Synthesis of a reported calpain inhibitor isolated from Streptomyces griseus

Article abstract of DOI:10.1016/S0960-894X(01)00524-8

The reported diketopiperazine calpain inhibitor, cis-L-L-3,6-bis-(4-hydroxybenzyl)-1,4-dimethylpiperazine-2,5-dione 1, and its analogues 3 and 4 were synthesized from the corresponding amino acids. The previously assigned structure of 1 is confirmed but n

Full text of DOI:10.1016/S0960-894X(01)00524-8

Bioorganic & Medicinal ChemistryLetters 11 (2001) 2647–2649
Synthesis of a Reported Calpain Inhibitor Isolated from
Streptomyces griseus
I. O. Donkor* and M. Lee Sanders
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
Received 7 June 2001; accepted 25 July2001
Abstract—The reported diketopiperazine calpain inhibitor, cis-l-l-3,6-bis-(4-hydroxybenzyl)-1,4-dimethylpiperazine-2,5-dione 1,
and its analogues 3 and 4 were synthesized from the corresponding amino acids. The previously assigned structure of 1 is confirmed
but neither synthetic 1 nor its N-methylphenylalanine analogues 3 and 4 inhibit porcine erythrocyte calpain I. # 2001 Elsevier
Science Ltd. All rights reserved.
Calpain (EC 3.4.22.17) is a unique cytosolic calcium-
activated cysteine protease that is widely distributed in
mammalian cells.1À3 The enzyme has been implicated in
various neurodegenerative disorders and several other
pathological conditions.4À7 Calpain has therefore
attracted considerable interest as a therapeutic target.
Several synthetic, semi-synthetic, as well as natural
products have been investigated as inhibitors of the
enzyme.8,9 Diketopiperazine 1 of N-methyltyrosine was
isolated from Streptomyces griseus (SC488) and repor-
ted to inhibit calpain (IC50=0.8 mM).10 Phevalin 2,
which bears some structural resemblance to diketopi-
perazine 1, was isolated from Streptomyces species and
also reported to inhibit calpain with an IC50 of
1.2 mM.11 These natural products attracted our atten-
tion as interesting leads for the discoveryof novel non-
peptide inhibitors of calpain. In this communication we
describe the total synthesis of diketopiperazine 1 and its
N-methylphenylalanine analogues 3 and 4 and their
12
calpain inhibitoryactivity. Marcuccio and Elix pre-
viouslyreported compounds 3 and 4 but no calpain
1
inhibitorydata were presented.
The diketopiperazine 1 of N-methyltyrosine and its
N-methylphenylalanine analogues 3 and 4 were syn-
thesized from the appropriatelyprotected amino acids
5a–5c and 6a–6c (see Scheme 1). Coupling of t-boc-
protected amino acids 5a–5c with amino acid methyl
esters 6a–6c followed bypurification of the crude pro-
duct bycolumn chromatography(silica gel) with
Scheme 1. (a) EDC/CH2Cl2/Et3N or CDI/anhyd THF/DIEA/N2; (b)
96% formic acid; (c) sec-butanol/toluene (4:1), reflux; (d) NaH/MeI/
DMF; (e) H2, 10% Pd/C, MeOH. For 1, R1=R2=Bzl (4-OH).
*Corresponding author. Tel.:+1-901-448-7736; fax:+1-901-448-6828;
e-mail: idonkor@utom.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00524-8

2648
I. O. Donkor, M. L. Sanders / Bioorg. Med. Chem. Lett. 11 (2001) 2647–2649
EtOAc/hexanes (1:1) as eluant afforded dipeptides
7a–7c.13 It was observed in the synthesis of 7a that
utilization of carbonyldiimidazole (CDI) instead of
ethylcarbodiimide hydrochloride (EDC) as the coupling
agent afforded higher yields of the corresponding
dipeptide. Following a modification of the procedure of
Nitecki et al.,14 compounds 7a–7c were selectively
deprotected with 96% formic acid and the resulting
dipeptide amino esters were transformed to diketo-
piperazines 8a–8c byrefluxing in a mixture of sec-
butanol/toluene (4:1).15 The diketopiperazines (8a–8c)
while 4 did not rotate plane polarized light due to its
dl-configuration.
