Current Medicinal Chemistry, 2000, 7, 1171-1188 1171

A Survey of Calpain Inhibitors

I. O. Donkor*

Department of Pharmaceutical Sciences, The University of Tennessee Health Sciences Center, Memphis,
TN 38163, USA

Abstract: Calpain is unique among the cysteine protease family of enzymes in that it combines thiol
protease activity with calmodulin-like activity. Despite its wide spread distribution the exact physiological
function(s) of calpain is yet to be deciphered. The enzyme is however, implicated in a number of
pathophysiological conditions. Due to the potential of calpain as a therapeutic target a number of inhibitors
have been described for the enzyme. In this article we have grouped calpain inhibitors into those derived from
natural sources, and those derived from chemical synthesis. Additionally, an overview of functional groups
that have been used as “warheadsö of calpain inhibitors is presented along with a discussion of the structure
activity relationship studies of the address region of peptidyl calpain inhibitors. Recent work in this area has led
to a better understanding of the structural requirements for tight binding of inhibitors to the active site of
calpain. A discussion of peptidomimetic calpain inhibitors, nonpeptide calpain inhibitors, and selectivity
of some calpain inhibitors are also presented. The recent disclosure of the crystal structure of a nonpeptide
calpain inhibitor bound to a hydrophobic pocket on the calcium-binding domain of calpain has opened the
door to future development of potent cell permeable nonpeptide calpain inhibitors.

I. INTRODUCTION et al. [4] which disclosed the crystal structure of the

Calpain is a member of the cysteine protease
family of enzymes. It is unique among this class of
proteins in that it requires Ca2+ for activation. At
least six isoforms of calpain have been identified
[1]. Two of the major calpain isoforms known as
calpain I (µ-calpain) and calpain II (m-calpain) are
widely distributed in mammalian cells. These
isoforms are very identical and differ mostly in
their Ca2+ requirement for activation. Calpain I is
activated by micro molar concentration of Ca2+ (1-
100 µM) whereas calpain II requires millimolar
concentration of Ca2+ (0.1-1 mM) for activation in
vitro. The presence of phospholipids make calpain
more sensitive to Ca2+ thus lowering the
concentration of Ca2+ required for activation in vivo
[2]. Structurally, calpain is a heterodimeric protein
consisting of a cysteine-proteinase domain coupled
to a calmodulin-like Ca2+-binding domain. Calpains
I and II consist of an 80 kDa and a 78 kDa catalytic
subunit (large subunit) respectively, with each
subunit coupled to a 30 kDa regulatory subunit
(small subunit). The crystal structure of the
regulatory subunit was the first to be solved [3].
This was recently followed by the work of Hosfield
*Address correspondence to this author at the University of Tennessee,
Memphis, 847 Monroe Avenue, Faculty Bldg. Room 327E, Memphis, TN
38163, USA; Phone: (901) 448-7736; Fax: (901) 448-6828; Email:
idonkor@utmem.edu
unactivated from calpain II. The crystal structure
revealed that the catalytic triad is not assembled.
Thus, Ca2+-binding must induce conformational
changes that lead to assembling of the activated
enzyme.
Calpain has a broad range of substrate specificity
and a number of cellular proteins are calpain
substrates. These include calmodulin-binding
proteins, cytoskeletal proteins (e.g., spectrin,
MAP-2), G-proteins, enzymes involved in signal
transduction (e.g., PKC, IP3 kinase), membrane
receptors (e.g., EGF receptor), and transcription
factors [5-10]. Most calpain substrates possess
hydrophilic residues such as proline, glutamic
acid, aspartic acid, serine, and threonine (i.e. the
PEST sequence) close to the cleavage site.
Calpain plays a role in several intracellular
signaling pathways mediated by Ca2+ but the
precise function(s) of the enzyme in vivo awaits
clarification. Calpain is implicated in a number of
pathological conditions and the enzyme has been
proposed as a potential therapeutic target for a
number of diseases including brain trauma, spinal
cord injury, Alzheimer’s disease, Parkinson’s
disease, subarachnoid hemorrhage, muscular
dystrophy, cardiac ischemia, restinosis, thrombotic
platelet aggregation, arthritis, and cataract. Several
review articles and books provide detailed

0929-8673/00 $19.00+.00 ¬ 2000 Bentham Science Publishers Ltd.

