Significance of hydrogen bonding at the S1′ subsite of calpain I

Article abstract of DOI:10.1016/S0960-894X(01)00301-8

α-Ketohydroxamates were synthesized as bioisosteres of α-ketoamides. The α-ketohydroxamates were generally more potent than the corresponding α-ketoamides. The potency of the compounds suggests that hydrogen bonding and steric bulk of substituents on the nitrogen atom of the ketoamide moiety influence calpain inhibition.

Full text of DOI:10.1016/S0960-894X(01)00301-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1753–1755
0
Significance of Hydrogen Bonding at the S1 Subsite of Calpain I
Isaac O. Donkor,* Xiaozhang Zheng, Jie Han, Calvin Lacy and Duane D. Miller
Department of Pharmaceutical Sciences, The University of Tennessee, Johnson Bldg. Room 327E,
847Monroe Avenue, Memphis, TN 38163, USA
Received 8February 2001; accepted 23 April 2001
Abstract—a-Ketohydroxamates were synthesized as bioisosteres of a-ketoamides. The a-ketohydroxamates were generally more
potent than the corresponding a-ketoamides. The potency of the compounds suggests that hydrogen bonding and steric bulk of
substituents on the nitrogen atom of the ketoamide moiety influence calpain inhibition. # 2001 Elsevier Science Ltd. All rights
reserved.
The implication of calpain I in neurological disorders
(e.g., stroke) and other pathological conditions (e.g.,
platelet aggregation and cardiac ischemia) has attracted
interest in the search for calpain inhibitors as potential
therapeutic agents.1ꢀ4 Most of the structure–activity
relationship studies of calpain inhibitors have focused
on modifications of the unprimed region of the inhibi-
tors to probe for structural requirements for binding to
the S subsites of calpain.5,6 A comparatively limited
number of studies have addressed the structural
requirements for binding to the S0 subsites of the
enzyme.7,8 In one such study, Li et al.7 demonstrated
tion of calpain inhibition. We also envisioned that N-
alkyl a-ketohydroxamates 7–9 should be better inhibi-
tors of calpain I compared to the tertiary a-ketoamide 2
if the oxygen atom served as a hydrogen bond acceptor
at the S1 subsite of calpain. While this work was in
progress Mallamo et al.9 disclosed compounds 5 and 6
in the patent literature.
0
Synthesis of the N,O-dialkyl hydroxylamines 14–16
required for the synthesis of compounds 7–9 is shown in
Scheme 1. The compounds were prepared from com-
mercially available di-t-butyl dicarbonate 10 and O-
alkyl hydroxylamine 11 in aqueous base. The resulting
carbamate 12 was treated with sodium hydride to effect
proton abstraction followed by the addition of iodo-
methane or bromoethane to give t-butyl-N-alkyl carba-
mate 13. Acidolysis of 13 gave the corresponding N,O-
dialkyl hydroxylamines 14–16.
0
that hydrogen bonding between an inhibitor and the S1
subsite of calpain is important for potent inhibition of
the enzyme. The workers showed that compounds 1 and
3, which are secondary a-ketoamides, are 380- and
1310-fold, respectively, more potent than compound 2,
which is a tertiary a-ketoamide. The difference in the
calpain inhibitory potency of the compounds was
attributed in part to the ability of 1 and 3 to undergo
hydrogen bonding interactions with the S0 subsite of
calpain and the inability of 2 to perform similar inter-
actions with the enzyme (Fig. 1). To further explore the
significance of hydrogen bonding interaction between
an inhibitor and the S0 subsite of calpain, we synthesized
and evaluated the calpain I inhibitory activity of com-
pounds 3–9. We reasoned that placing an oxygen atom
alpha to the amide bond nitrogen of compounds 3 and 4
as in a-ketohydroxamates 5 and 6, respectively, should
increase the strength of the hydrogen bond between the
inhibitor and the enzyme, and should result in potentia-
Compounds 3–9 were synthesized as shown in Scheme 2.
Boc-phenylalanine 17 was coupled with N,O-dimethyl
hydroxylamine to give Weinreb amide 18, which was
reduced to aldehyde 19 with LiAlH4.10 The aldehyde
was reacted with KCN to give cyanohydrin 20. Depro-
tection of 20 and hydrolysis of the cyano group gave a-
hydroxy-b-amino acid 21. The amino group of 21 was
Boc protected to give 22, which was coupled with either
O-alkyl hydroxylamine or N,O-dialkyl hydroxylamine
using EDC and HOBT as the coupling agent to give the
corresponding a-hydroxylamide or a-hydroxylhydro-
xamate 23. Cleavage of the Boc protecting group of 23
followed by coupling with Cbz-leucine and oxidation of
the hydroxyl group with Dess–Martin reagent gave
target compounds 3–9.
*Corresponding author. Tel.:+1-901-448-7736; fax: +1-901-448-
6828; e-mail: idonkor@utmem.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00301-8