Despite the similarityin the 1H NMR data of synthetic
diketopiperazine 1 and the natural product, 1 was
obtained as a solid while the natural product was
reported as an oil.10 Also, synthetic diketopiperazine 1
and its N-methylphenylalanine analogues 3 and 4
did not inhibit porcine erythrocyte calpain I up to
1 mM inhibitor concentration under our assaycon-
ditions.18 This is at odds with the reported calpain
inhibitoryactivity(IC 50=0.8 mM) of the diketopiper-
azine isolated from Streptomyces griseus. Casein and
calpain I isolated from purified human RBC were
used as the substrate and the enzyme, respectively, to
determine the calpain inhibitorypotencyof the diketo-
piperazine isolated from the natural product.10 Suc-
Leu-Tyr-AMC and calpain I isolated from porcine
erythrocytes were used as the substrate and the enzyme,
respectively, in the assay system that we used to
evaluate the calpain inhibitorypotencyof the synthetic
diketopiperazines 1, 3, and 4.18 We have previously
used our calpain assaysystem to duplicate the calpain
were N-methylated using NaH/MeI in DMF as described
12
byMarcuccio and Elix
to give the N-methylphenyl-
alanine analogues 3 and 4 as well as 9a.16 Catalytic
hydrogenolysis of 9a over 10% Pd/C at 60 psi afforded
the diketopiperazine 1 of N-methyltyrosine as a yellow
tinted solid.17
We expected that, like the diketopiperazine isolated
from S. griseus, 1 and 3 should have a cis or ll-config-
uration since theywere derived from dipeptides com-
posed of l-amino acids while 4 should have a trans or
dl-configuration since it was derived from a dipeptide
composed of d- and l-amino acids. Indeed, the 1H
NMR spectrum of 1 was identical to that of the diketo-
piperazine isolated from S. griseus.10 The spectra of 3
and 4 were also in agreement with literature values.12
Additionally, the splitting pattern and the resonance
positions of the geminal methylene protons and the
methine protons of 1 and 3 were similar and different
from those of 4, which has a trans or dl-configuration
(see Fig. 1). Furthermore, the specific rotation of the
compounds confirmed their assigned configuration.
Compounds 1 and 3 had specific rotations of À114.64 ꢀ
and À98.24 ꢀ, respectively, due to their ll-configuration
inhibitorypotencyof a known
a-ketoamide (Z-Leu-
Phe-CONHCH2CH2Ph) calpain inhibitor.19,20 Thus,
despite the differences in the assayprotocols the
synthetic diketopiperazines should have inhibited
calpain if theyare true calpain inhibitors. We suggest
that an unidentified contaminant of the diketopiper-
azine isolated from Streptomyces griseus is responsible
for the observed calpain inhibitoryactivityreported
10
byAlvarez et al.
Indeed, the fact that synthetic
diketopiperazine 1 is a solid and the diketopiperazine
isolated from the natural product is an oil is consistent
with the possibilityof an impurityin the natural
Figure 1. 1H NMR (500 MHz) spectra showing the aliphatic regions of 1, 3, and 4. S, solvent (DMSO-d6); W, water; a, methylene protons; b,
N–CH3 protons; c, methine protons.

I. O. Donkor, M. L. Sanders / Bioorg. Med. Chem. Lett. 11 (2001) 2647–2649
2649
product which maybe responsible for its calpain
inhibitoryaction.
18H, phenyl protons). Anal. calcd for C32H30N2O4: C, 75.87;
H, 5.97; N, 5.53. Found: C, 75.81; H, 6.04; N, 5.56. (b) cis-l-l-
3,6-Dibenzylpiperazine-2,5-dione 8b was obtained in 87%
yield. Mp>300 ꢀC. 1H NMR (TFA-d) d 2.25 (m, 2H,
PhCH2CH), 3.01 (d, 2H, PhCH2CH), 4.52 (s, 2H, PhCH2CH),
7.05–7.34 (m, 10H, phenyl protons). Anal. calcd for
C18H18N2O2: C, 73.45; H, 6.16; N, 9.52. Found: C, 73.32; H,
6.25; N, 9.43. (c) trans-d-l-3,6-Dibenzylpiperazine-2,5-dione
8c was obtained in 50% yield. Mp 294 ꢀC. 1H NMR (TFA-d) d
3.03–3.20 (m, 4H, PhCH2CH), 3.86 (d, 2H, PhCH2CH), 7.00–
7.28 (m, 10H, phenyl protons). Anal. calcd for C18H18N2O2:
C, 73.45; H, 6.16; N, 9.52. Found: C, 73.58; H, 6.22; N, 9.56.
16. (a) cis-l-l-3,6-Bis-(4-benzyloxybenzyl)-1,4-dimethylpiper-
azine-2,5-dione 9a was obtained in 59% yield as a colorless oil.
