Novel peptidyl aryl vinyl sulfones as highly potent and selective inhibitors of cathepsins L and B

Article abstract of DOI:10.1002/cmdc.201000109

Herein we present the design, synthesis, and evaluation of a structurally novel library of 20 peptidyl 3-aryl vinyl sulfones as inhibitors of cathepsins L and B. The building blocks, described here for the first time, were synthesized in a highly efficient and enantioselective manner, starting from 3-aryl-substituted allyl alcohols. The corresponding vinyl sulfones were prepared by a new approach, based on a combination of solid-phase peptide synthesis using the Fmoc/tBu strategy, followed by solution-phase coupling to the corresponding (R)-3-amino-3-aryl vinyl sulfones as trifluoroacetate salts. The inhibitory activity of the resulting compounds against cathepsins L and B was evaluated, and the compound exhibiting the best activity was selected for enzymatic characterization. Finally, docking studies were performed in order to identify key structural features of the aryl substituent.

Full text of DOI:10.1002/cmdc.201000109

MED
DOI: 10.1002/cmdc.201000109
Novel Peptidyl Aryl Vinyl Sulfones as Highly Potent and
Selective Inhibitors of Cathepsins L and B
Laura Mendieta,[a] Anna Picꢀ,[b] Teresa Tarragꢀ,[a] Meritxell Teixidꢀ,[a] Marcos Castillo,[b]
LlorenÅ Rafecas,[c] Albert Moyano,*[b] and Ernest Giralt*[a, b]
Herein we present the design, synthesis, and evaluation of a
structurally novel library of 20 peptidyl 3-aryl vinyl sulfones as
inhibitors of cathepsins L and B. The building blocks, described
here for the first time, were synthesized in a highly efficient
and enantioselective manner, starting from 3-aryl-substituted
allyl alcohols. The corresponding vinyl sulfones were prepared
by a new approach, based on a combination of solid-phase
peptide synthesis using the Fmoc/tBu strategy, followed by so-
lution-phase coupling to the corresponding (R)-3-amino-3-aryl
vinyl sulfones as trifluoroacetate salts. The inhibitory activity of
the resulting compounds against cathepsins L and B was eval-
uated, and the compound exhibiting the best activity was se-
lected for enzymatic characterization. Finally, docking studies
were performed in order to identify key structural features of
the aryl substituent.
Introduction
Cathepsins constitute a widespread group of proteases, a ma-
jority of which belong to the C1 family of the cysteine pro-
tease clan CA (CAC1).[1,2] Cathepsins are implicated in a variety
of processes, including protein turnover, enzyme activation,
hormone maturation, bone resorption, antigen presentation
and epidermal homeostasis.[2,3,4] On the basis of sequence simi-
larity, CAC1 cysteine proteases are divided into two groups:
cathepsin L-like (which includes mammalian cathepsins L, K,
and S, protozoan falcipains, rhodesain, cruzain, and CPA and
CPB Leishmania proteases), and cathepsin B-like (including
cathepsin B and the CPC Leishmania proteases).[2]
In healthy cells, cathepsins are localized in lysosomes.[2,4,5] In-
itially, they were thought to be housekeeping enzymes, but
they are now recognized to have other functions in healthy
cells: they process proteins in the nucleus and intracellular or-
ganelles, such as hormone secretory granules, and play specific
roles in physiological processes, such as bone remodeling, and
epidermal homeostasis.[6] Furthermore, they are highly regulat-
ed at several stages of the cell cycle, and their expression
levels vary depending on the tissue.[5]
and clearly evident in angiogenic islets and tumors, particularly
on the edges of the islet carcinomas.[7]
Cathepsin B degrades three of the most important basement
components,[7,9] resulting in proteolysis of surrounding tissues
and allowing cancer cell invasion.[10] Treatment of in vitro
human cancer cells[10] and of in vivo mice tumors with cathep-
sin inhibitors decreases the volume,[7] invasion[6] and growth of
tumors.[7,11] These data were confirmed by knockdown experi-
ments, with a decrease in cell proliferation observed in tumors
from cathepsin B (44% decrease) and cathepsin L (58%) knock-
out mice.[11] Furthermore, a decrease in vessel density of
tumors has been reported in response to treatment with cath-
epsin B and L inhibitors.[7] Additionally, selective inhibition of
the tumor-promoting cathepsins results in a decrease in tumor
invasiveness.[12]
The inhibition of cathepsins L and B has, therefore, recently
gained interest as a key strategy for the development of new
anticancer drugs. To date, many reversible and irreversible
cathepsin L and B inhibitors have been described. With respect
to reversible inhibitors, the reactive group can be an alde-
Cathepsins also play a key role in a number of human dis-
eases, such as rheumatoid arthritis and cancer. In cancer cells,
as well as in cytotoxic T lymphocytes and natural killer cells,
cathepsins are rerouted from lysosomes to the cell surface.[6] In
some types of cancer, cathepsins are also secreted to the exte-
rior.[6,7,8] Because the extracellular microenvironment of tumors
is generally acidic, cathepsins are still able to cleave other pep-
tides and proteins following translocation.[6] They also degrade
the basement and the extracellular matrix,[6,7,8] thereby pro-
moting angiogenesis and invasive tumor growth[7] and meta-
stasis.[7,8] The expression and activity of cathepsins are also
modified in cancer cells. There is a progressive increase in ca-
thepsin levels during tumorigenesis,[7,8] which correlates with a
higher malignancy of the tumor.[6,7,8] The activity is localized
[a] L. Mendieta, Dr. T. Tarragꢀ, Dr. M. Teixidꢀ, Prof. Dr. E. Giralt
Institute for Research in Biomedicine of Barcelona
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona (Spain)
Fax: (+34)934037126
E-mail: ernest.giralt@irbbarcelona.org
[b] Dr. A. Picꢀ, Dr. M. Castillo, Prof. Dr. A. Moyano, Prof. Dr. E. Giralt
Departament de Quꢁmica Orgꢂnica, Universitat de Barcelona
Martꢁ Franquꢃs, 1-11, 08028 Barcelona (Spain)
Fax: (+34)933397878
E-mail: amoyano@ub.edu
[c] L. Rafecas
ENANTIA S.L.
Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000109.
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Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
hyde,[13] an aldehyde-hydroxamic acid combination,[14] nonpep-
tidic cyanamide,[15] dipeptidyl nitrile[16] or b-lactam penam or
oxapenam compounds.[2] All of these are selective cathepsin L
inhibitors, except the dipeptidyl nitriles, which show higher af-
finity for cathepsin B. Irreversible inhibitors include E-64 ana-
logues[2] (mainly cathepsin B and L inhibitors) and alkyl vinyl
sulfones (cathepsin B, L, O2, and S inhibitors).[17,18,19] Peptidyl
vinyl sulfones comprise a group of potent, selective and cova-
lent inhibitors of cysteine proteases, with proven efficacy
against other cysteine proteases, such as cruzipain[2,20,21] and
cathepsins S,[22] K,[23] and F.[24]
merically pure 3-amino vinyl sulfones, all of which use a-amino
acids as starting materials and generally follow a synthetic
route that requires their reduction to a-amino aldehydes and a
subsequent Wadsworth–Emmons reaction with a sulfonylmeth-
yl phosphonate.[18] This imposes strong limitations on the
structural variability of the corresponding peptidyl vinyl sul-
fone derivatives. In particular, no aryl vinyl sulfone compounds
have been described in the literature as cathepsin inhibitors,
likely due to the easy racemization of the intermediate a-
amino-a-aryl aldehydes. Based on a general route to N-Boc-3-
amino vinyl sulfones previously developed in our laborato-
ries,[26] we herein report the synthesis of a structurally novel li-
brary of peptidyl 3-aryl vinyl sulfones as inhibitors of cathe-
psins L and B. These compounds were prepared by a novel
method using an Fmoc/tBu solid-phase synthesis strategy, fol-
lowed by solution-phase coupling to the corresponding 3-
amino-3-aryl vinyl sulfones. The inhibitory activities of these
compounds against cathepsins L and B were evaluated.