1172 Current Medicinal Chemistry, 2000, Vol. 7, No. 12
description of calpain structure, physiological
functions, and potential as a therapeutic target [6,
11-21]. In this Review Article calpain inhibitors
derived from natural and synthetic sources are
discussed with emphasis on the structure-activity
relationship (SAR) of the inhibitors.
II. CALPAIN INHIBITORS
The search for calpain inhibitors has been an on
going process following the discovery of the
enzyme over 30 years ago [22, 23]. General
protease inhibitors such as EDTA and EGTA (Ca2+
chelators), iodoacetic acid, 5,5’-dithiobis(2-
nitrobenzoic acid), N-ethylmaleimide, mersalyl,
isocoumarins, and diisopropyl phosphorofluoridate
are among the first inhibitors of the enzyme to be
identified. Most of these inhibitors are neither
selective nor potent which make them unattractive
as biomedical tools for studying calpain-mediated
events. Current knowledge about calpain substrate
specificity and the nature of the active site of the
enzyme have made possible the development of
potent and in some cases selective calpain
inhibitors.
A. Calpain Inhibitors Derived
Natural Sources
i. Calpain Inhibitors Derived
Mammals
Calpastatin is an endogenous polypeptide that
specifically inhibits both major isoforms of calpain
but not papain or cathepsin B [24]. The polypeptide
consists of an N-terminal domain (domain L) and
four repetitive domains (repeats 1-4). All four
repeats have similar inhibitory activity against
calpains I and II but domain L is devoid of
inhibitory action [25]. A synthetic 27-residue
peptide derivative based on a repeat region of
calpastatin is also an inhibitor of calpain [26]. Other
polypeptide inhibitors of calpain are α2-
macroglobulin [27] and the heavy chains of L- and
H-kininogen [28]. Puri et al. [29] employed the
highly conserved sequence, Gln-Val-Val-Ala-Gly-
NH2, present in domains II and III of human
kininogens as an address region to which they
appended a 3-nitro-2-pyridyl moiety via a disulfide
linkage to generate a weak irreversible inhibitor of
platelet calpain I.
ii. Calpain Inhibitors Derived from Plants
Streptomyces species is a rich source of calpain
inhibitors. Peptidyl aldehyde, diketopiperazine, and
I. O. Donkor
pyrazinone derivatives are among the calpain
inhibitors isolated from Streptomyces species.
Leupeptin (Ac-Leu-Leu-Arg-H) [30], antipain
([(S)-1-carboxy-2-phenylethyl]-carbamoyl-L-Val-
Arg-H) [31], strepin P-1 (N-i-valeryl-Tyr-Val-Arg-
H) [32] and the pentapeptides staccopins P-1 and
P-2 (Val-Val-Val-Val-Phe-H and Val-Val-Val-
Val-Tyr-H, respectively) [33] are examples of
peptidyl aldehyde calpain inhibitors isolated from
Streptomyces. The inhibitors inactivate calpain by
reacting reversibly with the active-site thiol of the
enzyme but they are not selective for calpain. For
example, leupeptin inhibits plasmin, trypsin,
papain, and cathepsin B; strepin P-1 inhibits
papain and trypsin; staccopins P-1 and P-2 are
however, not active against serine proteases but
they are potent inhibitors of calpain and papain.
The lack of selectivity and poor cellular
permeability make the inhibitors unattractive as
biomedical tools for investigating the role(s) of
calpain in cell function. In attempts to improve the
potency and cell permeability of peptidyl
aldehydes, SAR studies involving N-terminal
capping with lipophilic substituents were
undertaken. The studies led to the discovery of
calpeptin (Z-Leu-Nle-H) which possesses a
benzyloxycarbonyl moiety as the lipophilic N-
terminal agent [34]. The compound is cell
From permeable and inhibits human platelet calpain II with
an IC50 of 40 nM [35]. Incorporation of the
benzyloxycarbonyl group into Val-Phe-H gave
from MDL 28,170 (1), Fig. (1), which is also cell
permeable [36]. Using an acetyl moiety as the N-
terminal substituent, Sasaki et al. [37] discovered
calpain inhibitor I (Ac-Leu-Leu-Nle-H) and
calpain inhibitor II (Ac-Leu-Leu-Met-H). The
inhibitors are however, not specific for calpain.
Calpain inhibitor I is a better inhibitor of cathepsin
L (Ki = 0.5 nM) and calpain inhibitor II inhibits
cathepsin B (Ki = 100 nM). Sasaki and coworkers
substituted 4-phenylbutylryl on the N-terminal
nitrogen of Val-Phe-H to obtain compound 2
which inhibited calpain I and calpain II with Ki of
36 nM and 50 nM, respectively. Compound 2
weakly inhibited trypsin, chemotrypsin, and
cathepsin H [37]. Thus, starting with peptidyl
aldehydes isolated from Streptomyces, SAR
studies involving N-terminal capping led to
compounds with enhanced cell permeability and
calpain inhibitory potency. The compounds,
however, still lack specificity and are readily
oxidized under physiological conditions because
they are aldehydes [38].
Omura et al. [39] isolated cystamidin A (3) from
Streptomyces species KP-1241 culture. They
claimed that the compound is a calpain inhibitor but
provided no calpain inhibitory data. Bihovsky and
Pendrak [40] subsequently synthesized cystamidin
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1173

O O
H H
O N H N H
N N
H H
O O O O

M D L 28 ,1 70 ( 1) 2
HO
OH
O O
HN
O
NH 2 N
N
N
H N
O
3 4
5
O O

HO C HO

OH
O O O O O
H 7
N N O
N N OH
H H
O O
O
HO
6 HO
8
OH

Fig. (1). Calpain inhibitors derived from natural sources.

A (pyrrole-3-propanamide) and its regioiosmer
(pyrrole-2-propanamide) and found the compounds
to be inactive against recombinant human calpain I.
Alvarez et al. [41] isolated 3-benzyl-6-isopropyl
disubstituted 2(1)-prazinone 4 from Streptomyces
sp. The compound (which was termed Phevalin) is
presumably derived biosynthetically from
phenylalanine and valine. Phevalin inhibited calpain
activity in the casein hydrolysis assay with an IC50
of 1.3 µM. Diketopiperazine 5 (IC50 = 0.8 µM)
and tetrapeptide 6 (IC50 = 1.2 µM) are new calpain
inhibitors bearing N-methyl tyrosine residues that
were isolated from Streptomyces griseus (SC 488)
[42]. Damnacanthal (7), an anthraquinone
compound isolated from the root of Morinda
citrifolia effectively inhibited thrombin and calpain.
The compound is not selective since it inhibited
both serine and cysteine proteases [43]. Chung et
al. [44] isolated penicillide 8 having a 5H,7H-
dibenzo[b,g][1,5]dioxocin-5-one skeleton from the
culture broth of Penicillium species F60760 as a
nonpeptide inhibitor of calpain. The compound
inhibited calpain with an IC50 of 7.1 µM but it did
not inhibit papain at 200 µM.
Peptidyl epoxysuccinate inhibitors are the most
studied group of cysteine protease inhibitors
isolated from microbes. Trans-epoxysuccinyl-L-
leucylamido-4-guanidino-butane (E-64) (9, Table
1) was the first member to be reported. It was
isolated from Aspergillus japonicus TPR-64 by
Hanada et al. [45] and immediately followed by its
total synthesis and structure characterization [46].
E-64 and its derivatives inactivate cysteine
proteases by forming a covalent bond with the
active site thiolate. Epoxysuccinyl peptides are
highly selective for cysteine proteases and they
have been used as active site titrants for this class of
enzymes [45-50]. The compounds display no
selectivity for calpain relative to the other cysteine
proteases [47]. The mechanism for irreversible
inactivation of cysteine proteases by
epoxysuccinates and their mode of binding to
cysteine proteases have been studied by NMR [51]
1174 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

