Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I

Article abstract of DOI:10.1021/jm970197e

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P₁ site, Leu at P₂ is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe- CH₂F], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain 1 (with a second-order rate constant for inactivation of 276 000 M^{- 1} s^-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH₂F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC₅₀ values of 0.2 and 0.1 μM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Full text of DOI:10.1021/jm970197e

3820
J . Med. Chem. 1997, 40, 3820-3828
Syn th esis a n d Biologica l Activity of a Ser ies of P oten t F lu or om eth yl Keton e
In h ibitor s of Recom bin a n t Hu m a n Ca lp a in I
Sankar Chatterjee,*,† Mark A. Ator,*,‡ Donna Bozyczko-Coyne,*,‡ Kurt J osef,† Gregory Wells,†
Rabindranath Tripathy,† Mohamed Iqbal,† Ron Bihovsky,† Shobha E. Senadhi,‡ Satish Mallya,‡
Teresa M. O’Kane,‡ Beth Ann McKenna,‡ Robert Siman,‡ and J ohn P. Mallamo†
Departments of Chemistry and Biochemistry, Cephalon, Inc., 145 Brandywine Parkway,
West Chester, Pennsylvania 19380-4245
Received March 24, 1997X
Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration
following an episode of stroke. In this paper, we report on a series of potent dipeptide
fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies
revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2
is preferred. However, the nature of the N-terminal capping group has a significant effect on
the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-
2-yl)carbonyl-Leu-D,L-Phe-CH2F], having a tetrahydroisoquinoline containing urea as the
N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain
I (with a second-order rate constant for inactivation of 276 000 M-1 s-1) yet reported; tripeptide
4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study
displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine
proteases. Compounds which exhibited good inhibitory activity in the assay against isolated
rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell
assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 µM, respectively.
Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to
generate the corresponding dipeptide fluoromethyl ketone.
In tr od u ction
examine the system after exposure to the inhibitor to
see whether or not the postulated site of action was, in
fact, covalently labeled, an essential requirement for
understanding its mechanism of action.6 Recently we
disclosed a dipeptide fluoromethyl ketone, having a
tetrahydroisoquinoline-containing urea as the N-termi-
nal capping group as the most potent dipeptide fluo-
romethyl ketone irreversible inactivator of calpain I yet
described.7,8 We now present the full account of our
work describing the synthesis and the in vitro calpain
I inhibitory activity of a series of potent dipeptide
fluoromethyl ketones. We also present their inhibitory
activity against cathepsin B and cathepsin L, two other
related cysteine proteases, sensitive to inhibition by
fluoromethyl ketones. We demonstrate that the com-
pounds which display good activity in the assay for
calpain I inhibition also inhibit intracellular calpain I.
Calpains (I and II) are calcium-activated neutral
proteases belonging to a family of intracellular cyto-
plasmic cysteine proteases. Calpain I, thought to be the
predominant form activated during the pathological
conditions of nervous tissue, has been implicated in
several nervous system disorders including stroke,
Alzheimer’s disease, and epilepsy. We are interested
in selectively inhibiting calpain I to find new therapeu-
tics1 to treat stroke, one of the leading causes of
mortality in the western hemisphere. In the US alone,
more than half a million strokes occur each year, killing
more than one-third of its victims; remaining patients
survive, but only a fortunate third of these are left with
little or no physical or mental impairment.2 The major-
ity of all strokes occur when a blood vessel blocked by a
clot (or an air bubble) originating from the heart or an
atherosclerotic arterial plaque cuts off blood flow to a
region of the brain and induces localized anemia, known
as ischemia.2,3 An episode of stroke initiates a chain of
biochemical events resulting in the rise of intracellular
Ca2+ concentration, which in turn activates calpain.
Activated calpain degrades neuronal structural proteins,
resulting in neurodegeneration.
Ch em istr y
The syntheses of target compounds 4a -h are depicted
in Scheme 1. Acylated or sulfonylated leucine (1A-E)
was coupled with amino fluoro alcohols 2A-C to gener-
ate the dipeptide fluoro hydroxy compounds 3a -g.
Dess-Martin oxidation of 3a -g generated fluoromethyl
ketones 4a -g. Deprotection of the THP group in 4g
generated 4h . Compounds 1A,B are commercially
available. Compound 1C was synthesized by coupling
leucine with morpholinosulfonyl chloride.9 Compound
1D was obtained by reaction of benzyl isocyanate with
leucine tert-butyl ester hydrochloride salt, followed by
acidic hydrolysis. Compound 1E was synthesized by
coupling 1,2,3,4-tetrahydroisoquinoline, leucine methyl
ester hydrochloride salt and triphosgene10 followed by
basic hydrolysis. Compounds 2A-C were prepared
according to Scheme 2. Commercially available trans-
Potent peptide-based reversible4 and irreversible5
inhibitors of calpain I have been reported. Irreversible
inhibitors permanently inactivate a pathophysiological
enzyme; thus, for maximum benefit, they could poten-
tially be superior to reversible inhibitors which inacti-
vate in a transient manner. Additionally, the ability
of an irreversible inhibitor to label altered cellular
components gives the researcher an opportunity to
† Department of Chemistry.
‡ Department of Biochemistry.
X Abstract published in Advance ACS Abstracts, October 15, 1997.
S0022-2623(97)00197-0 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of Recombinant Human Calpain I
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3821
Sch em e 1a
Sch em e 3a
a
Reagents: (a) HCl(g), ether; (b) BOP, HOBt, NMM, DMF,
0-23 °C.
Sch em e 4a
a
Reagents: (a) general method A (IBCF, NMM, CH2Cl2 or THF,
DMF, -20 to 23 °C) or general method B (BOP, HOBt, NMM, DMF
0-23 °C); (b) Dess-Martin periodinane, CH2Cl2, 23 °C; (c) p-
toluenesulfonic acid monohydrate, CH3OH, 23 °C.
Sch em e 2a
a
Reagents: (a) N,O-dimethylhydroxylamine hydrochloride, Et3N,
BOP, CH2Cl2, 23 °C; (b) 3 M MeMgBr, THF, 0-23 °C; (c)
TBDMSOTf, Et3N, CH2Cl2, 0 °C; (d) F-TEDA-BF4, DMF, 23 °C.
ketone 9 via Weinreb amide 8; however during the
amide formation, the P1 site epimerized. Compound 9
was converted to the corresponding enol silyl ether
which on treatment with 1-(chloromethyl)-4-fluoro-1,4-
diazabicyclo[2.2.2]octane bis(tetrafluoroborate) (F-TE-
DA‚BF4, Selectflur reagent)13 generated compound 41.
To our knowledge, this is the first example of generation
of a dipeptide fluoromethyl ketone (albeit in low isolated
yield) by fluorination of a peptidic enol silyl ether.
Compounds 4a -l are a diastereomeric mixture, epimer-
ic at P1; compound 4g has an additional chiral center
(mixture of isomers) at the OTHP site. Compound 4m
is commercially available. Compound 4n is E-64 (trans-
epoxysuccinyl-L-leucylamido(4-guanidino)butane), a ref-
erence compound. Table 1 lists compounds 4a -m .
