Novel 2, 5-diketopiperazine derivatives as potent selective histone deacetylase 6 inhibitors: Rational design, synthesis and antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2019.111950

Histone deacetylase 6 (HDAC6) has gained popular attention for its wide participation in various pathological process recently. In this paper, a series of novel derivatives containing 2, 5-diketopiperazine (DKP) skeleton were developed as potent selective HDAC6 inhibitors (sHDAC6is). Most of these compounds exhibited low nanomolar IC₅₀ values toward HDAC6, and the best compound was 21b (IC₅₀ = 0.73 nM) which had 144–10941-fold selectivity over other HDAC isoforms. Western blot assay further validated these compounds to be sHDAC6is. Molecular simulation of 21b was conducted to rationalize the high binding affinity for HDAC6. In the cytotoxicity experiment, 18a, 18b and 18d gave superior or comparable influence on the growth of two multiple myeloma cells U266 and RPMI-8226 compared to ACY-1215. Moreover, the combination of 18a and adriamycin showed synergistic effect against non-small cell lung cancer cell A549. 18a and 18b also demonstrated appropriate drug metabolism in human liver microsome (HLM).

Full text of DOI:10.1016/j.ejmech.2019.111950

Journal Pre-proof
Novel 2, 5-diketopiperazine derivatives as potent selective histone deacetylase 6
inhibitors: Rational design, synthesis and antiproliferative activity
Xin Chen, Xinyang Chen, Raphael R. Steimbach, Tong Wu, Hongmei Li, Wenjia Dan,
Peidong Shi, Chenyu Cao, Ding Li, Aubry K. Miller, Zhixia Qiu, Jinming Gao, Yong
Zhu
PII:
S0223-5234(19)31102-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111950
EJMECH 111950
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 July 2019
Revised Date: 5 November 2019
Accepted Date: 2 December 2019
Please cite this article as: X. Chen, X. Chen, R.R. Steimbach, T. Wu, H. Li, W. Dan, P. Shi, C. Cao, D.
Li, A.K. Miller, Z. Qiu, J. Gao, Y. Zhu, Novel 2, 5-diketopiperazine derivatives as potent selective histone
deacetylase 6 inhibitors: Rational design, synthesis and antiproliferative activity, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.111950.
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2019 Published by Elsevier Masson SAS.

GRAPHICAL ABSTRACT
Cap
Linker
ZBG
O
O
O
O
H
NH
N
H
N
HN
HN
HN
OH
O
DKP scaffold
Compd. 21b
HDAC6 IC50 = 0.73 nM
44~10941-fold selectivity
1

Novel 2, 5-Diketopiperazine Derivatives as Potent Selective Histone Deacetylase 6
Inhibitors: Rational Design, Synthesis and Antiproliferative Activity
a
a
b, c
a
d
a
a
a
a
Xin Chen , Xinyang Chen , Raphael R. Steimbach , Tong Wu , Hongmei Li , Wenjia Dan , Peidong Shi , Chenyu Cao , Ding Li , Aubry
b, e
f
a,
d,
K. Miller , Zhixia Qiu , Jinming Gao *, Yong Zhu *
a
Shaanxi Key Labotory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University,
Yangling 712100, P. R. China
b
Cancer Drug Development Group, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
c
Biosciences Faculty, University of Heidelberg, 69120 Heidelberg, Germany
d
School of Science, China Pharmaceutical University, Nanjing, 210009, P. R. China
e
German Cancer Consortium (DKTK), 69120 Heidelberg, Germany
f
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, P. R. China
Corresponding Author
*
Jinming Gao
E-mail: jinminggao@nwsuaf.edu.cn; Fax: +87-29-87092335
Yong Zhu
*
E-mail: zhuyong@cpu.edu.cn; Fax: +86-25-86185182
Notes
The authors declare no competing interest.
Abstract
Histone deacetylase 6 (HDAC6) has gained popular attention for its wide participation in various
pathological process recently. In this paper, a series of novel derivatives containing 2, 5-diketopiperazine (DKP)
skeleton were developed as potent selective HDAC6 inhibitors (sHDAC6is). Most of these compounds exhibited
low nanomolar IC50 values toward HDAC6, and the best compound was 21b (IC50 = 0.73 nM) which had
1
44~10941-fold selectivity over other HDAC isoforms. Western blot assay further validated these compounds to
be sHDAC6is. Molecular simulation of 21b was conducted to rationalize the high binding affinity for HDAC6.
In the cytotoxicity experiment, 18a, 18b and 18d gave superior or comparable influence on the growth of two
multiple myeloma cells U266 and RPMI-8226 compared to ACY-1215. Moreover, the combination of 18a and
adriamycin showed synergistic effect against non-small cell lung cancer cell A549. 18a and 18b also demonstrated
appropriate drug metabolism in human liver microsome (HLM).
Key words:
HDAC6 inhibitor; DKP; Selectivity; Synthesis; Combination therapy
1
. Introduction
Protein lysine acetylation is a reversible posttranslational modification that is balanced by histone
acetyltransferases (HATs) and histone deacetylases (HDACs)[1]. During the past decades, HDACs as promise
targets had been studied extensively[2]. However, a lot of clinical trials indicate that the use of pan-HDACis leads
to undesirable side effects, such as fatigue, diarrhea, nausea, thrombocytopenia, and cardiotoxicity[3-5]. The
application of broad spectrum or class I selective HDACis is limited because of a narrow therapeutic window[6]. A
common view is that developing isoform-selective HDAC inhibitor (HDACi) would be advantageous over
pan-HDACis for lower toxicity and improved safety profile[7]. In contrast with the lethal effect of HDAC1-3

genetic ablation, mice with HDAC6-knocked out are viable and develop normally[8]. Among all the 11
2+
Zn -dependent HDAC members, HDAC6 consists of 1216 aa and uniquely has two functional domains, a dynein
motor binding domain and a zinc finger domain. HDAC6 is mainly localized in cytoplasm and plays an important
role in microtubule dynamics and chaperone activities[9]. The major substrates of HDAC6 are non-histone
proteins including α-tubulin, Hsp90, cortactin and peroxiredoxin, and this makes HDAC6 involved in many
cellular processes such as motility, adhesion, migration, growth of cells, immune synapse formation, and stress
granule formation[10, 11]. Moreover, HDAC6 is found to be highly expressed in many diseases including tumors
and neurological diseases; inhibition of HDAC6 suppresses microtubule dynamics and results in cell cycle arrest
and apoptosis, making HDAC6 become an attractive drug target in cancer therapy[12-14].
By so far, a lot of HDAC6is have been reported such as ricolinostat (ACY-1215)[15], HPOB[16], tubastatin
A[17] and et al[18-20], and they also have common pharmacophores: cap, linker and zinc binding group (ZBG).
However, the development of potent and efficient sHDAC6is for clinical treatment is still urgent. The intrinsic
biocompatibility and affinity for target binding of peptides have attracted chemists to obtain peptide inhibitors with
improved HDAC isoforms selectivity. The most famous peptide-based HDACis are cyclic tetrapeptides equipped
2+
with active Zn binding groups, such as the approved drug FK228 which displays class I selectivity[21]. Recently,
the use of peptide or peptoid as the capping group of HDACis arouses great interest (Fig. 1). Compound 1 is a
substrate mimic based on the histone H4 tail which incorporates a hydroxamic acid functionality, and inhibits
HDAC1 with an IC50 of 336 nM[22]. Compound 2 containing a peptoid capping group exhibits strong inhibition of
HDAC1-3 and HDAC6[23]. The dual HDAC-proteasome inhibitor 3, inhibits both HDAC6 and the
chymotrypsin-like proteasome, and shows potent and selective anticancer activity[24]. Trapoxin A (4), a microbial
cyclic tetrapeptide, is an essentially irreversible inhibitor of HDAC8, with Kd value of 3 nM[25].
Fig. 1 Reported peptide or peptoid HDAC inhibitors.
In consideration of the flexible conformation and instable physical and metabolite properties of peptides, a
conformation-restricted HDAC-interacting peptide bearing the ZBG group might provide a suitable scaffold for
novel HDACis. Wang et.al reported a series of stabilized peptide HDACis derived from HDAC1 substrate H3K56
which exhibited significantly increased safety window and better selectivity toward malignant cells than
SAHA[26]. Then, an inflexible capping group may improve selectivity against HDAC6 [27-29]. In present study,
we cyclized two adjacent peptide bonds (as exemplified by compound 1) into the rigid 2, 5-diketopiperazine
scaffold which exists in numerous bioactive natural products[30] and introduced the phenylhydroxamic acid group,
a key pharmacophore of sHDAC6i, to the N-1 of the DKP skeleton, producing a series of novel 1, 3-disubstituded
or 1, 3, 4-trisubstituded DKP derivatives (Fig. 2). Here, we reported the synthesis, structure and activity
relationship (SAR) study and anticancer evaluation of these derivatives.

