Solution-phase combinatorial synthesis and evaluation of piperazine-2,5- dione derivatives

Article abstract of DOI:10.1016/S0960-894X(99)00634-4

An efficient one-pot synthesis of a 61-membered combinatorial chemistry library of piperazine-2,5-diones was accomplished. Results of combinatorial synthesis, purification, analysis, and biological evaluation are described.

Full text of DOI:10.1016/S0960-894X(99)00634-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 91±94
Solution-Phase Combinatorial Synthesis and Evaluation of
Piperazine-2,5-dione Derivatives
Wendy A. Loughlin,* Raymond L. Marshall, Adelina Carreiro and Kathryn E. Elson
School of Science, Grith University, Brisbane, QLD 4111, Australia
Received 31 August 1999; accepted 21 September 1999
AbstractÐAn ecient one-pot synthesis of a 61-membered combinatorial chemistry library of piperazine-2,5-diones was accom-
plished. Results of combinatorial synthesis, puri®cation, analysis, and biological evaluation are described. # 2000 Elsevier Science
Ltd. All rights reserved.
Introduction
attack of amines on activated carbonyl groups, cycliza-
tion of N-pyruvoyl amino acid amides, intramolecular
Diels±Alder reaction, and synthesis from a-haloacyl
derivatives of amino acid esters.11 However, relatively
few methods a€ord an approach amenable to combi-
natorial synthesis that is not reliant on amino acid
building blocks. Therefore, we tested the suitability of
condensation of aldehydes with 1,4-diacetylpiperazine-
2,5-dione in the presence of base12 to generate symme-
trical and unsymmetrical derivatives in a one-pot
parallel solution-phase combinatorial synthesis, paying
particular attention to ease of isolation and purity of the
target product. The approach was modi®ed, as described
below, for the construction of a solution combinatorial
library.
Combinatorial chemistry1±3 is providing a new and
improved approach to the discovery of new and better
pharmacological agents. Small molecule libraries have
been rapidly accessed using a variety of methodologies
and techniques that have been developed for use in
solid-phase4,5 as well as solution-phase synthesis.6
Although solid-phase synthesis has been at the forefront
of combinatorial chemistry, parallel solution-phase
synthesis is an interesting alternative approach. The
advantages that characterize solution-phase synthesis
include validation time, facility of manipulations and
the diversity of reactions that can be performed.
The synthesis of piperazine-2,5-dione molecules has
received continuing interest because of their spectrum of
pharmacological activities. Examples of biologically
active 3,6-disubstituted piperazine-2,5-diones include
antibiotics such as terezine A,7 the safracins,8 and albo-
noursin,9 which also inhibits the growth of transplan-
table solid tumors in mice. Derivatives of piperazine-
2,5-diones have shown potential as therapeutic agents
and are used in medicine as antibiotics, synthetic vac-
cines, and in cancer therapy.10 In this paper we wish to
report the fast and ¯exible generation of an array of
piperazine-2,5-dione derivatives for the rapid identi®ca-
tion of potential cytotoxic agents.
Our approach to the design and synthesis of a piper-
azine-2,5-dione library consisted of the following steps:
(i) Retain the core piperazine-2,5-dione ring, a structural
element of several biologically active piperazine-2,5-
dione natural products. (ii) Develop a practical syn-
thetic procedure that allowed combinatorial synthesis.
(iii) Have a good diversity in the library by choosing a
variety of the aldehyde component such as aromatic,
heteroaromatic, aliphatic, and a,b-unsaturated alde-
hydes. (iv) Include aldehyde components that contain
polysubstituted aromatic functionality present in other
bioactive natural products such as lamellarin G, tri-
methyl ether.13 (v) Combine these elements to generate
three combinatorial sets of compounds yielding, ideally,
a small set of molecules suciently active to serve as
leads in further investigations. (vi) Gain some pre-
liminary biological data by testing members of the
library, using the brine shrimp lethality assay as an
antitumor prescreen.
