Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity as apoptosis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.09.078

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compo

Full text of DOI:10.1016/j.bmcl.2012.09.078

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7097–7099
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of enantiomerically pure perhydro-1,4-diazepine-2,5-dione
and 1,4-piperazine-2,5-dione derivatives exhibiting potent activity
as apoptosis inhibitors
Alejandra Moure a, Mar Orzáez b, Mónica Sancho b, Angel Messeguer a,
⇑
a Department of Chemical and Biomolecular Nanotechnology, Instituto de Química Avanzada de Cataluña, Consejo Superior de Investigaciones Científica, J. Girona 18,
08034 Barcelona, Spain
b Laboratory of Peptide and Protein Chemistry, Centro de Investigación Príncipe Felipe, E-46012 Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2012
Revised 19 September 2012
Accepted 22 September 2012
Available online 29 September 2012
Apoptosis is the process of programmed cell death and plays a fundamental role in several human
diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and
1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds
1 and 2 showed a potent
in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been
an issue in the development and use of pharmaceutical drugs, the present contribution reports the
synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as
apoptosis inhibitors is also reported.
Keywords:
Perhydro-1,4-diazepine-2,5-dione
1,4-Piperazine-2,5-dione
Apoptosis inhibitors
Chiral compounds
Ó 2012 Elsevier Ltd. All rights reserved.
Enantioselective synthesis
The apoptosis phenomenon embraces a highly regulated cellu-
lar pathway responsible for the elimination of cells that are not
longer required or that have suffered extensive damage.1 Diverse
apoptotic stimuli, such as activation of cell surface death receptors,
irradiation, anticancer agents, lack of survival factors, and ische-
mia2 induce signaling cascades that activate the caspase family
of cysteine-dependent aspartate-directed proteases. These
caspases are essential to the apoptotic process, as they are needed
for its initiation and execution.3 In particular, activation of caspase-
9 is consequence of the release of proapoptotic proteins from the
mitochondrial intermembrane space into the cytosol when apop-
tosis-inducing signals are detected by the cell.4 The formation of
the macromolecular complex, apoptosome, is a key event in this
pathway, which is also known as intrinsic apoptosis pathway.
The apoptosome is a holoenzyme multiprotein complex formed
by cytochrome c-activated Apaf-1, dATP, and procaspase-9.5 When
cytochrome c is released from mitochondria it binds to Apaf-1;
then Apaf-1 hydrolyzes the bound nucleotide and promotes the
oligomerization of the Apaf-1/cytochrome c complex.4,6
The hydrolyzed nucleotide is replaced by dATP/ATP, thus result-
ing in the formation of the apoptosome. In this multiprotein
complex the Apaf-1 caspase recruitment domain (CARD) domain
becomes now accessible to bind to the CARD domain of procas-
pase-9.4,6 Apoptosome-bound procaspase-9 is then activated and
proteolyzes the downstream effector caspases leading to the pro-
gression of cell death.
Defects in apoptosis regulation are at the origin of a variety of
diseases. The resistance to induction and execution of apoptosis
that cells acquire frequently correlates with cancer or autoimmune
diseases.7 In contrast, excessive apoptosis induces unwanted cell
death, which can promote pathological conditions, such as tissue
infarction, ischemia-reperfusion damage, degenerative diseases
or AIDS.8
Consequently, the formation of the apoptosome for the devel-
opment of apoptotic modulators has become a target of high ther-
apeutic interest. In this context, we have recently reported the
synthesis of conformationally constrained peptidomimetics that
have been identified as active compounds against the apoptosome
formation.9 Specifically, compounds 1 and 2 (Fig. 1) elicit a strong
antiapoptotic activity by interacting with Apaf-1.10–12
Compounds 1 and 2 have a chiral center. Since the three-
dimensional orientation of the functional groups of bioactive chiral
compounds is often crucial for the optimum fitting into its biolog-
ical target, the availability of the chiral enantiopure forms is
Abbreviations: DIC, N,N0-diisopropylcarbodiimide; TBTU, O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate; HOBt, 1-hydroxybenzotriazole;
DIPEA, N,N-diisopropylethylamine; AcOH, acetic acid; TFA, trifluoroacetic acid;
PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
⇑
Corresponding author. Tel.: +34 93 400 6100; fax: +34 93 204 5904.