Fig. 2 The design of novel DKP derivative as selective HDAC6 inhibitors.
2
2
. Results and discussion
.1 Chemistry
Although a lot of reports had described diverse synthesis of the DKP scaffold, a concise and convenient
synthetic procedure for 1, 3-disubstituted DKP is still rare, according to our literatures review. Here, we efficiently
constructed the 1, 3-disubstituted DKP skeleton through an one-pot synthesis in three steps from amino ester,
chloroacetyl chloride and substituted benzylamine in a good overall yield (Fig.3). The representative synthetic
process was as follows: (a) Amidation of amino ester with chloroacetyl chloride yielded intermediate A, using
triethylamine (Et N) as the base and acetonitrile as the solvent. (b) Methyl 4-(aminomethyl)benzoate was added to
3
give intermediate B when the temperature was ascending to reflux. (c) The mixture was concentrated in vacuo to
remove extra Et N and 10% silica gel was subsequently added to enhance the conversion of B to the cyclized 1,
3
3
-disubstituted DKP skeleton.
Fig 3. One-pot synthesis in three steps for 1, 3-disubstituted DKP skeleton.
At the beginning, the phenyl was chosen as the capping group because it was applied widely in many
HDACis. Stereochemistry of medicinal molecular had an important impact on pharmacological activity. Hence,
we synthesized both S- and R-configuration enantiomers 11a and 11b to investigate the effect of the C-3 chirality
on HDAC6 activity. The synthetic routes of compounds 11a-b were displayed in Scheme 1. Key DKP
intermediates 8a-b were synthesized following the one-pot procedure using 7a-b as starting materials. Hydrolysis
of 8a-b in the presence of sodium hydroxide at room temperature afforded 9a-b. Amidation of 9a-b with
O-(tetrahydro-2H-pyran-2-yl) hydroxylamine by 2-(7-Aza-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium
.
hexafluorophosphate (HATU) gave 10a-b. Deprotection of 10a-b in methanol with the catalytic p-TsOH H O
2
furnished target compounds 11a-b.
3 3
Scheme 1. Reagents and conditions: (a) 1. CH COCl, Et N, acetonitrile, 0 °C, 1 h; 2. Methyl 4-(aminomethyl)benzoate hydrochloride,
acetonitrile, reflux, 6 h; 3. 10% silica gel, reflux, overnight; (b) NaOH, THF/water (v/v) = 1:1, r.t., overnight; (c) HATU, N,

N-diisopropylethylamine (DIPEA), N, N-dimethylformamide (DMF), O-(tetrahydro-2H-pyran-2-yl) hydroxylamine, r.t., 6 h; (d)
.
p-TsOH H
2
O, MeOH, overnight.
To explore the SAR of ZBG group on different positions of phenyl linker, compound 13 with hydroxamic
acid on the m-position of phenyl linker was synthesized according to the method described in Scheme 1 with
starting material 7a.
3 3
Scheme 2. Reagents and conditions: (a) 1. CH COCl, Et N, acetonitrile, 0 °C, 1 h; 2. Methyl 3-(aminomethyl)benzoate hydrochloride,
acetonitrile, reflux, 6 h; 3. 10% silica gel, reflux, overnight; (b) NaOH, THF/water (v/v) = 1:1; (c) HATU, DIPEA, DMF,
.
O-(tetrahydro-2H-pyran-2-yl) hydroxylamine, r.t., 6 h; (d) p-TsOH H
2
O, MeOH, overnight.
Racemic compounds 15a-c were synthesized to preliminarily show the effect of different substituents on N-4
position of DKP on enzymatic activity. As showed in Scheme 3, nucleophilic reactions of 8a with various alkyl
halides gave intermediates 14a-c with sodium hydride (NaH) as the base. Then 14a-c were treated following the
same procedure as 8a-b to give desired compounds 15a-c.
3 3
Scheme 3. Reagents and conditions: (a) NaH, anhydrous tetrahydrofuran (THF), 60 °C, various alkyl halides, 3 h; (b) 1. CH COCl, Et N,
acetonitrile, 0 °C, 1 h; 2. Methyl 4-(aminomethyl)benzoate hydrochloride, acetonitrile, reflux, 6 h; 3. 10% silica gel, reflux, overnight; (c)
.
NaOH, THF/water (v/v) = 1:1; (d) HATU, DIPEA, DMF, O-(tetrahydro-2H-pyran-2-yl) hydroxylamine, r.t., 6 h; (e) p-TsOH H
2
O, MeOH,
overnight.
Based on the SAR of compounds 11a, 11b and 15a-c, R-configuration compounds 18a-e with different
substituents on N-4 position of DKP were synthesized. As shown in Scheme 4. Compound 7b was treated with
various aldehydes to afford Schiff’s bases, which were then in-situ reduced with sodium cyanoborohydride
(
NaBH CN) to generate intermediates 16a-e. Subsequently, 16a-e were reacted in the same procedure as shown in
3
Scheme 1 to give desired analogs 18a-e.
3 3 3
Scheme 4. Reagents and conditions: (a) 1. RCHO, r.t., 30 min; 2. MeOH, NaBH CN, 12 h; (b) 1. CH COCl, Et N, acetonitrile, 0 °C, 1 h;
2
. Methyl 4-(aminomethyl)benzoate hydrochloride, acetonitrile, reflux, 6 h; 3. 10% silica gel, reflux, overnight; (c) NaOH, THF/water
.
(
2
v/v) = 1:1; (d) HATU, DIPEA, DMF, O-(tetrahydro-2H-pyran-2-yl) hydroxylamine, r.t., 6 h; (e) p-TsOH H O, MeOH, overnight.
Besides, compounds 21a-b with indolyl capping group were also synthesized. By means of the same
procedure as 11a-b, compounds 21a-b were made from starting materials 19a-b, as described in Scheme 5.

3 3
Scheme 5. Reagents and conditions: (a) 1. CH COCl, Et N, acetonitrile, 0 °C, 1 h; 2. Methyl 4-(aminomethyl)benzoate hydrochloride,
acetonitrile, reflux, 6 h; 3. 10% silica gel, reflux, overnight; (b) NaOH, THF/water (v/v) = 1:1; (c) HATU, DIPEA, DMF,
.
O-(tetrahydro-2H-pyran-2-yl) hydroxylamine, r.t., 6 h; (d) p-TsOH H
2
O, MeOH, overnight.
2
.2 HDAC inhibition and SAR study of the target compounds
All prepared compounds were examined for their inhibitory ability against HDAC6, using Trichostatin A
TSA, a pan-HDAC inhibitor) and HPOB (sHDAC6i) as the positive controls. The preliminary selective profile
(
toward HDAC1 and HDAC8 were evaluated to guide the structural modification. IC50 values for inhibition of
these isoforms along with selectivity are summarized in Table 1. (S)-isomer 11a bearing a phenyl capping group,
preferentially inhibited HDAC6 and HDAC8 (IC , 17.5 and 15.6 nM respectively) with HDAC1 activity merely
50
in the micromolar range. Interestingly, by simply “switching” the chirality, the R stereoisomer 11b was 2-fold
more active than 11a on HDAC6 inhibition (IC50 = 8.62 nM). In addition, 11b also improved selectivity toward
HDAC6 versus HDAC1 (959-fold) and HDAC8 (4-fold). The shift of hydroxamic acid ZBG from C-4 to C-3 of
the phenyl linker (compound 11a vs 13) led to a sharp decrease of HDAC6 inhibitory potency and complete loss of
HDAC1 and -8 activities.
To our delight, racemic analogues 15a-c with different substituents on N-4 of DKP scaffold retained
enzymatic inhibitory potency. Methyl or phenyl substituent had a little better HDAC6 inhibitory activity than that
of allyl group, with IC50 values of 16.7, 16.3 and 25.4 nM, respectively. This result revealed that the cavity of
protein around N-4 of DKP could tolerate substitutions with various steric hindrances. Subsequently, further
structural modification of N-4 was performed based on 11b which had a better HDAC6 activity and selectivity.
Compared to 11b, compounds 18a-e with unsubstituted or substituted phenyl groups (-Cl and -OMe) all had
slightly reduced activities against HDAC6 with IC50 values in double-digit nanomolar range, but superior
selectivity over HDAC8. As shown in 18b-d, the relative positions of the -Cl substituent on the phenyl had no
obvious impact on activities.
The HDAC6 inhibitory activities were markedly influenced by substituents on C-3 position of DKP.
Replacement of the phenyl capping group with 3-indolyl substitution led to a 2.6-fold improvement of HDAC6
inhibition (21a vs 11a, IC50 = 6.64 and 16.7 nM, respectively). In comparison with 21a, the R-stereoisomer 21b
exhibited more than 9-fold better activity against HDAC6 with an IC50 value of 0.73 nM, and this trend was
consistent with those of 11a and 11b. Moreover, 21b exhibited nearly 10941-fold selectivity versus HDAC1 and
7
00-fold selectivity versus HDAC8. Compared to the sHDAC6 inhibitors HPOB (IC50 = 33.78 nM) and Tubastatin
A (IC50 = 15 nM), 21b also had 46-fold and 21-fold better enzymatic inhibitions on HDAC6 respectively, and
showed higher selectivity over HDAC1 and HDAC8.
Table 1 Inhibitory Profile of DKP Derivatives and Trichostatin A (TSA) against Human HDAC1, -6, and -8 (IC50, nM).
a
IC50
selectivity
1
Compound
R
R
Isomer
HDAC1
HDAC6
HDAC8
HDAC1/6
HDAC8/6
0.9
1
1a
H
H
S
7200± 51.92
17.50± 0.37
8.62± 0.41
15.60 ± 0.40
411
959
11b
R
8270± 143.11
30.90 ± 0.27
4
b
b
1
3
H
S
NA
4190± 93.67
16.70± 0.13
25.40± 0.26
NA
/
/
/
/
/
/
c
1
5a
5b
CH
3
(rac)-
(rac)-
-
-
-
1
-

