Synthesis and calpain inhibitory activity of α-ketoamides with 2,3-methanoleucine stereoisomers at the P2 position

Article abstract of DOI:10.1016/S0960-894X(00)00518-7

A series of novel ketoamides incorporating all four 2,3-methanoleucine stereoisomers at the P₂ position was synthesized. The compounds displayed a wide variation in K(i) values for inhibition of calpain I depending on the configuration of the P

Full text of DOI:10.1016/S0960-894X(00)00518-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2497±2500
Synthesis and Calpain Inhibitory Activity of ꢀ-Ketoamides with
2,3-Methanoleucine Stereoisomers at the P2 Position
Isaac O. Donkor,* Xiaozhang Zheng and Duane D. Miller
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
Received 3 August 2000; accepted 28 August 2000
AbstractÐA series of novel ketoamides incorporating all four 2,3-methanoleucine stereoisomers at the P2 position was synthesized.
The compounds displayed a wide variation in Ki values for inhibition of calpain I depending on the con®guration of the P2
methanoleucine residue. However, similar variation in cathepsin B inhibition was not observed suggesting that the S2 pocket of
calpain I is more stereosensitive than that of cathepsin B. # 2000 Published by Elsevier Science Ltd.
Introduction
interest in developing novel active site directed selective
inhibitors of calpain led us to study the e€ect of incor-
porating 2,3-methanoleucine units at the P2 position of
calpain inhibitors on the potency and selectivity of the
inhibitors. 2,3-Methanoamino acids are structurally
constrained and have only two possible side-chain rota-
tory angles, w=0ꢀ and w=120ꢀ corresponding to the Z-
and E-con®gurations, respectively. Incorporation of
such amino acids into a peptide e€ectively constrains
the proximal conformation of the peptide and also
makes the peptide resistant to proteolytic cleavage. In
this report we describe the synthesis and calpain I inhi-
bitory activity of a-ketoamides (1±4) incorporating 2,3-
methanoleucine stereoisomers (6±9) at the P2 position of
the inhibitors.
Calpain is a cytosolic calcium-activated neutral protease
that belongs to the papain superfamily of cysteine pro-
teases.1 Two major isoforms of calpain (calpains I and
II), which di€er in their calcium sensitivity under in
vitro conditions, are known.2 Excessive activation of
intracellular calpain occurs as a result of increased
intracellular calcium concentrations associated with
ischemic events.3 Calpain activation under ischemic
conditions may result in degradation of the structural
proteins. The enzyme has therefore been implicated in a
number of pathological conditions including neurologi-
cal disorders (e.g., stroke), cataract, cardiac ischemia,
and thrombotic platelet aggregation.1,3 5 The potential
involvement of calpain in a variety of disease states has
fueled the search for selective cell permeable calpain
inhibitors as biomedical tools for studying the cellular
role of calpain and as potential therapeutic agents for
treating conditions such stroke where considerable
evidence links overactivation of calpain to cellular
damage.6,7 Calpain inhibitors may be broadly grouped
into active site directed inhibitors (or domain II binding
inhibitors) and allosteric inhibitors (or domain IV
binding inhibitors). Several calpain inhibitors that inac-
tivate the enzyme by binding to the active site have been
reported.8 However, several of these inhibitors are not
selective for calpain since they also inhibit other cysteine
proteases such as cathepsin B and cathepsin L. Our
Synthesis of the P2-modi®ed analogues 1±4 required all
four 2,3-methanoleucine stereoisomers 6±9 (Fig. 1).
