Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

Article abstract of DOI:10.1016/j.bmcl.2008.10.007

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by α-methyl-γ,γ-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(α-methyl-γ,γ-diphenylallenecarbonyl)-l-phenylalanin e ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pK_a value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.

Full text of DOI:10.1016/j.bmcl.2008.10.007

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6202–6205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Possible involvement of radical intermediates in the inhibition of cysteine
proteases by allenyl esters and amides
a
b
b
Yoshio Takeuchi a, , Tomoya Fujiwara , Yoshihito Shimone , Hideki Miyataka ,
*
Toshio Satoh b, , Kenneth L. Kirk c, Hitoshi Hori d
a Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan
b Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
c Laboratory of Bioorganic Chemistry, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
d Department of Life System, Institute of Technology and Science, Graduate School, The University of Tokushima, Minamijosanjimacho-2, Tokushima 770-8506, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to investigate crystallographically the mechanism of inhibition of cysteine protease by a-methyl-
Received 17 April 2008
Revised 8 September 2008
Accepted 1 October 2008
Available online 5 October 2008
c
,
c-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we
synthesized N-( -methyl- -diphenylallenecarbonyl)- -phenylalanine ethyl ester 4. Reaction of 4 with
a
c,
c
L
thiophenol, the SH group of which has similar pKa value to that of cysteine protease, produced oxy-
gen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions.
Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of
oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical
mechanism.
Dedicated to the memory of Professor
Toshio Satoh of Tokushima Bunri University
who made many lasting contributions to the
field of medicinal chemistry.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Allene
Cysteine protease
Cysteine protease inhibitor
Oxygen
Radical
Cysteine proteases are an important class of peptide-processing
enzymes and, as the name implies, are characterized by having a
cysteine residue within the active site.1 These proteases are also
ubiquitous enzymes that play important roles in many biochemical
processes.1,2 The presence of cysteine proteases in pathogenic
microorganisms and roles in facilitating metastases of tumors are
examples of many factors that make selective cysteine protease
inhibitors important targets for drug design.1 As a consequence
of the wide therapeutic potential of these compounds, many cys-
teine protease inhibitors have been developed.3 In much of the de-
sign of these inhibitors, it has been assumed that the catalytic
mechanism of cysteine proteases is similar to that of serine prote-
ases. Thus, the cysteinyl and histidinyl residues present in the ac-
tive site form a thiolate/imidazolium ion pair that promotes the
formation of an S-acyl-enzyme intermediate that is necessary for
cleavage of the peptide bond.4 However, despite the similarities
of the proposed catalytic mechanism of cysteine and serine prote-
ases, important differences have been revealed. Thus, the replace-
ment of the active site serine with cysteine in two serine proteases
(trypsin and subtilisin) abolished activity.5 This result implies that
cysteine proteases have another reaction mechanism, for example,
a radical reaction mechanism that has been found in several enzy-
matic systems.6
Allenyl esters 2 derived from the hydrolysis and decarboxyl-
ation of diethyl a-alkynylmalonates 1 have been shown to inhibit
the catalytic activity of cathepsin B in vitro, but do not inhibit
* Corresponding author. Tel.: +81 76 434 7555.
E-mail address: takeuchi@pha.u-toyama.ac.jp (Y. Takeuchi).
Deceased, 6 July 2001.
Figure 1. Structures of compounds 1–3 and inhibitory activities of 3.10
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.007

Y. Takeuchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6202–6205
6203
in vivo (Fig. 1). This lack of in vivo inhibition was ascribed to deple-
tion of the inhibitor through competing reactions with endogenous
low molecular weight thiol compounds such as cysteine and re-
duced glutathione.7 As a result of our efforts to develop allene com-
pounds which would be stable and active in vivo, we found that a-
methyl-c,c-diphenylallenecarboxylic acid ethyl ester 3 could inhi-
bit the catalytic activities of cathepsin B8 (IC50 = 2.32 Â 10À4 M)
and urease9 (IC50 = 1.1 Â 10À6 M).10 Compound
3 was stable
in vivo and inhibited water immersion stress-induced ulcer forma-
tion in rats (Fig. 2).10b Thus we examined the reaction mechanism
of 3 with such thiol enzymes.