1
5c
8a
Ph
(rac)-
-
16.30± 0.55
-
/
/
1
Ph
R
R
R
R
R
9640.00±154.02 9.83± 0.21
8310.00±126.33 10.10± 0.11
9200.00±225.25 11.70± 0.34
8550.00±193.67 10.50± 0.32
145. 00± 2.10
162.00± 1.03
130.00± 1.16
150.00± 2.14
171.00± 1.22
981
823
786
838
591
15
16
11
14
12
1
8b
8c
8d
2-Cl-Ph
4-Cl-Ph
3-Cl-Ph
1
1
1
8e
3-OCH
3
-Ph
8100.00± 77.71
13.70± 0.09
2
1a
H
H
S
6390.00±101.50 6.64± 0.13
112.00±1.96
962
17
2
1b
R
8020.00± 36.73
0.73± 0.02
513.00±13.17
10941
700
TSA
HPOB
/
/
/
/
/
/
8.41± 0.25
5.56± 0.05
1160.00±49.90
1990.00±98.50
1.5
53
209
59
1780.00±67.20
33.78± 0.05
Tubastatin
d
d
d
/
/
/
16400
15
854
1093
57
d
A
a
IC50 values for enzymatic inhibition of HDAC enzyme. We ran experiments in duplicate. Assays were performed by Reaction Biology
b
c
d
Corporation (Malvern, PA, USA). NA: no inhibition activity. - : not tested. values for Tubastatin A come from reference[16].
To probe the selectivity of these DKP derivatives versus the broader family of HDAC isotypes, the most
potent compound 21b was selected for full HDAC profiling at all the class I, II and IV HDACs with SAHA (an
approved pan-HDACi) as the reference compound. As described in Table 2, 21b exhibited distinct selectivity over
class I HDAC1-3 (>10940-fold) and class IV HDAC11 (2456-fold). It also showed high level of selectivity over
other class II HDACs (4, 5, 7, 9) reaching >1000-fold. HDAC8 was inhibited with an IC50 of 513 nM, and this
might be contributed to its conformational flexibility to accommodate ligands with different structures including
phenyl-linker compounds. Besides, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay[31]
was performed to test the HDAC10 inhibitory activities of 21b and SAHA. 21b inhibited HDAC10 with an IC50
value of 105 nM which was much weaker than that of HDAC6. These results established 21b to be a potent and
sHDAC6i.
Table 2
a
Complete characterization of 21b at all 11 class I, II and IV HDAC enzymes (IC50 , nM).
Cpd.
21b
SAHA
Cpd.
21b
SAHA
b
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
8020±36.73
4.24±0.12
11.70±0.34
3.05±0.09
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
752.00±10.04
513.00 ±13.17
2560±51.52
NA
b
NA
1020±27.34
b
b
NA
NA
b
c
c
5620±178.56
4370±191.33
0.73± 0.02
NA
105
159
8750±126.78
7.10± 0.27
1800±25.06
952.00 ±31.47
a
IC50 values for enzymatic inhibition of HDAC enzymes. We ran experiments in duplicate. Assays were performed by Reaction Biology
b
c
Corporation (Malvern, PA, USA). NA: no inhibitory activity. HDAC10 activity was measured by the FRET assay.
2
.3 Western blot assay
Acetylation of α-tubulin is an important epigenetic marker for HDAC6 inhibition. Western blot assay was
performed to further validate the HDAC6 selectivity of the DKPs. HCT116 cells were treated with compound 21b,
SAHA and HPOB at 0.1, 1 and 10 µM for 24 h to measure the acetylation status of α-tubulin (HDAC6 dependent)

compared to total H3 acetylation (HDAC1-3 dependent) (Fig. 4). As expected, we observed a dose-dependent
increase in the level of Ac-tubulin when the cells were treated with compound 21b, but did not observe a
significant increase in Ac-H3 even at 10 µM. In contrast, SAHA induced an increase in Ac-tubulin as well as an
obvious increase in Ac-H3. These confirmed that cellular HDAC6 was selectively inhibited by the DKP 21b in a
robust manner.
Fig. 4 Western blot of 21b for Ac-H3 and Ac-Tub in HCT-116 cells with SAHA and HPOB as references.
2
.4 Molecular docking study
Molecular simulation was performed to understand the interaction between the DKPs and protein and guide
the SAR study, we docked the most potent compound 21b and reference compound HPOB into the human
HDAC6 catalytic domain 2 (PDB code: 5EDU) using the Cdocker software. As shown in Fig. 5(a), 21b fits
2+
perfectly into the hydrophobic channel of the binding pocket and exhibits a monodentate hydroxamate-Zn
2+
coordination with a Zn ···O separation of 1.96 Å. While the hydroxamate C=O group forms a hydrogen bond
2+
with the Zn -bound water molecule (O···H separation = 2.10 Å). This unusual zinc-binding mode has been
confirmed in many crystal structures of zHDAC6 complexed with the sHDAC6is[32]. A similar orientation of the
phenylhydroxamic acid group of HPOB was found (Fig. 5b). It was reported that the interaction between inhibitor
and the L1 loop was important for HDAC6 selectivity[33]. As illustrated in Fig. 5(a), the nitrogen atom of indolyl
group forms a hydrogen bond with a water molecule which interacts with the carboxyl residue of Asp497. And this
probably leads to the gap of enzymatic activities between 21b and 11b which bears a phenyl as the capping group.
Fig 5(a). Docking model of 21b (yellow) in complex with hHDAC6. Metal coordination and hydrogen bond interactions were indicated
by solid and dashed blue lines, respectively. Distances between the hydroxamate and zinc ion/water are given in Å. (b) Docking model of
HPOB (magenta) in complex with hHDAC6.The key residues were labeled in white. Zinc ion was labeled in brown. For clarity, all
hydrogen atoms were hidden except water molecule.
To rationalize the selectivity of 21b over other HDACs, especially the class I isoforms, we also performed
docking studies using the solved crystal structures of hHDAC1 (PDB code: 5ICN) and hHDAC8 (PDB code:

1
W22). As show in Fig. 6(a), the phenylhydroxamic acid group of 21b is not able to comfortably coordinate to the
catalytic zinc ion at the bottom of the narrow and deep binding pocket in hHDAC1. The distances between the
hydroxamate group and the zinc ion are obviously larger than those in zHDAC6. In the case of hHDAC8, the
interaction of indolyl and DKP scaffold with the pocket rim is unfavorable. Besides, the crowed space in hHDAC8
surface formed by shorter loop makes 21b in an awkward spatial conformation (Fig. 6b). Thus, these results reveal
the structural basis for the HDAC6 selectivity of the DKP analogues.
Fig 6. (a) The binding mode of 21b (green) in complex with hHDAC1. Metal coordination interactions were indicated by solid blue lines.
Distances between the hydroxamate and zinc ion are given in Å. (b) The binding mode of 21b (yellow) in complex with hHDAC8. The
key residues were labeled in white. Zinc ion was labeled in brown. For clarity, all hydrogen atoms were hidden.
2
.5 Antiproliferative activities of representative compounds
Representative compounds 21b and 18a were selected for antiproliferative screening against a set of 59 solid
or hematological tumor cell lines by NCI (termed NCI-60). The human tumor cell lines of the NCI-60 come from
different tissue origins including blood, lung, colon, central nervous system (CNS), melanoma, ovary, kidney,
prostate and breast. Concretely, compounds were screened at 10 µM concertration, and the growth percents (GP)
of the cells were calculated. As displayed in Table 3, the most potent compound 21b dramatically showed no or
weak antiproliferative activities against most cell lines (GP values > 80%) except HCT116 cell line (colon cancer)
with the GP value of 54.88%. Despite weaker HDAC6 and HDAC1 inhibitions than 21b, 18a seemed to have
overall superior activities against almost all the tumor cell lines. The probable cause of this division might be the
better lipophicity that made 18a penetrate the cell membrane more easily. However, the antiproliferative effects of
1
1
8a were also not remarkable. Among these, Leukemia cells such as RPMI-8226 and SR were more sensitive to
8a, with GP values of 58.59% and 63.55% respectively.
Table 3
In vitro antiproliferative screening against 60 cell lines of 21b and 18a (Growth percents at 10 µM concentration).
a
Origin of cancer
Leukemia
Cell line
CCRF-CEM
HL60 (TB)
K562
21b
18a
71.99
101.78
76.44
76.65
58.59
63.55
95.08
88.17
Origin of cancer
Cell line
M14
21b
18a
102.54
126.17
100.38
86.85
94.21
90.38
91.28
99.38
95.44
96.65
69.30
87.52
88.98
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
101.95
112.53
110.47
108.43
96.14
MOLT-4
RPMI-8226
SR
103.80
114.92
96.58
Non-Small Cell
Lung Cancer
A549/ATCC
EKVX
95.45
106.25
Ovarian Cancer
101.25

HOP-62
HOP-92
102.20
103.95
97.56
87.83
101.66
/
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
104.28
/
79.72
66.45
97.79
74.97
94.55
90.43
94.75
67.39
90.63
96.45
86.04
96.57
88.12
69.49
76.55
86.95
82.86
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
106.97
94.84
104.22
96.53
83.05
84.95
104.01
93.77
104.86
100.35
103.02
91.06
97.49
106.54
96.67
101.09
99.12
86.72
89.50
81.58
69.76
89.18
85.88
84.14
74.31
87.86
86.93
94.06
96.51
100.46
87.34
104.28
96.68
Renal Cancer
Colon Cancer
CNS Cancer
Melanoma
120.39
103.46
54.88
A498
ACHN
CAKI-1
103.26
104.25
102.18
103.39
99.26
RXF 393
SN12C
HT29
KM12
TK-10
SW-620
UO-31
SF-268
Prostate Cancer
Breast Cancer
PC-3
SF-295
105.35
107.20
DU-145
SF-539
MCF7
MDA-MB-231/A
TCC
SNB-19
96.85
85.43
107.24
95.19
SNB-75
U251
94.54
96.73
106.48
94.64
69.38
81.32
93.21
77.06
HS 578T
BT-549
103.99
98.22
82.00
102.82
69.11
79.92
LOX IMVI
MALME-3M
T-47D
87.29
MDA-MB-468
106.87
a
The values of cells growth percents were provided by NCI-60 program.
Based on the results in Table 3, compounds 11b, 18a, 18b, 18d and 21b were selected for the IC50 evaluation
against two multiple myeloma cells RPMI-8226 and U266, using the clinical sHDAC6i ACY-1215 and SAHA as
the positive compounds (Table 4). In comparison of ACY-1215, U266 cell was more sensitive to all five
compounds which had IC50 values in double-digit micromole range. Besides, 18a, 18b and 18d exhibited
antiproliferative activities against RPMI-8226 cell comparable to that of ACY-1215 with IC50 values of 11.650 µM,
1
2.130 µM and 13.420 µM, respectively. However, 11b and 21b, with better effects on HDAC6 inhibition, did not
show matched antiproliferative activities, and this might be due to their poor hydrophobicity. Compared to
sHDAC6is including DKPs and ACY-1215, nonselective SAHA had an obviously better activity. In addition, all
the DKP derivatives showed no influence on normal bone marrow cell HS-5.
Table 4
Growth inhibition (IC50, µM) of 18a, 18b, 21b and reference compounds ACY-1215, SAHA against RPMI-8226 and U266 cell lines.
Compound
1b
RPMI-8226
54.370± 3.055
11.650± 0.845
12.130± 1.220
13.420± 0.860
33.183± 2.140
9.260± 0.842
U266
HS-5
> 100
> 100
> 100
> 100
> 100
/
1
86.340± 8.900
25.882± 1.236
23.370± 2.105
23.100± 2.430
43.233± 5.474
> 100
18a
18b
18d
21b
ACY-1215

