Synthesis, docking study and inhibitory activity of 2,6-diketopiperazines derived from α-amino acids on HDAC8

Article abstract of DOI:10.1016/j.bioorg.2020.104080

Diketopiperazines (DKPs) have been regarded as an important scaffold from the viewpoint of synthesis due to their biological properties for the treatment of several diseases, including cancer. In this work, two novel series of enantiomeric 2,6-DKPs derived from α-amino acids were synthesized through nucleophilic substitution and intramolecular cyclization reactions. All the compounds were docked against histone deacetylase 8 (HDAC8), which is a promising target for the development of anticancer drugs. These compounds bound into the active site of HDAC8 in a similar way to Trichostatin A (TSA), which is an HDAC8 inhibitor. This study showed that the conformation of the 2,6-DKP ring, stereochemistry, and the type of substituent on the chiral center had an important role in the binding modes. The Gibbs free energies and dissociation constants values of HDAC8-ligand complexes showed that compounds (S)-4hBn, (S)-4m, (R)-4h, and (R)-4m were more stable and affine towards HDAC8 than TSA. The inhibitory activities of 4a, (S)-4h, (S)- and (R)-4(g, l, m) were evaluated in vitro on HDAC8. It was found that compounds (R)-4g (IC₅₀ = 21.54 nM) and (R)-4m (IC₅₀ = 10.81 nM) exhibited better inhibitory activities than TSA (IC₅₀ = 28.32 nM). These results suggested that 2,6-DKPs derivatives may be promising anticancer agents for further biological studies.

Full text of DOI:10.1016/j.bioorg.2020.104080

Journal Pre-proofs
Synthesis, docking study and inhibitory activity of 2,6-diketopiperazines de-
rived from α-amino acids on HDAC8
Flor Paulina Garrido González, Teresa Mancilla Percino
PII:
S0045-2068(20)31377-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.104080
YBIOO 104080
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18 December 2019
3 July 2020
4 July 2020
Please cite this article as: F. Paulina Garrido González, T. Mancilla Percino, Synthesis, docking study and
inhibitory activity of 2,6-diketopiperazines derived from α-amino acids on HDAC8, Bioorganic Chemistry
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Synthesis, docking study and inhibitory activity of 2,6-diketopiperazines derived from
α-amino acids on HDAC8
Flor Paulina Garrido González, Teresa Mancilla Percino*
Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, Apartado Postal 14-740, 07000, Ciudad de México, México
Keywords: 2,6-Diketopiperazines, enantiomers, HDAC8, Docking, Cancer.

Corresponding author.
Tel.: +52-555-747-3396; fax: +52-555-747-3889; e-mail: tmancill@cinvestav.mx
Abstract
Diketopiperazines (DKPs) have been regarded as an important scaffold from the viewpoint
of synthesis due to their biological properties for the treatment of several diseases, including
cancer. In this work, two novel series of enantiomeric 2,6-DKPs derived from α-amino acids
were synthesized through nucleophilic substitution and intramolecular cyclization reactions. All
the compounds were docked against histone deacetylase 8 (HDAC8), which is a promising target
for the development of anticancer drugs. These compounds bound into the active site of HDAC8
in a similar way to Trichostatin A (TSA), which is an HDAC8 inhibitor. This study showed that
the conformation of the 2,6-DKP ring, stereochemistry, and the type of substituent on the chiral
center had an important role in the binding modes. The Gibbs free energies and dissociation
constants values of HDAC8-ligand complexes showed that compounds (S)-4hBn, (S)-4m, (R)-
4
h, and (R)-4m were more stable and affine towards HDAC8 than TSA. The inhibitory activities
of 4a, (S)-4h, (S)- and (R)-4(g, l, m) were evaluated in vitro on HDAC8. It was found that
compounds (R)-4g (IC = 21.54 nM) and (R)-4m (IC = 10.81 nM) exhibited better inhibitory
5
0
50
activities than TSA (IC = 28.32 nM). These results suggested that 2,6-DKPs derivatives may be
5
0
promising anticancer agents for further biological studies.
1

1
. Introduction
Diketopiperazines (DKPs), also known as piperazinediones, are considered the smallest
cyclic peptides [1]. These heterocycles are characterized by having a piperazine ring and two
carbonyl groups, the position of which leads to three isomers, 2,3-, 2,5- and 2,6-DKPs. They are
an important scaffold in many natural products [2,3], which have shown diverse biological
properties, such as antihyperglycaemic [4], antibacterial [4-6], anticancer [4,7,8], and
neuroprotective [9] agents; oxytocin antagonists [10]; PAI-1 [4], and influenza virus inhibitors
[11]; calcium channels, opioid [12], and sigma receptor [13] binders; anticonvulsant [14],
cytotoxic [15], and trypanocidal [16,17] activities. Although 2,6-DKPs have been less studied for
the development of therapeutic agents than the other isomers, several researchers have focused
on the development of synthetic strategies to deliver quick access to the imide moiety, which is
responsible for the biological properties [18,19].
On the other hand, the 2,6-DKP framework is present in the bis(2,6-DKP) named ICRF-
1
54, as well as its derivatives ICRF-159, ICRF-187, ICRF-193, and MST-16, which inhibit the
topoisomerase II enzyme that is associated with the replication and transcription of DNA
20,21]. Moreover, these compounds exhibited antitumor activity against Lewis lung carcinoma,
[
sarcoma 180 and S-37, L1210 and P388 leukemia, B16 melanoma, malignant lymphoma, C-26
colon adenocarcinoma and C-38 human colon cancer [22–25]. ICRF derivatives reached clinical
trials [23,26,27], among which MST-16 showed to be particularly efficacious in the treatment of
adult T-cell leukemia and non-Hodgkin’s lymphoma [28,29].
It has been demonstrated that histone deacetylases (HDACs) are involved in cancer
development and progression. Because cancer is one of the leading causes of death worldwide
with increasing incidence and mortality rates, several researchers have been focused on the
development of new HDACs inhibitors [30]. It is well known that HDACs catalyze the
deacetylation of N-acetyl lysine residues of histones or non-histone proteins causing the
condensation of chromatin structure, thereby promoting transcriptional repression and silencing
of genes [31]. Thus far, eighteen HDAC enzymes have been identified and classified into two
2
+
major groups based on their structural similarity: classes I, II and IV (HDACs 1-11) are Zn -
+
dependent, and class III (sirtuins 1-7) is nicotinamide adenine dinucleotide (NAD )-dependent
[32,33].
2

It is known that the overexpression of HDAC8 has been linked with several types of
cancer, including colon, breast, lung, pancreas, and childhood neuroblastoma [34]. Knockdown
of HDAC8 has shown inhibition of cell proliferation [35], reduction of clonogenic growth, cell
cycle arrest and induction of cell differentiation without affecting global histone acetylation or
cellular HDAC activity [36]. With the aim to understand the catalytic mechanism of HDAC8, X-
ray crystal structures of several inhibitors complexed with this enzyme have been studied, thus
providing detailed insight into their binding modes [37–40]. At the same time, molecular
docking study has also been a helpful tool to design and investigate new HDAC8 inhibitors
(HDAC8i) [35,41-43].
Based on this background, our research group has been interested in the synthesis [18] and
docking studies of 2,6-DKPs derived from α-amino acids. In a previous work, we studied the
molecular docking of enantiomeric 2,6-DKPs as hTopo IIα inhibitors, where the binding modes
depended on the stereochemistry [44]. Also, we performed a docking study of some (S)-2,6-
DKPs and their precursors (S)-Methyl-2-(2-amino-2-oxoethylamino)-2-alkyl (MACs) on
HDAC8. It was found that 2,6-DKPs showed interactions with both active and catalytic sites of
the enzyme, while MACs only exhibited interactions with the active site. These results suggested
that 2,6-DKPs may be better inhibitors than their precursors, and they have not been published
yet.
In this work is presented the synthesis and the study of the binding modes of two series of
2
,6-DKPs derivatives within HDAC8 by docking. Furthermore, the evaluation of the inhibitory
activity of 4a, (S)-4h, (S)- and (R)-enantiomers of 4(g, l, m) on HDAC8 enzyme.
2
. Results and discussion
2
.1. Chemistry
The synthesis of two series of 2,6-DKPs derived from α-amino acids was carried out
through two synthetic pathways as is shown in Scheme 1. Compounds 4a, (S)-4i, (S)- and (R)-
4
(b, g, j, k, m) were synthesized in two steps by deprotonation of α-amino acid methyl ester
hydrochloride salts and one-pot tandem reaction; while compounds (S)-4e, (S)- and (R)-4(c, d, f,
l) were synthesized in three steps by deprotonation of α-amino acid methyl ester hydrochloride
3