In order to simplify the understanding of results, we employed
low weight thiol compounds as model compounds of cysteine pro-
tease. The reactions of 3 with L-cysteine and reduced glutathione
did not proceed in aqueous ethanol at 37 °C as we expected from
the in vivo stability of 3. These results were attributable to the rel-
atively higher pKa values of cysteine (pKa 8.3)11 and reduced gluta-
thione (pKa 8.7)11 compared to cysteine proteases (pKa 2.5–8.0)12
which results from the formation of the ion pair with the histidine
of the active site. We thus tried to examine the reaction of the con-
jugated allene system with the more acidic thiophenol (pKa 6.6)11
and to clarify the structure of the products unambiguously by X-
ray crystallographic analysis. Since it was rather difficult to crystal-
lize 3, we designed amide 4 as a suitable model compound for this
Scheme 1. Synthesis of amide 4 and reaction of 4 with thiophenol under ambient
air.
study because introduction of the L-phenylalanine ethyl ester unit
through the amide bond was expected to make crystallization of
the products easy due to the presence of the amide bond and the
additional aromatic group.
Compound 3 was hydrolyzed with sodium hydroxide in aque-
ous EtOH to afford the corresponding carboxylic acid 5 in 60% yield
(Scheme 1). Condensation of 5 with L-phenylalanine ethyl ester
was carried out with 1,3-dicyclohexylcarbodiimide (DCC) in
dichloromethane to give amide 4 in 60% yield. Preliminary biolog-
ical evaluation of 4 showed that compound 4 has moderate inhib-
itory activity (56% at 10À3 M) toward cathepsin B. Reaction of 4
with thiophenol in benzene proceeded smoothly to give a mixture
of diastereomers 6 and 7 isolated as crystals, as expected, in 43%
and 23% yields, respectively.13 The structure of 6 was confirmed
by X-ray crystallographic analysis (Fig. 3).14 If the addition of thio-
phenol to 4 occurs by simple nucleophilic addition, a proton will
Figure 3. ORTEP drawing of compound 6.
add to the
a-carbon of the allene carboxylic acid moiety of the
whereas the reaction under ambient air had gone to completion.
These results strongly indicate that the reaction of 4 with thiophe-
nol is, in fact, a radical process that is dependent on the presence of
oxygen. Indeed, Mueller and Griesbaum16 reported that addition
reaction of thiolate ions to allene derivatives may proceed via a
radical mechanism, not a nucleophilic mechanism, from the stud-
ies on reaction of thiolate ions with some allene derivatives. They
also indicated that reaction of thiolate ions to tetra-substituted al-
lene derivatives is somewhat difficult because, unlike mono-, di-,
and tri-substituted allene structures, a tetra-substituted allene
structure cannot be isomerized to an acetylene form, which is as-
sumed to be an active form to react with thiolate ions. Based on
this evidence, in vivo stability of our allene derivatives in contrast
to that of 2 may be due to their fully substituted structure.
product. The presence of the hydroxyl group at this position in
compounds 6 and 7 indicates that the reaction of 4 with thiophenol
is not a simple nucleophilic addition reaction. This product is indic-
ative of a radical reaction initiated by oxygen.
Based on the assumption that oxygen would be the initiator of
any radical process in this system, we next examined the same
reaction under an oxygen-free condition. Parallel reactions were
carried out, one under an oxygen-free argon atmosphere, and the
other in ambient air, and these were monitored by HPLC.15 After
6 h, there was no apparent reaction in the absence of oxygen,
Table 1
Catalytic activities of cathepsin B and urease under oxygen-free condition
SH-enzyme
% Catalytic activitya
Cathepsin Bb
Ureasec
72 ( 6.50)
60 ( 10.5)
a
Catalytic activity under ambient air taken as 100% (all values are means of at
least three experiments, standard deviation is given in parentheses).
b
The activity was measured by the method of Inubushi et al.21
Figure 2. Effect of 3 on ulcer formation induced by water immersion stress in
c
The activity was measured by the method of Smith et al.22
rats.10b

6204
Y. Takeuchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6202–6205
Scheme 2. Proposed mechanism for inhibition of cysteine protease by allenyl ester 3 (R = OEt) and amide 4 (R = NHCH(Bn)COOEt).
