Heterocyclic cellular lipid peroxidation inhibitors inspired by the marine antioxidant barettin

Article abstract of DOI:10.1016/j.bioorg.2018.11.024

The marine environment remains a rich source for the discovery and development of novel bioactive compounds. The present paper describes the design, synthesis and biological evaluation of a library of small molecule heterocyclic mimetics of the marine 2,5

Full text of DOI:10.1016/j.bioorg.2018.11.024

BioorganicChemistry84(2019)106–114
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Heterocyclic cellular lipid peroxidation inhibitors inspired by the marine
antioxidant barettin
Christophe Labrièrea,1, Jeanette H. Andersenb, Marte Albrigtsenb, Jørn H. Hansena,
⁎
Johan Svensona,c,
a Department of Chemistry, UiT The Arctic University of Norway, Breivika, N-9037 Tromsø, Norway
b Marbio, The Norwegian College of Fishery Science, UiT The Arctic University of Norway, Breivika, N-9037 Tromsø, Norway
c Department of Chemistry and Materials, RISE Research Institutes of Sweden, Box 857, SE-501 15 Borås, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords:
The marine environment remains a rich source for the discovery and development of novel bioactive com-
pounds. The present paper describes the design, synthesis and biological evaluation of a library of small mo-
lecule heterocyclic mimetics of the marine 2,5-diketopiperazine barettin which is a powerful natural antioxidant.
By mainly focusing on the influence from the brominated indole and heterocyclic core of barettin, a library of 19
compounds was prepared. The compounds comprised a heterocyclic core, either a 2,5 diketopiperazine, an
imidazolidinedione or a thioxothiazolidinone, which were mainly monosubstituted with ranging bulky sub-
stituents. The prepared compounds were screened for activity in a cellular lipid peroxidation assay using HepG2
cells. Several of the synthetic compounds showed antioxidant properties superior to the positive control barettin.
Two of the prepared compounds displayed inhibitory activity similar to commercial antioxidants with significant
inhibition at low µg/mL concentrations. The toxicity of the compounds was also investigated against MRC-5 lung
fibroblasts and none of the included compounds displayed any toxicity at 50 µg/mL.
2,5-Diketopiperazine
Barettin
Heterocycle
CLPAA
Structure–activity relationship
Marine natural products
1. Introduction
thetically to yield the improved analog plinabulin which has been in-
vestigated in a range of clinical studies against several types of tumors
The 2,5-diketopiperazine (DKP) motif is found in numerous bioac-
tive natural products, terrestrial and marine alike and represent the
smallest cyclic peptide. The bioactivity of natural DKPs and their syn-
thetic mimics differs vastly and was recently extensively reviewed by
Borthwick [1]. A highly relevant example from a drug discovery per-
spective is the 2,5-DKP phenylahistin initially isolated from Aspergillus
ustus NSC-F038 by Fukumoto and co-workers [2]. Phenylahistin ex-
hibits strong cell cycle inhibitory properties and it was modified syn-
[3–7]. Plinabulin and analogs thereof are potent microtubules depoly-
merisation agents with low nanomolar inhibitory concentrations [4]. A
related and also particularly interesting 2,5-DKP is barettin which was
originally isolated from the marine boreal deep-water sponge Geodia
barretti [8]. Barettin (cycloArg-(6-Br)-dehydro-Trp) has been ascribed
numerous bioactivities of which its ability to prevent settlement of
other marine micro- and macro organisms is the most thoroughly stu-
died and reported [9,10].
Abbreviations: DKP, diketopiperazine; CLPAA, cellular lipid peroxidation antioxidant assay; NMP, N-methyl-2-pyrrolidone; MTBE, methyl tert-butyl ether
⁎ Corresponding author at: Department of Chemistry and Materials, RISE Research Institutes of Sweden, Box 857, SE-501 15 Borås, Sweden.
E-mail address: Johan.svenson@ri.se (J. Svenson).
1 Present address: Université de Bretagne Occidentale, Biodimar/LEMAR UMR 6539, Rue Dumont d’Urville, 29280 Plouzané, France.
https://doi.org/10.1016/j.bioorg.2018.11.024
Received 27 September 2018; Received in revised form 1 November 2018; Accepted 17 November 2018
Availableonline22November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

C. Labrière et al.
BioorganicChemistry84(2019)106–114
In addition, barettin was recently shown by Lind and co-workers to
In order to add additional hydrophobic bulk to the indole, com-
display potent antioxidant and anti-inflammatory effects and it was
suggested that the compound may have an atheroprotective effect [11].
Only barettin and a debrominated analog were studied in the paper by
Lind et al. which limited the insights into the structure activity re-
lationship of the structural contributions to activity.
pounds 5 and 8 were further functionalised using Suzuki coupling [26].
Compound
5 was reacted with phenylboronic acid, 4-methox-
yphenylboronic acid and 4-chlorophenylboronic acid to yield 10, 11
and 12 respectively.
In a similar fashion, 8 was reacted with the same set of boronic acids
to afford compounds 13, 14 and 15 substituted in the indolic 7-posi-
tion.
Antioxidants can prevent the damage induced by oxidative stress
and free radicals and several diseases such as cardiovascular disease,
diabetes, cancer and inflammation have been associated with oxidative
damage [12,13]. Several studies have reported seemingly beneficial
effects with regards to the prevention or delayed progression of these
diseases by antioxidants but this has rarely been supported by larger
scale clinical studies [14,15]. While observations from clinical trials
into the actual effects of antioxidants remain inconsistent, several ob-
servational epidemiologic studies still imply that antioxidants can lower
the prevalence of certain diseases where oxidative damage is involved
[16,17].
Structurally simpler analogs of the brominated indole of barettin
were also included and prepared using 3 and a series of simple ben-
zaldehydes. Compound 3 was reacted with the aldehydes 16, 17 and 18
to afford the mono-acetyl derivatives 19 [27,28], 20 and 21 which
were deacetylated using hydrazine hydrate [29] to finally give 22 [28],
23 and 24 in high yields.
By reacting the bromoindole aldehydes 25 and 26 with hydantoin
(27), the central core of the barettin was replaced with a 2,4-imida-
zolidinedione core, to yield the derivatives 28 and 29 in a single step
condensation [30]. Indole hydantoin derivatives represent interesting
compounds and they have recently been shown to be promising p53-
MDM2 binding inhibitors with IC50 values down to 33 nM reported
[31]. Reaction of the same hydantoin 27 with the aldehyde 30 afforded
the derivative 31.
Based on the potent antioxidant activity of barettin we decided to
investigate this natural scaffold further with simplified synthetic com-
pounds in analogy to our ongoing work on transferring natural phar-
macophores onto DKP scaffolds [18]. The initial focus was on the in-
fluence of the heterocyclic core of barettin and also the role of the
brominated indole substituent. In Nature, Trp is often found in DKPs
and several hundred 2,5-DKPs natural product have been reported in
the literature [1]. For this purpose, a library of 19 analogs of ranging
structural complexity was designed and the compounds were evaluated
as inhibitors of the cellular lipid peroxidation in HepG2 cells. In addi-
tion, the potential cytotoxic effect of the prepared compounds was
further investigated using MRC-5 cells.
In addition, the thioxothiazolidinone derivatives 33 and 34 were
prepared as described in the literature by condensation of the aldehydes
25 and 26 with rhodanine 32 [32]. The thioxothiazolidinone core re-
presents a versatile scaffold that can be reacted with both nucleophiles
and electrophiles, that has been used to generate a range of different
biologically active compounds [33,34]. Following the same procedure,
the thioxothiazolidinone 35 was obtained by condensation of the al-
dehyde 30 with rhodanine 32 [35].