salts, nucleophilic substitution (S ) and intramolecular cyclocondensation. In the first step, the
N
commercially available α-amino acid methyl ester hydrochlorides 1a, (S)-1(b-m) or (R)-1(b-d, f,
g, j-m) reacted with KHCO under three different reaction conditions as described in the
3
experimental section to afford the corresponding α-amino esters 2a, (S)-2(b-m) and (R)-2(b-d, f,
g, j-m). The second step (ii) of the first route was a tandem reaction to form the N -C and N -C
1
2
4
5
bonds through a nucleophilic substitution (S ) and intramolecular cyclization of the α-amino
N
esters 2a, (S)-2i, (S)- or (R)-2(b, g, j-m) with 2-bromoacetamide in the presence of KHCO and
3
under reflux of acetonitrile for 24-30 h to obtain 4a, (S)-4i, (S)- and (R)-4(b, g, j-m) in good
yields (63-83%), except for 4a, which was of 40%.
O
O
H
N
R1
R2
1
O
O
R
i)
ii)
R1
R2
O
2
O
R
NH x HCl
NH2
N
H
2
1
a,(S)-1(b-m)
2a, (S)-2(b-m)
(R)-2(b-d, f, g, j-m)
4a, (S)-4(b-g, i-m)
(R)-4(b-d, f, g, j-m)
(
R)-1(b-d, f, g, j-m)
iii)
iv)
O
O
H
N
O
NH2
R1 R2
(
S)-3(c-f, l) and (R)-3(c, d, f, l)
1
2
a: R = R = H
1
2
2
1
2
(
S): R = b-m, R = H
(R): R = H, R = (b-d, f, g, j-m)
h: p-HO-C H CH
b: CH3
6
4
2
c: (CH ) CH
i: p-NH COCH O-C H CH
3
2
2
2
6
4
2
d: (CH ) CHCH
j: HOCH2
3
2
e: CH CH (CH )CH
k: CH (HO)CH
3
2
3
3
f: (CH ) (linked to N-4)
l: CH SCH CH
2
2
3
3
2
g: C H CH
2
m: (1H-indole)-3-CH2
6
5
Scheme 1. Synthetic pathways for 4a, (S)-4(b-g, i-m) and (R)-4(b-d, f, g, j-m). Reagents and
conditions: i) KHCO (1.2 equiv.), H O, rt; CH CN, rt; or reflux, 1.5 h; ii) 2-Bromoacetamide
3
2
3
(
1.2 equiv.), KHCO (2.5 equiv.), CH CN, Reflux, 24-30 h; iii) 2-Bromoacetamide (1.2 equiv.),
3 3
KHCO (1.5 equiv.), CH CN, Reflux, 6.5-9 h; iv) NaOH (1.0 equiv.), EtOH, rt, 10-20 min.
3
3
4

However, the synthesis of (S)-4e, (S)- and (R)-4(c, d, f, l) by the one-pot tandem reaction
was unsuccessful because 2,6-DKPs were obtained in low yields after a long reaction time (45
h). Then, these compounds were synthesized by the following alternative route, which involved
the S reaction between the corresponding α-amino ester 2 with 2-bromoacetamide in the
N
presence of 1.5 equiv. of KHCO and under reflux of acetonitrile for 6.5-9 h to give (S)-3e, (S)-
3
and (R)-3(c, d, f, l). Subsequently, these compounds formed the N -C bond by an intramolecular
1
2
cyclocondensation in the presence of NaOH for 10-20 min [45,46] to obtain the corresponding
,6-DKPs in 62-82% yield. In the case of synthesis of (S)-4hBn, (R)-4hBn, and (S)-4h more
reaction steps were considered [Scheme S1, supplementary information (SI)].
2
1
13
All the new 2,6-DKPs derivatives were characterized by spectroscopic data of H and C
NMR, infrared (FTIR), and High-resolution mass spectrometry (HRMS). H and 13C NMR
spectra of the target compounds confirmed the formation of the 2,6-DKP ring by the presence of
the characteristic signal of imide proton as a broad singlet at δ 8.45–10.95 ppm and the signals of
the carbonyl groups of the imide moiety at δ 171.8–175.1 ppm [18]. Their infrared spectra
1
-1
exhibited νN-H bands of amide and amine groups in the range of 3035-3411 cm , ν
and
C-H aliphatic
-1
bands in the range of 1678-1715 cm-1.
νC-H aromatic bands in the range of 2842-3198 cm and ν
C=O
The high-resolution mass spectra of the 2,6-DKPs exhibited the ion of the protoned molecules
+
[
M+H] , which were in agreement with their molecular formula (SI).
2
.2 Molecular Docking study
Molecular docking study was performed to investigate the binding modes of 4a, and the
pairs of enantiomers of 4(b-m, hBn) on the X-ray structure of HDAC8 (PDB ID: 1T64). This
enzyme has a single compact α/β domain composed of a central eight-stranded parallel β sheet
flanked by eleven α-helices, and a canonical secondary structure that link to the loops (L). The
active site is a long and narrow hydrophobic tunnel formed by His142, His143, Gly151, Phe152,
His180, Phe208, Met274, and Tyr306, which belong to several loops. At the bottom of this
2
+
tunnel, the catalytic site is found, which is constituted by Zn ion and the catalytic triad His142,
His143, and Tyr306 (Figure S1, SI) [37].
5

The docking protocol was validated through the re-docking of the co-crystallized TSA
within the HDAC8 structure. It showed that the inhibitor bound into the active site of the enzyme
providing an RMSD value of 0.91 Å. Therefore, the docking protocol was reliable for the
analysis of the binding modes of the target compounds with HDAC8.
From this analysis, it was observed that all the target compounds bound into the active site
of HDAC8. Additionally, some compounds bound with amino acid residues situated near this
site (Table S2, SI). Moreover, the Gibbs free energies (ΔG, kcal/mol), along with the
corresponding dissociation constants (K , µM) values were obtained for all the HDAC8-ligand
d
complexes in the range of -4.11 to -7.49 kcal/mol and 3.22-974.23 µM, respectively (Table 1).
These results indicated that (S)-4hBn, (S)-4m, (R)-4h, and (R)-4m showed higher stability and
affinity to HDAC8 than TSA. Among these compounds, (R)-4h exhibited the highest binding
affinity (K = 3.22 µM), and was approximately 5-fold better than (S)-4h (K = 14.31 µM).
d
d
Table 1
-1
Gibbs free energy ΔG (kcal.mol ) and dissociation constant K (µM) values of HDAC8-
d
ligand complexes
Compd.
ΔG
K
d
Compd.
ΔG
K
d
Compd.
ΔG
K
d
Compd.
ΔG
K
d
4
a
-4.81 297.54
(S)-4h
-6.61
-6.38
14.31 (R)-4h
21.14 (R)-4i
-7.49
-6.51
3.22
(
(
(
(
(
(
(
S)-4b
S)-4c
S)-4d
S)-4e
S)-4f
S)-4g
-4.76 322.30 (R)-4b
-5.24 143.12 (R)-4c
-4.69 364.99 (S)-4i
-5.44 102.09 (S)-4j
16.78
-4-42 576.44 (R)-4j
-4.32 683-81 (R)-4k
-5.43 104.73 (R)-4l
-4.11 974.23
-4.78 314.23
-5.82
54.13 (R)-4d
-6.51
-5.83
16.87 (S)-4k
53.64 (S)-4l
-5.18 159.77 (R)-4e
-4.78 314.84 (R)-4f
-6.10
34.03
4.81
-5.40 110.41 (S)-4m
-7.37
-7.09
3.93 (R)-4m -7.26
-6.94
8.19 (R)-4g
-6.76
11.16 TSA
6.31
S)-4hBn -7.36
4.01 (R)-4hBn -7.07
6.58
The study of binding modes of 2,6-DKPs and TSA with HDAC8 was based on the analysis
of the interactions, such as hydrogen bonds, π-π interactions, and coordination bonds. The
docking results showed that all the binding interactions of 2,6-DKPs with HDAC8 depended on
several effects, such as configuration, steric hindrance, and conformation of the 2,6-DKP ring.
The binding modes of the pairs of compounds whose effects mentioned above were more
pronouncedly are described as following: most of the compounds exhibited binding interactions
with the metal through imide and amine moieties, except for both enantiomers of 4i, whose
6

binding interactions were through the amide group of the substituent at the C-3 position. The
binding distances were in the range of 1.76-3.98 Å [47]. In the case of (S)- and (R)-enantiomers
of 4b, where the methyl group and H-4 were in axial position, it was observed that the binding
modes towards zinc were through N-1, O-2, and N-4, O-6, respectively, these results could be
explained based on the configuration. It was observed that (S)-2,6-DKPs with aliphatic
substituents chelated the zinc ion through O-2 and O-6 of imide moiety, this depended on the
steric hindrance of the substituent at the C-3 position. Thus, O-2 was closer to the metal as the
steric hindrance decreased, and the interaction through O-6 was favored as the volume of the
substituent increased. Fig. 1A shows this effect, where (S)-4b and (S)-4f interact through O-2
and O-6, respectively. In the case of (R)-enantiomers, where the aliphatic substituents were in
equatorial position, the chelation of the zinc ion was through O-2. Fig. 1B shows the effect
caused by the stereochemistry of (S)- and (R)-enantiomers of 4d, which interact with the metal
through O-6 and O-2, respectively.
Fig. 1. Binding modes of (S)-4b (yellow) and (S)-4f (gray) (A), and (S)-4d (orange) and (R)-4d
(
light blue) (B), with the amino acid residues of the active site of HDAC8 (light green) and Zn2+
orange).
(
In general, imide and amine moieties of all 2,6-DKPs presented hydrogen bonding
interactions with amino acid residues of the catalytic site, which bond lengths ranged from 1.69
to 3.10 Å [48].
7

