Synthesis and calpain inhibitory activity of peptidomimetic compounds with constrained amino acids at the P2 position

Article abstract of DOI:10.1016/j.bmcl.2008.07.094

The effect of incorporating α,α′-diethylglycine and α-aminocyclopentane carboxylic acid at the P₂ position of inhibitors on μ-calpain inhibition was studied. Compound 3 with α,α′-diethylglycine was over 20-fold more potent than 2 with α-aminocy

Full text of DOI:10.1016/j.bmcl.2008.07.094

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4806–4808
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and calpain inhibitory activity of peptidomimetic compounds
with constrained amino acids at the P2 position
*
Isaac O. Donkor , Rajani Korukonda
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The effect of incorporating
a
,
a0-diethylglycine and
a
-aminocyclopentane carboxylic acid at the P2 posi-
a0-diethylglycine was over
-aminocyclopentane carboxylic acid. Additionally, 3 was over 35-fold
-calpain compared to cathepsin B, while 2 was 3-fold selective for cathepsin B compared to
-calpain. Thus, the conformation induced by the P2 residue influenced the activities of the compounds
versus the closely related cysteine proteases, and suggests an approach to the discovery of selective
-calpain inhibitors.
Received 22 May 2008
Revised 21 July 2008
Accepted 24 July 2008
Available online 27 July 2008
tion of inhibitors on
20-fold more potent than 2 with
selective for
l-calpain inhibition was studied. Compound 3 with a,
a
l
l
Keywords:
Calpain
Cathepsin
l
Ó 2008 Elsevier Ltd. All rights reserved.
Cysteine proteases
Peptidomimetics
Constrained amino acids
Inhibitors
Calpain is a papain-like cytosolic cysteine protease that requires
calcium ions for activation.1 Several calpain isoforms have been re-
ported of which l-calpain and m-calpain are the most abundantly
tended conformation while those with a-aminocycloalkane car-
boxylic acids prefer a folded conformation.16 We demonstrated in
a previous study that incorporation of 2,3-methanoleucine stereo-
distributed in mammalian cells, and have been termed ubiquitous
calpains.1–3 Calpain is a promiscuous enzyme that catalyzes the
limited proteolysis of a broad range of substrates in vivo.2,3 The
physiological role of the enzyme is evolving, and it has been shown
to participate in signal transduction pathways.4–7 Calpain has at-
tracted considerable interest due in part to implication of the en-
isomers at the P2 position of peptidyl inhibitors influences
l-cal-
pain inhibition. Compound with 2,3-methanoleucine
1
a
stereoisomer of E-(2S,3S) configuration at the P2 position (Fig. 1)
was the most potent and selective member of the series albeit
zyme in
a
variety of pathological conditions including
Ph
neurological disorders (e.g., stroke and Alzheimer’s disease), cata-
ract, and cancer.8–13 This has led to the search for selective calpain
inhibitors as potential therapeutic agents and as biochemical
probes. Several compounds are known to inhibit the ubiquitous
calpains. However, most of the inhibitors are not specific for the
calpains because they also inhibit the closely related cysteine
cathepsins (e.g., cathepsin B).14,15 Hence there is a continuing need
for new calpain inhibitors with improved selectivity for the
enzyme.
Incorporation of constrained amino acids such as
glycines and -aminocycloalkane carboxylic acids into a peptide
restricts the conformational freedom of the peptide in the vicinity
of the constrained amino acid.16 This allows one to study the effect
of local conformational constraints on bioactivity because peptides
O
O
O
Ph
O
N
Ph
H
O
1, E-(2S,3S)-Methanoleucine isomer
Ph
O
H
Ph
N
H
H
a,
a0-dialkyl-
N
H
a
O
O
2
Ph
O
H
N
Ph
a,
a0-dialkylglycine residues generally adopt fully ex-
N
H
containing
O
O
3
* Corresponding author. Tel.: +1 901 448 7736; fax: +1 901 448 6828.
E-mail address: idonkor@utmem.edu (I.O. Donkor).