2. Results and discussion
3. Bioactivity testing
2.1. Compound design and synthesis
Following synthesis and purification, the compounds were in-
vestigated as inhibitors in a cellular lipid peroxidation antioxidant
assay employing HepG2 cells. In the CLPAA assay the lipid peroxidation
is initiated by the addition of cumene hydroperoxide and the inhibition
by the compounds can be evaluated using fluorescence after the addi-
tion of the reactive oxygen species marker C-11-BODIPY [36]. Initially
the compounds were screened at a concentration of 50 µg/mL to assess
their inhibitory potential. Compounds displaying sufficient inhibition
(< 50% remaining activity) were considered as active and were in-
cluded in a dose-response analysis to establish their EC50 values as
summarised in Table 1 and Fig. 2.
During the course of the present study, 19 heterocyclic compounds
were designed and synthesised (see Fig. 1). Their preparation (yields
reported solely for novel compounds) and biological evaluation is
presented in the following sections (see Schemes 1–7). While barettin
represents a natural promising antioxidant lead it still remains a
complicated compound to produce synthetically in significant
amounts. Relying on the isolation of natural products from natural
sources is rarely a viable option for extensive studies and the synthesis
of barettin was reported by Johnson et al. in 2004 [19]. However,
despite recent synthetic improvements such as those recently reported
by Kelley et al. [20] who employed iterative aldol condensations to
effectively generate monoalkylidene diketopiperazines, the en-
antioselective synthesis of barettin remains a low yielding synthesis.
From a rational drug design perspective a removal of the chiral
component of barettin would simplify the preparation significantly.
This strategy was also successfully employed during the transforma-
tion of phenylahistin into plinabulin [1]. As a removal of the charged
guanidine of barettin may also increase potential passive uptake
properties [21] it was decided to focus on compounds without a
charged chiral component with the main focus on the nature of the
brominated indole substituent. To further probe the structure activity
relationship of the prepared compounds, both imidazolidinedione and
thioxothiazolidinone cores where also investigated in addition to the
original 2,5-DKP scaffold.
The potential toxicity on human lung fibroblasts (MRC-5 cells) was
also evaluated using a commonly employed MTT-assay. Barettin has
previously been reported to display no reduction in viability at con-
centrations up to 100 µg/mL, (maximum concentration investigated)
against both HepG2 and MRC-5 cells. The prepared compounds were
thus investigated for toxic activity against MRC-5 cells in an analogous
fashion, initially at a concentration of 50 µg/mL. At 50 µg/mL, none of
the compounds induced a decrease in MRC-5 viability (data not shown)
and subsequently none of the compounds were evaluated in further
dose-response studies.
4. Structure-activity-relationship
The recent study on barettin and its debromo analog debromobar-
ettin revealed a CLPAA inhibition of 50% at 30 µg/mL (relative to the
positive control cumene hydroperoxide) [11]. Debromobarettin was
inactive as inhibitor implying that the influence of the halogen sub-
stituent is of major importance for the inhibitory ability of the dis-
ubstituted 2,5-DKP scaffold. Removal of the bromine has also been
observed to negatively affect the antifouling properties of barettin de-
rivatives [37]. The two compounds were also compared to the com-
mercially employed synthetic antioxidant butylated hydroxytoluene
Compound 1 was synthesised as described in the literature [22].
Compounds 4 and 7 were prepared from the reaction between the Boc-
protected aldehyde 2 or 6 and the 1,4-diacetylpiperazine-2,5-dione 3
[23]. Deprotection was carried out using hydrazine hydrate to afford 5
and 8 [24].
By changing the reaction conditions and also the number of
equivalents of 2, it was possible to also readily obtain the bis-indole
derivative 9 [25].
107

C. Labrière et al.
BioorganicChemistry84(2019)106–114
Fig. 1. Structures of the prepared compounds.
Scheme 1. Structure of compound 1. Preparation of compound 5 and 8. Reagents and conditions: (a) t-BuOK in THF, t-BuOH, NMP, RT, 24 h (4: 76%, 7: 50%); (b)
hydrazine hydrate, RT, 24 h (5: 73%, 8: 67%).
108

C. Labrière et al.
BioorganicChemistry84(2019)106–114
Scheme 2. Preparation of compound 9. Reagents and
conditions: (a) Cs2CO3, NMP, RT, 36 h; TFA, CH2Cl2,
RT, 2 h (63%).
Scheme 6. Preparation of compounds 28, 29 and 31. Reagents and conditions:
(a) piperidine, reflux, 1 h (28: 18%, 29: 10%, 31: 28%).
Scheme 3. Preparation of compound 10, 11 and 12. Reagents and conditions:
(a) PdCl2(PPh3)2, K2CO3, THF/H2O, 60 °C, 16 h (10: 79%, 11: 72%, 12: 44%).
Scheme 7. Preparation of compound 33, 34 and 35. Reagents and conditions:
(a) AcONa, ACOH, reflux, 2 h, (35: 47 %).
structures inspired by the structure of the naturally occurring barettin.
The prepared compounds are achiral and none of the compounds bear
the cationic arm of barettin. Instead focus lies on hydrophobic sub-
stituents, halogenation and alternative heterocyclic cores.
As shown in Table 1, 10 out of the 20 analysed compounds (in-
cluding barettin) displayed inhibition of cumene hydroperoxide in-
duced lipid peroxidation. It is interesting to see that several of these
simplified compounds are active despite the absence of the chiral ca-
tionic side chain found in natural barettin. Dose response analysis re-
vealed that several of the compounds (compounds 5, 9, 10, 12, 15, 28
and 31) displayed improved antioxidative activities in comparison to
Scheme 4. Preparation of compound 13, 14 and 15. Reagents and conditions:
(a) PdCl2(PPh3)2, K2CO3, THF/H2O, 60 °C, 48 h (13: 79%, 14: 72%, 15: 89%).
(BHT) which was significantly more potent with a 75% reduction of
oxidation at a concentration of 10 µg/mL [11].
The current library includes significantly simplified and rational
Scheme 5. Preparation of compounds 19–21 and 22–24. Reagents and conditions: (a) NEt3, NMP, RT, 16 h (20: 45%, 21: 80%); (b) hydrazine hydrate, RT, 2 h (23:
99%, 24: 92%).
109

C. Labrière et al.
BioorganicChemistry84(2019)106–114
Table 1
response analysis of compound 24 an EC50-value > 75 µg/mL was ob-
tained suggesting that this compound is not very active. The remaining
five active 2,5-DKPs display EC50 values between 5 and 25 µg/mL. Of
the smaller compounds 1, 5, 8, 22, 23 and 24 only compound 5 dis-
plays a pronounced activity with 10 µg/mL EC50-values. Compound 5 is
brominated in the 6-position whereas inactive compound 8 is bromi-
nated in the 7-position. In barettin, the 6-position is brominated and the
clear difference between 5 and 8 implies that this is a beneficial posi-
tion for activity. In analogy to 8, and also previous studies on debro-
mobarettin, the non brominated analog 1 is also inactive [11,37].
Compounds 22, 23, 24 were designed with smaller substituted phenyls
instead of indoles and none of them are regarded as active. The larger
compounds (10–15) prepared from 5 and 8 by additional Suzuki cou-
plings contained several active compounds. Both compounds 10 and 12
were active with a pronounced activity seen for the para-chlorinated
analog 12 (EC50 = 5 µg/mL). Compounds 13–15, substituted in the
indole 7-position from 8 are less active than their indole 6-analogs.
