removed
under reduced pressure. e product was purified via
column chromatography (silica, 1% MeOH in DCM) and
1
Synthesis of phenyl (3R)-3-(N-carbobenzyloxyphenylalanyl)
amino-4-phenylbut-1-enyl sulfone (10, Cbz-Phe-D-Phe-
VS-Ph) See the above procedure for peptide coupling
reaction using Cbz-Phe-OH. TFA salt 4 (TFA·D-Phe-
VS-Ph) was used as starting material. e product was
purified via column chromatography (silica, 1:1 Hex:
1
1
EtOAc) as a white powder with a 64.1% yield. H NMR
isolated as a white powder with an 86.1% yield. H NMR
(400 MHz, CDCl3) δ 2.61–3.14 (m, 4H, 2 × CH2Ph), 4.33
(q, 1H, α-H), 4.89–5.15 (m, 3H, α-H and CH2OPh), 5.34
(m, 1H, NH), 6.19 (d, 1H, NH), 6.32 (d, 1H, CH=CH-SO2),
6.80–7.44 (m, 16 H, 2 × CH2Ph, OPh, CH=CH-SO2), 7.47–
7.87 (m, 5H, Ph-SO2). 13C NMR (100 MHz, CDCl3) δ 38.16,
39.90, 40.16, 50.27, 56.54, 67.22, 127.21, 127.26, 127.67,
128.07, 128.11, 128.32, 128.60, 128.71, 128.81, 129.16,
129.27, 129.31, 131.15, 133.48, 135.28, 136.06, 140.03,
144.75, 170.48.
(400 MHz, CDCl3) δ 1.79–1.89 (m, 2H, CH2CH2Ph), 2.57
(m, 2H, CH2CH2Ph), 3.05 (m, 2H, CH2Ph), 3.28 (m, 4H,
CH2NCH2), 3.62 (m, 4H, CH2OCH2), 4.47 (q, 1H, α-H),
4.62 (s, broad, 1H, α-H), 4.96 (m, 1H, NH), 6.10 (dd, 1H,
CH=CH-SO2, J= 15 Hz, 2 Hz), 6.39 (d, 1H, NH), 6.80 (dd,
1H, CH=CH-SO2, J= 15 Hz, 8 Hz), 7.01–7.33 (m, 10H,
2 × Ph), 7.50–7.91 (m, 5H, Ph-SO2). 13C NMR (100 MHz,
CDCl3) δ 31.71, 35.68, 38.14, 43.96, 49.13, 56.19, 66.31,
126.35, 127.31, 127.68, 128.33, 128.58, 128.62, 128.92,
129.15, 129.21, 129.30, 130.65, 133.50, 136.62, 145.26,
157.13, 171.51.
General procedure for peptide coupling using Mp-Phe-OH.
Phenyl (3S)-3-(N-(N-methylpiperazine)-4-carboxamidophe-
nylalanyl)amino-5-phenylpent-1-enyl sulfone (11, Mp-Phe-
L-Hfe-VS-Ph) Compound 6 (Mp-Phe-OH) (0.874 g, 1.23
mmol) was dissolved in 3:1 THF/DMF (40 mL) at −10°C.
NMM (0.137 g, 1.35 mmol) was added and the reaction
was stirred for 20 min with the gradual addition of iBCF
(0.185 g, 1.35 mmol). e reaction was stirred for 1 h
maintaining a temp below −5°C. TFA salt 1 (TFA·L-Hfe-
VS-Ph) was added at −10°C. Additional NMM (0.137g,
1.35 mmol) was added and the reaction was stirred for
1 h at −10°C. e reaction was raised to room tempera-
ture and stirred overnight. Aq. NaHCO3 (10%, 200 mL)
was added and the product was extracted using EtOAc
(3 × 40 mL). e organic layers were combined and
washed with brine (2 × 50 mL) and dried (MgSO4). e
solvent was removed under reduced pressure and the
crude product was purified by column chromatography
(silica, 5% MeOH in DCM, 0.5% Et3N). e product was
Synthesis of phenyl (3R)-3-(N-morpholine-4-carboxami-
dophenylalanyl)amino-5-phenylpent-1-enyl sulfone. (8,
Mu-Phe-D-Hfe-VS-Ph) TFA salt 2 (TFA·L-Hfe-VS-Ph) was
used as the starting material and the general peptide
coupling procedure was followed. e product was puri-
fied via recrystallization (1:3 Hex: EtOAc) and isolated
as white crystals with an 84.6% yield. 1H NMR (400 MHz,
CDCl3) δ 1.68–1.82 (m, 2H, CH2CH2Ph), 2.41 (t, 2H,
CH2CH2Ph), 3.07 (m, 2H, CH2Ph), 3.25 (m, 4H, CH2NCH2),
3.63 (m, 4H, CH2OCH2), 4.42 (q, 1H, α-H), 4.62 (s, broad,
1H, α-H), 4.88 (d, 1H, NH), 6.18 (d, 1H, NH), 6.52 (d, 1H,
CH=CH-SO2), 6.79 (dd, 1H, CH=CH-SO2, J= 15 Hz, 5 Hz)
7.05–7.37 (m, 10H, 2 × Ph), 7.50–7.93 (m, 5H, Ph-SO2).
13C NMR (100 MHz, CDCl3) δ 31.69, 35.49, 37.86, 43.89,
49.20, 56.25, 66.29, 126.31, 127.21, 127.64, 128.34, 128.57,
128.84, 129.21, 129.29, 130.73, 133.44, 136.67, 140.26,
140.32, 145.42, 157.21, 171.54.
