Structure-based design, synthesis, and biological evaluation of novel piperine-resveratrol hybrids as antiproliferative agents targeting SIRT-2

Article abstract of DOI:10.1039/d1ra04061h

A series of novel piperine-resveratrol hybrids5a-hwas designed, synthesized, and structurally elucidated by IR, and¹H,¹³C, and¹⁹F NMR. Antiproliferative activities of5a-hwere evaluated by NCI against sixty cancer cell line

Full text of DOI:10.1039/d1ra04061h

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PAPER
Structure-based design, synthesis, and biological
evaluation of novel piperine–resveratrol hybrids as
antiproliferative agents targeting SIRT-2†
Cite this: RSC Adv., 2021, 11, 25738
abc
d
*
*
Ahmed H. Tantawy,
Xiang-Gao Meng, Adel A. Marzouk,e Ali Fouad,e
Ahmed H. Abdelazeem,fg Bahaa G. M. Youssif,
Hong Jiang
h
b
*
a
*
and Man-Qun Wang
A series of novel piperine–resveratrol hybrids 5a–h was designed, synthesized, and structurally elucidated
by IR, and 1H, 13C, and 19F NMR. Antiproliferative activities of 5a–h were evaluated by NCI against sixty
cancer cell lines. Compound 5b, possessing resveratrol pharmacophoric phenolic moieties, showed
a complete cell death against leukemia HL-60 (TB) and Breast cancer MDA-MB-468 with growth
inhibition percentage of ꢀ0.49 and ꢀ2.83, respectively. In addition, 5b recorded significant activity
against the other cancer cell lines with growth inhibition percentage between 80 to 95. New 5a–h
hybrids were evaluated for their inhibitory activities against Sirt-1 and Sirt-2 as molecular targets for their
antiproliferative action. Results showed that compounds 5a–h were more potent inhibitors of Sirt-2 than
Sirt-1 at 5 mm and 50 mm. Compound 5b showed the strongest inhibition of Sirt-2 (78 ꢁ 3% and 26 ꢁ 3%
inhibition at 50 mM and 5 mM, respectively). Investigation of intermolecular interaction via Hirschfeld
surface analysis indicates that these close contacts are mainly ascribed to the O–H/O hydrogen
bonding. To get insights into the Sirt-2 inhibitory mechanism, a docking study was performed where 5b
was found to fit nicely inside both extended C-pocket and selectivity pocket and could compete with
the substrate acyl-Lys. Another possible binding pattern showed that 5b could act by partial occlusion of
the NAD+ C-pocket. Collectively, these findings would contribute significantly to better understanding
the Sirt-2 inhibitory mechanism in order to develop a new generation of refined and selective Sirt-2
inhibitors.
Received 25th May 2021
Accepted 23rd June 2021
DOI: 10.1039/d1ra04061h
rsc.li/rsc-advances
1. Introduction
Since cancer is one of the world's leading major health prob-
lems causing death,1–3 nding and discovering new successful
aHubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory,
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan
430070, People's Republic of China. E-mail: mqwang@mail.hzau.edu.cn; ahmed.
tantawy@mail.hzau.edu.cn
anticancer drugs is one of the biggest challenges in drug
research. Sirtuins have attracted attention over the last decade
because they have participated in the regulation of many
processes that affect cancer cells,4,5 such as cellular metabo-
lism,6,7 chromatin structure control and genomic stability
maintenance.8,9 Sirtuins are part of a family of seven human
enzymes (SIRT1-7), which are NAD-related histone deacetylases
(HDACs).10,11 With NAD, SIRTs catalyze acetyl group removal
from N-acetyl lysine amino acid on histone substrates which
generates deacetylated proteins, nicotinamides and O-acetyl-
ADP-ribose molecules.12,13 Isotype Sirt-2 has taken part in
several cellular processes such as gene transcription, genome
constancies and cell cycle regulation during mitosis.14,15 Sirt-2 is
a key factor in the development of cancer and metastasis by
increasing cancer cell motility.16 In addition, Sirt-2 inhibition
showed an increase in tumor suppressor genes, including p53
and p21.17 Small molecules that can control sirtuin activities are
therefore considered potential therapeutics for the treatment of
bDepartment of Chemistry, College of Science, Huazhong Agricultural University,
Wuhan, 430070, China. E-mail: jianghong0066@126.com
cDepartment of Chemistry, College of Science, Benha University, Benha 13518, Egypt
dKey Laboratory of Pesticide and Chemical Biology, Ministry of Education, School of
Chemistry, Central China Normal University, Wuhan 430079, China. E-mail:
xianggao_meng@126.com
eDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University,
Assiut Branch, Assiut 71524, Egypt
fDepartment of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef 62514, Egypt
gDepartment of Pharmaceutical Sciences, College of Pharmacy, Riyadh Elm University,
Riyadh 11681, Saudi Arabia
hPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
† Electronic supplementary information (ESI) available. CCDC 1984255, 1984254,
2009516, 1984253, 1984251 and 1934954. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/d1ra04061h
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Fig. 1 The modification of piperine structure and the impact on the anticancer activity.