Para-chlorinated compound 15 still remains highly active
(EC50 = 10 µg/mL) but inferior to its indole 6-analog (12). Three bro-
minated boronic acids (2-, 3- and 4-bromophenylboronic acid) were
initially evaluated in the Suzuki couplings with an aim to generate
brominated analogs. However due to multiple side-reactions and
polymerisations, the strategy was abandoned in favor of the chlorinated
alternatives (12 and 15). To conclude the 2,5-DKP library, it appears
that halogenated compounds are more active. With regards to sub-
stitution patterns it also appears that the 6-position on the indole is
beneficial as actives 5, 9, 10 and 12 all share this substitution pattern
while the chlorinated 15 represents the only active 2,5-DKP substituted
in the 7-position. It also evident that the larger compounds are more
active than the smaller ones in this library.
Physicochemical properties of the compounds and their CLPAA inhibitory
properties.
Compound
Mw
ClogP1
Activity [50 µg/mL]
50% inhibition [µg/mL]3
2,5-Diketopiperazines
1
241.25
320.15
320.15
526.19
317.35
347.37
351.79
317.35
347.37
351.79
202.21
236.66
281.11
419.28
0.419
1.450
1.450
3.436
2.307
2.226
3.010
2.307
2.227
3.010
0.429
1.142
1.292
0.299
na2
a2
na
a
nt4
10
nt
5
8
9
12
25
nt
10
11
12
13
14
15
22
23
24
Barettin
a
na
a
5
na
na
a
nt
nt
10
nt
na
na
a
nt
> 75
30
a
Imidazolidinediones
28
29
31
306.12
306.12
277.28
2.023
2.023
2.166
a
a
a
20
> 75
2.5
Thioxothiazolidinones
33
34
35
339.23
339.23
310.39
2.823
2.823
2.966
na
na
na
nt
nt
nt
1
2
Calculated values using ChemBioDraw Ultra.
Not active or active. Active defined as > 50% inhibition and not active
as < 50% inhibition in the CLPAA assay.
3
4
Inhibition data generated from dose response analysis up to 75 µg/mL.
Not tested.
Moving over to hydantoin derivatives 28, 29 and 31, all displaying
an imidazolidinedione moiety, these derivatives all displayed antioxidant
activity in the initial screen at 50 µg/mL. This illustrates that this scaffold
is also suited for the generation of effective inhibitors. Both the 2,5-DKP
and the imidazolidinedione scaffold display a comparable ability to en-
gage in potential hydrogen bonding interactions and they appear equally
suited for the generation of this type of compounds. For compound 29,
the activity initially observed in the screen was determined to be
somewhat lower in the dose-response analysis in a similar fashion as for
24 with an EC50-value > 75 µg/mL. Worth noting is that compound 24
also is substituted in the 7-position on the indole which is in agreement
with the lower activity seen for the DKP library discussed in the previous
section. The 6-indole analog 28 was an active inhibitor (EC50 = 20 µg/
mL) which also correlates well with the SAR for the DKPs. Introduction of
the bulky aldehyde 30 generated compound 31 which is not haloge-
nated. Despite this, compound 31 represent the most active of all the
prepared compounds with a low EC50 of 2.5 µg/mL. This is superior to
compound 12 and suggest that these two compounds display antioxidant
activity equal to BHT in this assay [11].
those of barettin. The most potent inhibitors were compounds 12 and
31 which displayed low EC50 values of 5 and 2.5 µg/mL respectively.
Both of those compounds also revealed a 75% inhibition at 10 µg/mL,
which is similar to that of the commercial antioxidant BHT indicating a
particularly strong antioxidant activity [11]. These two compounds are
also superior in inhibition to the positive antioxidant control luteolin
which was included at 50 µg/mL (Table S1 in Supporting information)
in the CLPAA assay.
All compounds in the prepared library are noncharged, small, with
theoretical ClogP values ranging from 0.419 to 3.436. Compound 9
substituted with two 6-bromoindoles stands out as both the largest and
most hydrophobic compound in the library. Compound 9 is indeed an
active inhibitor but several of the other compounds display similar
activities while being both smaller and more hydrophilic so there ap-
pears to be no clear-cut link between size and polarity and activity.
As three different heterocyclic cores have been used to generate the
libraries it is also relevant to consider the SAR from this perspective.
Active compounds 5, 9, 10, 12, 15 and 24 are all 2,5-DKPs. Upon dose-
Fig. 2. Left: Dose-response analysis of the antioxidant properties of selected compounds in the CLPAA assay; -Δ- compound 5, -□- compound 15 and -×- compound
29. Right: Dose-response analysis of the antioxidant properties of compound 31 at a lower concentration range.
110

C. Labrière et al.
BioorganicChemistry84(2019)106–114
Compounds 33–35, represent thioxothiazolidinone derivatives of
stirred at room temperature for 24 h and then, diluted with water. The
precipitate was filtered, washed with ethanol, acetone and MTBE to
afford 5 as a pale yellow solid (140 mg, 73%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.00 (s, 2H), 6.94 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H),
7.60 (m, 1H), 7.61 (s, 1H), 7.94 (s, 1H), 8.94 (s, 1H), 9.54 (s, 1H), 11.74
(s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 44.9, 106.7, 108.1,
114.3, 114.6, 120.0, 122.6, 123.3, 126.0, 127.1, 136.5, 160.5, 164.5.
28, 29 and 31, and were prepared from rhodanine. Interestingly none
of these compounds were active in the initial screen which was un-
expected given that all the imidazolidinedione derivatives were active
in the initial screen and also generated the most active compound (31)
in this study. This observation implies that the sulfur containing
thioxothiazolidinone ring impairs the antioxidant capacity of the
compounds in comparison to the DKP and the imidazolidinedione
scaffolds.
FTMS (-ESI): calcd for
317.9876.
C
13H9N3O279Br−
: m/z = 317.9884, found:
5. Conclusions
6.2.3. tert-butyl (Z)-3-[(4-acetyl-3,6-dioxopiperazin-2-ylidene)methyl]-7-
bromo-1H-indole-1-carboxylate (7)
In the present study 19 synthetic heterocyclic antioxidants have
been prepared and evaluated for their ability to inhibit cumene hy-
droperoxide induced lipid peroxidation. The library contains com-
pounds inspired by the natural brominated marine antioxidant and
antifoulant barettin all of which are considerably simplified structu-
rally. Several of the prepared compounds displayed potent inhibitory
activities superior to barettin with two compounds exhibiting inhibitory
properties comparable to commercially employed antioxidants. The
high inhibitory potency observed for the most active compounds sug-
gest that these compounds should be further studied and refined in the
future. For the current library, the inhibitory activity is linked to the
nature and position of the indole substituents. The imidazolidinedione
scaffold compares well with natural 2,5-DKP scaffold while none of the
compounds incorporating a thioxothiazolidinone ring were active. The
study highlights how it is possible to generate highly active and
structurally simple synthetic antioxidants by employing a natural
marine template.
To a solution of 1,4-diacetylpiperazine-2,5-dione (2.5 g, 12.6 mmol,
1.5 eq.) and tert-butyl 7-bromo-3-formyl-1H-indole-1-carboxylate
(2.7 g, 8.3 mmol, 1.0 eq.) in a mixture of anhydrous tert-butanol
(20 mL) and NMP (20 mL) was added at 0 °C a solution of potassium
tert-butoxide in THF (1 M, 13 mL, 13.0 mmol, 1.6 eq.). The mixture was
stirred at room temperature for 24 h and then, diluted with water. The
resulting cloudy mixture was extracted with acetone and ethyl acetate,
and then, washed with a saturated ammonium chloride solution. The
organic layer was dried over Na2SO4, filtered through a silica gel pad
and evaporated under reduced pressure to afford, after trituration with
MTBE, 7 (1.9 g, 50%) as a pale yellow solid. 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 1.65 (s, 9H), 2.52 (s, 3H), 4.39 (s, 2H), 7.08 (s, 1H),
7.27 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz,
1H), 8.15 (s, 1H), 10.42 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ
(ppm): 26.8, 27.4, 45.8, 85.5, 107.3, 108.7, 112.3, 118.8, 124.9, 127.2,
129.9, 130.2, 132.7, 148.0, 161.3, 164.2, 171.9. FTMS (-ESI): calcd for
C
20H19O5N379Br−: m/z = 460.0514, found: 460.0523.