Superimposition of 4j enantiomers revealed that their rings were almost overlapped and
displayed similar hydrogen bonds with His143, Gly151, and Tyr306. However, their hydroxyl
groups adopted a different orientation, so that (S)-4j only presented a hydrogen bond with
Met274 (Fig. 2A).
Fig. 2. (A) Binding modes of (S)-4j (gray) and (R)-4j (pink), (B) π-π interactions of (S)-4m
(brown) and (R)-4m (gray), with the amino acid residues of the active site of HDAC8 (light
2
+
green). Zn is represented as an orange sphere.
2
,6-DKPs containing aromatic substituents exhibited π-π interactions (stacking and T-
shaped) with His142, His143, Phe152, His180, Phe207, Phe208, and Tyr306. The binding
distances were in the range of 3.55 to 4.44 Å for π-π stacking, and 4.13-5.93 Å for π-π T-shaped
interactions, which were similar to those described in the literature [49]. Fig. 2B shows that 4m
enantiomers exhibited a different orientation within HDAC8, where the (S)-enantiomer
interacted with Phe208 (3.55-4.30 Å) and the (R)-enantiomer with Phe152, His180, and Tyr306
(4.45-5.83 Å) by π-π stacking interactions. The latter enantiomer also showed π-π T-shaped
interaction with Phe208 at 4.01 Å.
It was found that (S)-4g showed binding modes similar to those observed for TSA. Fig. 3A
shows the orientation of (S)-4g and the re-docked pose of TSA within the active site of HDAC8,
where the imide and hydroxamic acid moieties of (S)-4g and TSA, respectively, showed
interactions with the metal. Therefore, the imide moiety acts as a Zn-binding group (ZBG). Also,
(S)-4g and TSA exhibited similar hydrogen bonds with His142, Gln263, and Gly304. Although
imide moiety of (R)-4g showed an opposite orientation to the ZBG of TSA, its aromatic group
8

presented π-π interactions with the catalytic triad that TSA did not show (Fig 3B). The binding
modes of the compounds mentioned above, along with their binding distances are shown in
Figures S2-S12, SI.
Fig. 3. (A) Hydrogen bonds and coordination bonds of (S)-4g (yellow) and TSA (gray), (B) π-π
interactions of (R)-4g (blue), and TSA (gray) with the amino acid residues of the active site of
2
+
HDAC8 (light green). Zn is represented as an orange sphere.
The binding modes of the 2,6-DKPs showed the importance of aromatic groups, imide, and
amine moieties for binding to HDAC8. Based on these findings, it could be considered that 2,6-
DKPs may be HDAC8 inhibitors and possible anticancer agents.
2
.3. HDAC8 inhibition assay
The in vitro inhibitory activity of 4a, (S)-4h, (S)- and (R)-enantiomers of 4(g, l, m) was
evaluated with the HDAC8 Fluorometric Drug Discovery Kit, using TSA as a positive control.
As shown in Table 2, the pair of enantiomers (S)-4m (IC = 51.11 ± 1.90 nM) and (R)-4m (IC
5
0
50
=
10.81 ± 0.18 nM) exhibited better inhibitory activities than the pairs of enantiomers (S)-4g
(
IC = 67.40 ± 0.58 nM) and (R)-4g (IC = 21.54 ± 0.79 nM); (S)-4l (IC = 88.43 ± 0.48 nM)
5
0
50
50
and (R)-4l (IC = 79.66 ± 0.51 nM). A comparison of the inhibitory effect of enantiomeric pairs
5
0
showed that (R)-enantiomers exhibited better inhibitory activities than (S)-enantiomers.
Furthermore, IC values revealed that (R)-4g and (R)-4m displayed higher inhibitory activities
5
0
9

than TSA. These results suggested that (R)-enantiomers of 4g and 4m may be good candidates
for further in vitro and in vivo studies.
Table 2
Inhibitory activity of 4a, (S)-4(g, h, l, m),
(R)-4(g, l, m) and TSA on HDAC8 enzyme
H
O
N
O
R1
R2
N
H
Compound R
IC50 (nM)
1
2
4
a
R , R = H
116.50 ± 1.67
67.40 ± 0.58
21.54 ± 0.79
82.05 ± 1.25
88.43 ± 0.48
79.66 ± 0.51
51.11 ± 1.90
10.81 ± 0.18
28.32 ± 1.23
1
2
(
(
(
(
(
(
(
S)-4g
R)-4g
S)-4h
S)-4l
R = C
6
H
5
CH
2
, R = H
1
2
R = H, R = C
6
H
5
CH
2
1
2
R = p-HO-C
6
H
4
CH
2
, R = H
1
2
R = CH
3
SCH
2
CH
2
, R = H
1
2
R)-4l
S)-4m
R)-4m
R = H, R = CH
3
SCH
2
CH
2
1
2
R = (1H-indole)-3-CH
2
, R = H
1
2
R = H, R = (1H-indole)-3-CH
2
TSA
TSA was used as a positive control.
Results are the mean ± standard deviation of three
separate determinations.
3
. Conclusion
In conclusion, two series of novel enantiomeric 2,6-DKPs were synthesized in good yields
by two synthetic routes. The molecular docking results suggested that binding modes of these
enantiomers within HDAC8 depended on several effects, such as the steric hindrance of the
substituent on the chiral center, configuration, and conformation of the 2,6-DKP ring. In general,
the imide and amine moieties of the target compounds interacted with the zinc ion and amino
acid residues of the active site of the enzyme, where the imide moiety was identified as a novel
Zn-binding group (ZBG). According to the inhibition assays, all the evaluated 2,6-DKPs
derivatives exhibited inhibitory activity on HDAC8. Among them, (R)-4g (IC = 21.54 nM) and
5
0
(
R)-4m (IC = 10.81 nM) showed better enzymatic inhibitory activities than TSA (IC = 28.32
5
0
5
0
1
0

nM). To sum up, these findings demonstrated that promising (R)-4g and (R)-4m may be possible
anticancer agents for further biological studies.
4
. Experimental section
4
.1. Materials and methods
®
All solvents and reagents were purchased from Sigma Aldrich Co and used without
further purification. Flash column chromatography (FC) was performed on Merck Silica gel 60
1
13
(
63-200 mesh ASTM). H and C NMR spectra were recorded on a JEOL ECA 500, JEOL
ECLIPSE 400 and Bruker Avance III HD 400 MHz spectrometers. Chemical shifts (δ) were
expressed in parts per million (ppm) relative to tetramethylsilane, which was used as the internal
reference. CDCl and DMSO-d were used as solvents. The following abbreviations were used to
3
6
describe the signal patterns: s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad). High-
resolution mass spectra were recorded on an Agilent G1969A HRMS-TOF or Bruker
micrOTOF-Q II spectrometers, both equipment with electrospray ionization (ESI). The Infrared
spectra were obtained on a Varian FTIR-640 spectrometer. Melting points were determined on a
Gallenkamp equipment MFB-595 with open capillary tubes and were uncorrected. HDAC8
®
Inhibitor Screening kit was purchased from Sigma Aldrich Co Catalog Number EPI007.
Fluorescence measurements were carried out using a Thermo Scientific Fluoroskan Ascent FL
spectrofluorometer. D-amino acid methyl ester hydrochlorides (R)-1(c, d, f, h, k) were prepared
according to the literature procedure [50,51]. 4a, (S)-4b, (R)-4b, (S)-4d and (S)-4f have been
already reported, and their NMR spectroscopic data were in agreement with the literature data
[18,45]. (S)-4(c-f), (S)-4l, (R)-4c, (R)-4d, (R)-4f and (R)-4l were prepared according to the
literature method [45,46].
4
.2. General procedure for the synthesis of α-amino acid methyl esters 2a, (S)-2(b-h, j-m)
and (R)-2(b-d, f-h, j-m)
In this section, we present the general procedure for the synthesis of 2a, (S)-2(b, f, j, k, l),
and (R)-2(b, f, j) by method A; (S)-2c, (R)-2c, (S)-2e, and (R)-2d by method B; and (S)-2d, (R)-
2
(k, l), (S)- and (R)-2(g, h, m) by method C.
1
1