Synthetic Chemistry
Methods for the synthesis of piperazine-2,5-diones
include cyclisation of dipeptide derivatives, intramolecular
*Corresponding author. Tel.: +61-7-3875-7567; fax: +61-7-3875-7656.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00634-4

92
W. A. Loughlin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 91±94
Commercial availability and appropriate functionalities
were selection factors used to generate three sets of
compounds consisting of (a) aliphatic, (b) mono-
substituted aromatic, and (c) heteroaromatic and poly-
substituted aromatic derivatives. The general procedure
developed for the preparation of a piperazine-2,5-diones
combinatorial library (Scheme 1) is now described. 1,4-
Diacetylpiperazine2,5-dione (2 mmol) was placed in a
library of quick-®t reacti-vials supported in an alumi-
nium reacti-block at 25 ꢀC. Dimethyl formamide (4 mL)
and the ®rst aldehyde (2 mmol) were added and the
mixture stirred until a homogenous solution resulted.
tert-Butyl alcohol (4 mL) and potassium tert-butoxide
(2 mmol) were added and the reaction mixture stirred
for 24 h at 25 ꢀC. The second aldehyde (2 mmol) was
added followed by further potassium tert-butoxide
(2 mmol). The reaction mixture was stirred for another
24 h at 25 ꢀC and then quenched with acetic acid (4 mL).
It was essential to alter the parallel puri®cation proce-
dure on the basis of the components used in the synth-
esis of the sets of compounds. The conditions used for
aromatic derivatives and aliphatic derivatives were ®rst
optimized on an individual target compound of the
library. The following parallel puri®cation procedures
were developed. For aromatic derivatives, the reaction
mixture was poured into a library of beakers, each con-
taining water (20 mL). The product that precipitated
was ®ltered o€ using a tandem ®ltration apparatus and
the residual solid dried in vacuo, analyzed, and tested.
For aliphatic derivatives, the reaction mixture was
extracted in the quick-®t micro-vial library using ethyl
acetate (3Â10 mL). The combined organic layers were
transferred to a library of beakers, dried (MgSO4), ®l-
tered into another quick-®t micro-vial library using a
tandem ®ltration apparatus, and the solvent removed in
vacuo using a multi-line vacuum adaptor. The dried
product was analyzed and tested.
synthesis conditions. This was noteworthy when para-
hydroxybenzaldehyde was used as an aldehyde compo-
nent. The phenol hydroxyl group must be acting as a
competing proton source and thus hindering the aldol
condensation reaction that forms the substituted piper-
azine-2,5-dione products. Yields of products varied and
were typically low in the aliphatic set of compounds and
high in the aromatic sets of compounds. The combina-
torial reaction conditions were not optimized for any
one individual compound yet rapidly provided 87% of
the expected di-substituted library formed under one
standard set of combinatorial reaction conditions. The
ease of scale of the solution combinatorial synthesis
provided sucient quantities of the piperazine-2,5-dione
derivatives for biological testing.
Biological Evaluation
In order to identify potential lead structures for further
cytotoxicity studies and identify the solubility of the
piperazine-2,5-dione derivatives for further biological
assays we carried out the brine shrimp assay for toxi-
city.15 A positive correlation exists between brine
shrimp toxicity and 9 KB cytotoxicity assay and the
brine shrimp assay is accurate in predicting in vivo
activity as cytotoxicity in a series of human solid-tumor
cell lines (p=0.033±0.334).16 We validated our assay
conditions using ca€eine15 and then tested the library of
disubstituted piperazine-2,5-diones. Artemia salina eggs
were kept for 24 h in arti®cial seawater as described
previously,15 and the nauplii (n=ꢁ20) were brought
into a 24-well micro-titre plate with brine (980 mL). The
compounds to be tested were added in various con-
centrations (1±100 mg dissolved in 20 mL of DMSO).
Each concentration was in quadruplicate. DMSO was
required to improve compound solubility. The max-
imum concentration tested was 100 mg/mL due to the
general insolubility of these compounds above this level.
After incubation at 26 ꢀC for 24 h, surviving larvae were
counted and compared to the DMSO controls. The
control deaths were <10%. The general levels of inhi-
bition are reported in Table 1. Thirteen compounds
(2aa, 2be, 2de, 2ee, 2ef, 2ms, 2nn, 2no, 2ns, 2ps, 2qr, 2qs,
and 2rr) caused a marked inhibition of all nauplii
movement at 100 mg/mL but did not cause nauplii
death. LD50s were determined for the three most potent
compounds, 2np, 2nq and 2oq and were calculated from
the dose±response curves by probit analysis,17 as being
60, 36, and 18 mg/mL, respectively.