Table 1. Epoxysuccinyl Derivatives have found that such cyclopropyl derivatives of E-

64c are devoid of calpain inhibitory activity
O
attesting to the importance of the epoxide ring for
the bioactivity of this class of compounds [56]. b).
R1O
R2 The stereochemistry of the epoxide group is
X important since the L-isomer of E-64 was a better
O
inactivator than the D-isomer [53]. c). Replacement
of the charged guanidino group at P3 with an
Compd. R1 X R2
isopentyl moiety as in Ep-475 did not affect
inhibitory potency but improved cellular
E-64 (9) H O Leu- permeability [53, 57]. d). N-Terminal capping with
NH(CH2)4NHC(NH)NH2 a benzyloxycarbonyl group, as in Ep-460 (15,
Ep-475 H O Leu-NH(CH2)2CH(CH3)2
Table 1), resulted in a dramatic increase in the rate
(10) of inactivation suggesting that calpain has a
hydrophobic binding site that can accommodate this
DC-11 (11, see structure below)
group. The significance of the carboxylate group
12 Et NH Leu-OBzl attached to the epoxide ring has been investigated.
13 Et N-Phe-Boc Leu-OBzl
Esterification of this group (as in compound 16)
resulted in 100-1000 fold decrease in calpain
14 H CH2 Leu-NH(CH2)2CH(CH3)2 inhibition. The esterified compounds however,
Ep-460 H O Leu-NH(CH2)4NH-Z readily permeated cells and were easily hydrolyzed
(15) to the active acid derivative [58]. Murata et al. [59]
16 Et O Leu-NH(CH2)2CH(CH3)2
disclosed E-64 derivative 17 as a poor inhibitor of
calpain II (IC50 = 200,000 nM) but an excellent
17 Et O Ile-Pro-OH inhibitor of cathepsin B (IC50 = 2.28 nM).
18 H O Phe-NH(CH2)2CH(CH3)2 Giordano et al. [60] synthesized epoxysuccinyl
derivatives with phenylalanine (18),
19 H O Tyr(I)-NH(CH2)2CH(CH3)2
monoiodotyrosine (19), and diiodotyrosine (20) as
20 H O Tyr(I2)-NH(CH2)2CH(CH3)2 the P2 substituent but the compounds were better
inhibitors of cathepsin B than calpain. Replacement
HO OC H O of the epoxide ring with a cyclic sulfate gave 1,3,2-
H
N dioxathiolane dioxide derivatives (21-23, Table 2)
H N which were better inhibitors of cathepsin B than
H
O
calpain [61, 62]. Indeed, cyclic sulfate 21 was
about 430-fold more potent against cathepsin B
D C - 1 1 ( 11 ) than calpain suggesting that the catalytic site of
calpain is sterically more hindered than that of
cathepsin B.
and X-ray crystallography [52]. SAR studies of E- Table 2. Cyclic Sulfate Epoxysuccinyl Derivatives
64 derivatives led to the following observations
(Table 1): a). The epoxide ring is important for
activity. E-64c [Ep-475, (10)] was found to be O
over a thousand fold more effective than DC-11 R OO C H
(11) in which the epoxide ring was replaced by N
N
a H
double bond [53]. Aziridine analogues of E-64c O O O
S
(e.g., 12) are generally poor calpain inhibitors. O O
Substitution on the aziridine nitrogen gave
compound 13, the R,R diastereomer of which was
2-fold and 7-fold selective for calpain I compared to µM)
IC50 (µ
calpain II and cathepsin H, respectively [54]. Compound R Calpain Cathepsin B
Kumar et al. [55] recently reported the
stereoselective synthesis of E-64 derivatives in 21 H 0.3 0.0007
which the cyclopropyl ring was used as a
bioisosteric replacement for the epoxide ring 22 Et 15.0 0.025
(compound 14) but gave no biological data. We 23 Bzl 6.0 0.009
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1175

B. Calpain Inhibitors Derived from Table 3. Reversible Calpain Inhibitor Warheads

Chemical Synthesis
Peptidyl calpain inhibitors consist of an address Warhead E.g. (Calpain inhibition)a Ref.b
region for enzyme recognition and a “warhead” for
covalent modification of the active site thiol. The H
inhibitors are generally designed by replacing the Z-Val-Phe-H (MDL 28170 [36]
scissile amide bond of calpain substrates with an O (IC50 = 0.01 µM)
electron deficient center (the warhead) that is
O
capable of reacting either reversibly or irreversibly
with the active site thiol. In reversible inhibitors the Z-Leu-Phe-COOH [75]
electron deficient center is usually an electrophilic OH
(Ki = 0.0085 µM)
carbonyl containing functional group (such as an O
aldehyde or a ketone). The inhibitors form
reversible covalent abducts (hemithioacetal or O
hemithioketal) with the active site thiolate of the R
Z-Leu-Phe-COOEt [75]
enzyme. The abducts resemble the transition states O
(Ki = 1.8 µM)
or reaction intermediates in substrate hydrolysis so O
they are tightly bound to the enzyme, hence they are
usually effective competitive inhibitors. O

i. Calpain Inhibitor Warheads N HR Z-Leu-Phe-CONH(CH2)2Ph [75]