6. Experimental section
6.2.4. (Z)-3-[(7-bromo-1H-indol-3-yl)methylene]piperazine-2,5-dione (8)
To a solution of 7 (900 mg, 2.0 mmol, 1.0 eq.) in NMP (4 mL) was
added hydrazine hydrate (0.76 mL, 15.6 mmol, 8.0 eq.). The mixture
was stirred at room temperature for 24 h and then, diluted with water.
The precipitate was filtered, washed with ethanol and MTBE to afford 8
as a yellow solid (140 mg, 67%). 1H NMR (DMSO‑d6, 400 MHz): δ
(ppm): 4.00 (s, 2H), 6.94 (s, 1H), 7.05 (m, 1H), 7.39 (d, J = 6.4 Hz,
1H), 7.66 (d, J = 6.4 Hz, 1H), 7.96 (s, 1H), 8.16 (s, 1H), 9.67 (s, 1H),
11.82 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 44.8, 104.5,
106.7, 109.1, 111.7, 121.2, 123.6, 124.6, 127.2, 128.7, 134.1, 160.5,
6.1. General
NMR spectra were acquired on a Varian 7000e 400 MHz spectro-
meter and HRMS was performed employing an LTQ Orbitrap XL Hybrid
Fourier Transform mass spectrometer from Thermo Scientific. Reagents
and solvents were obtained from commercial suppliers and used
without further purification. Air-sensitive reactions were carried out
under an argon atmosphere. Reaction were monitored by thin-layer
chromatography which was carried out on aluminum-backed plates
coated with silica gel and visualised under UV light at 254 nm and
ethanolic vanillin dip.
164.6. FTMS (-ESI): calcd for
found: 317.9887.
C
13H9N3O279Br−
: m/z = 317.9884,
6.2.5. (3Z,6Z)-3,6-bis[(6-bromo-1H-indol-3-yl)methylene]piperazine-2,5-
dione (9)
6.2. Synthesis
To a solution of 1,4-diacetylpiperazine-2,5-dione (0.24 g, 1.2 mmol,
1.0 eq.) and tert-butyl 6-bromo-3-formyl-1H-indole-1-carboxylate
(1.0 g, 3.1 mmol, 2.6 eq.) in NMP (6 mL) was added cesium carbonate
(1.3 g, 3.6 mmol, 3.0 eq.). The mixture was stirred at room temperature
for 36 h and then, diluted with water. The precipitate that formed was
filtered, taken up in acetone and the volatiles were removed under
reduced pressure. The residue was taken up in dichloromethane
(10 mL) and TFA (6 mL), and the resulting solution was stirred at room
temperature for 2 h. The mixture was quenched with water, basified
with NaOH 1 M and extracted with ethyl acetate and Et2O. The organic
layer was filtered and the precipitate washed with MTBE to afford 9 as a
dark yellow solid (400 mg, 63%). 1H NMR (DMSO‑d6, 400 MHz): δ
(ppm): 7.03 (s, 2H), 7.25 (dd, J = 8.5 Hz, J = 1.5 Hz 2H), 7.63 (d,
J = 1.5 hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 8.12 (s, 2H). 13C NMR
(DMSO‑d6, 100 MHz): δ (ppm): 107.0, 108.3, 114.4, 114.8, 120.1,
122.8, 1296.1, 126.3, 127.6, 136.6, 158.2. FTMS (-ESI): calcd for
6.2.1. tert-butyl (Z)-3-[(4-acetyl-3,6-dioxopiperazin-2-ylidene)methyl]-6-
bromo-1H-indole-1-carboxylate (4)
To a solution of 1,4-diacetylpiperazine-2,5-dione (1.4 g, 6.8 mmol,
2.0 eq.) and tert-butyl 6-bromo-3-formyl-1H-indole-1-carboxylate
(1.1 g, 3.4 mmol, 1.0 eq.) in a mixture of anhydrous tert-butanol (5 mL)
and NMP (10 mL) was added at 0 °C a solution of potassium tert-but-
oxide in THF (1 M, 5.1 mL, 5.1 mmol, 1.5 eq.). The mixture was stirred
at room temperature for 24 h and then, diluted with water. The re-
sulting precipitate was filtered, washed with ethanol and diethyl ether
to afford 4 as a pale yellow solid (1.2 g, 76%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 2.51 (s, 3H), 4.38 (s, 2H), 7.09 (s, 1H), 7.50 (dd,
J = 8.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 8.24 (d, 1H),
10.42 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 27.2, 28.0, 46.2,
85.7, 109.0, 113.1, 118.0, 118.2, 126.6, 127.6, 127.9, 128.9, 135.3,
148.9, 161.7, 164.6, 172.3. FTMS (-ESI): calcd for C20H19O5N379Br−
:
m/z = 460.0503, found: 460.0513.
C
22H13N4O279Br2−: m/z = 522.9411, found: 522.9407.
6.2.2. (Z)-3-[(6-bromo-1H-indol-3-yl)methylene]piperazine-2,5-dione (5)
To a solution of 4 (280 mg, 0.60 mmol, 1.0 eq.) in NMP (5 mL) was
added hydrazine hydrate (294 µL, 6.0 mmol, 10.0 eq.). The mixture was
6.2.6. (Z)-3-[(6-phenyl-1H-indol-3-yl)methylene]piperazine-2,5-dione
(10)
To a mixture of 5 (50 mg, 0.16 mmol, 1.0 eq.) in a mixture of
111

C. Labrière et al.
BioorganicChemistry84(2019)106–114
degassed THF (4 mL) and water (2 mL) were added, under an argon
atmosphere, phenylboronic acid (29 mg, 0.24 mmol, 1.5 eq.), K2CO3
(65 mg, 0.48 mmol, 3.0 eq.) and PdCl2(PPh3)2 (11 mg, 0.016 mmol,
0.1 eq.). The reaction was stirred at 60 °C for 16 h and then, diluted
with water after cooling to room temperature. The mixture was ex-
tracted with ethyl acetate, filtered and the precipitate was washed with
ethanol, dichloromethane and MTBE to afford 10 (40 mg, 79%) as a
dark yellow solid. 1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 4.01 (s, 2H),
7.02 (s, 1H), 7.34 (t, J = 7.0 Hz, 1H), 7.45 (m, 3H), 7.70 (m, 4H), 7.98
(s, 1H), 8.12 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 44.9,
107.4, 107.9, 109.7, 118.6, 119.2, 122.8, 126.7, 127.0, 128.9, 134.5,
136.3, 141.2, 160.6, 164.4. FTMS (-ESI): calcd for C19H14O2N3−: m/
z = 316.1081, found: 316.1098.
