Synthesis of a small library of diketopiperazines as potential inhibitors of calpain

Article abstract of DOI:10.1016/j.bmcl.2005.04.031

A small library of 2,5-diketopiperazines based on previously reported calpain inhibitors was synthesized. In addition, a concise total synthesis of the structurally related natural product phevalin (2) was accomplished. Despite literature reports that som

Full text of DOI:10.1016/j.bmcl.2005.04.031

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3034–3038
Synthesis of a small library of diketopiperazines as
potential inhibitors of calpain
a
a
a,b,
*
Yibin Zeng,a,b Qingshan Li, Robert P. Hanzlik and Jeffrey Aube
´
aDepartment of Medicinal Chemistry, 1251 Wescoe Hall Drive, University of Kansas, Lawrence, KS 66045-7582, USA
bKU Chemical Methodology and Library Development Center of Excellence, 1501 Wakarusa Drive,
University of Kansas, Lawrence, KS 66047, USA
Received 11 March 2005; revised 13 April 2005; accepted 18 April 2005
Available online 17 May 2005
Abstract—A small library of 2,5-diketopiperazines based on previously reported calpain inhibitors was synthesized. In addition, a
concise total synthesis of the structurally related natural product phevalin (2) was accomplished. Despite literature reports that some
of the compounds prepared were calpain inhibitors, none of the library members were found to have significant activity against
recombinant human calpain I.
Ó 2005 Elsevier Ltd. All rights reserved.
Calpains are a class of intracellular cytoplasmic non-
lysosomal cysteine proteases expressed ubiquitously in
mammalian cells. Among the 16 kinds of calpain identi-
fied thus far, l-calpain (or calpain I) and m-calpain (or
calpain II) are the two most thoroughly studied calpain
isoforms. The two enzymes differ in their sensitivity to
activation by calcium ions. Thus, calpain I is sensitive
to activation by micromolar concentrations of calcium,
whereas calpain II responds only to millimolar calcium
concentrations.1–3 Both isoforms are heterodimers made
up of identical 30 kDa subunits but different 80 kDa
subunits. Overactivation of calpain has been implicated
in many pathological conditions such as stroke,4 myo-
cardial infarction,4 AlzheimerÕs disease,5 ParkinsonÕs
disease5 and cancer.6 Accordingly, selective inhibitors
Figure 1. Some reported non-peptide inhibitors of calpain.
of calpain are of interest as pharmacological probes
and as potential therapeutics.7–9
and penicillide.12 The diketopiperazine dimer of N-
methyltyrosine, 1, has also been reported to inhibit cal-
pain,13 but this report has recently been questioned.14
As part of our ongoing study of cysteine proteases and
their associated medicinal chemistry, we were interested
in the identification of selective non-peptide inhibitors of
Despite some structural and/or binding site differences
calpain. Several groups have reported small molecules
having modest (micromolar) activity as calpain inhibi-
between these non-peptide calpain inhibitors, there are
also some similarities. Specifically, the presence of an
aromatic group two or three atoms away from either an-
other aromatic group or a hydrogen bonding substituent
suggests a possible motif for inhibitor design. In addi-
tion, the prominence of aromatic moieties in these mole-
tors (Fig. 1). These include the a-mercaptoacrylic acid
PD 150606 (which binds remotely to the catalytic site
of calpain)10 and the natural products phevalin (2)11
cules is consistent with the observed preference of
calpain for hydrophobic residues in small peptide sub-
strates and inhibitors.7
Keywords: Calpain; Peptidomimetic; Library diketopiperazine.
*
Corresponding author. Tel.: +1 785 864 4496; fax: +1 785 864
5326; e-mail: jaube@ku.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.031

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3034–3038
3035
Taking into consideration that both diketopiperazine
and phevalin are derived from 2,5-disubstituted piper-
azines (Fig. 2), we thought it possible to employ this com-
mon element as a scaffold for inhibitor synthesis. Thus,
a small library of diketopiperazines (Fig. 3) based on
compound 1 was synthesized. The library was specifi-
cally devised to examine the effects of N–Me versus
N–H substitution (1, 4, 6, 8, 10 vs 3, 5, 7, 9, 11), stereo-
chemistry (14, 15 vs 24, 25) and the hydrophobicity/
hydrophilicity of the side chain, on calpain inhibition
(e.g., 22, 23, 29, 30 vs 17–21).
