Benzoylalanine-derived ketoamides carrying vinylbenzyl amino residues: Discovery of potent water-soluble calpain inhibitors with oral bioavailability

Article abstract of DOI:10.1021/jm0210717

Novel benzoylalanine-derived ketoamides were prepared and evaluated for calpain I inhibition. Derivatives carrying vinylbenzyl amino residues in the P₂ - P₃ region inhibited calpain in nanomolar concentrations and thus represent a novel class of nonpeptidic calpain inhibitors. Selected examples exhibited an improved pharmacokinetic profile including improved watersolubility and metabolic stability. In particular, these calpain inhibitors showed oral bioavailability in rats as demonstrated by N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2- [E-2-(4-diethylaminomethylphenyl)ethen-1-yl]benzamide (5d). The closely related derivative N-(1-carbamoyl-1-oxohex-1-yl)-2-[E-2- (4-dimethylaminomethylphenyl)-ethen-1-yl]benzamide (5b) was evaluated for neuroprotective efficacy after experimental traumatic brain injury in a fluid percussion model in rats. When administered after injury, 5b reduced the number of damaged neurons by 41%, and this result would be in line with the suggested neuroprotective efficacy of calpain inhibition.

Full text of DOI:10.1021/jm0210717

2404
J . Med. Chem. 2003, 46, 2404-2412
Ben zoyla la n in e-Der ived Ketoa m id es Ca r r yin g Vin ylben zyl Am in o Resid u es:
Discover y of P oten t Wa ter -Solu ble Ca lp a in In h ibitor s w ith Or a l Bioa va ila bility
Wilfried Lubisch,* Edith Beckenbach, Sabina Bopp, Hans-Peter Hofmann, Arzu Kartal, Claudia Ka¨stel,
Tanja Lindner, Marion Metz-Garrecht, J utta Reeb, Ferdinand Regner, Michael Vierling, and Achim Mo¨ller
Neuroscience Discovery Research, Abbott GmbH & Co. KG, P.O. Box 210805, D-67008 Ludwigshafen, Germany
Received October 14, 2002
Novel benzoylalanine-derived ketoamides were prepared and evaluated for calpain I inhibition.
Derivatives carrying vinylbenzyl amino residues in the P2-P3 region inhibited calpain in
nanomolar concentrations and thus represent a novel class of nonpeptidic calpain inhibitors.
Selected examples exhibited an improved pharmacokinetic profile including improved water-
solubility and metabolic stability. In particular, these calpain inhibitors showed oral bio-
availability in rats as demonstrated by N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethyl-
aminomethylphenyl)ethen-1-yl]benzamide (5d ). The closely related derivative N-(1-carbamoyl-
1-oxohex-1-yl)-2-[E-2-(4-dimethylaminomethylphenyl)-ethen-1-yl]benzamide (5b) was evaluated
for neuroprotective efficacy after experimental traumatic brain injury in a fluid percussion
model in rats. When administered after injury, 5b reduced the number of damaged neurons
by 41%, and this result would be in line with the suggested neuroprotective efficacy of calpain
inhibition.
Mammalian calpains represent a family of nonlyso-
somal cysteine proteases of which 14 members have
been reported so far.1 The most familiar members of this
family, calpain I and calpain II, have been well-known
for many years.2 More recently discovered calpains have
attracted particular interest since their physiological
functions have been attributed to defined human dis-
eases. For example, calpain 10 has been postulated as
a susceptibility gene for type-2 diabetes,3 and, further-
more, the loss of activity of calpain 3 has been related
to limb-girdle muscular dystrophy 2A.4
therapeutic efficacy of calpain inhibitors in human
disease has been reported.17
Most of the known calpain inhibitors bind to the
catalytic center in a competitive manner and are derived
from small peptides which are structurally related to
the cleavage site of calpain substrates.18 Irreversible
calpain inhibitors are designed by assembling a back-
bone and a particular reactive moiety capable of enter-
ing an irreversible covalent bond with the cysteine
present in the catalytic center. Well-known irreversible
reactive moiety are epoxides, diazoketones, R-halo-
ketones, and vinyl sulfones. Most reversible calpain
inhibitors represent transition state mimetics consisting
of a backbone and an aldehyde or a ketone as the
reactive moiety which enter a reversible covalent bond
with the cysteine residue in the catalytic center. Two
of the well-known reversible inhibitors, MDL 28170 (1)19
and AK 295 (2),20 have the dipeptides Z-Val-Phe and
Z-Leu-Abu, respectively, as backbones and either an
aldehyde or a ketoamide moiety as a reactive moiety
(Figure 1). Some progress has been made in modifying
the peptidic backbone, and nonpeptidic calpain inhibi-
tors have been discovered.17,21 In general, using such
aldehyde and ketone moieties as reactive moiety can
give rise to problems with respect to stability, metabo-
lism, and selectivity. Accordingly, the peptidic aldehyde
MDL 28170 (1) shows typical structure-related dis-
advantages, such as decomposition during storage and
excessive metabolism.22 The ketoamide AK 295 (2),
which is one of the first water-soluble, reversible calpain
inhibitors, is only effective when administered via a
special route.20 Efforts to discover an appropriate sur-
rogate for the highly reactive aldehyde or ketone as
reactive moiety have been unsuccessful. Other func-
tional groups were examined as reactive moiety, but
most failed due to their modest inhibition of calpain.23,29
Moreover, there have been few reports of calpain inhibi-
Calpain I is activated by calcium ions and is also
called µ-calpain, according to micromolar calcium con-
centrations which are required for its activation in vitro.
Calpain I is an intracellular protease that is ubiqui-
tously found in man. Its physiological role is not yet fully
understood but many intracellular processes such as the
degradation of cytoskeleton proteins have been at-
tributed to calpain I.5 Over the past decade a number
of more specific roles of calpain I have been described
which suggest the involvement of calpain I in modulat-
ing cellular signal transduction,6 apoptotic processes,7
activation of platelet proteins,8 and degradation of
cyclin-dependent kinase cdck5 specific activator p35.9
Excessive activation of calpain I triggers serious cell
damage or even cell death,10 and calpain I is presumed
to contribute to the progress of a number of diseases.11
Indeed, the inhibition of calpain I has revealed beneficial
effects in experimental models of, for example, stroke,12
myocardial infarction,13 brain trauma,14 and multiple
sclerosis.15 Therefore calpain I has attracted interest in
drug discovery research.16 But, though substantial
efforts were focused on calpain inhibitors for many
years, up to now, no clinical trial investigating the
* To whom correspondence should be addressed. Phone: (49) 621
589 3445; fax: (49) 621 589 3398; e-mail: wilfried.lubisch@abbott.com.
10.1021/jm0210717 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/03/2003

Benzoylalanine-Derived Ketoamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2405
F igu r e 1.
tors exhibiting the oral activity which is required for
them to be used in some of the envisaged therapies.24
Hence, the use of the calpain inhibitors, so far reported,
is limited for one or more of the following reasons: poor
selectivity, poor metabolic stability, low cellular pen-
etration, poor kinetics, and, depending on the envisaged
therapeutic indication, low oral availability and low
water-solubility.17
F igu r e 2.