Figure 1. Structures of calpain inhibitors with constrained amino acids.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.094

I. O. Donkor, R. Korukonda / Bioorg. Med. Chem. Lett. 18 (2008) 4806–4808
4807
modest selectivity for
showed that the S2-subsite of
than that of cathepsin B, which suggested that compounds that
adopt different conformations would exhibit differential affinities
l
-calpain versus cathepsin B.17 This study
-calpain is more stereosensitive
Table 1
Inhibition of porcine erythrocyte
compounds 2 and 3
l-calpain and human liver cathepsin B by
l
Compound
l
-Calpain Kia
(l
M)
Cathepsin B Ki (
lM)
SRb
0.45
36.37
0.79
for
these closely related cysteine proteases with the goal of generating
selective -calpain inhibitors we studied the effect of incorporat-
ing -aminocyclopentane carboxylic acid (as in 2, Fig. 1) and
a0-diethylglycine (as in 3) at the P2 position on inhibition of
calpain and cathepsin B.
Compounds 2 and 3 were synthesized using general coupling
procedures as outlined in Schemes 1 and 2. 1-Amino-1-cyclopen-
tane carboxylic acid 4 (Scheme 1) or diethylglycine 9 (Scheme 2)
was refluxed in SOCl2 and MeOH for 24 h to give esters 5 and10,
respectively. The esters were coupled with 3-phenyl propionic
acid, using EDC, NMM, and HOBT, to give the dipeptide esters 6
and 11, which were hydrolyzed using 2 N NaOH to give the corre-
sponding dipeptide acids 7 and 12. Compound 7 was coupled with
l-calpain and cathepsin B. To further probe the active sites of
2
3
1.94 0.81
0.08 0.01
0.19 0.02
0.88 0.01
2.91 0.62
0.15 0.01
ALLNc
l
a
a
Ki values are means of triplicate determinations obtained by Dixon plots with
correlation coefficient of P0.95.
a,
l-
b
SR is selectivity ratio, which was determined by dividing the Ki value for
cathepsin B inhibition by that for calpain inhibition.
c
ALLN was purchased from Calbiochem.
ity of the compounds. Compound 3 with
P2 position inhibited -calpain with Ki of 0.08
fold better inhibitor of -calpain compared to 2 with
opentane carboxylic acid as the P2 residue. Furthermore, 3 was
over 35-fold selective for -calpain versus cathepsin B. Compound
2 on the contrary inhibited cathepsin B 3-fold better than 3.
Clearly, our data suggest that compounds that adopt extended con-
formation are better inhibitors of
with folded conformation. This is consistent with recent X-ray
crystallographic studies of the complexes of -calpain with bound
a
,
a0-diethylglycine at the
M. It was over 20-
-aminocycl-
l
l
l
a
l
L
-phenylalaninol in the presence of EDC, NMM, and HOBT to give
tripeptide alcohol 8. Compound 12 (Scheme 2) formed the cyclic
intermediate 13 in the presence of EDC. This was coupled with
L
-
l-calpain compared to those
phenylalaninol in the presence of EDC, NMM, and HOBT to give
the tripeptide alcohol 14. The tripeptide alcohols 8 and 14 were
oxidized using Dess–Martin periodinane reagent to afford the tar-
get compounds 2 (Scheme 1) and 3 (Scheme 2), respectively. The
final products were purified by flash column chromatography
and/or recrystallization from hexanes/CH2Cl2 (1:1).18 The com-
l
inhibitors, which showed that the inhibitors occupy the active site
pocket of the enzyme in extended conformation.20–22 Thus, unlike
l
-calpain, cathepsin B did not display significant preference for
any of the two compounds suggesting that the enzyme is not very
sensitive to the conformational differences induced by the con-
strained amino acids at the P2 position of the inhibitors. This is
consistent with our previous finding using compounds with 2,3-
methanoleucine stereoisomers as the P2 residue, which suggested
pounds were evaluated19 as inhibitors of porcine erythrocyte
l-
calpain (Calbiochem) and human liver cathepsin B (Calbiochem)
using ALLN as positive control. Table 1 shows the results of the
study. The nature of the P2 residue influenced the inhibitory activ-
O
O
O
O
a
b
c
H
N
H2N
H
N
OH
H2N
O
OH
O
7
O
O
4
6
d
5
O
O
H
N
H
N
e
OH
H
N
H
N
H
O
O
O
8
2
Scheme 1. Reagents: (a) SOCl2, MeOH; (b) 3-phenylpropionic acid, EDC, NMM, HOBT; (c) 2 N NaOH, MeOH; (d)
L
-phenylalaninol, EDC, NMM, HOBT; (e) Dess–Martin reagent.