There are a number of reported routes for the parallel
synthesis of diketopiperazines.16 In the course of meth-
odology verification, we found that a one-pot cycliza-
tion process of the dipeptide precursors could be
deployed in a straightforward parallel synthesis proto-
col.17 Thus the crude dipeptides were directly subjected
to treatment with TFA to release the Boc group and al-
low subsequent ring closure (Scheme 1). The diketopi-
perazines were obtained in 35–60% yield over three
steps. Twenty-four diketopiperazines were prepared in
this manner. When needed, methylation was carried
out using NaH/MeI in DMF,18 and debenzylation by
catalytic hydrogenation.19
Since no synthesis of phevalin (2) has been reported, we
also devised a concise total synthesis to obtain this natural
product. Because of conflicting reports13,14 on its activity,
the previously reported cyclic dimer of N-methyltyrosine
(1) was also prepared. The peptide aldehyde Boc-Val-
Phe-H (33) was prepared for use as a positive control in
inhibition assays. The related aldehyde Z-Val-Phe-H
(MDL 28170) is reported to be a slow tight-binding inhib-
itor of rat erythrocyte calpain (Ki = 10 nM).15
A short total synthesis of phevalin20 proceeded in a
similar fashion described for the diketopiperazines ex-
cept that an additional reduction of the Boc-Val-Phe-
OMe to the corresponding aldehydes was carried out be-
fore Boc-deprotection and cyclization (Scheme 2). The
in situ oxidation during the cyclization was a surprise
to us.
Figure 2. Structural similarity between diketopiperazine and phevalin.
Figure 3. Structural list of compounds in the initial library.

3036
Y. Zeng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3034–3038
Scheme 1. General synthesis of diketopiperazine analogues.
Scheme 2. Synthesis of phevalin.
Table 1. Inhibitory activity of analogues 1–32 against recombinant human calpain I
Compds
Highest conc
tested (lM)
Percent inhibition
observeda
Compds
Highest conc
tested (lM)
Percent inhibition
observeda
1
680
550
500
180
360
280
300
650
540
97
0
0
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
130
190
300
120
200
190
580
610
210
180
920
200
350
170
150
110
0
0
2
3
0
0
4
0
0
5
0
0
6
0
30
0
7
0
8
50
0
25
0
9
10
11
12
13
14
15
16
50
50
0
0
350
100
150
160
90
0
0
0
0
13
25
0
0
0
120
0
a Inhibition values are within 10%.
Compounds 1–32 were evaluated for inhibition of calpain
I in a continuous fluorescence assay essentially as de-
scribed by Croce et al.21 using recombinant calpain I pro-
duced by the baculovirus expression system22 and Suc-
Leu-Tyr-AMC23 as the fluorogenic substrate. The in-
crease in fluorescence was linear for at least 2 min, and
inhibition was assessed from the decrease in initial rate
compared to control incubations containing pure DMSO
instead of inhibitor dissolved in DMSO. Peptide aldehyde
33, used as a positive control compound, was found to de-
crease initial rates of substrate hydrolysis by 50% at a con-
centration of 1.2 lM in our assay system (see Table 1).

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3034–3038
3037
13. Alvarez, M. E.; Houck, D. R.; White, C. B.; Brownell, J.
E.; Bobko, M. A.; Rodger, C. A.; Stawicki, M. B.; Sun, H.
H.; Gillum, A. M.; Cooper, R. J. Antibiot. 1994, 47, 1195.
14. Donkor, I. O.; Sanders, M. L. Bioorg. Med. Chem. Lett.
2001, 11, 2647.
It was disappointing to find that phevalin (2), which had
previously been reported to inhibit calpain, showed no
detectable inhibition of recombinant human calpain I
in our assay system. Likewise, and in agreement with
Donkor and Sanders,14 we found that diketopiperazine
1 also had negligible calpain inhibitory activity. The
other diketopiperazine compounds synthesized and
tested at best showed slight inhibitory activity, the best
having IC50 values in the 0.1–1.0 mM range.
