Exploration of the importance of the P2-P3-NHCO-moiety in a potent di- or tripeptide inhibitor of calpain I: insights into the development of nonpeptidic inhibitors of calpain I.

Article abstract of DOI:10.1016/S0968-0896(98)00009-1

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydrogen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict requirement for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34 ((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity in the enzyme assay, also inhibited calpain I in a human cell line.

Full text of DOI:10.1016/S0968-0896(98)00009-1

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 509±522
Exploration of the Importance of the P2-P3 -NHCO-Moiety in
a Potent Di- or Tripeptide Inhibitor of Calpain I: Insights into
the Development of Nonpeptidic Inhibitors of Calpain Iy
Sankar Chatterjee,a,* Mohamed Iqbal,a Satish Mallya,b Shobha E. Senadhi,b
Teresa M. O'Kane,b Beth Ann McKenna,b Donna Bozyczko-Coyne,b
James C. Kauer,a Robert Simanb and John P. Mallamoa
aDepartment of Chemistry, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245, USA
bDepartment of Biochemistry, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245, USA
Received 20 October 1997; accepted 28 October 1997
AbstractÐCalpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an
episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene
inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydro-
gen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict require-
ment for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-
methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34
((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity
in the enzyme assay, also inhibited calpain I in a human cell line. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
calpain degrades neuronal structural or cytoskeletal
proteins resulting in lost synaptic contacts and neuro-
degeneration. Two major forms of calpains have been
identi®ed: calpain I and calpain II, which require low
and high micromolar Ca2+ concentrations for activation,
respectively. While calpain II is the predominant form in
many tissues, calpain I is thought to be the predominant
form activated during pathological conditions of ner-
vous tissue. We are interested in selectively inhibiting
calpain I to ®nd new therapeutics to treat stroke.3
Brain injury resulting from stroke is a major public
health problem worldwide. In the U.S., more than half a
million strokes occur each year, killing 150,000 people;
another 400,000 stroke victims survive, but only a for-
tunate third of these are left with little or no physical or
mental impairment.1 Major disability can result with
loss of the ability to communicate, ambulate, coordinate
or reason.2 The majority of all strokes occur when a
blood vessel blocked by a clot (or an air bubble) origi-
nating from the heart or atherosclerotic arterial plaque,
cuts o€ blood ¯ow to a region of the brain and induces
localized anemia, known as ischemia.1,2 An episode of
stroke initiates a chain of biochemical events resulting in
the rise of intracellular Ca2+ concentration, which, among
other e€ects, activates Ca2+-dependent enzymes includ-
ing calpain, a cytoplasmic cysteine protease. Activated
Potent peptide-based reversible4 and irreversible5 inhi-
bitors of calpain have been reported; however, to the
best of our knowledge, no full length X-ray crystal
structure of an enzyme±inhibitor complex has been
reported. Interestingly, in the reversible aldehyde series,
both tripeptides and dipeptides are potent inhibitors of
calpain I.4,5 This suggested to us that sucient binding
energy is obtained with occupancy of the S1 and S2
subsites of the enzyme.6 Takahashi, commenting on the
preferred substrates, noted that ``. . . an amino acid with
an aromatic or a bulky aliphatic side chain at the P3
position may to some extent increase the susceptibility
Key words: Stroke; calpain I; peptidomimetic; ketomethylene;
carbamethylene.
*Corresponding author.
yDedicated to Mrs Luxmi Chatterjee with respect and admiration.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00009-1

510
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
bamethylene (-CH2CH2-) moieties. In the process, we
would have access to a series of peptidomimetic inhibi-
tors of calpain I. In designing our target molecules, we
maintained an isobutyl group at the pseudo-P2 site to
mimic the P2-Leu of the corresponding peptidic inhibi-
tors but incorporated an aromatic moiety in the P3
region. We now present the full account of our work
describing the synthesis, and the in vitro recombinant
human calpain I inhibitory activity of the target com-
pounds.9 We also present the inhibitory activity of a
selected set of compounds against cathepsin B, a related
cysteine protease, and thrombin, a serine protease of
biological signi®cance. Finally, we present data demon-
strating that compounds which exhibit good activity in
the enzyme assay, also inhibit calpain I in a human
cell line.
Figure 1. Arrows indicate the critical sites along the peptide
inhibitor backbone which are engaged in hydrogen-bonding as
observed for peptide inhibitor binding to cysteine proteases:
papain superfamily (papain, cathepsin B, calpain I).
of the scissile bond to calpain''.7 Studies have shown
that calpain prefers Leu or Val at P2, but tolerates many
di€erent substituents at P1. Dolle et al. mentioned that
for a peptidic inhibitor of calpain I (a member of the
papain superfamily), the P2-NH moiety is a critical site
for hydrogen-bonding (Fig. 1).8 In order to probe the
importance of the P2-NH moiety in a tripeptide or a
dipeptide inhibitor of calpain I, we wished to replace
the P3-P2 amide bond in the tripeptide inhibitor, or
the carbamoyl/acyl moiety in the dipeptide inhibitor,
by corresponding ketomethylene (-COCH2-) and car-
Chemistry
Synthesis of the target compounds 7a±d is shown in
Scheme 1. Compounds 1a±d were converted to the cor-
responding b-ketoesters 2a±d, which on treatment with
sodium hydride and (R)-tri¯ate-ester (3), followed by
selective acidic hydrolysis and decarboxylation of the t-
butyl ester group, produced the g-ketoester 4a±d.10
Basic hydrolysis of 4a±d produced the corresponding g-
ketoacid 5a±d which was coupled with (S)-leucinol to
produce 6a±d. Compounds 6a,c,d were isolated as single
Scheme 1. Reagents: (a) 1,10-carbonyldiimidazole, THF, 0±23 ꢀC; (b) Li+ CH2COOtBu, THF; 78 ꢀC to 0 ꢀC; (c) 60% NaH, THF,
3, 23 ꢀC; (d) TFA, 23 ꢀC; (e) C6H6, re¯ux; (f) LiOH, MeOH-H2O; 70±75 ꢀC; (g) (S)-leucinol, BOP, HOBt, NMM, DMF 0±23 ꢀC; (h)
Pyr.SO3, Et3N, DMSO-CH2Cl2, 0±23 ꢀC.

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
511
isomers; compound 6b was a diastereomeric mixture.
Assuming that the alkylation of the b-ketoester 2a±d by
(R)-tri¯ate-ester 3 took place in an SN2 fashion,10 the
stereochemistry at the P2-site of the major product in
6a±d was tentatively assigned as (R). Oxidation of 6a±d
produced the desired aldehydes 7a±d. Similarly, com-
pounds 5a±b,d were coupled with 8a±b4c (Scheme 2) to
generate 9a±c, which on oxidation gave desired a-keto-
carboxamides 10a±c. Compounds 115a and 5d were
coupled to generate the compound 12 (Scheme 3) which
on oxidation gave ¯uoromethyl ketone 13 (diastereo-
meric mixture, epimeric at P1). Scheme 4 depicts the
synthesis of compounds 21a±b. Isocaproic acid (14) was
benzylated to generate the ester 15, which on alkylation
produced the racemic diester 16. Selective hydrolysis of
t-butyl ester in 16 gave 17, which was coupled with
1,2,3,4-tetrahydroisoquinoline to generate 18. Debenzyl-
ation of 18 produced 19. Compound 19 was coupled
with (s)-phenylalaninol to generate diastereomeric
20a,b, easily separable by silica±gel column chroma-
tography (20a being the faster moving isomer). Separated
diasteromers 20a and 20b were oxidized to generate
aldehydes 21a and 21b, respectively. The stereo-
chemistry at the pseudo-P2 site in 21a and 21b was ten-
tatively assigned as (R) and (S), respectively, based on
comparison of the calpain inhibitory activity of 21a,b
with that of a reference dipeptidyl aldehyde (Cbz-Val-
Phe-H, MDL 281704b). The syntheses of 30 and 31 are
depicted in Scheme 5. 2-Naphthalenethiol (22) was con-
verted to the ester 23, which on subsequent alkylation,
produced the corresponding racemic ester 24. Hydro-
lysis of 24 yielded the racemic acid 25, which was cou-
pled with (S)-leucinol to produce the diastereomeric
compounds 26 and 27, easily separable by silica gel col-
umn chromatography (26 being the faster-moving iso-
mer). Oxidation of 26 and 27 by m-chloroperbenzoic
acid generated the sulfonyl compounds 28 and 29, which
on further oxidation by sulfur trioxide-pyridine complex
in DMSO-CH2Cl2 produced the aldehydes 30 and 31.
The stereochemistry around the pseudo-P2 site in 30 and
31 was tentatively assigned as (S) and (R), respectively,
based on comparison of the calpain inhibitory activity
of 30 and 31 with that of the reference dipeptidyl alde-
hyde. While oxidation of compound 27 by sulfur tri-
oxide-pyridine complex in DMSO-CH2Cl2 produced the
aldehyde 32 (Scheme 6), oxidation by Davis' oxazir-
idine11 generated the corresponding sulfoxide 33, which
on further oxidation by sulfur trioxide±pyridine complex
in DMSO-CH2Cl2 gave the target aldehyde 34 (diaster-
eomeric mixture, epimeric at the sulfoxide center).
In vitro biology and discussion
The biological activities of the compounds were deter-
mined using recombinant human calpain I, prepared as
described by Meyer et al.12 with Suc-Leu-Tyr-MNA
Scheme 2. Reagents: (a) NMM, IBCF, THF-DMF, 20 to
23 ꢀC or BOP, HOBt, NMM, DMF, 0±23 ꢀC; (b) Dess±Martin
periodinane, CH2Cl2, 0±23 ꢀC.
Scheme 3. Reagents: (a) NMM, IBCF, 5d, CH2Cl2, 20 to
23 ꢀC or BOP, HOBt, NMM, DMF, 0±23ꢀC; (b) Dess±Martin
periodinane, CH2Cl2, 23 ꢀC.