O
O
b
c
O
O
a
H
H2N
H
N
OH
H2N
O
N
OH
O
O
12
O
9
11
10
d
O
e
f
O
O
O
H
N
H
N
N
OH
H
N
H
N
13
H
O
O
O
3
14
Scheme 2. Reagents: (a) SOCl2, MeOH; (b) 3-phenylpropionic acid, EDC, NMM, HOBT; (c) 2 N NaOH, MeOH; (d) EDC, DMF; (e)
L-phenylalaninol, EDC, NMM, HOBT; (f) Dess–
Martin reagent.

4808
I. O. Donkor, R. Korukonda / Bioorg. Med. Chem. Lett. 18 (2008) 4806–4808
93.5%). Mp 203–205 °C. 1H NMR (CDCl3, 300 MHz): d 8.98 (s, 2H), 3.84 (s, 3H),
2.29 (m, 4H), 2.13 (m, 2H), 1.89 (m, 2H).
1-(3-Phenyl-propionylamino)-cyclopentane carboxylic acid methyl ester (6).
that the S2-subsite of cathepsin B is not as stereosensitive as that of
l
-calpain.17
In summary, we have demonstrated using constrained amino
Compound
6 was obtained from 5 and commercially available 3-phenyl
propionic acid using the general coupling procedure. The product was obtained
as a white solid (0.76 g, 99.4%). Mp 65–67 °C 1H NMR (CDCl3, 300 MHz): d 7.25
(m, 5H), 5.86 (s, 1H), 3.70 (s, 3H), 2.93 (t, J = 9 Hz, 2H), 2.49 (t, J = 9 Hz, 2H), 2.20
(m, 2H), 1.87 (m, 2H), 1.72 (m, 4H).
acids as the P2 residue that peptidomimetic compounds that adopt
extended conformation are potentially potent and selective inhib-
itors of
l-calpain versus cathepsin B compared to related ana-
1-(3-Phenyl-propionylamino)-cyclopentane carboxylic acid (7). NaOH (2 N,
15 mL) was added to a solution of 6 (0.66 g, 3.67 mmol) in MeOH (15 mL),
and the mixture was stirred for 6 h at room temperature. The reaction mixture
was cooled to 0 °C, ethyl acetate (50 mL) was added and it was acidified with
5% HCl (pH 4.5). The mixture was extracted with EtOAc (2 ꢁ 30 mL). The
combined organic extracts were washed with brine (25 mL), dried with
Na2SO4, and evaporated to give 7 as a white solid (0.53 g, 55.3%). Mp 170–
172 °C. 1H NMR (CDCl3, 300 MHz): d 8.18 (s, 1H), 7.21 (m, 5H), 2.89 (t, J = 8 Hz,
2H), 2.47 (t, J = 7.2 Hz, 2H), 2.13 (m, 2H), 1.88 (m, 2H), 1.67 (m, 4H).
1-(3-Phenyl-propionylamino)-cyclopentane carboxylic acid (1-benzyl-2-hydroxy-
ethyl)-amide (8). Compound 8 was obtained from 7 and commercially available
logues that adopt folded conformation.
Acknowledgment
This work was supported by NIH Grant R15 HL083968-01 to
I.O.D.
References and notes
L
-phenylalaninol using the general coupling procedure. The product was
obtained as
a
white solid (0.69 g, 91.3%). Mp 50–52 °C. 1H NMR (CDCl3,
1. Croall, D. E.; Demartino, G. N. Physiol. Rev. 1991, 71, 813.
2. Suzuki, K.; Sorimachi, H.; Yoshizawa, T.; Kinbara, K.; Ishiura, S. Biol. Chem. 1995,
376, 523.