15. Angelastro, M. R.; Mehdi, S.; Burkhart, J. P.; Peet, N. P.;
Bey, P. J. Med. Chem. 1990, 33, 11.
16. For recent reviews on diketopiperazine synthesis see: (a)
Dinsmore, C. J.; Beshore, D. C. Tetrahedron 2002, 58,
3297; (b) Rajappa, S.; Natekar, M. V. Adv. Heterocycl.
Chem. 1993, 57, 187; Some recent examples of parallel
synthesis: (c) Wang, D.; Liang, M.; Tian, G.; Lin, H.; Liu,
H. Tetrahedron Lett. 2002, 43, 865; (d) Kennedy, A. L.;
Fryer, A. M.; Josey, J. A. Org. Lett. 2002, 4, 1167.
17. General procedure for the parallel synthesis of 3,6-
disubstituted-2,5-piperazinediones. To a scintillation vial
(20 mL) was added a Boc-protected amino acid (1 mmol),
DCC (1 mmol), HOBT (1 mmol), and an amino acid ester
(1 mmol), followed by addition of CH2Cl2 (5 mL), DMF
(2 mL) and Et3N (1 mmol). After flushing the vial with Ar,
the reaction vessel was capped and shaken for 15 h. The
mixture was then filtered through a 5 mL syringe tube
cotton plug and washed with a small amount of EtOAc.
The volume of the reaction mixture was reduced by
flushing with a stream of argon; water (10 mL) was added
and the mixture extracted with EtOAc. The organic layer
was washed with 10% citric acid, H2O, 5% NaHCO3 and
brine, dried (Na2SO4) and evaporated. The residue was
dissolved in CH2Cl2 (3 mL) and transferred to another
scintillation vial (20 mL), followed by the addition of TFA
(0.4 mL). After flushing the vial with argon, the reaction
mixture was capped and shaken for 2 h at room temper-
ature. After evaporation of solvent by flushing with argon,
2-butanol (2 mL), toluene (0.5 mL) and Et3N (1 mmol)
were added to the residue. After flushing with argon, the
vial was sealed with Teflon cap and shaken at 99 °C for
5 h. Most of 2,5-piperazinediones crystallized from the
reaction mixture and were collected by filtration and
washed with MeOH. Analytical samples were obtained by
drying the crystals in vacuo over P2O5. The non-crystalline
2,5-piperazinediones were flashed through a silica gel
column and evaporated to dryness to afford the desired
In summary, a small library of 2,5-diketopiperazines
was synthesized as potential calpain inhibitors. How-
ever, neither these constrained dipeptide analogues nor
phevalin showed any significant inhibition against re-
combinant human calpain I. On the other hand, the
dipeptidyl aldehyde analogue Boc-Val-Phe-H was, as ex-
pected, a potent inhibitor. We have therefore refocused
our efforts in the calpain inhibition area to structure-
based methods and to a high-throughput screening
approach.
Acknowledgments
We thank the American Heart Association and the NIH
National Institute of General Medical Sciences (KU-
CMLD Center, P50-GM069663) for support of this
work. We are also grateful to Professor Lester A. Mit-
scher and Dr. Yumi Ahn for their assistance in the ini-
tial set-up of the parallel synthesis apparatus; and to
Dr. Sherri Meyer, Cephalon for proving baculovirus
AcNPV-hCANPI-2-5.
References and notes
1. Sorimachi, H.; Ishiura, S.; Suzuki, K. Biochem. J. 1997,
328, 721.
2. Ono, Y.; Sorimachi, H.; Suzuki, K. Biochem. Biophys. Res.
Commun. 1998, 245, 289.
3. Inoue, J.; Nakamura, M.; Cui, Y.-S.; Sakai, Y.; Sakai, O.;
Hill, J. R.; Wang, K. K. W.; Yuen, P.-W. J. Med. Chem.
2003, 46, 868.
4. Goll, D. E.; Thompson, V. F.; Li, H.; Wei, W.; Cong, J.
Physiol. Rev. 2003, 83, 731.
compounds.