various human disorders, including cancer. Knockdown or sirtuins family with a great modulating potential. The well-
inhibition of Sirt-2 may disrupt the metabolism of cancer cells known stilbenoid polyphenol, resveratrol, was reported as
and thus prevent the spread and growth of cancer cells.18–21
a Sirt-1 and Sirt-5 activator while a weak Sirt-2 and Sirt-3
Natural products are one of the chief consistent sources of inhibitor.27–31 However, its metabolite piceatannol was found
lead drugs. Developments based on natural products including to act as an inhibitor to Sirt-2 protein.31 From the study of the
various anticancer drugs such as topotecan, docetaxel, vinde- reported SAR and the interactions with some SIRT proteins, it
sine, etoposide and vinorelbine have been published.22,23 was conceptualized that the phenolic moieties have a remark-
Natural products produced from bacterial, fungal, marine, able impact on their SIRT activities.27–31 Till now, there are no
plant and animal sources and natural product-inspired reported studies addressing the binding pattern and the key
compounds have wide benets in clinical trials as anti- interactions of resveratrol with Sirt-2 subtype despite the high
inammatory drugs, anticancer drugs, or other pharmaceu- degree of structural similarity between the conserved catalytic
tical agents.24 It is estimated that natural product-derived domains of the human sirtuins family. In order to explore this
compounds constitute more than 50% of anticancer agents; possible pattern and optimize its activity against Sirt-2 in
about 74% of anticancer compounds are either natural or particular, resveratrol was docked into the active site of Sirt-2
natural product-inspired compounds.25
(pdb code: 4RMG) co-crystallized with the SirReal2 ligand
Piperine, Fig. 1, is a nitrogenous alkaloid found in black using the Ligand Fit protocol found in Discovery Studio so-
pepper powder fruit that is widely used as a food avor in ware 2.5. It was obvious from the docking results that resvera-
a number of countries and used in many conventional food trol occupied a part of the extended C-site at the interface
preservation systems as well as in traditional medicines.26 between the Rossmann-fold domain and the zinc-binding
Piperine was proven to have anticancer activity with various domain without any clashing with either the acyl-Lys
mechanisms of action. The substitution of the piperidine substrate channel or the nicotinamide moiety of the co-factor
moiety with bulkier and extended groups has been reported to NAD+. It was involved in only one conventional H-bond with
signicantly enhance the potency as noted in compound 2, the Ile-118 residue and some hydrophobic interactions with
where the tryptophanyl moiety has been added.22
Leu-134 Leu-138, Tyr-139, Phe-143, and Phe-190 amino acids.
On the other hand, the polyphenolic compounds such as This interpretation suggests a considerable possibility for
resveratrol and piceatannol showed high affinity to the human further structural optimization, Fig. 2(A and B). This point can
Fig. 2 Docking of resveratrol into SIRT-2 3D structure (pdb code: 4RMG). (A) The disposition of resveratrol (orange) inside SIRT-2 active site
where the protein is represented as a solid surface colored according to atom charges; (B) the predicted binding pattern and interactions of
resveratrol within the active site of SIRT-2; (C) the suggested sites of structural modifications and elongation of resveratrol; green mesh
represents the Acyl-Lys substrate channel and the red mesh represents the C-pocket of nicotinamide moiety of NAD+ co-factor (cyan). The
poses were rendered as stick model and the residues are shown as smaller grey sticks. All hydrogens were removed for the purposes of clarity.
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Fig. 3 The design strategy of the novel piperine–resveratrol hybrids. In this design, the piperine backbone was tethered to 1,3-dihydroxyphenyl
moiety of resveratrol or piceatannol using an azomethine group as a linker via a fragment-based drug design approach to obtain the final
pharmacophore. Then, SAR studies was performed through replacing the phenolic moiety by with various electron-donating and electron-
withdrawing substituents.
be clearly rationalized by the small size of the resveratrol
Considering the aforementioned ndings, a new hybrid
structure. Inspirited by these ndings, we thought that the scaffold has been designed bearing the resveratrol pharmaco-
elongation or extension of the resveratrol structure to protrude phoric features bound to the piperine backbone using a frag-
into the hydrophobic acyl-lysine tunnel surrounded by the ment-based drug design approach in order to target the
highly conserved phenylanilines 119, 131, 234, 235 and Val233 sirtuins proteins, in particular Sirt-2, as molecular mechanism
or to partially occlude the NAD+ C-pocket would be a promising for the anticancer action of newly synthesized series, Fig. 3. To
approach to target and competitively inhibit the Sirt-2 isoform, extend our study and investigate the SAR, a series of piperine
Fig. 2(C). Thus, our strategy to increase the size of resveratrol or derivatives were synthesized by replacement of phenolic moiety
even utilizing its pharmacophoric phenolic moieties was with various electron donating and electron withdrawing
accomplished by hybridization with the piperine structure via substituents. The identity of the newly synthesized hybrids 5a–h
a hydrazino-linker in one scaffold, Fig. 3.