512
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
Scheme 4. Reagents: (a) Benzyl alcohol, p-toluenesulfonic acid monohydrate, benzene, re¯ux (Dean±Stark); (b) LDA, t-butyl bromo-
acetate, THF-hexane-HMPA, 78 ꢀC to 0 ꢀC; (c) 90% TFA, CH2Cl2, 23 ꢀC; (d) 1,2,3,4-tetrahydroisoquinoline, BOP-Cl, Et3N,
CH2Cl2, 23 ꢀC; (e) H2, 10% Pd-C (Degussa, H2O content 50%), MeOH; (f) BOP, HOBt, NMM, DMF, 0±23 ꢀC; (g) Pyr.SO3, Et3N,
DMSO-CH2Cl2, 0±23 ꢀC.
(Enzyme System Products, Dublin, CA) as substrate.
Table 1 displays the inhibitory data for the ketomethyl-
ene containing inhibitors. In the aldehyde series, in
order to probe the steric requirement of calpain I in the
P3 region, initially we attached a phenylbutyl chain to
the pseudo P2-site to generate compound 7a, a good
inhibitor. Shortening the phenylalkyl chain and having
an alkyl (ethyl) substitution at the carbon atom (pseudo
P3-site), a to the carbonyl group, gave 7b, which is ca.
2.5 times more potent than 7a. Interestingly, substitu-
tion of the ethyl group in 7b with a phenyl group to
generate 7c did not diminish the activity. Thus, it
was ca. 36 times more potent than the diastereomeric
compound 21b indicating the strict stereochemical
requirement of calpain I for the pseudo P2 site of this
class of inhibitor. Thus, it appeared that the -NHCO-
moiety at the P2-P3 region of a potent tripeptide (e.g.
Cbz-Leu-Leu-Leu-H: IC50 19 nM in this assay4a) or a
dipeptide inhibitor could e€ectively be replaced by a
-CH2CO- moiety. In order to explore the importance of
the -CO- moiety in the above ketomethylene series, we
wished to replace the ketomethylene moiety with a carba-
methylene (-CH2CH2-) moiety; at the same time, the
xanthene (or tetrahydroisoquinoline) moiety was
replaced by a naphthalene-S(O)n-moiety, a di€erent
aromatic system. The inhibitory data for this series of
carbamethylene containing inhibitors is shown in
Table 2. Compounds 31±34 were good inhibitors of
calpain I, compound 34 (as a diastereomeric mixture at
the sulfoxide centre) being equipotent to compounds 7d
and 21a. In this series also, compound 31 was 10 times
more potent than than diastereomeric compound 30,
revealing the importance of the correct stereochemistry
at the pseudo P2 site.
appeared that calpain might tolerate
a sterically
demanding group in the P3 region. Constraining the
aromatic rings of 7c into a xanthene moiety, produced
7d, a >®vefold more potent inhibitor than 7a. In a
similar way, in the a-ketocarboxamide series (10a±c),
xanthene containing compound 10c was ca. 65-fold
more potent than the parent phenylbutyl containing
10a; however, note that the P1 in 10a was Abu (R2=Et).
Finally, xanthene containing ¯uoromethyl ketone 13, an
irreversible inhibitor, also displayed good inactivation
of calpain I. In order to explore whether the P3-span-
ning xanthene moiety could be replaced by a di€erent
heterocycle, we generated diastereomeric (at the pseudo
Selectivity
P2)
compounds
21a±b,
containing
a
tetra-
Cathepsin B is a related cysteine protease which might
be sensitive to inhibition by the above set of com-
pounds. Thus, we tested compounds 7d, 10c, 13, 31, 32,
hydrohydroisoquinoline nucleus. Compound 21a was
equipotent to compound 7d. As shown, this compound

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
513
Scheme 6. Reagents: (a) Pyr.SO3, Et3N, DMSO-CH3Cl2, 0±
23 ꢀC; (b) Davis' oxaziridine (PhSO2N(O)CHPh), CH2Cl2,
23 ꢀC.
Scheme 5. Reagents: (a) 60% NaH, Br(CH2)3COOEt, THF, 0±
23 ꢀC; (b) LDA, 1-iodo-2-methylpropane, THF-hexane-
HMPA, 78 ꢀC to 23 ꢀC; (c) LiOH.H2O, EtOH-H2O, re¯ux;
(d) (S)-leucinol, BOP, HOBt, NMM, DMF, 0±23 ꢀC; (e) m-
CPBA, CH2Cl2, 0±23 ꢀC; (f) Pyr.SO3, Et3N, DMSO-CH2Cl2,
0±23 ꢀC.
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 calcium 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 ecacy.
Table 3 lists the cellular activities of those compounds
which displayed an IC50 4 50 nM in the enzyme assay.
As shown, the reversible inhibitors 7d, 31, and 34 are
cell-permeable and inhibit intracellular calpain I (IC50
<10 mM); irreversible inhibitor 13 is less potent.
and 34 against cathepsin B (substrate used Cbz-Phe-
Arg-AMC); Table 3 lists the inhibitory data. While,
compounds 7d, 10c, and 13 preferred calpain I by >17-
fold, ca. ninefold and 76-fold, respectively, over cathe-
psin B, compounds 31 and 34 preferred calpain I by
threefold and twofold, respectively, over cathepsin B.
The inhibitory activity of these compounds against
thrombin (substrate used: Cbz-Phe-Val-Arg-AMC), a
serine protease, is also shown in Table 3. Compounds
were less active against thrombin up to 10 mM.
Conclusion
We have described potent ketomethylene and carba-
methylene containing peptidomimetic inhibitors of
recombinant calpain I. Our study revealed, for the ®rst
time, that the -NHCO-moiety (possible hydrogen-bond-
ing site) at the P2-P3 region of a tripeptide or a dipeptide
Cellular activity
In order to probe the ability of these compounds to
penetrate cells and inhibit intracellular calpain I, we

514
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
Table 1. Recombinant human calpain I inhibitory activity of compounds 7a±d, 10a±c, 13, and 21a±ba,b,c,d
1
Compd
R1
R2
X
IC50 nM
kobs/I M 1 s
7a
7b
-(CH2)4Ph
-(s)-CH(Et)Ph
-CH(Ph)2
iBu
iBu
iBu
iBu
Et
H
H
138
55
Ð
Ð
7c
7d
H
50
25
Ð
Ð
Xanthen-9-yl
-(CH2)4Ph
H
10a
10be
10c
13
CONHEt
CONHEt
CONHEt
CH2F
8440
6685
130
-
Ð
Ð
-(s)-CH(Et)Ph
Xanthen-9-yl
Xanthen-9-yl
Et
iBu
Bn
Ð
76,000
21a
21b
Bn
Bn
H
H
28
Ð
Ð
1000
an=3, in all cases.
bFor compounds 7a±d, 10a±c and 21a±b, the stereochemistry at P1 is (S); 13 is diastereomeric mixture at P1.
cFor compounds 7a,c±d, 10a,c, 13, 21a, the stereochemistry at P2 is (R); for 21b, the stereochemistry at P2 is (S); 7b and 10b are
diasrereomeric mixture at P2.
dIn the same assay, the IC50 values for the reference compounds Cbz-Val-Phe-H (MDL 28170)4b and Cbz-Leu-Abu-CONHEt4c were
1
17 nM and 240 nM, respectively; kobs/I M 1 s for Cbz-Leu-Phe-CH2F5a was 136,000.
eElemental analysis: C24H36N2O4.1.25 H2O; calcd N 6.38; found N 7.22.
Synthesis of 2a±d: general procedure13
inhibitor of calpain I is not a strict requirement for the
inhibitory property. We have demonstrated that such an
-NHCO- moiety can e€ectively be replaced by a keto-
methylene or a carbamethylene moiety, provided an
aromatic system is employed in the P3 region. In the
absence of protein structural data, identi®cation of such
inhibitor binding motif can be used to design next gen-
eration nonpeptidic ligands. Work is currently under-
way to develop such inhibitors and will be reported in
due course.
To a cooled (0 ꢀC) solution of acid 1a±d (0.04±0.05 mol)
in anhydrous tetrahydrofuran (40±50 mL) was added
1,10-carbonyldiimidazole (1.05 equiv). The mixture was
stirred at 0 ꢀC for 0.5 h and then at room temperature
overnight. The next morning, this solution was added
slowly, over 1 h, to a cooled ( 78 ꢀC) solution of tert-
butyl lithioacetate (2.2 equiv, generated in situ from
t-butyl acetate and lithium diisopropylamide) in tetra-
hydrofuran (40±50 mL) and hexane (35±40 mL). The
mixture was stirred for an additional 0.5 h and quenched
with 1 N HCl (2.2 equiv), brought to 0 ꢀC and acidi®ed
with 1 N HCl to pH 3±4. The resulting aqueous solution
was extracted with ethyl acetate (2Â100 mL). The
Experimental
General methods
Thin-layer chromatography was done on silica gel plate
(MK6F 60A, size 1Â3 in, layer thickness 250 mm,
Whatman Inc.). Preparative chromatography was car-
ried out using Merck silica±gel, 40±63 mm, 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, USA.
Table 2. Recombinant human calpain I inhibitory activity of
compounds 30±31, 32, and 34a
Compd
IC50 nM
30
31
32
34
500
50
75
30
an=3, in all cases.