Our approach was to design novel calpain inhibitors,
which had fewer peptidelike features in order to obtain
more stable structures. First, we focused our efforts on
discovering novel nonpeptidic backbones appropriate for
reversible calpain inhibitors. Previously, we reported
substituted naphthoyl piperidines derived from keto-
amides as reactive moiety to be potent calpain inhibi-
tors.25 Proceeding these efforts we discovered the ben-
zoylalanine derivative 3 which was a moderately potent
calpain inhibitor (Figure 2). Optimization of the sub-
stitution pattern at the benzoyl moiety, in particular
in the ortho-position, has enabled us to design potent
calpain inhibitors such as 4.26 Interestingly, we noticed
that several ketoamide-derived inhibitors within this set
had improved metabolic stability in vitro and in vivo.
Encouraged by this finding we focused our efforts on
derivative 5 carrying a styryl residue at the benzoyl
moiety and a ketoamide as reactive moiety which,
ultimately, led us to the discovery of aminomethyl
derivatives such as 5d , which exhibit superior properties
such as water-solubility, potent calpain inhibition,
improved metabolic stability, and oral bioavailability.
The syntheses of the novel benzoylalanine-derived
ketoamides are outlined in Figures 3 and 4.
F igu r e 3. Route of synthesis for the P1 building block 11a .
(i) CH3OH, Et3N, rt.; (ii) AcOH, H2, Pd/C, 50 °C; (iii) CH3OH,
(Boc)2O, rt.; (iv) 1. THF/DMF, NH3, EDC, HOBt, rt.; 2. HCl.
and subsequently coupled to NH3 using the EDC/HOBt
coupling variant. After removal of the protection group
and recrystallization of the resulting amine, a single
diastereomer of amine 11a was obtained. The corre-
sponding phenyl derivative 11b was prepared according
Harbenson et al.27
The stilbene carboxylates 14 were prepared via Heck
reactions. Styrols 13 were added to o-bromobenzoic
carboxylate 12 in the presence of Pd[(o-tol3P)2Cl]2 at 100
°C to give the esters 14 in 50-100% yields. However,
the corresponding pyridine derivative 14c was only
obtained in lower yield (42%), which may be due to the
use of an aromatic chloride 12b. E-Configured stilbenes
14 were formed predominately in these Heck reactions.
The E-configuration of these stilbene derivatives was
determined according to the coupling constants (J )15-
17 Hz) between the two olefinic protons. In a final step,
The P1 building block 3-amino-2-hydroxyheptanoic
acid amide (11a ) was available starting from 1-nitro-
pentane (6) (Figure 3). Compound 6 was added to
glyoxalic acid at ambient temperature to give a dia-
stereomeric mixture of the nitro alcohol 8 in over 90%
yield. Hydrogenation of the crude 8 with Pd/C at an
elevated temperature (50 °C) gave the aminocarboxylate
9. The amino group in 9 was protected using (Boc)2O

2406 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Lubisch et al.
F igu r e 4. Route of synthesis for the envisaged calpain inhibitors 5. (i) DMF, Pd(OAc)2, (o-Tol)3P, 100 °C; (ii) THF/H2O, KOH;
(iii) DMF/CH2Cl2, EDC, HOBt, (iv) DMSO, DCC.
Ta ble 1. Synthesized Compounds and Their Results in Inhibition of Human Calpain I (Ki) and in Inhibition of the Degradation of
the Tyrosine Kinase Pp60src (IC50) in Human Platelets
calpain I
cathepsin B
pp60src
X
R1
R2
Ki (nM)b
Ki (nM)b
IC50 (µM)c
3
4a
4b
5a
5b
5c
5d
-
-
-
-
-
-
H
108026
nta
nta
nta
nta
1.4
0.8
1.0
0.7
0.7
CHdCH-Ph
2-naphthyl
2-naphthyl
4-(C6H4)-CH2N(CH3)2
4-(C6H4)-CH2N(CH3)2
4-(C6H4)-CH2N(CH2CH3)2
-
14026
nta
1526
8.4 ( 2.7
13.3 ( 1.5
18.3 ( 1.2
27.0 ( 2.5
15.5 ( 2.0
nta
CH
CH
N
CH
-
phenyl
CH2CH2CH3
phenyl
phenyl
-
99 ( 15
27 ( 6
83 ( 11
62 ( 9
18 ( 3
MDL 28170
a
b
Not tested. Mean ( SEM. c Mean of two or more independent experiments.
all esters 14 were hydrolyzed with diluted NaOH or
KOH to afford the carboxylates 15. These carboxylates
15 were coupled to the amines 11 by the EDC/HOBt
coupling variant to give the amides 16. These amides
16, all of which carry an secondary alcohol group, were
oxidized by DMSO/DCC at ambient temperature which
resulted in the envisaged ketoamides 5. Compound 5d
was converted into a methanesulfonate because of its
excellent solubility in water (>5%) whereas both 5b and
5c were used as hydrochlorides.
The prepared ketoamides 5 were evaluated for inhibi-
tion of calpain I in a common assay using human calpain
I isolated from erythrocytes and Suc-Leu-Tyr-AMC as
the fluorogenic substrate.28 The results are summarized
in Table 1.
All derivatives 5 carried a ketoamide as reactive
moiety and displayed a reversible mode of action (data
not shown). These four compounds were potent calpain
inhibitors, which confirmed the ketoamide moiety to be
eminently useful as a warhead in calpain inhibitor
design. The naphthalene 5a was the most potent inhibi-
tor within the present set and had a Ki of 8.4 nM.
Compounds 5b, 5c, and 5d displayed slightly dimin-
ished potency in calpain I inhibition. Their Kis were 13.3
nM, 18.3 nM, and 27.0 nM, respectively.
Our approach used the benzoyl derivative 3 as the
starting point carrying an aldehyde warhead. Derivate
3 showed only modest affinity for calpain I (Ki ) 1.08
µM).29 The insertion of a phenylvinyl or naphthylvinyl
moiety in the ortho-position of this benzoyl residue
resulted in 9-fold and 70-fold higher potency, respec-
tively. Interestingly, within this structural class both
aldehydes and ketoamides as reactive moiety exhibited
similar potency for calpain inhibition. For instance, the
naphthylvinyl ketoamide 5a displayed equally high
potency in calpain inhibition as the corresponding
aldehyde.26 A typical feature of these inhibitors may be
the ortho-substitution at the benzoyl moiety. The favor-
able long bulky residues attached to this benzoyl moiety
suggest a hydrophobic cave in the P2-P3 region at the
enzyme-binding pocket in which these residues fit well.