Ca lp a in I In h ibition a n d Discu ssion . The biologi-
cal activities of the compounds were determined using
rh calpain I, prepared as described by Meyer et al.14
with Suc-Leu-Tyr-MNA as the substrate. Table 2
displays the inhibitory data for compounds 4a -n . As
shown, P1-Phe is preferred over Abu and Ser (cf. 4a vs
4f and 4h ), respectively. Interestingly, protection of the
hydroxyl group of the P1-Ser residue as a tetrahydro-
pyran (THP) ether moiety generates an approximately
5 times more potent compound (cf. 4g vs 4h ). Thus, it
appears that the hydroxyl group of the P1-Ser residue
in 4h is involved in a destabilizing interaction with the
S1 site of the enzyme. This might also explain why the
inactivation rate of 17 000 M-1 s-1 determined by Shaw
a
Reagents: (a) in situ generated FCH2CHO (see text); (b) H2
(60 psi), absolute ethanol, Raney nickel (50% slurry in water,
catalytic amount).
â-nitrostyrene was reduced to compound 5A, which
underwent modified Henry nitro aldol condensation
with in situ generated fluoroacetaldehyde11,12 to gener-
ate compound 6A. Hydrogenation of 6A yielded 2A. It
should be noted that although we were able to separate
the erythro/threo isomers of 6A and convert them to
erythro/threo isomers of 2A (see the Experimental
Section), it was of no great advantage. Either isomer
of 2A was a mixture of inseparable enantiomers, as
there was no element of stereocontrol in the previous
nitroaldol step to generate 6A. Thus, reaction of either
the erythro or threo isomer of 2A to a P2 fragment and
eventual oxidation of the carbinol center would have
produced the same 1:1 mixture at the remaining epimer-
ic center, i.e. at P1. Thus, isomers of 2A were pooled.
Similarly, 1-nitropropane (5B) was converted to 2B via
6B. Finally 5C was transformed into 2C via 6C.
Syntheses of compounds 4i-k are shown in Scheme 3.
Hydrolysis of the t-Boc group of 4b generated 4i (HCl
salt), which was coupled with hydrocinnamic acid and
Cbz-Leu to generate 4j and 4k , respectively. During
this investigation, we also generated a dipeptide fluo-
romethyl ketone by fluorinating the corresponding
dipeptide methyl ketone (Scheme 4). Thus Cbz-Val-Phe-
OH (7) was converted to the corresponding methyl

3822 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Ta ble 1. List of Compounds 4a -m a
Chatterjee et al.
compd
P
R2
R1
RP HPLC tR (min)b
formula
anal.
4a
4b
4c
4d
4e
4f
4g
4h
4i
Cbz
t-Boc
iBu
iBu
iBu
iBu
iBu
iBu
iBu
iBu
iBu
iBu
iBu
iPr
Bn
Bn
Bn
Bn
Bn
Et
CH2OTHP
CH2OH
Bn
Bn
Bn
Bn
Me
27.64
26.87
22.39
24.30
26.56
24.16
31.95
21.29
13.80
25.91
29.08
30.80
f
C24H29FN2O4
C, H, N
C, H, N
c
C, H, N
C, H, N
C, H, N
d
C
C
C
C
C
21H31FN2O4
20H30FN3O5S
24H30FN3O3
26H32FN3O3
19H27FN2O4
morpholino-4-sulfonyl
(benzylamino)carbonyl
(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl
Cbz
Cbz
Cbz
H (HCl salt)
Ph(CH2)2CO
Cbz-Leu
Cbz
C23H33FN2O6
C18H25FN2O5
e
C
16H23FN2O2‚HCl
C, H
4j
4k
4l
C25H31FN2O3
C, H, N
C, H, N
C, H, N
C
C
30H40FN3O5
23H27FN2O4
4m
Cbz
Bn
a
Compounds 4a -l are diastereomeric mixture, epimeric at P1; compound 4g has an additional chiral center (mixture of isomers) at
b
OTHP site. HPLC conditions used: solvent A, water containing 0.1% TFA; solvent B, acetonitrile containing 0.1% TFA; detection, 215
nm; 10-100% B in 40 min; column, Zorbax Rx C8; flow, 1.2 mL/min. c High-resolution mass spectroscopy: calc 444.1968 (MH+), found
444.1973 (MH+). High-resolution mass spectroscopy: calc 453.2401 (MH+), found 453.2382 (MH+). e High-resolution mass spectroscopy:
d
calc 369.1826 (MH+), found 369.1837 (MH+). f Commercial sample.
Ta ble 2. Inhibitory Activities of Compounds 4a -n
k (M-1 s-1
a less active analog (4j), revealing that an additional
heteroatom in the region might be involved in an
energetically beneficial binding. Interestingly, con-
straining the benzylurea motif of 4d as part of a
tetrahydroisoquinolyl moiety generated the most potent
dipeptide fluoromethyl ketone inhibitor (4e, k ) 276 000
M-1 s-1) of calpain I yet reported; thus, it appears that
although the enzyme tolerates an heteroatom (O, N) in
the region spanning P3, it disfavors an NH moiety from
the benzylurea motif of 4d . However, note that the
tripeptide inhibitor Cbz-Leu-Leu-D,L-Phe-CH2F (4k , k
) 290 000 M-1 s-1) is equipotent to the dipeptide
inhibitor 4e. The reference compound 4n (E-64) dis-
plays an inactivation rate of 4700 M-1 s-1 in this assay,
which is comparable to the inactivation rate reported
in the literature using chicken gizzard calpain II (Pliura
et al.16 3700 M-1 s-1 and Parkes et al.17 7500 M-1 s-1),
demonstrating the validity of our assay method and the
similarity between rh calpain I and chicken gizzard
calpain II.
)
calpain cathepsin cathepsin
intact cell
compd
4a
4b
4c
4d
4e
4f
Ia
Ba
La
assayb IC50 (µM)
136300
68600
67200
67000
276000
24250
100000
21000
8000
300
150
100
5000
100
3200
0.2
42% at 10 µM
10
0.8
0.2
275
9400
7500
24500
9000
16300
25
48400c
64000
22600
31000
250
4g
4h
4i
1.3
4j
4k
4l
26600
290000
35000
d
600
4600
360
800
256000
21500
110000
0.1
4m
21000
4n (E-64) 4700
a
b
n g 3 in all cases. n g 2 in all cases. c A value of 72 000 was
reported in our preliminary communication.7
No significant
d
inhibition was observed up to 1 µM.
et al.7 for Cbz-Leu-D,L-Tyr-CH2F against chicken gizzard
calpain II is significantly lower than that of 136 300 M-1
s-1 for Cbz-Leu-D,L-Phe-CH2F (4a ), but comparable to
that of 21 000 M-1 s-1 for Cbz-Leu-D,L-Ser-CH2F (4h ).15
Thus, calpain I prefers a hydrophobic group at the P1-
site of this class of molecules. Previous studies indicate
that calpain prefers Leu or Val at P2;4,5 however, in this
series, compound 4a with P2-Leu is 4 times more potent
than compound 4l with P2-Val. It should be noted that
the commercial sample 4m with P2-Phe did not show
any significant inhibition of rh calpain I up to 1 µM,
revealing the importance of Leu or Val at P2 of a potent
calpain inhibitor.
Interestingly, the nature of the N-terminal capping
group plays a significant role in the potency of this series
of compounds. While Cbz, t-Boc, morpholinosulfonyl,
and (benzylamino)carbonyl are all well tolerated, Cbz
is preferred (4a vs 4b-d ). The lack of any capping
group, as in the free amine 4i (as the hydrochloride salt),
results in weak activity, indicating the importance of
the presence of an N-terminal capping group. Replace-
ment of the OCH2 moiety in the Cbz capping group of
compound 4a with the CH2CH2 moiety also generates
Mech a n ism of In h ibition . Inactivation of an en-
zyme by an inhibitor which involves initial complex
formation followed by covalent modification of the
enzyme typically displays time-dependent inactivation
kinetics. Inactivation of rh calpain I by the above series
of fluoromethyl ketones is time-dependent (data not
shown) and concentration-dependent. Replots of kobs
versus inhibitor concentration are linear (Figure 1),
allowing calculation of rates of inactivation. It should
be noted that no recovery of calpain activity is observed
when enzyme inactivated with 4a is diluted into an
assay mixture or when it is subjected to repeated cycles
of dilution and ultrafiltration, indicating the irreversible
nature of the inhibition of rh calpain I by 4a .