4-morpholinylcarbonyl-l-leucine-(1S)-(2-phenylethyl)-3-(phe-
nylsulfonyl)-(2E)-propenamide (Figure 1A), a peptidyl 3-alkyl
vinyl sulfone derived from the cathepsin S inhibitor homophe-
Results and Discussion
Library design and synthesis
The designed library was based on the scaffold presented in
Figure 2, in which X is either oxygen (making position 1 a mor-
pholinyl group) or an N-methyl group (resulting in a 4-methyl-
Figure 2. Proposed library of peptidyl 3-aryl vinyl sulfones.
piperazinyl group in position 1). R2 can be either isobutyl (so
that the amino acid in position 2 is Leu) or benzyl (yielding the
amino acid Phe in position 2). The highest variability was intro-
duced at the R1 position with five different groups: phenyl, 1-
naphthyl, 2-naphthyl, mesityl (2,4,6-trimethylphenyl), and 4’-bi-
phenyl, thereby enabling for the first time a systematic evalua-
tion of structure–activity relationships with respect to aryl
chain substituents in position 1 of the peptidyl vinyl sulfone.
The combination of all of the substituents in the scaffold gave
rise to 20 distinct structures for our library. It is worth noting
here that the structural diversity of this library can be easily in-
creased by sulfone substituent variations, which we restricted
to an unsubstituted phenyl group. To maintain the same chiral-
ity as known inhibitors, the absolute configuration of the two
stereogenic centers of this scaffold was S for the amino acid in
position 2 and R for the 3-amino-3-aryl vinyl sulfone.
Figure 1. Peptidyl vinyl sulfones previously described as cysteine protease
inhibitors.
nylalanine, has been identified as an antimalarial compound.[17]
Unfortunately, it was a potent but nonselective inhibitor.[17,25]
Compound N-(4-morpholinylcarbonyl)-l-phenylalanine-(1S)-(2-
phenylethyl)-3-(phenylsulfonyl)-(2E)-propenyl amide (Figure 1B)
was designed in a collaboration between pharmaceutical com-
panies.[25] Further modification of these original compounds
led to N-(4-methylpiperazin-1-ylcarbonyl)-l-phenylalanine-(1S)-
(2-phenylethyl)-3-(phenylsulfonyl)-(2E)-propenyl amide (Fig-
ure 1C), a potent cruzipain inhibitor[20] that is now under active
development as an anti-trypanosomal therapeutic compound.
The most common strategy for the preparation of peptidyl
vinyl sulfones relies on conventional methods of peptide syn-
thesis whereby the corresponding 3-amino vinyl sulfone is cou-
pled with amino acids or peptide derivatives. However, there
are very few methods available for the synthesis of enantio-
The peptidyl 3-aryl vinyl sulfones were synthesized in a
three-step strategy: coupling of the position 2 amino acid to
the resin, construction of the morpholineurea (Mu) or 4-meth-
ylpiperazineurea (Npipu) group, and solution-phase coupling
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E. Giralt et al.
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to 3-amino-3-aryl vinyl sulfones. The first two steps were done
in solid phase; the main advantage of this methodology is that
reactions can be driven to completion by use of excess re-
agents. Purification is performed after each step by simply
washing away the excess reagents. In contrast, more intensive
purification and characterization are required after each step in
solution-phase syntheses. In the synthesis of the peptidyl 3-
aryl vinyl sulfones, using the solid-phase method also enables
the preparation of a large quantity of the urea amino acid unit
that can later be divided for reactions with the various sulfones
in solution. The key N-Boc-(R)-3-amino-3-aryl vinyl sulfones 1–5
were prepared with good overall yields, complete diastereose-
lectivity (E configuration of the double bond), and excellent
enantiomeric purities (>95:5 er) using the synthetic sequence
outlined in Scheme 1. This sequence began with readily avail-
However, this protocol (using N-methylpiperazinocarbonyl
chloride) was not suitable for the synthesis of the N-methyl-
piperazine-derived ureas. Instead, these syntheses were ulti-
mately accomplished by reaction of the resin-bound amino
acid with carbonyl diimidazole in DMF, followed by treatment
with a large excess (20 equiv) of N-methylpiperazine. After
cleavage and purification, the target urea–amino acid conju-
gates Npipu-Phe-OH and Npipu-Leu-OH were isolated in 100
and 55% yield, respectively. The final solution-phase coupling
was carried out by reaction of the urea–amino acid fragments
with the (R)-3-amino-3-aryl vinyl sulfone trifluoroacetate salts
in the presence of DiPEA, using either PyBOP/HOAt (method A)
or N-cyclohexylcarbodiimide N’-methyl polystyrene HL (meth-
od B) in dichloromethane. The resulting peptidyl aryl vinyl sul-
fones 11–30 were purified by preparative HPLC (Scheme 2 and
Table 1).
Biological activity
The inhibitory activity of the library against cathepsins L and B
was evaluated. All inhibitors were analyzed fluorimetrically
using the human enzymes as well as substrate Z-Phe-Arg-AMC
for cathepsin L and Z-Arg-Arg-AMC for cathepsin B. The con-
centrations required for half-maximal inhibition (IC50) were cal-
culated as an initial screening assay and used to select the
most promising compounds.
All compounds exhibited greater inhibitory capacity toward
cathepsin L than cathepsin B (Table 2). The cathepsin inhibitory
efficacy of aryl vinyl sulfones, with regard to substituents, was
also determined. In general, compounds with a 4-methylpiper-
azinyl group in position 1 were more active than compounds
with a morpholinyl group. In position 2, Phe yielded better
Scheme 1. Reagents and conditions: a) PhSH, Et3N, CH2Cl2, CH3OH, reflux;
b) m-CPBA, CH2Cl2, RT (61–88% yield, two steps); c) morpho-CDI, cat. CuCl2,
CH3CN, RT (90–100% yield); d) 40% TFA in CH2Cl2, RT (quant).
able (E)-3-aryl-2-propenols and relied on the Sharp-
less catalytic epoxidation with d-(ꢀ)-diisopropyl tar-
trate as the chiral ligand for the establishment of the
absolute R configuration of the final compounds.[26]
Prior to the coupling step, N-Boc-(R)-3-amino-3-aryl
vinyl sulfones 1–5 were treated with a solution of tri-
fluoroacetic acid (TFA, 40% in dichloromethane) to
afford the corresponding trifluoroacetate salts 6–10.
For solid-phase synthesis of the urea–amino acid
moieties, we used a 2-chlorotrityl resin, following the
Fmoc/tBu strategy (0.7 equiv of Fmoc-aa-OH, 7 equiv
N,N-diisopropylethylamine (DiPEA) in dichlorome-
thane, room temperature; capped with methanol,
20% piperidine in N,N-dimethylformamide (DMF),
room temperature) to introduce the amino acid. The
coupling yields for Phe and Leu were 82 and 83%, re-
spectively. The Fmoc group was removed with 20%
piperidine in DMF. Condensation with 4-morpholino-
carbonyl chloride in the presence of DiPEA in DMF,
followed by cleavage from the resin with 2% TFA in Scheme 2. Reagents and conditions: a) Fmoc-aa-OH (0.7 equiv), DiPEA (1 equiv), CH2Cl2,
RT; b) 20% piperidine in DMF, RT; c) 4-morpholinocarbonyl chloride (1 equiv), DiPEA
dichloromethane, yielded the corresponding mor-
(2 equiv), DMF, RT; d) carbonyl diimidazole (5 equiv), N-methylpiperazine (20 equiv), DMF,
pholine-derived ureas. Purification by preparative
RT; e) 2% TFA in CH2Cl2, RT; f) method A: PyBOP (1.1 equiv), HOAt (2.1 equiv), DiPEA
HPLC afforded the desired compounds Mu-Phe-OH
(2.1 equiv), CH2Cl2, RT; g) method B: N’-methylpolystyrene HL (3 equiv), DiPEA (2.1 equiv),
and Mu-Leu-OH, in 70 and 76% yield, respectively. CH2Cl2, RT.