1754
I. O. Donkor et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1753–1755
Figure 1. Proposed hydrogen bonding interactions between the compounds and the S0 subsite of calpain. The calpain inhibitory potency of the
compounds mirror the hydrogen bonding pattern of the inhibitors at the S0 subsite of the enzyme.
Scheme 1. Reagents: (a) aqueous base; (b) NaH/DMF; (c) MeI or EtBr; (d) HCl.
Scheme 2. Reagents: (a) CDI, CH3NHOCH3; (b) LiAlH4; (c) NaHSO3; (d) KCN; (e) HCl/dioxane; (f) (t-ButOCO)2O, NaOH; (g) R1-NH-X-CH2-
R2, EDC, HOBT; (h) HCl/dioxane; (i) Cbz-leucine, EDC, HOBT; (j) Dess–Martin reagent.

I. O. Donkor et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1753–1755
1755
Table 1. Inhibition of porcine erythrocyte calpain I by compounds
3–9a
Acknowledgements
The study was supported in part by grant 5 K14
HL03536 from the US Public Health Service (I.O.D.)
and a University of Tennessee College of Pharmacy
Faculty Development Grant (I.O.D.).
Compound
R1
R2
Ki (mM)
1
2
3
4
5
6
7
8
9
H
Et
H
H
H
Et
Et
CH2Bn
CH2Et
OBn
OEt
OBn
OEt
OBn
0.200b
76.00b
0.072 (0.052)b
0.107
References and Notes
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5. Donkor, I. O. Curr. Med. Chem. 2000, 7, 1171.
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Med. Chem. 1993, 36, 3472.
0.045
0.062
0.673
0.750
28.00
H
CH3
CH3
CH2CH3
aKi values are the averages of triplicate determinations obtained by
Dixon plots where 1/n were plotted against I to give intersecting lines
with correlation coefficient >0.95. The assay mixture consisted of
50 mM Tris–HCl pH 7.4, 50 mM NaCl, 10 mM DTT, 1 mM EDTA,
1 mM EGTA, calpain I, and varying concentrations of inhibitors in
DMSO. Total DMSO concentration=2%.
8. Chatterjee, S.; Dunn, D.; Tao, M.; Wells, G.; Gu, Z.-Q.;
Bihovsky, R.; Ator, M. A.; Siman, R.; Mallamo, J. P. Bioorg.
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9. Mallamo, J. P.; Bihovsky, R.; Josef, K. A. International
Application (PCT) WO 00/16767, 2000.
The compounds were evaluated11 as inhibitors of cal-
pain I from porcine erythrocytes and the results are
shown in Table 1. Secondary a-ketohydroxamates 5 and
6 were marginally better inhibitors than the corres-
ponding secondary a-ketoamides 3 and 4, respectively.
Tertiary a-ketohydroxamates 7 and 8 were over 100-
fold more potent than tertiary a-ketoamide 2. However,
tertiary a-ketohydroxamate 9 was only about 3-fold
more potent than a-ketoamide 2. Thus, as the size of the
substituent on the ketoamide nitrogen was increased
from hydrogen (as in 5) to methyl (as in 7) to ethyl (as in
9) a significant decrease in calpain inhibitory potency
was observed (i.e., the calpain inhibitory potency of the
compounds decreased in the following order: 5>7>9).
The results suggest that, while hydrogen bonding may
contribute to the enhanced potency of the a-ketohydrox-
amates compared to0the a-ketoamides (Fig. 1), steric bulk
intolerance at the S1 subsite of the enzyme may also con-
tribute to the observed rank order of calpain inhibitory
potency of the compounds (i.e., 5>7>9).
10. Zheng, X.; Donkor, I. O.; Miller, D. D.; Ross, C. R., II
Chirality 2000, 12, 2.
11. 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 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 final
volume of 250 mL in a polystyrene microtiter plate. Assays
were initiated by addition of CaCl2 and the increase in fluo-
rescence (lex=370 nm, lem=440 nm) was monitored at ami-
bient temperature using a SPECTRAmax Gemini fluorescence
plate reader (Molecular Devices). Ki values were determined
by Dixon plots.12 The average of triplicate assays, plotted as 1/
n versus I, gave intersecting lines with correlation coefficient
ꢁ0.95. No other attempt was made to correct for slow bind-
ing or autolysis.
12. Dixon, M. Biochem. J. 1972, 129, 197.