The Bsmoc group as a novel scaffold for the design of irreversible inhibitors of cysteine proteases

Article abstract of DOI:10.1016/j.bmcl.2006.02.007

Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group. These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elastase, a serine protease.

Full text of DOI:10.1016/j.bmcl.2006.02.007

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2738–2741
The Bsmoc group as a novel scaffold for the design
of irreversible inhibitors of cysteine proteases
b,
b
b
c
Jim Iley,a, Rui Moreira, Luısa Martins, Rita C. Guedes and Claudio M. Soares
*
*
´
´
aChemistry Department, The Open University, Milton Keynes, MK7 6AA, UK
bCECF, Faculty of Pharmacy, University of Lisbon, Avenida das Forc¸as Armadas, 1600-083, Portugal
cInstituto de Tecnologia Quı´mica e Biolo´gicaUniversidade Nova de Lisboa, 6° Piso,
Avenida da Republica, Apartado 127, 2781-901 Oeiras, Portugal
Received 10 November 2005; revised 6 February 2006; accepted 6 February 2006
Available online 28 February 2006
Abstract—Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily
with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group.
These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-
order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elas-
tase, a serine protease.
Ó 2006 Elsevier Ltd. All rights reserved.
The Bsmoc, 1,1-dioxobenzo[b]thiophen-2-ylmethyl-oxy-
carbonyl, group is a novel, base-sensitive amino protect-
ing group, the usefulness of which is due to deblocking
and base scavanging occurring in a single step.1,2 Depro-
tection/base scavanging involve Michael addition of a
secondary amine, for example, piperidine, to form the
intermediate 2, which undergoes rearrangement to 3,
:Nu
Nu
OCONHR
Nu
S
S
S
O
O
O
O
O
O
3
1
2
1
as confirmed by H NMR spectroscopy (Scheme 1).1
Scheme 1. Mechanism for the deprotection of Bsmoc-amino acids with
nucleophiles (Nu, secondary amines).
The Bsmoc strategy has been successfully used in the
synthesis of a wide range of peptides and amino acid
drug derivatives under extremely mild conditions.2–5
The reactivity of 1 suggests the Bsmoc protecting group
to be a particularly attractive Michael acceptor for thiol
nucleophiles and thus a potential lead structure for the
design of irreversible cysteine protease inhibitors. Mi-
chael acceptor scaffolds, such as peptidyl vinylsulfones,
sulfonamides or sulfonates,6–8 have been used to design
cysteine protease inhibitors that irreversibly alkylate the
active site cysteine residue via conjugate addition.9 Cys-
teine proteases represent a broad class of proteolytic en-
zymes, widely distributed among mammals, protozoa,
bacteria, and viruses that are involved in a diverse range
of physiological processes, including microorganism–
host interaction mechanisms.Unregulated proteolysis
by cysteine proteases can lead to many disease states
and thus these enzymes are important targets for design-
ing inhibitors as potential therapeutic agents.10,11
The recent report12 of the inclusion of the Bsmoc moiety
in dihydroisoxazole inhibitors of human transglutamin-
ase 2, an enzyme believed to play a crucial role in several
pathological disorders, and that such inhibitors are
unstable in the presence of a thiol such as GSH, prompts
us to report our evaluation of the Bsmoc group as Mi-
chael acceptor for thiols and its ability to inhibit human
liver cathepsin B and papain in an irreversible manner.
Cathepsin B is a lysosomal cysteine protease that has
been implicated in rheumatoid arthritis and in the pro-
gression and invasion of tumors.10 Cathepsin B degrades
extracellular matrix components, either intracellularly
or extracellularly, thus enabling the tumor to progress.10
Keywords: Papain; Cathepsin B; Cysteine proteases; Bsmoc.