3. Sorimachi, H.; Ishiura, S.; Suzuki, K. Biochem. J. 1997, 328, 721.
4. Bano, D.; Young, K. W.; Guerin, C. J.; Lefeuvre, R.; Rothwell, N. J.; Naldini, L.;
Rizzuto, R.; Carafoli, E.; Nicotera, P. Cell 2005, 120, 275.
300 MHz): d 7.24 (m, 10H), 6.64 (s, 1H), 5.84 (s, 1H), 3.63 (m, 1H), 3.45 (m, 1H),
3.05 (m, 1H), 2.96 (m, 3H), 2.83 (m, 2H), 2.5 (m, 2H), 2.25 (m, 1H), 1.94 (m, 1H),
1.71 (m, 4H), 1.55 (m, 2H).
2-Amino-2-ethylbutyric acid methyl ester (10). SOCl2 (10 mL) was added
dropwise to
a solution of diethylglycine (0.7 g, 5.3 mmol) in methanol
(30 mL) at 0 °C, and the mixture was refluxed for 24 h followed by solvent
removal in vacuo. Further rinsing with MeOH/CH2Cl2 (2 ꢁ 30 mL) and
evaporation afforded 10 as a semi-viscous yellowish green oil (0.92 g, 95.6%).
1H NMR (CDCl3, 300 MHz): d 8.5 (s, 2H), 3.77 (s, 3H), 1.83 (q, J = 6 Hz, 4H), 0.877
(t, J = 7.2 Hz, 6H).
2-Ethyl-2-(3-phenyl-propionylamino)-butyric acid methyl ester (11). Compound
11 was obtained from 10 and commercially available 3-phenyl propionic acid
using the general coupling procedure. The product was obtained as viscous oil
(0.17 mg, 94.4%). 1H NMR (CDCl3, 500 MHz): d 7.25 (m, 5H), 6.32 (s, 1H), 3.74
(s, 3H), 2.96 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.2 Hz, 2H), 2.45 (m, 2H), 1.75 (m, 2H),
0.64 (t, J = 7 Hz, 6H).
5. Atencio, I. A.; Ramachandra, M.; Shabram, P.; Demers, G. W. Cell Growth Differ.
2000, 11, 247.
6. Dietrich, C.; Bartsch, T.; Schanz, F.; Oesch, F.; Wieser, R. J. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 10815.
7. Choi, Y. H.; Lee, S. J.; Nguyen, P.; Jang, J. S.; Lee, J.; Wu, M. L.; Takano, E.; Maki,
M.; Henkart, P. A.; Trepel, J. B. J. Biol. Chem. 1997, 272, 28479.
8. Zatz, A.; Starling, A. N. Engl. J. Med. 2005, 352, 2413.
9. Saido, T.; Sorimachi, H.; Suzuki, K. FASEB J. 1994, 8, 814.
10. Nixon, R. A. Ann. N.Y. Acad. Sci. 1989, 568, 198.
11. Wang, K. K. W.; Yuen, P.-W. Trends Pharm. Sci. 1994, 15, 412.
12. Wang, K. K. W.; Yuen, P.-W. Adv. Pharm. 1997, 37, 117.
2-Ethyl-2-(3-phenyl-propionylamino)-butyric acid (12). NaOH (2 N, 50 mL) was
added to a solution of 11 (0.8 g, 2.88 mmol) in MeOH (35 mL), and the mixture
was stirred for 36 h at room temperature. EtOAc (50 mL) was added to the
reaction mixture at 0 °C and then acidified with 5% HCl (pH 4.5). The mixture
was extracted with EtOAc (2 ꢁ 50 mL), and the combined organic extracts were
washed with brine, dried with Na2SO4 and evaporated to give 12 as a white
powdery solid (0.66 g, 87%). Mp 200–202 °C. 1H NMR (DMSO, 300 MHz): d 7.49
(s, 1H), 7.21 (m, 5H), 2.78 (t, J = 9 Hz, 2H), 2.43 (t, J = 9 Hz, 2H), 1.80 (s, 1H), 1.78
(m, 4H), 0.62 (t, J = 6 Hz, 6H).
