Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor

Article abstract of DOI:10.1021/jm0201924

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (8,

Full text of DOI:10.1021/jm0201924

868
J . Med. Chem. 2003, 46, 868-871
Brief Articles
Str u ctu r e-Activity Rela tion sh ip Stu d y a n d Dr u g P r ofile of
N-(4-F lu or op h en ylsu lfon yl)-L-va lyl-L-leu cin a l (SJ A6017) a s a P oten t Ca lp a in
In h ibitor
J un Inoue,*,† Masayuki Nakamura,† Ying-She Cui,† Yusuke Sakai,† Osamu Sakai,† J eanette R. Hill,‡
Kevin K. W. Wang,§ and Po-Wai Yuen§
Kobe Creative Center, Senju Pharmaceutical Co., LTD, 1-5-4 Murotani, Nishi-ku, Kobe, Hyogo 651-2241, J apan,
Ricerca, LLC, 7528 Auburn Road PO Box 1000 Concord, Ohio 44077, and Neuroscience Therapeutics,
Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received May 3, 2002
Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from
the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated
in vitro. Among them, N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (8, SJ A6017) potently
inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases
(interleukin 1â-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor
VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively
safe profile.
In tr od u ction
In this article, we describe the substituent effect of
N-arylsulfonyldipeptidyl aldehydes on calpain inhibition
and the safety, permeability, and metabolic profiles of
N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (8), the most
potent inhibitor.
The calpains (EC 3.4.22.17), which belong to the
cysteine protease family, exist ubiquitously in cytosol
and are activated by calcium. There are 16 kinds of
calpain identified in mammalian tissue which can be
divided into two groupsscalpains that are tissue-specific
and calpains that are ubiquitously expressed.1 Among
them, two representative types of calpain isoform have
been well studied, µ-calpain (or calpain I) and m-calpain
(or calpain II), which differ in their in vitro requirements
for calcium. The distribution and biochemical property
of calpains have been the subject of several reviews.2
Overactivation of calpain has also been implicated in a
variety of disorders such as muscular dystrophy, Alzhe-
imer’s disease, stroke, ischemic disease, and cataract.3
As one of the first potent peptidyl aldehyde cysteine
protease inhibitors, leupeptin was found in culture
filtrates of various species of actinomycetes.4 Unfortu-
nately, this compound showed poor cell penetration and
lacked enzyme specificity. Chemical modification studies
of leupeptin as the lead compound show that peptidyl
aldehyde type calpain inhibitors, calpeptin or MDL28170,
were potent cell penetrative compounds.5 It has been
known that calpains favorably cleaved the C-terminal
side (P1 position) of tyrosine, methionine, or arginine
next to leucine or valine (P2 position).6 And additionally,
Harris7 reported N-benzyloxycarbonyl-leucyl-tyrosinal
and N-benzyloxycarbonyl-valyl-tyrosinal were potent
calpain inhibitors, and Iqbal8 also reported N-benzyl-
oxycarbonyl-leucyl-valinal, N-benzyloxycarbonyl-tert-bu-
tylglycyl-leucinal, and N-benzyloxycarbonyl-leucyl-(O-
benzyl)tyrosinal were the most potent calpain inhibitors.
Resu lts a n d Discu ssion
Ch em istr y. The series of N-sulfonyldipeptide alde-
hyde was prepared as outlined in Scheme 1. Amino
acids 1 were coupled with arylsulfonyl chloride by the
Shotten-Baumann method to generate the sulfonylated
amino acids 2. Dipeptidyl alcohols 4 were synthesized
by the active ester 3 prepared from 2 and various amino
alcohols. A DMSO oxidation9 of compound 4 using sulfur
trioxide-pyridine complex with triethylamine gave
dipeptidyl aldehydes (5).