3. (a) Otto, H. H.; Schirmeister, T. Chem. Rev. 1997, 97, 133; (b) Leung-Toung, R.; Li,
There are several reports describing the oxidative inhibition of
caspases,17 a family of cysteine proteases, with oxidants such as ni-
tric oxide,18 hydrogen peroxide,19 or peptidyl peroxide.20 However,
we have found no report that describes the oxidative activation of
cysteine proteases with an exogenous oxidant. Activation resulting
from the presence of oxygen should necessarily result from oxygen
functioning as an initiator of a radical-mediated reaction. However,
to our knowledge, such a radical-mediated reaction of cysteine
proteases previously has never been considered. If the reaction of
cysteine proteases with 3 were to proceed via a mechanism similar
that of thiophenol, then a decreased catalytic activity of the prote-
ase should be observed under oxygen-free conditions. Thus, we
examined the catalytic reaction of cathepsin B under an oxygen-
free argon atmosphere. Under the oxygen-free condition, the cata-
lytic activity of cathepsin B was, in fact, lowered, but not totally
lost (Table 1). This result strongly indicates that a radical interme-
diate, which is initiated with oxygen, is involved in the catalytic
reaction of cysteine proteases. The fact that oxygen depletion re-
sults in only partial inactivation also suggests that cysteine prote-
ases may include both radical and nucleophilic components to the
mechanism of the catalytic reaction, although it is also possible
that residual traces of oxygen in the solution may be a factor in this
result. It is also interesting that the catalytic activity of urease was
lowered under an oxygen-free argon atmosphere (Table 1).
In summary, based on similarity of the pKa value of the SH
group to cysteine protease, we employed thiophenol as a model
compound to investigate the mechanism of the inhibition of cys-
teine protease by allenyl ester 3. Reaction of amide 4, a more highly
crystalline derivative of allene compound 3, with thiophenol pro-
ceeded by a mechanism involving radical species, formation of
which was initiated by oxygen. It should be noted that the catalytic
activities of cathepsin B and urease also depended on the presence
or absence of oxygen. These results suggest that cysteine proteases
can act as radical catalysts using oxygen as an initiator. On the ba-
sis of these results and on information from the literature,23 we
propose the mechanism for inhibition of cysteine proteases by 3
and 4 in Scheme 2. Further studies on the involvement of radical
species in reaction of cysteine protease, including direct observa-
tion of radical intermediates by ESR, are currently underway.
W.; Tam, T. F.; Karimian, K. Curr. Med. Chem. 2002, 9, 979.
4. Storer, A. C.; Menard, R. Methods Enzymol. 1994, 244, 486.
5. (a) Neet, K. E.; Koshland, D. E. Proc. Natl. Acad. Sci. U.S.A. 1966, 56, 1606; (b)
Polgar, L.; Bender, M. L. Biochemstry 1967, 6, 610; (c) Neet, K. E.; Nanci, A.;
Koshland, D. E. J. Biol. Chem. 1968, 243, 6392; (d) Yokosawa, H.; Ojima, S.; Ishii,
S. J. Biochem. 1977, 82, 869; (e) Higaki, J. N.; Evnin, L. B.; Craik, C. S. Biochemistry
1989, 28, 9256.
6. (a) Stubbe, J.; van der Donk, W. A. Chem. Rev. 1998, 98, 705; (b) Kim, J.; Darley,
D. J.; Buckel, W.; Pierik, A. J. Nature 2008, 452, 239.
7. (a) Nagao, Y.; Kim, K.; Sano, S.; Kakegawa, H.; Lee, W. S.; Shimizu, H.; Shiro, M.;
Katunuma, N. Tetrahedron Lett. 1996, 37, 861; (b) Sano, S.; Shimizu, H.; Kim, K.;
Lee, W. S.; Shiro, M.; Nagao, Y. Chem. Pharm. Bull. 2006, 54, 196.
8. Musil, D.; Zucic, D.; Turk, D.; Engh, R. A.; Mayr, I.; Huber, R.; Popovic, T.; Turk,
V.; Towatari, T.; Katunuma, N.; Bode, W. EMBO J. 1991, 10, 2321.
9. Sirko, A.; Brodzik, R. Acta. Biochim. Pol. 2000, 47, 1189.
10. (a) Satoh, T.; Miyataka, H. Jpn. Kokai Tokkyo Koho JP2001354628, 2001.; (b)
Satoh, T. Jpn. Kokai Tokkyo Koho JP10182451, 1998. Manuscript on SAR studies
of the derivatives of 3 is under preparation.