The size of this pocket might be huge since the large
distal vinyl naphthalene residue seemed favorable for
affinity. This is in agreement with other studies dem-
onstrating favorable effect on affinity by large lipohilic
residues in P2-P3.30 Most of the reported transition-
state mimetic calpain inhibitors carry polar groups such
as an amide or a sulfonamide within the P2-P3 region,
which are considered to be just as strongly favorable in
calpain inhibition.21 Surprisingly, the above-mentioned
derivatives such as 5a do not carry any polar moieties
within this region, which indicates that the incorpora-
tion of an optimized lipophilic side chain in P2-P3
overcomes the lack of such an important polar ligand-
enzyme interaction.
Insertion of aminomethyl moiety into the para-posi-
tion at the phenylvinyl residue in 4 also resulted in
calpain inhibitors which were 5- to 10-fold superior in
potency. This finding appears to be contradictory to the
above results since both the insertion of lipophilic
residues, such as the annellated benzene ring (5a ), and
a highly polar residue, such as a basic amino group (5b-
d ), as substituents at the distal phenylvinyl moiety was
favorable. It is unclear whether the amines 5b-d fit in

Benzoylalanine-Derived Ketoamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2407
F igu r e 6. Dose-dependent inhibition of the pp60src degrada-
tion in human platelets by calpain inhibitor 5d . Shown are
percent inhibition of the cleavage of pp60src versus the
inhibitor concentration.
F igu r e 5. Correlation of inhibition constants Ki of calpain I
inhibition and either actin-binding protein (ABP) degradation
(IC50, open circles, gray line) or pp60src degradation (IC50
,
squares, black line) of a set of calpain inhibitors in the human
platelet assay. The set includes AK295, MDL 28170, the herein
presented inhibitors, and a number of corresponding deriva-
tives (data not shown). A linear regression analyses was used
to determine the correlation coefficient r.
IC50 values, as demonstrated by 5d (Figure 6). The IC50
values in the pp60src assay were generally 5- to 10-fold
lower than in the ABP assay.
We also evaluated these results of the same set of
calpain inhibitors by a linear regression analysis (Figure
5) and obtained a good correlation (r ) 0.90, p < 0.001)
between enzymatic calpain inhibition (Ki) and cellular
inhibition of pp60src degradation (IC50). Therefore this
assay appeared to be useful to examine the cellular
penetration of calpain inhibitors.
this enzyme-binding pocket in the same manner as the
naphthalene 5a . Further studies with the recently
reported X-ray structure of µ-calpain might help to
elucidate these findings.31,32
Since calpain I is an intracellular enzyme, the capa-
bility of inhibitors to cross cell membranes is of consid-
erable importance. In general, the penetration of calpain
inhibitors into cells has been estimated by their inhibi-
tion of calpain-mediated intracellular protein degrada-
tion. Several cytoskeletal proteins, such as spectrin,
MAP2, actin-binding protein (ABP), talin etc., were
evaluated, and extracellular applicated calpain inhibi-
tors attenuated or blocked their degradation.32 In a first
approach we examined the calpain-mediated degrada-
tion of actin binding protein (ABP) and talin in human
platelets.33 A number of peptidic and nonpeptidic calpain
inhibitors were evaluated and, indeed, they inhibited
the formation of the corresponding degradation products
(data not shown). However, most compounds exhibited
rather similar cellular efficacy (expressed as IC50),
raising questions on the correlation of these efficacies
with calpain inhibition. We examined the correlation of
the cellular activity (IC50) and calpain inhibition (Ki) by
a linear regression analysis and, in fact, observed no
correlation (r ) -0.02, p ) 0.47, Figure 5). Of course,
the differential ability of the compounds to penetrate
into cells might impede any correlation. However even
structurally very closely related derivatives displayed
similar potencies in this assay inspite of substantial
differences in their Ki values for calpain inhibition.
Interestingly, similar small variations in the cellular
efficacy of calpain inhibitors were reported when using
another cytoskeleton protein, spectrin, as a marker for
calpain activity.34 To overcome this problem we searched
for other calpain degraded proteins and selected the
tyrosine kinase pp60src which was identified as calpain
substrate by Oda et al.35 In an adapted assay, calpain
in human platelets was activated by adding the calcium-
ionophore A23187 and calcium, and the pp60src deg-
radation products with molecular weights of 52kDa and
47kDa were identified by Western blotting. The com-
pounds exhibited concentration-dependent inhibition of
the pp60src degradation, which enabled us to determine
The calpain inhibitors 5a -d were evaluated in this
pp60src-assay and exhibited IC50s ranging from 0.6 and
1.4 µM, which demonstrates that all four inhibitors
penetrated into cells (Table 1), even though there was
a more than 30-fold decrease in cellular activity (IC50)
compared to enzymatic inhibition (Ki). This decrease is
in line with the results reported elsewere by other
groups using spectrin as a marker.34 At first glance,
these differences in activity may indicate poor or moder-
ate ability of the inhibitors to penetrate into cells. But
the inhibitors were present within cells immediately and
were equipotent when the time of incubation was varied
(data not shown). Unspecific intracellular protein bind-
ing of the inhibitors may have impact on their free
concentration and therefore on the IC50s in this assay.
However, the inhibitors 5b-d showed protein binding
below 90% which are acceptable values ruling out
significant problems due to high protein binding (data
not shown). Both the good correlation of the pp60src
assay and the similar magnitude of declines of cellular
potency suggest the involvement of other unknown
factors in these cellular calpain assays. It is unclear
whether these declines in cellular activity may have
impact on the results in vivo.
We also evaluated whether the novel calpain 5a -d
inhibitors block other cysteine proteases such as cath-
epsin B, papain, and caspase 3 (data not shown). All
four compounds 5a -d inhibit cathepsin B with Kis
ranging from 20 to 100 nM. No inhibition was seen for
papain and caspase 3 up to 10 µM. Therefore these
inhibitors exhibit a similar profile like many other
reported transition state mimetic calpain inhibitors
blocking both calpain and cathepsin B whereas good
selectivity versus other cysteine proteases was observed.
One major issue in our search for novel calpain
inhibitors was to improve their pharmacokinetic profile.
Therefore, the novel inhibitors 5 were evaluated for

2408 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Lubisch et al.
physicochemical and pharmacokinetic parameters in-
cluding oral bioavailability.
With the exception of 5a , the present inhibitors 5
showed considerable water-solubility as salts at pH 5
to 5.5 which enabled the intravenous administration of
aqueous solutions. The compound 5d as a methane-
sulfonate even exhibited a solubility of over 5% in water.
At pH values of around 7, only 5c displayed considerable
water-solubility which may be due to its pyridine
moiety.
At pH 7, the log P values of 5b, 5c, and 5d were
estimated to be 3.4, 2.9, and 2.8, respectively, which are
fairly good values for compounds to cross cell mem-
branes.36
F igu r e 7. Efficacy of 5b on the survival rate of neurons after
experimental traumatic brain injury performed by the lateral
fluid percussion method in rats. 40 mg/kg (15 min) and 20 mg/
kg (120 min) of 5b were administered ip after inducing injury.