1
isolated as a clear colorless syrup with a 32.2% yield. H
NMR (400 MHz, CDCl3) δ 1.74 (m, 1H, CH2CH2Ph), 1.85
(m, 1H, CH2CH2Ph), 2.25 (s, 3H, CH3N), 2.31 (m, 4H,
CH2N(CH3)CH2), 2.54 (m, 2H, CH2CH2Ph), 3.00 (m, 2H,
CH2Ph), 3.28 (m, 4H, CH2NCH2), 4.58 (broad, 2H, α-H,
α-H), 5.23 (d, 1H, NH), 6.10 (dd, 1H, CH=CH-SO2, J= 15
Hz, 2 Hz), 6.77 (dd, 1H, CH=CH-SO2, J= 15 Hz, 8 Hz),
7.01–7.33 (m, 11H, 2 × Ph, NH), 7.50–7.91 (m, 5H, Ph-SO2).
13C NMR (100 MHz, CDCl3) δ 31.77, 35.67, 38.57, 43.73,
46.02, 49.07, 54.45, 55.96, 126.22, 127.08, 127.63, 128.38,
128.52, 128.64, 129.28, 129.30, 130.32, 133.48, 136.67,
140.18, 140.47, 145.83, 156.95, 172.01. MS (ESI) m/z 575.2
[M+H]+.
General procedure for peptide coupling reaction using Cbz-
Phe-OH. Phenyl (3S)-3-(N-carbobenzyloxyphenylalanyl)
amino-4-phenylbut-1-enyl sulfone (9, Cbz-Phe-L-Phe-
VS-Ph) TFA salt 3 (TFA·L-Phe-VS-Ph) (0.946 g, 2.36
mmol) was dissolved in DCM (75 mL). Et3N (0.572 g, 2.76
mmol) was added, followed by Cbz-Phe-OH (0.776 g, 2.59
mmol), EDCl (0.497 g, 2.59 mmol) and HOBT (0.350 g,
2.59 mmol). Additional Et3N was added to adjust the pH
to between 9 and 10. e reaction was stirred overnight.
e reaction was washed with citric acid (10%, 3 × 25 mL),
aq. NaHCO3 (10%, 3 × 25 mL), brine (2 × 25 mL) and dried
(MgSO4). e solvent was removed under reduced
© 2013 Informa UK, Ltd.

472 B.D. Fennell et al.
Synthesis of phenyl (3R)-3-(N-(N-methylpiperazine)-4-car-
boxamidophenylalanyl)amino-5-phenylpent-1-enyl sulfone
(12, Mp-Phe-D-Hfe-VS-Ph) See general procedure for
peptide coupling using Mp-Phe-OH. TFA salt 2 (TFA·D-
Hfe-VS-Ph) was used as the starting material. e crude
product was purified using column chromatography
(silica, 5% MeOH in DCM, 0.5% Et3N). e product was
CDCl3) δ 2.71 (t, 2H, CH2CH2Ph), 2.87 (t, 2H, CH2CH2Ph),
3.21 (m, 2H, CH2Ph), 3.37 (m, 4H, CH2NCH2), 3.60 (dd,
2H, CH2SO2, J= 2 Hz, 8 Hz), 3.67 (m, 4H, CH2OCH2), 4.64
(m, 2H, α-H and C=CH), 4.84 (d, 1H, NH), 7.13–7.40 (m,
10H, 2 × Ph), 7.45–7.66 (m, 5H, Ph-SO2), 8.67 (s, broad,
1H, NH). 13C NMR (100 MHz, CDCl3) δ 33.71, 35.77, 37.72,
43.79, 54.47, 56.29, 66.30, 103.83, 126.00, 127.41, 128.27,
128.43, 128.63, 129.07, 129.13, 129.28, 133.83, 136.59,
138.33, 141.15, 145.45, 156.89, 171.05. MS (ESI) m/z 562.2
[M+H]+.
1
isolated as a clear colorless syrup with a 22% yield. H
NMR (400 MHz, CDCl3) δ 1.68 (m, 1H, CH2CH2Ph), 1.80
(m, 1H, CH2CH2Ph), 2.32 (s, 3H, CH3N), 2.41 (m, 6H,
CH2N(CH3)CH2 and CH2CH2Ph), 3.07 (m, 2H, CH2Ph),
3.32 (m, 4H, CH2NCH2), 4.41 (m, 1H, α-H), 4.62 (m, 1H,
α-H), 4.91 (d, 1H, NH), 6.33 (d, 1H, NH) 6.52 (dd, 1H,
CH=CH-SO2, J= 15 Hz, 2 Hz), 6.85 (dd, 1H, CH=CH-
SO2,J= 15 Hz, 8 Hz), 7.01–7.33 (m, 10H, 2× Ph), 7.50–7.91
(m, 5H, Ph-SO2). 13C NMR (100 MHz, CDCl3) δ 31.7, 35.52,
37.76, 43.65, 46.00, 49.17, 54.41, 56.28, 60.41, 126.27,
127.16, 127.64, 128.36, 128.56, 128.82, 129.23, 129.28,
130.68, 133.43, 136.76, 140.37, 145.5, 157.04, 171.68.