*
Corresponding authors. Tel.: +44 1908652513; fax: +44 1908858327
(J.I); tel.: +351 217946477; fax: +351 217946470 (R.M.); e-mail
addresses: j.n.iley@open.ac.uk; rmoreira@ff.ul.pt
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.007

J. Iley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2738–2741
2739
R
(i)
R
O
H
(i)
N
O
XNH
S
S
OH
LG
BocNH
N
O
O
O
O
O
O
O
4a-h
6, X = Boc
5
(ii)
Scheme 2. Reagents: (i) RNCO (for 4a–e) or RCOCl (for 4f–h), TEA,
CH2Cl2.
7, X = H
(iii)
Papain is a cysteine protease with a broad range of spec-
ificity, sharing sequence and structural homology with
other mammalian cysteine proteases.13
S
OCOCl
O
4i,j
O
8
Scheme 3. Reagents: (i) Me2CHCH2CH2NH2, TEA, DCM; (ii) TFA,
DCM; (iii) 8, TEA, THF.
On the basis of the known chemistry of the Bsmoc group
(Scheme 1), we rationalized that compounds 4 (see
Scheme 2) with a good leaving group (LG) would inac-
tivate papain via conjugate addition of a cysteine SH
group to form 2 (NuH = CysSH) or, by rearrangement,
3, either of which contain a second Michael acceptor
capable of reacting with a second active site nucleophile
and thus leading to irreversible inactivation.
enzyme inactivation, we studied their reaction with
phenylmethanethiol, dithiothreoitol (DTT), and N-ace-
tylcysteine. In contrast to the reaction of Bsmoc-protect-
ed amino acids with piperidine, which leads to the
rearrangement product 3, compounds 4 undergo Mi-
chael addition with phenylmethanethiol to form 915
along with the corresponding amine or carboxylic acid
(Scheme 4). The second-order rate constants for 4a–d
correlate (Fig. 1) with the Hammett r values for the sub-
stituents in the arylamino moiety, yielding a q value of
0.3 (r2 = 0.91). This indicates that reactivity increases
with electron-withdrawing substituents, the relatively
low value being consistent with rate-limiting addition
of thiol to the cyclic vinylsulfone moiety rather than
the carbonyl carbon or exocyclic methylene positions.
A similar q value can be determined for reaction of
4a–d with DTT (Table 1). Furthermore, the second-
order rate constant for the reaction of 4d with N-acetyl-
cysteine (44.4 MÀ1 sÀ1) is some 20 times greater than the
The carbamic acid (4a–e) or carboxylic acid (4f–h)
Bsmoc derivatives were prepared in good yield by reac-
tion of 1,1-dioxobenzo[b]thiophen-2-ylmethanol (4,
LG = OH) with the appropriate isocyanate or benzoyl
chloride, respectively (Scheme 2 and Table 1). The Leu-
i-Am, 4i, and Phe-i-Am, 4j, carbamates (Table 1) were
also prepared to probe the potential interaction of the
amino acid with the S2 subsite of papain and cathepsin
B. Cystein proteases of the papain superfamily prefer
substrates with lipophilic amino acids, for example,
Phe, in the P2 position.9 Moreover, the Leu side chain
in the epoxysuccinyl-Leu-i-Am inactivator, E64c, has
been shown to interact with the S2 subsite in papain.9
Compounds 4i,j were prepared from the corresponding
Boc-protected aminoacid N-hydroxysuccinimide ester,
5, and iso-amylamine, to yield the amides 6 (Scheme
3).14 After removal of the Boc group, the intermediates
7 reacted with 1,1-dioxobenzo[b]thiophen-2-ylmethyl
chloroformate, 8, to give 4i,j.
S
Ph
(i)
Me
S
S
LG
O
O
O
O
4
9
To assess the intrinsic reactivities of these derivatives,
and as a measure of the irreversible chemical step of
Scheme 4. Reagents: (i) PhCH2SH, TEA, CH2Cl2.