After 14 days efficacy was determined as survival ratio, which
is a ratio of intact neurons of the ipsilateral side and the
undamaged contralateral dentate gyrus and is expressed as
mean percentage ( SEM (p < 0.001).
The metabolic stability in vitro was evaluated using
porcine liver subfractions.37 After incubation of 5b, 5c,
and 5d in the presence of microsomes for 30 min at 37
°C, >95%, >95%, and >85% of the parent compounds,
respectively, were still unchanged which indicated poor
or moderate metabolic degradation by liver enzymes.
Therefore these compounds were further evaluated for
their pharmacokinetic profile in rats after intravenous
and oral administration.44 Representative data for 5d
may be summarized as follows. There was a linear
correlation between dosages and the plasma AUCs after
oral administration of the compound. Intravenous ad-
ministration of 5d gave a half-life of 1.4 h, a clearance
rate CL of 1.7 L/h/kg, and a distribution volume of 3.6
L/kg. After oral administration, the timepoint of maxi-
mum concentration tmax was reached at 2 h, and half-
life t1/2 was 3.3 h. The clearance rate CL was 1.9 L/h/kg
and oral bioavailability was determined as F ) 89%.
Accordingly, 5d showed an acceptable half-life and high
bioavailability after oral administration. The enhanced
distribution volume prompted us to evaluate tissue
levels as the inhibitor concentration in the tissue and
cells might be most closely related to the efficacy of
calpain inhibition. Indeed, we observed considerably
higher levels of 5d in tissues such as the heart, lung,
liver, and kidney, which reached 5- to 10-fold higher
levels than in the plasma.
Calpain inhibitors were suggested as useful agents
in the treatment of traumatic brain injury (TBI). After
experimental traumatic brain injury, substantial loss
of cytoskeletal proteins was observed which was at-
tributed to enhanced calpain activity.38 Furthermore,
the calpain inhibitor AK 295 (2) attenuated motor and
cognitive deficits following experimental brain injury in
rats.39 However, these studies gave rise to controversial
data since injury-induced spectrin breakdown and corti-
cal lesion size were not affected by AK 295. This is
remarkable since a decrease in spectrin degradation is
often used as a marker of calpain inhibition in vitro and
in vivo.40
brains. Fourteen days later the animals were sacrificed
and the brains removed. After Toluidine Blue staining,
the intact hilus cells of the dentate gyrus were counted.
For evaluation of the efficacy we examined both the
ipsilateral and the contralateral side using the contra-
lateral side as a control. The percentage of the neurons
which survived (survival ratio) was calculated by the
ratio of the number of the intact neurons within the
ipsilateral and the contralateral side. In control animals,
the survival ratio was 49% ( 3 which means that about
one-half of all neurons in the dentate gyrus died within
the observation period of 14 days. To evaluate the
calpain inhibitor 5b, the compound was administered
intraperitoneally (ip) at 15 min (40 mg/kg) and 120 min
(20 mg/kg) after inducing the lesion (Figure 7). The
survival ratio of neurons in these treated animals rose
to 70% ( 4 (p < 0.001). Therefore, the treatment by 5b
reduced the cell death in the dentate gyrus by 41%.
Consequently, the calpain inhibitor 5b demonstrated
significant efficacy in protecting neurons from cell death
after experimental traumatic brain injury even when
administered after inducing injury and thus would
be in line with the suggestion that calpain inhibition
may be beneficial in the treatment of traumatic brain
injury.
In summary, we envisaged superior calpain I inhibi-
tors using benzoylalanine-derived backbones and keto-
amides as warheads. Recognizing the significance of
o-phenylvinyl residues at the benzoyl moiety we de-
signed novel potent calpain inhibitors. The insertion of
an aminomethyl residue as para-substituents at the
distal phenylvinyl moiety resulted in potent calpain
inhibitors 5. All three derivatives, 5b (A-705239), 5c (A-
558693), and 5d (A-705253), exhibited substantially
improved pharmacokinetic profiles, in particular water-
solubility and metabolic stability. These inhibitors also
showed oral bioavailability as demonstrated by 5d . The
derivative 5b was evaluated for neuroprotective efficacy
after experimental traumatic brain injury in the lateral
fluid percussion rat model. Derivative 5b reduced neu-
ronal cell death by 41% and revealed significant neuro-
protective efficacy even when administered after induc-
ing the injury. This result would be in line with the
proposed use of calpain inhibitors in the treatment of
traumatic brain injury.14 The evaluation of these calpain
inhibitors in further pharmacological models designed
We evaluated 5b for efficacy in experimental trau-
matic brain injury using the lateral fluid percussion
method (FP method) in rats. The lateral fluid percussion
method was developed to create a model of traumatic
brain injury in the rat for the evaluation of potential
neuroprotective compounds.41 This technique induces a
controlled and consistent injury to tissue underlying the
site of impact, including the neocortex, hippocampus,
and thalamus. Rats were subjected to brain injury by a
burst of fluid pressure (2.5-2.9 bar) on the exposed

Benzoylalanine-Derived Ketoamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2409
1
afford 29.3 g (59%) of 14a as oil. H NMR (DMSO-d6) δ 1.35
to investigate therapeutic efficacy in human diseases
will be reported elsewhere.
(t, 3H), 4.38 (q, 2H), 7.38 (d, J ) 16 Hz, 1H), 7.4-8.2 (m, 12H).
MS m/z: 302 (M+).
2-[E-2-(4-Dim eth yla m in om eth ylph en yl)eth en -1-yl]ben -
zoic a cid m eth yl ester (14b): yield 74%. H NMR (DMSO-
Exp er im en ta l Section
1
1H NMR spectra were recorded either on a Varian Innova
400 MHz, a Bruker Avance 400 MHz, or a Bruker Avance 500
MHz spectrometer with tetramethylsilane as an internal
standard. 13C NMR spectra were recorded on a Bruker Avance
500 MHz spectrometer. A coupling constant J is given in detail
when it it is necessary for determination the configuration.
Electrospray ionization mass spectra (ESI) in a low-resolution
mode or fast atom bombardment (FAB) mass spectra were
recorded on a Finnigan Mat 90 (EI) or a Finnigan TSQ 700
(ESI) spectrometer. Elementary analyses were performed on
a LECO 1000. Melting points were determined on a Bu¨chi 530
and were uncorrected.
d6) δ 2.10 (s, 6H) 3.86 (s, 3H), 7.19 (d, 1H) 7.30 (d, 2H) 7.40 (t,
1H), 7.53 (d, 2H), 7.62 (t, 1H) 7.80-7.90 (m, 2H). MS m/z )
309 (M+).
2-[E -2-(4-Dim e t h yla m in om e t h ylp h e n yl)e t h e n -1-yl]-
n icotin ic a cid eth yl ester d ih yd r och lor id e (14c): yield
42%. Mp 197-198 °C. 1H NMR (DMSO-d6) δ 1.37 (t, 3H), 2.60
(d, 6H), 4.30 (d, 2H), 4.38 (q, 2H), 7.63 (dd, 1H), 7.66 (d, 2H),
7.73 (d, 2H), 7.93 (d, J ) 16 Hz, 1H), 8.20 (d, J ) 16 Hz, 1H),
8.32 (d, 1H), 8.81 (d, 1H), 11.2 (broad, 1H, N+H). MS m/z: 310
(M+).