6.2.10. (Z)-3-{[7-(4-methoxyphenyl)-1H-indol-3-yl]methylene}
piperazine-2,5-dione (14)
To a mixture of 8 (100 mg, 0.32 mmol, 1.0 eq.) in a mixture of de-
gassed THF (8 mL) and water (4 mL) were added, under an argon at-
mosphere, 4-methoxyphenylboronic acid (97 mg, 0.64 mmol, 2.0 eq.),
K2CO3 (130 mg, 0.47 mmol, 3.0 eq.) and PdCl2(PPh3)2 (22 mg,
0.031 mmol, 0.1 eq.). The reaction was stirred at 60 °C for 48 h and
then, diluted with water after cooling to room temperature. The pre-
cipitate was filtered and the residue was washed with ethanol, di-
chloromethane and MTBE to afford 14 (80 mg, 72%) as a dark yellow
solid. 1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 3.85 (s, 3H), 4.00 (s, 2H),
7.03 (s, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.15 (m, 1H), 7.20 (t, J = 7.4 Hz,
1H), 7.57 (d, J = 8.6 Hz, 2H), 7.61 (d, J = 7.4 Hz, 1H), 7.90 (s, 1H),
8.12 (s, 1H), 9.63 (s, 1H), 11.44 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz):
δ (ppm): 44.9, 55.3, 107.5, 108.2, 114.4, 116.9, 120.5, 122.0, 122.8,
125.6, 127.0, 127.9, 129.5, 132.9, 158.7, 160.9, 164.6. FTMS (-ESI):
calcd for C20H16O3N3−: m/z = 346.1197, found: 346.1192.
6.2.7. (Z)-3-{[6-(4-methoxyphenyl)-1H-indol-3-yl]methylene}piperazine-
2,5-dione (11)
To a mixture of 5 (50 mg, 0.16 mmol, 1.0 eq.) in a mixture of de-
gassed THF (4 mL) and water (2 mL) were added, under an argon at-
mosphere, 4-methoxyphenylboronic acid (36 mg, 0.23 mmol, 1.5 eq.),
K2CO3 (65 mg, 0.47 mmol, 3.0 eq.) and PdCl2(PPh3)2 (11 mg,
0.016 mmol, 0.1 eq.). The reaction was stirred at 60 °C for 16 h and
then, diluted with water after cooling to room temperature. The mix-
ture was extracted with ethyl acetate, filtered and the precipitate was
washed with ethanol, dichloromethane and MTBE to afford 11 (40 mg,
72%) as a grey solid. 1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 3.80 (s,
3H), 4.01 (s, 2H), 7.02 (m, 3H), 7.38 (d, J = 8.0 Hz, 1H), 7.61 (m, 3H),
7.68 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 8.12 (s, 1H), 9.49 (s, 1H), 11.67
(s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 44.9, 55.2, 107.5,
6.2.11. (Z)-3-{[7-(4-chlorophenyl)-1H-indol-3-yl]methylene}piperazine-
2,5-dione (15)
To a mixture of 8 (100 mg, 0.32 mmol, 1.0 eq.) in a mixture of de-
gassed THF (8 mL) and water (4 mL) were added, under an argon at-
mosphere, 4-chlorophenylboronic acid (100 mg, 0.64 mmol, 2.0 eq.),
K2CO3 (130 mg, 0.47 mmol, 3.0 eq.) and PdCl2(PPh3)2 (22 mg,
0.031 mmol, 0.1 eq.). The reaction was stirred at 60 °C for 48 h and
then, diluted with water after cooling to room temperature. The pre-
cipitate was filtered and the residue was washed with ethanol, di-
chloromethane and MTBE to afford 15 (100 mg, 89%) as a grey solid.
1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 4.00 (s, 2H), 7.02 (s, 1H), 7.23
(m, 2H), 7.60 (d, J = 8.2 Hz, 2H), 7.66 (m, 3H), 7.93 (s, 1H), 8.13 (s,
1H), 9.64 (s, 1H), 11.53 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ
(ppm): 44.9, 107.2, 108.4, 117.9, 120.5, 122.3, 123.0, 124.5, 127.1,
128.1, 129.0, 130.2, 132.2, 132.8, 137.2, 160.7, 164.6. FTMS (-ESI):
calcd for C19H13O2N335Cl−: m/z = 350.0702, found: 350.0697.
107.8, 109.1, 114.3, 118.5, 119.0, 122.7, 125.9, 126.8, 127.8, 133.6,
−
134.3, 136.4, 158.4, 160.7, 164.5. FTMS (-ESI): calcd for C20H16O3N3
:
m/z = 346.1197, found: 346.1194.
6.2.8. (Z)-3-{[6-(4-chlorophenyl)-1H-indol-3-yl]methylene}piperazine-
2,5-dione (12)
To a mixture of 5 (50 mg, 0.16 mmol, 1.0 eq.) in a mixture of de-
gassed THF (4 mL) and water (2 mL) were added, under an argon at-
mosphere, 4-chlorophenylboronic acid (38 mg, 0.23 mmol, 1.5 eq.),
K2CO3 (65 mg, 0.47 mmol, 3.0 eq.) and PdCl2(PPh3)2 (11 mg,
0.016 mmol, 0.1 eq.). The reaction was stirred at 60 °C for 16 h and
then, diluted with water after cooling to room temperature. The mix-
ture was extracted with ethyl acetate, filtered and the precipitate was
washed with ethanol, dichloromethane and MTBE to afford 12 (25 mg,
44%) as a grey solid. 1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 4.02 (s,
2H), 7.02 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 2H),
7.68 (s, 1H), 7.73 (d, J = 8.0 Hz, 3H), 8.00 (s, 1H), 9.54 (s, 1H), 11.76
(s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 44.9, 107.3, 107.9,
109.8, 118.7, 119.1, 122.9, 126.7, 127.3, 128.5, 128.8, 131.5, 133.1,
6.2.12. (Z)-1-acetyl-3-(4-chlorobenzylidene)piperazine-2,5-dione (20)
To a solution of 1,4-diacetylpiperazine-2,5-dione (1.0 g, 5.0 mmol,
1.0 eq.) and 4-chlorobenzaldehyde (703 mg, 5 mmol, 1.0 eq.) in NMP
(3 mL) was added triethylamine (0.7 mL, 5.0 mmol, 1.0 eq.). The mix-
ture was stirred at room temperature for 16 h and then, diluted with
water. The resulting precipitate was filtered, washed with acetone and
MTBE to afford 20 as a beige solid (800 mg, 45%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.36 (s, 2H), 6.93 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H),
7.60 (d, J = 8.4 Hz, 2H), 10.43 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz):
δ (ppm): 26.7, 45.6, 117.4, 127.5, 128.6, 131.3, 132.0, 133.0, 161.5,
164.1, 171.8. FTMS (-ESI): calcd for
C
13H10O3N235Cl−
:
m/
z = 277.0385, found: 277.0387.
136.3, 140.0, 160.7, 164.5. FTMS (-ESI): calcd for C19H13O2N335Cl−
:
m/z = 350.0702, found: 350.0699.