4,4-Diethyl-2-phenethyl-4H-oxazol-5-one (13). EDC (0.087 g, 0.46 mmol) was
added to a solution of 12 (0.8 g, 2.88 mmol) in CH2Cl2/DMF (10:2) at 0 °C, and
the mixture was stirred for 24 h at room temperature. The reaction mixture
was diluted with CH2Cl2 (60 mL), washed with saturated NaHCO3, 0.5 N HCl,
and water successively, and dried with Na2SO4. Evaporation of the solvent gave
13 as colorless oil (0.92 g, 98.69%). Mp 180–182 °C. 1H NMR (CDCl3, 300 MHz):
d 7.27 (m, 5H), 3.05 (m, 2H), 2.87 (m, 2H), 1.76 (q, J = 6 Hz, 4H), 0.71 (t, J = 6 Hz,
6H).
13. Bartus, R. T.; Chen, E. Y.; Lynch, G.; Kordower, J. H. Exp. Neurol. 1999, 155, 315.
14. Donkor, I. O. Curr. Med. Chem. 2000, 12, 1171.
15. Neffe, A. T.; Abell, A. D. Curr. Opin. Drug Discov. Devel. 2005, 6, 684.
16. Wolff, M. E., Ed.; Burger’s Medicinal Chemistry and Drug Discovery. Vol. 1:
Principles and Practice, 5th ed.; John Wiley and Sons, Inc.: New York, NY,1995; p
817–181.
17. Donkor, I. O.; Zheng, X.; Miller, D. D. Bioorg. Med. Lett. 2000, 10, 2497.
18. General coupling method. A solution of the carboxylic acid (1 equiv) in CH2Cl2
was cooled in an ice-bath and EDC (1.05 equiv), HOBT (1.05 equiv), NMM
(3 equiv), and the appropriate amine (1 equiv) were added consecutively. The
reaction mixture was stirred overnight at room temperature. Water was added
to the reaction mixture and extracted with CH2Cl2. The combined organic
extracts were washed successively with saturated NaHCO3solution (30 mL),
0.5 N HCl (30 mL), and then water (30 mL), and dried over Na2SO4. Evaporation
of the solvent followed by column chromatographic purification afforded the
desired amide.
General oxidation method. Dess–Martin periodinane reagent (1.05 equiv) was
added to a solution of the appropriate alcohol (1 equiv) in CH2Cl2 (20 mL), and
the reaction mixture was stirred for 2 h at room temperature. Na2S2O3ꢀ5 H2O
(14–28 equiv) in saturated NaHCO3 solution was added and stirred for
additional 10 min. The mixture was extracted with CH2Cl2 (3 ꢁ 30 mL). The
combined CH2Cl2 extracts were washed with 0.5 N HCl (30 mL), water (30 mL),
and dried over Na2SO4. The solvent was evaporated and crude product was
purified by column chromatography.
1-(3-Phenyl-propionylamino)-cyclopentane carboxylic acid (1-benzyl-2-oxo-
ethyl)-amide (2). Compound 8 was transformed to 2 as described under the
general method for oxidation. The product was obtained as a white crystalline
solid (0.5 g, 77%). Mp 148–150 °C. 1H NMR (CDCl3, 300 MHz): d 9.57 (s, 1H),
7.29 (m, 11H), 5.46 (s, 1H), 4.55 (q, J = 9 Hz, 1H), 3.10 (m, 2H), 2.93 (t, J = 9 Hz,
2H), 2.63 (t, J = 7.5 Hz, 2H), 2.16 (m, 2H), 1.79 (m, 2H), 1.66 (m, 2H), 1.53 (m,
2H). Anal. (C24H28N2O3), C, H, N. Calcd: C 73.44, H 7.19, N 7.14. Found: C 73.21,
H 7.24, N 7.04. ESI MS: m/z 447 (M + Na + CH3OH)+.