Synthesis of phenyl (Z)-3-(N-morpholine-4-carboxamido
phenylalanyl)amino-5-phenylpent-2-enyl sulfone (15, Z-Mu-
Phe-Hfe-AS-Ph) See above procedure for vinyl sulfone
isomerization. Compound 8 (Mu-Phe-D-Hfe-VS-Ph)
was used as the starting material. e crude product was
purified via column chromatography (silica, 1% MeOH in
DCM) and isolated as a white powder with an 8.6% yield.
1H NMR (400 MHz, CDCl3) δ 2.71 (t, 2H, CH2CH2Ph), 2.87
(t, 2H, CH2CH2Ph), 3.21 (m, 2H, CH2Ph), 3.37 (m, 4H,
CH2NCH2), 3.60 (dd, 2H, CH2SO2, J= 1 Hz, 8 Hz), 3.67 (m,
4H, CH2OCH2), 4.64 (m, 2H, α-H and C=CH), 4.84 (d, 1H,
NH), 7.13–7.40 (m, 10H, 2 × Ph), 7.45–7.66 (m, 5H, Ph-
SO2), 8.67 (s, broad, 1H, NH). 13C NMR (100 MHz, CDCl3)
δ 33.71, 35.77, 37.72, 43.79, 54.47, 56.29, 66.30, 103.83,
126.00, 127.41, 128.27, 128.43, 128.63, 129.07, 129.13,
129.28, 133.83, 136.59, 138.33, 141.15, 145.45, 156.90,
171.06. MS (ESI) m/z 562.2 [M+H]+.
Synthesis of phenyl (3S)-3-(N-(N-methylpiperazine)-4-
carboxamidophenylalanyl)amino-5-phenylbut-1-enyl sul-
fone (13, Mp-Phe-L-Phe-VS-Ph) See general procedure for
peptide coupling using Mp-Phe-OH. TFA salt 3 (TFA·L-
Phe-VS-Ph) was used as the starting material. e crude
product was purified using column chromatography
(silica, 5% MeOH in DCM, 0.5% Et3N). e product was
1
isolated as a clear colorless syrup with a 45.1% yield. H
NMR (400 MHz, CDCl3) δ 2.33 (m, 7H, CH2N(CH3)CH2
and CH2N(CH3)CH2), 2.85 (dd, 2H, CH2Ph, J= 6 Hz and 3
Hz), 3.01 (m, 2H, CH2Ph), 3.28 (m, 4H, CH2NCH2), 4.41 (q,
1H, α-H), 4.76 (d, 1H, NH), 4.93 (m, 1H, α-H), 6.03 (dd,
1H, CH=CH-SO2, J= 15 Hz, 2Hz), 6.22 (d, 1H, NH), 6.84
(dd, 1H, CH=CH-SO2), J= 15 Hz, 5 Hz), 7.01–7.30 (m, 10H,
2 × Ph), 7.50–7.83 (m, 5H, Ph-SO2). 13C NMR (100 MHz,
CDCl3) δ 37.97, 40.17, 43.67, 54.48, 55.89, 127.07, 127.22,
127.68, 128.62, 128.68, 128.87, 129.16, 129.24, 129.29,
129.35, 130.86, 133.44, 135.51, 136.73, 140.11, 144.76,
156.78, 171.36.
Synthesis of phenyl (E)-3-(N-carbobenzyloxyphenylalanyl)
amino-4-phenylbut-2-enyl sulfone (16, E-Cbz-Phe-Phe-
AS-Ph) See above procedure for vinyl sulfone isomer-
ization. Compound 9 (Cbz-Phe-L-Phe-VS-Ph) was used
as the starting material. e reaction was stirred for
70 min at −20°C. e crude product was purified via col-
umn chromatography (silica, 2:1 Hex: EtOAc). e final
compound was isolated as a white powder with a 15.8%
1
yield. H NMR (400 MHz, CDCl3) δ 3.03 (m, 2H, CH2Ph),
3.67–3.89 (m, 4H, PhCH2C=CHCH2SO2), 4.35 (d, 1H, α-H),
4.83 (t, 1H, C=CH), 5.12 (s, broad, 3H, NH, OCH2Ph),
7.09–7.39 (m, 15H, 3 × Ph), 7.45–7.75 (m, 5H, Ph-SO2). 13
C
Preparation of allyl sulfones: General procedure for vinyl
sulfone isomerization. Phenyl (E)-3-(N-morpholine-4-carbox
amidophenylalanyl)amino-5-phenylpent-2-enyl sulfone (14,
E-Mu-Phe-Hfe-AS-Ph) To dry THF (40 mL), t-BuOOH
(4.01 mmol, 1.2 mL, 3.3 M in toluene) was added drop-
wise under argon at −78°C, followed by n-BuLi (1.02
mmol, 0.65 mL, 1.6 M in hexane). Compound 7 (Mu-Phe-
L-Hfe-VS-Ph) (0.500 g, 0.890 mmol) was dissolved in
dry THF (5 mL) and added dropwise. e reaction was
allowed to warm to −20°C and stirred for 9 h. e reac-
tion was quenched with aq. NH4Cl (10%, 50 mL) and the
aqueous layer was extracted with EtOAc (3 × 50 mL). e
organic layers were combined and washed with water
(3 × 50 mL), brine (2 × 50 mL) and dried (MgSO4). e
solvent was removed under reduced pressure and the
crude product was purified via column chromatography
(silica, 1% MeOH in DCM). e product was isolated as
NMR (100 MHz, CDCl3) δ 37.75, 40.14, 54.47, 56.77, 67.40,
106.61, 126.76, 127.29, 128.24, 128.26, 128.29, 128.48,
128.53, 128.98, 129.16, 129.17, 129.24, 133.93, 135.95,
135.97, 137.36, 138.31, 144.45, 156.02, 170.11.