SAHA
0.684± 0.083
0.590± 0.012
/
A lot of researches had reported the combined treatment of HDAC6 inhibitor and anticancer agents in
various tumors. In this research, combination of 18a and adriamycin was performed to determine whether the
synergistic antiproliferative effect could occur in solid tumor non-small cell lung cancer cell A549. As shown in
Table 5, compound 18a and adriamycin exhibited significant antiproliferative influence on A549 cell with
combination index (CI ) of 0.676. This finding suggested that combined DKP derivative 18a and adriamycin
50
warranted attention for cancer treatment.
Table 5
Combined cytotoxic effect of 18a and adriamycin on A549 cell line.
Compound
8a
IC50 for individual use
100 µM
IC50 for combination
8.17 µM
1
adriamycin
255 nM
152 nM
a
CI50
0.676
a
The calculation of CI50 value was as shown in experimental section.
2
.6 Microsomal stability study
Preliminary stability evaluation of compounds 18a and 18b in human liver microsome was performed to
determinate half-life (T1/2). As shown in Table 6, the elimination T1/2 values of 18a and 18b were 9.7 h and 10.1 h
in HLM, better than that of ACY-1215 (7.5 h).
Table 6
In vitro stabilities of compounds 18a, 18b and ACY-1215 toward HLM.
Compound
HLM Concentration
0.8 mg/mL
Substrate Concentration
0.5 µM
T1/2
9.7 h
10.1 h
7.5 h
1
8a
8b
1
0.8 mg/mL
0.5 µM
ACY-1215
0.8 mg/mL
0.5 µM
3
. Conclusion
In summary, we designed a series of novel HDAC6 inhibitors bearing natural DKP scaffold and developed a
convenient synthesis to prepare them. Most of these derivatives exhibited distinct inhibitory activities and
selectivity toward HDAC6. The most potent compound 21b showed an IC50 value of 0.73 nM against HDAC6 and
1
0941-fold selectivity over HDAC1. Although weaker enzymatic activity was observed for 18a, it performed
better than 21b in the NCI antitumor screening toward 59 cell lines. Compared to ACY-1215, 18a, 18b and 18d
displayed superior or comparable antiproliferative activities against two multiple myeloma cells with IC50 values
in the low micromolar range. Moreover, the combination of 18a and adriamycin was more potent than either
compound alone. Besides, 18a and 18b also had favorable metabolic stability in HLM. Our results demonstrated
that these DKP derivatives deserved further structural modifications and in vivo pharmacological evaluation.
4
. Experimental section
4
.1 Chemistry
All reagents and solvents were reagent grade or were purified by standard methords before used in the lab.

Isolation and purification of the compounds were performed by flash column chromatography on silica gel 60
230-400 mesh). Analytical thin-layer chromatography (TLC) was conducted on fluka TLC plates (silica gel 60
(
1
13
F254, aluminum foil). The structures of synthesized compounds were characterized by H NMR, C NMR and
MS. The structural information and detailed synthetic process for the intermediates and target compounds are
shown in this section. Melting points were measured using an X-4 melting-point apparatus with a microscope
1
13
(
Beijing Tech Instrument) and were not corrected. H and C NMR spectra were recorded in DMSO-d by a 500
6
MHz spectrometer: chemical shifts (δ) are given in parts per million, coupling constants (J) in Hz. MS were
determined by a Nicolet 2000 FT-IR mass spectrometer and MAT-212 mass spectrometer. All of the target
compounds were examined by HPLC, and the purity of every case was ≥ 95%. Reverse-phase HPLC was
performed on an Agilent Technologies 1260 Infinity, which was equipped with a C18 column (Agilent Zorbax
SB-C18, 5 µM, 4.6 mM × 150 mM). Mobile phase A was acetonitrile, and mobile phase B was water.
4
.1.1 General procedure for the synthesis of DKP scaffolds 8a-b, 12, 17a-e and 20a-b.
Various amino acid esters (0.1 mol) and triethylamine (69.3 mL, 0.5 mol) were added in 250 mL anhydrous
acetonitrile at 0 °C. Then resulting mixture was stirred for 10 minutes. 2-chloroacetyl chloride (8 mL, 0.1 mol) was
added to the reaction solution and the resulting mixture was stirred for additional 1 hour at 0 °C, and treated with
methyl 4-(aminomethyl)benzoate hydrochloride (20.17 g, 0.1 mol). The reaction mixture was heated to reflux for 6
h, then concentrated in vacuo to remove needless triethylamine. To the mixture was added 10% silica gel (5.5 g)
and 200 mL additional acetonitrile, then the mixture was refluxed overnight and monitored end by TLC. The
product was obtained as a white solid by chromatography on a silica gel column.
4
.1.1.1 Methyl (S)-4-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)benzoate (8a). 81.7% isolated yield; white solid;
+
1
MS (ESI, m/z): 375.4 [M+Na] ; Mp: 118-120 °C. H-NMR (500 MHz, DMSO-d ) δ: 8.40 (s, 1H), 7.78 (d, J =
6
8
7
.22 Hz, 2H), 7.56 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 7.06 (s, 1 H), 7.00 (t, J =
.1 Hz, 1H), 6.96 (d, J = 8.1 Hz, 2H), 4.44 (d, J = 15.2 Hz, 1H), 4.28 (d, J = 15.2 Hz 2H), 3.87 (s, 3H), 3.50-3.36
(
m, 2H), 3.07 (dd, J = 14.4 Hz, J = 4.1 Hz, 1H), 2.75 (t, J = 13.6 Hz, 1H).
1 2
4
.1.1.2 Methyl (R)-4-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)benzoate (8b). 85.6% isolated yield; white solid;
+
1
MS (ESI, m/z): 375.4 [M+Na] ; Mp: 119-121°C. H-NMR (500 MHz, DMSO-d ) δ: 8.40 (s, 1H), 7.92 (d, J = 8.2
6
Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 7.2 Hz, 1H), 7.16 (t, J = 7.4 Hz, 2H), 7.10 (d, J = 7.1 Hz, 2H), 4.67
(
d, J = 14.9 Hz, 1H), 4.29 (d, J = 15.0 Hz, 2H), 3.89 (s, 3H), 3.54 (d, J = 17.2 Hz, 1H), 3.18 (dd, J = 13.5 Hz, J =
1 2
3
.8 Hz, 1H), 2.93 (dd, J = 13.5 Hz, J = 4.7 Hz, 1H), 2.74 (t, J = 17.3 Hz, 1H).
1 2
4
.1.1.3 Methyl (S)-3-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)benzoate (12). 92.3% yield, white solid. MS (ESI,
+
1
m/z): 375.4 [M+Na] ; Mp: 115-117 °C. H-NMR (500 MHz, DMSO-d ) δ: 8.26 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H),
6
7
.52 (s, 1H), 7.38 (s, 1H), 7.19 (s, 1H), 7.14 (t, J = 7.3 Hz, 2H), 7.12 (d, J = 7.5 Hz, 2H), 4.92 (d, J = 13.8 Hz, 1H),
.46 (d, J = 15.2 Hz, 2H), 3.89 (s, 3H), 3.55 (d, J = 17.9 Hz, 1H), 3.19 (dd, J = 13.1 Hz, J = 3.8 Hz, 1H), 2.92 (dd,
4
1
2
J = 12.8 Hz, J = 4.9 Hz, 1H), 2.75 (t, J = 17.2 Hz, 1H).
1
2
4
.1.1.4 methyl (R)-4-((3,4-dibenzyl-2,5-dioxopiperazin-1-yl)methyl)benzoate (17a) 66.5% yield, white solid. MS
+
1
1
(
ESI, m/z): 443.5 [M+H] ; Mp: 165-167 °C. H-NMR (500 MHz, DMSO-d ) δ: H-NMR (500 MHz, DMSO-d ) δ:
6
6
7
7
1
.99 (s, 1H), 7.97 (s, 1H), 7.39 (dd, J = 15.1, J = 7.8 Hz, 4H), 7.33 (d, J = 7.8 Hz, 3H), 7.20 (d, J = 7.4 Hz, 1H),
1 2
.05 (t, J = 7.6 Hz, 2H), 6.97 (d, J = 7.3 Hz, 2H), 5.17 (d, J = 14.9 Hz, 1H), 4.71 (d, J = 14.6 Hz, 1H), 4.25 (d, J =
4.5 Hz, 1H), 4.16 (d, J = 7.2 Hz, 1H), 3.90 (s, 3H), 3.61 (d, J = 17.1 Hz, 1H), 3.13-3.02 (m, 2H), 2.48 (d, J = 17.1