N-(1-Benzyl-2-oxo-ethyl)-2-ethyl-2-(3-phenyl-propionylamino)-butyramide (3).
Compound 14 was transformed to 3 as described under the general method
for oxidation. The product was obtained as a white crystalline solid (0.3 mg,
98.3%). Mp 98–100 °C. 1H NMR (CDCl3, 300 MHz): d 9.62 (s, 1 H), 7.26 (m, 10H),
6.48 (s, 1H), 6.25 (d, J = 6.6 Hz, 1H), 4.74 (q, J = 6.9 Hz, 1H), 3.12 (d, J = 7.2 Hz,
2H), 2.94 (t, J = 7.5 Hz, 2H), 2.48 (m, 4H), 1.48 (m, 1H), 1.38 (m, 1H), 0.644 (t,
J = 7.2 Hz, 3H), 0.54 (t,J = 7.2 Hz, 3H). Anal. (C24H30N2O3), C, H, N. Calcd: C 73.07,
N-(1-Benzyl-2-hydroxy-ethyl)-2-ethyl-2-(3-phenyl-propionylamino)-butyramide
(14). Compound 14 was obtained from 13 and commercially available
phenylalaninol using the general coupling procedure. The product was
obtained as
white crystalline solid (0.6 mg, 94.6%). 1H NMR (CDCl3,
L-
a
300 MHz): d 7.28 (m, 10H), 6.06 (s, 1H), 6.09 (d, J = 7.2 Hz, 1H), 4.25 (m,
1H), 3.65 (m, 2H), 3.15 (s, 1H), 2.95 (m,3H), 2.81 (m, 1H), 2.53 (t, J = 6 Hz, 2H),
2.33 (m, 2H), 1.53 (m, 1H), 1.38 (m, 1H), 0.63 (t, J = 6 Hz, 3H), 0.45 (t, J = 6 Hz,
3H).
19. Calpain inhibition assay. The Ki values for inhibition of porcine erythrocyte
l-
calpain (Calbiochem) activity was monitored as previously reported23 in a
buffer consisting of 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
concentrations of the inhibitor in DMSO (2% total concentration), and 5 mM
CaCl2 in a final volume of 250 L in a microtiter plate. Cathepsin B inhibition
lg of calpain from porcine erythrocyte (Calbiochem), varying
l
assay. The Ki values for inhibition of human liver cathepsin B (Calbiochem)
activity was determined as described for calpain using a reaction mixture
containing 1 nM human liver cathepsin B (Calbiochem), 50 mM NaOAc (pH
6.0), 1 mM EDTA, 0.5 mM DTT, 50
and varying concentrations of inhibitor in DMSO (2% total concentration) in a
final volume of 250 L.
lM or 250 lM of substrate (Z-Arg-Arg-AMC),
l
20. Cuerrier, D.; Moldoveanu, T.; Inoue, J.; Davies, P. L.; Campbell, R. L. Biochemistry
2006, 45, 7446.
H
7.66,
(M + Na + CH3OH)+.
1-Amino-cyclopentane carboxylic acid methyl ester (5). SOCl2 (1 mL) was added
dropwise to solution of 1-amino-1-cyclopentane carboxylic acid (0.4 g,
N 7.10. Found: C 72.99, H 7.75, N 7.10. ESI MS: m/z 449
21. Li, Q.; Hanzlik, R. P.; Weaver, R. F.; Schonbrunn, E. Biochemistry 2006, 45, 701.
22. Moldoveanu, T.; Campbell, R. L.; Cuerrier, D.; Davies, P. L. J. Mol. Biol. 2004, 343,
1313.
a
23. Donkor, I. O.; Korukonda, R.; Huang, T. L.; LeCour, L., Jr. Bioorg. Med. Chem. Lett.
2003, 13, 783.
3.1 mmol) in methanol (10 mL) at 0 °C, and the mixture was refluxed for 3 h. It
was cooled and evaporated in vacuo to give the product as a white solid (0.52 g,