Method A: A mixture of the corresponding α-amino acid methyl ester hydrochloride (1.0
equiv.) and potassium bicarbonate (1.5 equiv.) in acetonitrile (60 mL) was stirred at room
temperature for 1.5 h. Then, the mixture was filtered and concentrated under reduced pressure to
afford the corresponding α-amino acid methyl ester as a yellow oil. Yield: 60-100% (Table S1,
SI). The crude product was used in the next step without further purification.
Method B: A mixture of the corresponding α-amino acid methyl ester hydrochloride (1.0
equiv.) and potassium bicarbonate (1.5 equiv.) in acetonitrile (60 mL) was stirred under reflux
for 1.5 h. The reaction mixture was cooled, filtered, and concentrated under reduced pressure to
give the corresponding α-amino acid methyl ester as a yellow oil. Yield: 79-100% (Table S1,
SI). The crude product was used in the next step without further purification.
Method C: A solution of the corresponding α-amino acid methyl ester hydrochloride (1.0
equiv.) and potassium bicarbonate (1.5 equiv.) in H O (20 mL) was stirred at room temperature
2
for 1.5 h. The reaction mixture was extracted with chloroform (3 x 30 mL). The combined
organic layers were dried over anhydrous Na SO , filtered, and evaporated under reduced
2
4
pressure to afford the corresponding α-amino acid methyl ester as a yellow oil. Yield: 85-100%
Table S1, SI). The crude product was used in the next step without further purification.
(
4
.3. Synthesis of (S)-4(c, e, g, i-m) and (R)-4(c, d, f, g, j-m)
2
,6-DKPs were prepared by two procedures, one of them involved the SN and
intramolecular cyclocondensation of the corresponding α-amino esters (S)-2i, (S)- or (R)-2(g, j,
k, m) with 2-bromoacetamide by the one-pot tandem reaction, and the other one involved the SN
of the corresponding α-amino esters (S)-2e, (S)- or (R)-2(c, d, f, l) with 2-bromoacetamide
followed by intramolecular cyclocondensation of (S)-3(c-f, l) or (R)-3(c, d, f, l) [45,46].
4
.3.1. Synthesis of (S)-4g, (S)-4(i-k, m), (R)-4g and (R)-4(j, k, m)
The methodology described below is a general procedure for the synthesis of (S)-4g, (S)-
(i-k, m), (R)-4g and (R)-4(j, k, m) with some variations in reaction time and purification, which
4
are indicated in their synthesis.
1
2

4
.3.1.1. Synthesis of (3S)-3-benzylpiperazine-2,6-dione (S)-4g
A mixture of (S)-2g (1.00 g, 5.58 mmol), KHCO (1.40 g, 13.95 mmol) and 2-
3
bromoacetamide (0.94 g, 6.70 mmol) in acetonitrile (60 mL) was stirred under reflux for 30 h.
After cooling to room temperature, the mixture was filtered and concentrated under reduced
pressure. The product was precipitated using a mixture of CHCl /acetone (5:1, 20 mL) and
3
filtered under vacuum to afford (S)-4g as a white powder. Yield 0.72 g (63%); m.p. 174-176 °C.
1
H NMR (500 MHz, DMSO-d ): δ 2.72 (1H , dd, J = 14.2 Hz, JBX = 9.7 Hz), 2.85 (1H, m),
6
B
AB
3
.13 (1H , dd, J = 14.2 Hz, J = 3.8 Hz), 3.32 (1H overlapped with the signal of H O from
A AB AX B, 2
DMSO-d ), 3.41 (1H , dd, J = 17.5 Hz, JAX = 5.5 Hz), 3.54 (1H , td, J = 9.7 Hz, JAX = 3.8
6
A
AB
X
BX
1
3
Hz), 7.14-7.24 (5H, m), 10.83 (1H, s). C NMR (125 MHz, DMSO-d ): δ 35.7, 48.3, 58.7,
6
-1
1
26.6, 128.5, 129.8, 139.1, 173.9, 174.9. ATR-FTIR (ν, cm ): 3281, 2984, 1711, 1689. HRMS
+
(
ESI): [M+H] calcd for C H N O 205.09715, found 205.09743.
1
1
13
2
2
4
.3.1.2. (3R)-3-benzylpiperazine-2,6-dione (R)-4g
The procedure was the same as described above for the synthesis of (S)-4g to afford (R)-4g
1
as a white powder. Yield 0.89 g (78%); m.p. 174-176 °C. H NMR (400 MHz, DMSO-d ): δ
6
2
3
3
.73 (1H , dd, J = 14.0 Hz, JBX = 9.8 Hz), 2.85 (1H, m), 3.13 (1H , dd, J = 14.0 Hz, JAX =
B AB A AB
.8 Hz), 3.32 (1H , dd, J = 17.6 Hz, J = 9.0 Hz), 3.41 (1H , dd, J = 17.6 Hz, J = 5.4 Hz),
B
AB
BX
A
AB
AX
1
3
.54 (1H , td, J = 9.8 Hz, J = 3.8 Hz), 7.14-7.24 (5H, m), 10.82 (1H, s). C NMR (100
X
BX
AX
MHz, DMSO-d ): δ 35.7, 48.3, 58.8, 126.6, 128.6, 129.8, 139.2, 173.9, 174.9. ATR-FTIR (ν,
6
-1
+
cm ): 3281, 2985, 1712, 1692. HRMS (ESI): [M+H] calcd for C H N O 205.09715, found
1
1
13
2
2
2
05.09737.
4
.3.1.3. (3S)-2-(4-((3,5-dioxopiperazin-2-yl)methyl)phenoxy)acetamide (S)-4i
A mixture of (S)-2h (1.44 g, 7.38 mmol), KHCO (2.60 g, 25.82 mmol) and 2-
3
bromoacetamide (2.28 g, 16.23 mmol) in acetonitrile (90 mL) was stirred under reflux for 27 h.
The crude product was washed with MeOH (15 mL) and filtered. The remaining solid was
o
washed with CH CN (10 mL) at 50 C and filtered under vacuum to afford (S)-4i as a yellow
3
1
3

1
powder. Yield: 1.37 g (67%); m.p. 192-194 °C. H NMR (500 MHz, DMSO-d ): δ 2.67 (1H ,
6
B
dd, J = 14.1 Hz, J = 9.6 Hz), 2.80 (1H, m), 3.05 (1H , dd, J = 14.1 Hz, J = 3.6 Hz), 3.31
AB
BX
A
AB
AX
(
1H , dd, J = 17.7 Hz, J = 9.2 Hz), 3.40 (1H , dd, J = 17.7 Hz, J = 5.3 Hz), 3.47 (1H ,
B AB BX A AB AX X
td, J = 9.6 Hz, J = 3.6 Hz), 4.34 (2H, s), 6.80 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.5 Hz),
BX
AX
1
3
7
6
2
.35 (1H, s), 7.46 (1H, s), 10.80 (1H, s). C NMR (125 MHz, DMSO-d ): δ 34.7, 48.2, 58.8,
6
-1
7.2, 114.8, 130.8, 131.5, 156.7, 170.6, 173.9, 174.9. ATR-FTIR (ν, cm ): 3398, 3283, 3198,
+
993, 1698, 1660. HRMS (ESI): [M+H] calcd for C H N O 278.11353, found 278.11335.
1
3
16
3
2
4
.3.1.4. (3S)-3-(hydroxymethyl)piperazine-2,6-dione (S)-4j
From (S)-2j (2.00 g, 16.78 mmol), KHCO (4.22 g, 41.97 mmol) and 2-bromoacetamide
3
(2.84 g, 20.15 mmol) in acetonitrile (100 mL); (S)-4j was purified by precipitation with EtOH
1
(
45 mL). Pink powder; yield 1.89 g (78%); m.p. 158-160 °C. H NMR (500 MHz, DMSO-d ): δ
6
2
.93 (1H, br s), 3.32-3.38 (1H, 1H , overlapped with the signal of H O), 3.48 (1H , d, J = 17.4
B 2 A AB
1
3
Hz), 3.60 (1H, m), 3.78 (1H, m), 4.88 (1H, t, J = 5.3 Hz), 10.79 (1H, s). C NMR (125 MHz,
-1
DMSO-d ): δ 48.4, 59.1, 61.1, 174.0, 174.1. ATR-FTIR (ν, cm ): 3302, 3232, 3078, 2947,
6
+
1
702. HRMS (ESI): [M+H] calcd for C H N O 145.06077, found 145.06095.
5
v
9
2
3
4
.3.1.5. (3R)-3-(hydroxymethyl)piperazine-2,6-dione (R)-4j
From (R)-2j (2.00 g, 16.78 mmol), KHCO (4.22 g, 41.97 mmol) and 2-bromoacetamide
3
(2.84 g, 20.15 mmol) in acetonitrile (100 mL); the product was purified in the same way as
1
described above for (S)-4j. Pink powder; yield 1.89 g (78%); m.p. 158-160 °C. H NMR (400
MHz, DMSO-d ): δ 3.03 (1H, br s), 3.36 (1H, overlapped with the signal of H O), 3.42 (1H , d,
6
2
B
JAB = 17.6 Hz), 3.53 (1H , d, J = 17.6 Hz), 3.65 (1H , d, J = 10.3 Hz), 3.83 (1H , d, J =
AB
A
AB
B
AB
A
1
3
1
1
0.3 Hz), 4.90 (1H, s), 10.83 (1H, s). C NMR (100 MHz, DMSO-d ): δ 48.4, 59.1, 61.3, 173.9,
6
-1
+
74.1. ATR-FTIR (ν, cm ): 3300, 3230, 3086, 2950, 1702. HRMS (ESI): [M+H] calcd for
C H N O 145.06077, found 145.06084.
5
9
2
3
1
4