11. DeCollo, T. V.; Lees, W. J. J. Org. Chem. 2001, 66, 4244.
12. (a) Berti, P. J.; Storer, A. C. J. Mol. Biol. 1995, 246, 273; (b) Turk, B.; Turk, V.; Turk,
D. Biol. Chem. 1997, 378, 141; (c) Pinitglang, S.; Watts, A. B.; Patel, M.; Reid, J.
D.; Noble, M. A.; Gul, S.; Bokth, A.; Naeem, A.; Patel, H.; Thomas, E. W.;
Sreedharan, S. K.; Verma, C.; Brocklehurst, K. Biochemistry 1997, 36, 9968.
13. To a solution of 4 (2.00 g, 4.7 mmol) in dry benzene (20 mL) was added
thiophenol (5.0 mL, 47 mmol). After stirring at room temperature for 48 h, the
resulting mixture was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography (benzene/EtOAc 10/1) to
give 6 (1.05 g, 43%) and 7 (0.56 g, 23%). (S)-2-Hydroxy-2-methyl-4,4-diphenyl-
3-thiophenyl-3-butenecarbonyl-L-phenylalanine ethyl ester (6): White prisms;
20
mp 127–129 °C; [
a
]
D
À4° (c 1.35, CHCl3); IR (KBr) 3057, 1740, 1657 cmÀ1
;
1H
NMR (400 MHz, CDCl3) d 1.22 (3H, t, J = 7.1 Hz), 1.58 (3H, s), 3.06 (2H, m), 3.32
(1H, s), 4.14 (2H, q, J = 7.3 Hz), 4.41 (1H, q, J = 6.2 Hz), 6.94–7.31 (20H, m); 13C
NMR (100 MHz, CDCl3) d 14.1, 27.9, 29.7, 37.8, 53.6, 61.5, 78.5, 126.7, 127.1,
127.6, 127.9, 128.5, 129.1, 129.3, 135.6, 136.1, 137.3, 142.1, 143.9, 167.7, 171.2,
174.1; MS (EI) m/z 551 (M+). (R)-2-Hydroxy-2-methyl-4,4-diphenyl-3-
thiophenyl-3-butenecarbonyl-
L
-phenylalanine ethyl ester (7): White prisms;
mp 146–149 °C; [ ;
a
]
D
20 +37° (c 0.91, CHCl3); IR (KBr) 3406, 1738, 1658 cmÀ1
1H
NMR (400 MHz, CDCl3) d 1.23 (3H, t, J = 7.3 Hz), 1.57 (3H, s), 3.01 (2H, d,
J = 5.9 Hz), 3.60 (1H, s), 4.17 (2H, q, J = 7.2 Hz), 4.53 (1H, q, J = 5.9 Hz), 6.99–
7.26 (20H, m); 13C NMR (100 MHz, CDCl3) d 14.1, 27.9, 29.7, 38.0, 53.4, 61.4,
78.9, 125.9, 126.8, 127.1, 127.6, 127.9, 128.5, 129.1, 129.4, 135.7, 136.0, 137.5,
141.8, 144.0, 170.9, 173.8, 195.9; MS (EI) m/z 551 (M+).
14. Crystal data of 6: C34H33NO4S, orthorhombic, P212121, M = 551.705, a = 5.9840
(3) Å, b = 20.514 (2) Å, c = 24.393 (3) Å, V = 2994.4 (5) Å3, Z = 4, Dc = 1.224 Mg/
m3,
l(MoK
a
) = 0.146 mmÀ1, T = 298 K, colorless prism (0.35 Â 0.2 Â 0.15 mm),
4253 measured, 4242 independent, R = 0.073, wR = 0.157 for 2477 observed
reflections [I > 2
r
(I)]. CCDC 699505 contains the supplementary
crystallographic data for this molecule. These data can be obtained free of
charge
via
www.ccdc.cam.ac.uk/data_request/cif,
or by contacting
by
The
emailing
Cambridge
data_request@ccdc.cam.ac.uk,
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033.
Acknowledgments
15. Compound 4 (7.7 mg, 0.018 mmol) was dissolved in dry benzene (0.77 mL)
under an oxygen-free argon atmosphere or ambient air. The oxygen-free
condition was obtained by bubbling argon through the solution. Thiophenol
We thank T. Kimoto and C. Nakamura for experimental assis-
tance. K.L.K. acknowledges support from the intramural research
funds of NIDDK.