(3S,6S)-3-Benzyl-6-(40-benzyloxy-benzyl)-
2,5-piperazinedione (7): From N-a-Boc-tyrosine-O-benzyl
ether and phenylalanine methyl ester (200 mg, 50%) as a
white crystal, mp 267–269 °C. 1H NMR (400 MHz,
DMSO-d6) d 2.18 (dd, 1H, J = 6.0 Hz, 13.6 Hz), 2.28
(dd, 1H, J = 6.0 Hz, 13.6 Hz), 2.51–2.59 (m, 2H), 3.92 (br
s, 1H), 3.98 (br s, 1H), 5.06 (s, 2H), 6.90–6.93 (m, 4H),
7.04–7.39 (m, 10H), 7.91 (d, 2H, J = 8.8 Hz, D2O
5. Takuma, K.; Baba, A.; Matsuda, T. Prog. Neurobiol.
2004, 72, 111.
exchangeable); 13C NMR (100.6 MHz, DMSO-d6)
d
6. Guicciardi, M. E.; Gores, G. J. Canc. Biol. Ther. 2003, 2,
153.
7. Donkor, I. O. Curr. Med. Chem. 2000, 7, 1171.
8. Yuen, P.-W.; Wang, K. K. W. Drugs Future 1998, 23,
741.
39.3, 56.3, 56.4, 69.9, 115.5, 127.3, 128.3, 129.0, 129.2,
129.4, 130.7, 131.7, 137.5, 138.0, 158.0, 167.0, 167.1; IR
(KBr) 1676, 1659 cmꢀ1; MS (FAB) m/e 401 (M++1);
HRMS calcd for C25H25N2O3 (M++H): 401.1865. Found:
401.1858. [a]D ꢀ113 (c 0.65, DMSO).
9. Wells, G. J.; Bihovsky, R. Exp. Opin. Ther. Pat. 1998, 8,
1707.
18. The 1,4-dimethyl-2,5-piperazinediones were obtained by
the following representative procedure. 1,4-Dihydro-2,5-
piperazinedione (0.5–1 mmol) was suspended in DMF
(10 mL) and cooled in an ice bath under Ar, NaH (60%,
3 equiv) was added and the mixture was stirred for 0.5 h
followed by addition of MeI (5 equiv). The reaction
mixture was allowed to warm up to room temperature
over 3 h. Water (30 mL) was added and the reaction
mixture extracted with EtOAc, which was sequentially
washed with H2O and brine. The organic layer was dried
(Na2SO4) and concentrated; chromatography afforded the
desired 1,4-dimethyl-2,5-piperazinediones. (3S,6S)-1,4-Di-
methyl-3-benzyl-6-(40-benzyloxy-benzyl)-2,5-piperazinedione
10. Wang, K. K. W.; Nath, R.; Posner, A.; Raser, K. J.;
Buroker-Kilgore, M.; Hajimohammadreza, I.; Probert, A.
W., Jr.; Marcoux, F. W.; Ye, Q.; Takano, E.; Hatanaka,
M.; Maki, M.; Caner, H.; Collins, J. L.; Regus, A.; Lee, K.
S.; Lunney, E. A.; Hays, S. J.; Yuen, P.-W. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 6687.
11. Alvarez, M. E.; White, C. B.; Gregory, J.; Kydd, G. C.;
Harris, A.; Sun, H. H.; Gillum, A. M.; Cooper, R. J.
Antibiot. 1995, 48, 1165.
12. Chung, M.-C.; Lee, H.-J.; Chun, H.-K.; Kho, Y.-H. J.
Microbiol. Biotechnol. 1998, 8, 188.