2-[E-2-(4-Diet h yla m in om et h ylp h en yl)et h en -1-yl]b en -
zoic a cid eth yl ester (14d ): yield 100%. 1H NMR (DMSO-
d6) δ 0.97 (t, 6h), 1.35 (t, 3H), 2.45 (q, 4H), 3.53(s, 2H), 4.37
(q, 2H), 7.17 (d, J ) 16 Hz, 1H), 7.33 (d, 2H), 7.40 (t, 1H), 7.50
(d, 2H), 7.60 (t, 1H), 7.80 (d, J ) 16 Hz, 1H), 7.85 (t, 2H). MS
m/z: 337 (M+).
3-Am in o-2-h yd r oxyh ep ta n oic a cid a m id e (11a ): 118.5
g (1 mol) of 1-nitropentane and 204.4 g (2 mol) of Et3N were
dissolved in 400 mL of CH3OH. To this solution was added
93.1 g (1 mol) of glyoxalic acid hydrate gradually over 2h, and
afterward the reaction mixture was stirred for 16 h at ambient
temperature. The solvent was removed in vacuo. The resulting
residue was dissolved in H2O and washed with ether. The pH
of the aqueous phase was adjusted to 5 by adding citric acid
and extracted with CH2Cl2. The organic phase was dried and
evaporated in vacuo to yield 196 g of the crude 3-amino-2-
hydroxyheptanoic acid (8a ) as a mixture of both diastereomers.
8a (196 g) was dissolved in 1100 mL of glacial acetic acid, and,
after adding 20 g Pd/C, the reaction mixture was hydrogenated
at 50°C and 1 atm. The reaction mixture was filtered, and the
solvent of the filtrate was removed in vacuo. The residue was
treated with 2-propanol, and the resulting precipitate was
collected and dried to give 80 g of 3-amino-2-hydroxyheptanoic
acid (9a ) as a white crystalline solid. 9a (79 g) and 200 g of
Et3N were dissolved in 500 mL of CH3OH, and 118.5 g (Boc)2O
was added. The reaction mixture was stirred for 16h at
ambient temperature. The solvent was removed in vacuo. The
residue was poured into 1000 mL of H2O, and this aqueous
phase was extracted with ether. The ether phase was dis-
carded. The pH value of the aqueous phase was adjusted to
3-4 by adding 2 M HCl, and the resulting phase was extracted
with CH2Cl2. This organic phase was dried, and the solvent
was removed in vacuo to afford 139 g of the crude product
3-tert-butoxycarbonylamino-2-hydroxyheptanoic acid (10a ).
Crude 10a (122.5 g) was dissolved in 700 mL of THF/DMF
(6/1). HOBt (63.3 g) was added, and the reaction mixture was
stirred for 15min. After cooling the solution to 10 °C, 90 g of
EDC was added, and the reaction mixture was stirred for
another 30 min. Then, gaseous NH3 was bubbled through the
reaction mixture for 2 h. Next, the reaction solution was stirred
for 16 h at ambient temperature. The solvent was removed in
vacuo. The resulting residue was poured into H2O and
extracted with EtOAc. The organic phase was washed with
aqueous 2 M NaHCO3 and H2O and dried, and the solvent was
removed in vacuo. This residue (49 g) was suspended in
2-propanol. 2-Propanolic HCl was added, and the suspension
was stirred for several hours at ambient temperature. The
precipitate was collected and dried to give 35.5 g of a single
diastereomer 11a as a hydrochloride: mp 174-176 °C. 1H
NMR (D2O) δ 0.9 (t, 3H), 1.25-1.4 (m, 4H), 1.6-1.7 (m, 2H),
1.78 (m, 1H), 3.63 (m, 1H), 4.43 (d, 1H). MS m/z: 160 (M+).
Anal. (C7H16N2O2‚0.25 HCl‚0.5H2O) C, H, Cl, N.
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
Ca r boxyla tes 15. E-2-[2-(Na p h th -2-yl)eth en -1-yl]ben zoic
a cid (15a ): 29 g (95.9 mmol) of 14a was dissolved in 200 mL
of THF, and a solution of 10.8 g (192 mmol) of KOH in 150
mL of H2O was added. The reaction mixture was refluxed for
12 h. Then the organic solvent was removed in vacuo. The
resulting aqueous solution was diluted by adding 200 mL of
H2O and filtered. This solution was cooled on ice and acidified
by adding 2 M HCl. The resulting precipitate was collected
1
and dried to obtain 26.6 g (100%) of 15a . Mp 211-212 °C. H
NMR (DMSO-d6) δ 7.36 (d, J ) 16 Hz, 1H), 7.4-7.65(m,4H),
7.8 (dd, 1H), 7.85-8.05(m,4H), 8.12 (d, J ) 16 Hz, 1H). MS
m/z: 274 (M+).
2-[E-2-(4-Dim eth yla m in om eth ylph en yl)eth en -1-yl]ben -
zoic a cid (15b): yield 98%. Mp 215-216 °C. 1H NMR (DMSO-
d6) δ 2.23 (s, 6H) 3.53 (s, 2H) 7.13 (d, J ) 15 Hz, 1H),
7.35(m,3H) 7.55(m,3H), 7.80 (d, 2H), 7.95 (d, J ) 15 Hz, 1H).
MS m/z: 281 (M+).
2-[E -2-(4-Dim e t h yla m in om e t h ylp h e n yl)e t h e n -1-yl]-
n icotin ic a cid (15c): yield 76%. Mp 180-181 °C. 1H NMR
(DMSO-d6) δ 2.45 (s, 6H), 2.92 (s, 2H), 7.30(m, 1H), 7.55 (d,
2H), 7.65 (d, 2H), 7.82 (d, J ) 16 Hz, 1H), 8.10 (dd, 1H), 8.25
(d, J ) 16 Hz, 1H), 8.60(m, 1H). MS m/z: 282 (M+).
2-[E-2-(4-Diet h yla m in om et h ylp h en yl)et h en -1-yl]b en -
zoic a cid (15d ): yield 87%. Mp 180-181 °C. 1H NMR (DMSO-
d6) δ 1.0 (t, 3H), 2.53 (q, 4H), 3.61 (s, 2H), 7.13 (d, J ) 17 Hz,
1H), 7.36 (m, 3H), 7.52 (m, 3H), 7.8 (d, 2H), 7.92 (d, J ) 17
Hz, 1H). MS m/z: 309 (M+).