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Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
Table 1. Structures and yields for solution-phase coupling and purifica-
tion of the library compounds.
Compd
X
R1
R2
Method[a]
Yield [%][b]
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
O
O
O
O
O
O
O
O
O
Phenyl
Bn
Bn
Bn
Bn
Bn
iBu
iBu
iBu
iBu
iBu
Bn
Bn
Bn
Bn
Bn
iBu
iBu
iBu
iBu
iBu
A
A
A
A
B
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
78
10
6
1-Naphthyl
2-Naphthyl
Mesityl
4’-Biphenyl
Phenyl
1-Naphthyl
2-Naphthyl
Mesityl
4’-Biphenyl
Phenyl
1-Naphthyl
2-Naphthyl
Mesityl
4’-Biphenyl
Phenyl
1-Naphthyl
2-Naphthyl
Mesityl
18
44
56
28
16
30
43
26
33
61
29
38
28
81
57
46
51
Figure 3. Time-course experiment of compound 30 against cathepsin L.
O
therefore defined as an irreversible inhibitor of cathepsin L
with a second-order rate constant (k2) of 181.420 sꢀ1 mꢀ1. Kinet-
ic experiments for compounds 30 (against cathepsin L) and 23
(against cathepsin B) were performed in order to further study
the inhibitory mechanisms of action; Lineweaver–Burk plots
(Supporting Information figure 1) showed competitive inhibi-
tion of the respective proteases.
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
With regard to the interaction between inhibitors and pro-
teins, peptidyl aryl vinyl sulfones are well established covalent
inhibitors of cysteine proteases. As such, they undergo a Mi-
chael addition with the thiol group of the active site cysteine
to form an irreversible thioether bond.[17,18,19,27,28] This is also
our mechanistic hypothesis for the compounds described
herein.
4’-Biphenyl
[a] Method A: PyBOP as coupling agent; method B: N’-methyl polystyrene
HL as coupling agent. [b] Yield of coupling step after purification by prep-
arative HPLC.
results than Leu for cathepsin B, while cathepsin L did not dis-
criminate between these two residues. Finally, in position 3,
phenyl group compounds were preferred for cathepsin L while
2-napthyl compounds exhibited higher potency for cathe-
psin B. Compound 30 was not only the strongest inhibitor of
cathepsin L (IC50 =2.6 nm), but it also presented the highest se-
lectivity toward this protease (IC50 CatB/IC50 CatL =404). Compound
28 was also a potent inhibitor of cathepsin L (IC50 =4.9 nm).
The inhibitor with highest activity toward cathepsin B was
compound 23, although this compound did not demonstrate
selectivity (IC50 =304 nm; IC50 CatB/IC50 CatL =17). As a control, IC50
values of E-64 were calculated for cathepsins L (IC50 =8.3 nm)
and B (IC50 =2.3 nm).
Selectivity of peptidyl aryl vinyl sulfones for the cathepsins
was evaluated against serine proteases prolyl oligopeptidase
(POP) and dipeptidyl peptidase IV (DPP IV). Compounds 23 and
30 were tested at 100 and 500 mm, with no significant inhibi-
tion observed for DPP IV. In the case of POP, the IC50 values
were higher than 100 mm. (Supporting Information table 1).
Docking
To provide a structural basis for the data with our new peptidyl
aryl vinyl sulfones, we performed computational docking stud-
ies. Compounds 30 and 29 were docked to human cathepsin L
(PDB ID: 1CS8) and compound 23 to human cathepsin B (PDB
ID: 1HUC).[29] No covalent binding was considered in the dock-
ing studies. The protein was set as rigid; therefore, reported
distances may be smaller due to its flexibility as potential hy-
drogen bonds may be favored following first approach and
contact of the compound with the protein.
Docking of compound 30 in the cathepsin L active site
showed that the inhibitor extended from S2 to S2’ (Figure 4).
Aryl vinyl sulfone 30 interacts with active site Cys25 through
two potential hydrogen bonds, one with the oxygen atom of
position 2 and one with the oxygen atom of the sulfone group
(Figure 5A). Cys25 is also adjacent to the b-vinyl carbon; this
proximity may favor nucleophilic attack of the sulfur atom and
subsequent formation of a covalent bond (Figure 5B). Com-
pound 30 also interacts with active site His163 through a hy-
drogen bond (Figure 5C), while two other hydrogen bonds
might be formed after the inhibitor approaches the protein.
The sulfone, located near S2, is one of the groups that ap-
pears to interact most strongly with cathepsin, through two
hydrogen bonds and three more potential bonds involving
Cys25, Trp26, and Gly68 (Supporting Information figure 2). The
4’-biphenyl substituent is surrounded by Asn66 and Gln21 (Fig-
Elucidation of the inhibition mechanism
Time-course experiments of compound 30 against cathepsin L
were carried out to study the mode of inhibition and charac-
terize the kinetic parameters. The representation of enzyme ac-
tivity versus time confirmed that aryl peptidyl vinyl sulfone 30
was a time-dependent inhibitor (Figure 3). Compound 30 was
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E. Giralt et al.
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ure 5D). Other hydrogen bonds
may be possible in the interac-
tion between compound 30 and
cathepsin L, involving residues
Asp162, Gln19 and Trp189,
which may form favorable con-
tacts between these molecules.
Finally, the 4-methylpiperazinyl
group, located near S2’, may
form three hydrogen bonds with
residues Gln19 and Cys22 (Sup-
porting Information figure 3).
Table 2. Inhibition of cathepsins L and B by the library of peptidyl 3-aryl vinyl sulfones.
Compd
Substituents
R2
IC50 [nm]
X
R1
Cathepsin L
32
Cathepsin B
865
11
To further understand the dif-
ferences in inhibition rates, com-
pound 29 was docked against
cathepsin L. Compound 29 dif-
fers from compound 30 only at
position 2 (mesityl group versus
4’-biphenyl, respectively). In
spite of this single change, the
effectiveness of the inhibitor de-
creases 500-fold. This can be at-
tributed to the large volume oc-
cupied by the mesityl group, in
comparison with the 4’-biphenyl
group. The bulkiness of the me-
sityl group prevents it from fit-
ting into subsite S1, the area de-
lineated by Asn66 and Gln21,
whereas compound 30 fits very
well into this same site. As a
result, the backbone of com-
pound 29 cannot be accommo-
dated near the cathepsin sur-
face, increasing the distances of
potential hydrogen bonds and
decreasing the binding energy
12
13
>105
>105
1000–1500
860
14
15
>105
>105
100–1000
415
16
17
860
1700
(Supporting
figure 4).
Information
5ꢂ106
>105
The catalytic residues of cath-
epsin B include Cys29, His199,
and Asn219. Compound 23
demonstrates the capacity to
form hydrogen bonds with
His199 (Figure 6B) and Cys29
18
19
10
703
(Supporting
Information
figure 5). The sulfone group of
compound 23 is likely to form a
total of five hydrogen bonds
with Gly27, Cys29, His199, and
Ala200 (Supporting Information
figure 6) while the 2-naphthyl
group is surrounded by His111,
Val176, Leu181, and Met196 (Fig-
ure 6C). Finally, the 4-methylpi-
perazinyl group forms one hy-
drogen bond and has three
105
>105
20
930
660
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Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
other potential ones with resi-
dues His110, His111, and Trp221
Table 2. (Continued)
(Supporting
figure 7).