3038
Y. Zeng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3034–3038
(8): From 7 (420 mg, 98%), as light yellow syrup: 1H NMR
99%) in a similar fashion as a light yellow crystals, mp
207–208 °C (lit.14 208 °C); [a]D ꢀ118 (c 1.0, MeOH) (lit.14
ꢀ114, c 1, MeOH). All other physical data matched those
reported by Donkor and Sanders.14
(400 MHz, CDCl3) d 2.20–2.26 (m, 2H), 2.76–2.89 (m,
8H), 4.01–4.04 (m, 1H), 4.06–4.09 (m, 1H), 5.03 (s, 2H),
6.95–7.12 (m, 6H), 7.28–7.36 (m, 8H); 13C NMR
(100.6 MHz, CDCl3) d 34.0, 38.5, 39.5, 64.7, 64.8, 70.4,
115.6, 127.7, 127.7, 128.4, 129.0, 129.3, 129.5, 130.0, 131.1,
137.2, 137.4, 158.5, 165.9, 165.9; IR (neat) 1658,
1512 cmꢀ1; MS (FAB) m/e 429 (M++1); HRMS calcd
for C27H29N2O3 (M++H): 429.2178. Found: 429.2188.
[a]D ꢀ98 (c 0.8, MeOH).
20. Physical data for phevalin: Rf = 0.75 (95:5 CHCl3/MeOH);
mp 117–118 °C. 1H NMR (400 MHz, CDCl3) d 1.26
(d, 6H, J = 6.7 Hz), 3.44 (m, 1H), 3.85 (s, 2H), 7.26–7.38
(m, 6H); 13C NMR (100.6 MHz, CDCl3) d 20.4, 30.5, 37.0,
122.8, 127.7, 129.3, 129.6, 136.4, 137.2, 157.9, 162.4; IR
(neat) 1643, 1610 cmꢀ1; MS (FAB) m/e 229 (M++1);
HRMS calcd for C14H17N2O (M++H): 229.1341. Found:
229.1316.
21. (a) Sasaki, T.; Kikuchi, T.; Yumoto, N.; Yoshimura, N.;
Murachi, T. J. Biol. Chem. 1984, 259, 12489; (b) Croce,
K.; Flaumenhaft, R.; Rivers, M.; Furie, B.; Furie, B. C.;
Herman, I. M.; Potter, D. A. J. Biol. Chem. 1999, 274,
36321.
22. Meyer, S. L.; Bozyczko-Coyne, D.; Mallya, S. K.; Spais,
C. M.; Bihovsky, R.; Kaywooya, J. K.; Lang, D. M.;
Scott, R. W.; Siman, R. Biochem. J. 1996, 314, 511,
Baculovirus AcNPV-hCANPI-2-5 was kindly provided by
Dr. Sherri Meyer, Cephalon.
23. A typical assay condition: To a cuvette was added 6 mL
substrate (8 mM) + 460 mL buffer solution (Tris pH 7.3,
50 mM; EDTA, 0.5 mM; b ME, 10 mM) + 4 mL inhibitor
or DMSO + 10 mL calpain (1 mg/mL) + 20 mL CaCl2
(40 mM). kex = 380 nm, kem = 460 nm.
19. Debenzylation was carried out in CHCl3/MeOH at 45–50
psi H2 pressure for 6 h with 10% Pd–C as catalyst. After
filtration through Celite and concentration, the residue
was recrystallized from MeOH/EtOAc to afford the
desired debenzylated compounds. (3S,6S)-1,4-Dimethyl-
3-benzyl-6-(40-hydroxybenzyl)-2,5-piperazinedione
(4):
From 8 (290 mg, 97%), as a white crystal, mp 238–
1
239 °C. H NMR (400 MHz, DMSO-d6) d 2.11–2.21 (m,
2H), 2.50–2.63 (m, 2H), 2.65 (s, 3H), 2.71 (s, 3H), 4.06–
4.11 (m, 2H), 5.03 (s, 2H), 6.72 (d, 2H, J = 8.0 Hz), 6.84
(d, 2H, J = 8.0 Hz), 7.05–7.33 (m, 5H), 9.31 (s, 1H, D2O
exchangeable); 13C NMR (100.6 MHz, DMSO-d6) d 33.3,
33.4, 37.8, 39.0, 63.7, 63.9, 116.2, 127.5, 127.8, 129.3,
130.3, 131.4, 138.4, 157.2, 165.6, 165.7; IR (KBr) 1667,
1644 cmꢀ1; MS (FAB) m/e 339 (M++1); HRMS calcd for
C20H23N2O3 (M++H): 339.1709. Found: 339.1700. [a]D
ꢀ91 (c 1.0, DMSO). Compound 1 was obtained (180 mg,