N -(1-B e n zy l-2-c a r b a m o y l-2-h y d r o x y e t h y l)-2-[E -2-
(n a p h th -2-yl)eth en -1-yl]ben za m id e (16a ): 1.52 g (5.6 mmol)
of 15a , 1.54 g (5.5 mmol) of 3-amino-2-hydroxy-3-phenyl-
butanoic acid amide (11b),27 and 2.8 g (27.8 mmol) of Et3N
were dissolved in 200 mL of CH2Cl2/DMF (3/1) and stirred at
ambient temperature for 30 min. HOBt (0.75 g, 5.6 mmol) was
added, and the reaction solution was cooled to 0 °C. Then, 1.1
g (5.6 mmol) of EDC was added, and the reaction mixture was
stirred for another 60 min at 0 °C. Next, the solution was
stirred for 16 h at ambient temperature. The reaction solution
was concentrated in vacuo, and the resulting residue was
redissolved in 500 mL of H2O. The aqueous solution was
rendered alkaline by adding 2 M NaHCO3 solution, which
caused the precipitation of a white solid. The solid was
collected and dried to give 1.86 g (74%) of 16a . Mp 232-234
°C. 1H NMR (DMSO-d6) δ 2.84 (dd, 1H), 2.96 (dd, 1H), 3.99
(dd, 1H), 4.57 (m, 1H), 5.76 (d, 1H, OH), 7.17 (m, 1H), 7.25-
7.36 (m, 8H), 7.42-7.55 (m, 4H), 7.63 (dd, 1H), 7.86 (d, 1H),
7.89-7.95 (m, 4H), 8.2 (d, 1H). MS m/z: 448 (M+).
Rep r esen ta tive P r oced u r e for th e P r ep a r a tion of
Ester s 14. E-2-[2-(Na p h th -2-yl)eth en -1-yl]ben zoic a cid
eth yl ester (14a ): 25.5 g (0.17mol) of 2-vinylnaphthalene,
29.7 g (0.13mol) of 2-bromobenzoic acid ethyl ester (12a ), 22.5
mL (0.16mol) of Et3N, 0.54 g of Pd(OAc)2, and 1.44 g of tri-o-
tolylphosphine were added to 200 mL of DMF. After further
adding 3 mL of H2O, the reaction mixture was stirred at 100
°C for 3 h. Next, the mixture was poured into H2O, and the
aqueous solution was extracted with EtOAc. The EtOAc phase
was washed several times with H2O and dried, and the solvent
was removed in vacuo. The residue was purified chromato-
graphically on silica gel (eluant: heptane/EtOAc ) 5/1) to
N-(1-Car bam oyl-1-h ydr oxyh ex-1-yl)-2-[E-2-(4-dim eth yl-
a m in om eth ylp h en yl)eth en -1-yl]ben za m id e (16b ): 11.3 g
(40.2 mmol) of 11a and 12.1 g (120 mmol) of Et3N were
dissolved in 300 mL of dry DMF. Then, 5.4 g (39.2 mmol) of
HOBt was added. After the reaction mixture was stirred for
15 min, 7.9 g (40.2 mmol) of 11a was added, and stirring was

2410 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12
Lubisch et al.
continued for 30 min. The reaction mixture was cooled to 10
°C, and 8.4 g (43.8 mmol) of EDC was added portionwise to
the solution. Next, the solution was stirred at ambient tem-
perature for 16 h. Then, the reaction solution was poured into
450 mL of a aqueous 4 M NaCl solution, and the resulting
precipitate was collected, washed with H2O and ether, and
finally dried to afford 14.3 g (84%) of 16b. Mp 195-196 °C. 1H
NMR (DMSO-d6) δ 0.85 (t, 3H) 1.1-1.4 (m, 4H) 1.5-1.7 (m,
2H) 2.15 (s, 6H) 3.35 (s, 2H) 4.0 (m, 1H) 4.3 (m, 1H) 5.7 (d,
1H, OH) 7.15-7.50 (m, 11H) 7.85 (m, 1H) 8.05 (d, 1H). MS
m/z: 485 (M+). Anal. (C25H33N3O3‚0.75H2O) C, H, N.
organic phase was separated and immediately acidified by
adding 2-propanolic HCl. The solvent was removed in vacuo,
and the resulting solid was crystallized from acetone/EtOH
(4:1) to obtain 5.5 g (52%) of the hydrochloride of 5b. Mp 132-
134 °C.
(m, 2H), 1.58 (m, 1H), 1.81 (m, 1H), 2.66 (s, 6H), 4.22 (s, 2H),
5,17 (ddd, 1H), 7.30 (d, J ) 16.4 Hz, 1H), 7.38 (dd, 1H), 7.39
(d, 1H), 7.48 (d, J ) 16.4 Hz, 1H), 7.49 (t, 1H), 7.56 (d, 2H),
7.63 (d, 2H), 7.83 (broad, 1H, NH), 7.88 (d, 1H), 8.12 (broad,
1H, NH), 8.4 (broad, 1H, NH), 10.7 (broad, 1H, NH). MS m/z:
421 (M+). Anal. (C25H31N3O3‚HCl‚H2O) C, H, Cl, N.
1H NMR (DMSO-d6) δ 0.84 (t, 3H), 1.30 (m, 2H), 1.39
N-(1-Ben zyl-2-ca r ba m oyl-2-h yd r oxyeth yl)-2-[E-2-(4-d i-
m eth ylam in om eth ylph en yl)eth en -1-yl]n icotin am ide (16c):
12.5 g (44.3 mmol) of 15c, 10 g (44.3 mmol) of 3-amino-2-
hydroxy-3-phenylbutanoic acid amide (11b),28 2 g (14.8 mmol)
of HOBt, and 21.5 mL (155 mmol) of Et3N were added
successively to 500 mL of DMF and stirred at ambient
temperature for 30 min. EDC (7.7 g, 44.3 mmol) was added in
portions, and the reaction mixture was stirred for 16 h. Then
the solvent was removed in vacuo. After suspending the
residue in 1000 mL of H2O, the aqueous phase was made
alkaline by adding 45 mL of 2 M NaOH. The solution was
cooled on ice, and the resulting precipitate was collected,
washed with H2O, dried, and crystallized from EtOH to yield
N-(1-Ben zyl-2-ca r ba m oyl-2-oxoeth yl)-2-[E-2-(4-d ieth yl-
a m in om eth ylp h en yl)eth en -1-yl]n icotin a m id e (5c): 2.0 g
(4.36 mmol) of 15c and 9.0 g (43.6 mmol) of DCC were
dissolved in 40 mL of CH2Cl2/DMSO (2/1). After the solution
was cooled to 10 °C, 1.44 mL (17.45 mmol) of Cl2CHCOOH
was dropped into the reaction solution. Then the reaction
mixture was stirred for 1 h at ambient temperature. The
reaction solution was poured into ice and water, and the
resulting precipitate was filtered off. The resulting aqueous
solution was diluted with 400 mL of H2O and washed several
times with EtOAc. The pH of the aqueous phase was adjusted
to pH 8 by adding 2 M NaHCO3. This aqueous phase was
extracted several times with EtOAc. The combined EtOAc
phases were washed with H2O and brine and dried, and the
solvent was removed in vacuo. The resulting solid was
suspended in 200 mL of water and redissolved by adding 5.7
mL of 2 M HCl. The solution was filtered, and the filtrate was
frozen and freeze-dried in vacuo to give a solid. This solid was
recrystallized from ethanol to afford 1.5 g (69%) of 5c as a
1
14.7 g (73%) of 16c: Mp 212-213 °C. H NMR (DMSO-d6) δ
2.16 (s, 6H), 2.79 (d, 1H), 2.80 (s, 1H), 3.41 (s, 2H), 4.11 (dd,
1H), 4.58 (m, 1H), 5.98 (d, OH), 7.07 (d, J ) 15.7 Hz, 1H),
7.12 (m, 1H), 7.2-7.45 (m, 11H), 7.60 (dd, 1H), 7.73 (d, J )
15.7 Hz, 1H), 8.48 (d, 1H), 8.59 (dd, 1H). MS m/z: 458 (M+).
Anal. (C29H34N4O3) C, H, N.