Furthermore, covalent binding of 4a to rh calpain I
was directly assessed using radiolabeled compound
[14CH2]Cbz-Leu-D,L-Phe-CH2F (4a *). It is generally
accepted that in the presence of calcium ion, the 80 kDa
subunit of calpain I undergoes autolysis to a 78 kDa
and subsequently to a 76 kDa form which represents
the active enzyme species.18 In order to determine to
which Mr form 4a binds, rh calpain I and 4a * were
incubated under conditions in which calpain would

Inhibitors of Recombinant Human Calpain I
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3823
Thus, 4a * gained access to the active site of calpain I
not only in the 76 kDa species but in the 78 kDa species
as well.
Selectivity. Cathepsins B and L are two related
cysteine proteases which are sensitive to inhibition by
dipeptide fluoromethyl ketones.6,8b Thus, we examined
the inhibitory activity of the compounds against isolated
cathepsins B and L, using the fluorogenic substrate Cbz-
Phe-Arg-AMC. Table 2 includes the inhibitory data for
the compounds against cathepsins B and L.
Ca th ep sin B. A number of fluoromethyl ketones
presented in this study displayed excellent selectivity
for rh calpain I over cathepsin B. Compound 4a with
P1-Phe is >450 times selective for calpain I over cathe-
psin B; however, compound 4f with P1-Abu is equipotent
both for calpain I and cathepsin B. Interestingly, such
an effect is less profound between 4g with P1-Ser(THP)
and 4h with P1-Ser; while the former inhibits calpain I
11 times faster than cathepsin B, the activity of the
latter compound is comparable against both of the
enzymes. The nature of the N-terminal capping group
again plays a significant role in the compound selectiv-
ity. While compounds 4a ,b are >450 times more
selective for calpain I over cathepsin B, compounds 4c,d
are >670 times and >240 times selective for calpain I
over cathepsin B, respectively. Compounds 4e and 4k ,
the two most potent compounds against isolated rh
calpain I, are moderately selective (37 times and 63
times, respectively) over cathepsin B.
F igu r e 1. Recombinant human calpain I was incubated with
substrate and varying concentrations of inhibitor (0-100 nM).
Progress curves were obtained and values of kobs were calcu-
lated for each inhibitor concentration as described in the
Experimental Section. Replot of kobs versus inhibitor concen-
tration was linear and was used to calculate the second-order
rate constant for inactivation given in Table 2.
Ca th ep sin L. The selectivity of the compounds for
rh calpain I over cathepsin L is modest. Compound 4a
with P1-Phe is >27 times selective for calpain I over
cathepsin L; however, note that compound 4f with P1-
Abu prefers (>2.5 times) cathepsin L over calpain I.
Among various N-capping groups, t-Boc-containing 4b
prefers calpain I over cathepsin L by >680 times.
Between compounds 4e and 4k , the two most potent
compounds in the calpain I assay, the former prefers
calpain I by ca. 6 times over cathepsin L, while the latter
is equipotent in both assays.
Cellu la r Activity. In order to probe the ability of
these compounds to penetrate cells and inhibit intrac-
ellular calpain I, we tested a set of compounds in an
intact cell assay system. Treatment of Molt 4 cells
(human leukemic T cells) with calcium ion and an
ionophore results in the elevation of intracellular cal-
cium which, in turn, activates calpain I. This is followed
by calpain I-mediated cleavage of cytoskeletal proteins,
including spectrin. Inhibition of formation of spectrin
breakdown products (SBDPs) inside the cell by a
compound measures its efficacy. Table 2 lists the
inhibitory activities of a set of compounds which showed
good activity (k > 60 000 M-1 s-1) in the enzyme assay.
While compounds 4a ,d -e,g,k also displayed good activ-
ity in this assay, compounds 4b,c were less potent.
Compounds 4e and 4k inhibited calpain I with IC50
values of 0.2 and 0.1 µM, respectively. Thus, the
compounds are cell-permeable and inhibit intracellular
calpain I.
F igu r e 2. (A) Immunoblot of rh calpain I. (B) Phosphoimage
of rh calpain I. Lane 1: rh calpain I incubated for 5 min with
a 10-fold molar excess of 4a * in the absence of calcium ion.
Lane 2: rh calpain I incubated for 5 min with a 10-fold molar
excess of 4a * in the presence of calcium ion. Lane 3: rh calpain
I incubated with calcium ion for 5 min, followed by incubation
with a 10-fold molar excess of 4a * for an additional 5 min.
Lane 4: rh calpain I incubated with calcium ion for 5 min,
followed by incubation with a 10-fold and 100-fold molar excess
of 4a * and Cbz-Val-Phe-H, respectively, for an additional 5
min.
remain unautolyzed, be partially autolyzed, or be fully
autolyzed. Following incubation in the presence of
calcium ion, 0.8-0.9 mol of 4a * was incorporated per
mole of calpain I. Minimal (0.01 mol of 4a */mol of
calpain I) incorporation was detected when calpain I was
incubated in the absence of calcium ion. In a similar
way, minimal (0.03 mol of 4a */mol of calpain I) incor-
poration was also observed when calpain I was incu-
bated in the presence of calcium ion and saturating
concentrations of Cbz-Val-Phe-H (MDL 28170),19
a
known active site-directed inhibitor. Thus, the data are
consistent with near-stoichiometric binding of 4a * to the
active site of calpain I.
Figure 2 illustrates the Mr forms of rh calpain I to
which 4a * covalently bound. Predominant radiolabeling
of the 76 kDa species was clearly evident. A relatively
minor amount of radiolabel was observed in both the
80 and 78 kDa species. It is not known whether this
labeling represents inhibitor specifically bound to the
active site of the enzyme. Interestingly, the relative
percent incorporation (based upon the total quantity of
protein) of radiolabel into the 78 kDa species repre-
sented a significant fraction of the total labeling.
Incorporation into the 80 kDa species remained minor.
Con clu sion
In this paper, we described an account of our work
on a series of potent and selective fluoromethyl ketone
inhibitors of recombinant human calpain I. While
compound 4e emerged as the most potent (k ) 276 000
M-1 s-1) dipeptide fluoromethyl ketone inhibitor of

3824 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chatterjee et al.
pylethylamine (20 mL, 0.11 mol) in methylene chloride (30
mL). The reaction mixture was stirred for another 0.5 h,
concentrated, and taken into ethyl acetate (200 mL). The
organic layer was washed with 2% citric acid (2 × 100 mL),
2% NaHCO3 (2 × 100 mL), water (2 × 50 mL), and brine (2 ×
50 mL) and dried (MgSO4). Solvent evaporation gave a crude
material which was purified by flash chromatography (elu-
ant: hexane-ethyl acetate, 2:1) to generate [(1,2,3,4-tetrahy-
droisoquinolin-2-yl)carbonyl]leucine methyl ester (6.50 g, 43%)
as a viscous oil: 1H NMR (CDCl3) δ 0.90 (6H, d), 1.60 (3H, m),
2.90 (2H, broad t), 3.70 (2H, m), 3.80 (3H, s), 4.55 (1H, m),
4.60 (2H, s), 4.90 (1H, d), 7.20 (4H, m).
calpain I yet reported, compound 4c displayed excellent
selectivity (>670 times) for calpain I over cathepsin B.