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
Table 3. Selectivity and intact cell assay data for selected compoundsa
515
Compound
Calpain I
(IC50 nM)
Cathepsin B
(IC50 nM)
Thrombin
(% inh at 10 mM)
Intact cell assay
(% inh of SBDP at 10 mM)
7d
10c
13
31
32
34
25
130
440
1150
1,000b
150
25
13
9
65
Ð
24
80
Ð
68
76,000b
50
75
1
60
60
5
30
2
aFor calpain I, cathepsin B, and thrombin, n ˆ 3; for intact cell assay, n ˆ 2.
bRate of inactivation (kobs/I M 1 s 1).
organic layer was washed with brine (1Â40 mL), dried
over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure. Puri®cation of the
crude material by ¯ash chromatography (silica gel,
eluant: 5±6% ethyl acetate in hexanes) gave the desired
product 2a±d in 60±70% yield.
(8±10 mL) and stirred at room temperature for 1±2 h.
Excess tri¯uoroacetic acid was removed and the residue
was taken into benzene (30±40 mL) and heated at re¯ux
for 1±2 h. The solvent was removed under reduced
pressure and the crude product was puri®ed by ¯ash
chromatography (silica±gel, eluant: 4±5% ethyl acetate
in hexanes) to give the desired compounds 4a±d in 35±
45% yield over three steps.
t-Butyl 3-oxo-7-phenylheptanoate (2a). Colorless oil; Rf
(10% ethyl acetate in hexane): 0.37; 1H NMR (CDCl3) d
7.30±7.10 (m, 5H), 3.35 (s, 2H), 2.60 (m, 2H), 2.50 (m,
2H), 1.60 (m, 4H), 1.45 (s, 9H).
Methyl 2-isobutyl-4-oxo-8-phenyloctanoate (4a). Color-
less oil; Rf (10% ethyl acetate in hexane): 0.34; 1H NMR
(CDCl3) d 7.35±7.15 (m, 5H), 3.65 (s, 3H), 2.90±2.80 (m,
2H), 2.60 (m, 2H), 2.50±2.30 (m, 3H), 1.70±1.50 (m,
6H), 1.25 (m, 1H), 1.95 (d, 3H), 1.85 (d, 3H).
t-Butyl 3-oxo-4-phenylhexanoate (2b). Colorless oil; Rf
(10% ethyl acetate in hexane): 0.49; 1H NMR (CDCl3) d
7.38±7.18 (m, 5H), 3.70 (t, 1H), 3.35 (d, 1H), 3.20 (d,
1H), 2.10 (m, 1H), 1.70 (m, 1H), 1.45 (s, 9H), 0.85 (t,
3H).
Methyl 2-isobutyl-4-oxo-(S)-5-phenylheptanoate (4b).
Colorless oil; Rf (10% ethyl acetate in hexane): 0.48;
1H NMR (CDCl3) d 7.40±7.18 (m, 5H), 3.60 (s, 3H),
3.50 (t, 1H), 2.85 (m, 1H), 2.75 (m, 1H), 2.45 (dd, 1H),
2.05 (m, 1H), 1.70 (m, 1H), 1.45 (m, 2H), 1.15 (m, 1H),
0.90±0.70 (m, 1H).
t-Butyl 3-oxo-4,4-diphenylbutanoate (2c). Colorless oil;
Rf (10% ethyl acetate in hexane): 0.47; 1H NMR
(CDCl3) d 7.40±7.20 (m, 10H), 5.35 (s, 1H), 3.45 (s, 1H),
1.45 (s, 9H).
Methyl 2-isobutyl-4-oxo-5,5-biphenylpentanoate (4c).
White solid, mp 64.5±65.5 ꢀC; Rf (10% ethyl acetate in
hexane): 0.41; H NMR (CDCl3) d 7.40±7.15 (m, 10H),
5.15 (s, 1H), 3.65 (s, 3H), 3.00±2.85 (m, 2H), 2.60 (q,
1H), 1.50 (m, 2H), 1.20 (m, 1H), 0.90 (d, 3H), 0.80 (d,
3H).
t-Butyl 3-oxo-4-(9-xanthenyl)propionoate (2d). White
solid, mp 101±103 ꢀC; Rf (10% ethyl acetate in hexane):
0.46; 1H NMR (CDCl3) d 7.40±7.00 (m, 8H), 5.00 (s,
1H), 3.20 (s, 2H), 1.40 (s, 9H).
1
Synthesis of 4a±d: General procedure10. A solution of the
keto-ester 2a±d (0.02±0.03 mol) in anhydrous tetra-
hydrofuran (20±25 mL) was slowly added, at room
temperature, to a slurry of sodium hydride (60% in oil,
1.05 equiv) in anhydrous tetrahydrofuran (10±15 mL).
After the evolution of hydrogen gas ceased, the solution
was treated with the tri¯ate-ester 3 (1.2±1.3 equiv, gen-
erated from the corresponding (R)-hydroxyester and
tri¯ic anhydride in the presence of 2,6-lutidine). The
reaction mixture was stirred overnight, diluted with
ether (100±150 mL), washed with water (30±40 mL),
dried over magnesium sulfate and concentrated under
reduced pressure to give the crude diester intermediate.
This material was dissolved in tri¯uoroacetic acid
Methyl 2-isobutyl-4-oxo-4-(9-xanthenyl)butanoate (4d).
Colorless oil; Rf (10% ethyl acetate in hexane): 0.40;
1H NMR (CDCl3) d 7.40±7.18 (m, 8H), 4.90 (s, 1H),
3.55 (s, 3H), 2.80±2.60 (m, 2H), 2.30 (dd, 1H), 1.30 (m,
2H), 1.00 (m, 1H), 0.80 (d, 3H), 0.70 (d, 3H).
Synthesis of 5a±d: General procedure. A mixture of the
ester 4a±d (0.005±0.006 mol), lithium hydroxide-mono-
hydrate (1.3±1.4 equiv), methanol (25±30 mL) and water
(8±10 mL) was gently heated at 70±75 ꢀC for 1.5±2.0 h.
Methanol was removed under reduced pressure. The
aqueous layer was washed with diethyl ether (20±
25 mL), acidi®ed at 0 ꢀC with 1 N HCl and then extracted