(Ki = 0.0052 µM)
O
Electrophilic carbonyl containing functional
groups that have been used as warheads in O
OR
reversible calpain inhibitors include aldehydes P
[34-37, 63-74] and α-keto carbonyl compounds OR Z-Leu-Leu-P(O)(OCH3)2 [85]
such as α-ketoacids, α-ketoamides, α-ketoesters (IC50 = 0.43 µM)
O
[75-84], α-diketones [76], and α-keto phosphorus
compounds [85] (Table 3 and Fig. 2). Li et al. [75] O
studied a series of α-keto carbonyl containing P
OR

calpain inhibitors and observed that calpain R Z-Leu-Leu-P(O)(Ph)OEt [85]

inhibitory potency follows the order: α-ketoacids > (IC50 = 0.37 µM)
O
α-ketoamides > α-ketoesters. Using N,N-
disubstituted α-ketoamides they demonstrated that O
the difference in activity between α-ketoamides and P
R

α-ketoesters is due to the inability of α-ketoesters R Z-Leu-Leu-P(O)(CH3)2 [86]

to form a critical H-bond interaction with the (IC50 > 10 µM)
O
enzyme. α-Keto heterocycles such as 24 (IC50 =
90 nM vs. calpain II) and 25 (IC50 = 33 nM) [86-
90] were designed as prodrugs to circumvent the 24 (IC50 = 0.09 µM vs. [86]
inherent metabolic instability of aldehydes in vivo m-calpain)
and also to increase localization of the inhibitor in HO O
neuronal cells. The amphiphilic properties of the
cyclopropenone ring led Ando et al. [91] to design O
cysteine protease inhibitors incorporating this ring
26 (IC50 = 0.35 µM) [91]
as a warhead (e.g. 26, IC50 = 350 nM).
Replacement of the scissile bond of calpain
substrates with epoxysuccinate and its derivatives a
The enzyme is calpain I except where indicated otherwise.
(e.g., aziridine and dioxathiolane) [45-50, 61, 92], b
Selected references.
ketomethylenes [38, 93-100], methylsulfonium
salts [38, 101, 102], vinyl sulfones [103], and calpain relative to serine proteases. The basis for
disulfide linkages [29] afford irreversible inhibitors selectivity may be attributed to the greater
of the enzyme (Table 4 and Fig. 2). Peptidyl nucleophilicity of cysteine (thiolate) versus serine.
epoxysuccinates, acyloxymethyl ketones, and Peptidyl chloromethyl ketones on the other hand
diazomethyl ketones are selective inhibitors of
1176 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

O
H
( H 2 C ) 1 4H 3C N O
S N
O O H
OH O
OH
N
O
O N
H
24 O

M D L 1 04 ,90 3 ( 25 )

O
O
H
N
S N
O O H
OH

26
O
O
H O
O N P
N O
O
H
O O

27

O O

HN +
O N N S
H H
O O

28
O N N
H
O N N
N O
H
O O

29

O O O
O O
H H H
N N O H 3C N N S
N S N
H O H
O O O

O S O

30 31

Fig. (2). Examples of reversible and irreversible calpain inhibitors.

Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1177

Table 4. Irreversible Calpain Inhibitor Warheads

Table 4. Irreversible Calpain Inhibitor Warheads

Warhead E.g. (Calpain inhibition)aa Ref.

Warhead E.g. (Calpain inhibition) Ref.
H OO C
H OO C
Ep-475 (7,450) [53]
Ep-475 (7,450) [53]
O
O
HO OC
HO OC
12 (inactive) [54]
N 12 (inactive) [54]
N
H
H
H OO C
H OO C

O O 21 (IC50 = 0.3 µM) [61,62]

O O 21 (IC50 = 0.3 µM) [61,62]
S
S
O O
O O

N2
N2 Z-Leu-Leu-Tyr-CH=N2 (230,000) [97]
O Z-Leu-Leu-Tyr-CH=N2 (230,000) [97]
O
F
F Z-Leu-Leu-Phe-CH2F (290,000) [94]
O Z-Leu-Leu-Phe-CH2F (290,000) [94]
O
Cl
Cl Z-Leu-Leu-Phe-CH2Cl (9,500) [107]
O Z-Leu-Leu-Phe-CH2Cl (9,500) [107]
O
O
O

O Ar Z-Leu-Phe-CH2OCO-2,6-Cl2-Ph (11,000) [97]