Information
Conclusions
Compd
21
Substituents
R2
IC50 [nm]
For the first time, we have ap-
plied a new methodology that
combines solid-phase and solu-
tion syntheses for the produc-
tion of a structurally novel pep-
tidyl 3-aryl vinyl sulfone library.
We screened the library and
identified powerful inhibitors of
cathepsins L and B. The most
active compound was further
studied and determined to be
an irreversible covalent competi-
tive inhibitor. Finally, docking
studies were performed, with
analysis of the results corrobo-
rating the experimental data. Be-
cause cathepsins L and B are
currently key targets for the
treatment of cancer, the inhibi-
tors described herein constitute
interesting and promising candi-
dates for the development of
new anticancer drugs.
X
R1
Cathepsin L
100
Cathepsin B
520
22
5ꢂ104
5ꢂ104–105
23
24
17.5
304
>105
>105
25
8.5
300
Experimental Section
Materials and methods
Melting points were determined in
an open capillary tube by means
of a Gallenkamp apparatus and
were not corrected. Specific rota-
tions were determined at room
temperature (258C) in a PerkinElm-
er 241 MC instrument; concentra-
26
27
82
886
536
>105
tions are given in g(100 mL)ꢀ1
.
NMR spectra (1H, 13C) were record-
ed using a Varian-Gemini 200 (1H
200 MHz) or a Mercury 400 instru-
ment (1H 400 MHz). Signal multi-
plicity in 13C NMR spectra was es-
tablished by means of DEPT or
HSQC experiments. Mass spectra
using CI, ESI, or FAB techniques
were recorded on a Hewlett–Pack-
ard HP-5988A instrument. MALDI-
TOF spectra, using an ACH (a-
28
29
5
690
>1000
>105
cyano-4-hydroxycinnamic
acid)
matrix, were determined in a Voy-
ager-DE RP instrument (PE Biosys-
30
2.6
1050
tems) with
a 337 nm nitrogen
laser. Analytical HPLC was per-
formed using a Waters 1525 instru-
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1561

E. Giralt et al.
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treated with an aqueous NaOH solution (0.8 mL, 10% in brine) and
diluted with Et2O (9 mL). The temperature was then raised to 108C,
and anhydrous MgSO4 (0.8 g) and Celite (0.1 g) were added. After
stirring for 15 min, the resulting mixture was filtered through
Celite, washed with Et2O, and the solvents were removed under re-
duced pressure. The crude product was purified by chromatogra-
phy (silica gel column, hexanes/EtOAc mixtures of increasing polar-
ity) to afford (2S,3R)-2,3-epoxy-3-(2-naphthyl)propanol (0.52 g, 68%
yield) with a 98:2 er (Mosher’s ester).
A mixture of this epoxy alcohol (3.1 g, 15.5 mmol), LiClO4 (41.2 g,
0.39 mol), and NaN3 (5.0 g, 78 mmol) in dry CH3CN (75 mL) was
stirred at 658C for 3 h. After cooling to room temperature, the re-
action mixture was poured over H2O (950 mL) and extracted thor-
oughly with Et2O. After drying the mixture over MgSO4, evapora-
tion of the solvents afforded 3.6 g (96% yield) of (2S,3S)-3-azido-3-
(2-naphthyl)propane-1,2-diol. Without further purification, this
compound (3.4 g, 14 mmol) was dissolved in EtOAc (36 mL) and
hydrogenated at atmospheric pressure (balloon) using 10% Pd/C
as a catalyst (0.34 g) in the presence of di-tert-butyl dicarbonate
(4.0 g, 18 mmol). After stirring the compound for 27 h at room
temperature and filtering over Celite, evaporation of the solvents
was followed by chromatographic purification (silica gel column,
hexanes/EtOAc mixtures of increasing polarity) to give 2.7 g (61%
yield) of (2S,3S)-3-(tert-butoxycarbonylamino)-3-(2-naphthyl)pro-
pane-1,2-diol.
A
solution of the above diol (2.5 g, 8.0 mmol), PPh3 (2.3 g,
8.6 mmol) and diisopropyl azodicarboxylate (1.7 mL, 8.6 mmol) in
anhydrous CHCl3 (42 mL) was heated at reflux for 4.5 h. After cool-
ing to room temperature, the solvent was eliminated under re-
duced pressure and the residue subjected to column chromatogra-
phy (silica gel, hexanes/EtOAc mixtures of increasing polarity) to
afford 2.0 g (84% yield) of (S)-2-[(S)-1-(tert-butoxycarbonylamino)-1-
(2-naphthyl)methyl]oxirane. A solution of this oxirane (0.90 g,
3.0 mmol), Et3N (0.43 mL, 3.0 mmol) and PhSH (0.31 mL, 3.0 mmol)
in anhydrous CH3OH (30 mL), under a N2 atmosphere, was heated
at reflux for 1 h. Evaporation of the solvent under reduced pressure
gave 1.3 g (quantitative yield) of (2S,3S)-3-(tert-butoxycarbonylami-
no)-3-(2-naphthyl)-1-phenylthio-2-propanol. Without further purifi-
cation, this compound (3.0 mmol) was dissolved in dry CH2Cl2
(60 mL), and a solution of purified m-chloroperbenzoic acid (1.30 g,
7.5 mmol) was added dropwise. After stirring the solution for 1.5 h
at room temperature, excess peroxy acid was destroyed by wash-
ing with 10% aqueous Na2SO3. The organic phase was washed
with aqueous saturated NaHCO3 and brine then dried over MgSO4.
The solvent was removed by rotary evaporation. Purification of the
crude product by column chromatography (Et3N-pretreated silica
gel, hexanes/EtOAc mixtures of increasing polarity) to afford 0.93 g
(70% yield) of (2S,3S)-3-(tert-butoxycarbonylamino)-3-(2-naphthyl)-
1-phenylsulfonyl-2-propanol.
A solution of the above hydroxysulfone (0.70 g, 1.6 mmol), mor-
pholinocarbonyldiimidazole (morpho-CDI, 1.35 g, 3.2 mmol) and
anhydrous CuCl2 (22 mg, 0.16 mmol) in anhydrous CH3CN (40 mL)
was stirred at 708C for 30 min. After cooling to room temperature,
the reaction mixture was filtered through Celite to give, upon re-
moval of the solvent, 0.69 g (quantitative yield) of (3R,E)-3-(tert-bu-
toxycarbonylamino)-3-(2-naphthyl)-1-phenylsulfonyl-1-propene (3).
(3R,E)-3-(tert-Butoxycarbonylamino)-3-phenyl-1-phenylsulfonyl-
1-propene (1): See ref. [26] for spectral data for this compound.
(3R,E)-3-(tert-Butoxycarbonylamino)-3-(1-naphthyl)-1-phenylsul-
fonyl-1-propene (2): See the Supporting Information.
Figure 4. Docking of compound 30 in cathepsin L subsites. C atoms are
shown in light green; N, O, and S atoms are colored by atom type.
ment (photodiode array Waters 996 detection) or in a Waters Alli-
ance 2795 instrument (MS-ESP ZQ detection). Preparative HPLC pu-
rifications were performed with a Waters 600 instrument, using au-
tomatic injection (Waters 2700), UV/Vis two-channel detection
(Waters 2847), and automatic fraction collection (Waters Fraction
Collector II), with a Symmetry C18 (100ꢂ30 mm) column.