516
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
into diethyl ether (3Â20 mL). The organic layer was
washed with brine (1Â10 mL) and dried over anhydrous
sodium sulfate. Solvent evaporation at reduced pressure
yielded the intermediates 5a±d in 85±90% yield, which
were used without further puri®cation.
(CDCl3) d 7.40±7.10 (m, 5H), 5.85 (m, 1H), 4.00±3.80
(m, 1H), 3.70±3.40 (m, 3H), 3.00±2.70 (m, 2H), 2.50±
2.30 (td, 1H), 2.00 (m, 1H), 1.80±1.20 (m, 8H), 1.00±0.70
(m, 15H).
(R)-2-Isobutyl-4-oxo-5,5-biphenylpentanoic acid ((S)-1-
hydroxymethyl-3-methyl)butyl amide (6c). White solid,
mp 111±112 ꢀC; Rf (5% methanol in methylene chloride)
2-Isobutyl-4-oxo-8-phenyloctanoic acid (5a). Colorless
oil; 1H NMR (CDCl3) d 7.35 (m, 5H), 3.00±2.70 (m,
2H), 2.60 (m, 2H), 2.50±2.30 (m, 3H), 1.70±1.50 (m,
6H), 1.35±1.20 (m, 1H), 1.95 (d, 3H), 1.85 (d, 3H).
1
0.45; H NMR (CDCl3) d 7.40±7.10 (m, 10H), 5.85 (d,
1H), 5.10 (s, 1H), 3.85 (m, 1H), 3.60 (m, 1H), 3.50 (m,
1H), 3.00 (q, 1H), 2.80±2.70 (m, 2H), 2.30 (dd, 1H),
1.60±1.00 (m, 6H), 1.00±0.80 (m, 12H).
2-Isobutyl-4-oxo-(S)-5-phenylheptanoic acid (5b). White
solid, mp 61±63 ꢀC; H NMR (CDCl3) d 7.40±7.10 (m,
1
5H), 3.60 (m, 1H), 2.90 (m, 1H), 2.75 (m, 1H), 2.50 (m,
1H), 2.05 (m, 1H), 1.70 (m, 1H), 1.50 (m, 2H), 1.15 (m,
1H), 0.90-0.70 (m, 9H).
(R)-2-Isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-
hydroxymethyl-3-methyl)butyl amide (6d). White solid,
mp 168±169 ꢀC; Rf (5% methanol in methylene chloride)
0.55; 1H NMR (CDCl3) d 7.40±7.00 (m, 8H), 5.70 (d,
1H), 4.90 (s, 1H), 3.85 (m, 1H), 3.60 (m, 1H), 3.50 (m,
1H), 2.80 (t, 1H), 2.70 (q, 1H), 2.50 (m, 1H), 2.30 (dd,
1H), 1.60 (m, 1H), 1.50±1.20 (m, 5H), 1.00-0.60 (4 sets
of doublets, 12H).
2-Isobutyl-4-oxo-5,5-biphenylpentanoic acid (5c). White
solid, mp 81.5±83.5 ꢀC; H NMR (CDCl3) d 7.40±7.10
1
(m, 10H), 5.15 (s, 1H), 3.00±2.85 (m, 2H), 2.60±2.70 (m,
1H), 1.60±1.40 (m, 2H), 1.30±1.20 (m, 1H), 0.90 (d, 3H),
0.80 (d, 3H).
Synthesis of 7a±d: General procedure. To a cooled (0 ꢀC)
solution of alcohol 6a±d (0.05±0.10 mmol) in anhydrous
methylene chloride (2±3 mL) and anhydrous dimethyl
sulfoxide (2±3 mL) was added triethylamine (3 equiv).
Sulfur trioxide-pyridine complex (3 equiv) was slowly
added to the stirred mixture over a period of 5 min
and the ice-bath was removed. The mixture was stirred
for another 1 h, poured into water (10 mL) and extrac-
ted into ether (3Â10 mL). The organic layer was washed
with 2% citric acid solution (2Â5 mL), saturated
sodium bicarbonate solution (2Â5 mL), brine (1Â5 mL)
and dried over anhydrous magnesium sulfate. Solvent
evaporation gave a residue that was washed with n-
pentane (5±8 mL) and recrystallized (ethyl acetate-
hexanes) to produce compounds 7a±d in 50±60%
yield.
2-Isobutyl-4-oxo-4-(9-xanthenyl)butanoic
acid
(5d).
White solid, mp 125±127 ꢀC; H NMR (CDCl3) d 7.40±
7.00 (m, 8H), 4.95 (s, 1H), 2.80±2.60 (m, 2H), 2.30 (dd,
1H), 1.35 (m, 1H), 1.00 (m, 1H), 0.80 (d, 3H), 0.70 (d, 3H).
1
Synthesis of 6a±d: General procedure. To a cooled (0 ꢀC)
solution of 5a±d (0.0005±0.001 mol) in anhydrous N,N-
dimethylformamide (3±4 mL) was added N-methylmor-
pholine (3 equiv) followed by 1-HOBt (1 equiv) and
BOP (1 equiv). The mixture was stirred for 15 min and
to it was added (S)-leucinol (1.3±1.4 equiv). The cooling
bath was removed and the mixture was stirred for 1±2 h,
poured into water (5 mL) and extracted into ethyl acet-
ate (3Â10 mL). The organic layer was washed with 2%
citric acid solution (2Â5 mL), saturated sodium bicar-
bonate solution (2Â5 mL), brine (1Â5 mL) and dried
over anhydrous sodium sulfate. Solvent evaporation
under reduced pressure gave a crude material that was
puri®ed by ¯ash column chromatography (silica gel,
eluant: 4±5% methanol-methylene chloride) to produce
6a±d in 40±60% yield.
(R)-2-Isobutyl-4-oxo-8-phenyloctanoic acid ((S)-1-formyl-
3-methyl)butyl amide (7a). White solid, mp 64±65 ꢀC; Rf
1
(30% ethyl acetate in hexanes): 0.48; H NMR (CDCl3)
d 9.55 (s, 1H), 7.30±7.10 (m, 5H), 6.10 (d, 1H), 4.50 (m,
1H), 3.00±2.80 (m, 2H), 2.60 (m, 1H), 2.40 (m, 4H),
1.80±1.60 (m, 8H), 1.50±1.10 (m, 2H), 1.00±0.80 (m,
12H). MS m/e 388 (M+H), 410 (M+Na). Anal.
(C24H27NO3) C, H, N.
(R)-2-Isobutyl-4-oxo-8-phenyloctanoic acid ((S)-1-hydroxy-
methyl-3-methyl)butyl amide (6a). Viscous oil; Rf (5%
methanol in methylene chloride) 0.40; 1H NMR
(CDCl3) d 7.40±7.10 (m, 5H), 5.85 (d, 1H), 3.85 (m, 1H),
3.60 (m, 1H), 3.50 (m, 1H), 3.00±2.80 (m, 2H), 2.70 (m,
1H), 2.60 (m, 2H), 2.50±2.30 (m, 3H), 1.70±1.50 (m,
8H), 1.45±1.20 (m, 2H), 1.00±0.80 (m, 12H).
(R)-2-Isobutyl-4-oxo-(S)-5-phenylheptanoic acid ((S)-1-
formyl-3-methyl)butyl amide (7b). Viscous liquid: Rf
1
(30% ethyl acetate in hexanes): 0.52; H NMR (CDCl3)
d 9.55 and 9.45 (2 singlets, 3:2), 7.40±7.10 (m, 5H), 6.10
(m, 1H), 4.60±4.30 (2 sets of multiplate, 3:2, 1H), 3.00±
2.70 (m, 2H), 2.50±2.30 (td, 1H), 2.00 (m, 1H), 1.80±1.20
(m, 8H), 1.00±0.70 (m, 15H). MS m/e 374 (M+H).
Anal. (C23H35NO3.0.4 H2O) C, H, N.
(R)-2-Isobutyl-4-oxo-(S)-5-phenylheptanoic acid ((S)-1-
hydroxymethyl-3-methyl)butyl amide (6b). White gum;
Rf (5% methanol in methylene chloride) 0.36; H NMR
1