The diversely substituted products (entries 1±70, Table
1)14 obtained from the solution combinatorial synthesis
were identi®ed by a combination of HPLC/EI±MS and
1H NMR techniques. Analysis of the samples using
mass spectroscopy was an ecient approach, which
provided unambiguous evidence that the combinatorial
synthesis was very e€ective and indicated that all iden-
ti®ed products were obtained in greater than 85% pur-
ity. All samples gave a mass spectral result that was
assigned to either the expected product (87% of the
library), or a mono or disubstituted by-product (10% of
the library). Only two cases failed to react under the
standard combinatorial reaction conditions (entries 13
and 18, Table 1). Isolation of mono or disubstituted by-
products was a result of the nonreactivity of one of the
aldehyde components under the standard combinatorial
From the general levels of inhibition and the LD50
values the mono-functionalised aromatic analogues
(entries 22±42, Table 1) displayed no levels of inhibition
at the maximal concentration used in the assay. By
contrast, members of the aliphatic and poly-functional,
heteroaromatic libraries (entries 1±21 and 43±70, Table
1) displayed varying degrees of activity. The optimal
length and branching for an aliphatic side chain appears
to be when R=C4H9 as this is the common element of
four of the ®ve aliphatic analogues the displayed inhi-
bition in Table 1. Incorporation of a pyridyl side chain,
promoted inhibition or death of the nauplii in ®ve of the
Scheme 1.

W. A. Loughlin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 91±94
Table 1. Summary of piperazine-2,5-dione derivativesa
93
No.
Yieldb (%)
ES±MSc
Observed mass
ion (MW)
Inhibition leveld
(mg/mL)e or
No.
Yield (%)
ES±MSc
Observed mass
ion (MW)
Inhibition leveld
(mg/mL)e or
LD50 (mg/mL)e
LD50 (mg/mL)e
1
2
3
4
5
6
7
8
2aa18
2ab
15
46
0
17
21
14
23
10
24
51
18
83
41
88
51
6
47
0
36
35 1f
30 1f
82
49
48
76
95
75
81
35 2hh
83
83
165 (166)
181* (180)
100
>100
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
2il
26 2ll
76
54
26
68
56
55
64
38
95
69
87
13
41
51
56
64
73
44
50
51
34
93
35
90
77
60
24
48
66
31
60
95
35
85
357f (340)
379 (380)
379 (380)
368 (369)
379 (380)
368 (369)
357 (358)
367 (368)
331* (330)
336* (335)
388 (389)
388 (389)
426h (419)
418 (419)
293* (292)
298* (297)
350 (351)
352* (351)
382* (381)
380 (381)
301 (302)
355 (356)
357* (356)
387* (386)
385 (386)
409 (410)
409 (410)
439 (440)
439 (440)
409 (410)
441* (440)
439 (440)
471* (470)
469 (470)
469 (470)
2jj12
2jk
>100y
>100y
>100y
>100y
>100y
>100y
>100y
>100y
>100y
>100y
>100y
>100y
100{
2ac
2ad19
2ae
191 (192)
221 (222)
277g (254)
193 (194)
207 (208)
205 (206)
235 (236)
267 (268)
223* (222)
219 (220)
249 (250)
281 (282)
217 (218)
247 (248)
>100y
>100
>100y
>100y
>100
>100
100
>100y
>100
>100
>100
>100
>100y
100
2jl
2kk20,21
2kl
2af15
2bb
2ll22
2mm
2mn
2mo
2mp
2mq
2mr
2ms
2nn23
2no
2np
2nq
2nr
2bc
9
2bd15
2be
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
2bf15
2cc
2cd15
2ce
2cf15
2dd16
2de
100y
100{
60y
2df
2ee
36y
285i (278)
255j (310)
269k (342)
289 (290)
319 (320)
329l (306)
334 (335)
334 (335)
323 (324)
349 (350)
351m (336)
364 (365)
364 (365)
353 (354)
259n (322)
379o (351)
379p (351)
100y
>100
100{
>100{
>100y
18y
2ef
2ns
20
2€
2oo24
2op
2oq
2or
2gg12,25
2gh26,27
2gi
>100y
>100
>100
>100{
>100y
>100
>100y
>100{
>100y
>100y
>100{
>100y
100y
2gj12
2gk
2gl
2hh29
2hi
2hj
2hk
2hl
2os
2pp28
2pq
2pr
2ps
>100{
>100y
>100y
2qq30
2qr
>100y
100{
65
2qs
2rr
2rs
2ss31
100{
2ii
2ij
2ik
6 1i
17 2jj
50 2kk
100y
>100y
>100y
aa=CH3, b=CH2CH3, c=CHꢂCH3†2, d=CHˆCHCH3, e=CH2CH2CH2CH2CH3, f=CHˆCHPh, g=Ph, h=p-CH3OC6H4, i=p-HOC6H4, j=p-
O2NC6H4, k=o-O2NC6H4, l=p-ClC6H4, m=3-Indolyl, n=2-pyridyl, o=thienyl, p=3,4-ꢂCH3O†2±C6H4, q=3,5-(CH3O)2±C6H4, r=2,4,6-
(CH3O)3±C6H4, s=3,4,5-(CH3O)3±C6H4..