Others and us have previously reported the asymmetric
synthesis and stereochemical purity of the stereo-
isomers.9,10 Compounds 1±5 were synthesized as out-
lined in Scheme 1. Boc-protected phenylalanine 10
was coupled with O,N-dimethylhydroxylamine hydro-
chloride using ethylcarbodiimide (EDC) as the coupling
agent in the presence of N-methyl morpholine (NMM)
to give Weinreb amide 11, which was reduced with LAH
to give Boc-protected amino aldehyde 12 as previously
reported.11 Aldehyde 12 was transformed to cyano-
hydrin 13, the nitrile group of which was hydrolyzed to
give a-hydroxy-b-amino acid 14. Boc protection of
the amino group of 14 followed by coupling with
phenethylamine and subsequent deprotection gave a-
hydroxy-b-amino amide 17. Coupling of 17 with either
*Corresponding author. Tel.: +1-901-448-7736; fax: +1-901-448-
6828; e-mail: idonkor@utmem.edu
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00518-7

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I. O. Donkor et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2497±2500
Cbz-protected l-leucine or Cbz-protected 2,3-methano-
leucine derivatives 6±9 followed by Dess±Martin oxi-
dation of the secondary alcohol gave the corresponding
a-ketoamides 1±5.12
2,3-methanoleucine stereoisomers had pronounced
e€ects on inhibition of calpain I compared to cathepsin
B. By analyses of Dixon plots,15 the inhibition was
found to be competitive with Ki values for inhibition of
calpain I ranging from 0.75 to 40 mM. The Ki values for
inhibition of cathepsin B on the other hand did not
show a similar wide variation (Ki=1.26±6.78 mM) sug-
gesting that the S2 pocket of cathepsin B did not dis-
criminate between the stereoisomers. Additionally, the
E-isomers (3 and 4) were better inhibitors of calpain I
than the Z-isomers (1 and 2). Indeed, 3 with an E-
(2S,3S) con®guration at the P2-position was over 50-
fold a better inhibitor of calpain I than 2 with a P2 Z-
(2R,3S) con®guration. On the contrary, with respect to
cathepsin B inhibition, no such dramatic con®gurational
Results and Discussion
The compounds were evaluated as inhibitors of calpain
I from porcine erythrocytes and cathepsin B from
human liver.13 Table 1 shows the results of the study.
Compound 5 was previously reported by Li et al.14
as
a potent a-ketoamide inhibitor of calpain I
(Ki=0.052 mM). In our assay system 5 inhibited calpain
I with a Ki value of 0.07 mM. The con®guration of the
Figure 1. Structures of target compounds 1±5 and 2,3-methanoleucine stereoisomers 6±9 from which they were derived.

I. O. Donkor et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2497±2500
2499
Scheme 1. Reagents: (a) CH3NHOCH3/EDC/NMM. (b) LiAlH4. (c) NaHSO3. (d) NaCN. (e) HCl/Dioxane. (f) (t-BuOCO)2O/NaOH. (g)
PhCH2CH2NH2/EDC/HOBt. (h) HCl/Dioxane. (i) X/EDC/HOBt (X=Cbz-l-leucine, 6, 7, 8, or 9). (j) Dess±Martin reagent.
Acknowledgements
Table 1. Inhibition of porcine erythrocyte calpain I and human liver
cathepsin B by compounds 1±5a
The study was supported in part by grant 5 K14
HL03536 from the U. S. Public Health Service (IOD)
and a University of Tennessee College of Pharmacy
Faculty Development Grant (IOD).
Ki (mM)
Compound
Calpain Ib
Cathepsin Bc
Selectivity ratiod
1 [Z-(2S,3R)]
2 [Z-(2R,3S)]
3 [E-(2S,3S)]
4 [E-(2R,3R)]
5
11.13
40.00
0.75
6.15
0.07
5.05
4.56
6.78
1.26
0.30
0.5
0.1
9.0
0.2
4.0
References and Notes
aKi values were determined by Dixon plots using the average of tripli-
cate assays and plotting 1/n versus I to give intersecting lines with
correlation coecient ꢁ0.95.
1. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
2. Croall, D. E.; Demartino, G. N. Physiol. Rev. 1991, 71,
813.
3. Wang, K. K. W.; Yuen, P. W. Trends Pharmacol. Sci. 1994,
15, 412.
b50 mM Tris HCl pH 7.4, 50 mM NaCl, 10 mM DTT, 1 mM EDTA,
1 mM EGTA, 2% DMSO.
c20 mM NaOAc pH 5.2, 0.5 mM DTT, 2% DMSO.
dSelectivity ratios were determined by dividing the Ki values for
cathepsin B inhibition by those for calpain I inhibition.
4. Saido, T.; Sorimachi, H.; Suzuki, K. FASEB J. 1994, 8,
814.
5. Wang, K. K.; Yuen, P.-W. Adv. Pharmcol. 1997, 37, 117.
6. Markgraf, C. G.; Velayo, N. L.; Johnson, M. P. Stroke
1998, 29, 152.