Solvents (DMF, CH2Cl2, CH3CN, CHCl3, CH3OH) were purchased from
SDS and were dried as necessary by conventional techniques.[30]
General synthetic reagents were purchased from Sigma–Aldrich,
Fluka, Merck, or SDS, and were used as received. Fmoc-Phe-OH
and Fmoc-Leu-OH were acquired from Iris Biotech; benzotriazol-1-
yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP),
1-hydroxy-7-azabenzotriazole (HOAt), and N-cyclohexylcarbodii-
mide-N’-methyl polystyrene HL were purchased from Novabio-
chem; the 2-chlorotrityl resin was purchased from CBL Patras.
Reactions run under anhydrous conditions were performed in
flame-dried glassware, by means of septum and syringe tech-
niques, under an atmosphere of purified N2.
Synthesis of (R)-N-Boc-3-amino-3-aryl vinyl sulfones 1–5
Representative procedure: synthesis of (3R,E)-3-(tert-butoxycar-
bonylamino)-3-(2-naphthyl)-1-phenylsulfonyl-1-propene (3): d-
(ꢀ)-DIPT (90 mg, 0.40 mmol), Ti(OiPr)4 (60 mL, 0.20 mmol), and
tBuOOH (4.0 mL of a 2m solution in isooctane; 8.0 mmol) were
added sequentially to a cold (ꢀ208C), stirred suspension of 4 ꢃ
powdered molecular sieves (previously activated, 120 mg) in anhy-
drous CH2Cl2 (4 mL) under N2 atmosphere. After stirring 1 h at
ꢀ208C, a solution of (2E)-3-(2-naphthyl)-2-propen-1-ol[31] (0.70 g,
4.0 mmol) in anhydrous CH2Cl2 (2 mL) was added via syringe. After
stirring for 3 h at the same temperature, the reaction mixture was
(3R,E)-3-(tert-Butoxycarbonylamino)-3-(2-naphthyl)-1-phenylsul-
fonyl-1-propene (3): Colorless solid, mp: 150–1528C; [a]D =+45.1
(c=1.4, acetone); 1H NMR (200 MHz, CDCl3): d=1.39 (s, 9H), 4.95
(br, 1H), 5.64 (brm, 1H), 6.55 (dd, J=15.0 Hz, J’=1.8 Hz, 1H), 7.21
1562
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Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
Figure 5. Docking images of compound 30 with cathepsin L. A) Interaction between Cys25 and compound 30. B) Interaction between the sulfur atom of the
active site cysteine and compound 30. C) Interaction between the active site histidine and compound 30. D) Cleft defined by Asn66 and Gln21 into which the
4’-biphenyl substituent projects.
(dd, J=15.0 Hz, J’=4.8 Hz, 1H), 7.32 (dd, J=8.4 Hz, J’=1.8 Hz, 1H),
7.52–7.64 (m, 6H), 7.82–7.90 ppm (m, 5H); 13C NMR (50 MHz,
CDCl3): d=28.2 (CH3), 55.1 (CH), 80.5 (C), 124.7, 126.2, 126.6, 126.7,
127.6, 127.9, 129.2, 129.3, 131.3 (CH), 133.0, 133.2 (C), 133.4 (CH),
135.2, 140.0 (C), 145.2 (CH), 154.5 ppm (C); MS (CI, NH3): m/z=441
([M+18], 77%), 385 ([Mꢀ38], 100%).
(3R,E)-3-Amino-3-(2-naphthyl)-1-phenylsulfonyl-1-propene
tri-
1
fluoroacetate salt (8): H NMR (400 MHz, CD3OD): d=5.42 (d, J=
6.4 Hz, 1H), 6.95 (dd, J=15.2 Hz, J’=1.2 Hz, 1H), 7.22 (dd, J=
15.2 Hz, J’=6.4 Hz, 1H), 7.45 (dd, J=8.4 Hz, J’=1.8 Hz, 1H), 7.55–
7.63 (m, 4H), 7.71 (m, 1H), 7.87–7.91 (m, 5H), 7.98 ppm (d, J=
8.4 Hz, 1H).
(3R,E)-3-(tert-Butoxycarbonylamino)-3-(2,4,6-trimethylphenyl)-1-
phenylsulfonyl-1-propene (4): See the Supporting Information.
(3R,E)-3-Amino-3-(2,4,6-trimethylphenyl)-1-phenylsulfonyl-1-pro-
pene trifluoroacetate salt (9): See the Supporting Information.
(3R,E)-3-(tert-Butoxycarbonylamino)-3-(4’-biphenyl)-1-phenylsul-
fonyl-1-propene (5): See the Supporting Information.
(3R,E)-3-Amino-3-(4’-biphenyl)-1-phenylsulfonyl-1-propene
fluoroacetate salt (10): See the Supporting Information.
tri-
Synthesis of (R)-3-Amino-3-aryl vinyl sulfone trifluoroacetate
salts 6–10: A solution of the N-Boc amino vinyl sulfone 1–5
(500 mmol) in 40% TFA in CH2Cl2 (1 mL) was stirred at room tem-
perature for 30 min. The solvent and excess TFA were removed
under a stream of N2, and the resulting product was lyophilized
and characterized by 1H NMR spectroscopy.
Incorporation of Fmoc-amino acids to the chlorotrityl chloride
resin: The resin (1.0 g, functionalization 1.54 mmolgꢀ1) was condi-
tioned with CH2Cl2 and subsequently treated with the Fmoc-amino
acid (0.7 equiv) in the presence of DiPEA (1.8 mL, 110 mmol,
7.0 equiv) for 1 h at room temperature. The resin was then treated
with CH3OH (10 min at room temperature) and washed with
CH2Cl2. Removal of the Fmoc group was effected by treatment
with DMF (5ꢂ1 min), 20% piperidine in DMF (2ꢂ10 min), and DMF
(5ꢂ1 min). Final functionalization was determined by spectropho-
tometric analysis of the piperidine–dibenzofulvene adduct in solu-
(3R,E)-3-Amino-3-phenyl-1-phenylsulfonyl-1-propene trifluoro-
acetate salt (6): See the Supporting Information.
(3R,E)-3-Amino-3-(1-naphthyl)-1-phenylsulfonyl-1-propene
fluoroacetate salt (7): See the Supporting Information.
tri-
ChemMedChem 2010, 5, 1556 – 1567
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1563

E. Giralt et al.
MED
Figure 6. Docking images of compound 23 and cathepsin B. A) Interaction between the active site cysteine and compound 23. B) Interactions between
His199 and compound 23. C) Amino acid environment of the 2-naphthyl group. D) Compound 23 docked into the active site of cathepsin B.
tion (Fmoc-Phe-OH: functionalization 0.884 mmolgꢀ1, 82% yield;
Fmoc-Leu-OH: functionalization 0.892 mmolgꢀ1, 83% yield).
Morpholineurea–leucine conjugate, Mu-Leu-OH: 180 mg of a col-
orless solid, 83% yield. tR =6.11 min; MS (ESP+): m/z=245.4
[M+1]+.
Synthesis of morpholineurea conjugates: The resin-bound amino
acid obtained above (1.0 g each) was conditioned with DMF and
treated with 4-morpholinocarbonyl chloride (230 mL, 2.0 mmol) and
with Hꢄnig’s base (510 mL, 3.0 mmol) for 16 h at room temperature.
The coupling was controlled by the Kaiser test (ninhydrin), and the
urea–amino acid conjugate was cleaved from the resin by treat-
ment with 2% TFA in CH2Cl2 (3ꢂ10 min). The resin was washed
with CH2Cl2 and the resulting solution concentrated by a stream of
N2 prior to lyophilization. The crude product was analyzed by HPLC
(Symmetry C18 column, 5 mm, 4.6ꢂ150 mm, F=1 mLminꢀ1, H2O!