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
517
(R)-2-Isobutyl-4-oxo-5,5-biphenylpentanoic acid ((S)-1-
formyl-3-methyl)butyl amide (7c). White solid, mp 85±
95 ꢀC (softening to melt); Rf (30% ethyl acetate in hex-
(broad, 1H), 6.35±6.20 (m, 1H), 5.35±5.20 (m, 1H),
5.20±5.00 (m, 1H), 3.60±3.50 (m, 1H), 3.40±3.20 (m,
3H), 2.90±2.70 (m, 2H), 2.40 (dt, 1H), 2.10±1.90 (m,
2H), 1.80±1.60 (m, 2H), 1.50±1.30 (m, 1H), 1.25±1.10
(m, 3H), 1.00±0.70 (m, 12H). Slower moving isomer:
white solid, mp 115±130 ꢀC (softening to melt); Rf (5%
methanol in methylene chloride) 0.35; 1H NMR
(CDCl3) d 7.40±7.10 (m, 5H), 7.00±6.80 (broad, 1H),
6.35±6.20 (m, 1H), 5.35±5.20 (m, 1H), 5.20±5.00 (m,
1H), 3.60±3.50 (m, 1H), 3.40±3.20 (m, 3H), 2.90±2.70
(m, 2H), 2.40 (dt, 1H), 2.10±1.90 (m, 2H), 1.80±1.60 (m,
2H), 1.50±1.30 (m, 1H), 1.25±1.10 (m, 3H), 1.00±0.70
(m, 12H).
1
anes): 0.45; H NMR (CDCl3) d 9.55 (s, 1H), 7.40±7.10
(m, 10H), 6.10 (d, 1H), 5.10 (s, 1H), 4.50 (m, 1H), 3.00
(q, 1H), 2.90±2.70 (m, 1H), 2.60 (dd, 1H), 1.80±1.00 (m,
6H), 1.00±0.80 (m, 12H). MS m/e 422 (M+H). Anal.
(C27H35NO3.0.5H2O) C, H, N.
(R)-2-Isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-
1-formyl-3-methyl)butyl amide (7d). White solid, mp
115±125 ꢀC (softening to melt); Rf (30% ethyl acetate in
1
hexanes): 0.45; H NMR (CDCl3) d 9.50 (s, 1H), 7.40±
7.00 (m, 8H), 6.00 (d, 1H), 4.90 (s, 1H), 4.40 (m, 1H),
2.80±2.65 (q, 1H), 2.60 (m, 1H), 2.30 (dd, 1H), 1.80±1.20
(m, 6H), 1.00-0.60 (4 sets of doublets, 12H). MS m/e 435
(M+H). Anal. (C27H33NO4.0.5H2O) C, H, N.
N-((R)-2-Isobutyl-4-oxo-4-(9-xanthenyl))butanoyl-3-(S)-
amino-2-(R,S)-hydroxy-5-methylhexanoic acid ethyl amide
(9c). Mixture of diasteromers (at the hydroxy center):
white solid, mp 196±201 ꢀC (softening to melt); Rf (5%
methanol in methylene chloride) 0.47; 1H NMR
(CDCl3) d 7.40±7.00 (m, 8H), 6.95±6.85 (broad, 1H),
6.05 (d, 1H), 5.35 (d, 1H), 4.90 (s, 1H), 4.05 (d, 1H),
4.00±3.90 (m, 1H), 3.40±3.20 (m, 1H), 3.20±3.10 (m,
1H), 2.65±2.60 (dd, 1H), 2.60±2.45 (m, 1H), 2.30±2.20
(dd, 1H), 1.90±1.70 (m, 2H), 1.70±1.50 (m, 1H), 1.45±
1.20 (m, 3H), 1.15 (t, 3H), 0.95 (t, 3H), 0.85 (t, 3H), 0.80
(t, 3H), 0.70 (t, 3H).
Synthesis of 9a±c: General procedure. To a cooled
( 20 ꢀC) solution of 5a±b,d (0.50±1.00 mmol) in anhy-
drous tetrahydrofuran (3±4 mL) was added N-methyl-
morpholine (3.3 equiv) followed by isobutyl
chloroformate (1.1 equiv). The mixture was stirred for
15 min by which time the temperature rose to 0 ꢀC. A
solution of 8a,b (1 equiv) in anhydrous N,N-dimethyl-
formamide (3±4 mL) was added to the reaction mixture.
The cooling bath was removed and the mixture was
stirred for another 2 h. It was then poured into water (5±
8 mL) and extracted into ethyl acetate (3Â15 mL). The
organic layer was washed with 2% citric acid solution
(2Â5 mL), saturated sodium bicarbonate solution
(2Â5 mL), brine (1Â5 mL) and dried over anhydrous
sodium sulfate. Solvent evaporation under reduced
pressure gave a crude material that was puri®ed by
¯ash chromatography (silica gel, eluant: 2±3% metha-
nol-methylene chloride) to produce 9a±c in 25±30%
yield. The coupling reaction was also successful in the
presence of BOP/HOBt/NMM, as described previously.
Synthesis of 10a±c: General procedure. To a cooled
(0 ꢀC) solution of 9a±c (0.05±0.10 mmol) in anhydrous
methylene chloride (2±3 mL) was added Dess±Martin
periodinane reagent (3±4 equiv). The cooling bath was
removed and the mixture was stirred for an additional
30±45 min. It was then diluted with methylene chloride
(10±15 mL) and washed with 10% sodium thiosulfate
solution (5Â5 mL), saturated sodium bicarbonate solu-
tion (2Â5 mL) and brine (1Â5 mL). Drying over anhy-
drous sodium sulfate and solvent removal under
reduced pressure gave a material that was washed with
n-pentane (5±8 mL) and recrystallized (ethyl acetate-
hexanes) to generate the desired targets 10a±c in 50±
60% yield.
N-((R)-2-Isobutyl-4-oxo-8-phenyl)octanoyl-3-(S)-amino-2-
(R,S)-hydroxypentanoic acid ethyl amide (9a). Mixture
of diastereomers (at the hydroxy centre); white solid, mp
97±99 ꢀC; Rf (5% methanol in methylene chloride) 0.47;
1H NMR (CDCl3) d 7.30±7.10 (m, 5H), 6.95±6.80
(broad, 1H), 6.30 (d, 1H), 5.80±5.50 (broad, 1H), 4.15
(s, 1H), 3.90±3.80 (m, 1H), 3.40±3.10 (m, 2H), 2.90±2.70
(m, 2H), 2.65±2.55 (broad, 2H), 2.45±2.35 (m, 3H),
2.00±1.70 (m, 2H), 1.65±1.40 (m, 7H), 1.20±1.00 (m,
3H), 0.95-0.75 (m, 9H).
N-((R)-2-Isobutyl-4-oxo-8-phenyl)octanoyl-3-(S)-amino-
2-oxopentanoic acid ethyl amide (10a). White solid, mp
123±124 ꢀC; Rf (5% methanol in methylene chloride)
1
0.50; H NMR (CDCl3) d 7.30±7.10 (m, 5H), 6.95±6.80
(broad, 1H), 6.30 (d, 1H), 5.30±5.10 (m, 1H), 3.40±3.30
(m, 2H), 2.90±2.70 (m, 2H), 2.65±2.55 (broad, 2H),
2.45±2.35 (m, 3H), 2.10±1.90 (m, 1H), 1.75±1.40 (m,
8H), 1.30±1.00 (m, 3H), 0.95±0.75 (m, 9H). MS m/e 431
(M+H), 453 (M+Na). Anal. (C25H38N2O4.0.25 H2O)
C, H, N.
N-((R)-2-Isobutyl-4-oxo-(S)-5-phenyl)heptanoyl-3-(S)-
amino-2-(R,S)-hydroxypentanoic acid ethyl amide (9b).
Diastereomers (at the hydroxy centre) were separated;
faster moving isomer: white solid, mp 150±160 ꢀC (soft-
ening to melt); Rf (5% methanol in methylene chloride)
N-((R)-2-Isobutyl-4-oxo-(S)-5-phenyl)heptanoyl-3-(S)-
amino-2-oxopentanoic acid ethyl amide (10b). White
solid, mp 105±115 ꢀC (softening to melt); Rf (5%
1
0.37; H NMR (CDCl3) d 7.40±7.10 (m, 5H), 7.00±6.80