Str u ctu r e-Activity Rela tion sh ip . Inhibitory activ-
ity of synthesized compounds was evaluated by the
reported method10 summarized in Table 1. When the
less bulky group (Me) was incorporated in the P3
position (17), calpain inhibitory activity decreased com-
pared to the aromatic ring, and when alanine was
incorporated into the P1 position (11), the activity
decreased. These results suggest that an aromatic group
at the P3 position and a certain size of bulky amino acid
residual aldehyde at the P1 position are required for
potent calpain inhibition. When the P2 position was
changed from valine to norvaline (18) or norleucine (19),
the activity also decreased. A long chain was not
acceptable at the P2 position. Many studies have been
done to identify the preferences at the P1 and P2 position
of a peptidyl inhibitor. Harris revealed that a certain
size of sterically hydrophobic amino acid at P1 or P2
position was important.7 Iqbal mentioned that Leu and
Phe were preferred at P2/P1 for calpain.8 Our results
basically agreed with the previous results. Also, P2 N-H
was required, probably bound to calpain with hydrogen
* To whom correspondence should be addressed. Phone:+81-78-997-
1010. Fax:+81-78-997-1016. E-mail: jun-inoue@senju.co.jp.
† Senju Pharmaceutical Co.
‡ Ricerca, LLC.
§ Pfizer Global Research and Development.
10.1021/jm0201924 CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/01/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 869
Sch em e 1. A General Synthetic Procedure for N-Arylsulfonyldipeptide Aldehyde
Ta ble 1. Variation of the P1 and P3 Substituent on
N-sulfony-L-valylpeptidyl Aldehydes and Their Inhibitory
Activities against µ-Calpain
One possible reason is that we use commercially avail-
able porcine enzyme instead of the human recombinant
enzyme reported in the previous paper by Iqbal.8 8 was
about 10 to 50 times more potent against µ-calpain, and
2 to 20 times more potent against m-calpain than the
known calpain inhibitors. 8 was the most potent calpain
inhibitor tested.
configuration
IC50 (nM)
En zym e Sp ecificity. Compound 8 inhibited µ- (IC50
) 7.5 nM) and m-calpain (IC50 ) 78 nM) and cathepsin
B (IC50 ) 15 nM) and L (IC50 ) 1.6 nM) potently but
did not inhibit other cysteine proteases (interleukin 1â-
converting enzyme) (>100 µM), serine proteases (throm-
bin (>100 µM), factor VIIa (>100 µM), factor Xa (>60
µM), trypsin (>100 µM), chymotrypsin (>200 µM)), or
proteasome (>200 µM). Generally speaking, peptidyl
aldehyde inhibitors broadly inhibit both cysteine and
serine proteases.15,16 Recently, Wells found that N-(3,4-
dihydro-6,7-ethylenedioxy-2-ethyl-2H-1,2-benzothiazine-
3-carbonyl)-L-phenylalaninal 1,1-dioxide was a selective
inhibitor of calpain I vs cathepsin B because of its rigid
structure.17 But, it is unclear whether inhibiting cathe-
psin B and L causes critical side effects for humans.
compd
P3
R
P2
P1
A
B
µ-calpain
6
7
8
2-Nap
2-Nap
H
L-Val L-Leu-H
L-Val L-Phe-H
L-Val L-Leu-H
L-Val L-Phe-H
L-Val L-Trp-H
L-Val L-Ala-H
L-Val L-Leu-H
L-Val L-Phe-H
L-Val L-Trp-H
L-Val L-Leu-H
L-Val L-Phe-H
L-Val L-Leu-H
L-Nval L-Leu-H
L-Nle L-Leu-H
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
D
D
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
D
L
D
10
14
7.5
27
23
630
31
14
13
28
18
830
130
H
H
H
H
H
H
H
H
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4-Me-Ph H
4-Me-Ph H
Me
H
H
H
4-F-Ph
4-F-Ph
260
4-F-Ph CH3 L-Val L-Leu-H
21000
14000
42000
1000000
4-F-Ph
4-F-Ph
4-F-Ph
H
H
H
L-Val L-Leu-H
L-Val L-Leu-H
L-Val L-Leu-H
Toxicity. Cytotoxicity of 8, 6, and MDL28170 were
evaluated by using human neuroblastoma SY5Y cells
and rat cerebellar granule neurons. The 50% lethal
concentrations (LD50) of 8, 6, and MDL28170 were over
1000 µM, 9.8 µM, and 45.9 µM with SY5Y cells,
respectively. 8 was approximately over 100 times less
cytotoxic than 6 and 20 times less cytotoxic than
MDL28170. The fact of 8 cell penetration was already
reported with lens culture.18,19 So 8 was relatively less
cell toxic but could penetrate into cells. 8 exhibited no
bacterial reverse mutation up to 5000 µg/plate from an
Ames test result. Acute toxicity (rat, i.p. and p.o.) and
repeat toxicity test (rat, p.o., for 2 weeks) of 8 were done.