Table 1. Second-order rate constants for the reaction of 4 with DTT (k2) and for papain inactivation by 4 (kobsd/[I])
Compound
LG
k2 (MÀ1 sÀ1
)
kobsd/[I] (MÀ1 sÀ1
)
Papain
Cathepsin B
PPE
4a
4b
4c
4d
4e
4f
OCONHC6H4-4-OMe
OCONHC6H5
4.68
5.68
8.67
9.65
3.67
7.82
7.68
7.80
6.65
7.10
2.15
1.24
0.64
NDb
0.89
2.48
0.45
1.60
0.93
0.65
18.7
0.22
0.46
0.035
ND
NIa
NI
OCONHC6H4-4-CF3
OCONHC6H4-4-NO2
OCONHCH2C6H5
OCOC6H5
NI
NI
0.45
0.030
0.10
0.24
0.14
0.085
NI
NI
4g
4h
4i
OCOC6H4-4-OMe
OCOC6H4-2-OMe
OCO-Leu-NH-iAm
OCO-Phe-NH-iAm
NI
NI
ND
ND
4j
Ac-Phe-NH-CH2-CH@CH-SO2Mec
a No inhibition.
b Not determined.
c Ref. 16.

2740
J. Iley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2738–2741
-0.6
-0.7
-0.8
-0.9
-1
[I], neither KI nor kinact (the first-order rate constant for
4d
the chemical inactivation step) could be determined for
papain or cathepsin B. However, the corresponding sec-
ond-order rate constants for inactivation, k2 (=kinact/KI),
were determined from the slopes of the plots of kobsd
versus [I] and are expressed as kobsd/[I], ranging from
0.35 to 2.48 MÀ1 sÀ1 for papain (Table 1). These values
indicate that both carboxylate and carbamic acid leav-
ing groups lead to active compounds and contrast with
the value of 0.08 MÀ1 sÀ1 determined for the alcohol
starting material (4, LG = OH). Such 6- to 35-fold dif-
ference between the second-order inactivation constants
for compounds 4a–h, when compared to the alcohol
counterpart, can be ascribed, in part, to the increased
electron-withdrawing effect of the carbonyloxymethyl
moiety when compared to the hydroxyl substituent
(the Taft r* values for OCOC6H5 and OH are 2.57
and 1.34, respectively). However, the most potent papa-
in inhibitor in the carbamate series is the 4-MeO substi-
tuted compound 4a, which is ca. 3 more active than the
4-CF3 analogue 4c. Unfortunately, introducing the Leu-
i-Am and Phe-i-Am side chains, (4i and 4j, respectively)
did not improve the inhibitory potency when compared
to carbamates and esters 4a–h. Despite being efficient
Michael acceptors, Bsmoc derivatives 4a–j are ca. 8
to 40 times less potent than vinylsulfone Ac-
PheNHCH2CH@CHSO2Me (Table 1).16
4c
4b
4a
-0.4
0
0.4
0.8
σ
Figure 1. Hammett plot for the reaction of Bsmoc derivatives 4a–d
with PhCH2SH at 25 °C.
corresponding value for the reaction with piperidine
(2.1 MÀ1 sÀ1), the reagent recommended for removing
the Bsmoc protecting group, indicating a significantly
greater reactivity of the Bsmoc scaffold with the thiol
functionality than with an amine. Additionally, there
is no major difference between the reactivity of carba-
mates 4a–e and esters 4f–h, as would be expected if reac-
tion were occurring at the ring C-3, rather than the
carbonyl, position. Indeed, the sterically hindered 2-
MeO-substituted ester 4h reacts with DTT at an almost
identical rate to that of its 4-MeO counterpart 4g (Table
1), which is also consistent with conjugate addition of
the thiol to the vinylsulfone.