O Ar Z-Leu-Phe-CH2OCO-2,6-Cl2-Ph (11,000) [97]
O
O
O Ar
O O Ar
P O
O P O
O O 27 (365,000) [98,108]
Ar 27 (365,000) [98,108]
O Ar
O
+
+
S
S 28 (200,000) [101]
O 28 (200,000) [101]
O
H et
O H et
O 29 (230,000) [99,100]
O 29 (230,000) [99,100]
O
O O
O S O
S Z-Leu-Leu-Tyr-VSPh (24,300) [103]
Z-Leu-Leu-Tyr-VSPh (24,300) [103]
O2 N
O2 N
O
O
S S Phe-Gln-Val-Val-Cys-Npys (5,850) [29]
S S Phe-Gln-Val-Val-Cys-Npys (5,850) [29]
RH N
RH N
a
Calpain inactivation rate is expressed as M-1s-1 except as indicated for compound 21. VSPh is (vinylsulfonyl)benzene. Npys is 3-nitro-2-sulfenylpyridine.
a proteases calpain inhibition by peptidyl
Calpain inactivation rate is expressed as M -1 s -1 except as indicated for compound 21. VSPh is (vinylsulfonyl)benzene. Npys is 3-nitro-2-sulfenylpyridine.
chloromethyl ketones is due to alkylation of the
inactivate both cysteine and serine proteases. The active site thiolate [104] while inactivation of serine
mechanism of inactivation is however, different for proteases involves alkylation of the active site
the proteases. In concert with other cysteine histidine [105]. In attempts to generate selective
protease inhibitors, peptidyl fluoromethyl ketones
1178 Current Medicinal Chemistry, 2000, Vol. 7, No. 12
were investigated as enzyme inhibitors [106].
Compounds incorporating fluoromethyl ketone
warheads were found to be potent inhibitors of
calpain compared to the corresponding
chloromethyl ketone analogues. For example, Z-
Leu-Leu-Phe-CH2Cl is a weak inhibitor of calpain
(9,500 M-1s-1) [107] while Z-Leu-Leu-Phe-CH2F
(290,000 M-1s-1) is a potent calpain inhibitor [94].
Within the diazomethyl ketone class of inhibitors,
tripeptide derivatives are more potent than dipeptide
derivatives. For example, Z-Leu-Leu-Tyr-CH=N2
is a potent inhibitor of calpain (230,000 M-1s-1)
while Z-Leu-Tyr-CH=N2 (1,470 M-1s-1) is a weak
calpain inhibitor [97]. Dipeptides with phosphorous
containing ketomethylene warheads (i.e. methyl
phosphinates, phosphonates, and phosphates) such
as 27 (365,000 M-1s-1) [98, 108] and tripeptides
with dimethylsulfonium methyl ketone warheads
such as 28 [101] are potent irreversible calpain
inhibitors. Compared to these compounds,
acyloxymethyl ketones are weaker inhibitors of
calpain while aryloxymethyl ketones are practically
inactive [98, 101, 109]. Sulfonium methyl ketones
are among the most potent inhibitors of calpain
known [101]. Compared to other warheads, the
order of reactivity towards calpain is: sulfonium
methyl ketone > fluoromethyl ketone, diazomethyl
ketone >> acyloxymethyl ketone [101]. The
sulfonium methyl ketone warhead is a good motif
for controlling selectivity through an affinity for the
S’ subsite of calpain by varying the substituents on
the sulfur atom. This possibility is however,
virtually unexplored. Heteroaryloxymethyl ketones
including pyridyloxy-, pyrimidinyloxy-, 2-
oxodihydrofuran-4-yl-oxy, benzotriazolyloxy-
methyl ketones (e.g. 29, 230,000 M-1s-1) have
been reported as cysteine protease inhibitors [99,
100]. According to Palmer et al. [103] dipeptide
vinyl sulfones such as 30 do not inhibit calpain and
tripeptide derivatives such as 31 (8,400 M-1s-1) are poor
inhibitors of the enzyme.
ii. SAR of the Address Region of Calpain
Inhibitors
Various researchers have investigated the
address region of calpain inhibitors in attempts to
enhance potency, selectivity and cellular
permeability of the inhibitors. Most of the studies
have centered on the unprimed region of the
inhibitors. Li et al. [75] used the α-ketoamide
warhead to access both the primed and unprimed
subsites of calpain. In this study, ketoamides 32
and 33 Fig. (3) showed 28-fold and 18-fold
selectivity for calpain II over calpain I, respectively.
This finding suggested that calpain isoform
selective agents might be obtained via SAR
I. O. Donkor
studies of the primed region of calpain inhibitors.
The authors also demonstrated that hydrogen
bonding between calpain and the P1-P1’ amide
moiety of α-ketoamides is important for enzyme
inhibition. For example, monosubstituted α-
ketoamide 33 was 380-fold more effective than
N,N-disubstituted α-ketoamide 34. The difference
in calpain inhibitory potency was attributed to the
inability of 34 to form a critical hydrogen bond at
the active site of the enzyme. In a recent study,
Chatterjee et al. [110] reported a series of potent
calpain I inhibitors such as 35 and 36 with
substituents spanning the S’ subsite of calpain.
The S1 subsite of calpain can tolerate a wide
variety of amino acids at the P1 position of
inhibitors [75, 94, 111]. For example, P1-Phe (37,
136,300 M-1s-1) is preferred over Abu (38, 24,250
M-1s-1), and serine (39, 21,000 M-1s-1) [94].
Protection of the P1-Ser residue as a
tetrahydropyranyl ether as in 40 (100,000 M-1s-1)
increased calpain I inhibition 5-fold. Introduction of
Tyr(Bn) and Lys(SO2-Ph) at P1 as in compounds
41 (Ki = 4 nM) and 42 (Ki = 2 nM) resulted in 8-
fold and 12-fold more potent inhibitors than 43 (Ki
= 36 nM), respectively [63]. The results suggest
that the S1 subsite of calpain can accommodate
large hydrophobic groups from the P1 position of
inhibitors to enhance calpain inhibition. The data
also show that the presence of polar groups such as
the hydroxyl moiety of serine in the S1 pocket is
detrimental to binding. This polar residue
intolerance at the S1 subsite might account for the
dramatic drop in activity observed by Angliker et al.
[112] in their study of Z-Leu-D,L-Tyr-CH2F
(17,000 M-1s-1) and Z-Leu-D,L-Phe-CH2F
(136,300 M-1s-1) and the similar activities of Z-
Leu-D,L-Tyr-CH2F and Z-Leu-D,L-Ser-CH2F
(21,000 M-1s-1) as inhibitors of chicken gizzard
calpain II.
Until recently, it was a generally held view that
to obtain a potent peptidyl calpain inhibitor the P2
residue must be either L-Val or L-Leu. However,
Chatterjee and coworkers [63] used a variety of D-
amino acids including D-Ser(Bn) (44, Ki = 8 nM),
D-Thr(Bn) (45, Ki = 9 nM), D-Phe (46, Ki = 11
nM), D-Trp (47, Ki = 10 nM), and D-(thiophen-2-
yl)-Ala (48, Ki = 11 nM) to demonstrate that
calpain can tolerate D-amino acids at the P2 position
of inhibitors. They also showed that 49 with L-
Ser(Bn) (Ki = 41 nM) at P2 is more than 5-fold less
potent than 44 with D-Ser(Bn) at P2 attesting to the
importance of the D-configuration at P2. Tripathy et
al. [113] studied the effect of introducing constraint
in the P2 residue on calpain inhibition using proline
as the P2 substituent. The enzyme accommodated
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1179