Hz, 2H).
4
.1.1.5 Methyl (R)-4-((3-benzyl-4-(2-chlorobenzyl)-2, 5-dioxopiperazin-1-yl)methyl)benzoate (17b) 78.1% yield,
+
1
white solid. MS (ESI, m/z): 477.2 [M+H] ; Mp: 178-180 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.96 (s, 1H), 7.80
6
(d, J = 7.8 Hz, 2H), 7.34 (t, J = 8.4 Hz, 3H), 7.20 (t, J = 7.0 Hz, 1H), 7.11 (t, J = 7.3 Hz, 2H), 7.02 (d, J = 7.2 Hz,
2
3
H), 5.22 (d, J = 15.5 Hz, 1H), 4.65 (d, J = 14.5 Hz, 1H), 4.31 (d, J = 14.6 Hz, 1H), 4.25-4.10 (m, 2H), 3.89 (s,
H), 3.63 (s, 1H), 3.27 (dd, J = 13.5 Hz, J = 4.4 Hz, 1H), 3.16-3.06 (m, 1H), 2.56 (d, J = 17.2 Hz, 1H).
1
2
4
.1.1.6 Methyl (R)-4-((3-benzyl-4-(4-chlorobenzyl)-2, 5-dioxopiperazin-1-yl)methyl)benzoate (17c) 85.2% yield,
+
1
white solid. MS (ESI, m/z): 477.2 [M+H] ; Mp: 196-198 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.97 (s, 1H), 7.80
6
(d, J = 7.7 Hz, 2H), 7.43 (s, 1H), 7.37 (d, J = 7.5 Hz, 2H), 7.30 (d, J = 7.7 Hz, 2H), 7.19 (t, J = 6.9 Hz, 1H), 7.04 (t,
J = 7.1 Hz, 2H), 6.96 (d, J = 7.1 Hz, 2H), 5.16 (d, J = 15.0 Hz, 1H), 4.66 (d, J = 14.4 Hz, 1H), 4.38-4.00 (m, 3H),
3
1
.90 (s, 3H), 3.57 (d, J = 17.1 Hz, 1H), 3.27 (d, J = 9.8 Hz, 1H), 3.11 (d, J = 11.9 Hz, 1H), 2.43 (d, J = 16.9 Hz,
H).
4
.1.1.7 Methyl (R)-4-((3-benzyl-4-(3-chlorobenzyl)-2,5-dioxopiperazin-1-yl)methyl)benzoate (17d) 81.6% yield,
+
1
white solid. MS (ESI, m/z): 477.2 [M+H] ; Mp: 183-185 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.96 (s, 1H), 7.81
6
(d, J = 7.8 Hz, 2H), 7.41 (t, J = 6.9 Hz, 2H), 7.32 (s, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.05 (t, J = 7.3 Hz, 2H), 6.95 (d,
J = 7.3 Hz, 2H), 5.14 (d, J = 15.1 Hz, 1H), 4.67 (d, J = 14.5 Hz, 1H), 4.24 (dd, J = 27.0 Hz, J = 11.4 Hz, 3H),
1
2
3
.90 (s, 3H), 3.59 (d, J = 17.1 Hz, 1H), 3.26 (dd, J = 13.4 Hz, J = 4.1 Hz, 1H), 3.11 (d, J = 11.2 Hz, 1H), 2.46 (d,
1 2
J = 17.2 Hz, 1H).
4
.1.1.8 Methyl (R)-4-((3-benzyl-4-(3-methoxybenzyl)-2, 5-dioxopiperazin-1-yl)methyl)benzoate (17e) 82.0% yield,
+
1
white solid. MS (ESI, m/z): 495.2 [M+Na] ; Mp: 176-178 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.97 (s, 2H),
6
7
.80 (d, J = 7.7 Hz, 2H), 7.31 (d, J = 7.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H), 7.06 (t, J = 7.2 Hz, 2H), 6.98 (d, J = 7.2
Hz, 2H), 6.90 (s, 2H), 5.19 (d, J = 15.0 Hz, 1H), 4.65 (d, J = 14.5 Hz, 1H), 4.25 (d, J = 14.5 Hz, 1H), 4.21 (s, 1H),
4
2
.12 (d, J = 14.9 Hz, 1H), 3.91 (s, 3H), 3.76 (s, 3H), 3.61 (s, 1H), 3.34-3.20 (m, 1H), 3.10 (d, J = 11.1 Hz, 1H),
.49 (d, J = 17.1 Hz, 1H).
4
.1.1.9 Methyl (S)-4-((3-((1H-indol-3-yl)methyl)-2, 5-dioxopiperazin-1-yl)methyl)benzoate (20a) 89.0% yield,
+
1
white solid. MS (ESI, m/z): 392.2 [M+H] ; Mp: 156-158 °C. H-NMR (500 MHz, DMSO-d ) δ: 9.06 (s, 1H), 8.41
6
(d, J = 2.3 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.53 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.06 (d,
J = 2.1 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 8.1 Hz, 2H), 4.31 (d, J = 3.6 Hz, 2H), 4.29-4.26 (m, 1H),
3
1
.91 (s, 3H), 3.37 (s, 1H), 3.36 (d, J = 6.7 Hz, 1H), 3.08 (dd, J = 14.4 Hz, J = 4.3 Hz, 1H), 2.73 (d, J = 17.1 Hz,
1 2
H).
4
.1.1.10 Methyl (R)-4-((3-((1H-indol-3-yl)methyl)-2, 5-dioxopiperazin-1-yl)methyl)benzoate (20b) 90.3% yield,
+
1
white solid. MS (ESI, m/z): 392.2 [M+H] ; Mp: 160-162 °C. H-NMR (500 MHz, DMSO-d ) δ: 9.04 (s, 1H), 8.37
6
(
s, 1H), 7.57 (dd, J = 14.2, J = 7.9 Hz, 3H), 7.41 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.05 (s, 1H), 7.02 (t,
1 2
J = 7.4 Hz, 1H), 6.95 (d, J = 7.8 Hz, 2H), 4.30 (d, J = 8.5 Hz, 2H), 4.28 (s, 1H), 3.43-3.38 (m, 1H), 3.91 (s, 3H),
3
.20 (d, J = 4.8 Hz, 1H), 3.07 (dd, J = 14.3, J = 3.7 Hz, 1H), 2.74 (d, J = 17.2 Hz, 1H).
1 2
4
.1.2 General procedure for the preparation of intermediates 14a-c.
To a mixture of 8a (35.2 mg, 0.1mmol) in anhydrous THF (25 mL) was slowly added NaH (4.4 mg, 0.11

mmol) in batches at 0 °C. After 5 min, halohydrocarbon (0.1 mmol) was added. Then, the mixture was heated to
reflux for 6 h. The reaction was monitored by TLC until the maximum conversion. Methanol was added to quench
the reaction, then the mixture was poured into water (30 mL) and extracted with EtOAc (3×30 mL). The
combined organic extracts were washed with brine (2×30 mL), dried over anhydrous Na SO and concentrated in
2
4
vacuo. The product was obtained by chromatography on a silica gel column.
4
.1.2.1 Methyl 4-((3-benzyl-4-methyl-2, 5-dioxopiperazin-1-yl)methyl)benzoate (14a) 45.3% yield, white solid. MS
+
1
(
ESI, m/z): 367.2 [M+H] ; Mp: 120-122 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.96 (d, J = 7.0 Hz, 2H), 7.36 (d, J
6
=
7.1 Hz, 2H), 7.20 (d, J = 6.8 Hz, 1H), 7.07 (t, J = 6.7 Hz, 2H), 6.97 (d, J = 6.8 Hz, 2H), 4.58 (d, J = 14.6 Hz, 1H),
.41 (s, 1H), 4.33 (d, J = 14.5 Hz, 1H), 3.89 (s, 3H), 3.47 (d, J = 17.1 Hz, 1H), 3.18 (d, J = 12.9 Hz, 1H), 3.10 (d, J
12.9 Hz, 1H), 2.96 (s, 3H), 2.35 (d, J = 17.1 Hz, 1H).
4
=
4
.1.2.2 Methyl 4-((4-allyl-3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)benzoate (14b) 61.0% yield, white solid. MS
+
1
(
ESI, m/z): 443.2 [M+H] ; Mp: 173-175 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.97 (d, J = 7.2 Hz, 2H), 7.38 (d, J
6
=
7.0 Hz, 2H), 7.20 (d, J = 6.2 Hz, 1H), 7.05 (d, J = 6.0 Hz, 2H), 6.98 (d, J = 6.0 Hz, 2H), 5.84 (d, J = 5.5 Hz, 1H),
5
3
.32-5.13 (m, 2H), 4.66 (d, J = 14.5 Hz, 1H), 4.52 (d, J = 15.1 Hz, 1H), 4.29 (d, J = 20.0 Hz, 2H), 3.89 (s, 3H),
.59 (dd, J = 14.5 Hz, J = 5.9 Hz, 1H), 3.53 (d, J = 17.1 Hz, 1H), 3.14 (dd, J = 59.2 Hz, J = 13.0 Hz, 2H), 2.42
1
2
1
2
(d, J = 17.0 Hz, 1H).
4
.1.2.3 Methyl 4-((3,4-dibenzyl-2,5-dioxopiperazin-1-yl)methyl)benzoate (14c) 58.0% yield, white solid. MS (ESI,
+
1
m/z): 393.2 [M+H] ; Mp: 154-156 °C. H-NMR (500 MHz, DMSO-d ) δ: 7.98 (d, J = 7.0 Hz, 2H), 7.49-7.27 (m,
6
7
1
H), 7.19 (d, J = 6.4 Hz, 1H), 7.04 (s, 2H), 6.96 (d, J = 6.0 Hz, 2H), 5.17 (d, J = 15.1 Hz, 1H), 4.72 (d, J = 14.4 Hz,
H), 4.24 (d, J = 14.5 Hz, 1H), 4.14 (d, J = 16.2 Hz, 2H), 3.90 (s, 3H), 3.61 (d, J = 17.0 Hz, 1H), 3.26 (d, J = 10.4
Hz, 1H), 3.07 (d, J = 13.0 Hz, 1H), 2.46 (d, J = 17.0 Hz, 1H).
4
.1.3 General procedure for the preparation of intermediates 16a-e.
To a solution of D-phenylalanine methyl ester hydrochloride (21.6 mg, 0.1 mmol) in MeOH (15 mL) was
added various aromatic aldehydes (0.1 mmol). The resulting mixture was stirred vigorously at room temperature
for 30 min. After confirmation of reaction completion by TLC, NaBH CN was added slowly at 0 °C. The mixture
3
was stirred at room temperature for 12 h. After removal of the solvent, H O was added to dissolve inorganic salt,
2
and the mixture was extracted with EtOAc (3×20 mL). The organic layers were combined, washed with brine,
and dried by anhydrous MgSO . The solvent was removed in vacuo, and the residue was purified by column
4
chromatography.
4
4
7
.1.3.1 Methyl benzyl-D-phenylalaninate (16a)
+
1
1.0% yield, white solid, MS (ESI, m/z): 270.3 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 7.48-7.43 (1H, m),
6
.41 (1H, m), 7.30 (3H, t, J = 7.2 Hz), 7.28-7.22 (3H, m), 7.20 (2H, m), 3.77 (1H, t, J = 16.4 Hz), 3.66-3.59 (2H,
m), 3.59 (3H, s), 3.51-3.42 (1H, m), 3.01-2.84 (2H, m).
4
3
3
2
.1.3.2 Methyl (2-chlorobenzyl)-D-phenylalaninate (16b)
+
1
2.5% yield, white solid, MS (ESI, m/z): 303.2 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 7.15-7.48 (m, 9H),
6
.69 (d, J = 13.5 Hz, 1H), 3.64 (d, J = 13.5 Hz, 1H), 3.55 (s, 3H), 3.52 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 6.2 Hz, 2H),
.44 (s, 1H).