518
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
methanol in methylene chloride) 0.53; 1H NMR
(CDCl3) d 7.40±7.10 (m, 5H), 7.00±6.80 (broad, 1H),
6.35±6.20 (m, 1H), 5.35±5.20 (m, 1H), 5.20±5.00 (m,
1H), 3.60±3.50 (m, 1H), 3.40±3.20 (m, 3H), 2.90±2.70
(m, 2H), 2.40 (dt, 1H), 2.10±1.90 (m, 2H), 1.80±1.60 (m,
2H), 1.50±1.30 (m, 1H), 1.25±1.10 (m, 3H), 1.00±0.70
(m, 12H). MS m/e 417 (M+H), 439 (M+Na). Anal.
(C24H36N2O4.1.25 H2O) C, H; N: calcd, 6.38; found, 7.22.
tion (5Â5 mL), saturated sodium bicarbonate solution
(2Â5 mL) and brine (1Â5 mL). Drying over anhydrous
sodium sulfate and solvent removal under reduced
pressure gave a material that was washed with n-pen-
tane (10 mL) and dried under vacuum to generate
0.020 g (74%) of 13 (diastereomeric mixture) as a white
1
solid, Rf (30% ethyl acetate in hexanes): 0.40; H NMR
(CDCl3) d 7.30±6.95 (m, 13H), 6.05±5.85 (2 sets of
doublet, 1H), 5.00±4.45 (m, 4H), 3.10±2.80 (m, 3H),
2.60±2.15 (m, 3H), 1.30±1.10 (m, 1H), 1.00±0.75 (m,
2H), 0.70±0.50 (m, 6H). MS m/e 502 (M+H), 524
(M+Na). Anal. (C31H32FNO4.0.5H2O) C, H, N.
N-(R)-2-Isobutyl-4-oxo-4-(9-xanthenyl))butanoyl-3-(S)-
amino-5-methyl-2-oxohexanoic acid ethyl amide (10c).
White solid, mp 182±183 ꢀC; Rf (5% methanol in
methylene chloride) 0.70; 1H NMR (CDCl3) d 7.40±7.00
(m, 8H), 6.85±6.75 (broad, 1H), 6.15 (d, 1H), 5.25±5.15
(m, 1H), 4.90 (s, 1H), 3.40±3.25 (m, 2H), 2.75±2.65 (dd,
1H), 2.60±2.50 (m, 1H), 2.40±2.25 (dd, 1H), 2.00 (m,
1H), 1.80±1.60 (m, 4H), 1.45±1.20 (m, 2H), 1.15 (t, 3H),
0.90 (t, 6H), 0.80 (t, 3H), 0.70 (t, 3H). MS m/e 507
(M+H), 529 (M+Na). Anal. (C30H38N2O5) C, H, N.
Benzyl isocaproate (15). A mixture of isocaproic acid
(14, 12.31 g, 0.106 mol), benzyl alcohol (10.88 g,
0.10 mol) and p-toluenesulfonic acid monohydrate
(1.00 g) in dry benzene (100 mL) was re¯uxed using a
Dean±Stark water separator for 2 h. After cooling, ben-
zene was removed and the mixture was diluted with
ether (50 mL) and washed successively with saturated
NaHCO3 solution (2Â20 mL), brine (1Â20 mL), dried
(MgSO4) and solvent evaporated to give 15 (20.00 g,
97%) as a colorless oil; Rf (5% ethyl acetate in hexanes) :
0.46; 1H NMR (CDCl3) d 7.40±7.20 (m, 5H), 5.10 (s,
2H), 2.35 (t, 2H), 1.55 (m, 3H), 0.90 (d, 6H).
(R)-2-Isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid (R,S)-
1-benzyl-3-¯uoro-(R,S)-2-hydroxypropyl amide (12). To
a cooled ( 20 ꢀC) solution of 5d (0.34 g, 1.00 mmol) in
anhydrous methylene chloride (4 mL) was added N-
methylmorpholine (0.212 g, 2.10 mmol) followed by iso-
butyl chloroformate (0.143 g, 1.05 mmol). The mixture
was stirred for 15 min by which time the temperature
rose to 0 ꢀC. A solution of 11 (0.184 g, 1.00 mmol) in
anhydrous methylene chloride (6 mL) was added to the
reaction mixture. The cooling bath was removed and the
mixture was stirred for another 2 h, poured into water
(8 mL) and extracted into ethyl acetate (3Â15 mL). The
organic layer was washed with 2% citric acid solution
(2Â5 mL), saturated sodium bicarbonate solution
(2Â5 mL), brine (1Â5 mL) and dried over anhydrous
sodium sulfate. Solvent evaporation under reduced
pressure gave a crude material that was puri®ed by
¯ash silica gel column chromatography (eluant: 2%
methanol in methylene chloride) to produce 0.14 g
(27%) of 12 (diastereomeric mixture) as a white solid.
The coupling reaction was also successful in the pre-
sence of BOP/HOBt/NMM, as described previously; Rf
(5% methanol in methylene chloride) 0.47; 1H NMR
(CDCl3) d 7.40±6.95 (m, 13H), 5.85±5.65 (2 sets of
doublet, 1H), 4.80 (d, 1H), 4.60±4.20 (m, 3H), 4.10±3.75
(m, 2H), 3.10±2.60 (m, 3H), 2.50±2.15 (m, 2H), 1.30±
1.10 (m, 1H), 1.00±0.75 (m, 2H), 0.70±0.50 (m, 6H).
t-Butyl 3-benzoyloxycarbonyl-5-methylhexanoate (16).
To a cooled ( 78 ꢀC) solution of lithium diisopropyla-
mide (12 mmol, obtained in situ from diisopropylamine
and n-butyllithium) in a mixture of tetrahydrofuran
(20 mL) and hexane (4.8 mL) was added slowly the
compound 15 (2.06 g, 10 mmol) in anhydrous tetra-
hydrofuran (8 mL). The mixture was stirred for 30 min
and tert-butyl bromoacetate (2.34 g, 12 mol) in hexa-
methylphosphoramide (2.09 mL) was added to the ¯ask.
The mixture was stirred at 78 ꢀC for 30 min, slowly
brought to 0 ꢀC over a period of 2 h and quenched by
the cautious addition of saturated ammonium chloride
(50 mL). The mixture was extracted into ether
(3Â50 mL) and the combined organic layer was washed
with brine (1Â25 mL), dried over anhydrous MgSO4
and concentrated to give a crude material. Puri®cation
of this material by ¯ash chromatography over silica gel
(eluant: 2% ethyl acetate in hexanes) gave 2.00 g (63%)
of racemic 16 as a colorless oil; Rf (5% ethyl acetate in
1
hexanes): 0.34; H NMR (CDCl3) d 7.40±7.30 (m, 5H),
5.15 (q, 2H), 2.90 (m, 1H), 2.60 (q, 1H), 2.35 (q, 1H),
1.60±1.20 (m, 3H), 1.40 (s, 9H), 1.00±0.80 (2 sets of
doublet, 6H).
(R)-2-Isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid (R,S)-
1-benzyl-3-¯uoro-2-oxopropyl amide (13). To a cooled
(0 ꢀC) solution of 12 (0.030 g, 0.054 mmol) in anhydrous
methylene chloride (4 mL) was added Dess±Martin per-
iodinane reagent (0.045 g, 0.108 mmol). The cooling
bath was removed and the mixture was stirred for an
additional 30 min, diluted with methylene chloride
(15 mL) and washed with 10% sodium thiosulfate solu-
3-Benzoyloxycarbonyl-5-methylhexanoic acid (17).
A
mixture of 16 (0.54 g, 1.673 mmol) and 90% tri-
¯uoroacetic acid (1.5 mL) in methylene chloride (3 mL)
was stirred at room temperature for 1.5 h. Solvent and
excess reagent were removed under reduced pressure to
give 0.44 g of crude 17. An 1H NMR (CDCl3) of this

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
519
material showed no detectable peak for the tert-butyl
ester moiety at d 1.40. This material was used without
further puri®cation.
(R/S)-2-Isobutyl-4-oxo-4-(2-(1,2,3,4-tetrahydroisoquino-
linyl))butanoic acid ((S)-(1-formyl-2-phenyl))ethyl amide
(21a,b). These compounds were synthesized following
the same synthetic procedure as described before for the
syntheses of the compounds 7a±d. Thus, oxidation of
0.10 g (0.24 mmol) of 20a generated 0.05 g (50%) of 21a
and oxidation of 0.075 g (0.177 mmol) of 20b generated
0.035 g (48%) of 21b. Compound 21a: White solid, mp
45±65 ꢀC (softening to melt); Rf (70% ethyl acetate in
Benzyl 2-isobutyl-4-oxo-4-(2-(1,2,3,4-tetrahydroisoquino-
linyl))butanoate (18). To
a stirred mixture of 17
(0.95 g, 3.60 mmol) and 1,2,3,4-tetrahydroisoquinoline
(0.48 g, 3.60 mmol) in anhydrous methylene chloride
(8 mL) at room temperature, was added triethylamine
(0.80 g, 7.92 mmol) followed by BOP-Cl (0.92 g,
3.60 mmol). The mixture was stirred overnight, diluted
with methylene chloride (10 mL) and washed succes-
sively with water (1Â5 mL), saturated NaHCO3
(2Â5 mL) and brine (1Â5 mL). Drying over Na2SO4 and
solvent evaporation gave a crude material that was
puri®ed by ¯ash chromatography over silica (eluant:
20% ethyl acetate in hexanes) to give 0.89 g (65%) of 18,
as a colorless oil; Rf (20% ethyl acetate in hexane): 0.70;
1H NMR (CDCl3) d 7.40±7.00 (m, 9H), 5.10 (q, 2H),
4.70 (q, 2H), 3.90±3.60 (m, 2H), 3.20±3.00 (m, 1H),
2.90±2.70 (m, 3H), 2.50±2.40 (m, 1H), 1.70±1.50 (m,
2H), 1.40±1.30 (m, 1H), 1.00±0.80 (m, 6H).
1
hexane): 0.58; H NMR (CDCl3) d 9.60 (s, 1H), 7.30±
7.10 (m, 9H), 6.75 (d, 1H), 4.70±4.50 (m, 3H), 3.90±3.50
(m, 2H), 3.20±2.60 (m, 6H), 2.40±2.30 (m, 1H), 1.80±
1.20 (m, 3H), 1.00±0.80 (m, 6H). MS m/e 421 (M+H).
Anal. (C26H32N2O3.H2O) C, H, N. Compound 21b:
1
White foam, Rf (70% ethyl acetate in hexane): 0.41; H
NMR (CDCl3) d 9.50 (d, 1H), 7.40±7.10 (m, 9H), 6.60
(t, 1H), 4.70±4.50 (m, 3H), 3.90±3.50 (m, 2H), 3.20±2.60
(m, 6H), 2.40±2.30 (m, 1H), 1.70±1.10 (m, 3H), 1.00±
0.80 (m, 6H). MS m/e 421 (M+H), 443 (M+Na). Anal.
(C26H32N2O3.2.2H2O) C, H, N.
Ethyl 4-(2-thionaphthyl)butyrate (23). To a stirred mix-
ture of NaH (60% in oil, 2.04 g, 0.05 mol) in anhydrous
THF (20 mL) at 0 ꢀC was added slowly a solution of 2-
naphthalenethiol (22, 7.79 g, 0.048 mol) in anhydrous
THF (20 mL). The mixture was stirred for 30 min. A
solution of ethyl 4-bromobutyrate (10.43 g, 0.053 mol)
in anhydrous THF (20 mL) was slowly added to the
reaction ¯ask. The mixture was stirred for another 1 h,
by which time temperature changed to room tempera-
ture. The reaction mixture was then poured into a ice-
water mixture (100 mL) and extracted into ether
(3Â100 mL). The combined organic layer was washed
with water (2Â25 mL), brine (1Â20 mL), dried (MgSO4),
and concentrated to give a crude product that was pur-
i®ed by ¯ash silica gel column chromatography (eluant:
3% ethyl acetate in hexanes) to give 12.20 g (92%) of 23
as a colorless oil; Rf (5% ethyl acetate in hexanes): 0.25;
1H NMR (CDCl3) d 7.80 (t, 4H), 7.50 (m, 3H), 4.15 (q,
2H), 3.10 (t, 2H), 2.50 (t, 2H), 2.00 (2 overlapping t,
2H).
2-Isobutyl-4-oxo-4-(2-(1,2,3,4-tetrahydroisoquinolinyl))-
butanoic acid (19). A mixture of the benzyl ester 18
(0.88 g, 2.32 mmol) and 10% Pd-C (0.30 g, Degussa,
H2O content 50%) in methanol (30 mL) was hydro-
genated for 2 h in a Parr apparatus (40±30 psi). The
reaction mixture was then ®ltered through a celite pad
and concentrated to give 0.67 g (100%) of 19 as a pale-
yellow solid, mp 95±105 ꢀC (softening to melt); 1H
NMR (CDCl3) d 7.40±7.00 (m, 4H), 5.00±4.80 (q, 2H),
4.00±3.60 (m, 2H), 3.20±3.00 (m, 1H), 2.90±2.70 (m,
3H), 2.60±2.40 (m, 1H), 1.80±1.60 (m, 2H), 1.40±1.30
(m, 1H), 1.00±0.80 (m, 6H).
(R/S)-2-Isobutyl-4-oxo-4-(2-(1,2,3,4-tetrahydroisoquino-
linyl))butanoic acid ((S)-(1-hydroxymethyl-2-phenyl))ethyl
amides (20a,b). These compounds were generated fol-
lowing the same synthetic procedure as described before
for the syntheses of the compounds 6a±d. Thus, the
coupling between 0.26 g (0.885 mmol) of 19 and 0.17 g of
(S)-phenylalaninol gave 20a,b, that were separated by
¯ash silica gel column chromatography (eluant: ethyl
acetate) to give 0.11 g (28%) of 20a and 0.10 g (27%) of
20b, respectively. Compound 20a: White foam, Rf (ethyl
Ethyl 2-isobutyl-4-(2-thionaphthyl)butyrate (24). To a
cooled ( 78 ꢀC) solution of lithium diisopropylamide
(0.053 mol, prepared in situ from corresponding nBuLi
and diisopropylamine) in THF (40 mL) hexane
(21 mL) was added slowly a solution of compound 23
(12.13 g, 0.044 mol) in anhydrous THF (20 mL). The
mixture was stirred for 1 h and to it was added a solu-
tion of 1-iodo-2-methylpropane (9.76 g, 0.053 mol) in
hexamethylphosphoramide (9.23 mL). The mixture was
stirred for another 3 h, by which time the temperature
slowly changed to room temperature. The reaction
mixture was quenched with saturated NH4Cl (50 mL),
diluted with water (50 mL) and extracted into ether
(3Â100 mL). The combined organic layer was washed
1
acetate): 0.62; H NMR (CDCl3) d 7.30±7.10 (m, 9H),
6.60 (d, 1H), 4.70±4.50 (q, 2H), 4.50 (s, 1H), 4.00 (m,
1H), 3.90±3.50 (m, 4H), 3.00±2.60 (m, 6H), 2.40±2.30
(m, 1H), 1.70±1.60 (m, 1H), 1.60±1.40 (m, 1H), 1.20 (m,
1H), 1.00±0.80 (m, 6H). Compound 20b: White foam; Rf
1
(ethyl acetate): 0.44; H NMR (CDCl3) d 7.30±7.00 (m,
9H), 6.00 (m, 1H), 4.80±4.50 (q, 2H), 4.60 (s, 1H), 4.30
(m, 1H), 3.90±3.40 (m, 4H), 3.00±2.60 (m, 6H), 2.40±
2.20 (m, 1H), 1.60±1.50 (m, 1H), 1.30±1.20 (m, 1H), 1.00
(m, 1H), 0.80±0.60 (m, 6H).