Ph Ph
O O O O
H H
O N O N
N N N N
H H H H
O O O O

33
32
Ph Ph
O O Cl O O
H
O N N H S O 2X
N N N NH
H H
O O O
Cl

34
CN
3 5, X =
S

O R
H
O N
N F 36 , X = N HA c
H S
O O

3 7, R = B n; 3 8, R = Et;
3 9, R = C H 2 O H ; 40 , R = C H 2 O T H P

O R O
(D)
H (D)
R H
O N
H N
H
HN O
NH O
S O2 C H3 C H 3S O 2

4 4, R = C H 2 O B n ; 4 5, R = ( S) ( C H 3 ) C H O B n
4 1, R = C H 2 C 6H 4- p - B n
4 6, R = C H 2 P h; 47 , R = C H 2 - ( 3- i nd ol e )
4 2, R = ( C H 2 )4 N H S O 2P h
4 3, R = C H 2 C H 3 4 8, R = C H 2 - ( 2- th io ph e ne ) ; 49 , R = L - C H 2 O B n

S O2 C H3 S O 2C H 3
N N
H H
N CH O N C HO
H H
O O

50 51

Fig. (3). Examples of compounds used to study calpain active site.

both L-proline (50, IC50 = 45 nM) and D-proline
(51, IC50 = 53 nM) derivatives at the P2 position
further supporting the suggestion that L-Leu and L-
Val are not obligatory substituents at the P2-
position of a potent calpain inhibitor. Giordano et
al. [59] synthesized a series of EP-475 derivatives
incorporating Phe (18), Tyr(I) (19), and Tyr(I2)
(20) in place of the P2-Leu residue of compound
10 (Table 1). This study suggested that the S2
subsite of calpain I is a better accommodator of
bulky hydrophobic substituents than that of
calpain II as reflected by the apparent second order
rate constant of inactivation of the compounds.
Further investigation of the bulk tolerance of the
S2 pockets of the enzymes may lead to the
discovery of calpain isoform selective agents.
1180 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

O O
H H N
O H
O N N N
H N
N Br H
H N
O O H O
O

52 53 54
O

O N O N
O
H
N H
N NH 2
H N N
H O
O H O
N O
H
O

55 56 57

O O

O O
N O O H
H H N
N N H
H H
O O
O O

59
58 62

C H 3S O 2N H
O
H H N
O N N S O
(D ) C H2 C H2 N HS O2 H
+ N
O O S H

O O

60 63

F
O
H H N O
N N S
H
C H 2 C H 2N H S O 2 O N H

F O O
O O

61 64

Fig. (4). Examples of peptidomimetic calpain inhibitors.