was proved using 1H, 13C and 19F NMR. Furthermore, the single-
Moreover, several reports showed that piperine enhanced crystal structures of 5a–f were unambiguously elucidated by X-
the in vivo bioavailability and ADME properties of resveratrol.32 ray crystallography. The anticancer activities of 5a–h were
Hence, a commercial combination of piperine with resveratrol evaluated by NCI against sixty cancer cell lines of nine different
is currently available in the market and it is promoted to have tissues.
many health benets in terms of improving strength and
endurance. Additionally, this combination is used as antioxi-
dant, cancer protective and for weight loss. Recently, it was
2. Results and discussion
found that the combination of resveratrol with the polyphenolic
compounds such as piperine and curcumin has a signicant
activity against estrogen receptor-positive MCF-7 breast cancer
cells however, the authors proposed the action was accom-
plished through reducing glyoxalase-1 (GLO1) activity.32,33
2.1. Chemistry
Scheme 1 outline the synthetic procedures for key intermedi-
ates 2, 3, and target compounds 5a–h. According to Scheme 1,
piperic acid 2 was formed by hydrolyzing piperine (1) with
alcoholic KOH,34 followed by a reaction with oxalyl chloride in
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the presence of DMF as a catalyst to yield piperic acid chloride pyridazinone ring (NHCO) and the other at 3.73–3.86 ppm
3.35 Carbonyl compounds were treated with hydrazine hydrate associated with the benzylic protons. The 13C NMR spectra of
to yield the corresponding hydrazones 4a–h, which were then Va–f showed the characteristic benzylic carbon at 35.1 ppm and
reacted with piperic acid chloride to yield the corresponding the (C]O) at 168 ppm. The olefenic and aromatic carbons
compounds 5a–h. The synthesized compounds were elucidated appeared at their expected chemical shis.36–38 Interestingly, the
by 1H, 13C and 19F NMR.
prepared compounds are soluble in common organic solvents,
The IR spectrum of 5b revealed a broad band at 3432– such as CHCl3, tetrahydrofuran, and Et2O, and partially soluble
3300 cmꢀ1 related to (OH) and strong stretching band at in methanol and ethanol. Compounds 5a–f easily yielded X-ray
1651 cmꢀ1 related to (C]O), which is consistent with the quality crystals from slow evaporation of a mixture solution of
proposed structure. In the 1H NMR of compound 5b, two methanol and CH2Cl2 with the appropriate amount of 5a–f at
common singlet signals one at 13.10–13.18 ppm related to room temperature.
Scheme 1 Synthetic route for the synthesis of novel piperine–resveratrol hybrids 5a–h Reagents and conditions: (a) KOH, 95% methanol, reflux,
48 h, 93%; (b) (COCl)2, DCM room temperature, 16 h; (c) NH2NH2, THF, reflux 24 h (d) DCM, 0 ꢂC, 64–82%.
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respectively) than compound 5b (Table 3). Notably, 5h, con-
taining 4-bromophenyl moiety, showed promising potency with
75 ꢁ 3% and 22 ꢁ 5% SIRT2 inhibition at 50 mM and 5 mM,
respectively.
We then evaluated the 5b, 5e and 5h IC50 values against
SIRT2, all of which inhibited SIRT2 by more than 75% at 50 mM
compared to the SIRT2 selective inhibitor AGK2 as a reference.
The results are listed in Table 4. Compound 5b was the most
potent one among the tested derivatives, with IC50 value of 21 ꢁ
3 mM in comparison to the reference AGK2 (IC50 ¼ 13.9 ꢁ 1
mM).39
2.2. Biology
2.2.1. Evaluation of in vitro antiproliferative activity for
compounds 5a–h. The antiproliferative activity of the target
compounds 5a–h was screened against a panel of 60 cancer cell
lines according to NCI-guidelines at concentration of 10 mM.