Similarly, compound 4b preferred calpain I over cathe-
psin L by >680 times. Compounds which exhibited good
inhibitory activity in the enzyme assay also inhibited
intracellular calpain I. Work is now continuing to
determine the behavior of this class of molecules in
several animal models of stroke and will be reported in
due course.
Exp er im en ta l Section
A mixture of the above ester (5.50 g, 0.018 mol), LiOH‚H2O
(2.3 g, 0.054 mol), THF (50 mL), and H2O (20 mL) was stirred
at room temperature for 2.5 h. THF was removed in vacuo,
and the residue was diluted with water (50 mL). The aqueous
layer was washed with ether (1 × 50 mL), acidified with dilute
acid (pH 3 ∼ 4), and extracted into ethyl acetate (3 × 100 mL).
The organic layer was washed with water (2 × 10 mL) and
brine (2 × 20 mL) and dried (MgSO4). Solvent evaporation
generated 1E (5.00 g, 95%) as a white solid: mp 120-125 °C
(softening at 100 °C); 1H NMR (CDCl3) δ 0.90 (6H, t), 1.60 (1H,
m), 1.80 (2H, m), 2.90 (2H, t), 3.60 (2H, m), 4.40 (1H, m), 4.60
(2H, dd), 4.90 (1H, d), 6.00 (1H, broad), 7.20 (4H, m); MS m/e
291 (M + H), 313 (M + Na).
Ch em istr y. Gen er a l Meth od s. Thin layer chromatog-
raphy was done on silica gel plates (MK6F 60A, size 1 × 3 in.,
layer thickness 250 µm, Whatman Inc.). Preparative chro-
matography was carried out using Merck silica gel, 40-63 µm,
230-400 mesh. 1H NMR spectra were recorded on a GE QE
Plus instrument (300 MHz) using tetramethylsilane as inter-
nal standard. Electrospray mass spectra were recorded on a
VG platform II instrument (Fisons Instruments). Elemental
analyses were performed by Quantitative Technologies Inc. of
Whitehouse, NJ . High-resolution mass spectroscopy was
performed by M-Scan Inc. of West Chester, PA. Compounds
1A,B were obtained from Advanced ChemTech, Louisville, KY,
and compound 4m was available from Enzyme Systems
Products, Dublin, CA.
Mor p h olin o-4-su lfon ylleu cin e (1C). A mixture of mor-
pholine hydrochloride (60 g, 0.49 mol), chloroform (200 mL),
and sulfuryl chloride (800 mL) was refluxed for 4 h. After
cooling to room temperature, additional sulfuryl chloride (200
mL) was added to the flask, and the mixture was refluxed for
another 3 h. Solvent evaporation gave a dark residue which,
on distillation under high vacuum (2 mmHg, 90 °C), generated
morpholinosulfonyl chloride as a colorless thick oil (74.60 g,
83%).
In a separate flask, leucine (40 g, 0.30 mol) was dissolved
in 3 N NaOH (600 mL), and to it was slowly added morpholi-
nosulfonyl chloride (57.20 g, 0.30 mol) in THF (120 mL). The
reaction mixture was stirred for 4 h and filtered. The filtrate
was acidified with dilute acid and extracted into ethyl acetate
(3 × 500 mL). The organic layer was washed with water (1 ×
50 mL), dried (Na2SO4), and concentrated to give 1C (29.61 g,
35%), which was used without any further purification: 1H
NMR (CDCl3) δ 1.00 (6H, q), 1.60 (1H, m), 1.85 (1H, m), 3.20
(4H, t), 3.75 (4H, t), 4.00 (1H, m), 5.10 (1H, d), 6.40 (1H, b);
MS m/e 281 (M + H), 303 (M + Na).
1-F lu or o-3-n itr o-4-p h en yl-2-bu ta n ol (6A). To a stirred
mixture of trans-â-nitrostyrene (5.25 g, 0.035 mol) and silica
gel (10 g, 230-400 mesh) in chloroform (400 mL) and 2-pro-
panol (75 mL) at room temperature was slowly added sodium
borohydride (5.50 g, 0.145 mol) over a period of 45 min. The
reaction mixture was stirred for an additional 15 min and
carefully quenched with 10% hydrochloric acid (20 mL). The
separated solid was filtered and washed with chloroform (50
mL). The organic layer was washed with water (2 × 10 mL)
and brine (2 × 10 mL) and dried (Na2SO4). Solvent evapora-
tion at reduced pressure gave a crude material which was
purified by flash chromatography (silica gel, 8% ethyl acetate-
hexane) to give 2.86 g of 1-nitro-2-phenylethane (5A): colorless
1
oil; Rf (10% ethyl acetate in hexane) 0.40; H NMR (CDCl3) δ
7.40-7.20 (5H, m), 4.60 (2H, t), 3.30 (2H, t).
In a separate flask, to a cooled (-78 °C) solution of oxalyl
chloride (2 M) in methylene chloride (11.60 mL, 0.0232 mol)
was added slowly dimethyl sulfoxide (3.65 g, 3.32 mL, 0.0467
mol). The reaction mixture was stirred for 15 min. A solution
of 2-fluoroethanol (1.16 g, 0.0181 mol) in methylene chloride
(10 mL) was slowly introduced into the reaction flask. After
another 15 min of stirring, the reaction mixture was diluted
with anhydrous methylene chloride (180 mL), and triethyl-
amine (9.20 g, 12.63 mL, 0.090 mol) was added to it. Stirring
was continued for another 2 h by which time the temperature
changed to room temperature. A solution of compound 5A
(2.74 g, 0.0181 mol) in anhydrous methylene chloride (10 mL)
was added to the reaction mixture, and stirring was continued
overnight. The mixture was then washed with water (2 × 20
mL), 4% hydrochloric acid (3 × 20 mL), water (2 × 20 mL),
saturated sodium bicarbonate solution (2 × 20 mL), and brine
(2 × 20 mL). Drying (Na2SO4) of the organic phase and solvent
evaporation gave a crude material which was purified by flash
chromatography (silica gel, 25% ethyl acetate-hexane) to give
6A (isomers I and II) as erythro/threo isomers. The combined
yield was 3.01 g (78%).
6A (isomer I): white solid, mp 71-73 °C; Rf (30% ethyl
acetate in hexane) 0.46; 1H NMR (CDCl3) δ 7.40-7.10 (m, 5H),
4.90 (m, 1H), 4.60 (m, 1H), 4.50-4.30 (m, 2H), 3.45-3.25 (m,
2H), 2.70 (d, 1H); MS m/e 214 (M + H), 236 (M + Na).
6A (isomer II): colorless oil; Rf (30% ethyl acetate in hexane)
0.42; 1H NMR (CDCl3) δ 7.40-7.15 (m, 5H), 4.90 (m, 1H), 4.65
(m, 1H), 4.50 (m, 1H), 4.20 (m, 1H), 3.40-3.30 (m, 2H), 2.90
(d, 1H); MS m/e 214 (M + H), 236 (M + Na).
1-F lu or o-3-n itr o-2-p en ta n ol (6B). This compound (dias-
teromeric mixture) was prepared from 1-nitropropane (5B)
according to the procedure described above for 6A: pale yellow
liquid (52% yield); bp 130-140 °C (0.3 mmHg); 1H NMR
(CDCl3) δ 1.05 (3H, t), 2.00 (2H, m), 4.20 (1H, m), 4.40 (1H,
m), 4.60 (2H, m).