bOverall yields from diacetylglycine are given.
cMass ions are generally [M H] and obtained in the negative mode. * indicates that mass ion is [M+H] obtained in positive mode.
dLowest value at which severe inhibition of nauplii movement was observed.
ey or { indicates that the compound was partially insoluble at 100 mg/mL or at 10 mg/mL, respectively.
f[M H] for 2ll.
g[M+Na].
h[M+Li].
i[M+Li].
j[M CH3] 255, calculated mass for 1f is 270.
k[M H] 269, calculated mass for 1f is 270.
l[M+Na].
m[M+H] for 2hh.
n[M H] 259, calculated mass for 1i is 260.
o[M H] for 2jj.
p[M H] for 2kk.
six analogues bearing this group. Polyether function-
ality also gave rise to inhibition or death, however, no
discernable pattern was present in these analogues, as to
the position or degree of substitution of the aromatic
ring. Although the 3,5-dimethoxy group was present in
four active analogues, combination with itself (2qq,
entry 65, Table 1) provided no activity and combination
with the thienyl group, which did not display activity in
other analogues within the library provided surprisingly
the most active analogue in the current library, 2oq. The
identi®cation of 2oq as the most active analogue serves
to illustrate the power of combinatorial chemistry to
identify nonrationally designed lead compounds that
may have potential therapeutic value.
Conclusions
In summary, we have developed a strategy to quickly
generate a library of piperazine-2,5-dione with high
purity by solution-phase parallel chemistry. The prac-
tical ease with which piperazine-2,5-dione libraries can
be prepared is worth stressing. The majority of reported
combinatorial library synthesis involves specialized

94
W. A. Loughlin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 91±94
solid supported methodologies, and equipment. In con-
trast the use of solution-phase synthesis described herein
would require little specialized knowledge or ®nancial
investment. While the three sets of compounds descri-
bed above are only of small size, their purpose is purely
illustrative. The range of functionalised sidechains
included in the piperazine-2,5-dione core for this small
library demonstrates the versatility and scope of this
procedure. Given the range of aldehydes used, one can
readily visualize a diverse range of potential library
substrates and products. More importantly the strategy
will be applicable to the synthesis of other biologically
active piperazine-2,5-diones, such as calpain inhibi-
tors,32 which bear N-substitutents and saturation at C2
and 6. Through the screening procedure we have iden-
ti®ed key side chains that are potential pharmacophores
for the generation of the next library of compounds. We
have identi®ed three piperazine-2,5-dione derivatives,
2np, 2nq and 2oq that are toxic compounds towards
brine-shrimp and thus potential lead compounds for
more speci®c cytotoxicity assays. We are currently pur-
sing the combinatorial synthesis of other analogues
based on the current results. Further screening of ana-
logues against speci®c assays to eliminate toxic analo-
gues that are not antitumor compounds and identify
which analogues are antitumor compounds will be
carried out in the future.
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MHz, CDCl3, TFA-d): 0.90±0.97 (m, 6H, 2ÂCH3), 1.25±1.40
(m, 8H, 4ÂCH2), 2.28±3.20 (m, 4H, 2ÂCH2), 6.42 (t, JCH,CH2
=
7.5 Hz, 2ÂCH), NH not observed. 3-(3,4-dimethoxy-benzyl-
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3.92 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 6.60 (s, 2H, o-C6H2
(OCH3)3), 6.82±7.10 (m, 5H, C=CHC6H3(OCH3)2, C=CH
C6H2(OCH3)3, o-C6H3(OCH3)2, m-C6H3(OCH3)2), 8.2 (br s,
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