No death and no pathological damage were observed in
all tests up to 100 mg/kg (i.p.) and 1000 mg/kg (p.o.).
bonding since N-methylation dramatically decreased the
inhibitory activity (20 vs 8). The importance of P2 N-H
has been reported by Iqbal.8 Dolle also mentioned the
importance of P2 N-H on not only calpain but on other
members of the papain superfamily and interleukin-1â
converting enzyme.11 Chatterjee, however, reported that
the NHCO moiety at the P2-P3 region of peptide
inhibitor is not a strict requirement for enzyme recogni-
tion.12 Diastereomers and the enantiomer of 8 were
distinctly weak inhibitors in comparison with 8 (vs 21,
22, 23). It is important to keep the absolute configura-
tion of each amino acid residue the same as the natural
type amino acid for calpain inhibition. Yasuma13 also
reported the importance of S-configuration on cathepsin
L inhibition. Chatterjee, however, revealed the impor-
tance of a D-configuration, which is unnatural amino
acid configuration, at P2 in their series of methansul-
fonydipeptidyl aldehydes.14 To clarify the reasons for
these two significant differences, further investigations
are required such as computational docking study or
crystallization of the enzyme-inhibitor complex.
P er m ea bility w ith Ca co-2 Cells. The apparent
permeability of 8 was analyzed by a LC-MS/MS method,
and Papp was calculated to be 1.41 × 10-6, which
suggests a relatively low permeability for this com-
pound. However, only about 33% of dosed 8 was
recovered, indicating that compound was bound to the
cells or to the membrane. Even though the low recovery
of 8 complicates interpretation of the experiment, the
calculated Papp is likely indicative of low permeability
for this compound.
P oten cy. Potency of 8 compared to known calpain
inhibitors is shown (see detail in Supporting Informa-
tion). The IC50s of reference compounds reported here
are approximately 5 times greater than literature IC50s.
P r otein Bin d in g to Mou se, Ra t, Mon k ey, Dog,
a n d H u m a n P la sm a . 8 was incubated with plasma

870 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Brief Articles
from mouse, rat, monkey, dog, and human, and the
filtrate was analyzed. At 10 µM and 100 µM, respec-
tively, 8 was 95.4% and 91.6% bound to human plasma,
89.9% and 90.2% bound to mouse plasma, 89.9% and
85.4% bound to rat plasma, 75.8% and 66.8% bound to
dog plasma, and 95.1% and 90.5% bound to monkey
plasma.
injury in retina,21 and protection on functional recovery
after delayed administration in diffuse head injury.22
Although 8 was the best candidate compound for clini-
cally therapeutic use, there are problems remaining
such as bioavailability or protein binding that need to
be overcome by pharmaceutical formulation techniques
or structural modifications.
CYP 450 In d u ction in Hu m a n Hep a tocytes. Hu-
man hepatocytes were incubated with 1 and 10 µM of
8. Incubation with 8 did not induce CYP450 1A2, 2C9,
or 2C19 activity. However, 8 at 1 µM and at 10 µM
increased 3A4 activity by 1.7 and 2.2-fold, respectively.
CYP 450 In h ib it ion . Inhibition assays for cyto-
chromes CYP450 1A2, 2C9, 2C19, 2D6, and 3A4 were
performed separately. 8 does not inhibit CYP450 1A2
(IC50 > 1000 µM) but slightly inhibits 2C9 (IC50 ) 492
µM), 2C19 (IC50 ) 177 µM), 2D6 (IC50 ) 291 µM), and
3A4 (IC50 ) 81 µM with BFC as substrate, IC50 ) 531
µM with BQ as substrate). However, 8 does not inhibit
CYP450 1A2, 2C9, 2C19, 2D6, or 3A4 at concentrations
less than 50 µM, which is generally considered to be the
limit of physiologically relevant inhibition of CYP450
activity.
Com p a r ison of In ter sp ecies Meta bolism in Hu -
m a n , Mou se, Dog, Mon k ey, a n d Ra t Micr osom es.