4
3
7
.1.3.3 Methyl (4-chlorobenzyl)-D-phenylalaninate (16c)
+
1
4.1% yield, white solid, MS (ESI, m/z): 303.2 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 7.63-7.49 (m, 4H),
6
.27-7.15 (m, 5H), 3.79 (d, J = 12.9 Hz, 1H), 3.63 (d, J = 13.7 Hz, 1H), 3.56 (s, 3H), 3.45 (d, J = 6.6 Hz, 1H), 3.17
(d, J = 6.6 Hz, 2H), 2.44 (s, 1H).
4
4
3
2
.1.3.4 Methyl (3-chlorobenzyl)-D-phenylalaninate (16d)
+
1
2.7% yield, white solid, MS (ESI, m/z): 303.2 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 7.16-7.60 (m, 9H),
6
.80 (d, J = 13.7 Hz, 1H), 3.61 (d, J = 13.9 Hz, 1H), 3.53 (s, 3H), 3.52 (d, J = 6.5 Hz, 1H), 3.07 (d, J = 6.4 Hz, 2H),
.48 (s, 1H).
4
4
6
3
.1.3.5 Methyl (3-methoxybenzyl)-D-phenylalaninate (16e)
+
1
5.8% yield, white solid, MS (ESI, m/z): 300.2 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 7.35-7.14 (m, 7H),
6
.90 (d, J = 8.8 Hz, 2H), 3.78 (d, J = 13.6 Hz, 1H), 3.64 (d, J = 13.4 Hz, 1H), 3.53 (s, 3H), 3.52 (d, J = 6.7 Hz, 1H),
.02 (d, J = 6.6 Hz, 2H), 2.35 (s, 1H).
4
.1.4 General procedure for the hydrolysis of esters to yield various acids
To a solution of various esters (1 mmol) in MeOH and H O (10 mL, v/v = 1: 1) was added lithium hydroxide
2
(95.8 mg, 4 mmol) at 0 °C. Then, the mixture was allowed to stir at room temperature. The reaction was monitored
by TLC until the maximum conversion. After neutralized with acetic acid, the formed precipitate was collected by
filtration, washed with water, and dried in vacuo to give the title compounds.
4
3
7
4
2
.1.4.1 (S)-4-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)benzoic acid (9a) 78.2% yield, white solid, MS (ESI, m/z):
+
1
39.1 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 12.12 (s, 1H), 8.41 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.58 (d, J =
6
.2 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.09 (s, 1 H), 7.03 (t, J = 7.1 Hz, 1H), 6.70 (d, J = 8.1 Hz, 2H),
.44 (d, J = 15.2 Hz, 1H), 4.28 (d, J = 15.2 Hz 2H), 3.52-3.37 (m, 2H), 3.08 (dd, J = 14.2 Hz, J = 4.4 Hz, 1H),
1
2
.79 (m, 1H).
4
3
7
1
1
.1.4.2 (R)-4-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzoic acid (9b) 69.3% yield, white solid, MS (ESI, m/z):
+
1
39.1 [M+H] . H-NMR (500 MHz, DMSO-d ) δ: 12.04 (s, 1H), 8.41 (s, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.28 (d, J =
6
.9 Hz, 2H), 7.21 (s, 1H), 7.17 (t, J = 7.5 Hz, 2H), 7.10 (d, J = 7.2 Hz, 2H), 4.69 (d, J = 15.1 Hz, 1H), 4.30 (d, J =
5.0 Hz, 2H), 3.58 (d, J = 17.1 Hz, 1H), 3.20 (dd, J = 13.3 Hz, J = 3.6 Hz, 1H), 2.96 (m, 1H), 2.75 (t, J = 17.2 Hz,
1
2
H).
4
.1.5 General procedure for the amidation of various acids with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine
To a solution of acid (1 mmol) in DMF (10 mL) were added DIPEA (0.66 mL, 4 mmol), HATU (353.2 mg,
.1 mmol) at 0 °C. After 30 min, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (117.2 mg, 1 mmol) were added.
1
The mixture was stirred for 5 h. After completion of the reaction as monitored by TLC, the mixture was poured
into water (30 mL) and extracted with EtOAc (3×30 mL). The combined organic extracts were washed with brine
(
2 × 30 mL), dried over anhydrous Na SO and concentrated in vacuo. The product was obtained by
2 4
chromatography on a silica gel column.
4
.1.5.1 4-(((S)-3-benzyl-2,5-dioxopiperazin-1-yl)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (10a)
1
5
3.6% yield, white solid. Mp: 193-195 °C. H-NMR (500 MHz, DMSO-d ) δ: 9.01 (s, 1H), 8.40 (s, 1H), 7.72 (d, J
6
=
8.02 Hz, 2H), 7.13-7.17 (m, 5H), 7.08 (d, J = 7.4 Hz, 2H), 5.70 (s, 1H), 4.63 (d, J = 14.5 Hz, 1H), 4.30 (d, J =

2
2
.1 Hz, 1H), 4.21 (d, J = 14.3 Hz, 1H), 3.74-3.64 (m, 2H), 3.50 (d, J = 17.1 Hz, 1H), 3.17 (d, J = 3.9 Hz, 1H),
.99-2.90 (m, 1H), 2.70 (d, J = 17.2 Hz, 1H), 1.82-1.79 (m, 2H), 1.74-1.56 (m, 4H).
4
.1.5.2 4-(((R)-3-benzyl-2,5-dioxopiperazin-1-yl)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (10b)
1
4
3.7% yield, white solid. Mp: 201-203 °C. H-NMR (500 MHz, DMSO-d ) δ: 9.03 (s, 1H), 8.37 (s, 1H), 7.70 (d, J
6
=
7.9 Hz, 2H), 7.28-7.13 (m, 5H), 7.08 (d, J = 7.1 Hz, 2H), 5.72 (s, 1H), 4.59 (d, J = 14.3 Hz, 1H), 4.30 (s, 1H),
4
2
.25 (d, J = 14.5 Hz, 1H), 3.75-3.68 (m, 2H), 3.50 (d, J = 17.1 Hz, 1H), 3.19 (dd, J = 13.2 Hz, J = 3.5 Hz, 1H),
1
2
.92-2.85 (m, 1H), 2.80-2.65 (m, 1H), 1.80-1.56 (m, 6H).
4
.1.6 General procedure for deprotection of tetrahydro-2H-pyran to yield hydroxylamines
.
To a solution of compound (1 mmol) in methanol (10 mL) was added TsOH H O (0.2 mmol), and the mixture
2
was stirred at room temperature for overnight. After removal of volatiles, residues were purified by
chromatography on a silica gel column.
4
2
7
4
.1.6.1 (S)-4-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (11a) 73.5% yield, white solid. Mp:
1
26-128 °C. H-NMR (500 MHz, DMSO-d ) δ: 11.28 (s, 1H), 9.09 (s, 1H), 8.41 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H),
6
.16 (td, J = 7.2, J = 3.1 Hz, 5H), 7.09 (d, J = 7.2 Hz, 2H), 4.65 (d, J = 14.7 Hz, 1H), 4.31 (d, J = 2.2 Hz, 1H),
1
2
.23 (d, J = 14.7 Hz, 1H), 3.51 (d, J = 17.2 Hz, 1H), 3.17 (d, J = 3.9 Hz, 1H), 2.98-2.90 (m, 1H), 2.70 (d, J = 17.2
13
Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 166.00, 165.27, 145.98, 138.25, 136.03, 130.55, 128.57, 128.54,
6
2
D
0
1
27.46, 127.26, 126.01, 56.01, 49.08, 48.91, 48.67. [α]
= −0.49 (1.22 mg/mL, MeOH). HR-MS (ESI, m/z):
+
+
Calcd for: 354.1448. (C H N O , [M+H] ). Found: 354.1449.
19
20
3
4
4
.1.6.2 (R)-4-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (11b) 72.1% yield, white solid. Mp:
11-212 °C. 11b (R-configuration) showed almost the same proton resonances as the enantiomer 11a
2
(
S-configuration). In the lower field, nine proton signals indicated two groups of aryl protons resonance.
Higher-field signals showed three pairs of methylene groups with coupling constants 14.7, 17.2 and 13.3 Hz. The
1
proton signal of chiral C-3 on DKP skeleton is consistent with 11a. H-NMR (500 MHz, DMSO-d ) δ: 11.23 (s,
6
1
1
H), 9.04 (s, 1H), 8.36 (s, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.28-7.13 (m, 5H), 7.09 (d, J = 7.1 Hz, 2H), 4.64 (d, J =
4.7 Hz, 1H), 4.31 (m, 1H), 4.24 (d, J = 14.7 Hz, 1H), 3.52 (d, J = 17.2 Hz, 1H), 3.18 (dd, J = 13.3 Hz, J = 3.6
1
2
13
Hz, 1H), 2.99-2.88 (m, 1H), 2.80-2.68 (m, 1H). C-NMR (126 MHz, DMSO-d ) δ: 165.97, 165.27, 145.98,
6
2
D
5
1
39.37, 136.02, 130.55, 128.58, 128.55, 127.52, 127.27, 55.99, 48.92, 48.66. [α]
= −0.47 (1.06 mg/mL, MeOH).
+
+
HR-MS (ESI, m/z): Calcd for: 354.1448. (C H N O , [M+H] ). Found: 354.1449.
19
20
3
4
4
1
7
7
1
1
4
.1.6.3 (S)-3-((3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (13) 62.1% yield, white solid. Mp:
1
76-178 °C. H-NMR (500 MHz, DMSO-d ) δ: 11.32 (s, 1H), 9.12 (s, 1H), 8.40 (s, 1H), 7.71 (d, J = 7.0 Hz, 1H),
6
.66 (s, 1H), 7.53 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.1 Hz, 1H), 7.28 (d, J = 6.5 Hz, 1H), 7.15 (s, 1H), 7.10 (s, 1H),
.06 (s, 1H), 4.61 (d, J = 14.4 Hz, 1H), 4.26 (d, J = 15.2 Hz, 2H), 3.50 (d, J = 17.2 Hz, 1H), 3.17 (d, J = 13.1 Hz,
13
H), 2.93 (d, J = 13.4 Hz, 1H), 2.65 (d, J = 17.2 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 166.03, 165.37,
6
45.87, 138.32, 136.52, 135.93, 131.57, 130.50, 128.99, 128.60, 128.48, 127.68, 127.21, 126.48, 126.01, 55.99,
2
D
5
+
+
8.81. [α]
= −0.67 (1.16 mg/mL, MeOH). HR-MS (ESI, m/z): Calcd for: 354.1448. (C H N O , [M+H] ).
19 20 3 4
Found: 354.1449.
4
.1.6.4 4-((3-benzyl-4-methyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (15a) 83.3% isolated yield;
1
white solid. Mp: 190-192 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.26 (s, 1H), 9.07 (s, 1H), 7.75 (d, J = 8.1 Hz,
6