The results for each compound (Table 1) were recorded as the
percent of growth inhibition of treated cells compared to
untreated control cells. Compound 5b (R1 ¼ H, R2 ¼ 2,4-dihy-
droxyphenyl) was the most active among the tested compounds
which directly reects the inuences of phenolic moieties on
the antiproliferative activity of the title scaffold. 5b possessing
a complete cell death against leukemia HL-60 (TB) and Breast
cancer MDA-MB-468 with growth inhibition percentage of
ꢀ0.49 and ꢀ2.83, respectively. In addition, 5b recorded signif-
icant activity against leukemia cancer cell lines K-562 and
MOLT-4, Non-small cell lung cancer cell lines NCI-H322M and
NCI-H460, Colon cancer cell lines COLO 205, HCT-15 and HCT-
116, CNS cancer cell lines SF-295 and SF-539, Melanoma cancer
cell lines LOX IMVI and UACC-62, Ovarian cancer cell lines NCI/
ADR-RES, OVCAR-3, and OVCAR-4, Renal cancer cell lines CAKI-
1, ACHN, and UO-31, and Breast cancer MCF7 cell line with
growth inhibition percentage between 80 to 95. On the other
hand, compound 5a (R1 ¼ H, R2 ¼ 4-hydroxyphenyl) showed
moderate activity against the cancer cell lines studied with
growth inhibition percentage between 17 to 73, Table 1.
Compounds 5e (R1 ¼ H, R2 ¼ 2-hydroxy-4-diethylaminophenyl)
and 5h (R1 ¼ CH3, R2 ¼ 4-bromophenyl) showed good activities
against most of cancer cell lines with growth inhibition
percentage between 40 to 97, Table 1. Compounds 5c, 5f and 5g
2.2.3. Cell cycle analysis and apoptosis assay
2.2.3.1. Cell cycle analysis. Cell cycle analysis was conducted
for the most active compound 5b against human pancreatic
cancer cell line MCF-7. The percentage of MCF-7 cells in the G0/
G1 phase of the control cell cycle was 53.64%, with a signicant
decrease to 35.08% following treatment with compound 5b,
while the percentage of cells in the S phase was marginally
reduced with compound 5b (35.56%) compared to the control
(36.41%) (Fig. 4). The percentage of MCF-7 human pancreatic
cancer cell line in the G2/M phase increased to 34.36%
following treatment with compound 5b. In addition, it is clear
that the percentage of apoptotic cells in the pre-G1 process
increased from 1.79% for control of untreated MCF-7 human
pancreatic cancer cells to 17.34% and 22.17% for controlled 5b
and doxorubicin cells, respectively (Fig. 4, Table 5). According to
the above results, compound 5b exhibited mainly cell cycle
arrest during the Pre-G1 and G2/M phases. In addition, it is
clear that the compound studied is not cytotoxic but anti-
proliferative, triggering programmed cell death and cell cycle
arrest.
bearing
3-methoxy-4-hydroxyphenyl,
(bis(4-uorophenyl)
methylene) and 3,5-di-tert-butyl-2-hydroxyphenyl, respectively
were found to be least effective against the studied cancer cell
lines.
2.3. Description of crystals 5a–f and Hirschfeld surface
analysis
2.2.2. Sirtuins inhibitory activity of 5a–h hybrids. Sirtuin-
TK assay was performed to estimate the Sirtuins inhibitory
potential of 5a–h (Tables 2 and 3). The results of this test
strongly complemented the ndings of cancer cell-based
assessments. Generally, the piperine–resveratrol 5a–h hybrids
were found to be stronger inhibitors of Sirt2 than Sirt1 at 5 mM
and 50 mM. Of the 8 compounds evaluated, 3 analogs (5b, 5e and
5h) were more likely to inhibit Sirt2 (>70% inhibition) than Sirt1
(<50% inhibition) at 50 mM. The other compounds were poor
inhibitors of both enzymes. Of these compounds studied, 5b (R1
¼ H, R2 ¼ 2,4-dihydroxyphenyl) showed a potent inhibition of
SIRT2 (78 ꢁ 3% and 26 ꢁ 3% inhibition at 50 mM and 5 mM,
respectively). Compared with compound 5b, which has 2,4-di-
hydroxyphenyl moiety, compound 5a, containing 4-hydrox-
yphenyl moiety, showed lower potency to inhibit SIRT2 (72 ꢁ
3% and 19 ꢁ 3% inhibition at 50 mM and 5 mM, respectively).
Replacement of the 4-hydroxy group in compound 5b with 4-
diethylamino in compound 5e resulted in a slight reduction of
the inhibitory SIRT2 values (74 ꢁ 2% and 24 ꢁ 3% inhibition at
50 mM and 5 mM, respectively). A further analysis of the
compound pairs [5b vs. 5c] showed that 5c (containing 4-
hydroxy-3-methoxyphenyl) would likely have less potent SIRT2
inhibition (58 ꢁ 3% and 17 ꢁ 3% inhibition at 50 mM and 5 mM,
The Hirschfeld surfaces and ngerprint plots can be utilized to
identify a type and region of intermolecular interactions, which
are capable of being generated using Crystal-Explorer so-
ware.40 Molecular Hirschfeld surface in a crystal structure is
constructed based on the electron distribution. Its normalized
contact distance (dnorm) based on both de, di and van der Waals
radii of the atom is listed in the following equation. Then,
intermolecular contacts in the crystal can be analyzed by
a combination of de and di in the form of a 2D ngerprint plot as
listed in the below equation.41 Complementary regions are
visible in the ngerprint plots where one molecule acts as
a donor (de > di) and the other as an acceptor (de < di). The
ngerprint plots can be divided into highlighting particular
close interactions between the two atoms.42 This decomposition
enables the separation of contributions from different interac-
tion types in the full ngerprint.