SAR studies aimed at generating peptidomimetic bioisosteric replacements of the backbone amide of
replacements for peptide-based enzyme inhibitors dipeptide and tripeptide inhibitors of the enzyme.
have attracted considerable attention due to Most of these changes have focused on the P2 and
pharmacokinetic problems associated with peptidic P3 regions of the inhibitors. The critical role that
inhibitors. The general approach to the design of the P1-P2 amide linkage plays in calpain inhibition
peptidomimetic calpain inhibitors involves [114, 115] generally precludes this region from
Calpain Inhibitors
extensive modification. Nonetheless, several
oxygen-containing heterocyclic compounds (e.g.
25) with modified P1-P2 amide linkage have been
reported as useful peptidomimetic cysteine protease
inhibitors. Such compounds are potent aldehyde
prodrugs with excellent oral bioavailability and
cellular permeability [89, 116, 117].
Azapeptide-based peptidomimetics have been
reported as calpain inhibitors. In these compounds
the alpha carbon of the P1 amino acid residue is
replaced with a nitrogen atom. Such a modification
reduces the electrophilicity of the P1 carbonyl group
and changes the geometry of the alpha position
from tetrahedral to trigonal (i.e. planar, achiral)
[118]. This makes azapeptides more resistant to
enzymatic hydrolysis than their normal peptide
analogues. The reduced reactivity of the carbonyl
group also makes azapeptides with poor leaving
groups poor inhibitors of serine proteases [119,
120-123]. However, due to the greater
nucleophilicity of the thiolate at the active site of
cysteine proteases, these enzymes including calpain
are inhibited by azapeptides [124]. Thus,
azapeptide α-halomethyl ketones such as 52 are
moderate inhibitors of calpain (1,500 M-1s-1) and
poor inhibitors of cathepsin B (875 M-1s-1) [125].
Peptidomimetic calpain inhibitors incorporating a
variety of heterocylces at the P2 position have been
reported in the recent literature [see Fig. (4) for
examples]. Peptide mimetic constructs that have
been introduced at the P2 position include
substituted quinolinecarboxamides such as 53
(IC50 = 12 nM), which is equipotent with standard
L,L-dipeptide aldehydes [81, 126];
indolecarboxamides (e.g. 54, IC 50 = 30 nM)
[127]; and ketobenzamides (e.g. 55, Ki < 10 µM)
[84, 128]. 1,4-Disubstituted piperidines (e.g. 56
and 57 and cyclohexanes (e.g. 58) have been used
as scaffolds for the P2-P3 bridge [67, 71, 83].
Aryl-substituted benzoyl groups (e.g. 59) and
other aryl-substituted benzoheterocycles have been
used as P2 mimetic constructs [82, 129, 130].
These examples clearly demonstrate that
benzoheterocycles are good peptide mimetic
replacements for Leu or Val at P2. Additionally, it
has recently been demonstrated that replacement of
the P2 residue of α-ketoamides such as 60 (Ki =
10 nM) with a 2,6 difluorobenzoyl group as in 61
(Ki = 14 nM) affords equipotent calpain inhibitors
[110]. This study revealed that the binding affinity
offered by substituents spanning the P’ address
region of α-ketoamide inhibitors of calpain can
adequately offset the loss in binding affinity due to
the absence of a P2-amino acid residue. Using a
series of ketomethylene (e.g. 62, IC50 = 25 nM)
Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1181
and carbamethylene (e.g. 63, IC50 = 30 nM)
inhibitors of recombinant human calpain I
Chatterjee et al. [73] reported that the P2-P3 amide
bond of a potent dipeptide or tripeptide inhibitor of
calpain is not a strict requirement for calpain
inhibition. This finding is at odds with the
results of Dolle and colleagues [131] who
concluded that the P2-NH moiety is a critical
hydrogen-bonding site, which must be maintained in
a peptidic calpain inhibitor. Unlike the P2-P3
amide linkage, the P1-P2 amide bond is very
important for calpain inhibition. Thus,
ketomethylene derivative 64 is 250-fold less potent
than the parent compound, MDL 28,170 (1)
against chicken gizzard smooth muscle calpain
[114]. The loss in potency of 64 reflects the lack of
a critical hydrogen bond interaction between the
NH of the P1 amide and calpain [115].
N-terminal capping of the P3 and P4 residues of
calpain inhibitors prevent metabolism by
aminopeptidases, improve cellular permeability,
and serve as additional recognition elements for
binding to the S3 and S4 subsites of calpain. Amino
acid protecting groups such as benzyloxycarbonyl
[34, 35], acetyl [37], and 4-phenylbutylryl [37]
have been employed as N-terminal capping agents
of synthetic analogues of calpain inhibitors derived
from microbes as discussed above. Other groups
that have been used as N-terminal capping agents
[see Fig. (5)] include aryl- (or alkyl) sulfonyl
groups (e.g. 65-68, 66, 14, 15) [63, 64, 87-89,
132, 133]; alkanoyl groups (e.g. 69, 70, [65, 66,
132]; various substituted benzoyl, naphthoyl, and
several heterocyclic acyl groups [64, 132]; xanthine
(e.g. 62) [72, 73]; thionaphthalene (e.g. 63) [74,
75]; and cyclic sulfonamides like 3,4-
dihydrobenzothiazines (e.g. 71) and related
heterocycles [134].
C. Nonpeptide Calpain Inhibitors
Inorganic lead and nonpeptide organic
compounds of diverse structure are calpain
inhibitors. Lead (Pb2+) is a viable substitute for
Ca2+ in a variety of intracellular processes but it
also acts as a noncompetitive inhibitor of calpain
depending on the concentrations of both ions [135].
A series of α-mercaptoacrylates of which 72
(PD150606) and 73 (PD151746) [Fig. (6)] are
examples were disclosed by Wang et al. [136,
137] as potent, selective and cell permeable
uncompetitive (with respect casein) inhibitors of
calpain. X-ray crystal studies revealed that 72
inhibit calpain by binding to a hydrophobic pocket
on domain VI of calpain small subunit [3].
1182 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

F
O
H O
N H H
S N H3 C N H
O O H S N
O O H
O
O
O
65

66

HO
O O
O
H
N H H
N N
S N
H S N
O O H
O O O

67 68

C H 3( C H 2) 7 CH=CH NH O
O O CH 3
H
H3 C N N H
N N
OH H H
O O
C O NH 2
69

O
C O NH 2
OH O O O H
H N
N H H
C H 3- ( C H 2) 9 N N N O
H H O S
O O O O
C O NH 2
70
71

Fig. (5). Examples of peptidomimetic calpain inhibitors with various N-terminal capping groups.

Chloromethyl ketone 74 was described as a weak calpain and the kinetics of inhibition of most of the
irreversible nonpeptide inhibitor of calpain [138]. nonpeptide inhibitors are yet to be deciphered. It is
Isocoumarin analogues such as 75 are good conceivable that the compounds may inhibit calpain
inhibitors of serine proteases but are weak by binding to an allosteric site on the enzyme.
inhibitors of calpain (IC50 of 10 µM) [139].
Clavam-3-carboxamide derivatives such as 76
(23,500 M-1s-1) are irreversible inhibitors of D. Selectivity of Calpain Inhibitors
calpain [140]. Aurintricarboxylic acid 77 was Most peptidyl calpain inhibitors lack selectivity
demonstrated by Posner et al. [141] as a weak (IC50 for the enzyme. This lack of selectivity is inherent
of 22 µM) nonselective cell permeable calpain in the design of the inhibitors since the warheads of
inhibitor. Quinolinecarboxamides including 78 calpain inhibitors are identical to those found in
(IC50 of 510 nM) are fairly potent noncompetitive other proteases inhibitors. In the design of calpain
inhibitors of calpain [142]. Nonpeptide calpain inhibitors it was envisioned that appending general
inhibitors such as phevalin 4 (41), diketopiperazine protease inhibitor warheads to calpain preferring
5 (42), damnacanthal 7 (43) and penicillide 8 (44), subsite residues would generate selective inhibitors
which are obtained from natural sources were of the enzyme. However, this approach to selective
discussed earlier. The site of interaction with calpain inhibitors has enjoyed limited success
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1183

HO O OC H3
O OH
HN CH 3 CH O
O
N
N CH 3 OH

N O
O
4 5 7

O O H
N
H 3C O
CH 3 I SH
SH

O C O OH
HO C O OH F
HO
72 73
8

O
H
N N Cl
N
H
O
H
N O O
H2N O
O NH
N N (C H2 )6
O H
O S NH 2

Cl
74 75 76

O
O
C O OH
C O NH 2

N
H OO C C O OH
N Cl

HO OH
77 78
OH

Fig. (6). Examples of nonpeptide calpain inhibitors.