1
dihydrochloride. Mp 189-190 °C. H NMR (DMSO-d6) δ 2.71
N-(1-Ben zyl-2-ca r ba m oyl-2-h yd r oxyeth yl)-2-[E-2-(4-d i-
eth yla m in om eth ylp h en yl)eth en -1-yl]ben za m id e (16d ):
20 g (64.6 mmol) of 15d , 15 g (64.6 mmol) of 3-amino-2-
hydroxy-3-phenylbutanoic acid amide (11b),27 and 2.9 g (21.5
mmol) of HOBt were added to 1400 mL of dry DMF. Et3N (32
mL, 226 mmol) was added, and the resulting solution was
cooled to 0 °C. Then 12.4 g (64.6 mmol) of EDC was added,
and the solution was stirred at ambient temperature for 16 h.
The solvent was removed under reduced pressure which
afforded an oily residue. This oil was redissolved in EtOAc.
This solution was subsequently washed with 2 M NaHCO3 and
H2O and dried, and the solvent was removed in vacuo. The
resulting residue was dissolved in CH2Cl2/MeOH, and the
subsequent careful addition of n-pentane caused a precipita-
tion of a white solid which was collected by filtration to afford
(d, 6H), 2.87 (dd, 1H), 3.27 (dd,1H), 4.32 (d,4H), 5.47 (m, 1H),
7.25 (t, 1H), 7.27-7.36 (m,6H), 7.47 (dd,1H), 7.63 (dd, 4H),
7.70 (d, J ) 15 Hz, 1H), 7.87 (d,1H), 7.92(broad,1H, NH), 8.26
(broad,1H,NH), 8.68 (dd, 1H), 9.25 (d,1H), 10.9(broad, 1H). 13
C
NMR (DMSO-d6) δ 34.6 (t), 41.4, 56.0, 58.7, 121.4, 123.4, 126.5,
127.7, 128.2, 128.9, 131.5, 131.6, 132.2 136.1, 136.6, 137.4,
139.9, 146.5, 148.7, 162.7, 165.8, 196.3. MS m/z: 474 (M+
+
H2O), 456 (M+). Anal. (C29H32N4O3‚2HCl‚3H2O) C, H, Cl, N.
N-(1-Ben zyl-2-ca r ba m oyl-2-oxoeth yl)-2-[E-2-(4-d ieth yl-
a m in om eth ylp h en yl)eth en -1-yl]ben za m id e (5d ): A solu-
tion of 30 g (61 mmol) of 16d in 110 mL of DMSO and 127.2
g (61 mmol) of DCC in 330 mL of CH2Cl2 were combined. At
15 °C, 31.9 g (150 mmol) of dichloroacetic acid was added, and
stirring was continued for 1 h. Then the reaction mixture was
filtered, and the filtrate was poured into 1.8 L of ice/water.
The aqueous phase was extracted three times with EtOAc, and
these organic phases were discarded. The pH of the resulting
aqueous phase was adjusted to 8 by adding 2 M NaHCO3. Then
the aqueous phase was again extracted with EtOAc. The
organic phase was washed with H2O and brine and dried, and
the solvent was removed in vacuo. The residue was dissolved
in CH2Cl2/acetone, and 4.6 g of methanesulfonic acid was
added. Ether was carefully added to cause precipitation of a
solid which was collected and dried to obtain 21.8 g (61%) of
the methanesulfonate of 5d . Mp 109-111 °C. 1H NMR (DMSO-
d6) δ 1.23 (t, 3H), 2,34 (s, 3H), 2.86 (dd, 1H), 3.23 (dd, 1H),
3.08 (q, 4H), 4.29 (s, 2H), 5.42 (m, 1H), 7.1-7.6 (m, 9H), 7.4-
7.7(6H), 7.85 (d, 1H), 7.92 (s, 1H), 8.20 (s, 1H), 8.97 (d, 1H).
13C NMR (DMSO-d6): δ ) 8.5, 34.5, 39.3, 46.0, 54.7, 55,8,
123.5, 124.5, 125.1, 126.3, 126.6, 126.8, 127.3, 127.5, 128.0,
128,1, 128.4, 128.9, 129.5, 129.7, 130.3, 131.2, 132.7, 135.7,
137,8, 139.0, 163.0, 168.7, 196.8. MS m/z: 501 (M+ + H2O),
483 (M+). Anal. (C30H33N3O3‚1.25CH3SO3H) C, H, N, S.
1
22.5 g (72%) of 16d . Mp 195-196 °C. H NMR (DMSO-d6) δ
0.98 (t, 6H), 2.45 (q, 4H), 2.80 (m, 2H), 3.53 (s, 2H), 4.10 (m,
1H), 4.58 (m, 1H), 5.93 (d, 1H, OH), 7.0-7.5 (m,16H), 7.78 (d,
1H), 7.23 (d, 1H). MS m/z ) 485 (M+). Anal. (C30H35N3O3‚
0.5H2O) C, H, N.
N-(1-Ben zyl-2-ca r ba m oyl-2-oxoeth yl)-2-[E-2-(n a p h th -2-
yl)eth en -1-yl]ben za m id e (5a ): 1.83 g (4.1 mmol) of 16a and
4.11 g (50.8 mmol) of Et3N were dissolved in 75 mL of dry
DMSO. Py‚SO3 (6.5 g, 40.6 mmol) was added, and the resulting
reaction mixture was stirred for 16 h at ambient temperature.
The reaction solution was poured into 500 mL of H2O, and
the aqueous solution was extracted twice with EtOAc. The
combined EtOAc phases were washed with H2O and dried, and
the solvent was removed in vacuo. The residue was crystallized
from EtOAc/n-pentane to afford 1.66 g (71%) of 5a as a white
solid. Mp 155-159 °C. 1H NMR (DMSO-d6) δ 2.9 (dd, 1H), 3.26
(dd, 1H), 5.45 (m, 1H), 7.10-7.60 (m,13H), 7.73 (d, 1H), 7.8-
8.0 (m, 5H), 8.23 (s, 1H), 8.98 (d, 1H). MS m/z: (448, M+). Anal.