6.2.13. (Z)-1-acetyl-3-(4-bromobenzylidene)piperazine-2,5-dione (21)
To a solution of 1,4-diacetylpiperazine-2,5-dione (1.0 g, 5.0 mmol,
1.0 eq.) and 4-bromobenzaldehyde (925 mg, 5 mmol, 1.0 eq.) in NMP
(3 mL) was added triethylamine (0.7 mL, 5.0 mmol, 1.0 eq.). The mix-
ture was stirred at room temperature for 16 h and then, diluted with
water. The resulting precipitate was filtered, washed with acetone and
MTBE to afford 21 as a yellow solid (1.3 g, 80%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.36 (s, 2H), 6.93 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H),
7.60 (d, J = 8.4 Hz, 2H), 10.28 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz):
δ (ppm): 26.7, 45.6, 117.4, 127.5, 128.6, 131.3, 132.0, 133.0, 161.5,
6.2.9. (Z)-3-[(7-phenyl-1H-indol-3-yl)methylene]piperazine-2,5-dione
(13)
To a mixture of 8 (100 mg, 0.32 mmol, 1.0 eq.) in a mixture of de-
gassed THF (8 mL) and water (4 mL) were added, under an argon at-
mosphere, phenylboronic acid (78 mg, 0.64 mmol, 2.0 eq.), K2CO3
(130 mg, 0.47 mmol, 3.0 eq.) and PdCl2(PPh3)2 (22 mg, 0.031 mmol,
0.1 eq.). The reaction was stirred at 60 °C for 48 h and then, diluted
with water after cooling to room temperature. The precipitate was fil-
tered and the residue was washed with ethanol, dichloromethane and
MTBE to afford 13 (80 mg, 79%) as a grey solid. 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.00 (s, 2H), 7.02 (s, 1H), 7.21 (m, 2H), 7.44 (m,
1H), 7.55 (d, J = 7.4 Hz, 2H), 7.63 (m, 3H), 7.90 (s, 1H), 8.12 (s, 1H),
9.64 (s, 1H), 11.49 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm):
44.9, 107.3, 108.2, 117.3, 120.5, 122.3, 122.9, 125.7, 127.0, 127.4,
128.0, 128.3, 129.0, 132.8, 138.3, 160.8, 164.6. FTMS (-ESI): calcd for
164.1, 171.8. FTMS (-ESI): calcd for C13H10O3N279Br−
:
m/
z = 320.9880, found: 320.9880.
6.2.14. (Z)-3-(4-chlorobenzylidene)piperazine-2,5-dione (23)
To a mixture of 20 (200 mg, 0.72 mmol, 1.0 eq.) in NMP (2 mL) was
added hydrazine hydrate (52 µL, 1.08 mmol, 1.5 eq.). The reaction was
stirred at room temperature for 2 h. Water was added and the insoluble
was filtered, washed with water and triturated with acetone and MTBE to
C
19H14O2N3−: m/z = 316.1092, found: 316.1085.
112

C. Labrière et al.
BioorganicChemistry84(2019)106–114
6.2.19. (Z)-5-[(1H-benzo[g]indol-3-yl)methylene]-2-thioxothiazolidin-4-
afford 23 as a pale yellow solid (170 mg, 99%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.00 (s, 2H), 6.65 (s, 1H), 7.44 (m, 2H), 7.50 (m,
2H), 8.32 (s, 1H), 10.04 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm):
44.8, 112.6, 127.6, 128.6, 131.0, 132.2, 132.4, 159.8, 164.7. FTMS
(-ESI): calcd for C11H8O2N235Cl−: m/z = 235.0280, found: 235.0279.
one (35)
A mixture of rhodanine (136 mg, 1.02 mmol, 1.0 eq.), sodium
acetate (251 mg, 3.06 mmol, 3.0 eq.) and 1H-benzo[g]indole-3-carbal-
dehyde (200 mg, 1.02 mmol, 1.0 eq.) in acetic acid (2 mL) was refluxed
during 2 h. The mixture was cooled to room temperature, then diluted
with water and the precipitate was filtered to afford, after trituration
with ethanol and MTBE, 35 as a red solid (150 mg, 47%). 1H NMR
(DMSO‑d6, 400 MHz): δ (ppm): 7.50 (t, J = 7.4 Hz, 1H), 7.62 (t,
J = 7.4 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), 8.01 (d,
J = 8.2 Hz, 1H), 8.04 (m, 2H), 8.47 (d, J = 7.4 Hz, 1H), 13.15 (s, 1H),
13.61 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 112.5, 118.1,
6.2.15. (Z)-3-(4-bromobenzylidene)piperazine-2,5-dione (24)
To a mixture of 21 (200 mg, 0.62 mmol, 1.0 eq.) in NMP (2 mL) was
added hydrazine hydrate (45 µL, 0.93 mmol, 1.5 eq.). The reaction was
stirred at room temperature for 2 h. Water was added and the insoluble
was filtered, washed with water and triturated with acetone and MTBE
to afford 24 as pale yellow solid (160 mg, 92%). 1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 4.00 (s, 2H), 6.63 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H),
7.58 (d, J = 8.2 Hz, 2H), 8.32 (s, 1H), 10.05 (s, 1H). 13C NMR
(DMSO‑d6, 100 MHz): δ (ppm): 45.2, 113.1, 121.2, 128.0, 131.7, 131.9,
133.2, 160.2, 165.1. FTMS (-ESI): calcd for C11H8O2N279Br−: m/
z = 278.9775, found: 278.9774.
119.0, 120.8, 121.8, 122.1, 123.1, 124.7, 126.1, 127.2, 128.5, 130.5,
−
131.3, 169.1, 194.8. FTMS (-ESI): calcd for
C
16H9N2OS2
:
m/
z = 309.0162, found: 309.0159.
7. Cellular lipid peroxidation method
6.2.16. (E)-5-[(6-bromo-1H-indol-3-yl)methylene]imidazolidine-2,4-dione
(28)
HepG2 cells were cultured and assayed in MEM supplemented with
Earle’s salt (E-MEM, Biochrom) further supplied with fetal bovine
serum (10% FBS, Merck), L-alanyl-L-glutamine (2 mM, Merck) and
gentamycin (10 µg/mL Biochrom, A2712). The cells were seeded in
black 96-well microtiter plates with clear bottom (Corning, 3603) at
approximately 90,000 cells/well and incubated overnight in 5% CO2 at
37 °C. The cell media was replaced and cells were washed with PBS.
Total reaction volume was 100 μL. The cells were subsequently labelled
with C11-BODIPY (Invitrogen, Eugene, OR, USA) for 30 min and in-
cubated with ranging concentrations of the test compounds for 1 h. The
compounds were prepared from a DMSO stock by addition of MQ-
water, from which 20 µL were added to 80 µL of MEM to reach the
desired final concentration (maximum final DMSO concentration was
0.5%) in the wells. Cumene hydroperoxide (50 µM CumOOH final
concentration, Sigma-Aldrich) was added to initiate lipid peroxidation
and the plate was immediately installed in a Victor3 Plate Reader. Both
red (590/7 nm (excitation), 632/45 nm (emission)) and green (485/
14 nm, 520/10 nm) fluorescence were recorded to follow the lipid
peroxidation. Percent inhibition was calculated relative to the positive
control (50 µM CumOOH without test compound). The antioxidant ef-
fect of known antioxidant luteolin at 50 µg/mL was used as positive
antioxidant control and Hank’s saline solution without CumOOH was
used as negative control to ensure the quality of the in vitro assay.
A mixture of hydantoin (446 mg, 4.46 mmol, 2.5 eq.) and 6-bro-
moindole-3-carboxaldehyde (400 mg, 1.78 mmol, 1.0 eq.) in piperidine
(2 mL) was refluxed during 1 h. The mixture was cooled to room tem-
perature, then diluted with acetone and ethyl acetate. The organic layer
was washed with a 1 M HCl, dried over Na2SO4 and then evaporated
under reduced pressure. The residue was triturated with ethyl acetate,
ethanol and MTBE to afford 28 as an orange solid (250 mg, 18%). 1H
NMR (DMSO‑d6, 400 MHz): δ (ppm): 6.70 (s, 1H), 7.22 (dd, J = 8.4 Hz,
J = 1.6 Hz, 1H), 7.61 (d, J = 1.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 8.13
(s, 1H), 10.14 (bs, 1H), 11.01 (bs, 1H), 11.92 (s, 1H). 13C NMR
(DMSO‑d6, 100 MHz): δ (ppm): 100.9, 108.6, 114.4, 115.0, 120.1,
122.9, 124.3, 125.9, 127.5, 136.6, 155.2, 165.3. MS: FTMS (-ESI): calcd
for C12H7N3O279Br−: m/z = 303.9727, found: 303.9726.