E-mail address: angel.messeguer@iqac.csic.es (A. Messeguer).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.078

7098
A. Moure et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7097–7099
Cl
We examined whether the reductive amination should be run
in two steps: imine-formation and then reduction.16 The
corresponding resin-bound intermediate was stirred with 3,3-
diphenylpropanal in 1% AcOH in DMF for 1.5 h. The imine group
was stable enough to the filtration and washing procedures.
Subsequently, the corresponding resin-bound imines were trea-
ted with a solution of NaCNBH3 in 1:1 DMF-MeOH. The mixtures
were stirred for 2 h to render the corresponding amine. Then, the
amino group was acylated with bromoacetic acid and treated with
2,4-dichlorophenethylamine to give the enantiopure compounds
(R)-5, (S)-5 and (R)-7, (S)-7. Noticeably, HPLC monitoring of (R)-7
and (S)-7 derivatives indicated the spontaneous deprotection of
the acid and further cyclisation to give the desired 1,4-
piperazine-2,5-dione compounds (R-2 and S-2) in high purity after
cleavage from the resin with a TFA/CH2Cl2/H2O cocktail. The low
energetic barriers to promote the deprotection and cyclization to
generate six-membered rings usually observed in peptide chemis-
try can account for this result.17,18
Cl
O
O
O
N
N
N
H2N
O
N
H2N
Cl
O
N
O
N
Cl
O
Cl
O
Cl
Cl
(R)-1
(R)-2
Cl
Cl
Cl
O
O
O
In the case of the perhydro-1,4-diazepine-2,5-dione (R-1 and S-
1), the acid deprotection was carried out by treatment with
Pd(PPh3)4 in the presence of PhSiH3, and the solid-phase cyclisa-
tion was promoted with PyBOP and HOBt in the presence of DIPEA.
Finally, the desired compounds were obtained after release from
the resin.
N
N
N
H2N
Cl
O
N
H2N
Cl
O
N
O
N
Cl
O
O
Cl
Cl
The residues obtained were purified by semipreparative RP-
HPLC to render the enantiopure compounds (R)-1, (S)-1, (R)-2
and (S)-2 with good overall yields, 29%, 26%, 29% and 36% ‘respec-
tively’, and enantiomeric excess values of 98%, 99%, 98% and 98%,
‘respectively’. These ee values correspond to those of the respective
starting aspartic acid substrates, which indicate that no detectable
racemization occurred during the different synthetic steps. In addi-
tion, for the case of the 1,4-piperazine-2,5-diones 2, this synthetic
procedure rendered the desired compounds with better yields than
the procedure employed previously for the racemic compounds.9
Specifically, less steps were needed, all 3 diversity sources could
be inserted sequentially followed by the spontaneous cyclization
to the final heterocyclic skeleton. In contrast, our previous syn-
thetic route involved the insertion of the third diversity source
by a solid-phase reaction following the induced formation of the
1,4-piperazine-2,5-dione intermediate bearing 2 diversity sources
in solution.9
Table 1 shows the results of the biological activity of enantio-
pure compounds (R)-1, (S)-1, (R)-2 and (S)-2 as apoptosis inhibi-
tors determined in an in vitro (reconstitution of apoptosome
from recombinant proteins) and a cell extract (reconstitution of
apoptosome in cellular extracts by addition of recombinant Apaf-
1) assays (for details see Supplementary data). Values obtained
for the corresponding racemates are indicated for comparison pur-
poses. Interestingly, there are no significant differences between
(R) and (S) enantiomers for both peptidomimetic families 1 and
2. The same behaviour was observed for the two independent as-
says; therefore we correlate the higher activity elicited by the race-
mic mixture with its higher solubility. On the other hand, the
behaviour of the racemic mixtures as aggregating inhibitors was
discarded using the enzymatic b-lactamase activity assay previ-
ously described as a method to avoid the selection of promiscuous
inhibitors.19 Finally, both compounds were unable to inhibit b-
lactamase activity.20
(S)-1
(S)-2
Cl
Figure 1. Chemical structures of the perhydro-1,4-diazepine-2,5-dione (R-1 and S-1)
and 1,4-piperazine-2,5-dione (R-2 and S-2) apoptosis inhibitors.