dnorm¼ (di– rvi dw)/rivdw + (de ꢀ revdw)/rvedw
Crystals of 5b were determined at room temperature. X-ray
experiments indicate that there are each one complete 5b and
one methanol solvent molecule in its asymmetric unit. The
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Table 1 One dose assay of nine different cancer cell types of compounds 5a–ha
Growth% inhibition
Subpanel cancer cell lines
5a
5b
5c
5d
5e
5f
5g
5h
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
32.40
44.10
58.83
23.50
16.87
64.02
81.02
ꢀ0.49
88.37
86.66
52.34
78.74
12.13
—
10.48
—
15.33
—
20.48
—
56.11
—
—
62.05
78.30
97.35
82.42
81.71
46.39
82.38
—
—
—
—
—
36.26
—
—
—
—
—
—
44.26
15.88
40.06
25.29
19.41
93.31
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
—
—
—
—
—
—
18.48
—
22.76
63.94
79.77
72.86
46.60
62.41
70.00
82.25
86.03
78.87
—
—
—
—
18.28
—
14.74
—
—
—
—
—
—
—
—
—
46.69
47.83
71.47
51.62
50.53
50.33
66.83
80.25
87.48
—
—
—
13.77
—
—
—
—
—
—
—
—
—
10.06
—
—
—
—
12.49
24.99
33.23
11.51
24.10
—
36.51
60.40
19.91
21.98
11.67
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
—
—
—
39.67
—
86.03
61.09
91.33
83.03
51.91
76.61
73.5
—
—
—
—
—
—
—
—
—
—
24.23
—
60.08
45.98
92.14
78.73
32.24
69.83
67.68
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10.78
12.34
36.30
—
24.89
25.44
33.35
19.77
14.25
—
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
17.15
—
17.56
—
26.97
—
75.57
86.23
85.66
50.87
35.98
68.74
—
—
15.33
—
—
—
—
—
—
25.68
—
66.32
58.30
77.28
38.99
41.19
56.75
—
—
—
—
16.64
—
—
—
—
—
32.95
—
32.46
—
12.47
14.48
68.09
16.04
—
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
UACC-257
UACC-62
38.53
—
17.10
73.02
—
—
—
28.50
90.31
59.69
69.73
67.38
47.24
57.87
71.52
85.74
—
—
—
—
—
—
—
11.32
11.11
—
22.24
70.14
—
—
—
16.06
81.77
43.47
63.64
51.12
34.33
47.95
75.82
70.87
—
—
—
—
—
—
—
12.19
12.05
—
—
—
—
—
—
—
38.29
28.67
26.74
13.60
11.01
14.25
12.00
28.51
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
24.72
—
12.67
—
—
—
71.10
90.27
90.76
57.55
77.21
94.85
73.00
—
—
—
—
—
—
—
10.14
—
—
—
—
61.80
95.06
84.74
39.11
67.09
82.45
60.49
12.09
—
—
—
—
14.13
—
—
—
—
29.67
24.42
65.24
10.69
24.67
33.86
—
—
—
—
—
—
—
—
Renal cancer
786-0
ACHN
CAKI-1
RXF 393
SN12C
—
—
25.08
—
—
70.98
85.61
90.35
42.15
67.24
—
—
—
—
—
—
—
24.55
—
—
52.78
74.37
70.70
27.41
45.91
—
—
12.79
—
—
—
—
—
—
—
14.63
24.09
28.09
—
12.40
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Table 1 (Contd.)
Growth% inhibition
Subpanel cancer cell lines
5a
5b
5c
5d
5e
5f
5g
5h
TK-10
UO-31
—
39.21
69.04
88.57
—
14.03
—
23.71
62.17
75.03
—
18.30
—
27.88
—
44.91
Prostate cancer
PC-3
DU-145
19.78
—
62.05
50.01
—
—
15.03
—
59.10
46.82
—
—
—
—
30.92
43.99
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578 T
BT-549
T-47D
28.77
17.21
—
21.48
18.59
—
85.87
65.61
29.82
72.73
76.14
ꢀ2.83
20.13
—
—
13.62
19.80
—
43.56
—
—
—
—
80.48
56.75
31.90
48.23
73.68
86.64
—
—
—
—
—
—
—
—
—
—
—
—
38.66
21.03
86.45
—
65.01
29.28
MDA-MB-468
—
a
(ꢀ): complete cell death, (—): not calculated.