[(Ben zyla m in o)ca r b on yl]leu cin e (1D). To a stirred
solution of benzyl isocyanate (0.45 g, 0.0034 mol) in methylene
chloride (5 mL) at 0 °C was added slowly a mixture of Leu-
OtBu hydrochloride salt (0.75 g, 0.0034 mol) and diisopropyl-
ethylamine (0.46 g, 0.0036 mol) in methylene chloride (5 mL).
The cooling bath was removed, the mixture was stirred for
another 0.5 h, and solvent was removed. The residue was
taken into ethyl acetate (15 mL) and was washed with 2%
citric acid solution (2 × 10 mL), saturated NaHCO3 solution
(1 × 10 mL), and brine (1 × 10 mL), and dried (Na2SO4).
Solvent evaporation gave [(benzylamino)carbonyl]leucine tert-
1
butyl ester as a white solid (0.90 g, 90%): mp 73-76 °C; H
NMR (CDCl3) δ 0.90 (6H, t), 1.40-1.80 (3H, m), 1.45 (9H, s),
4.40 (3H, m), 4.90 (2H, m), 7.30 (5H, m); MS m/e 321 (M + H),
343 (M + Na).
To 0.40 g of the above material was added a mixture of 90%
TFA (3 mL) and methylene chloride (3 mL), and the resulting
mixture was stirred at room temperature for 3 h. Excess
reagents were removed under high vacuum to give 0.35 g of
1
1D, which was directly used in the next step; H NMR of an
aliquot showed the absence of a peak at δ 1.45 for t-Boc group.
[(1,2,3,4-Te t r a h yd r oisoq u in olin -2-yl)ca r b on yl]le u -
cin e (1E). To a solution of triphosgene (7.00 g, 0.024 mol) in
methylene chloride (20 mL) at room temperature was added
a mixture of leucine methyl ester hydrochloride (9.10 g, 0.05
mol) and diisopropylethylamine (20 mL, 0.11 mol) in methyl-
ene chloride (100 mL) over a period of 2.5 h. The mixture was
stirred for another 0.5 h, and to it was added a solution of
1,2,3,4-tetrahydroisoquinoline (7.50 g, 0.05 mol) and diisopro-

Inhibitors of Recombinant Human Calpain I
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3825
+
1-F lu or o-3-n it r o-4-[(t et r a h yd r op yr a n -2-yl)oxy]-2-b u -
ta n ol (6C). This compound (diasteromeric mixture) was
prepared from 2-(2-nitroethoxy)tetrahydropyran (5C) accord-
ing to the procedure described above for 6A: pale yellow
viscous oil (29% yield, purification by flash chromatography,
(CDCl3 20% DMSO-d6) δ 0.85 (6H, m), 1.25 (2H, m), 1.60 (1
H, q), 2.82-3.15 (6H, m), 3.65 (5H, m), 3.85 (1H, m), 4.22 (1
H, m), 4.26-4.6 (2H, m), 4.96 (1H, d), 6.95 (1H, d), 7.15-7.30
(5H, m), 7.35 (1H, d); MS m/e 446 (M + H), 468 (M + Na).
3-[N-[(Ben zyla m in oca r bon yl)leu cyl]a m in o]-1-flu or o-4-
p h en yl-2-bu ta n ol (3d ). Method A: white solid; 1H NMR
(DMSO-d6) δ 0.70 and 0.80 (6H, 2 sets of t), 1.00-1.50 (3H, a
series of m), 2.60 (1H, m), 3.10 (1H, m), 3.60 (1H, m), 3.80
(1H, m), 4.00-4.50 (5H, a series of m), 5.40 (1H, m), 6.00 (1H,
m), 6.40 (1H, m), 7.20 (10H, m), 7.80 and 8.00 (1H, 2 sets of
d); MS m/e 430 (M + H), 452 (M + Na).
3-[N-[[(1,2,3,4-Tet r a h yd r oisoq u in olin -2-yl)ca r b on yl]-
leu cyl]a m in o]-1-flu or o-4-p h en yl-2-bu ta n ol (3e). Method
B: white solid; 1H NMR (CDCl3) δ 0.70-1.00 (6H, m), 1.40
(2H, m), 1.60 (1H, m), 2.90 (2H, m), 3.00 (2H, m), 3.50 (1H,
m), 3.70 (1H, m), 3.90-4.60 (10H, m), 4.80 and 4.90 (1H, 2
sets of d), 6.70 and 7.00 (1H, 2 sets of d), 7.20 (9H, m); MS m/e
456 (M + H), 478 (M + Na).
3-[N-[(Ben zyloxyca r b on yl)leu cyl]a m in o]-1-flu or o-2-
p en ta n ol (3f). Method B: white solid; 1H NMR (CDCl3) δ
0.90 (9H, m), 1.42-1.75 (5H, m), 3.70-4.60 (6H, m), 5.15 (3H,
m), 6.30 (1H, br), 7.35 (5H, s); MS m/e 369 (M + H), 391 (M +
Na).
1
silica gel, eluant: 35% ether in hexanes); H NMR (CDCl3) δ
1.60 (6H, m), 3.42-4.88 (10H, m); MS m/e 236 (M - H).
3-Am in o-1-flu or o-4-p h en yl-2-bu ta n ol (2A). A mixture
of intermediate 6A (isomer I, 0.48 g, 2.25 mmol), absolute
ethanol (20 mL), and Raney nickel (50% slurry in water,
catalytic) was hydrogenated (60 psi) in a Parr apparatus for 5
h. Filtration through a Celite pad and solvent evaporation
gave 0.41 g of 2A (isomer I), which was used without further
purification. Similar treatment of 6A (isomer II, 0.80 g, 3.75
mmol) gave 0.51 g of 2A (isomer II).
2A (isomer I): white solid; mp 64-67 °C; 1H NMR (CDCl3)
1.70-2.20 (3H, m), 2.50 (1H, q), 2.95 (1H, dd), 3.20 (1H, m),
3.80 (1H, m), 4.50 (1H, d), 4.70 (1H, d), 7.30 (5H, m); MS m/e
184 (M + H), 206 (M + Na).
2A (isomer II): white solid; mp 67-70 °C; 1H NMR (CDCl3)
δ 1.65-2.20 (3H, m), 2.55 (1H, q), 2.95 (1H, dd), 3.10 (1H, m),
3.60 (1H, m), 4.55 (1H, d), 4.70 (1H, d), 7.20 (5H, m); MS m/e
184 (M + H), 206 (M + Na).
3-Am in o-1-flu or o-2-p en ta n ol (2B). This compound (di-
astereomeric mixture) was prepared according to the procedure
3-[N-[(Ben zyloxyca r b on yl)leu cyl]a m in o]-1-flu or o-4-
[(tetr a h yd r op yr a n -2-yl)oxy]-2-bu ta n ol (3g). Method B:
viscous oil; 1H NMR (CDCl3) δ 0.90 (7H, m), 1.55 (4H, m), 1.75
(4H, m), 3.45-4.60 (12H, m), 5.10 (2H, s), 5.20 (1H, br), 7.37
(5H, s); MS m/e 455 (M + H), 477 (M + Na).
1
described above for 2A: pale yellow oil (69% yield); H NMR
(CDCl3) δ 0.95 (3H, t), 1.35 (1H, m), 1.60 (1H, m), 2.05 (2H,
br), 2.80 (1H, m), 3.65 (1H, m), 4.53 (3H, m); MS m/e 122 (M
+ H).