required.13–15 Indeed, the use of chiral drugs in enantiopure form is
now a requirement for virtually every new chemical entity and the
development of methods to synthesise enantiopure compounds
has become a key goal in medicinal chemistry.13–15 In this context,
the present contribution reports on an efficient solid-phase syn-
thetic approach that leads to both peptidomimetics 1 and 2 as
enantiopure compounds. In addition, preliminary data on the
activity of 1 and 2 enantiomers as apoptosis inhibitors are also
reported.
We investigated the possibility of using enantiopure starting
materials to obtain the desired compounds. To this aim, a straight-
forward approach was to use aspartic acid derivatives. Aspartic
acid is readily available in enantiopure form containing either
the
our strategy involved the use of N-alpha-(9-fluorenylmethyloxy-
carbonyl)- - and -aspartic acid beta-allyl ester ( - and -Fmoc-
Asp(OAll)-OH), to synthesise the enantiomers of 1 and N-alpha-
(9-fluorenylmethyloxycarbonyl)- - and -aspartic acid alpha-allyl
ester, ( - and -Fmoc-Asp(OAll)), to prepare the corresponding
a- or b-carboxylic acid moiety protected as allyl ester. Thus,
L
D
L
D
L
D
L
D
enantiomers of 2.
The desired compounds were synthesised using solid-phase
chemistry (Scheme 1). In our case, this methodology showed sev-
eral indisputable advantages over solution chemistry. The se-
quence route started by the deprotection of the Fmoc group of
the polystyrene AM RAM resin; then, the free amine was acylated
with bromoacetic acid in the presence of DIC, followed by the cou-
pling of 2,4-dichlorophenethylamine to the bromo derivative to
yield 3. Next, 3 was treated with the appropriate aspartic acid.
In summary, enantiopure peptidomimetics (R)-1, (S)-1, (R)-2
and (S)-2 have been synthesized by using an efficient solid-phase
methodology. The activities of these compounds as apoptosis
inhibitors suggest that the chiral center present in these two fam-
ilies are not a determining factor for their binding to Apaf-1. From
this information, we have turned our attention to the cis/trans con-
formers present in the exocyclic tertiary amide bond and the re-
sults obtained will be reported elsewhere.
Thus,
L
-Fmoc-Asp(OAll)-OH,
Asp(OAll) and
D
-Fmoc-Asp(OAll)-OH,
-Fmoc-Asp(OAll) were introduced using TBTU in
L-Fmoc-
D
the presence of HOBt and DIPEA, followed by removal of the Fmoc
group to give the enantiopure derivatives (R)-4, (S)-4, (R)-6 and
(S)-6. These resin-bound intermediates were subjected to reduc-
tive amination with 3,3-diphenylpropanal (for preparation see
Supplementary data).

A. Moure et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7097–7099
7099
1.
O
R3
R3
N
O
HN
O
O
HO
O
O
O
O
HN
O
Fmoc
N
NH2
R1
R2
O
O
L-Fmoc-Asp(OAll)-OH
N
O
N
R3
O
R2
O
N
2. Piperidine 20% in DMF
N
R3
O
HN
O
HN
)-4
H2N
(
(
)-5
(
S
)-1
1. Piperidine 20% in DMF
2. BrCH2COOH, DIC
3. R1NH2, Et3N
S
S
1. Removal of allyl ester
2. Cyclization
3. TFA:DCM:H2O
( 60:40:2)
H
N
H
N
1. 3,3-diphenylpropanal
2. NaCNBH3
Fmoc
NH
R1
R3
HN
3. BrCH2COOH, DIC
O
4. R3NH2, Et3N
O
N
O
3
R2
R3
O
N
R2
N
O
O
O
NH2
O
O
O
O
1.