0.045% TFA/CH3CN!0.036% TFA, gradient from 0 to 100% in
15 min, 258C) and by HPLC–MS, and purified by preparative HPLC
Synthesis of the N-methylpiperazineurea conjugates: The resin-
bound amino acid (1.0 g each) was conditioned with DMF and
treated with carbonyl diimidazole (810 mg, 5.0 mmol) for 3 h at
room temperature. After filtration and washing of the resin with
DMF, N-methylpiperazine (2.2 mL, 20 mmol) was added; stirring in
DMF was maintained for 1 h at room temperature. The coupling
was controlled by the Kaiser test (ninhydrin), and the urea–amino
acid conjugate was cleaved from the resin by treatment with 2%
TFA in CH2Cl2 (3ꢂ10 min). The resin was washed with CH2Cl2 and
the resulting solution concentrated by a stream of N2 prior to lyo-
philization. The crude product was analyzed by HPLC (Symmetry
C18 column, 5 mm, 4.6ꢂ150 mm, F=1 mLminꢀ1, H2O!0.045%
TFA/CH3CN!0.036% TFA, gradient from 0 to 100% in 15 min,
258C) and by HPLC–MS, and purified by preparative HPLC (Symme-
try C18 column, 5 mm, 100ꢂ30 mm, F=10 mLminꢀ1, H2O!0.1%
TFA/CH3CN!0.05% TFA).
(Symmetry C18 column, 5 mm, 100ꢂ30 mm, F=10 mLminꢀ1
H2O!0.1% TFA/CH3CN!0.05% TFA).
,
Morpholineurea–phenylalanine conjugate, Mu-Phe-OH: 195 mg
of a colorless solid, 79% yield; tR =6.41 min; MS (ESP+): m/z=
279.4 [M+1]+.
N-Methylpiperazineurea–phenylalanine conjugate, Npipu-Phe-
1
OH: 290 mg of a colorless solid, 100% yield; tR =5.02 min; H NMR
1564
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Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
(400 MHz, [D6]DMSO+[D]TFA): d=2.80 (s, 3H), 2.88–3.00 (m, 5H),
3.09 (dd, J=13.6 Hz, J’=4.4 Hz, 1H), 3.37–3.41 (m, 2H), 4.03–4.11
(m, 2H), 4.29 (dd, J=10.4 Hz, J’=4.4 Hz, 1H), 7.20–7.28 ppm (m,
5H); 13C NMR (100 MHz, [D6]DMSO+[D]TFA): d=37.3 (CH2), 41.5
(CH2), 42.8 (CH3), 52.8 (CH2), 56.1 (CH), 126.9 (CH), 128.7 (CH), 129.8
(CH), 138.9 (C), 157.4 (C), 174.6 ppm (C); MS (MALDI): m/z=292.1
[M+1]+, 314.1 [M+23]+, 330.1 [M+39]+.
127.8, 127.9, 129.0, 129.2, 129.3 (CH, CHar), 131.6 (CH, =CHS), 133.0,
133.2 (C, Car), 133.6 (CH, CHar), 134.7, 136.5, 140.1 (C, Car), 144.2 (CH,
CH=), 157.0 (C, C=O-urea), 171.3 ppm (C, C=O); MS (MALDI):
m/z=584.2 [M+1]+, 606.2 [M+23]+, 622.2 [M+39]+; HPLC tR =
10.55 min (gradient 0!100% CH3CN in 15 min).
(S)-N-((R,E)-1-(2,4,6-Trimethylphenyl)-3-(phenylsulfonyl)allyl)-2-
morpholino-3-phenylpropanamide (14): See the Supporting Infor-
mation.
N-methylpiperazineurea–leucine
conjugate,
Npipu-Leu-OH:
140 mg of a colorless solid, 61% yield. tR =4.65 min; 1H NMR
(400 MHz, [D6]DMSO+[D]TFA): d=0.83 (d, J=6.4 Hz, 3H), 0.88 (d,
J=6.4 Hz, 3H), 1.44–1.67 (m. 3H), 2.81 (s, 3H), 2.93–3.06 (m, 4H),
3.41 (brd, 2H), 4.06–4.16 ppm (m, 3H); 13C NMR (100 MHz,
[D6]DMSO+[D]TFA): d=21.7 (CH3), 23.4 (CH3), 25.0 (CH), 41.5 (CH2),
42.7 (CH3), 52.6 (CH), 52.9 (CH2), 157.6 (C), 175.7 ppm (C); MS
(MALDI): m/z=258.1 [M+1]+.
N-((S)-1-((R,E)-1-(Biphenyl-4-yl)-3-(phenylsulfonyl)allylamino)-1-
oxo-3-phenylpropan-2-yl)morpholine-4-carboxamide (15): See
the Supporting Information.
N-((S)-4-Methyl-1-oxo-1-((R,E)-1-phenyl-3-(phenylsulfonyl)allyl-
amino)pentan-2-yl)morpholine-4-carboxamide (16): See the Sup-
porting Information.
N-((S)-4-Methyl-1-((R,E)-1-(naphthalen-1-yl)-3-(phenylsulfonyl)-
allylamino)-1-oxopentan-2-yl)morpholine-4-carboxamide
(17):
See the Supporting Information.
Solution-phase coupling: construction of the peptidyl 3-aryl
vinyl sulfone library
N-((S)-4-Methyl-1-((R,E)-1-(naphthalen-2-yl)-3-(phenylsulfonyl)-
allylamino)-1-oxopentan-2-yl)morpholine-4-carboxamide
(18):
Method A: Colorless solid (8.0 mg, yield 16%); 1H NMR (400 MHz,
CDCl3): d=0.89 (d, J=6.4 Hz, 3H, CH(CH3)2), 0.92 (d, J=6.4 Hz, 3H,
CH(CH3)2), 1.50–1.64 (m, 3H, CH2CH(CH3)2), 3.21 (m, 4H, 2CH2N),
3.53 (m, 4H, 2CH2O), 4.00 (brs, 1H, NH), 4.44 (m, 1H, NH), 5.08
(brs, 1H, NH), 5.88 (m, 1H, 2-NphCHN), 6.50 (dd, J=15.0 Hz, J’=
2.0 Hz, 1H, =CHS), 7.17 (dd, J=15.0 Hz, J’=5.2 Hz, 1H, CH=), 7.22
(dd, J=8.4 Hz, J’=2.0 Hz, Har), 7.47–7.88 ppm (m, 11H, Har);
13C NMR (100 MHz, CDCl3): d=22.2 (CH3, CH(CH3)2), 22.8 (CH3,
CH(CH3)2), 24.9 (CH, CH(CH3)2), 40.8 (CH2, CH2CH), 43.9 (CH2, CH2N),
53.0 (CH, CHN), 53.6 (CH, 2-NphCHN), 66.1 (CH2, CH2O), 124.6,
126.3, 126.7, 127.6, 127.7, 128.0, 129.1, 129.4, 129.5 (CH, CHar),
131.9 (CH, =CHS), 133.0, 133.2 (C, Car), 133.6 (CH, CHar), 134.5, 140.0
(C, Car), 144.3 (CH, CH=), 157.4 (C, C=O-urea), 173.2 ppm (C, C=O);
MS (MALDI): m/z=550.2 [M+1]+, 572.2 [M+23]+, 588.2 [M+39]+;
HPLC tR =10.47 min (gradient 0!100% CH3CN in 15 min).
Method A (PyBOP): PyBOP (28.6 mg, 55 mmol), HOAt (15 mg,
110 mmol) and DiPEA (9.4 mL, 55 mmol) were added sequentially to
a magnetically stirred solution of the urea–amino acid conjugate
(55 mmol) in dry CH2Cl2 (3 mL). After stirring for 5 min at room tem-
perature, the amino vinyl sulfone salt 5–10(50 mmol) was added,
followed by an equimolar amount of DiPEA (8.5 mL, 50 mmol). The
resulting mixture was stirred for 30 min, then the solvent was re-
moved by a stream of N2 and the residue was lyophilized. The
crude product was analyzed by HPLC (Symmetry C18 column, 5 mm,
4.6ꢂ150 mm, F=1 mLminꢀ1, H2O!0.045% TFA/CH3CN!0.036%
TFA, gradient from 0 to 100% in 15 min, 258C) and by HPLC–MS,
and purified by preparative HPLC (Symmetry C18 column, 5 mm,
100ꢂ30 mm, F=10 mLminꢀ1
,
H2O!0.1% TFA/CH3CN!0.05%
TFA).