Synthesis of phenyl (Z)-3-(N-carbobenzyloxyphenylalanyl)
amino-4-phenylbut-2-enyl sulfone (17, Z-Cbz-Phe-Phe-
AS-Ph) See above procedure for vinyl sulfone isomeriza-
tion. Compound 10 (Cbz-Phe-D-Phe-VS-Ph) was used as
the starting material. e reaction was stirred for 70min
at −20°C. e product was purified by column chromatog-
raphy (silica, 2:1 Hex: EtOAc) and isolated as a white pow-
der with a 16.7% yield. 1H NMR (400 MHz, CDCl3) δ 3.03
(m, 2H, CH2Ph), 3.67–3.89 (m, 4H, PhCH2C=CHCH2SO2),
4.35 (d, 1H, α-H), 4.83 (t, 1H, C=CH), 5.12 (s, broad, 3H,
NH, OCH2Ph), 7.09–7.39 (m, 15H, 3 × Ph), 7.45–7.75 (m,
5H, Ph-SO2). 13C NMR (100 MHz, CDCl3) δ 37.76, 40.16,
1
a white powder with a 6.7% yield. H NMR (400 MHz,
Journal of Enzyme Inhibition and Medicinal Chemistry

Optimization of peptidyl allyl sulfones 473
54.49, 56.80, 67.38, 106.67, 126.77, 127.28, 128.24, 128.29,
128.48, 128.54, 128.60, 128.62, 128.97, 129.16, 129.18,
129.25, 133.94, 136.00, 137.37, 138.31, 144.38, 156.07,
170.1.
α-H and NH), 7.06–7.41 (m, 10H, 2 × Ph), 7.46–7.85 (m,
5H, Ph-SO2), 8.45 (s, 1H, NH). 13C NMR (100 MHz, CDCl3)
δ 37.58, 40.20, 43.60, 52.93, 54.40, 54.63, 56.25, 105.78,
126.65, 127.16, 128.29, 128.42, 128.89, 129.12, 129.21,
129.24, 133.84, 136.64, 137.57, 138.45, 144.39, 156.83,
171.15. MS (ESI) m/z 561.2 [M+H]+.
Synthesis of phenyl (E)-3-(N-(N-methylpiperazine)-4-
carboxamidophenylalanyl)amino-5-phenylpent-2-enyl
sulfone (18, E-Mp-Phe-Hfe-AS-Ph) See above procedure
for vinyl sulfone isomerization. Compound 11 (Mp-Phe-
L-Hfe-VS-Ph) was used as the starting material. e reac-
tion was stirred for 8h at −20°C and stored at −20°C for
an additional 12h. e crude product was purified via
column chromatography (silica, 3% MeOH in DCM, 0.5%
Et3N) and isolated as a white powder with a yield of 8.2%.
1H NMR (400MHz, CDCl3) δ 2.29 (s, 3H, CH2N(CH3)CH2),
2.37 (m, 4H, CH2N(CH3)CH2), 2.69 (t, 3H, CH2CH2Ph),
2.83 (t, 3H, CH2CH2Ph), 3.18 (m, 2H, CH2Ph), 3.41 (m. 4H,
CH2NCH2), 3.59 (m, 2H, CH2-SO2), 4.61 (q, 1H, α-H), 4.67
(t, 1H, C=CH), 4.86 (d, 1H, NH), 7.13–7.40 (m, 10H, 2×Ph),
7.44–7.68 (m, 5H, Ph-SO2), 8.61 (s, 1H, NH).13C NMR
(100MHz, CDCl3) δ 33.73, 35.88, 37.72, 43.62, 46.06, 54.44,
54.49, 56.37, 103.91, 125.97, 127.32, 128.30, 128.42, 128.62,
129.01, 129.10, 129.32, 133.78, 136.68, 138.44, 141.16,
145.20, 156.84, 171.13. MS (ESI) m/z 575.2 [M+H]+.
Procedure for elimination of Mu-Phe-Hfe-VS-Ph. (2S)-N-
(5-phenylpent-1-en-3-ylidene)-2-(N-morpholine-4-car-
boxamidophenylalanyl)amino-3-phenylpropanamide. (21,
Mu-Phe-Hfe-Elim) See general procedure for isomeriza-
tion of the vinyl sulfone. Compound 7 was used as the
starting material. e reaction was allowed to warm to
room temperature and instead of adding both n-BuLi and
t-BuOOH at −78°C, only n-BuLi was added. e product
waspurifiedbycolumnchromatography(silica,3%MeOH
in DCM) and isolated as a white powder with a 6.4% yield.