R3
O
N
R3
O
N
R1
N
Fmoc
O
HN
O
O
HO
H2N
HN
HN
O
L-Fmoc-Asp-OAll
2. Piperidine 20% in DMF
(S)-6
R1 = R3 = 2,4-dichlorophenethyl, R2 = 3,3-diphenylpropyl
Scheme 1. Synthesis of enantiopure perhydro-1,4-diazepine-2,5-dione (S)-1 and 1,4-piperazine-2,5-dione (S)-2. The compounds (R)-1 and (R)-2 were obtained following the
(S)-7
(S)-2
same route using D-Fmoc-Asp(OAll)-OH and D-Fmoc-Asp-OAll, respectively.
References and notes
1. Pérez-Payá, E.; Orzáez, M.; Mondragón, L.; Wolan, D.; Wells, J. A.; Messeguer,
A.; Vicent, M. J. Med. Res. Rev. 2011, 31, 649.
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3. Mahrus, S.; Trinidad, J. C.; Barkan, D. T.; Sali, A.; Burlingame, A. L.; Wells, J. A.
Cell 2008, 134, 866.
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Wang, X. Cell 1997, 91, 479.
6. Acehan, D.; Jiang, X.; Morgan, D. G.; Heuser, J. E.; Wang, X.; Akey, C. W. Mol. Cell
2002, 9, 423.
Table 1
Apoptosome inhibition activity of enantiomers (R)-1, (S)-1, (R)-2 and (S)-2 and their
corresponding racemates
Compound
In vitro assay EC50
(l
M)
Cell extract Assay EC50 (lM)
0
7.9
5.0
3.2
6.2
10.6
9.6
16.8
14.4
14.0
17.0
29.9
23.9
(R)-1
(S)-1
2
(R)-2
(S)-2
7. Debatin, K. M.; Fulda, S. Apoptosis and Cancer Therapy; Wiley-VCH: Weinheim,
2006.
8. Reed, J. C. Nat. Rev. Drug Disc. 2002, 1, 111.
9. Moure, A.; Sanclimens, G.; Bujons, J.; Masip, I.; Alvarez-Larena, A.; Pérez-Payá,
E.; Alfonso, I.; Messeguer, A. Chem. Eur. J. 2011, 17, 7927.
10. Mondragón, L.; Orzáez, M.; Sanclimens, G.; Moure, A.; Armiñán, A.; Sepulveda,
P.; Messeguer, A.; Vicent, M. J.; Pérez-Payá, E. J. Med. Chem. 2008, 51, 521.
11. Malet, G.; Martin, A. G.; Orzaez, M.; Vicent, M. J.; Masip, I.; Sanclimens, G.;
Ferrer-Montiel, A.; Mingarro, I.; Messeguer, A.; Fearnhead, H. O.; Perez-Paya, E.
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Moure, A.; Messeguer, A.; Pérez-Payá, E.; Kroemer, G. Apoptosis 2009, 14, 182.
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Germany, 2006.
Acknowledgments
We thank Professer Enrique Pérez-Payá for valuable discussions
on the manuscript. Support from MICINN (Grants SAF2008-00048,
SAF2011-30542-C02-01 and BIO2007 60066), and a JAE-CSIC Fel-
lowship to A.M. are acknowledged.
16. Salvatore, R. N.; Yoon, C. H.; Jung, K. W. Tetrahedron 2001, 57, 7785.
17. Fischer, P. M. J. Pept. Sci. 2003, 9, 9.
Supplementary data
18. Carpenter, K. A.; Weltrowska, G.; Wilkes, B. C.; Schmidt, R.; Schiller, P. W. J. Am.
Chem. Soc. 1994, 116, 8450.
19. Seidler, J.; McGovern, S. L.; Doman, T. N.; Shoichet, B. K. J. Med. Chem. 2003, 46,
4477.
Supplementary data (the details of the synthesis, analytical data
and biological activity assays are described) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2012.09.078.
20. Results to be published elsewhere.