1H NMR (CDCl3) d 2.22 (dd, 2H, 4-BzlOPhCH2CH, J=6.6,
14.2), 2.75 (s, 6H, NCH3), 2.80 (dd, 2H, 4-BzlOPhCH2CH,
J=4.0, 14.2), 4.01 (dd, 2H, 4-BzlOPhCH2CH, J=4.0, 6.6),
5.02 (s, 4H, PhCH2O), 6.91–7.02 (m, 8H, phenyl protons),
7.27–7.37 (m, 10H, phenyl protons). Anal. calcd for
Acknowledgements
The studywas supported in part bygrant 5 K14
HL03536 from the US Public Health Service (I.O.D.)
and a Universityof Tennessee College of Pharmacy
FacultyDevelopment Grant (I.O.D.).
References and Notes
1. Sorimachi, H.; Saido, T. C.; Suzuki, K. FEBS Lett. 1994,
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2. Suzuki, K.; Ohno, S. Cell Struct. Funct. 1990, 156, 1.
3. Lin, G.-D.; Chattopahhyay, D.; Maki, M.; Wang, K. K. W.;
Carson, M.; Jin, L.; Yuen, P.-W.; Takano, E.; Hatanaka, M.;
DeLucas, L. J.; Narayana, S. V. L. Nat. Struct. Biol. 1997, 4,
539.
C34H34N2O4 0.2EtOAc: C, 75.69; H, 6.50; N, 5.07. Found: C,
75.31; H, 6.47; N, 5.02. (b) cis-l-l-3,6-Dibenzyl-1,4-dimethyl-
piperazine-2,5-dione 2 was obtained in 94% yield. Mp 144–
146 ꢀC. H NMR (CDCl3) d 2.22 (dd, 2H, PhCH2CH, J=6.5,
1
14.2), 2.76 (s, 6H, NCH3), 2.84 (dd, 2H, PhCH2CH, J=4.2,
14.2) 4.06 (dd, 2H, PhCH2CH, J=4.2, 6.5), 7.07–7.35 (m,
10H, phenyl protons); 13C NMR (CDCl3) d 33.5 (NCH3), 39.0
(CH2), 64.3 (CH), 127.2, 128.8, 129.0, 129.6, 137.0 (Ar), 165.4
(CO). [a]2D3 À98.24 ꢀ (c 1, CH3OH). Anal. calcd for
C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.26; H,
6.92; N, 8.72. (c) trans-d-l-3,6-Dibenzyl-1,4-dimethylpiper-
azine-2,5-dione 3 was obtained in 89% yield. Mp 184–187 ꢀC.
1H NMR (CDCl3) d 2.84 (s, 6H, NCH3), 2.96 (dd, 2H,
PhCH2CH, J=4.2, 14.1 Hz), 3.21 (dd, 2H, PhCH2CH, J=3.1,
14.1 Hz), 3.39 (t, 2H, PhCH2CH, J=3.6), 6.95–7.27 (m, 10H,
phenyl protons); 13C NMR (CDCl3) d 32.2 (NCH3), 36.9
(CH2), 61.7 (CH), 127.3, 128.3, 129.6, 134.8 (Ar), 165.2 (CO).
[a]2D6 +0.05 ꢀ (c 1, CH3OH). Anal. calcd for C20H22N2O2: C,
74.51; H, 6.88; N, 8.69. Found: C, 74.58; H, 6.93; N, 8.74.
17. cis-l-l-3,6-Bis-(4-hydroxybenzyl)-1,4-dimethylpiperazine-
2,5-dione 1 was obtained in 88% yield as a yellow tinted solid.
Mp 208 ꢀC. 1H NMR (CH3OH-d4) d 2.14 (dd, 2H, 4-
OHPhCH2CH, J=6.4, 14.2 Hz), 2.75 (s, 6H, NCH3), 2.76 (dd,
2H, 4-OHPhCH2CH, J=4.2, 14.2 Hz), 4.09 (dd, 2H, 4-
OHPhCH2CH, J=4.2, 6.4 Hz), 6.75 (d, 4H, phenyl protons),
6.91 (d, 4H, phenyl protons). 1H NMR (DMSO-d6) d 2.03 (dd,
2H, 4-OHPhCH2CH, J=6.0, 14.2 Hz), 2.48 (dd, 2H, 4-
OHPhCH2CH, J=4.5, 14.2 Hz), 2.58 (s, 6H, NCH3), 3.96 (t,
2H, 4-OHPhCH2CH, J=5.3), 6.61–6.78 (dd, 8H, phenyl pro-
tons), 9.22 (s, 2H, PhOH); 13C NMR (DMSO-d6) d 32.6
(NCH3), 37.3 (CH2), 63.1 (CH), 115.3, 127.2, 130.4, 156.3
(Ar), 164.9 (CO); MS: calcd for C20H22N2O4+H=355.4;
Found: 355.3. [a]2D5 À114.64 ꢀ (c 1, CH3OH). Anal. calcd for
4. Sorimachi, H.; Ishiura, S.; Suzuki, K. Biochem. J. 1997,
328, 721.