520
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
with brine (1Â30 mL), dried (MgSO4), and concentrated
to give a crude product that was puri®ed by ¯ash silica
gel column chromatography (eluant: 2% ethyl acetate in
hexanes) to give 4.60 g (32%) of 24 as a colorless oil; Rf
(5% ethyl acetate in hexanes): 0.37; 1H NMR (CDCl3) d
7.80 (t, 4H), 7.50 (m, 3H), 4.15 (q, 2H), 3.00 (m, 2H),
2.65 (m, 1H), 2.00 (m, 1H), 1.80 (m, 1H), 1.60 (m, 3H),
1.25 (t, 3H), 0.90 (t, 6H).
tion generated 0.070 g (100%) of compound 28, that
was used without any further puri®cation. In a similar
way, 0.065 g of 27 was converted to 0.070 g (100%) of
29. Compound 28: White solid, mp 112±114 ꢀC; Rf
1
(60% ethyl acetate in hexanes): 0.35; H NMR (CDCl3)
d 8.50 (s, 1H), 8.00 (m, 3H), 7.85 (d, 1H), 7.65 (m, 2H),
6.05 (d, 1H), 4.15 (m, 1H), 3.80 (dd, 1H), 3.50 (m, 1H),
3.40 (m, 1H), 3.20 (m, 1H), 2.80 (m, 2H), 2.00 (m, 2H),
1.60-1.40 (m, 5H), 1.20 (m, 1H), 0.90 (m, 12H). Com-
pound 29: White solid, mp 112±115 ꢀC; Rf (60% ethyl
2-Isobutyl-4-(2-thionaphthyl)butyric acid (25). A mixture
of compound 24 (4.53 g, 0.014 mol), lithium hydroxide
monohydrate (2.30 g, 0.055 mol), ethanol (60 mL) and
water (15 mL) was gently re¯uxed for 3 h. Ethanol was
removed in vacuo and the basic aqueous layer was
washed with ether (2Â20 mL), acidi®ed with 5 N HCl
and extracted into ethyl acetate (3Â50 mL). The com-
bined organic layer was washed with brine (1Â20 mL),
dried (MgSO4), and concentrated to give 3.80 g (92%)
of 25 as a white solid, mp 76±78 ꢀC; 1H NMR (CDCl3) d
7.80 (t, 4H), 7.40 (m, 3H), 3.00 (m, 2H), 2.70 (m, 1H),
2.00 (m, 1H), 1.80 (m, 1H), 1.60 (m, 2H), 1.25 (m, 1H),
0.90 (t, 6H).
1
acetate in hexanes): 0.32; H NMR (CDCl3) d 8.50 (s,
1H), 8.00 (m, 3H), 7.85 (d, 1H), 7.70 (m, 2H), 6.00 (d,
1H), 4.05 (m, 1H), 3.65 (m, 1H), 3.55 (m, 1H), 3.20 (m,
2H), 2.65 (m, 2H), 2.00 (q, 2H), 1.60 (m, 3H), 1.40 (m,
2H), 1.20 (m, 1H), 0.90 (m, 12H).
(S)/(R)-2-Isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)-
1-formyl-3-methyl)butyl amides (30,31). These com-
pounds were synthesized following the synthetic proce-
dure as described above for the syntheses of the
compounds 7a±d. Thus, the oxidation of 28 (0.063 g,
0.145 mmol) gave 0.040 g (60%) of compound 30. In a
similar way, 0.063 g of 29 was converted to 0.034 g
(54%) of 31. Compound 30: White solid, mp 104±109 ꢀC
(softening to melt); Rf (70% ethyl acetate in hexanes):
0.87; 1H NMR (CDCl3) d 9.60 (s, 1H), 8.55 (s, 1H), 8.00
(m, 4H), 7.70 (m, 2H), 6.40 (d, 1H), 4.65 (m, 1H), 3.65
(m, 1H), 3.20 (m, 1H), 2.80 (m, 1H), 2.10 (m, 1H), 1.90
(m, 1H), 1.60±1.40 (m, 4H), 1.20 (m, 1H), 0.90 (m, 12H).
MS m/e 432 (M+H). Anal. (C24H33NO4S) C, H, N.
Compound 31: White foam, Rf (70% ethyl acetate in
hexanes): 0.83; 1H NMR (CDCl3) d 9.60 (s, 1H), 8.45 (s,
1H), 8.00 (m, 3H), 7.85 (d, 1H), 7.70 (m, 2H), 6.20 (d, 1H),
4.50 (m, 1H), 3.20 (m, 2H), 2.70 (m, 1H), 2.00 (q, 2H),
1.80±1.40 (m, 5H), 1.20 (m, 1H), 0.90 (m, 12H). MS
m/e 432 (M+H). Anal. (C24H33NO4S.0.5 H2O) C, H,
N.
(S)/(R)-2-Isobutyl-4-(2-thionaphthyl)butyric acid ((S)-1-
hydroxymethyl-3-methyl)butyl amide (26,27). These
compounds were generated following the synthetic pro-
cedure as described above for the syntheses of the com-
pounds 6a±d. Thus, the coupling between 25 (3.35 g,
0.011 mol) and (S)-leucinol (1.70 g, 0.0143 mol), in the
presence of BOP/HOBt/NMM, gave a mixture of com-
pounds that were separated by column chromato-
graphy (silica gel, 30% ethyl acetate in hexanes) to
produce 0.95 g (21%) of 26 and 1.10 g (25%) of 27,
respectively. Compound 26: White solid, mp 96±98 ꢀC;
Rf (50% ethyl acetate in hexanes): 0.51; 1H NMR
(CDCl3) d 7.80 (t, 4H), 7.45 (m, 3H), 5.65 (d, 1H), 4.05
(m, 1H), 3.60 (m, 1H), 3.40 (m, 1H), 3.15 (m, 1H), 3.00
(m, 1H), 2.80 (b, 1H), 2.50 (m, 1H), 2.00 (m, 1H), 1.80±
1.10 (m, 7H), 0.90 (m, 12H). Compound 27: White
solid, mp 85±90 ꢀC; Rf (50% ethyl acetate in hexanes):
(R)-2-Isobutyl-4-(2-thionaphthyl)butyric acid ((S)-1-formyl-
3-methyl)butyl amide (32). This compound was pre-
pared from compound 27, following the same procedure
as described above for the synthesis of 7a±d. Thus,
0.23 g of 27 was oxidized to generate 0.12 g (53%) of 32
as a white solid, mp 75±76 ꢀC; Rf (70% ethyl acetate in
1
0.40; H NMR (CDCl3) d 7.80 (m, 4H), 7.45 (m, 3H),
5.70 (d, 1H), 4.05 (m, 1H), 3.65 (m, 1H), 3.55 (m, 1H),
3.15 (m, 1H), 2.90 (m, 2H), 2.50 (m, 1H), 2.00 (m, 1H),
1.80-1.40 (m, 4H), 1.40±1.10 (m, 3H), 0.90 (t, 6H), 0.80
(t, 6H).
1
hexanes): 0.85; H NMR (CDCl3) d 9.60 (s, 1H), 7.80
(m, 4H), 7.45 (m, 3H), 5.90 (d, 1H), 4.60 (m, 1H), 3.20
(m, 1H), 2.90 (m, 1H), 2.60 (m, 1H), 2.00 (m, 1H), 1.80±
1.40 (m, 5H), 1.40 (m, 1H), 1.20 (m, 1H), 0.90 (m, 12H).
MS m/e 400 (M+H), 422 (M+Na). Anal. (C24H33
NO2S) C, H, N.
(S)/(R)-2-Isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)-
1-hydroxymethyl-3-methyl)butyl amide (28,29). To a
solution of compound 26 (0.065 g, 0.1618 mmol) in
methylene chloride (3 mL) at 0 ꢀC was added m-chlor-
operbenzoic acid (95%, 0.062 g, 0.356 mmol) in methyl-
ene chloride (2 mL). The cooling bath was removed, the
mixture was stirred for another 30 min and washed suc-
cessively with 5% sodium thiosul®te solution (2Â5 mL),
water (1Â5 mL), 3% NaHCO3 solution (2Â5 mL), and
brine (1Â5 mL). Drying (Na2SO4) and solvent evapora-
(R)-2-Isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-
hydroxymethyl-3-methyl)butyl amide (33). To a stirred
solution of 27 (0.23 g, 0.573 mmol) in methylene chloride
(4 mL) at room temperature was added a solution of
Davis' oxaziridine (0.17 g, 0.58 mmol). The mixture was