Syn th eses of 4a -g: Gen er a l Meth od . To a solution of
3a -g (1 equiv) in anhydrous methylene chloride at 0 °C was
slowly added Dess-Martin periodinane (2-3 equiv). The
cooling bath was removed, and the mixture was stirred for
1-16 h, diluted with methylene chloride, and washed several
times with 10% Na2S2O3 solution followed by saturated
NaHCO3 solution, water, and brine. Drying (MgSO4) and
solvent evaporation gave the product which was further
purified by flash chromatography using hexanes-ethyl acetate
mixture as eluant, yield 75-95%.
Cbz-Leu -D,L-P h e-CH2F (4a ): white solid; 1H NMR (CDCl3)
δ 0.90 (6H, m), 1.50 (3H, m), 3.00 (1H, m), 3.20 (1H, m), 4.20
(1H, m), 4.80 (2H, m), 5.00 (2H, m), 5.10 (2H, s), 6.60 (1H, d),
7.10 (2H, d), 7.40 (8H, m); MS m/e 429 (M + H), 451 (M +
Na). Anal. (C24H29N2O4F) C, H, N.
tBoc-Leu -D,L-P h e-CH2F (4b): white solid; 1H NMR (CDCl3)
δ 0.91 (6H, m), 1.43 (9H, s), 1.56 (3H, m), 3.10 (2H, ddd), 4.04
(1H, m), 4.80 (4H, m), 6.60 (1H, dd), 7.15 (2H, d), 7.29 (3H,
m); MS m/e 395 (M + H), 417 (M + Na). Anal. (C21H31N2O4F)
C, H, N.
Mor p h olin o-4-su lfon yl-Leu -D,L-P h e-CH2F (4c): white
solid; 1H NMR (CDCl3) δ 0.88 (6H, m), 1.43 (2H, m), 1.78 (1H,
q), 2.92-3.35 (6H, m), 3.60-3.82 (5H, m), 4.70-5.25 (4H, m),
6.36 (1H, q), 7.15-7.30 (5H, m); MS m/e 444 (M + H); HRMS
calc 444.1968 (MH+), found 444.1973 (MH+).
[(Ben zyla m in o)ca r bon yl]-Leu -D,L-P h e-CH2F (4d ): white
solid; 1H NMR (CDCl3) δ 0.80 (6H, m), 1.40 (3H, m), 2.90 (1H,
m), 3.10 (1H, m), 4.10 (1H, m), 4.30 (2H, m), 4.70 (3H, m),
5.50 (1H, m), 5.65 and 5.75 (1H, 2 sets of d), 7.20 (10H, m),
7.45 and 7.60 (1H, 2 sets of d); MS m/e 428 (M + H), 450 (M
+ Na). Anal. (C24H30N3O3F) C, H, N.
[(1,2,3,4-Tet r a h yd r oisoq u in olin -2-yl)ca r b on yl]-Leu -
D,L-P h e-CH2F (4e): white solid; 1H NMR (CDCl3) δ 0.90 (6H,
m), 1.60 (3H, m), 2.90 (2H, t), 3.00 (1H, m), 3.20 (1H, m), 3.60
(2H, m), 4.40 (1H, m), 4.50 (2H, m), 4.80 (4H, m), 6.90 and
7.10 (1H, 2 sets of d), 7.20 (9H, m); MS m/e 454 (M + H), 476
(M + Na). Anal. (C26H32N3O3F) C, H, N.
Cbz-Leu -D,L-Abu -CH2F (4f): white solid; 1H NMR (CDCl3)
δ 0.90 (9H, m), 1.60 (4H, m), 1.95 (1H, m), 4.20 (1H, m), 4.85
(2H, m), 5.05 (1H, m), 5.15 (3H, m), 6.60 (1H, m), 7.33 (5H,
m); MS m/e 367 (M + H), 389 (M + Na). Anal. (C19H27N2O4F)
C, H, N.
Cbz-Leu -D,L-Ser (OTHP )-CH2F (4g): waxy solid; 1H NMR
(CDCl3) δ 0.95 (7H, m), 1.50 (4H, m), 1.70 (4H, m), 3.50 (1H,
br), 3.75 (2H, m), 4.20 (3H, m), 4.40 (1H, br), 4.90 (3H, m),
5.12 (2H, s), 5.20 (1H, m), 7.35 (5H, m); MS m/e 453 (M + H),
3-Am in o-1-flu or o-4-[(tetr a h yd r op yr a n -2-yl)oxy]-2-bu -
ta n ol (2C). This compound (diastereomeric mixture) was
prepared according to the procedure described above for 2A:
pale yellow oil (86% yield); 1H NMR (CDCl3) δ 1.18-1.90 (9H,
m), 3.10 (1H, m), 3.50 (2H, m), 3.85 (3H, m), 4.55 (3H, m); MS
m/e 208 (M + H).
Syn th eses of 3a -g: Gen er a l P r oced u r e. Compounds
3a -g (diastereomeric mixture) were synthesized following
either of two general methods, A or B.
Gen er a l Meth od A. To a cooled (-20 °C) solution of 1A-E
(1 equiv) in methylene chloride or THF was added N-methyl-
morpholine (2.1 equiv) followed by isobutyl chloroformate (1.05
equiv). The mixture was stirred for 10 min, the cooling bath
was replaced by an ice bath, and compound 2A-C (1 equiv)
in methylene chloride or DMF was introduced into the reaction
flask. Stirring was continued for 1 h by which time the
temperature changed to room temperature. The reaction
mixture was diluted with ethyl acetate. The organic layer was
washed with 2% citric acid, 2% NaHCO3, water, and brine and
dried (MgSO4). Solvent evaporation gave a crude material
which was purified by flash chromatography (eluant: hexane-
ethyl acetate), yield 40-60%.
Gen er a l Meth od B. To a cooled (0 °C) solution of 1A-E
(1 equiv) in DMF was added N-methylmorpholine (3-4 equiv)
followed by 1-HOBt (1.05 equiv) and BOP (1.05 equiv). The
mixture was stirred for 10 min, compound 2A-C (1.1 equiv)
was introduced into the reaction flask, and the ice bath was
removed. Stirring was continued for 1-16 h. The reaction
mixture was poured into a mixture of ice-water and extracted
into ethyl acetate. The organic layer was washed with 2%
citric acid, 2% NaHCO3, water, and brine and dried (MgSO4).
Solvent evaporation gave a crude material which was purified
by flash chromatography (eluant: hexane-ethyl acetate), yield
80-90%.
3-[N-[(Ben zyloxyca r b on yl)leu cyl]a m in o]-1-flu or o-4-
p h en yl-2-bu ta n ol (3a ). Method A: white solid; 1H NMR
(CDCl3) δ 0.80 (6H, m), 1.30 (2H, m), 1.60 (2H, m), 2.90 (2H,
m), 3.50 (1H, 2 sets of d), 4.00 (2H, m), 4.30-4.90 (3H, m),
5.10 (2H, d), 6.10 and 6.30 (1H, 2 sets of d), 7.30 (10H, m); MS
m/e 431 (M + H), 453 (M + Na).
3-[N-[(ter t-Bu t oxyca r b on yl)leu cyl]a m in o]-1-flu or o-4-
p h en yl-2-bu ta n ol (3b). Method B: white solid; 1H NMR
(CDCl3) δ 0.91 (6H, m), 1.43 (9H, s), 1.56 (3H, m), 2.94 (2H,
m), 3.95 (2H, m), 4.39 (4H, m), 5.03 (1H, m), 6.78 (1H, dd),
7.25 (5H, m); MS m/e 397 (M + H), 419 (M + Na).