4
.3.1.6. (3S)-3-(hydroxyethyl)piperazine-2,6-dione (S)-4k
From (S)-2k (1.47 g, 11.04 mmol), KHCO (2.78 g, 27.62 mmol) and 2-bromoacetamide
3
(1.87 g, 13.26 mmol) in acetonitrile (90 mL); (S)-4k was purified by precipitation with EtOH (45
mL), washed with MeOH (20 mL) and filtered under vacuum. White powder; yield 1.41 g
1
(
81%); m.p.153-154 °C. H NMR (400 MHz, DMSO-d ): δ 1.19 (3H, d, J = 6.4 Hz), 2.82 (1H,
6
br s), 3.15 (1H, br), 3.36 (1H , d, J = 17.5 Hz), 3.56 (1H , d, J = 17.5 Hz), 4.27 (1H, br m),
B
AB
A
AB
1
3
4
6
.84 (1H, d, J = 4.3 Hz), 10.77 (1H, s). C NMR (100 MHz, DMSO-d ): δ 21.1, 48.0, 62.2,
6
-1
+
6.0, 174.1, 174.7. ATR-FTIR (ν, cm ): 3314, 3035, 2844, 1679. HRMS (ESI): [M+H] calcd
for C H N O 159.0764, found 159.0737.
6
11
2
3
4
.3.1.7. (3R)-3-(hydroxyethyl)piperazine-2,6-dione (R)-4k
From (R)-2k (1.47 g, 11.04 mmol), KHCO (2.78 g, 27.62 mmol) and 2-bromoacetamide
3
(1.87 g, 13.26 mmol) in acetonitrile (90 mL); the product was purified in the same way as
1
described above for (S)-4k. White Powder; yield 1.22 g (70%); m.p. 152-154 °C. H NMR (400
MHz, DMSO-d ): δ 1.19 (3H, d, J = 6.4 Hz), 2.81 (1H, br s), 3.15 (1H, br), 3.38 (1H , d, J =
AB
6
B
1
8.0 Hz), 3.56 (1H , d, J = 18.0 Hz), 4.27 (1H, br m), 4.84 (1H, d, J = 4.9 Hz), 10.76 (1H, s).
A AB
1
3
-1
C NMR (100 MHz, DMSO-d ): δ 20.9, 48.0, 61.9, 65.9, 174.1, 174.7. ATR-FTIR (ν, cm ):
6
+
3
314, 3036, 2842, 1678. HRMS (ESI): [M+H] calcd for C H N O 159.0764, found 159.0736.
6
11
2
3
4
.3.1.8. (3S)-3-((1H-indol-2-yl)methyl)piperazine-2,6-dione (S)-4m
From (S)-2m (1.00 g, 4.58 mmol), KHCO (1.15 g, 11.45 mmol) and 2-bromoacetamide
3
(
0.77 g, 5.50 mmol); (S)-4m was purified by precipitation with CHCl (20 mL). White powder;
3
1
yield 0.93 g (83%); m.p. 183-185 °C. H NMR (500 MHz, DMSO-d ): δ 2.73 (1H, m), 2.93
6
(
1H , dd, J = 14.6 Hz, J = 8.6 Hz), 3.24 (1H , d, J = 14.6 Hz), 3.34 (1H , overlapped with
B AB BX A AB B
the signal of H O), 3.43 (1H , d, J = 17.3 Hz), 3.59 (1H, m), 6.94 (1H, t, J = 7.1 Hz), 7.03 (1H,
2
A
AB
1
3
t, J = 7.1 Hz), 7.14 (1H, s), 7.31 (1H, d, J = 8.0 Hz), 7.52 (1H, d, J = 8.0 Hz), 10.82 (2H, s). C
NMR (125 MHz, DMSO-d ): δ 25.7, 48.5, 58.3, 110.8, 111.8, 118.8, 119.0, 121.3, 124.4, 128.0,
6
1
5

-1
1
36.6, 173.9, 175.1. ATR-FTIR (ν, cm ): 3411, 3283, 2979, 1711, 1691. HRMS (ESI):
+
[
M+H] calcd for C H N O 244.10805, found 244.10846.
1
3
14
3
2
4
.3.1.9. (3R)-3-((1H-indol-2-yl)methyl)piperazine-2,6-dione (R)-4m
From (R)-2m (1.00 g, 4.58 mmol), KHCO (1.15 g, 11.45 mmol) and 2-bromoacetamide
3
(0.77 g, 5.50 mmol); the product was purified in the same way as described above for (S)-4m.
1
White powder; yield 0.76 g (68%); m.p. 182-184 °C. H NMR (500 MHz, DMSO-d ): δ 2.73
6
(
1H, m), 2.92 (1H , dd, J = 14.8 Hz, JBX = 8.6 Hz), 3.23 (1H , dd, J = 14.8 Hz, JAX = 4.0
B AB A AB
Hz), 3.35 (1H , dd, J = 17.6 Hz, JBX = 8.6 Hz), 3.42 (1H , dd, J = 17.6 Hz, JAX = 5.4 Hz),
B
AB
A
AB
3
.58 (1H , td, J = 8.6 Hz, J = 4.0 Hz), 6.93 (1H, t, J = 7.1 Hz), 7.02 (1H, t, J = 7.1 Hz), 7.13
X BX AX
1
3
(
1H, s), 7.31 (1H, d, J = 8.0 Hz), 7.50 (1H, d, J = 8.0 Hz), 10.81 (1H, s), 10.82 (1H, s). C NMR
(
125 MHz, DMSO-d ): δ 25.7, 48.5, 58.3, 110.8, 111.8, 118.7, 118.9, 121.3, 124.4, 128.0, 136.6,
6
-1
+
1
73.8, 175.1. ATR-FTIR (ν, cm ): 3411, 3282, 2977, 1711, 1691. HRMS (ESI): [M+H] calcd
for C H N O 244.10805, found 244.10826.
1
3
14
3
2
4
.3.2. Synthesis of methyl-2-(2-amino-2-oxoethylamino)-2-alkylacetates (S)-3(c-f, l) and
(R)-3(c, d, f, l)
The methodology described below is a general procedure for the synthesis of methyl-2-(2-
amino-2-oxoethylamino)-2-alkylacetates with some variations in reaction time and purification,
which are indicated in their synthesis.
4
.3.2.1. Synthesis of (S)-methyl 2-((2-amino-2-oxoethyl)amino)-3-methylbutanoate (S)-3c
A mixture of L-valine methyl ester (S)-2c (1.00 g, 7.62 mmol), KHCO (1.15 g, 11.43
3
mmol) and 2-bromoacetamide (1.29 g, 9.15 mmol) in acetonitrile (60 mL) was stirred under
refluxed for 8.5 h. After cooling to room temperature, the mixture was filtered and concentrated
under reduced pressure. The residue was diluted with water (20 mL) and extracted with
chloroform (3 x 30 mL). The combined organic layers were dried over anhydrous Na SO ,
2
4
filtered and evaporated under reduced pressure. The procedure carried out to remove (S)-2c that
1
6