The metabolic stability of 8 after incubation with
microsomes in the presence and absence of NADPH was
measured by LC-MS/MS. 8 was almost completely meta-
bolized following incubation with human, mouse, mon-
key, and rat microsomes. After 60 min, only 2.5%, 2.1%,
0%, and 6.4% of parent compound remained with
human, mouse, monkey, and rat microsomes, respec-
tively. The control for monkey incubations with no
NADPH also shown a loss of 82% of parent, suggesting
that metabolism of 8 by monkey microsomes may be due
to factors other than CYP450 activity. Interestingly,
metabolism by dog microsomes appears to be less
efficient with 68% of the parent compound remaining
after 60 min. The structures of the metabolite have not
been determined yet, but the appearance of diastre-
omers and enantiomers were determined in serum at
PK/PD study.
Exp er im en ta l Section
Gen er a l P r ep a r a t ion for Su lfon yl Am in o Acid (2):
N-(4-F lu or op h en ylsu lfon yl)-L-va lin e. To a solution of L-
valine (11.7 g, 100 mmol) in 1 N NaOH (100 mL) and water
(150 mL) were dropwise added 4-fluorobenzenesulfonyl chlo-
ride (17.5 g, 90 mmol) in THF (100 mL) and 1 N NaOH (100
mL) at the same time under an ice-cooled condition and stirred
for 18 h at room temperature. The reaction mixture was made
(pH ) 2 to 3) by the addition of concd HCl thereto and
extracted with EtOAc. After concentration, the residue was
washed with hexane-EtOAc to give the object compound
(15.5 g, 56%) as a white crystalline solid.
Gen er a l P r ep a r a tion for N-Hyd r oxysu ccin im id e Ester
(3): N-(4-F lu or op h en ylsu lfon yl)-L-va lin e N-Hyd r oxysu c-
cin im id e Ester . To a solution of the above compound (15.0
g, 55 mmol) and N-hydroxysuccinimide (7.6 g, 66 mmol) in
THF (200 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (12.6 g, 66 mmol) in CH2Cl2 (200
mL) under an ice-cooled condition. The mixture was stirred
for 4 h at room temperature. Then evaporation of solvent,
EtOAc was added to the residue and the solution was washed
with dilute HCl and sat. aq NaHCO3. After concentration, the
residue was washed with hexane-EtOAc to give the target
compound (17.6 g, 87%) as a white crystalline solid.
Gen er a l P r oced u r e for Dip ep tid yl Alcoh ol (4) P r ep a -
r a tion : N-(4-F lu or op h en ylsu lfon yl)-L-va lyl-L-leu cin ol. To
a solution of the above ester (2.0 g, 5.4 mmol) and L-leucinol
(0.82 g, 7.0 mmol) in CH2Cl2 (50 mL) was added Et3N (1.6 g,
16 mmol) and stirred for 2 h at room temperature. Then the
reaction mixture was washed with dilute HCl and sat. aq
NaHCO3. After concentration, the residue was washed with
hexane-EtOAc to give the target compound (1.9 g, 94%) as a
white crystalline solid.
Gen er a l P r oced u r e for Dip ep tid yl Ald eh yd e P r ep a r a -
tion : N-(4-F lu or op h en ylsu lfon yl)-L-va lyl-L-leu cin a l (8).
To a solution of the above alcohol (10 g, 27 mmol) in DMSO
(100 mL) and CH2Cl2 (50 mL) was added Et3N (16 g, 160
mmol). Purified sulfur trioxide-pyridine complex (17 g, 110
mmol) in DMSO (70 mL) was added thereto, and then the
mixture was stirred for 40 min at 0-5 °C. EtOAc was added
to the mixture and washed with dilute HCl and sat. aq
NaHCO3. After concentration, the residue was recrystallized
from EtOAc to give 8 (1.9 g, 17%).
Con clu sion
We confirmed that an aromatic group at the P3
position and a certain size of bulky amino acid residual
aldehyde at the P1 position is required for potent calpain
inhibition in N-arylsulfonyldipeptidyl aldehydes. Our
SAR study disagrees with previous studies on some
points (P2 N-H, stereo configuration). It is likely each
inhibitor interacts with the enzyme by hydrogen bond-
ing or hydrophobic interactions, and these differences
are defined by the combination of P1 and P2 amino acid
residues. It is difficult to determine which amino acids
work best for P1 and P2 position, but the balance
between them is important.