3-[N-[(Mor p h olin o-4-su lfon yl)leu cyl]a m in o]-1-flu or o-
1
4-p h en yl-2-bu ta n ol (3c). Method B: white solid; H NMR

3826 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Chatterjee et al.
475 (M + Na); HRMS calc 453.2401 (MH+), found 453.2382
(MH+).
TBDMSOTf (0.4 mL, 2.07 mmol). The mixture was stirred
for 1.5 h, diluted with ethyl acetate (l0 mL), and washed
successively with saturated NaHCO3 (2 × 5 mL) and brine (2
× 5 mL). Drying and solvent evaporation gave a crude product
which was purified by chromatography (hexanes-ethyl ac-
etate, 3:1, with 5 drops of triethylamine per 100 mL of solution)
to give the enolsilyl ether as a white solid (0.21 g, 87%).
A mixture of above enolsilyl ether (0.19 g, 0.36 mmol),
1-(chloromethyl)-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tet-
rafluoroborate) (F-TEDA-BF4, 0.14 g, 0.39 mmol) and DMF (4
mL) was stirred at room temperature overnight, diluted with
a mixture of ether-ethyl acetate (2:1, 20 mL), and washed
successively with water (2 × 10 mL) and brine (2 × 10 mL).
Drying (MgSO4) and solvent evaporation gave a crude product
which was contaminated with compound 9. Rigorous purifica-
tion by column chromatography (eluant: CH2Cl2-CH3CN, 95:
5) generated 4l as a white solid (diastereomeric mixture, 0.015
g, 10%): 1H NMR (CDCl3) δ 0.90 (6H, m), 2.10 (1H, m), 3.00
(1H, m), 3.15 (1H, m), 3.95 (1H, t), 4.70-5.15 (6H, m), 6.40
(1H, t), 7.30 (10H, m); MS m/e 415 (M + H), 437 (M + Na).
Anal. (C23H27N2O4F) C, H, N.
Cbz-Leu -D,L-Ser -CH2F (4h ). A solution of 4g (0.035 g, 0.08
mmol) in methanol (2 mL) was treated with p-toluenesulfonic
acid monohydrate (0.014g, 0.08 mmol). The mixture was
stirred at ambient temperature for 2 h. Solvent was evapo-
rated in vacuo, and the residue was dissolved in ethyl acetate
(10 mL). The organic phase was washed with saturated
sodium bicarbonate (2 × 5 mL), water (2 × 5 mL), and brine
(2 × 5 mL). Drying (MgSO4) and solvent evaporation gave a
crude product which was purified by flash chromatography to
give 4h as a glassy amorphous solid (0.013 g, 45%): 1H NMR
(CDCl3) δ 0.95 (7H, m), 1.65 (3H, m), 4.00 (2H, m), 4.15 (1H,
m), 4.90 (2H, m), 5.15 (4H, m), 7.00 (1H, m), 7.35 (5H, m); MS
m/e 369 (M + H), 391 (M + Na); HRMS calc 369.1826 (MH+),
found 369.1837 (MH+).
Leu -D,L-P h e-CH2F -HCI (4i). A stirred suspension of 4b
(0.094 g, 0.24 mmol) in ether was saturated with HCl gas. The
resulting solution was resaturated with HCl gas after 20 min.
The solvent and excess HCl were evaporated after 40 min,
affording the product as a light yellow solid (0.080 g, 100%
yield): 1H NMR (D2O) δ 0.74 (6H, d), 1.65 (3H, m), 2.74 (1H,
m), 3.28 (1H, m), 3.79 (1H, m), 4.46 (2H, m), 7.34 (5H, m); MS
m/e 295 (M + H). Anal. (C16H23N2O2F‚HCl) C, H.
P h CH2CH2CO-Leu -D,L-P h e-CH2F (4j). Compound 4i and
hydrocinnamic acid were coupled according to general method
B, as described before, to generate 4j as a white amorphous
solid (100% yield): 1H NMR (CDCl3) δ 0.80 (3H, m), 1.22 (3H,
m), 2.50 (2H, m), 2.96 (3H, m), 3.16 (1H, dt), 4.42 (1H, quintet),
4.60-5.03 (3H, m), 5.91 (0.4H, d), 6.07 (0.6H, d), 7.01-7.32
(11H, m); MS m/e 427 (M + H), 449 (M + Na). Anal.
(C25H31N2O3F) C, H, N.
Cbz-Leu -Leu -D,L-P h e-CH2F (4k ). Compound 4i and Cbz-
Leu-OH were coupled according to general method B, as
described before, to generate 4k as a white solid (71% yield):
1H NMR (CDCl3) δ 0.87 (6H, m), 1.39 (4H, m), 1.59 (4H, m),
2.97 (1H, dt), 3.20 (1H, dt), 4.20 (1H, m), 4.41 (1H, m), 4.60-
5.18 (5H, m), 5.41 (0.4H, d), 5.59 (0.6H, d), 6.69 (0.4H, d), 6.76
(0.6H, d), 7.05-7.38 (11H, m); MS m/e 542 (M + H), 564 (M +
Na). Anal (C30H40N3O5F) C, H, N.
Cbz-Va l-D,L-P h e-N(Me)OMe (8). To a stirring solution of
Cbz-Val-Phe-OH (7, 2.00 g, 5.00 mmol) and triethylamine (0.57
g, 0.79 mL, 5.64 mmol) in methylene chloride (25 mL) at room
temperature was added BOP reagent (2.5 g, 5.64 mmol). After
10 min, N,O-dimethylhydroxylamine hydrochloride (0.61 g,
6.20 mmol) was added to the flask, followed by triethylamine
(0.62 g, 0.86 mL, 6.20 mmol). Stirring was continued for 3 h.
The reaction mixture was diluted with methylene chloride (50
mL) and washed successively with dilute acid (2 × 30 mL),
saturated NaHCO3 (2 × 20 mL), H2O (2 × 20 mL), and brine
(2 × 30 mL). Drying (MgSO4) and solvent evaporation gave a
crude product which was purified by flash chromatography
(hexanes-ethyl acetate, 35:65) to give a white foam (diaster-
eomeric mixture, 1.72 g, 78%): 1H NMR (CDCl3) δ 0.80 (6H,
m), 2.00 (1H, m), 2.90 (1H, m), 3.10 (1H, m), 3.20 (3H, m) 3.70
(3H, d), 4.10 (1H, m), 5.10 (2H, m), 5.30 (2H, m), 6.60 (1H, m),
7.30 (10H, m); MS m/e 442 (M + H), 464 (M + Na).
Cbz-Va l-D,L-P h e-CH3 (9). A solution of compound 8 (1.56
g, 3.53 mmol) in anhydrous THF (25 mL) was added slowly to
a cooled (0 °C) solution of 3 M MeMgBr in ether (7 mL, 21.00
mmol) diluted with anhydrous THF (20 mL). The cooling bath
was removed, and the reaction mixture was stirred overnight.
The next day, the reaction mixture was quenched at 0 °C with
a mixture of saturated NH4Cl and water (1:1, 30 mL) and
extracted into ethyl acetate (3 × 50 mL). The combined
organic layer was washed with brine (2 × 30 mL), dried
(MgSO4), and concentrated to give a residue which was purified
by flash chromatography (EtOAc-hexanes, 65:35) to generate
a white solid (diastereomeric mixture, 1.21 g, 86%): 1H NMR
(CDCl3) δ 0.90 (6H, m), 2.10 (1H, m), 2.15 (3H, m), 3.00 (1H,
m), 3.15 (1H, m), 4.00 (1H, m), 4.80 (1H, m), 5.15 (2H, d), 5.30
(1H, t), 6.50 (1H, broad), 7.30 (10H, m); MS m/e 397 (M + H),
419 (M + Na).