Method B (DCCI resin): N-Cyclohexylcarbodiimide-N’-methyl poly-
styrene HL resin (80 mg, functionalization 1.9 mmolgꢀ1, 150 mmol)
and Hꢄnig’s base (9.4 mL, 55 mmol) were added sequentially to a
magnetically stirred solution of the urea–amino acid conjugate
(55 mmol) in dry CH2Cl2 (3 mL). After stirring for 5 min at room tem-
perature, the amino vinyl sulfone salt (5–10; 50 mmol) was added,
followed by an equimolar amount of DiPEA base (8.5 mL, 50 mmol).
The resulting mixture was stirred for 30 min, the solvent was re-
moved by a stream of N2 and the residue lyophilized. The crude
product was analyzed by HPLC (Symmetry C18 column, 5 mm, 4.6ꢂ
150 mm, F=1 mLminꢀ1, H2O!0.045% TFA/CH3CN!0.036% TFA,
gradient from 0 to 100% in 15 min, 258C) and by HPLC–MS, and
purified by preparative HPLC (Symmetry C18 column, 5 mm, 100ꢂ
30 mm, F=10 mLminꢀ1, H2O!0.1% TFA/CH3CN!0.05% TFA).
N-((S)-1-Oxo-3-phenyl-1-((R,E)-1-phenyl-3-(phenylsulfonyl)allyl-
amino)propan-2-yl)morpholine-4-carboxamide (11): See the Sup-
porting Information.
(S)-N-((R,E)-1-(2,4,6-Trimethylphenyl)-3-(phenylsulfonyl)allyl)-4-
methyl-2-morpholinopentanamide (19): See the Supporting Infor-
mation.
N-((S)-1-((R,E)-1-(Biphenyl-4-yl)-3-(phenylsulfonyl)allylamino)-4-
methyl-1-oxopentan-2-yl)morpholine-4-carboxamide (20): See
the Supporting Information.
4-Methyl-N-((S)-1-oxo-3-phenyl-1-((R,E)-1-phenyl-3-(phenylsulfo-
nyl)allylamino)propan-2-yl)piperazine-1-carboxamide (21): See
the Supporting Information.
4-Methyl-N-((S)-1-((R,E)-1-(naphthalen-1-yl)-3-(phenylsulfonyl)-
allylamino)-1-oxo-3-phenylpropan-2-yl)piperazine-1-carbox-
amide (22): See the Supporting Information.
4-Methyl-N-((S)-1-((R,E)-1-(naphthalen-2-yl)-3-(phenylsulfonyl)-
allylamino)-1-oxo-3-phenylpropan-2-yl)piperazine-1-carbox-
amide (23): Method B: Colorless solid (18.2 mg, yield 61%);
1H NMR (400 MHz, CDCl3): d=2.28 (br, 2H, CH2N), 2.43 (s, 3H,
CH3N), 3.03 (m, 2H, CH2Ph), 3.10 (br, 4H, 2CH2N), 3.79 (br, 2H,
CH2N), 4.58 (m, 1H, CHN), 5.83 (m, 1H, CHN-2Nph), 6.17 (brd, 1H,
NH), 6.21 (dd, J=15.0 Hz, J’=2.4 Hz, 1H, =CHS), 7.03 (dd, J=
15.0 Hz, J’=4.8 Hz, 1H, CH=), 7.15–7.20 (m, 6H, Har), 7.41–
7.80 ppm (m, 12H, 11Har, NH); 13C NMR (100 MHz, CDCl3): d=38.5
(CH3, CH2Ph), 41.0 (CH2, CH2N), 43.0 (CH3, CH3N), 52.6 (CH2, CH2N),
53.5 (CH, CHN-2Nph), 56.7 (CH, CHN), 124.6, 126.1, 126.7, 127.2,
127.6, 127.7, 127.9, 128.7, 129.1, 129.2, 129.4 (CH, CHar), 131.5 (CH,
=CHS), 132.8, 133.1 (C, Car), 133.7 (CH, CHar), 135.0, 136.6, 139.7 (C,
Car), 144.7 (CH, CH=), 156.5 (C, C=O-urea), 172.2 ppm (C, C=O); MS
(MALDI): m/z=597.2 [M+1]+, 619.2 [M+23]+, 635.2 [M+39]+;
HPLC tR =9.02 min (gradient 0!100% CH3CN in 15 min).
N-((S)-1-((R,E)-1-(Naphthalen-1-yl)-3-(phenylsulfonyl)allylamino)-
1-oxo-3-phenylpropan-2-yl)morpholine-4-carboxamide (12): See
the Supporting Information.
N-((S)-1-((R,E)-1-(Naphthalen-2-yl)-3-(phenylsulfonyl)allylamino)-
1-oxo-3-phenylpropan-2-yl)morpholine-4-carboxamide
(13):
Method A: Colorless solid (3.0 mg, yield 6%); 1H NMR (400 MHz,
CDCl3): d=3.06 (m, 2H, CH2Ph), 3.21 (m, 4H, 2CH2N), 3.55 (m, 4H,
2CH2O), 4.57 (m, 1H, CHN), 5.05 (brd,1H, NH), 5.85 (m, 1H, CHN-
2Nph), 6.20 (dd, J=15.2 Hz, J’=1.8 Hz, 1H, =CHS), 6.83 (brd, 1H,
NH), 7.04 (dd, J=15.2 Hz, J’=4.8 Hz, 1H, CH=), 7.14–7.27 (m, 7H,
Har), 7.47–7.88 ppm (m, 10H, Har); 13C NMR (100 MHz, CDCl3): d=
38.5 (CH2, CH2Ph), 43.9 (CH2, CH2N), 53.5 (CH, 2NphCHN), 56.0 (CH,
CHN), 66.2 (CH2, CH2O), 124.6, 126.3, 126.7, 126.8, 127.4, 127.7,
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(S)-N-((R,E)-1-(2,4,6-Trimethylphenyl)-3-(phenylsulfonyl)allyl)-2-
(4-methylpiperazin-1-yl)-3-phenylpropanamide (24): See the Sup-
porting Information.
N-((S)-1-((R,E)-1-(Biphenyl-4-yl)-3-(phenylsulfonyl)allylamino)-1-
oxo-3-phenylpropan-2-yl)-4-methylpiperazine-1-carboxamide
(25): See the Supporting Information.
4-Methyl-N-((S)-4-methyl-1-oxo-1-((R,E)-1-phenyl-3-(phenylsulfo-
nyl)allylamino)pentan-2-yl)piperazine-1-carboxamide (26): See
the Supporting Information.
4-Methyl-N-((S)-4-methyl-1-((R,E)-1-(naphthalen-1-yl)-3-(phenyl-
sulfonyl)allylamino)-1-oxopentan-2-yl)piperazine-1-carboxamide
(27): See the Supporting Information.