7. Wang, K. K.; Nath, R.; Posner, A.; Raser, K. J.; Buroker-
Kilgore, M.; Ye, Q.; Takano, E. K. S.; Lunney, E. A.; Hays, S.
J.; Yuen, P.-W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6687.
8. Wells, G. J.; Bihovsky, R. Exp. Opin. Ther. Pat. 1998, 8,
1707.
9. Donkor, I. O.; Zheng, X.; Han, J.; Miller, D. D. Chirality
2000, 12, 551.
10. Burgess, K.; Li, W. Tetrahedron Lett. 1995, 36, 2725.
11. Zheng, X.; Donkor, I. O.; Miller, D. D.; Ross, C. Chir-
ality 2000, 12, 2.
discrimination was observed. Replacement of the P2-
leucine residue of 5 with the 2,3-methanoleucine
stereoisomers resulted in at least a 10-fold decrease in
calpain I inhibitory potency. However, 3, which was the
most potent calpain I inhibitor of the series, was 9-fold
selective for calpain I over cathepsin B. Compound 5
displayed only 4-fold selectivity between the two
enzymes. Thus, despite the 10-fold decrease in potency,
incorporation of the E-(2S,3S) 2,3-methanoleucine
stereoisomer at the P2-position of 5 enhanced selectivity
for calpain I over cathepsin B.

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I. O. Donkor et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2497±2500
12. All of the novel ketoamides had satisfactory analytical
data, 1H, 13C NMR, and elemental analyses. Compound 1:
35.37, 37.24, 39.64, 40.35, 40.42, 55.95, 67.23, 126.77, 127.05,
128.09, 128.28, 128.58, 128.63, 128.69, 128.77, 129.26, 135.82,
135.95, 138.09, 156.31, 159.30, 170.26, 195.26. Anal.
Mp 194.0±194.8 ꢀC. H NMR (CDCl3) d 0.79 (s, 1H, (CH3)2-
1
(C33H37N3O5) C, H, N. Compound 4: Mp 154.0±154.9 ꢀC. H
1
CHCHC(CH2)), 0.99 (d, 3H, J=6.5 Hz, (CH3)2CHCH-
C(CH2)), 1.06 (d, 3H, J=6.5 Hz, (CH3)2CHCHC(CH2)), 1.15±
1.30 (m, 1H, (CH3)2CHCHC(CH2)), 1.43±1.52 (m, 1H, (CH3)2-
CHCHC(CH2)), 1.70±C(CH2)), 1,70±1.78 (m, 1H, (CH3)2-
CHCHC(CH2)), 2.84±2.96 (m, 2H, PhCH2CH2NH), 3.06±3.16
(m, 1H, PhCH2CHCO), 3.30±3.41 (m, 1H, PhCH2CHCO),
3.51±3.72 (m, 2H, PhCH2CH2NH), 5.03±5.19 (m, 3H,
PhCH2OCONH, PhCH2CHCO), 5.44±5.57 (m, 1H, CONH),
6.91 (s, 1H, CONH), 6.99 (s, 1H, CONH), 7.04±7.37 (m, 15H,
Ph-H); 13C NMR (CDCl3) d 22.14, 22.68, 28.02, 35.35, 35.87,
37.10, 39.83, 40.43, 55.89, 126.80, 127.03, 128.33, 128.49,
128.59, 128.68, 128.79, 129.43, 135.67, 135.82, 138.02, 159.07,
171.51, 194.98. Anal. (C33H37N3O5) C, H, N. Compound 2:
Mp 155.2±156.0 ꢀC. 1H NMR (CDCl3) d 0.85±1.04 (m, 6H,
(CH3)2CHCHC(CH2)), 1.11±1.21 (m, 1H, (CH3)2CHCHC-
(CH2)), 1.38±1.70 (m, 3H, (CH3)2CHCHC(CH2)), 2.84±2.93
(m, 2H, PhCH2CH2NH), 3.01±3.10 (m, 1H, PhCH2CHCO),
3.22±3.37 (m, 1H, PhCH2CHCO), 3.51±3.73 (m, 2H,
PhCH2CH2NH), 5.09 (s, 2H, PhCH2OCONH), 5.20 (s, 1H,