did not react with 2-bromoacetamide is described below: The residue (yellow oil) was treated
with hexane (30 mL) and stirred for 15 min. After this time, the mixture was decanted and dried
under reduced pressure to give a viscous oil. Then, the residue was treated with a mixture of
hexane/diethyl ether (1:1, 30 mL) and stirred for 10 min. After this time, the mixture was
1
decanted and dried under reduced pressure to give (S)-3c as a yellow oil. Yield 1.28 g (89%). H
NMR (400 MHz, CDCl ): δ 0.95 (3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 7.0 Hz), 1.97 (1H, m),
3
2
3
5
.02 (1H, s), 2.98 (1H , d, J = 17.5 Hz), 3.02 (1H, d, J = 6.0 Hz), 3.43 (1H , d, J = 17.5 Hz),
B AB A AB
1
3
.73 (3H, s), 6.15 (1H, br s), 7.07 (1H, br s). C NMR (100 MHz, CDCl ): δ 18.1, 19.4, 31.4,
3
-1
1.3, 51.7, 67.5, 174.5, 174.9. ATR-FTIR (ν, cm ): 3440, 3326, 3198, 2961, 1729, 1673.
+
HRMS (ESI): [M+H] calcd for C H N O 189.12337, found 189.12345.
8
17
2
3
4
.3.2.2. (R)-methyl 2-((2-amino-2-oxoethyl)amino)-3-methylbutanoate (R)-3c
The procedure was the same as described above for the synthesis of (S)-3c to afford (R)-3c
1
as a yellow oil. Yield 1.28 g (89%). H NMR (400 MHz, CDCl ): δ 0.93 (3H, d, J = 7.0 Hz),
3
0
.97 (3H, d, J = 7.0 Hz), 1.90 (1H, br s), 1.97 (1H, m), 2.97 (1H , d, J = 17.3 Hz), 3.00 (1H, d,
B AB
1
3
J = 6.0 Hz), 3.42 (1H , d, J = 17.3 Hz), 3.70 (3H, s), 5.84 (1H, br s), 7.06 (1H, br s). C NMR
A
AB
-1
(
100 MHz, CDCl ) δ: 18.2, 19.5, 31.5, 51.4, 51.9, 67.6, 174.5, 175.0. ATR-FTIR (ν, cm ):
3
+
3
436, 3333, 3195, 2960, 1728, 1668. HRMS (ESI): [M+H] calcd for C H N O 189.12337,
8
17
2
3
found 189.12377.
4
.3.2.3. (S)-methyl 2-((2-amino-2-oxoethyl)amino)-4-methylpentanoate (S)-3d
A mixture of L-Leucine methyl ester (S)-2d (1.00 g, 6.89 mmol), KHCO (1.04 g, 10.33
3
mmol) and 2-bromoacetamide (1.16 g, 8.26 mmol) in acetonitrile was stirred under reflux for 9
1
h. (S)-3d was only purified by extractions. Yellow oil; yield 1.27 g, (91%). H NMR (400 MHz,
CDCl ): δ 0.89 (3H, d, J = 6.5 Hz), 0.91 (3H, d, J = 6.5 Hz), 1.46 (2H, m), 1.74 (1H, m), 2.09
3
(
1H, br s), 3.00 (1H , d, J = 17.3 Hz), 3.25 (1H, m), 3.37 (1H , d, J = 17.3 Hz), 3.69 (3H, s),
B AB A AB
1
3
6
5
.48 (1H, br s), 7.04 (1H, br s). C NMR (100 MHz, CDCl ): δ 21.9, 22.8, 24.8, 42.5, 50.8,
3
-1
1.8, 60.1, 174.7, 175.7. ATR-FTIR (ν, cm ): 3437, 3321, 3165, 2955, 2870, 1732, 1672.
+
HRMS (ESI): [M+H] calcd for C H N O 203.13902, found 203.13909.
9
19
2
3
1
7

4
.3.2.4. (R)-methyl 2-((2-amino-2-oxoethyl)amino)-4-methylpentanoate (R)-3d
A mixture of D-Leucine methyl ester (R)-2d (1.00 g, 6.89 mmol), KHCO (1.04 g, 10.33
3
mmol) and 2-bromoacetamide (1.16 g, 8.26 mmol) in acetonitrile was stirred under reflux for 9
h. (R)-3d was extracted with chloroform, and purified by washing with hexane. Yellow oil; yield
1
1
1
.16 g (83%). H NMR (400 MHz, CDCl ): δ 0.93 (3H, d, J = 6.6 Hz), 0.95 (3H, d, J = 6.6 Hz),
3
.49 (2H, m), 1.77 (1H, m), 1.96 (1H, br s), 3.04 (1H , d, J = 17.3 Hz), 3.28 (1H, dd, J = 8.3
B
AB
1
3
Hz, J = 6.0 Hz), 3.40 (1H , d, J = 17.3 Hz), 3.73 (3H, s), 6.00 (1H, br s), 7.06 (1H, br s). C
A
AB
NMR (100 MHz, CDCl ): δ 21.9, 22.8, 24.9, 42.8, 50.9, 51.8, 60.1, 174.5, 175.7. ATR-FTIR (ν,
3
-1
+
cm ): 3442, 3329, 3199, 2955, 2872, 1732, 1672. HRMS (ESI): [M+H] calcd for C H N O
3
9
19
2
2
03.13902, found 203.13900.
4
.3.2.5. (2S,3S)-methyl-2-((2-amino-2-oxoethyl)amino)-3-methylpentanoate (S)-3e
A mixture of L-isoleucine methyl ester (S)-2e (1.00 g, 6.89 mmol), KHCO (1.04 g, 10.33
3
mmol) and 2-bromoacetamide (1.16 g, 8.26 mmol) in acetonitrile was stirred under reflux for 11
h. (S)-3e was purified in the same way as described above for (S)-3c. Yellow oil; yield 1.27 g
1
(
91%). H NMR (400 MHz, CDCl ): δ 0.89 (3H, d, J = 7.5 Hz), 0.93 (3H, d, J = 7.5 Hz), 1.19
3
(
1H, m), 1.51 (1H, m), 1.72 (1H, m), 2.09 (1H, s), 2.98 (1H , d, J = 17.3 Hz), 3.09 (1H, d, J =
B
AB
1
3
6
.0 Hz), 3.41 (1H , d, J = 17.3 Hz), 3.72 (3H, s), 6.24 (1H, br s), 7.06 (1H, br s). C NMR
A AB
-1
(
100 MHz, CDCl ): δ 11.4, 15.8, 25.2, 38.1, 51.3, 51.7, 66.5, 174.6, 174.8. ATR-FTIR (ν, cm ):
3
+
3
2
435, 3334, 3197, 2962, 2922, 2876, 1729, 1672. HRMS (ESI): [M+H] calcd for C H N O
9
19
2
3
03.13902, found 203.13937.
4
.3.2.6. (S)-methyl-1-((2-amino-2-oxoethyl)pyrrolidine)-2-carboxylate (S)-3f
A mixture of L-proline methyl ester (S)-2f (1.00 g, 7.74 mmol), KHCO (1.17 g, 11.61
3
mmol) and 2-bromoacetamide (1.31 g, 9.29 mmol) in acetonitrile was stirred under reflux for 8
h. (S)-3f was extracted with chloroform, and purified by washing with a mixture of
1
hexane/diethyl ether (60:40, 30 mL). White powder; yield 1.37 g (95%); m.p. 113-116 °C. H
NMR (500 MHz, CDCl ): δ 1.87 (2H, m), 1.95 (1H, m), 2.16 (1H, m), 2.55 (1H, m), 3.16 (1H ,
3
B
1
8

d, J = 16.6 Hz), 3.20 (1H, m), 3.38 (1H , d, J = 16.6 Hz), 3.41 (1H, dd, J = 9.2 Hz, J = 5.3
AB
A
AB
1
3
Hz), 3.70 (3H, s), 5.61 (1H, br s), 7.41 (1H, br s). C NMR (120 MHz, CDCl ): δ 24.3, 29.9,
3
-
1
5
1
2.1, 54.8, 58.4, 66.0, 174.2, 174.9. ATR-FTIR (ν, cm ): 3415, 3200, 2984, 2953, 2927, 2803,
+
746, 1632. HRMS (ESI): [M+H] calcd for C H N O 187.10772, found 187.10782.
8
15
2
3
4
.3.2.7. (R)-methyl-1-((2-amino-2-oxoethyl)pyrrolidine)-2-carboxylate (R)-3f
A mixture of D-proline methyl ester (R)-2f (1.00 g, 7.74 mmol), KHCO (1.17 g, 11.61
3
mmol) and 2-bromoacetamide (1.31 g, 9.29 mmol) in acetonitrile was stirred under reflux for 8
h. (R)-3f was purified in the same way as described above for (S)-3f. White powder; yield 1.31 g
1
(
91%); m.p. 112-115 °C. H NMR (500 MHz, CDCl ): δ 1.80 (2H, m), 1.90 (1H, m), 2.11 (1H,
3
m), 2.50 (1H, m), 3.11 (1H , d, J = 16.8 Hz), 3.14 (1H, m), 3.34 (1H , d, J = 16.8 Hz), 3.37
B
AB
A
AB
1
3
(
1H, dd, J = 9.2 Hz, J = 5.3 Hz), 3.65 (3H, s), 6.21 (1H, br s), 7.33 (1H, br s). C NMR (125
-1
MHz, CDCl ): δ 24.2, 29.8, 52.0, 54.7, 58.2, 65.9, 174.4, 174.8. ATR-FTIR (ν, cm ): 3415,
3
+
3
191, 2983, 2954, 2802, 1746, 1632. HRMS (ESI): [M+H] calcd for C H N O 187.10772,
8
15
2
3
found 187.10787.
4
.3.2.8. (S)-methyl 2-((2-amino-2-oxoethyl)amino)-4-(methylthio)butanoate (S)-3l
A mixture of L-Methionine methyl ester (S)-2l (1.00 g, 6.13 mmol), KHCO (0.92 g, 9.19
3
mmol) and 2-bromoacetamide (1.03 g, 7.35 mmol) in acetonitrile was stirred under reflux for 6.5
h. (S)-3l was extracted with ethyl acetate, and purified by column chromatography on silica gel
(hexane/ethyl acetate 95:5 followed by 90:10, 80:10, and 60:40). Yellow oil, yield 0.59 g (51%).
1
H NMR (500 MHz, CDCl ): δ 1.78-1.86 (2H, m), 1.99 (1H, m), 2.09 (3H, s), 2.61 (2H, m), 3.05
3
(
1H , d, J = 17.3 Hz), 3.40 (1H, dd, J = 8.5 Hz, J = 5.3 Hz), 3.42 (1H , d, J = 17.3 Hz), 3.73
B
AB
A
AB
1
3
(
3H, s), 5.78 (1H, br s), 7.03 (1H, br s). C NMR (125 MHz, CDCl ): δ 15.5, 30.6, 32.3, 51.0,
3
-1
5
2.2, 60.4, 174.1, 174.9. ATR-FTIR (ν, cm ): 3451, 3303, 3197, 2952, 2917, 1731, 1670.
+
HRMS (ESI): [M+H] calcd for C H N O S 221.09544, found 221.09524.
8
17
2
3
1
9