1H NMR (400 MHz, CDCl3) δ 2.13 (m, 1H, CH2CH2Ph),
2.44 (m, 2H, 1H of CH2CH2Ph, 1H of CH2CH2Ph), 2.63
(m, 1H, CH2CH2Ph), 2.92 (m, 2H, CH2NCH2), 3.11 (m, 1H,
CH2Ph), 3.26 (m, 3H, 1H of CH2Ph, 2H, of CH2NCH2), 3.52
(m, 2H, CH2OCH2), 3.63 (m, 2H, CH2OCH2), 4.50 (t, 1H,
α-H), 5.02 (m, 2H, CH=CH2), 6.11 (dd, 1H, CH=CH2, J= 19
Hz, 11 Hz), 7.10–7.35 (m, 10H, 2 × Ph), 7.60 (s, broad, 1H,
NH). 13C NMR (100 MHz, CDCl3) δ 29.60, 37.41, 37.71,
46.80, 61.97, 66.38, 78.62, 114.24, 126.25, 127.02, 128.32,
128.60, 129.77, 129.82, 136.55, 140.60, 140.97, 159.09,
172.24. MS (ESI) m/z 442.2 [M+Na]+.
Synthesis of phenyl (Z)-3-(N-(N-methylpiperazine)-4-
carboxamidophenylalanyl)amino-5-phenylpent-2-enyl
sulfone (19, Z-Mp-Phe-Hfe-AS-Ph) See above procedure
for vinyl sulfone isomerization. Compound 12 (Mp-Phe-
D-Hfe-VS-Ph) was used as the starting material. e reac-
tion was stirred for 8h at −20°C and stored at −20°C for
an additional 12h. e crude product was purified via
column chromatography (silica, 5% MeOH in DCM, 0.5%
Et3N) and isolated as a white powder with a 6.7% yield.
1H NMR (400MHz, CDCl3) δ 2.28 (s, 3H, CH2N(CH3)CH2),
2.37 (m, 4H, CH2N(CH3)CH2), 2.68 (t, 3H, CH2CH2Ph),
2.83 (t, 3H, CH2CH2Ph), 3.18 (m, 2H, CH2Ph), 3.39 (m, 4H,
CH2NCH2), 3.59 (m, 2H, CH2-SO2), 4.61 (q, 1H, α-H), 4.67
(t, 1H, C=CH), 4.86 (d, 1H, NH), 7.13–7.40 (m, 10H, 2×Ph),
7.44–7.68 (m, 5H, Ph-SO2), 8.61 (s, 1H, NH). 13C NMR
(100MHz, CDCl3) δ 33.74, 35.88, 37.71, 43.62, 45.92, 53.44,
54.44, 56.37, 103.89, 125.97, 127.32, 128.30, 128.42, 128.62,
129.01, 129.10, 129.32, 133.78, 136.68, 138.45, 141.16,
145.20, 156.84, 171.12. MS (ESI) m/z 575.2 [M+H]+.
Enzyme assays
General assay parameters
All assays used the incubation method to measure the
irreversible inhibition of the specified enzyme49. Enzyme
activity was observed via monitoring hydrolysis of vari-
ous fluorogenic 7-amino-4-methylcoumarin (AMC) sub-
strates using a BioTek Flx800 Plate Reader (λex = 360 nm,
λem = 460 nm). Enzyme activity was measured at the spec-
ified incubation temperature. All assay conditions con-
tained less than 8.3% dimethylsulfoxide (DMSO). Pseudo
first-order inactivation rate constants were obtained from
ln vt/v0 versus time plots.
Cathepsin B Assay40 Human liver cathepsin B (0.259 mg/
mL) was purchased from Calbiochem. A D-10 dilution of
cathepsin B was prepared with a 5 µL aliquot of the stock
enzyme and 45 µL of deionized water. A D-100 activation
dilution was prepared by combining 267 µL kinetic buffer
(0.1 M potassium phosphate, 1.25 mM EDTA, 0.01% Brij
35 at pH 6.0), 30 µL of the D-10 solution and 3 µL of 0.1M
dithiothreitol (DTT) (freshly prepared). e D-100 acti-
vation dilution was allowed to activate for 1 h at 0°C. e
incubation solution contained 30 µL of inhibitor stock (10
µM-5 mM in DMSO), 300 µL kinetic buffer and 30 µL of
D-100 enzyme prep at 25°C. Aliquots (50 µL) of the incu-
bation solution were withdrawn at varying intervals and
combined with 200 µL of substrate buffer (500 µM Cbz-
Arg-Arg-AMC [Calbiochem Cathepsin B Substrate III],
Synthesis of phenyl (E)-3-(N-(N-methylpiperazine)-4-
carboxamidophenylalanyl)amino-5-phenylbut-2-enyl sul-
fone (20, E-Mp-Phe-L-Phe-AS-Ph) See above procedure
for vinyl sulfone isomerization. Compound 13 (Mp-Phe-
L-Phe-VS-Ph) was used as the starting material. e
reaction was stirred for 3 h at −20°C. e product was
purified via column chromatography (silica, 3% MeOH
in DCM, 0.5% Et3N) and isolated as a white powder with
1
a yield of 7.2%. H NMR (400 MHz, CDCl3) δ 2.27 (s, 3H,
CH2N(CH3)CH2), 2.34 (m, 4H, CH2N(CH3)CH2), 2.93–3.18
(m, 2H, CH2Ph), 3.32 (m, 4H, CH2NCH2), 3.61–3.91 (m,
4H, CH2-SO2 and CH2Ph), 4.46 (q, 1H, α-H), 4.79 (m, 2H,
© 2013 Informa UK, Ltd.

474 B.D. Fennell et al.
0.25% DMSO v/v, 0.1 M potassium phosphate, 1.25 mM
EDTA, 0.01% Brij at pH 6.0,) for assaying.