Table 2 Inhibitory activities of 5a–h against human SIRT1
the light red spots are corresponding to C–H/O and C–H/p
interactions. The O/H/H/O intermolecular interactions
(30.4%) appear as distinct spikes in the 2D ngerprint plot
(Fig. 5d). In the ngerprint plots, there are two sharp spikes in
the lower le of the plots due to the O/N–H/O hydrogen bonds
(Fig. 5c). The proportion of O/H/H/O interactions comprises
30.4% of the total Hirschfeld surfaces. The points in the (di, de)
regions around (1.168, 1.107) from the ngerprint plots belong
to C–H/p interactions (17.2%) (Fig. 5e) which is mainly exist-
ing between the benzene ring and the methanol solvent at (2 ꢀ
x, 1 ꢀ y, z). p/p interactions are not represented because there
are no typical ‘wings’ at the top le and bottom right of the two-
dimensional ngerprint plot (Fig. 5f), occupying ca. 8.3% of the
total Hirschfeld surface. For the crystal packing, the molecules
of 5b are linked by these O/N–H/O hydrogen bonds into a two-
dimensional layer structure parallel to the (001) plane. These
% inhibition of SIRT1
Compound
5 mm
50 mm
5a
5b
5c
5d
5e
5f
13 ꢁ 3
18 ꢁ 3
12 ꢁ 1
11 ꢁ 2
20 ꢁ 5
14 ꢁ 4
9 ꢁ 2
42 ꢁ 3
45 ꢁ 3
38 ꢁ 3
48 ꢁ 3
44 ꢁ 2
39 ꢁ 3
35 ꢁ 1
44 ꢁ 3
5g
5h
16 ꢁ 5
Table 3 Inhibitory activities of 5a–h against human SIRT2
% inhibition of SIRT2
(001) layer structures are further linked into
dimensional network.
In the crystal structure of 5a (Fig. S9†), there is one complete
molecule and one methanol molecule in its asymmetric unit in
a three-
Compound
5 mm
50 mm
72 ꢁ 3
5a
5b
5c
5d
5e
5f
19 ꢁ 3
26 ꢁ 3
17 ꢁ 3
18 ꢁ 2
24 ꢁ 3
20 ꢁ 4
19 ꢁ 2
22 ꢁ 5
78 ꢁ 3 which the component ions are linked into a one-dimensional
58 ꢁ 3
70 ꢁ 3
74 ꢁ 2
69 ꢁ 3
55 ꢁ 1
75 ꢁ 3
hydrogen-bonded chain running along the [010] axis. A
Hirschfeld surface analysis indicates that the H/O (25.6%),
H/C (22.1%), C/C (5.9%) and H/H (38.7%) contacts in 5a are
comparable to those in crystal structure of 5b.
5g
5h
Table 4 IC50 values for the inhibitory activity of compounds 5b, 5e
and 5h against SIRT2 enzyme
whole molecule shows a nearly at conguration due to these
several double bonds. The Hirschfeld surfaces of compound 5b Compound
have been mapped over dnorm (Fig. 5a) and shape index (Fig. 5b).
IC50 (mm)
5b
21 ꢁ 3
23 ꢁ 2
The intermolecular interactions mainly originated from
hydroxyl oxygen and acyl hydrazone nitrogen atoms can be seen
5e
5h
26 ꢁ 3
in the Hirschfeld surface as the bright red areas in Fig. 5a, and
AGK2 (ref. 39)
13.9 ꢁ 1
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Fig. 4 Cell cycle analysis of 5b in human pancreatic cancer cell line MCF-7.
In the crystal structure of 5c, there is one complete molecule bonded network parallel to the (001) plane. A Hirschfeld
in its asymmetric unit in which the component ions are linked surface analysis for the host molecule indicates that the H/O,
into
a two-dimensional hydrogen-bonded layer structure H/C and C/C contacts including their reciprocal contacts are
parallel to the plane (20ꢀ1). A Hirschfeld surface analysis 19.2%, 27.1% and 3.2% of the total surface, respectively
indicates that the contacts of H/O, H/C, C/C and H/H in (Fig. S11†).
the crystal packing of 5c are 22.8%, 28.8%,1.4% and 35.3%
In 5e, there is each one host molecule and one methanol
respectively (Fig. S10†). In 5d, there is one complete molecule molecule in its asymmetric unit. Analysis indicates the
and two water molecules in its asymmetric unit in which the component ions are rstly linked into a one-dimensional
component ions are linked into a two-dimensional hydrogen- hydrogen-bonded chain running along the [010] axis. These
neighboring [010] chains are linked into a three-dimensional
network by a combination of C–H/p and p/p interactions.