Compounds 4 are also time-dependent inhibitors of
cathepsin B,20 but the corresponding kobsd/[I] values
are 2–80 times smaller than those for papain. The most
potent compounds in the series, 4a and 4f, are also the
most selective for papain. Interestingly, the most active
compound against papain, 4f, is the least active against
cathepsin B. As for papain, the Leu-i-Am and Phe-i-Am
side chains did not improve inhibition against cathepsin
B. The Bsmoc derivatives 4 were also tested against por-
cine pancreatic elastase (PPE),21 a serine protease, using
the dilution assay. None of the Bsmoc derivatives inhib-
ited PPE up to a concentration of 10 mM, which sug-
The inhibitory potency of compounds 4 against papain
and cathepsin B was assessed using Kitz and Wilson’s
incubation method.17 Both Bsmoc carbamate, 4a–e and
4i,j, as well as ester, 4f–h, derivatives, were found to be
time-dependent inactivators of papain (Fig. 2) and
cathepsin B. Pseudo-first-order rate constants of inacti-
vation, kobsd values, were obtained from plots of % activ-
ity (v/v0) against time.18 For each assay, the concentration
of each Bsmoc derivative was normalized to take account
of the competitive reaction with DTT.18,19 The irrevers-
ible nature of the inactivation was shown in a routine as-
say, when no reactivation of enzyme activity was detected
even after 4d of dialysis at 25 °C.
gests that 4 are selective inhibitors for cysteine
proteases (Table 1).
We have yet to examine the precise nature of the inter-
action of the enzyme and the Bsmoc derivatives using
mass spectrometry. However, modeling the interaction
of 4f with papain (pdb: 1PPN) using Autodock soft-
ware22 reveals the preferred conformation of the inhibi-
tor to sit in the active site such that the pro-S sulfonyl
oxygen atom forms a hydrogen bond to the NH of
Gly-66, while the benzoate moiety sits in the S2 pocket
of the enzyme. This orientation presents C-3 of the ben-
zothiophene-1,1-dioxide system to the sulfur atom of
Cys-25 of the Asn-175/His-159/Cys-25 catalytic triad,
as would be expected for Michael addition chemistry
as described above (Fig. 3).
For the enzyme inhibition experiments, due to solubility
problems at the higher values of inhibitor concentration
8
6
4
2
0
The results herein presented show that the Bsmoc scaf-
fold provides a novel and unexplored approach for
achieving cysteine protease inhibiton. The Bsmoc-
derived carbamates and esters 4 react with thiols via
conjugated addition to the cyclic vinylsulfone moiety.
This pathway is likely to be identical to that of
0
2
4
6
8
104[I]/M
Figure 2. Plot of the first-order rate constant, kobsd, for the inactiva-
tion of papain 4b against inhibitor concentration.

J. Iley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2738–2741
2741
6. Palmer, J. T.; Rasnik, D.; Klaus, J. L.; Bro¨mme, D.
J. Med. Chem. 1995, 38, 3193.
7. Roush, W. R.; Gwaltney, S. L.; Cheng, J.; Scheidt, K. A.;
McKerrow, J. H.; Hansell, E. J. Am. Chem. Soc. 1998,
120, 10994.
8. Reddick, J. J.; Cheng, J.; Roush, W. R. Org. Lett. 2003, 5,
1967.
¨
9. Powers, J. C.; Asgian, J. L.; Ekicu, O. D.; James, K. E.
Chem. Rev. 2002, 102, 4639.
10. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
11. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem.
2000, 43, 305.
12. Choi, K.; Siegel, M.; Piper, J. L.; Yuan, L.; Cho, E.;
Strnad, P.; Omary, B.; Rich, K. M.; Khosla, C. Chem.
Biol. 2005, 12, 469.
13. Meara, J. P.; Rich, D. H. J. Med. Chem. 1996, 39, 3357.
14. Roush, W. R.; Hernandez, A. A.; McKerrow, J. H.;
Selzer, P. M.; Hansell, E.; Engel, J. C. Tetrahedron 2000,
56, 9747.