especially regarding selective inhibition of calpain calpain selectivity of dipeptide ketoamides by using
over cathepsin L. Leu-Phe or Leu-Abu at the P2-P1 position.
Chatterjee et al. [73] observed that incorporation Calpain is unique among the cysteine protease
of a xanthine group at the P3 position of family of enzymes in that it possesses a calcium-
ketomethylene calpain inhibitors such as 62 binding domain. Inhibition of calpain via
increased selectivity for calpain over cathepsin B by interaction of compounds with this domain
about 17-fold. In their study of a series of α-keto presents an attractive route to calpain selective
carbonyl containing calpain inhibitors Li et al. [75] agents. Indeed, Wang et al. [3, 136] have disclosed
noted that a number of the inhibitors were equally α-mercaptoacrylates (e.g. 72 and 73) as the first
potent with the calpains but some were an order of calpain inhibitors that inhibit the enzyme by binding
magnitude less effective with cathepsin B (Table 5). to the calcium domain. The compounds are highly
These investigators found that they could increase selective for calpain versus cathepsin B (Table 5).
1184 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

Table 5. Examples of Selective Inhibitors of Calpain

µM)
Ki (µ

Compounda Calpainb Cat. B (SR)c Ref.

Z-Leu-Abu-CONH(CH2)8CH3 0.12 150 (1,250) [75]

Z-Leu-Phe-COOH 0.0085 4.5 (529) [75]
Z-Leu-Phe-COOH 0.0057 (Cal. II) 4.5 (790) [75]
Z-Leu-Phe-COOEt 1.8 3.40 (189) [75]
Z-Leu-Phe-COOEt 0.40 (Cal. II) 3.40 (850) [75]
Z Leu-Phe-CONH(CH2)2Ph 0.052 9.3 (179) [75]
Z Leu-Phe-CONH(CH2)2Ph 0.024 (Cal. II) 9.3 (388) [75]
72 0.21 127.8 (609) [136]
73 0.26 >200 (>769) [136]
78 0.5 25 (50) [142]

Rate (M-1s-1)

t-Boc-Leu-Phe(DL)-CH2F 68,600 150 (457) [94]

Z-(D)-Ala-Leu-Phe-CH2-DFB 31,000 100 (310) [97]
Z-Leu-Leu-Phe-CH2S+(CH3)2Br- 200,000 (Cal. II)b 3,500 (57) [101]
Z-Leu-Leu-Tyr-CHN2 230,000 (Cal. II) b
1,300 (177) [143]
a
Z = Benzyloxycarbonyl; DFB =2,6-Di-fluorobenzene.
b
Calpain I was used except where indicated as calpain II.
c
Selectivity Ratios equal to or greater than 50 are reported.

Quinazolinecarboxamides such as 78 also show Leu-Leu-Phe-CH2 OCO[2,6-(CF3 )2 ]Ph, 1000 M -

over 50-fold selectivity for calpain over both s ) are poor inhibitors of calpain. The
1 -1

cathepsin B and cathepsin L. The compounds are
not active site directed inhibitors of calpain and
hence may inhibit the enzyme by binding to a yet to
be identified allosteric site [142].
Chatterjee and coworkers [94] demonstrated that
t-Boc-Leu-Phe(DL)-CH2F is over 450-fold and
680-fold selective for calpain I over cathepsins B
and L, respectively. Harris and others [97] studied
a series of dipeptide and tripeptide
arylacyloxymethyl ketones and found that none of
the dipeptides were selective for calpain versus
cathepsin L but a tripeptide with a P3 D-Ala residue
was over 100-fold selective for calpain over
cathepsins B and L. Pliura and coworkers [101]
demonstrated that peptidyl sulphonium methyl
ketones (e.g. Z-Leu-Leu-Phe-CH2S+(CH3)2.Br-,
200,000 M-1s-1) are selective inhibitors of calpain
over cathepsin B (3500 M-1s-1). Unlike sulfonium
methyl ketones, acyloxymethyl ketones (e.g. Z-
difference in calpain inhibitory activity between
these compounds was attributed to the steric bulk
of the arylacyloxy leaving group, which cannot be
tolerated by the active site of calpain. Crawford et
al. [143] showed that Z-Leu-Leu-Tyr-CHN2 is over
170-fold selective for calpain II versus cathepsin B
but the compound is about 7-fold, a better inhibitor
of cathepsin L than calpain II.
III. CONCLUSION
A variety of peptidyl calpain inhibitors derived
from mammalian, plant, and synthetic sources have
appeared in the literature. Majority of these
inhibitors are active site directed reversible or
irreversible agents with limited selectivity for
calpain over other cysteine proteases. Most of the
SAR studies undertaken to enhance the potency and
selectivity of the inhibitors involved modifications
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1185

to enhance binding to the unprimed subsite of [12]
calpain. Though potency enhancement has been
realized to some degree, selectivity via this
[13]
approach still remains a formidable challenge.
Compared to the unprimed subsite, the primed [14]
subsite of calpain has been marginally investigated.
Future SAR studies should therefore concentrate
efforts at investigating the primed subsite of the [15]
enzyme. The calcium-binding domain also affords [16]
an excellent target for the design of calpain selective
agents. Though, this site has not been adequately
explored, it will undoubtedly be the focus of future
attempts at developing novel selective cell
permeable nonpeptide calpain inhibitors. [17]
Otto, H-H.; Schirmeister, T. Chem. Rev. 1997, 97,
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IV. ACKNOWLEDGEMENT
[19] Wang, K.K.W. Trends Pharm. Sci. 1990, 11, 139-
The author thanks NHLBI for grant 1 K14 142.
HL03536 to develop selective calpain inhibitors,
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