5. Saido, T.; Sorimachi, H.; Suzuki, K. FASEB J. 1994, 8, 814.
6. Bartus, R. T. Neuroscientist 1997, 3, 314.
7. Wang, K. K. W.; Yuen, P.-W. Adv. Pharm. 1997, 37, 117.
8. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
9. Donkor, I. O. Curr. Med. Chem. 2000, 7, 1171.
10. Alvarez, M. E.; Houck, D. R.; White, C. B.; Brownell,
J. E.; Bobko, M. A.; Rodger, C. A.; Stawick, M. B.; Sun,
H. H.; Gillum, A. M.; Cooper, R. J. Antibiot. 1994, 47, 1195.
11. Alvarez, M. E.; Houck, D. R.; White, C. B.; Brownell,
J. E.; Bobko, M. A.; Rodger, C. A.; Stawick, M. B.; Sun,
H. H.; Gillum, A. M.; Cooper, R. J. Antibiot. 1995, 48, 1165.
12. Marcuccio, S. M.; Elix, J. A. Aust. J. Chem. 1984, 37,
2397.
1
13. The H NMR spectra of all the compounds in this study
were recorded on a Bruker 300 MHz instrument. (a) t-Boc-l-
tyrosyl(O-benzyl)-l-tyrosine(O-benzyl)methyl ester 7a was
obtained in 96% yield as a white solid of melting point 163–
165 ꢀC. H NMR (CDCl3) d 1.41 (s, 9H, t-Boc), 2.92–3.0 (m,
1
4H, PhCH2CH), 3.66 (s, 3H, OCH3), 4.28 (m, 1H,
CHCONH), 4.73 (dd, 1H, CHCOOCH3), 5.01 (d, 4H,
PhCH2O), 6.81–7.12 (m, 8H, phenyl protons), 7.27–7.42 (m,
10H, phenyl protons). Anal. calcd for C38H42N2O7: C, 71.45;
H, 6.63; N, 4.39. Found: C, 71.64; H, 6.57; N, 4.31. (b) t-Boc-
l-phenylalanyl-l-phenylalanine methyl ester 7b was obtained
in 60% yield as a white solid of melting point 116–117 ꢀC. H
1
NMR (CDCl3) d 1.39 (s, 9H, t-Boc), 2.98–3.12 (m, 4H,
PhCH2CH), 3.67 (s, 3H, OCH3), 4.32 (m, 1H, CHCONH),
4.78 (dd, 1H, CHCOOCH3), 6.95–7.30 (m, 10H, phenyl pro-
tons). Anal. calcd for C24H30N2O5: C, 67.59; H, 7.09; N, 6.57.
Found: C, 67.34; H, 7.03; N, 6.47. (c) t-Boc-d-phenylalanyl-l-
phenylalanine methyl ester 7c was obtained in 63% yield as a
.
C20H22N2O4 1H2O: C, 64.50; H, 6.50; N, 7.52. Found: C,
64.27; H, 6.49; N, 7.51.
18. Calpain activitywas 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
fluorescence (gex=370 nm, gem=440 nm) was monitored at
ambient temperature using a SPECTRAmax Gemini fluor-
escence plate reader (Molecular Devices).
white solid of melting point 124–126 ꢀC. H NMR (CDCl3) d
1
1.38 (s, 9H, t-Boc), 2.90–3.11 (m, 4H, PhCH2CH), 3.67 (s, 3H,
OCH3), 4.35 (m, 1H, CHCONH), 4.83 (dd, 1H,
CHCOOCH3), 6.94–7.30 (m, 10H, phenyl protons). Anal.
calcd for C24H30N2O5: C, 67.59; H, 7.09; N, 6.57. Found: C,
67.34; H, 7.04; N, 6.54
14. Nitecki, D. E.; Halpern, B.; Westley, J. W. J. Org. Chem.
1968, 33, 864.
15. (a)
cis-l-l-3,6-Bis-(4-benzyloxybenzyl)piperazine-2,5-
19. Donkor, I. O.; Zheng, X.; Miller, D. D. Bioorg. Med.
Chem. Lett. 2000, 10, 2497.
20. Donkor, I. O.; Zheng, X.; Han, J.; Lacy, C.; Miller, D. D.
Bioorg. Med. Chem. Lett. 2001, 11, 1753.
dione 8a was obtained in 84% yield. Mp>260 ꢀC. H NMR
(TFA-d) d 2.15 (m, 2H, PhCH2CH), 2.87 (d, 2H, PhCH2CH),
4.47 (s, 2H, PhCH2CH), 5.19 (s, 4H, PhCH2O), 6.96–7.35 (m,
1