Figure 3. Autodock-generated active site interaction of papain and 4f
(green). His-159 (blue) and Cys-25 (yellow) are also highlighted. Figure
generated using Pymol software: DeLano, W.L. The PyMOL Molec-
ular Graphics System (2002) DeLano Scientific, San Carlos, CA, USA.
http://www.pymol.org.
15. Salient data for 9 are as follows: 1H NMR (300 MHz,
CD3CN) 1.91 (3H, s, CH3Ar), 3.96 (2H, s, CH2Ph), 7.08–
7.75 (9H, m, ArH); 13C NMR (75 MHz, CDCl3) 8.41
(CH3Ar), 38.73 (CH2Ph), 121.05, 122.80, 127.58, 128.57,
128.64, 129.36, 132.32, 132.60, 133.52, 135.64, 136.50,
144.95 (Ar). C16H12O2S2 requires: C, 63.55; H, 4.67%.
Found: C, 63.50; H, 4.70%.
alkylation of the active site cysteine that leads to papain
and cathepsin B inactivation.
Acknowledgments
16. Liu, S.; Hanzlik, R. P. J. Med. Chem. 1992, 35, 1067.
17. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
18. Papain (EC 3.4.22.2, from Sigma) assays were per-
formed as described in Zhao, G.; Zhou, Z. S. Bioorg.
Med. Chem. Lett. 2001, 11, 2331, Gradients from
inhibitor runs were normalized with respect to the
first-order exponential decrease in Bsmoc inhibitor
concentration due to DTT-catalyzed decomposition at
the pH of the incubation.19 Observed pseudo-first-order
rate constants of inactivation, kobsd, were obtained from
plots of % activity against time.
19. Hinchliffe, P. S.; Wood, J. M.; Davis, A. M.; Austin,
R. P.; Beckett, R.; Page, M. I. Org. Biomol. Chem.
2003, 1, 67.
20. Human cathepsin B (EC 3.4.22.1, from Calbiochem)
assays were carried out as described for papain using pH
5.5, 50 mM acetate buffer (2 mM DTT, 2 mM EDTA).
The substrate was Z-Arg-Arg-NA (0.1 mM).
We thank FCT, POCTI, and FEDER (Portugal) for
financial support and a grant to L.M. (SFRH/BD/
6499/2001).
References and notes
1. Carpino, L. A.; Philbin, M.; Ismail, M.; Truran, G. A.;
Mansour, E. M. E.; Iguchi, S.; Ionescu, D.; El Faham, A.;
Riemer, C.; Warrass, R.; Weiss, M. S. J. Am. Chem. Soc.
1997, 119, 9915.
2. Carpino, L. A.; Ismail, M.; Truran, G. A.; Mansour, E.
M. E.; Iguchi, S.; Ionescu, D.; El Faham, A.; Riemer, C.;
Warrass, R. J. Org. Chem. 1999, 64, 4324.
3. Greenwald, R. B.; Zhao, H.; Reddy, P. J. Org. Chem.
2003, 68, 4894.
4. Carpino, L. A.; Krause, E.; Sferdean, C. D.; Schumann,
¨
M.; Fabian, H.; Bienert, M.; Beyermann, M. Tetrahedron
Lett. 2004, 45, 7519.
5. Carpino, L. A.; Ghassemi, S.; Ionescu, D.; Ismail, M.;
Sadat-Aalaee, D.; Truran, G. A.; Mansour, E. M. E.;
Siwruk, G. A.; Eynon, J. S.; Morgan, B. Org. Process Res.
Dev. 2003, 7, 28.
21. PPE (EC 3.4.21.36, from Sigma) inhibition assays were
carried out as described in Ref. 19.
22. AutoDock 3.0; automated docking using a lamarckian
genetic algorithm and empirical binding free energy
function Morris, G. M.; Goodsell, D. S.; Halliday, R. S.;
Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comp.
Chem. 1998, 19, 1639.