A Hirschfeld surface analysis indicates that the H/O (19.7%),
H/C (17.4%), C/C (5.4%) and H/H (51.5%) contacts are also
Table 5 Apoptosis induction analysis of compound 5b
comparable to those in crystal structures above mentioned
(Fig. S12†). In the crystal of 5f (Fig. S13†), a dimer is formed by
a pair of complementary N–H/O hydrogen bonds. A Hirschfeld
surface analysis indicates that the H/O (including the recip-
rocal contacts) contacts comprise 13.9% of the total surface.
Apoptosis
% total
%
Sample code
Cell line
% early
% late
necrosis
5b
Control
MCF-7
MCF-7
6.16
1.72
2.74
0.88
2.29
0.23
1.13
0.61
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Fig. 5 Hirshfeld surfaces in 5b mapped with (a) dnorm; (b) shape index, (c) full fingerprint plot; (d) H/O contacts (O–H/O and N–H/O); (e)
H/C contacts (C–H/p) and (f) C/C contacts (p/p).
The H/C (C–H/p, 18.9%) and C/C (p/p, 5.3%) are both the top-ranked pose of compound 5b demonstrated several
comparable to those in 5b.
plausible molecular interactions and various binding patterns,
From the Hirschfeld analysis, we can obviously see that the Fig. 6A. It was reported that the active site of Sirt-2 could be
O/N–H/O hydrogen bonds are the main driving forces in the divided into several sites. The C-pocket accommodates the
crystal packing. The C–H/p interactions also play an impor- nicotinamide moiety of the co-factor NAD+ while the acetyl-Lys
tant role in the crystal aggregates. However, the p/p interac- substrate binding channel is formed by several hydrophobic
tions comprise the least of the total Hirschfeld surface in these phenylalanine's. In addition, there is a pocket close to the C-
ve structures. We can also nd that the lateral hydroxyl oxygen pocket and link the inducible selectivity pocket in Sirt-2 with
and acyl hydrazone nitrogen atoms are in favor of hydrogen- the acyl-Lys tunnel called extended C-site, Fig. 6(B).47,48
bonding to some other acceptors. To sum up above
From a precise inspection of the results, it was conceptual-
mentioned discussion, one can nd that the O/N–H/O ized that there are two possible different hypotheses or binding
hydrogen bonds are preferred in the crystallization state. patterns can be used to rationalize the inhibitory activity of our
Although a crystallization state cannot be completely a repre- compound 5b against SIRT-2: (I) our newly synthesized hybrid
sentation of a solution state, it can still give us some hints when molecule 5b adopts a very similar binding mode consistent with
these molecules are used an antiproliferative inhibitors they that of the co-crystallized ligand, SirReal2 where it binds to the
may rstly interact with the target position of a protein through extended C-site as well as to the reported selectivity pocket that
hydrogen bonds (vide infra).
formed by two loops of the hinge region connecting the
Rossmann-fold and zinc-binding domains. The binding pattern
analysis revealed that 1,3-dihydroxyphenyl moiety of 5b can be
accommodated into the lipophilic selectivity pocket in
a manner similar to the dimethyl mercapto-pyrimidine residue
of SirReal2 maintaining the same p–p stacking with Phe190. In
addition, two hydrogen bonds were formed between the two
hydroxyl groups of 5b and Ala135 and Ile169 residues. The rest
of the 5b kinked conformation occupies the Extended C-site
adjacent to the C-pocket and it is oriented towards the acetyl-
lysine substrate tunnel. The benzodioxolyl moiety of 5b
protrudes into the substrate channel forming hydrophobic
interactions with Phe119, Phe131, Ile232, Val233 and Phe234
amino acids. This bulky benzodioxolyl moiety equivalent to the
2.4. Molecular docking study
In an attempt to rationalize the obtained in vitro Sirt-2 assay
results,
a molecular docking study of the most potent
compound 5b into the active site of human Sirt-2 was per-
formed to elucidate the possible underlying inhibitory mecha-
nisms and to predict the binding mode and the interactions
that can be formed. The 3D crystal structure of Sirt-2 (PDB code:
4RMG) in complex with SirReal2 ligand and Co-factor NAD+ was
used aer preparation. The docking simulation was carried out
using Ligand Fit embedded in Discovery Studio soware 2.5
(San Diego, USA) according the reported method.43–46 Analysis of
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Fig. 6 Docking of compound 5b into SIRT-2 3D structure (pdb code: 4RMG). (A) Overlay of the top docked poses 5b (green and violet) and
SirReal-2 (yellow) as a co-crystallized ligand into the Sirt-2 binding site where the Sirt-2 protein is represented as secondary structure displayed in
a flat ribbon style (cartoon) colored in black-white; (B) labelling of the different binding pockets of Sirt-2 protein. The NAD+ co-factor (cyan) is
displayed in stick.