2
H), 7.28 (d, J = 8.1 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 2H), 6.96 (d, J = 7.3 Hz, 2H), 4.56 (d, J
14.5 Hz, 1H), 4.40 (t, J = 4.1 Hz, 1H), 4.28 (d, J = 14.5 Hz, 1H), 3.45 (d, J = 17.0 Hz, 1H), 3.14 (m, 2H), 2.95 (s,
=
1
3
3
1
H), 2.32 (d, J = 17.0 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 166.01, 163.98, 139.17, 135.58, 132.56, 130.15,
6
29.02, 128.67, 127.59, 127.52, 62.84, 48.68, 48.60, 36.63, 31.99. HR-MS (ESI, m/z): Calcd for: 368.16048.
+
+
(
C H N O , [M+H] ). Found: 368.16068.
20 22 3 4
4
.1.6.5 4-((4-allyl-3-benzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (15b) 79.2% isolated yield;
1
white solid. Mp: 194-195 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.27 (s, 1H), 9.08 (s, 1H), 7.76 (d, J = 7.6 Hz,
6
2
H), 7.30 (d, J = 7.7 Hz, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 7.2 Hz, 2H), 6.97 (d, J = 7.2 Hz, 2H), 5.95-5.74
(m, 1H), 5.35-5.17 (m, 2H), 4.62 (d, J = 14.4 Hz, 1H), 4.52 (d, J = 14.4 Hz, 1H), 4.25 (d, J = 13.8 Hz, 2H), 3.59
(
dd, J = 15.3 Hz, J = 6.6 Hz, 1H), 3.51 (d, J = 17.0 Hz, 1H), 3.20 (dd, J = 13.6, J = 4.2 Hz, 1H), 3.09 (d, J =
1
2
1
2
1
3
1
1
3.0 Hz, 1H), 2.41 (d, J = 17.0 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 166.05, 164.42, 164.09, 139.22,
6
35.51, 133.12, 132.61, 130.28, 129.08, 128.67, 127.58, 118.67, 60.47, 48.80, 48.74, 45.91, 36.85. HR-MS (ESI,
+
+
m/z): Calcd for 394.17613. (C H N O [M+H] ). Found 394.17633.
22
24
3
4
4
.1.6.6 4-((3, 4-dibenzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (15c) 91.2% isolated yield; white
1
solid. Mp: 218-220 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.27 (s, 1H), 9.08 (s, 1H), 7.98 (s, 1H), 7.77 (d, J =
6
8
7
1
3
1
4
.2 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.34 (d, J = 2.0 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H),
.05 (t, J = 7.6 Hz, 2H), 6.96 (d, J = 7.2 Hz, 2H), 5.17 (d, J = 15.0 Hz, 1H), 4.67 (d, J = 14.4 Hz, 1H), 4.21 (d, J =
4.4 Hz, 1H), 4.18-4.14 (m, 2H), 4.13 (s, 1H), 3.59 (d, J = 17.1 Hz, 1H), 3.26 (dd, J = 13.8 Hz, J = 5.2 Hz, 1H),
1
2
13
.08 (dd, J = 13.8, J = 3.7 Hz, 1H), 2.47 (d, J = 17.1 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 165.98, 164.59,
1
2
6
62.79, 139.17, 137.07, 135.49, 132.62, 130.28, 129.21, 129.10, 128.69, 128.52, 128.11, 127.60, 127.57, 60.61,
+
+
8.84, 48.80, 46.53, 36.76. HR-MS (ESI, m/z): Calcd for 444.19178. (C H N O [M+H] ). Found 444.19186.
26
26
3
4
4
.1.6.7 (R)-4-((3, 4-dibenzyl-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (18a) 61.3% isolated yield;
1
white solid. Mp: 220-222 °C; H NMR (500 MHz, DMSO-d ) δ: 11.27 (s, 1H), 9.07 (s, 1H), 7.77 (d, J = 7.9 Hz,
6
2
H), 7.41 (t, J = 7.3 Hz, 2H), 7.33 (d, J = 7.1 Hz, 3H), 7.30 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.06 (t, J
7.5 Hz, 2H), 6.96 (d, J = 7.5 Hz, 2H), 5.17 (d, J = 15.0 Hz, 1H), 4.67 (d, J = 14.5 Hz, 1H), 4.21 (d, J = 14.5 Hz,
H), 4.16 (s, 2H), 4.13 (s, 1H), 3.59 (dd, J = 17.1 Hz, J = 9.0 Hz, 1H), 3.25 (d, J = 4.9 Hz, 1H), 3.13-3.02 (m,
=
1
1
1
−
1
1
13
H), 2.47 (d, J = 16.9 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 165.99, 164.59, 162.80, 139.18, 137.06, 135.50,
6
2
D
5
32.63, 130.28, 130.00, 129.21, 129.09, 128.69, 128.52, 128.11, 127.60, 60.63, 48.85, 48.80, 46.55, 36.27. [α]
=
+
+
2.02 (1.37 mg/mL, MeOH). HR-MS (ESI, m/z): Calcd for 444.19178. (C H N O [M+H] ). Found 444.19186.
26
26
3
4
4
.1.6.8 (R)-4-((3-benzyl-4-(2-chlorobenzyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (18b) 63.5%
1
isolated yield; white solid. Mp: 225-227 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.38 (s, 1H), 9.20 (s, 1H), 7.97 (s,
6
1
H), 7.81 (d, J = 7.8 Hz, 2H), 7.33 (t, J = 8.4 Hz, 3H), 7.22 (t, J = 7.0 Hz, 1H), 7.10 (t, J = 7.3 Hz, 2H), 7.03 (d, J
7.2 Hz, 2H), 5.23 (d, J = 15.5 Hz, 1H), 4.64 (d, J = 14.5 Hz, 1H), 4.32 (d, J = 14.5 Hz, 1H), 4.26-4.11 (m, 2H),
=
13
3
.62 (s, 1H), 3.28 (dd, J = 13.5 Hz, J = 4.4 Hz, 1H), 3.18-3.08 (m, 1H), 2.57 (d, J = 17.2 Hz, 1H). C-NMR (126
1 2
MHz, DMSO-d ) δ: 165.99, 164.81, 162.88, 139.28, 135.38, 133.84, 133.15, 132.47, 130.30, 130.16, 129.97,
6
2
D
5
1
29.03, 128.97, 128.77, 127.95, 127.66, 127.60, 60.94, 48.82, 48.75, 44.94, 36.97. [α]
= 3.46 (1.20 mg/mL,
+
+
MeOH). HR-MS (ESI, m/z): Calcd for 478.15281. (C H ClN O [M+H] ). Found 478.15289.
26
25
3
4
4
.1.6.9 (R)-4-((3-benzyl-4-(4-chlorobenzyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (18c) 58.0%
1
isolated yield; white solid. Mp: 205-207 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.38 (s, 1H), 9.20 (s, 1H), 7.97 (s,
6