[
14CH2]Cbz-Leu -D,L-P h e-CH2F (4a *). Radiosynthesis was
performed by Chemsyn Science Laboratories, Lenexa, KS.
[14CH2]Benzyl chloroformate (5.0 mCi, 0.086 mmol, 58 mCi/
mmol, prepared from [14CH2]benzyl alcohol and phosgene in
toluene) and iPr2NEt (35 µL, 0.20 mmol) were added to a
solution of compound 4i (0.028 g, 0.085 mmol) in methylene
chloride (0.4 mL) at 0 °C. After 80 min at 0 °C, the mixture
was diluted with methylene chloride (5 mL), rinsed with water,
1 M HCl, saturated Na2CO3, and saturated NaCl, and then
dried over MgSO4. The crude product was purified by chro-
matography on silica gel (EtOAc-hexanes, 35:65) to afford
pure product 4a * (16.9 mg, 2.3 mCi, 58 mCi/mmol) which
coeluted with authentic 4a on silica gel TLC (EtOAc-hexanes,
50:50, and CHCl3-MeOH, 90:10) and HPLC (4.6 × 250 mm
Zorbax Rx C18 column; 1 mL/min flow rate; 90:10:0.1 H2O-
CH3CN-TFA for 5 min; linear gradient from 10% to 100% of
99.9:0.1 CH3CN-TFA over 45 min; detection at 215 nm).
Bioch em istr y. Ca lp a in I In a ctiva tion Assa y. Assays
for inactivation of calpain contained 50 mM Tris-Cl (pH 7.5),
50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM â-mercapto-
ethanol, 0.2 mM Suc-Leu-Tyr-MNA (Enzyme Systems Prod-
ucts, Dublin, CA), 10 nM recombinant human calpain I, 3%
DMSO, and varying concentrations of inhibitor and were
initiated by the addition of 5 mM CaCl2. Reactions were
performed at ambient temperature in single cuvettes with the
increase in fluorescence (λex
) 340 nm, λem ) 425 nm) recorded
continuously on a Perkin-Elmer LS50B spectrofluorimeter
(Norwalk, CT) and were monitored until there was no further
product generated in inhibitor-containing assays. In some
experiments, inactivation rates were determined in 96-well
plates (Dynatech) in
a final volume of 200 µL using a
Fluoroskan II plate reader, with kinetic data acquired using
DeltaSoft software (BioMetallics Inc., Princeton, NJ ). Inhibi-
tor concentrations were at least 10-fold greater than the
enzyme concentration in all cases.
The second-order rate constant for inactivation of calpain I
was calculated using published methods.20a,b The simplest
kinetic model for irreversible inactivation involves formation
of an initial reversible complex (EI) followed by irreversible
formation of the inactivated enzyme (EI*). The pseudo-first-
order rate constant for inactivation, kobs, is dependent on
inhibitor concentration (It) and the individual rate constants,
as shown in eq 1,20a when inhibitor is in excess over enzyme.
Equation 2, resulting from rearrangement of eq 1, permits
calculation of the second-order rate constant for inactivation,
referred to as k.
k1
k3
E + I
\
98 EI*
k2
kobs ) [k1k3It/(k2 + k3)]/(1 + S/Km)
(1)
Cbz-Va l-D,L-P h e-CH2F (41). To a stirred solution of
compound 9 (0.19 g, 0.475 mmol) in methylene chloride (3 mL)
at 0 °C was added excess triethylamine (0.4 mL) followed by
Values of kobs were obtained from plots of fluorescence vs
time by nonlinear regression (Sigma Plot or GraphPad Prism)

Inhibitors of Recombinant Human Calpain I
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3827
and the samples were heated for 15 min at 65 °C. Supernatant
protein concentrations were determined by the BCA assay
(Pierce, Inc., Rockford, IL). Samples (equal total protein) were
resolved on 6% SDS-PAGE, transferred to nitrocellulose, and
analyzed by immunoblot using a polyclonal antibody specific
for calpain I-mediated spectrin breakdown products (SBDPs),24
followed by an alkaline phosphatase conjugated goat anti-
rabbit antibody (BIORAD, Inc.) and an alkaline phosphatase
detection system (BIORAD, Inc.). To determine the percent
inhibition, the integrated optical density (IOD) of the SBDPs
in the presence and absence of inhibitor was determined using
a BIOQUANT-OS/2 image analysis system (R&M Biometrics,
Inc., Nashville, TN). IC50 values were calculated from the
percent inhibition of SBDPs at varying inhibitor concentrations
using nonlinear regression analysis.
k ) (kobs/It)(1 + S/Km)
(2)
(3)
to the exponential equation (3)20b
y ) Ae(-k t) + B
obs
where y is the fluorescence at time t (Ft), A is the amplitude
of the reaction (F0 - F∞), and B is the maximal amount of
product formed when the enzyme is completely inactivated
(F∞). The apparent second-order rate constant for inactivation,
k, was calculated according to eq 2 from a replot of kobs vs I. A
Km value of 0.5 mM was employed in these calculations. In
all cases, linear replots were obtained, without evidence for
saturation kinetics in any case.
Because calpain I undergoes an autolytic loss of activity
under assay conditions, progress curves for control reactions
lacking inhibitor demonstrate a small first-order deviation
from linearity (kobs typically <0.0005 s-1). Values of kobs were
calculated for control reactions and included in plots of kobs vs
I, where they fell directly on the line defined by inhibitor-
containing assays.
Ack n ow led gm en t. The authors would like to thank
Dr. J effry Vaught for his continuing support and
encouragement and Dr. J ames C. Kauer for critical
review of the manuscript. We also thank Dr. Zi-Qiang
Gu for technical assistance. We are grateful to Dr. G.
Sankar Lal of Air Products (Allentown, PA) for a
generous gift of F-TEDA-BF4 (Selectflur) reagent. We
are pleased to acknowledge SmithKline Beecham Phar-
maceuticals for partial support of this research.
Bin d in g Assa y w ith 4a *. Recombinant human calpain I
(2.2 µM) was incubated at 37 °C with an ca. 10 fold molar
excess of 4a * (1.28 × 108 dpm /µmol) in the absence or presence
of calcium ion (5 mM) in 20 mM Tris, 50 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 5 mM â-mercaptoethanol. To specifically
capture the intermediate autolyzed 78 kDa species of calpain
I, inhibitor was added simultaneous to calcium ion addition.
To capture the 76 kDa autolyzed species of calpain I, inhibitor
was added 5 min following calcium ion addition. One reaction
mixture also contained Cbz-Val-Phe-H (250 µM). Following
addition of all reagents, incubations were continued for 5, 15,
or 30 min. All reactions were stopped by diluting 10-fold with
incubation buffer. Each mixture was then subjected to ultra-
filtration on a Microcon 10 filter (Amicon) for 30 min at 4 °C,
yielding a 10-fold concentration of the enzyme. The enzyme
was then subjected to four additional cycles of redilution with
assay buffer and concentration. Incorporation of radiolabel
into calpain I was determined by scintillation counting, with
the enzyme quantitated by its absorbance at 280 nm using an
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Samples were also
.
run on 10% SDS-PAGE21 and transfered to nitrocellulose22
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In ta ct Cell Assa y. Molt-4 cells (human leukemic T cells)
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removed by centrifugation and the supernatant collected.
Then 10% SDS was added to yield a final concentration of 1%,
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