(12.8 lL, 0.125 mmol) was added to the solution and aliquots were taken after
stirring at room temperature for 1, 3, and 6 h and analyzed by HPLC using a
Chiralcel OD-R column (4.6 Â 250 mm) (Daicel Chemical Industries, Ltd.,
Tokyo, Japan), a mixture of acetonitrile/water (6/4) was used for the mobile
phase. The UV absorptions of the products were measured at 254 nm.
16. Mueller, W. H.; Griesbaum, K. J. Org. Chem. 1967, 32, 856.
References and notes
17. Denault, J. B.; Salvesen, G. S. Chem. Rev. 2002, 102, 4489.
18. Mannick, J. B.; Hausladen, A.; Liu, L.; Hess, D. T.; Zeng, M.; Miao, Q. X.; Kane, L.
S.; Gow, A. J.; Stamler, J. S. Science 1999, 284, 651.
1. Lecaille, F.; Kaleta, J.; Brömme, D. Chem. Rev. 2002, 102, 4459.
2. Chapman, H. A.; Riese, R. J.; Shi, G. P. Annu. Rev. Physiol. 1997, 59, 63.

Y. Takeuchi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6202–6205
6205
19. Borutaite, V.; Brown, G. C. FEBS Lett. 2001, 500, 114.
condition was obtained by bubbling argon through all solutions except the
20. Hampton, M. B.; Morgan, P. E.; Davies, M. J. FEBS Lett. 2002, 527, 289.
water solution of urease. After incubating at 37 °C for 10 min, a solution of
21. Cathepsin
ethylenediaminetetraacetate–0.4 M sodium acetate buffer (pH 5.5) in
water (250 L) was added water (350 L), 0.2 M -cysteine (40 L), 2 U/
mL cathepsin B (10 L, BioPur, Bubendorf, Switzerland) under an oxygen-
B
enzyme assay: To
a
solution of 4 mM sodium
2 mg/mL urea in water (100
incubated at 37 °C for 15 min. To the mixture was added solutions of 10%
potassium carbonate in water (500 L) and 0.5% dansyl chloride in acetone
(600 L). The mixture was incubated at 55 °C for 90 min, and then a solution of
lL) was added to the mixture. The mixture was
l
l
L
l
l
l
l
free argon atmosphere or ambient air. The oxygen-free condition was
obtained by bubbling argon through all solutions except the water solution
of cathepsin B. After incubating at room temperature for 3 min, a solution
5% glycine in water (3 mL) was added. After incubating at 55 °C for 30 min,
water (5 mL) was added to the mixture. The mixture was allowed to cool to
room temperature and extracted with chloroform (2 mL). The organic layer
was concentrated to give dansyl amide. The obtained dansyl amide was
of 80 lM Z-Phe-Arg-MCA (Peptide Institute, Osaka, Japan) in water (250 lL)
was added to the mixture. Aliquots were taken after incubating at 37 °C for
5 min, and then monochloroacetic acid (1 mL, pH 4.3) was added to
terminate the reaction. The fluorescence intensity of the liberated 7-amino-
4-methylcoumarin in the mixture was measured at excitation 370/emission
dissolved in a solution of 1
Aliquots were taken and analyzed by HPLC using
l
M dansyl methyl amide in acetonitrile (1 mL).
a
YMC-Pack ODS-A
(6.0 Â 150 mm) (Yamazen Co., Osaka, Japan), a solution of 50% acetonitrile in
17.2 mM phosphate buffer (pH 7.2) was used for the elution phase. The
fluorescence intensity of the obtained dansyl amide was measured at
excitation 340/emission 530 nm on a JASCO FP-920 fluorescence detector
(Tokyo, Japan). See also: Smith, J. R. L.; Smart, A. U.; Hancock, F. E.; Twigg, M. V.
Chem. Ind. 1989, 353.
460 nm on
a HITACHI F-3000 fluorescence spectrophotometer (Tokyo,
Japan). See also: Inubushi, T.; Kakegawa, H.; Kishino, Y.; Katunuma, N. J.
Biochem. 1994, 116, 282.
22. Urease enzyme assay: To a solution of 2.4 U/mL urease (Toyobo Co. Ltd., Osaka,
Japan) in water (100
under an oxygen-free argon atmosphere or ambient air. The oxygen-free
l
L) was added 0.1 M phosphate buffer (650
l
L, pH 7.0)
23. Wang, X.; Ni, Z.; Lu, X.; Hollis, A.; Banks, H.; Rodriguez, A.; Padwa, A. J. Org.
Chem. 1993, 58, 5377.