Calpain I Assay40 Calpain I from porcine erythrocytes
(1.9 mg/mL) was purchased from Calbiochem. e
incubation buffer was prepared from 2883 µL of HEPES
pH 7.5 buffer, 39 µL of CaCl2 (0.5 M) and 78 µL of 0.5 M
cysteine (freshly prepared). e incubation solution con-
tained 30 µL inhibitor stock (10 µM–5 mM in DMSO), 30
µL enzyme stock (1.9 mg/mL) and 300 µL of incubation
buffer at 25°C. Aliquots (50 µL) of the incubation solution
were withdrawn at varying intervals and combined with
200 µL of substrate buffer (9524 µL HEPES pH 7.5 buf-
fer, 80 µL 0.2 M Suc-Leu-Tyr-AMC in DMSO [Calbiochem
Calpain I Substrate II], 214 µL 0.5 M cysteine and 107 µL
0.5 M CaCl2) for assaying.
Scheme 2. Isomerization equilibrium and elimination
mechanism.
2). Subsequent protonation of the carbon adjacent
to the sulfone furnishes allyl sulfones 14–2052. It was
found that the choice of base, temperature and reac-
tion duration were all significant factors affecting the
isomerization. Previously reported methods of isom-
erization used KOtBu52, DBU53 or n-BuLi/t-BuOOH40.
e use of 1 eq. of n-BuLi and 4.5 eq. of t-BuOOH at
−20°C resulted in nearly complete isomerization while
the other bases, regardless of duration or temperature,
produced a mixture of compounds which were not
purified further. Hine et al.52,54 demonstrated that with
a relatively low free energy barrier of isomerization,
an equilibrium exists between the two isomers (vinyl
and allyl) resulting in incomplete isomerization. Along
with this equilibrium, we observed an additional side
product, formed by elimination of the sulfonyl moiety
(Scheme 2). Elimination occurs after formation of the
allyl sulfone, when the adjacent nitrogen is deproton-
ated, eliminating the sulfonyl group and forming an
extended conjugated system. Higher temperatures
(>−20°C) or fewer equivalents of t-BuOOH favoured
elimination. Elimination was only observed for the
Hfe-based compounds7,8,11–13. We purified one ana-
logue side product (Mu-Phe-Hfe-Elim) and added it to
the selection of compounds to be tested for inhibitor
potency with the various proteases.
Cruzain Assay e enzyme stock (50 µM) was diluted
2000 fold (25 nM) using assay buffer (100 mM sodium
acetate pH 5.5, 5 mM DTT, 0.001% Triton X-100, freshly
prepared). e incubation solution contained 12.5 µL
inhibitor stock (10 µM-5 mM in DMSO), 12.5 µL D-2000
enzyme solution and 350 µL assay buffer. Aliquots (50
µL) of the incubation solution were withdrawn at varying
intervals and combined with 200 µL of substrate buffer
(4998 µL assay buffer and 2 µL 20 mMCbz-Phe-Arg-AMC
in DMSO [Enzo Life Sciences]) for assaying.
Trypsin Assay50 Trypsin from bovine pancreases was
acquired from Sigma (Milwaukee, WI). e incubation
solution contained 230 µL of buffer (50mM sodium phos-
phate buffer, pH 8.0), 60 µL of trypsin (2.5 µM in buffer)
and 10 µL inhibitor stock (10 µM-5mM in DMSO) at 37°C.
Aliquots (50 µL) of the incubation solution were withdrawn
at varying intervals and combined with 200 µL of substrate
buffer (4 µL of 22.7 mMCbz-Phe-Arg-AMC in DMSO [Enzo
Life Sciences] in 2400 µL of buffer) for assaying.
Enzyme expression and purification
Cruzain
Cruzain was expressed and purified using a modified
version (Lee G., Craik CS, unpublished results) of a pre-
viously published protocol51, activated and purified as
recently described32.
To prevent self-degradation, active cruzain was inhib-
ited with S-methyl methanethiosulfonate (MMTS), a
covalent reversible inhibitor. MMTS inhibited cruzain
was further concentrated to 50 µM in 100 mM MES pH
5.8, 500 mMNaCl and stored at −80 °C.
We suspect that the acidic property of the t-BuOOH
partially attenuates the strength of the n-BuLi, giv-
ing rise to a milder, buffered environment. is has
not previously been reported in the literature. e
mild conditions seem to favour the formation of the
allyl sulfone while minimizing further elimination of
the sulfonyl group. Addition of n-BuLi in absence of
t-BuOOH exclusively results in complete elimination of
the sulfonyl group, with no allyl sulfone intermediate
observed. Chelation of Li+55 as well as the “syn-effect,”
forming a homoaromatic system between Li+ and the
delocalized anionic intermediate53,56, may be factors
that explain the requirement of n-BuLi instead of DBU
or KOtBu, assisting in the stabilization of the delocal-
ized intermediate.
Results and discussion
Formation of the allyl sulfone
One of the key challenges in synthesizing the inhibi-
tors was finding the correct vinyl-allyl sulfone isom-
erization conditions. e proposed mechanism
of allyl isomerization occurs by deprotonation of the
α-proton forming a delocalized intermediate (Scheme
Reaction time was also a key factor in successful
isomerization. Compounds 9, 10 and 13 (R-Phe-Phe-
VS-Ph) with a Phe residue in the P1 position required less
Journal of Enzyme Inhibition and Medicinal Chemistry

Optimization of peptidyl allyl sulfones 475
than two hours to isomerize while the remaining vinyl
sulfones (7, 8, 11 and 12; R-Phe-Hfe-VS-Ph) with a Hfe
residue in the P1 position required more than nine hours.
is variation in reaction rate has been primarily attrib-
uted to sterics. e increased mobility introduced by the
additional methylene of the Hfe residue, compared to the
Phe side chain, allows the phenyl ring to impede access
of the base to the α-proton, slowing the reaction rate and
extending the required isomerization time.