As the consequence, we found the best inhibitor
against calpains was N-(4-fluorophenylsulfonyl)-L-valyl-
L-leucinal (8) in this series of compounds based on
potency and specificity. 8 was less toxic and had no
activity on both P450 induction and inhibition, but
Caco-2 permeability was poor and protein binding was
relatively high. However, several previously reported in
vivo studies regarding 8 have shown amelioration of
cataract formation,20 protection on ischemia-reperfusion
Ack n ow led gm en t. The authors wish to thank Drs.
Yutaka Kawamatsu, Mitsuyoshi Azuma, Yukuo Yoshida
(Senju), and Drs. Frank W. Marcoux, Michael F. Raf-
ferty, and Thomas G. Heffner (Pfizer) for their helpful
advice and encouragement.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mental details, all biological test protocol and data, and
elemental analyses. This material is available free of charge
via the Internet at http://pubs.acs.org.
Refer en ces
(1) (a) Sorimachi, H.; Saido, T. C.; Suzuki, K. New area of calpain
research: discovery of tissue-specific calpains. FEBS Lett. 1994,
343, 1-5. (b) http://ag.arizona.edu/calpains/index.html.
(2) (a) Wang, K. K.; Villalobo, A.; Roufogalis, B. D. Calmodulin-
binding proteins as calpain substrates. Biochem. J . 1989, 262,
693-706. (b) Croall, D. E.; DeMartino, G. N. Calcium-Activated
Neutral Protease (Calpain) System: Structure, Function, and
Regulation. Physiol. Rev. 1991, 71, 813-847. (c) Saido, T. C.;
Sorimachi, H.; Suzuki, K. Calpain: New perspectives in molec-
ular diversity and physiological-pathological involvement. FASEB.
J . 1994, 8, 814-822.

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 871
-NHCO-Moiety in Potent Di- or Tripeptide inhibitor of
Calpain I: Insights into the Development of Nonpeptidic Inhibi-
tors of Calpain I. Bioorg. Med. Chem. 1998, 6, 509-522.
(13) Yasuma, T.; Oi, S.; Choh, N.; Nomura, T.; Furuyama, N.;
Nishikawa, A.; Fujisawa, Y.; Sohda, T. Synthesis of peptide
aldehyde derivatives as selective inhibitors of human cathepsin
L and their inhibitory effect on bone resorption. J . Med. Chem.
1998, 41, 4301-4308.
(14) Chatterjee, S.; Gu, Z.-Q.; Dunn, D.; Tao, M.; J osef, K.; Tripathy,
R.; Bihovsky, R.; Senadhi, S. E.; O’Kane, T. M.; McKenna, B.
A.; Mallya, S.; Ator, M. A.; Bozyczko-Coyne, D.; Siman, R.;
Mallamo, J . P. D-Amino Acid Containing, High-Affinity Inhibi-
tors of Recombinant Human Calpain I. J . Med. Chem. 1998, 41,
2663-2666.
(15) Aoyagi, T.; Umezawa, H. Structures and activities of protease
inhibitors of microbial origin. In Protease and Biological Control;
Reich, E., Rifkind, D., and Shaw, E., Eds.; Cold Spring Harbor
Laboratory: New York, 1975; pp 429-454.
(16) Mykles, D. L. Proteinase families and their inhibitors. Methods
Cell Biol. 2001, 66, 247-287.
(17) Wells, G. J .; Tao, M.; J osef, K. A.; Bihovsky, R. 1,2-Benzothiazine
1,1-dioxide P2-P3 peptide Mimetic aldehyde calpain I inhibitors.
J . Med. Chem. 2001, 44, 3488-3503.
(18) Fukiage, C.; Azuma, M.; Nakamura, Y.; Tamada, Y.; Nakamura,
M.; Shearer, T. R. SJ A6017, a newly synthesized peptide alde-
hyde inhibitor of calpain: amelioration of cataract in cultured
rat lenses. Biochim. Biophys. Acta 1997, 1361, 304-312.
(19) Fukiage, C.; Azuma, M.; Nakamura, Y.; Tamada, Y.; Shearer
T. R. Nuclear cataract and light scattering in cultured lenses
from guinea pig and rabbit. Curr. Eye Res. 1998, 17, 623-
635.
(20) Tamada, Y.; Fukiage, C.; Mizutani, K.; Yamaguchi, M.; Naka-
mura, Y.; Azuma, M.; Shearer, T. R. Calpain inhibitor, SJ A6017,
reduces the rate of formation of selenite cataract in rats. Curr.