4
.3.2.9. (R)-methyl 2-((2-amino-2-oxoethyl)amino)-4-(methylthio)butanoate (R)-3l
A mixture of D-Methionine methyl ester (R)-2l (1.00 g, 6.13 mmol), KHCO (0.92 g, 9.19
3
mmol) and 2-bromoacetamide (1.03 g, 7.35 mmol) in acetonitrile was stirred under reflux for 6.5
h. (R)-3l was purified in the same way as described above for (S)-3l. Yellow oil, yield 0.58 g,
1
(
50%). H NMR (400 MHz, CDCl ): δ 1.84 (1H, m), 1.97-2.05 (2H, m), 2.10 (3H, s), 2.63 (2H,
3
m), 3.07 (1H , d, J = 17.2 Hz), 3.42 (1H, dd, J = 8.8 Hz, J = 5.2 Hz), 3.44 (1H , d, J = 17.2
B
AB
A
AB
1
3
Hz), 3.74 (3H, s), 6.04 (1H, br s), 7.05 (1H, br s). C NMR (100 MHz, CDCl ): δ 15.4, 30.6,
3
-
1
3
1
2.3, 50.9, 52.1, 60.3, 174.2, 174.8. ATR-FTIR (ν, cm ): 3442, 3322, 3198, 2951, 2917, 2840,
+
731, 1668. HRMS (ESI): [M+H] calcd for C H N O S 221.09544, found 221.09547.
8
17
2
3
4
.3.3. Synthesis of (S)-4(c, e, l) and (R)-4(c, d, f, l)
The methodology described below is a general procedure for the synthesis of (S)-4(c, e, l)
and (R)-4(c, d, f, l) with some variations in reaction time and purification, which are indicated in
their synthesis.
4
.3.3.1 Synthesis of (3S)-3-isopropyl-piperazine-2,6-dione (S)-4c
To a stirred solution of (S)-3c (830 mg, 4.41 mmol) in absolute EtOH (22 mL), NaOH
(176 mg, 4.41 mmol) was added at room temperature. After dissolution of the hydroxide, the
mixture was stirred for 10 min and quenched with a saturated aqueous solution of ammonium
chloride (20 mL). The resulting cloudy solution was extracted with EtOAc (3 x 30 mL). The
combined organic layers were washed with water (20 mL), dried over anhydrous Na SO ,
2
4
filtered and concentrated under reduced pressure. The residue was precipitated with diethyl ether
(20 mL), filtered and dried under vacuum to afford (S)-4c as a white powder. Yield 544 mg
1
(
79%); m.p. 117-118 °C. H NMR (400 MHz, CDCl ): δ 0.97 (3H, d, J = 7.0 Hz), 1.10 (3H, d, J
3
=
7.0 Hz), 1.83 (1H, br s), 2.50 (1H, m), 3.28 (1H, d, J = 4.6 Hz), 3.58 (1H , d, J = 18.0 Hz),
B AB
1
3
3
4
.79 (1H , d, J = 18.0 Hz), 8.52 (1H, br s). C NMR (100 MHz, CDCl ): δ 17.2, 19.5, 27.8,
A AB 3
-
1
8.2, 62.9, 172.2, 173.1. ATR-FTIR (ν, cm ): 3289, 3275, 2978, 2963, 1681. HRMS (ESI):
+
[
M+H] calcd for C H N O 157.09715, found 157.09727.
7
13
2
2
2
0

4
.3.3.2. Synthesis of (3R)-3-isopropyl-piperazine-2,6-dione (R)-4c
The procedure was the same as described above for the synthesis of (S)-4c. The residue
was extracted with chloroform, and purified by precipitation with hexane (20 mL) to give (R)-4c.
1
White powder; yield 482 mg (70%); m.p. 117-119 °C. H NMR (400 MHz, CDCl ): δ 0.98 (3H,
3
d, J = 7.0 Hz), 1.11 (3H, d, J = 7.0 Hz), 1.78 (1H, br s), 2.50 (1H, m), 3.29 (1H, d, J = 4.7 Hz),
1
3
3
.59 (1H , d, J = 18.0 Hz), 3.80 (1H , d, J = 18.0 Hz), 8.45 (1H, br s). C NMR (100 MHz,
B AB A AB
-1
CDCl ): δ 17.2, 19.5, 27.8, 48.3, 63.0, 172.0, 173.0. ATR-FTIR (ν, cm ): 3290, 3276, 2978,
3
+
2
963, 1681. HRMS (ESI): [M+H] calcd for C H N O 157.09715, found 157.09743.
7
13
2
2
4
.3.3.3. (3R)-3-(2-methylpropyl)piperazine-2,6-dione (R)-4d
From (R)-3d (500 mg, 2.47 mmol), absolute EtOH (12.5 mL) and NaOH (98.9 mg, 2.47
mmol); the residue was extracted with CH Cl (3 x 25 mL), and purified by precipitation with
2
2
1
hexane (15 mL) to give (R)-4d. White powder; yield 300 mg (71%); m.p. 117-119 °C. H NMR
(
400 MHz, CDCl ): δ 0.94 (3H, d, J = 6.3 Hz), 0.98 (3H, d, J = 6.3 Hz), 1.54 (1H, m), 1.78-1.92
3
(
3H, m), 3.47 (1H, dd, J = 10.0 Hz, 4.0 Hz), 3.58 (1H , d, J = 18.0 Hz), 3.73 (1H , d, J
=
AB
B
AB
A
1
3
1
1
8.0 Hz), 8.82 (1H, br s). C NMR (100 MHz, CDCl ) δ: 21.2, 23.2, 24.5, 38.4, 47.5, 55.7,
3
-1
+
72.4, 174.6. ATR-FTIR (ν, cm ): 3306, 2959, 2870, 1707, 1682. HRMS (ESI): [M+H] calcd
for C H N O 171.11280, found 171.11308.
8
15
2
2
4
.3.3.4. (3S)-3-(1-methylpropyl)piperazine-2,6-dione (S)-4e
From (S)-3e (500 mg, 2.47 mmol), absolute EtOH (12.5 mL) and NaOH (98.9 mg, 2.47
mmol); (S)-4e was purified in the same way as described above for (R)-4c. White powder, yield
1
2
60 mg (62%); m.p. 79-80°C. H NMR (500 MHz, CDCl ): δ 0.90 (3H, t, J = 7.0 Hz), 1.04 (3H,
3
d, J = 7.0 Hz), 1.21 (1H, m), 1.45 (1H, m), 1.80 (1H, br s), 2.16 (1H, br m), 3.29 (1H, d, J = 4.0
1
3
Hz), 3.52 (1H , d, J = 17.8 Hz), 3.73 (1H , d, J = 17.8 Hz), 8.58 (1H, br s). C NMR (125
B
AB
A
AB
-1
MHz, CDCl ): δ 11.9, 16.2, 24.8, 34.6, 48.3, 62.7, 172.2, 173.3. ATR-FTIR (ν, cm ): 3282,
3
+
2
1
964, 2932, 2876, 1714, 1680. HRMS (ESI): [M+H] calcd for C H N O 171.11280, found
8
15
2
2
71.11266.
2
1