We performed 2D NOESY experiments on the P1 Hfe-
based allyl sulfones (14, 15, 18 and 19). is method
could not be applied to the P1 Phe-based compounds (16,
17 and 20). With only a single methylene group directly
adjacent to the allyl sulfone double bond, long range
J-coupling between the vinyl proton and benzyl methy-
lene of the Phe made the NOE indistinguishable from the
residual COSY cross peaks. However, with an additional
carbon present between the vinyl proton and the benzyl
methylene in the P1 Hfe-based compounds, long range
J-coupling did not disrupt the resolution of the NOE.
We focused on the cross peak magnitude between the
2.71 and 4.64ppm resonances, representing the spatial
coupling between the protons of benzyl methylene of the
Hfe residue and the vinyl proton, respectively. e differ-
ence in spectra is shown in Figure 3 (14 in blue and 15 in
red). eindividualpeakswereintegratedusingequivalent
contour levels and normalized against multiple residual
COSY peaks present in each spectrum (Table 1). e allyl
sulfones formed from the L-Hfe vinyl sulfones 14 and 18
display a 30% weaker NOE compared with their D-Hfe ana-
logues 15 and 19. is allowed us to conclude that the allyl
sulfones derived from the D-Hfe vinyl sulfone analogues
exhibit Z configuration while those derived from the L-Hfe
vinyl sulfone analogues possess E configuration.
Stereochemistry of allyl sulfones
Following the synthesis of allyl sulfones 14 and 15
(Mu-Phe-L/D-Hfe-AS-Ph), the compounds were assayed
for inhibitory potency with cathepsin B and cruzain.
Although the two compounds have identical 1D 1H
NMR spectra (all spectroscopic data is provided in the
Supporting Appendix), they differ in their ability to
inhibit the enzymes by an order of magnitude, indica-
tive of a significant difference in structure (see Enzyme
Kinetics section, Table 2). e only variation in structure
1
that would be undetectable by H NMR is the stereo-
chemistry of the allyl sulfone double bond. Because of
the presence of only a single proton on the double bond,
no distinctive J-coupling is produced with either E or Z
configurations. is difference in stereochemistry arises
during the isomerization process. With a different spatial
orientation, the steric bulk of the P1 residue side chain
can either favour E or Z configuration.
Enzyme kinetics
e synthesized dipeptidyl allyl sulfones 14–20 were
assayed with cathepsin B, cruzain, calpain I and trypsin
using fluorogenic enzyme assays and the incubation
method. Pseudo-first order kinetics were applied to
calculate kobs/[I] values (Table 2)59. e Cbz-containing
compounds 16,17 unfortunately were insoluble in the
In an effort to deduce the stereochemistry of both ana-
logues, we used Nuclear Overhauser Effect Spectroscopy
(NOESY) experiments based on the difference in the spa-
tial arrangement around the double bond, which should
produce unique spatial magnetic coupling. e E and Z
isomers of the Mu-Phe-Hfe-AS-Ph structure were mod-
elled using molecular dynamics calculations by MOE
to predict Nuclear Overhauser Effect (NOE) potentials.
Jones et al.57 have demonstrated that using energetically
minimized models to predict NOE effects produced less
than 4% error from the experimentally observed NOE. An
MMFF94x potential was used to minimize the energetics
ofthemodelmoleculesusingasolvationparameterbased
on distance with a relative dielectric constant of 4.801 to
match the solvation of the samples in chloroform during
NMR acquisition. Distances were measured between the
alkene vinyl proton and the benzyl methylene of the Hfe
side chain. Inter-proton distances of 4.01 and 2.22 Å were
calculated for the E and Z isomers, respectively (Figure
2). Based on this information, a weak NOE should be
observed for the E-isomer (3.5–5 Å) while the Z-isomer
should display a strong NOE (<2.5 Å)58.
Figure 2. Energy minimized models of the E and Z isomers of
Mu-Phe-Hfe-AS-Ph.
Table 1. Quantification of relative NOE magnitude and the deduced stereochemistry.
Compound
Structure
Vinyl sulfone precursor
Mu-Phe-L-Hfe-VS-Ph
Mu-Phe-D-Hfe-VS-Ph
Mp-Phe-L-Hfe-VS-Ph
Mp-Phe-D-Hfe-VS-Ph
Normalized NOE
Configuration
14
15
18
19
Mu-Phe-Hfe-AS-Ph
Mu-Phe-Hfe-AS-Ph
Mp-Phe-Hfe-AS-Ph
Mp-Phe-Hfe-AS-Ph
0.688
1.00
E
Z
E
Z
0.749
1.00
© 2013 Informa UK, Ltd.

476 B.D. Fennell et al.
assays. All the remaining compounds 14,15,18–21 dis-
played time dependent, irreversible inhibition.
cruzain compared with the Mp group (6080 1390 M−1s−1
for 14 versus 2310 230 M−1s−1 for 18).
All inhibitors 14,15,18–21 demonstrated specificity
for cysteine proteases, with no inhibition observed for
trypsin, a serine protease with similar substrate specific-
ity as cruzain. e same fluorogenic substrate, Cbz-Phe-
Arg-AMC, was used to assay both enzymes. Additionally,
specificity for clan CA, family C1 was observed with
negligible inhibition (<10 M−1s−1) of calpain I (family C2).