S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
521
stirred for another 30 min and concentrated in vacuo to
give a crude product. It was puri®ed by silica gel column
chromatography (eluant: 10% ethyl acetate in hexanes
followed by 50% ethyl acetate in hexanes) to give 0.21 g
(86%) of 33 (diastereomeric mixture at the sulfoxide
center), as a white solid, mp 138±140 ꢀC; Rf (ethyl acet-
ate): 0.51; 1H NMR (CDCl3) d 8.20 (s, 1H), 7.90 (m,
3H), 7.60 (m, 2H), 7.50 (m, 1H), 6.20 (q, 1H), 4.10 (m,
1H), 3.70 (m, 1H), 3.55 (m, 1H), 3.20 (q, 1H), 3.10±2.80
(m, 2H), 2.60 (m, 1H), 2.10±1.00 (a series of m, 8H),
0.90 (m, 12H).
where y is the ¯uorescence at time t (Ft), A is the
amplitude of the reaction (F0 F ), and B is the max-
1
imal amount of product formed when the enzyme is
completely inactivated (F ). The apparent second-order
1
rate constant for inactivation was calculated from the
slope of a plot of kobs versus inhibitor concentration as
(kobs/I)* (1+S/Km), correcting for the e€ect of substrate
on the inactivation rate.
Intact cell assay
Molt-4 cells (human leukemic T cells) were suspended in
HEPES Bu€ered Saline (HBS, 20 mM HEPES, pH 7.2±
7.5, 5.4 mM KCl, 120 mM NaCl, 25 mM glucose,
1.5 mM MgSO4, 1 mM Na-pyruvate) at 2Â107 cells per
mL, 50 mL of which was added to the wells of a 96-well
plate. To this, 50 mL of inhibitor solution, at twice the
®nal desired concentration, in HBS was added. Follow-
ing a 10 min incubation at 37 ꢀC, on a nutator (Clay
Adams1 Brand), 100 mL of HBS containing the ®nal
desired inhibitor concentration, 10 mM Ca+2 and
40 mM Ionomycin was added. The suspension was
incubated further for 30 min at 37 ꢀC on a nutator, after
which cells were harvested by centrifugation. The cell
pellet was then solubilized by the addition of lysis bu€er
containing 20 mM Tris (pH 8.2), 137 mM NaCl, 13 mM
EDTA and 1% Triton X-100, 10 mg/mL leupeptin,
10 mg/mL aprotinin, 10 mg/mL pepstatin A and 1 mM
Pefabloc. After a 30±60 min incubation on ice, with
periodic vortexing, insoluble material was removed by
centrifugation and the supernatant collected. 10% SDS
was added to yield a ®nal concentration of 1% and the
samples heated for 15 min at 65 ꢀC. Supernatant protein
concentrations were determined by the BCA assay
(Pierce, Inc., Rockford, IL, USA). Samples (equal total
protein) were resolved on 6% SDS±PAGE, transferred
to nitrocellulose and analyzed by immunoblot using a
polyclonal antibody speci®c for calpain I-mediated spectrin
breakdown products (SBDPs), followed by an alkaline
phosphatase conjugated goat anti-rabbit antibody
(BIORAD, Inc., Hercules, CA, USA) and an alkaline phos-
phatase 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, USA).
(R)-2-Isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-
formyl-3-methyl)butyl amide (34). This compound was
prepared from compound 33, following the synthetic
procedure as described above for the synthesis of 7a±d.
Thus 0.160 g of 33 was oxidized to 0.060 g (38%) of 34
(diastereomeric mixture at the sulfoxide center), as a
white solid, mp 134±144 ꢀC (softening to melt); Rf (70%
ethyl acetate in hexanes): 0.49; 1H NMR (300 MHz,
CDCl3) d 9.55 (d, 1H), 8.20 (d, 1H), 7.95 (m, 3H), 7.60
(m, 3H), 6.50 and 6.40 (2 sets of d, 1H), 4.50 (m, 1H),
3.10±2.80 (m, 2H), 2.70 (m, 1H), 2.10 (m, 1H), 1.90±1.40
(m, 6H), 1.25 and 1.10 (2 sets of m, 1H), 0.90 (m, 12H).
MS m/e 416 (M+H), 438 (M+Na). Anal.
(C24H33NO3S.0.1 H2O) C, H, N.
In vitro calpain assay
For reversible inhibitors, the reaction mixture contained
50 mM Tris HCl (pH 7.5), 50 mM NaCl, 0.2 mM Suc-
Leu-Tyr-MNA (Enyme Systems Products, Dublin, CA),
1 mM EDTA, 1 mM EGTA, 5 mM b-mercaptoethanol,
10 nM recombinant human calpain I, varying con-
centrations of inhibitor and 5 mM CaCl2 in a ®nal
volume of 200 mL in a polystyrene microtiter plate.
Assays were initiated by addition of CaCl2 and the
increase in ¯uorescence (lex=340 nm, lem=430nm) was
monitored at ambient temperature using a Fluoroskan
II ¯uorescence plate reader. Values of IC50 s were cal-
culated from velocities determined from the linear por-
tion of reaction progress curves. For irreversible
inhibitors, reactions were performed at ambient tem-
perature in single cuvettes with the increase in ¯uores-
cence
continuously on
(lex=340 nm,
l
Perkin±Elmer LS50B spectro-
em=425 nm)
recorded
a
¯uorimeter (Norwalk, CT, USA) and were monitored
until there was no further product generated in inhi-
bitor-containing assays. Inhibitor concentrations were
at least 10-fold greater than the enzyme concentration.
Value of kobs, the pseudo ®rst-order rate constant for
inactivation, were calculated from plots of ¯uorescence
vs. time by non-linear regression (Sigma Plot) to the
exponential eq (1).14
Acknowledgements
The authors would like to thank Dr Je€ry Vaught for
his continuing support and encouragement. We also
thank Drs Ron Bihovsky, Mark A. Ator, Radindranath
Tripathy, Ming Tao and Gregory Wells for many help-
ful discussions. We are pleased to acknowledge
SmithKline Beecham Pharmaceuticals for partial sup-
port of this research.
y ˆ Aeꢀ
‡ B
ꢀ1†
kobsÃt†

522
S. Chatterjee et al./Bioorg. Med. Chem. 6 (1998) 509±522
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