4-Methyl-N-((S)-4-methyl-1-((R,E)-1-(naphthalen-2-yl)-3-(phenyl-
sulfonyl)allylamino)-1-oxopentan-2-yl)piperazine-1-carboxamide
(28): Method B: Colorless solid (16.0 mg, yield 57%); 1H NMR
(400 MHz, CDCl3): d=0.80 (d, J=6.0 Hz, 3H, CH(CH3)2), 0.83 (d, J=
6.0 Hz, 3H, CH(CH3)2), 1.50–1.59 (m, 3H, CH2CH(CH3)2), 2.48 (br, 2H,
CH2N), 2.53 (s, 3H, CH3N), 3.18 (br, 4H, 2CH2N), 3.99 (br, 2H, CH2N),
4.32 (m, 1H, CHN), 5.93 (m, 1H, CHN-2Nph), 6.25 (brd, 1H, NH),
6.57 (dd, J=15.2 Hz, J’=2.0 Hz, 1H, =CHS), 7.16 (dd, J=15.2 Hz,
J’=4.8 Hz, 1H, =CH), 7.24 (dd, J=8.4 Hz, J’=1.6 Hz, 1H, Har-2Nph),
7.38–7.52 (m, 4H, Har), 7.59–7.72 (m, 4H, Har), 7.79–7.81 (m, 2H,
Har), 8.13 ppm (d, J=8.4 Hz, 1H, Har-2Nph); 13C NMR (100 MHz,
CDCl3): d=21.9 (CH3, CH(CH3)2), 22.7 (CH3, CH(CH3)2), 24.8 (CH,
CH(CH3)2), 40.7 (CH2, CH2CH(CH3)2), 41.0 (CH2, CH2N), 43.1 (CH3,
CH3N), 52.9 (CH2, CH2N), 53.3 (CH, CHN-2Nph), 54.3 (CH, CHN),
124.8, 126.2, 126.7, 127.5, 127.6, 127.9, 129.0, 129.4 (CH, CHar),
131.6 (CH, =CHS), 132.8, 133.1 (C, Car), 133.8 (CH, CHar), 135.0, 139.6
(C, Car), 145.0 (CH, CH=), 156.9 (C, C=O-urea), 173.7 ppm (C, C=O);
MS (MALDI): m/z=563.2 [M+1]+, 585.2 [M+23]+, 601.2 [M+39]+;
HPLC tR =8.95 min (gradient 0!100% CH3CN in 15 min).
(S)-N-((R,E)-1-(2,4,6-Trimethylphenyl)-3-(phenylsulfonyl)allyl)-4-
methyl-2-(4-methylpiperazin-1-yl)pentanamide (29): See the Sup-
porting Information.
enzyme was at a concentration of 70 ngmLꢀ1, cathepsin B enzyme
at 6.65 ngmLꢀ1, POP at 10.6 ngmLꢀ1, and DPP IV at 0.187 ngmLꢀ1
,
with compound stock solutions prepared in DMSO at a final con-
centration of 5 mm or 25 mm. Dilutions from the stock solution
were used in order to perform the inhibition curves.
Cathepsin enzymatic assays were performed at room temperature
using 139 mL buffer, 3 mL compound stock solution, 3 mL enzyme,
and 5 mL of 3 mm substrate per well. After the addition of buffer,
enzyme, and compound, the mixture was pre-incubated for 15 min
at 378C. Next, the substrate was added, and the mixture was incu-
bated for 30 min at 378C. The reaction was stopped by the addi-
tion of 150 mL of 1m NaOAc (pH 4) to each well. The plate was
read in the fluorimeter at lexcitation =360/40 nm and lemission =485/
40 nm.
Time-course experiments were performed at room temperature
using 139 mL buffer, 3 mL compound stock solution, 3 mL enzyme,
and 5 mL of 3 mm substrate per well. Pre-incubation was done for
0, 5, 10, 15, 30, 60, 90, and 120 min. After pre-incubation, substrate
was added, and plates were incubated at 378C for 30 min. Follow-
ing this incubation, the reaction was stopped and fluorescence
readings were taken.
POP and DPP IV enzymatic assays were performed at room temper-
ature using 135 mL buffer, 3 mL compound stock solution, 2 mL
enzyme, and 10 mL of 3 mm substrate per well. After the addition
of buffer, enzyme and compound, the mixture was pre-incubated
for 15 min at 378C. Next, the substrate was added and the mixture
was incubated for 60 min at 378C. The reaction was stopped by
the addition of 150 mL of 1m NaOAc (pH 4) to each well. The plate
was read in the fluorimeter at lexcitation =360/40 nm and lemission
=
485/40 nm.
Docking
Compounds were drawn using MarvinSketch and translated to
three-dimensional viewing with MarvinView. The X-ray crystal struc-
ture corresponding to PDB ID: 1CS8 (1.80 ꢃ resolution) was used
as the target structure for cathepsin L, with the water molecules,
as well as the propeptide sequence, removed from the file. For
cathepsin B, the X-ray crystal structure corresponding to PDB ID:
1HUC (2.10 ꢃ resolution) was used, with the water molecules elimi-
nated.
N-((S)-1-((R,E)-1-(Biphenyl-4-yl)-3-(phenylsulfonyl)allylamino)-4-
methyl-1-oxopentan-2-yl)-4-methylpiperazine-1-carboxamide
(30): See the Supporting Information.
Enzyme assays
Cathepsin L (3.4.22.15) and cathepsin B (3.4.22.1), both from
human liver, and DPP IV (3.4.14.5) from porcine kidney, were pur-
chased from Sigma–Aldrich (Deisenhofen, Germany). POP was ob-
tained by recombinant expression in E. coli as previously de-
scribed.[32] The cathepsin L substrate used in experiments was Z-
Phe-Arg-AMC, the cathepsin B substrate was Z-Arg-Arg-AMC, the
POP substrate was Z-Gly-Pro-AMC, and the DPP IV substrate was H-
Gly-Pro-AMC. All of them were obtained from Bachem (Bubendorf,
Switzerland). NaOAc, Triton X-100, and DTT were purchased from
Fluka Chemika (Buchs, Switzerland), EDTA was obtained from USB
Corporation (Staufen, Germany), acetic acid was purchased from
Carlo Erba Reagenti (Italy), sodium phosphate monobasic and diba-
sic were acquired from Sigma–Aldrich (Deisenhofen, Germany) and
DMSO was provided by Panreac Quꢅmica S.A.U. (Barcelona, Spain).
Fluorescence was measured using a BIO-TEK FL600 Microplate
Docking calculations were performed with AutoDock version 4.[33]
The protein was set as rigid while the compounds were defined as
flexible. A grid map of 60ꢂ60ꢂ60 points with a point spacing of
0.375 ꢃ (generated using AutoGrid, version 4) was placed in the
active center of the enzyme, using AutoDock tools. For each com-
pound, 100 docking runs were carried out using 50 individuals and
the Lamarckian genetic algorithm. After calculations, the results
were evaluated, and the best docked conformations were selected
for further analysis.
Acknowledgements
The authors thank ENANTIA S.L. for their enthusiasm and support
of this project. This work was funded by MCYT-FEDER (Bio2008-
00799), MEC (AYA2006-15648-C02-01), and the Generalitat de
Catalunya (XRB 2009 and Grup Consolidat 2009SGR1005).
Fluorescence
Reader
(Bio-Tek
Instruments,
VT,
USA).
For inhibition assays, 96-well microplates from Costar (Corning Life
Sciences, NY, USA) were used. The cathepsin L buffer was 100 mm
NaOAc, containing 0.01% Triton X-100, 5 mm EDTA and 5 mm DTT,
with the pH adjusted to 5.5 by the addition of acetic acid. The
buffer for cathepsin B was 100 mm sodium phosphate, pH 6.2,
1 mm EDTA and 1 mm DTT. The buffer used for POP and DPP IV
was 100 mm sodium/potassium phosphate, pH 8. The cathepsin L
Keywords: aryl vinyl sulfones
·
cathepsins
·
cysteine
proteases · enzymes · medicinal chemistry
1566
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1556 – 1567

Aryl Vinyl Sulfone Inhibitors of Cathepsins L and B
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Received: March 17, 2010
Revised: June 8, 2010
Published online on July 22, 2010
ChemMedChem 2010, 5, 1556 – 1567
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1567