Within clan CA, all of the allyl sulfones (14, 15, 18 and
20) showed approximately 10-fold selectivity for cruzain
over cathepsin B except compound 19 (Z-Mp-Phe-Hfe-
AS-Ph), which demonstrated no selectivity between
the two enzymes (200 40 M−1s−1 for cathepsin B and
130 10 M−1s−1 for cruzain).
e allyl sulfone double bond stereogenicity also con-
tributed significantly to inhibitory potency. Examining
E-allyl sulfones (14 and 18) and Z-allyl sulfones (15 and
19), the E stereochemistry resulted in an order of magni-
tude greater kobs/[I] values for all cysteine proteases tested.
Comparing isomers 14 and 15, kobs/[I] values for cruzain
were 6080 1390M−1s−1 and 260 80M−1s−1, respectively. A
similar difference in potency was observed for cathepsin B.
Diastereomers 18 and 19 displayed the same trend as well.
e P1 amino acid residue selection also played a key
role in inhibitor potency. Although the stereochemistry
of compound 20 (Mp-Phe-Phe-AS-Ph) could not be
determined using the previously discussed NOE method
(section 2.2), the kinetic data provides a clear indica-
tion that regardless of double bond stereogenicity, the
Phe residue in the P1 position resulted in a decrease in
potency. For cruzain, compound 20 had an inhibition
rate constant of 45 1 M−1s−1. With the assumption that
compound 20 would follow the same isomerization
mechanism as other allyl sulfones formed from vinyl sul-
fones with L-stereochemistry in their P1 residue, it should
likely have E configuration. In that case, it is 100-fold less
potent than its P1 Hfe analogue 18 (2310 230 M−1s−1).
Even if the above assumption is not valid and compound
20 adopted Z stereochemistry, the Z-Hfe analogue 19 still
remains more potent (130 10 M−1s−1). e same trends
were observed with cathepsin B with an even greater
decrease in potency with the presence of the P1 Phe resi-
due. Further optimization of additional analogues with
P1 Phe residues as well as investigation into absolute
stereochemistry was abandoned.
Comparing the potencies of inhibitors 14 (P3 = Mu)
versus 18 (P3 = Mp), the P3 group exhibited almost neg-
ligible effect on potency for cathepsin B (420 110 M−1s−1
for 14 versus 570 20 M−1s−1 for 18). In contrast, the Mu
substituent achieved a 3-fold increase in potency for
e most potent of the synthesized inhibitors was
compound 14 (E-Mu-Phe-Hfe-AS-Ph) with a kobs/[I] of
6080 1390M−1s−1 for cruzain. For comparative purposes,
compound 11 (Mp-Phe-L-Hfe-VS-Ph, also K11777)42 was
assayed with cruzain (221000 8000M−1s−1) and displayed
10-fold selectivity over cathepsin B (39300 1300M−1s−1).
Although none of the inhibitors were able to achieve the
potency of K11777, equivalent selectivity was maintained.
e precise mechanism of inhibition of the allyl sul-
fone moiety remains unknown. We have previously pro-
posed possible irreversible mechanisms (Scheme 3)40
Figure 3. NOESY comparison of 14 (blue) and 15 (red).
Table 2. Inhibitor potencies for proteases.
k
obs/[I] (M−1s−1)
Cruzain
6080 1390
260 80
NS
Compound
Inhibitor
Trypsin
NI
Cathepsin B
420 110
<10
Calpain I
<10
14
15
16
17
18
19
20
21
E-Mu-Phe-Hfe-AS-Ph
Z-Mu-Phe-Hfe-AS-Ph
Cbz-Phe-Phe-AS-Ph (isomer A)
Cbz-Phe-Phe-AS-Ph (isomer B)
E-Mp-Phe-Hfe-AS-Ph
Z-Mp-Phe-Hfe-AS-Ph
Mp-Phe-Phe-AS-Ph (isomer A)
Mu-Phe-Hfe-Elim
<10
NI
NS
NS
NS
NS
NI
NS
NS
NS
570 20
200 40
2310 230
130 10
<10
<10
NI
13
1
45
1
<10
NI
< 10
< 10
<10
NI
NS, not soluble; NI, no inhibition.
isomer A=from L-P1 vinyl sulfone residues, isomer B=from D-P1 vinyl sulfone residue.
Journal of Enzyme Inhibition and Medicinal Chemistry

Optimization of peptidyl allyl sulfones 477
B will be necessary to extend our understanding of the
allyl sulfone inhibition mechanism. In addition, further
modification of the current design will be necessary to
achieve or exceed the potency of K11777.
Acknowledgments
We would like to thank James McKerrow and his lab at
the University of California − San Francisco for providing
cruzain for enzyme inhibition analysis.
Declaration of interest
Scheme 3. Potential mechanisms of inhibition.
is material is based upon work supported by the
National Science Foundation under Grant No. 0922775.
e Howard Hughes Medical Institute provided addi-
tional research funding.
including a SN2 attack by the active site thiolate on the
carbon adjacent to the sulfone, resulting in the elimina-
tion of the sulfone (a); a Michael addition to the double
bond and the resulting elimination of the sulfone (b);
and the direct elimination of the sulfone group, forming
the enamide, which is then subject to a Michael addition
via the thiolate on the terminal alkene carbon (c).
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