4
.3.3.5. (3R)-3-Tetrahydropyrrolo[1,2-a]pyrazine-1,3(2H,4H)-dione (R)-4f
From (R)-3f (500 mg, 2.69 mmol), absolute EtOH (13 mL), and NaOH (107 mg, 2.69
mmol); (R)-4f was purified in the same way as described above for (R)-4c. White powder; yield
1
2
2
1
2
90 mg (70%); m.p. 138-140 °C. H NMR (500 MHz, DMSO-d ): δ 1.68 (2H, m), 1.94 (2H, m),
6
.55 (1H, m), 2.66 (1H, m), 3.33 (1H, overlapped with the signal of H O), 3.45 (1H , d, J =
AB
2
B
1
3
7.0 Hz), 3.55 (1H , d, J = 17.0 Hz), 10.95 (1H, br s). C NMR (125 MHz, DMSO-d ): δ
A
AB
6
-1
2.1, 26.0, 52.1, 53.5, 62.5, 172.4, 174.2. ATR-FTIR (ν, cm ): 2971, 2915, 1715, 1688. HRMS
+
(
ESI): [M+H] calcd for C H N O 155.08150, found 155.08156.
7
11
2
2
4
.3.3.6. (3S)-3-(2-(methylthio)ethyl)piperazine-2,6-dione (S)-4l
From (S)-3l (1.00 g, 4.54 mmol), a mixture of CH CN/absolute EtOH (80:20, 20 mL) and
3
NaOH (0.18 g, 4.54 mmol); (S)-4l was purified in the same way as described above for (S)-4c.
1
White powder, yield 0.70 g (82%); m.p. 64-66 °C. H NMR (400 MHz, CDCl ): δ 1.90-1.97
3
(
2H, m), 2.13 (3H, s), 2.34 (1H, m), 2.72 (2H, m), 3.61 (1H , d, J = 18.0 Hz), 3.67 (1H, dd, J =
B AB
1
3
8
1
1
.8 Hz, 4.1 Hz), 3.77 (1H , d, J = 18.0 Hz), 8.56 (1H, br s). C NMR (100 MHz, CDCl ): δ
A AB 3
-1
5.2, 28.7, 30.3, 48.1, 56.3, 171.9, 173.5. ATR-FTIR (ν, cm ): 3281, 3163, 3037, 2949, 2916,
+
686. HRMS (ESI): [M+H] calcd for C H N O S 189.06923, found 189.06930.
7
13
2
2
4
.3.3.7. (3R)-3-(2-(methylthio)ethyl)piperazine-2,6-dione (R)-4l
From (R)-3l (1.00 g, 4.54 mmol), a mixture of CH CN/absolute EtOH (80:20, 20 mL) and
3
NaOH (0.18 g, 4.54 mmol); (R)-4l was purified in the same way as described above for (S)-4c.
1
White powder; yield 0.68 g (80%); m.p. 65-67 °C. H NMR (400 MHz, CDCl ): δ 1.89-1.98
3
(
2H, m), 2.13 (3H, s), 2.34 (1H, m), 2.72 (2H, m), 3.61 (1H , d, J = 18.0 Hz), 3.66 (1H, dd, J =
B AB
1
3
8
1
1
.7 Hz, 4.3 Hz), 3.77 (1H , d, J = 18.0 Hz), 8.49 (1H, br s). C NMR (100 MHz, CDCl ): δ
A AB 3
-1
5.2, 28.7, 30.3, 48.1, 56.3, 171.8, 173.5. ATR-FTIR (ν, cm ): 3288, 3160, 3045, 2949, 2914,
+
687. HRMS (ESI): [M+H] calcd for C H N O S 189.06923, found 189.06939.
7
13
2
2
2
2

4
.3.4 Procedure for preparation of (3S)-3-(4-hydroxybenzyl)piperazine-2,6-dione (S)-4h
The synthetic pathway for the synthesis of (S)-4h is shown in Scheme S1 (SI). Herein, we
only described the debenzylation procedure of (S)-4hBn, which is the last step in the pathway.
To a solution of (S)-3-(4-(benzyloxy)benzyl)piperazine-2,6-dione (S)-4hBn (250 mg, 0.806
mmol) in a mixture of MeOH/CH CN (80:20, 10 mL) was added Pd/C (10%, 25.0 mg). The
3
resulting solution was stirred under hydrogen atmosphere for 2.5 h at room temperature After
.
reaction completion, the mixture was filtered through Celite to remove the Pd/C catalyst, and the
filtrate was concentrated under reduced pressure [52]. The resulting solid was washed with a
mixture of DCM/EtOAc (50:50, 10 mL), filtered and dried under vacuum to afford (S)-4h as a
1
white powder. Yield 126 mg (71%); m.p. 171-174 °C. H NMR (400 MHz, DMSO-d ): δ 2.68
6
(
1H , dd, J = 14.3 Hz, J = 9.3 Hz), 2.78 (1H, br m), 3.03 (1H , dd, J = 14.3 Hz, J = 3.4
B AB BX A AB AX
Hz), 3.34 (1H, s, overlapped with the signal of H O), 3.41 (1H, br), 3.47 (1H, br m), 6.85 (2H, d,
2
1
3
J = 8.0 Hz), 7.02 (2H, d, J = 8.0 Hz), 9.16 (1H, br s), 10.81 (1H, br s). C NMR (100 MHz,
-1
DMSO-d ): δ 34.8, 48.2, 58.9, 115.3, 128.8, 130.7, 156.1, 173.8, 174.8. ATR-FTIR (ν, cm ):
6
+
3
2
399, 3287, 3044, 2828, 1694. HRMS (ESI): [M+H] calcd for C H N O 221.09207, found
1
1
13
2
3
21.09209.
4
.4. Molecular Docking
Molecular Docking of the 2,6-DKPs and TSA (reference compound) was performed on the
X-ray crystal structure of human HDAC8 enzyme, which was retrieved from the RCSB Protein
Data Bank (PDB ID: 1T64) [37,38]. The crystal structure was optimized by removing the water
molecules and the co-crystallized ligands using PyMOL, whereas hydrogen atoms were added
using AutoDockTools-1.5.6. All the compounds were drawn using GaussView 5.0.8, and were
geometrically optimized by density functional theory calculations at the B3LYP/6-31G** level
using Gaussian 09W software [53]. Docking calculations were carried out using AutoDock
4
1
.2.6® software [54]. The grid box covered the entire volume of the HDAC8 (126 Å x 126 Å x
26 Å dimensions) with a grid spacing of 0.375 Å. The main Lamarckian Genetic Algorithm
[55] parameters were set to 100 runs, a population size of 100, and a maximum number of 1 x
1
07 energy evaluations. The co-crystallized TSA was re-docked on HDAC8 to validate the
2
3

docking protocol. The re-docked result was evaluated by calculation of root mean standard
deviation (RMSD) using Chimera software [56]. Docking poses of the target compounds and
TSA were analyzed based on the best energy and were visualized using Discovery studio 3.5
software v19.1.0.18287 [57].
4
.5. HDAC8 inhibition assay
The inhibitory activity of 4a, (S)-4h, (S)- and (R)-4(g, l, m) on HDAC8 was evaluated by
detection of lysyl deacetylase activity of the recombinant human HDAC8, using HDAC8
Fluorometric Drug Discovery Kit according to manufacturer’s instructions (Sigma Aldrich Co®
Catalog No EPI007). The stock solutions of the compounds and TSA (inhibitor control) were
prepared in DMSO and diluted in assay buffer to reach the final concentrations in the range of
2
00 to 6.5 nM. The final concentration of DMSO was less than 0.1% and did not affect the
activity of the assays. The assays were performed by pre-incubating the enzyme with the test
o
compounds, including TSA, at 37 C for 10 min. Then, the substrate was added and the mixture
o
was incubated at 37 C for 60 min. After, 10 µL of developer were added and the mixture was
o
incubated at 37 C for 5 min. The Fluorescence signal was measured using a fluorescence plate
reader (Thermo Scientific Fluoroskan Ascent FL) at excitation and emission wavelengths of 390
and 485 nm, respectively. All experiments were performed in triplicate. The IC values were
5
0
calculated using nonlinear regression with normalized dose-response fit in Prism GraphPad
software.
Conflict of interest
The authors have declared no conflicts of interest.
Acknowledgments
The authors would like to thank Consejo Nacional de Ciencia y Tecnología (Conacyt-
México) for scholarship to FPGG (261706). The authors are also grateful for the computing
granted by LANCAD and Conacyt in the Supercomputer Hybrid Cluster “Xiuhcoatl” at General
Coordination of Information and Communications Technologies (CGSTIC) of Cinvestav. The
authors wish to thank Dr. Patricia Talamás-Rohana for allowing us to use the Thermo Scientific
2
4

Fluoroskan Ascent FL spectrofluorometer from the Department of Infectomics and Molecular
Pathogenesis at Cinvestav and also thank MSc. Juan Carlos Osorio-Trujillo and Biol. José Daniel
Morales-Mora for their support in the development of the enzymatic assays.
Appendix A. Supplementary data
1
13
Supplementary information consists of H, C NMR and HRMS spectra of the novel 2,6-
DKPs.
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Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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