(C25H27N3O3‚0.5H2O) C, H, N.
Ca lp a in I In h ibition . Calpain I was purified from human
erythrocytes according to the method of Gabrijelcic-Geiger et
al.42 In brief, after four chromatographic steps (DEAE-
Sepharose, Phenyl-Sepharose, Superdex 200, and Blue-
Sepharose), the enzyme was obtained at a purity of >95%, as
assessed by SDS-PAGE and HPLC analysis. Its composition
was determined by endoproteinase Lys-C cleavage, peptide
mapping, and protein sequencing. The buffer solution used in
experiments contained 50 mM Tris-HCl, pH 7.5; 0.1 M NaCl;
1 mM dithiothreithol; 0.11 mM CaCl2. Suc-Leu-Tyr-AMC
N -(1-Ca r b a m oyl-1-oxoh e x-1-yl)-2-[E -2-(4-d im e t h yl-
a m in om eth ylp h en yl)eth en -1-yl]ben za m id e (5b): 9.7 g
(22.9 mmol) of 16b and 43.9 g (229 mmol) of EDC were
dissolved in 400 mL of DMSO. At ambient temperature, 11.8
g (91.5 mmol) of dichloroacetic acid was dropped into the
stirred solution. The resulting reaction mixture was stirred
for further 30 min at ambient temperature. Then the reaction
mixture was poured into 1200 mL of H2O, and the pH of this
aqueous solution was adjusted to 8 by adding 2 M NaHCO3.
The resulting solution was extracted with EtOAc, and this

Benzoylalanine-Derived Ketoamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 12 2411
(Bachem/Switzerland) was used as fluorogenic calpain sub-
strate.27,28,43 Fluorescence of the cleavage product 7-amino-4-
methylcoumarin (AMC) was measured continuously in a Spex-
Fluorolog fluorimeter at λex ) 380 nm and λem ) 460 nm. The
fluorescence signal was linear over a time period of 60 min.
The inhibitors and the calpain substrate were dissolved in
DMSO, but the DMSO concentration in the final experimental
solutions did not exceed 2%.
predetermined height to create a burst of fluid pressure on
the surface of the rat’s brain (2.5-2.9 bar). Fourteen days later,
the rat was killed and the brain removed. After cryoprotection,
the brain was cut to 30 µm slices (coronal slices) on a freezing
microtome. After Toluidine Blue staining, the extent of injury
was measured by counting the hilar neurons of the dentate
gyrus, both on the side of the injury and the contralateral side.
In a typical experiment, 10 µL of substrate (250µM final
concentration) and then 10 µL of µ-calpain (µg/mL final
concentration, i.e., 18 nM) were added to 1 mL of buffer
solution. After 15 min, 10 µL of the inhibitor solution (50 or
100 µM solution in DMSO, i.e., 500 or 1000 nM final concen-
tration) were added, and the experiment was continued for
40 min. All this time, fluorescence was recorded over the time.
Ki values were determined using the classical equation for
reversible inhibition, under steady-state conditions and calpain
concentrations much smaller than inhibitor concentrations,
i.e., Ki ) I/(vo/vi) - 1; where I ) inhibitor concentration, vo )
initial velocity before adding the inhibitor; vi ) reaction
velocity at equilibrium. A study was done to compare the
autocatalytic effect at different temperatures and calcium
concentrations. The decrease in rate (autocatalysis) over time
is virtually absent at 12 °C and 0.11 mM Ca2+ (data not shown)
and all studies were therefore conducted at these conditions.
Ca lp a in -Med ia ted Degr a d a tion of Tyr osin e Kin a se
p p 60sr c in P la telets. The method is described more in detail
by Oda et al.35 Fresh human, citrate-treated blood was
centrifuged at 200g for 15 min to obtain the platelet-rich
plasma which was pooled and subsequently diluted with the
same volume of a buffer (68 mM NaCl, 2.7 mM KCl, 0.5 mM
MgCl2‚6H2O, 0.24 mM NaH2PO4‚H2O, 12 mM NaHCO3, 5.6
mM glucose, 1 mM EDTA, pH 7.4). After centrifugation and
washing with the buffer, the pellet was diluted with buffer to
adjust the platelet count to 107 cells/mL. The experiments were
carried out at 37 °C.
In a typical experiment, 5 × 106 platelets were preincubated
with the inhibitor (as DMSO solution) for 5 min. 1 µM
ionophore A23187 (Calbiochem) and 5 mM CaCl2 were added
to activate the platelet calpain. After 5 min, the platelets were
briefly centrifuged at 13000 rpm, and the pellet was taken up
in SDS sample buffer (20 mM Tris-HCl, 5 mM EDTA, 5 mM
EGTA, 1 mM DTT, 0.5 mM PMSF, 5 µg/mL leupeptin, 10 µM
pepstatin, 10% glycerol, and 1% SDS). The proteins were
fractionated on a 12% gel. To examine pp60src and its 52 kDa
and 47 kDa cleavage products, proteins were transferred
electrophoretically from the gel onto a nitrocellulose membrane
and identified by Western blotting. The polyclonal rabbit anti-
Cys-src (pp60src) antibody employed was obtained from Bio-
mol-Feinchemikalien (Hamburg). This primary antibody was
detected by using a goat HRP-coupled second antibody (Roche,
Germany). Cleavage of pp60src was quantified densitometri-
cally. Nonactivated platelets (control 1: no cleavage) and
platelets treated with ionophore and calcium (control 2:
corresponds to 100% cleavage) were used as controls. The ED50
values were determined as the concentration of inhibitor at
which the intensity of the color reaction of the 60 kDa band
was 50% of the mean of intensities of control 1 minus control
2.
Tr a u m a tic Br a in In ju r y in th e Ra t. A group of male
Sprague Dawley rats (180-280 g) were used and divided into
a treated and a control group (each 20 animals). The rats were
anaesthetized with halothane (1-2%, 4% for initial induction)
in a mixture of 70% N2O and 30% O2 delivered through a loose
fitting face mask. The right temporo-parietal region of the head
was exposed, a small craniotomy was made, and a male Luer-
Lock fitting was fixed over the opening using dental cement.
After hardening of the cement, the rat was attached to a fluid
percussion device. The device consisted of a saline-filled
cylinder, one end of which was connected, via a pressure
transducer, to a female Luer-Lock fitting which was connected
to the rat’s skull, thus creating an airtight fluid-filled chamber.
The other end of the cylinder was stoppered with a piston. A
heavy metal pendulum was dropped onto the piston from a
Refer en ces
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disease. Trends Mol. Med. 2001, 7, 355-362. (b) Dear, T. N.;
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localization, and expression pattern of Capn12, a new member
of the calpain large subunit gene family. Genomics 2000, 68,
152-160.
(2) J ohnson, P. Calpains (intracellular calcium-activated cysteine
proteinases): structure-activity relationships and involvement
in normal and abnormal cellular metabolism. Int. J . Biochem.
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