Eye Res. 2001, 22, 280-285.
(21) Sakamoto, Y.; Nakajima, T.; Fukiage, C.; Sakai, O.; Yoshida,
Y.; Azuma, M.; Shearer T. R. Involvement of calpain isoforms
in ischemia-reperfusion injury in rat retina. Curr. Eye Res. 2000,
21, 571-580.
(22) Kupina, N. C.; Nath, R.; Bernath, E. E.; Inoue, J .; Mitsuyoshi,
A.; Yuen, P. W.; Wang, K. K.; Hall, E. D. The novel calpain
inhibitor SJ A6017 improves functional outcome after delayed
administration in a mouse model of diffuse brain injury. J .
Neurotrauma 2001, 18, 1229-1240.
(3) Yuen, P. W.; Wang, K. K. Calpain inhibitors: novel neuropro-
tectants and potential anticataract agents. Drugs Future 1998,
23, 741-749.
(4) Aoyagi, T.; Takeuchi, T.; Matsuzaki, A.; Kawamura, K.; Kondo,
S.; Hamada, M.; Maeda, K.; Umezawa, H. Leupeptins, new
protease inhibitors from Actinomycetes. J . Antibiot. 1969, 22,
283-286.
(5) (a) Wang, K. K.; Yuen, P. W. Calpain inhibition: An overview of
its therapeutic potential. Trends Pharmacol. Sci. 1994, 15, 412-
419. (b) Mehdi, S. Cell-Penetrating Inhibitors of Calpain. Trends
Biochem. Sci. 1991, 16, 150-153.
(6) Sasaki, T.; Kikuchi, T.; Yumoto, N; Yoshimura, N.; Murachi, T.
Comparative specificity and kinetic studies on porcine calpain I
and calpain II with naturally occurring peptides and synthetic
fluorogenic substrates. J . Biol. Chem. 1984, 259, 12489-12494.
(7) Harris, A. L.; Gregory, J . S.; Maycock, A. L.; Graybill, T. L.; Osifo,
I. K.; Schmidt, S. J .; Dolle, R. E. Characterization of a continuous
fluorogenic assay for calpain I. Kinetic evaluation of peptide
aldehydes, halomethyl ketones and (acyloxy)methyl ketones as
inhibitors of the enzyme. Bioorg. Med. Chem. Lett. 1995, 5, 393-
398.
(8) Iqbal, M.; Messina, P. A.; Freed, B.; Das, M.; Chatterjee, S.;
Tripathy, R.; Tao, M; J osef, K. A.; Dembofsky, B.; Dunn, D.;
Griffith, E.; Siman, R.; Senadhi, S. E.; Biazzo, W.; Bozyczko-
Coyne, D.; Meyer, S. L.; Ator, M. A.; Bihovsky, R. Subsite
requirements for peptide aldehyde inhibitors of human calpain
I. Bioorg. Med. Chem. Lett. 1997, 7, 539-544.
(9) Hamada, Y.; Shioiri, T. New Methods and Regents in Organic
Synthesis. 29. A Practical Method for the Preparation of Opti-
cally Active N-Protected R-Amino Aldehydes and Peptide Alde-
hydes. Chem. Pharm. Bull. 1982, 30, 1921-1924.
(10) Buroker-Kilgore, M.; Wang, K. K. W. A Coomassie Brilliant Blue
G-250-Based Colorimetric Assay for Measuring Activity of
Calpain and Other Proteases. Anal. Biochem. 1993, 208, 387-
392.
(11) Dolle, R. E.; Singh, J .; Rinker, J .; Hoyer, D.; Prasad, C. V. C.;
Graybill, T. L.; Salvino, J . M.; Helaszek, C. T.; Miller, R. E.; Ator,
M. A. Aspartyl alpha-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-
yl)oxy)methyl ketones as interleukin-1 beta converting enzyme
inhibitors. Significance of the P1 and P3 amido nitrogens for
enzyme-peptide inhibitor binding. J . Med. Chem. 1994, 37,
3863-3866, and the references therein.
a
(12) Chatterjee, S.; Iqbal, M.; Mallya, S.; Senadhi, S. E.; O′Kane, T.
M.; McKenna, B. A.; Bozyczko-Coyne, D.; Kauer, J . C.; Siman,
R.; Mallamo, J . P. Exploration of the Importance of the P2-P3
J M0201924