PhCH2CHCO), 5.47±5.61 (m, 1H, CONH), 6.93 (s, 1H,
CONH), 7.05 (s, 1H, CONH), 7.21±7.39 (m, 15H, Ph-H); 13C
NMR (CDCl3) d 20.10, 22.09, 22.14, 26.66, 35.38, 36.99,
39.56, 40.46, 55.61, 67.28, 126.76, 127.04, 128.20, 128.39,
128.61, 128.70, 128.75, 129.28, 135.81, 138.11, 156.42, 159.19,
170.01, 195.25. Anal. (C33H37N3O5) C, H, N. Compound 3:
1H NMR (CDCl3) d 0.75±0.96 (m, 6H, (CH3)2CHCHC(CH2)),
1.01±1.07 (m, 1H, (CH3)2CHCHC(CH2)), 1.12±1.30 (m, 1H,
(CH3)2CHCHC(CH2)), 1.35±1.52 (m, 2H, (CH3)2CHCHC-
(CH2)), 2.84±2.93 (m, 2H, PhCH2CH2NH), 3.00±3.08 (m, 1H,
PhCH2CHCO), 3.23±3.33 (m, 1H, PhCH2CHCO), 3.51±3.64
(m, 2H, PhCH2CH2NH), 5.09±5.16 (m, 2H, PhCH2OCONH),
5.21 (s, 1H, PhCH2CHCO), 5.47±5.56 (m, 1H, CONH), 6.88
(s, 1H, CONH), 7.03 (s, 1H, CONH), 7.21±7.36 (m, 15H, Ph-
H); 13C NMR (CDCl3) d 20.72, 22.06, 22.12, 26.54, 28.08,
NMR (CDCl3) d 0.73±0.79 (m, 1H, (CH3)2CHCHC(CH2)),
0.96 (d, 3H, J=6.4 Hz, (CH3)2CHCHC(CH2)), 1.04 (d, 3H,
J=6.4 Hz, (CH3)2CHCHC(CH2)), 1.13±1.33 (m, 1H, (CH3)2-
CHCHC(CH2)), 1.43±1.51 (m, 1H, (CH3)2CHCHC(CH2)),
1.68±1.78 (m, 1H, (CH3)2CHCHC(CH2)), 2.84±2.92 (m, 2H,
PhCH2CH2NH), 3.16±3.36 (m, 2H, PhCH2CHCO), 3.50±3.70
(m, 2H, PhCH2CH2NH), 4.95±5.25 (m, 3H, PhCH2OCONH,
PhCH2CHCO), 5.46±6.80±6.91 (m, 2H, CONH), 7.04±7.37
(m, 15H, Ph-H, 1H, CONH); 13C NMR (CDCl3) d 22.05,
22.13, 28.08, 35.38, 35.94, 37.39, 39.82, 40.42, 55.75, 67.39,
126.80, 127.08, 128.09, 128.16, 128.27, 128.38, 128.58, 128.63,
128.68, 128.78, 129.27, 129.47, 135.53, 135.85, 138.06, 156.26,
159.19, 171.69, 195.41. Anal. (C33H37N3O5) C, H, N.
13. Calpain activity was monitored in a reaction mixture
containing 50 mM Tris HCl (pH 7.4), 50 mM NaCl, 10 mM
dithiothreitol, 1 mM EDTA, 1 mM EGTA, 0.2 mM or 1.0 mM
Suc-Leu-Tyr-AMC (Calbiochem), 2 mg porcine erythrocyte
calpain I (Calbiochem), varying concentrations of inhibitor
dissolved in DMSO (2% total concentration) and 5 mM CaCl2
in a ®nal volume of 250 mL in a polystyrene microtiter plate.
Assays were initiated by addition of CaCl2 and the increase in
¯uorescence (lex=370 nm, lem=440 nm) was monitored at
amibient temperature using a SPECTRAmax Gemini ¯uores-
cence plate reader (Molecular Devices). Ki values were deter-
mined by Dixon plots.15 The average of triplicate assays,
plotted as 1/n versus I, gave intersecting lines with correlation
coecient ꢁ0.95. No other attempt was made to correct for
slow binding or autolysis.
14. Li, Z.; Patil, G. S.; Golubski, Z. E.; Hori, H.; Tehrani, K.;
Foreman, J. E.; Eveleth, D. D.; Bartus, R. T.; Powers, J. C. J.
Med. Chem. 1993, 36, 3472.
15. Dixon, M. The graphical determination of Km and Ki.
Biochem. J. 1972, 129, 197.