6.2.17. (E)-5-[(7-bromo-1H-indol-3-yl)methylene]imidazolidine-2,4-dione
(29)
A mixture of hydantoin (223 mg, 2.23 mmol, 2.5 eq.) and 7-bro-
moindole-3-carboxaldehyde (200 mg, 0.89 mmol, 1.0 eq.) in piperidine
(1 mL) was refluxed during 1 h. The mixture was cooled to room tem-
perature, then diluted with acetone and ethyl acetate. The organic layer
was washed with 1 M HCl, dried over Na2SO4 and then evaporated
under reduced pressure. The residue was triturated with ethanol, di-
chloromethane, heptane and acetone to afford 29 as a dark yellow solid
(70 mg, 10%). 1H NMR (DMSO‑d6, 400 MHz): δ (ppm): 6.70 (s, 1H),
7.07 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz,
1H), 8.21 (s, 1H), 10.25 (s, 1H), 11.08 (s, 1H), 11.99 (s, 1H). 13C NMR
(DMSO‑d6, 100 MHz): δ (ppm): 100.8, 104.5, 109.6, 117.7, 121.5,
124.5, 124.9, 127.7, 128.6, 134.3, 155.3, 165.4. FTMS (-ESI): calcd for
8. Cytotoxicity testing
The potential cytotoxic activity of the compounds was investigated
at a concentration of 50 µg/mL against human normal lung fibroblasts
(MRC5). MRC5 cells were cultured and assayed in MEM (Biochrom)
supplied with fetal bovine serum (10% FBS, Merck), L-alanyl-L-gluta-
mine (2 mM, Biochrom), non-essential amino acids (Biochrom), sodium
pyruvate (1 mM Biochrom), NaHCO3 (0.15% Biochrom) and genta-
mycin (10 µg/mL Biochrom). The cells were seeded in 96-well micro-
titer plates (Thermo Fisher Scientific) at 4000 cells/well and incubated
for 24 h in 5% CO2 at 37 °C to allow the cells to adhere before the cell
media was replaced and the compounds added. The plates were left to
incubate with the compounds for 72 h before 10 µL of MTS solution
(CellTiter 96 Aqueous One Solution Reagent, Promega) was added to
each well, and the cells were additionally incubated for 1 h at 37 °C. The
absorbance at 485 nm was measured using a DTX multimode detector
(Beckman Coulter). The negative control was defined as cells assayed
with their respective cell media supplemented with 1% DMSO (1% was
the highest DMSO concentration used in wells with compounds), and
positive control as cells assayed with 0.5% Triton X-100 (Sigma-
Aldrich). Cell viability was calculated as follows: Cell survival
(%) = (Absorbance treated wells – absorbance positive control)/(ab-
sorbance negative control – absorbance positive control) × 100.
C
12H7N3O279Br−: m/z = 303.9727, found: 303.9727.
6.2.18. (E)-5-[(1H-benzo[g]indol-3-yl)methylene]imidazolidine-2,4-dione
(31)
A mixture of hydantoin (256 mg, 2.56 mmol, 2.5 eq.) and 1H-benzo
[g]indole-3-carbaldehyde (200 mg, 1.02 mmol, 1.0 eq.) in piperidine
(1 mL) was refluxed during 2 h. The mixture was cooled to room tem-
perature, then diluted with acetone and ethyl acetate. The organic layer
was washed with water, dried over Na2SO4 and then evaporated under
reduced pressure. The residue was triturated with ethanol and MTBE to
afford 31 as a dark red solid (80 mg, 28%).1H NMR (DMSO‑d6,
400 MHz): δ (ppm): 6.84 (s, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.57 (m, 1H),
7.59 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.97 (d, J = 7.8 Hz,
1H), 8.20 (m, 1H), 8.38 (d, J = 7.8 Hz, 1H), 10.18 (s, 1H), 11.07 (s,
1H), 12.66 (s, 1H). 13C NMR (DMSO‑d6, 100 MHz): δ (ppm): 101.7,
110.2, 118.1, 120.6, 120.9, 121.8, 122.8, 124.1, 124.2, 124.5, 127.8,
−
128.4, 130.1, 130.5, 155.3, 165.4. FTMS (-ESI): calcd for C16H10N3O2
:
m/z = 276.0778, found: 276.0778.
113

C. Labrière et al.
BioorganicChemistry84(2019)106–114
Acknowledgments
[15] E.R. Greenberg, J.A. Baron, T.D. Tosteson, D.H. Freeman Jr, G.J. Beck, J.H. Bond,
T.A. Colacchio, J.A. Coller, H.D. Frankl, R.W. Haile, A clinical trial of antioxidant
vitamins to prevent colorectal adenoma, New Engl. J. Med. 331 (1994) 141–147.
[16] K.B. Pandey, S.I. Rizvi, Plant polyphenols as dietary antioxidants in human health
and disease, Oxid. Med. Cell. Longev. 2 (2009) 270–278.
This work was partly supported with grants from the Norwegian
Research Council (ES508288) and J.S. and C.L. are grateful for the
support. J.S. was further supported by a VINNMER M.C. incoming grant
from VINNOVA (grant. 2014-01435). J.H.H. wishes to acknowledge
NORINNOVA and the Department of Chemistry at UiT for funding parts
of this project. Dr. Truls Ingebrigtsen at UiT is acknowledged for as-
sistance during the manuscript revision.
[17] A. Rietveld, S. Wiseman, Antioxidant effects of tea: evidence from human clinical
trials, J. Nutr. 133 (2003) 3285S–3292S.
[18] C. Labrière, N. Kondori, J.S. Caous, M. Boomgaren, K. Sandholm, K.N. Ekdahl,
J.H. Hansen, J. Svenson, Development and evaluation of cationic amphiphilic an-
timicrobial 2, 5-diketopiperazines, J. Pept. Sci. (2018) e3090.
[19] A.L. Johnson, J. Bergman, M. Sjögren, L. Bohlin, Synthesis of barettin, Tetrahedron
60 (2004) 961–965.
[20] E.W. Kelley, S.G. Norman, J.R. Scheerer, Synthesis of monoalkylidene diketopi-
perazines and application to the synthesis of barettin, Org. Biomol. Chem. 15
(2017) 8634–8640.
Appendix A. Supplementary material
[21] P.A. Wender, D.J. Mitchell, K. Pattabiraman, E.T. Pelkey, L. Steinman,
J.B. Rothbard, The design, synthesis, and evaluation of molecules that enable or
enhance cellular uptake: peptoid molecular transporters, Proc. Nat. Acad. Sci. 97
(2000) 13003–13008.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2018.11.024.
[22] A.L. Johnson, T. Janosik, J. Bergman, Synthesis of the diketopiperazine dipodazine,
Arkivoc 8 (2002) 57–61.
References
[23] L. Bohlin, J. Bergman, M. Sjogren, U. Goransson, Google Patents, EP1888560A1,
2005.
[1] A.D. Borthwick, 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry,
and bioactive natural products, Chem. Rev. 112 (2012) 3641–3716.
[2] K. Kanoh, S. Kohno, T. Asari, T. Harada, J. Katada, M. Muramatsu, H. Kawashima,
H. Sekiya, I. Uno, (−)-Phenylahistin: a new mammalian cell cycle inhibitor pro-
duced by Aspergillus ustus, Bioorg. Med. Chem. Lett. 7 (1997) 2847–2852.