1
1
4
H), 7.80 (d, J = 7.7 Hz, 2H), 7.43 (s, 1H), 7.37 (d, J = 7.5 Hz, 2H), 7.31 (d, J = 7.7 Hz, 2H), 7.19 (t, J = 6.9 Hz,
H), 7.05 (t, J = 7.1 Hz, 2H), 6.96 (d, J = 7.1 Hz, 2H), 5.15 (d, J = 15.0 Hz, 1H), 4.67 (d, J = 14.4 Hz, 1H),
.38-4.00 (m, 3H), 3.57 (d, J = 17.1 Hz, 1H), 3.27 (d, J = 9.8 Hz, 1H), 3.11 (d, J = 11.9 Hz, 1H), 2.44 (d, J = 17.1
13
Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 165.95, 164.68, 162.87, 139.19, 136.10, 135.34, 132.78, 132.46,
6
2
D
5
1
30.37, 130.25, 129.09, 128.68, 127.61, 60.77, 48.85, 48.78, 46.05, 36.79. [α]
= 0.86 (1.14 mg/mL, MeOH).
+
+
HR-MS (ESI, m/z): Calcd for 478.15281. (C H ClN O [M+H] ). Found 478.15289.
26
25
3
4
4
.1.6.10 (R)-4-((3-benzyl-4-(3-chlorobenzyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (18d) 78.2%
1
isolated yield; white solid. Mp: 231-233 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.36 (s, 1H), 9.17 (s, 1H), 7.97 (s,
6
2
2
1
H), 7.80 (d, J = 7.8 Hz, 2H), 7.41 (t, J = 6.9 Hz, 2H), 7.32 (s, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.05 (t, J = 7.3 Hz,
H), 6.96 (d, J = 7.3 Hz, 2H), 5.14 (d, J = 15.1 Hz, 1H), 4.67 (d, J = 14.5 Hz, 1H), 4.24 (dd, J = 27.0 Hz, J =
1
2
1.4 Hz, 3H), 3.59 (d, J = 17.1 Hz, 1H), 3.27 (dd, J = 13.4 Hz, J = 4.1 Hz, 1H), 3.12 (d, J = 11.2 Hz, 1H), 2.45 (d,
1
2
13
J = 17.1 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 165.97, 164.79, 162.85, 139.73, 139.20, 135.36, 133.81,
6
2
D
5
1
1
32.53, 131.00, 130.26, 129.07, 128.70, 128.26, 128.04, 127.62, 127.10, 60.98, 48.83, 48.78, 46.29, 36.88. [α]
=
+
+
.18 (1.13 mg/mL, MeOH). HR-MS (ESI, m/z): Calcd for 478.15281. (C H ClN O [M+H] ). Found 478.15289.
26
25
3
4
4
.1.6.11 (R)-4-((3-benzyl-4-(3-methoxybenzyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (18e) 48.6%
1
isolated yield; white solid. Mp: 200-202 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.38 (s, 1H), 9.21 (s, 1H), 7.97 (s,
6
2
H), 7.80 (d, J = 7.7 Hz, 2H), 7.31 (d, J = 7.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H), 7.06 (t, J = 7.2 Hz, 2H), 6.98 (d, J
7.2 Hz, 2H), 6.91 (s, 2H), 5.19 (d, J = 15.0 Hz, 1H), 4.65 (d, J = 14.5 Hz, 1H), 4.26 (d, J = 14.5 Hz, 1H), 4.20 (s,
H), 4.12 (d, J = 14.9 Hz, 1H), 3.76 (s, 3H), 3.61 (s, 1H), 3.34-3.20 (m, 1H), 3.10 (d, J = 11.1 Hz, 1H), 2.49 (d, J =
=
1
1
1
−
13
7.1 Hz, 1H). C-NMR (126 MHz, DMSO-d ) δ: 166.07, 164.65, 162.88, 160.03, 139.22, 138.52, 135.45, 132.52,
6
2
D
5
30.33, 130.27, 129.06, 128.69, 127.61, 120.50, 114.26, 113.33, 60.64, 56.27, 55.44, 48.82, 46.48, 36.76. [α]
=
+
+
1.51 (1.02 mg/mL, MeOH). HR-MS (ESI, m/z): Calcd for 474.20235. (C H N O [M+H] ). Found 474.20245.
27
28
3
5
4
.1.6.12 (S)-4-((3-((1H-indol-3-yl)methyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (21a) 53.0%
1
isolated yield; white solid. Mp: 198-200 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.22 (s, 1H), 11.05 (s, 1H), 9.06
6
(
s, 1H), 8.41 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.53 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.5 Hz,
H), 7.05 (d, J = 2.1 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.1 Hz, 2H), 4.31 (d, J = 3.6 Hz, 2H), 4.29-4.26
m, 1H), 3.38 (s, 1H), 3.36 (d, J = 6.7 Hz, 1H), 3.08 (dd, J = 14.4 Hz, J = 4.3 Hz, 1H), 2.73 (d, J = 17.1 Hz, 1H).
1
(
1
2
13
C-NMR (126 MHz, DMSO-d ) δ: 166.64, 165.11, 145.91, 139.44, 138.30, 136.48, 132.29, 128.60, 127.97,
6
2
D
5
1
27.57, 126.01, 125.19, 121.48, 119.20, 118.97, 111.83, 108.50, 56.14, 49.01, 48.42. [α]
= 0.92 (1.20 mg/mL,
+
+
MeOH). HR-MS (ESI, m/z): Calcd for 393.15573. (C H N O [M+H] ). Found 393.15594.
21
21
4
4
4
.1.6.13 (R)-4-((3-((1H-indol-3-yl)methyl)-2, 5-dioxopiperazin-1-yl)methyl)-N-hydroxybenzamide (21b) 61.5%
1
isolated yield; white solid. Mp: 189-191 °C; H-NMR (500 MHz, DMSO-d ) δ: 11.16 (s, 1H), 10.97 (s, 1H), 9.04
6
(
s, 1H), 8.37 (s, 1H), 7.58 (dd, J = 14.2, J = 7.9 Hz, 3H), 7.40 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.06 (s,
1 2
1
3
H), 7.01 (t, J = 7.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 2H), 4.31 (d, J = 8.5 Hz, 2H), 4.29 (s, 1H), 3.43-3.38 (m, 1H),
13
.20 (d, J = 4.8 Hz, 1H), 3.07 (dd, J = 14.3, J = 3.7 Hz, 1H), 2.74 (d, J = 17.2 Hz, 1H). C-NMR (126 MHz,
1
2
DMSO-d ) δ: 166.63, 165.14, 164.56, 139.46, 136.47, 132.33, 128.00, 127.89, 127.54, 125.19, 121.52, 119.20,
6
2
D
5
119.00, 111.80, 108.55, 56.13, 49.09, 49.01, 48.43. [α]
= 2.16 (1.16 mg/mL, MeOH). HR-MS (ESI, m/z): Calcd
+
+
for 393.15573. (C H N O [M+H] ). Found 393.15594.
21
21
4
4
4
.2 In vitro HDAC enzymatic assay

The inhibitory activity of all HDAC isoforms except HDAC10 were conducted by the Reaction Biology
Corporation, Malvern , PA using HDAC fluorescent activity assay based on the unique Fluor de Lys™ substrate
and developer combination. Compounds were dissolved in DMSO and tested in at least 10-dose IC50 mode with
3
-fold serial dilution starting at 50 µM. GraphPad Prism 5.0 software was used to calculate the IC50 values for each
compound.
The assay for HDAC10 activity was performed in the Cancer Drug Development group at the German Cancer
Research Center (69210 Heidelberg, Germany). The detailed protocol can be found in reference [30].
4
.3 Western Blotting
HCT116 cells (1×10 ) were seeded overnight and then incubated with indicated concentrations of compounds
6
for 24 h. Cell extract was prepared by lysing cultured cells with a mammalian protein extraction reagent
supplemented with EDTA-free protease inhibitor for 15 min. Supernatants were collected following centrifugation
of lysed cells at 15000 g for 10 min at 4 °C. To analyze the cell lysate, 30 µg of total protein per sample was
resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes with immobilized proteins
were probed with antibodies for H3 acetylation. The reactive protein bands were visualized using an ECL
detection system.
4
.4 Growth Percent on 60 Cell Lines by NCI.
All detailed test method is described on the NCI Web site (https://dtp.cancer.gov/discovery_development
/
nci-60/methodology.htm).
4
.5 In vitro cell growth inhibitory activity.
Culture medium and culture condition of cell lines. The cells were cultured in IMDM medium with 20% FBS,
00 U/mL penicillin and 100 ug/mL streptomycin. All cells were maintained at 37 °C in a humidified atmosphere
1
of 5% CO in air.
2
Cell Growth Inhibitory Assay. The protocol using Alamar blue reagent for antiproliferative activitives was as
follows: 1. plated 100 µL cell suspension or completed medium into 96-well plate using a Matrix 12-channel
pipettor. And filled residual wells with 200 µL PBS per well; 2. drugs were added to each well of 96-well plate; 3.
placed plate into an incubator with corresponding culture condition for 72 h; 4. pipetted 22 µL Alamar blue
solution (1 mM) into each well of 96-well plate; 5. returned plate to incubator and leave for 5~6 h; 6. shook plate
for 10 seconds and recorded fluorescence at 530/590 nm.
Combination index was calculated by applying the equation CI50 = IC50Ac/IC50As + IC50Bc/IC50Bs, where IC50As
and IC50Bs are concentrations of inhibitors A and B required to produce 50% effect when used as single agents, and
IC50Ac and IC50Bc are concentrations of inhibitors A and B required to produce 50% effect when used in
combination.
4
.6 Computational Methods
All computational work was performed in Discovery Studio 3.0 software (BIOVIA, 5005 Wateridge Vista
Drive, San Diego, CA92121 USA). Docking was conducted using cdocker module based on the cocrystals of
human HDAC6 (PDB code: 5EDU), human HDAC1 (PDB code: 5ICN) and human HDAC8 (PDB code: 1W22).
The cavities of the cocrystals occupied by their ligands were selected as the binding sites. Key waters molecules in
the binding pocket were reserved. The energy minimization for compound was performed by Powell's method for
1
000 iterations using tripos force field and with Gasteiger-Hückel charge. The other docking parameters were kept
as default.

4
.7 Microsomal stability assay
Human liver microsome was purchased from Research Institute for Liver Disease (Shanghai) Co., Ltd.
NADPH was purchased from Roche. The assay was as follows: each incubated mixture contained 0.8 mg/mL
human liver microsome, 50 µL magnesium chloride, 60 µL potassium phosphate buffer (pH 7.4) and 0.5 µM test
compound in a total volune of 200 µL. After prewarming at 37 °C for 5 min, 50 µL NADPH was added to initiate
the reaction. The reaction was terminated after 0, 5, 10, 15, 30, 60 or 90 min by adding 400 µL ice-cold ethyl
acetate into 200 µL of incubation mixture. The sample was then centrifuged at 4000 rpm for 10 min at 4 °C. The
supernatant was then analyzed by LC-MS/MS.
Acknowledgment
We thank the NCI Division of Cancer Treatment & Diagnosis for the NCI-60 screening of our compounds.
This work was supported by the National Natural Science Foundation of China (81903464 and 21503168),
Chinese Universities Scientific Fund (2452017174) and the Natural Science Foundation of Jiangsu Province
(BK20180573).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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HIGHLIGHTS
ò Potent and highly selective HDAC6 inhibitors based on the natural 2, 5-diketopiperazine scaffold.
ò Computational simulations suggested the key interactions between DKPs and HDAC6.
ò Combination of 18a and adriamycin exhibited synergistic antiproliferative influence on A549 cell.

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.