[3] K. Kanoh, S. Kohno, J. Katada, Y. Hayashi, M. Muramatsu, I. Uno, Antitumor ac-
tivity of phenylahistin in vitro and in vivo, Biosci. Biotech. Bioch. 63 (1999)
1130–1133.
[24] P.P. Molesworth, M.G. Gardiner, R.C. Jones, J.A. Smith, R.S. Tegg, C. Wilson,
Synthesis and phytotoxicity of structural analogues of thaxtomin natural products,
Aust. J. Chem. 63 (2010) 813–820.
[25] S. Marcuccio, J. Elix, Pyrazine chemistry. II. Reduction of 3,6-dibenzylidenepiper-
azine-2,5-diones, Aust. J. Chem. 37 (1984) 1791–1794.
[26] H.L. Yeung, K.C. Sham, C.S. Tsang, T.C. Lau, H.L. Kwong, A chiral iron-sexipyridine
complex as a catalyst for alkene epoxidation with hydrogen peroxide, Chem. Comm.
(2008) 3801–3803.
[4] B. Nicholson, G.K. Lloyd, B.R. Miller, M.A. Palladino, Y. Kiso, Y. Hayashi,
S.T. Neuteboom, NPI-2358 is a tubulin-depolymerizing agent: in vitro evidence for
activity as a tumor vascular-disrupting agent, Anticancer Drugs 17 (2006) 25–31.
[5] Y. Hayashi, S. Orikasa, K. Tanaka, K. Kanoh, Y. Kiso, Total synthesis of anti-mi-
crotubule diketopiperazine derivatives: phenylahistin and aurantiamine, J. Org.
Chem. 65 (2000) 8402–8405.
[27] A.R. Katritzky, W.Q. Fan, M. Szajda, Q.L. Li, K.C. Caster, Conjugated systems de-
rived from piperazine-2,5-dione, J. Heterocyclic Chem. 25 (1988) 591–597.
[28] L.M. Yang, W. Rong-Yang, A.T. McPhail, T. Yokoi, K.H. Lee, Neihumicin, a new
cytotoxic antibiotic from Micromonospora neihuensis, J. Antibiot. 41 (1988)
488–493.
[29] C. Gallina, A. Liberatori, Condensation of 1,4-diacetylpiperazine-2,5-dione with
aldehydes, Tetrahedron 30 (1974) 667–673.
[6] M.M. Mita, M.A. Spear, L.K. Yee, A.C. Mita, E.I. Heath, K.P. Papadopoulos,
K.C. Federico, S.D. Reich, O. Romero, L. Malburg, Phase 1 first-in-human trial of the
vascular disrupting agent plinabulin (NPI-2358) in patients with solid tumors or
lymphomas, Clin. Cancer Res. 16 (2010) 5892–5899.
[30] W. Balansa, R. Islam, D.F. Gilbert, F. Fontaine, X. Xiao, H. Zhang, A.M. Piggott,
J.W. Lynch, R.J. Capon, Australian marine sponge alkaloids as a new class of gly-
cine-gated chloride channel receptor modulator, Bioorg. Med. Chem. 21 (2013)
4420–4425.
[7] A. Mita, R. Heist, O. Aren, P. Mainwaring, L. Bazhenova, S. Gadgeel, R. Blum,
J. Polikoff, J. Biswas, M. Spear, Phase II study of docetaxel with or without plina-
bulin (NPI-2358) in patients with non-small cell lung cancer (NSCLC), J. Clin.
Oncol. 28 (2010) 7592-7592.
[31] B. Graves, T. Thompson, M. Xia, C. Janson, C. Lukacs, D. Deo, P. Di Lello, D. Fry,
C. Garvie, K.S. Huang, Activation of the p53 pathway by small-molecule-induced
MDM2 and MDMX dimerization, Proc. Nat. Acad. Sci. 109 (2012) 11788–11793.
[32] S.E. Adams, C. Parr, D.J. Miller, R.K. Allemann, M.B. Hallett, Potent inhibition of Ca
2+-dependent activation of calpain-1 by novel mercaptoacrylates, MedChemComm.
3 (2012) 566–570.
[8] G. Lidgren, L. Bohlin, J. Bergman, Studies of Swedish marine organisms VII. A novel
biologically active indole alkaloid from the sponge Geodia baretti, Tetrahedron Lett.
27 (1986) 3283–3284.
[9] K.Ø. Hanssen, G. Cervin, R. Trepos, J. Petitbois, T. Haug, E. Hansen, J.H. Andersen,
H. Pavia, C. Hellio, J. Svenson, The bromotyrosine derivative ianthelline isolated
from the Arctic marine sponge Stryphnus fortis inhibits marine micro-and macro-
biofouling, Mar. Biotechnol. 16 (2014) 684–694.
[33] M.G. Bursavich, A.M. Gilbert, S. Lombardi, K.E. Georgiadis, E. Reifenberg,
C.R. Flannery, E.A. Morris, Synthesis and evaluation of aryl thioxothiazolidinone
inhibitors of ADAMTS-5 (Aggrecanase-2), Bioorg. Med. Chem. Lett. 17 (2007)
1185–1188.
[10] M. Sjögren, U. Göransson, A.L. Johnson, M. Dahlström, R. Andersson, J. Bergman,
P.R. Jonsson, L. Bohlin, Antifouling activity of brominated cyclopeptides from the
marine sponge Geodia barretti, J. Nat. Prod. 67 (2004) 368–372.
[11] K.F. Lind, E. Hansen, B. Østerud, K.E. Eilertsen, A. Bayer, M. Engqvist, K. Leszczak,
T.Ø. Jørgensen, J.H. Andersen, Antioxidant and anti-inflammatory activities of
barettin, Mar. Drugs. 11 (2013) 2655–2666.
[34] M. Stuible, L. Zhao, I. Aubry, D. Schmidt-Arras, F.D. Böhmer, C.J. Li,
M.L. Tremblay, Cellular inhibition of protein tyrosine phosphatase 1B by uncharged
thioxothiazolidinone derivatives, Chembiochem. 8 (2007) 179–186.
[35] T.B. Parsons, C. Ghellamallah, L. Male, N. Spencer, R.S. Grainger, Regioselective
dibromination of methyl indole-3-carboxylate and application in the synthesis of 5,
6-dibromoindoles, Org. Biomol. Chem. 9 (2011) 5021–5023.
[12] N.S. Dhalla, R.M. Temsah, T. Netticadan, Role of oxidative stress in cardiovascular
diseases, J. Hypertens. 18 (2000) 655–673.
[36] T. Hofer, T.E. Eriksen, E. Hansen, I. Varmedal, I.-J. Jensen, J. Hammer-Andersen,
R.L. Olsen, Studies on Experimental Models, Springer, 2011, pp. 637–657.
[37] R. Trepos, G. Cervin, C. Hellio, H. Pavia, W. Stensen, K. Stensvåg, J.S. Svendsen,
T. Haug, J. Svenson, Antifouling compounds from the sub-arctic ascidian Synoicum
pulmonaria: Synoxazolidinones A and C, pulmonarins A and B, and synthetic ana-
logues, J. Nat. Prod. 77 (2014) 2105–2113.
[13] B. Halliwell, Antioxidants in human health and disease, Annu. Rev. Nutr. 16 (1996)
33–50.
[14] D.P. Vivekananthan, M.S. Penn, S.K. Sapp, A. Hsu, E.J. Topol, Use of antioxidant
vitamins for the prevention of cardiovascular disease: meta-analysis of randomised
trials, Lancet 361 (2003) 2017–2023.
114