naphthyl moiety of SirReal2, is thought to force the acetyl-lysine
(II) Another considerable binding mode was observed where
out of its physiological position in a competitive inhibitory 5b binds in an inverted fashion to what was adopted in the
manner. Interestingly, the 5b structure is longer than sirReal2 former hypothesis. It was found that the benzodioxolyl moiety
one which in turn will likely compete with the acyl-Lys substrate induces the formation of the selectivity pocket instead of the
in a stronger way. In this hypothesis, 5b does not interfere with dihydroxyphenyl in a similar manner to that of sirReal2 forming
the C-pocket where nicotinamide moiety of NAD+ binds, the reported p–p stacking with Phe190. However, the dihy-
Fig. 7A–C.
droxyphenyl moiety was oriented downwards toward the C-
Fig. 7 The first hypothesis of binding pattern of 5b inside Sirt-2 active site; (A) overlay of the top docked poses 5b (green and violet) and SirReal-2
(yellow) as a co-crystallized ligand into the secondary structure of Sirt-2 active pocket; (B) the interactions and binding mode of 5b (green) inside
the Sirt-2 active sites (extended C-pocket and selectivity pocket); (C) superimposition of 5b (green) and SirReal2 (yellow). The poses were
rendered as stick model. p–p interactions were represented as orange solid line. Hydrogen bonds were represented as dashed green lines.
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Fig. 8 The second hypothesis of binding pattern of 5b inside Sirt-2 active site; (A) overlay of the top docked poses 5b (green and violet) and
SirReal-2 (yellow) as a co-crystallized ligand into the secondary structure of Sirt-2 active pocket; (B) the interactions and binding mode of 5b
(violet) inside the Sirt-2 active sites (Extended C-pocket and selectivity pocket); (C) the window shows magnification of the possible clash
between 5b (violet) and the NAD+ co-factor (cyan). The rest of NAD+ was removed for clarity. The poses were rendered as stick model. p–p
interactions were represented as orange solid line. Hydrogen bonds were represented as dashed green lines.
pocket where the nicotinamide moiety of NAD+ binds and cancer cell lines of nine different tissues in compliance with the
initializes the deacetylation reaction. This conformation is NCI protocol has been utilized for the evaluation of 5a–h hybrids.
stabilized by forming a hydrogen bond with His187. Another Compounds 5b, 5e and 5h showed signicant antiproliferative
hydrophobic interaction between the unsaturated alkenyl chain activity. 5a–h were found to be more potent inhibitors of Sirt-2
of 5b and the gate keeper, Phe96 residue, which helps nicotin- than Sirt-1. Results of cell cycle investigation showed that 3.64%
amide to release and prevents the backward reaction was of pre-G1 apoptosis was induced by compound 5b on MCF-7 with
observed. It was also noted that 5b was highly distorted from the a high percentage of cell accumulation in G2-M phase. In these
extended C-site or the substrate tunnel and did not have strong complexes with the hydroxyl groups, the component ions are
hydrophobic contact with Phe119, Phe131, Ile232, Val233 and mainly linked into a 3D framework by a combination of O–H/O
Phe234 amino acids residue, Fig. 8A–C. This hypothesis sug- hydrogen bonds, C–H/O, C–H/p and p/p interactions.
gested that 5b might partially occupy the C-pocket and occlude Hirschfeld surface analysis indicates that O–H/O hydrogen
or compete with nicotinamide moiety of NAD+ in contrary to the bonds consist of ca. 20% of the total surface, which indicate that in
sirReal2 mechanism which a is partially non-competitive the biological system the O–H/O interaction may be mainly
towards NAD+. We think that the former hypothesis is a much attributed to their biological characteristics. A docking study of the
more reasonable explanation for the inhibitory activity of 5b most active compound 5b was carried out to get insights into the
against Sirt-2 where it shows the highest ranked binding pattern Sirt-2 inhibitory mechanism and justify the in vitro results. The
with the lowest energy score. Moreover, targeting both selec- results revealed two possible binding patterns of 5b that could be
tivity pocket and the substrate channel by linking two distinct used to rationalize its Sirt-2 inhibitory activity by either competi-
moieties in one scaffold is much more acceptable and signi- tion with acyl-Lys substrate or partially blocking co-factor NAD+ C-
cant mechanism for inhibition of Sirt-2. Indeed, we will need pocket. These ndings highlight the importance of Sirt-2 as
further kinetic competition studies to distinguish and prove a promising anticancer target and open a new avenue to develop
one of these two inhibitory mechanisms. However, these results novel superior and selective Sirt-2 inhibitors.
support the hypothesis that targeting either the acyl-Lys
substrate binding site or NAD+ co-factor C-pocket represents
a useful approach to develop novel and potent SIRT2 competi-
tive inhibitors.
4. Experimental
4.1. Chemistry
General details and crystallographic data (Table S1): see ESI†
and Appendix A.
3. Conclusion
In this study, a series of piperine–resveratrol hybrids 5a–h was
4.1.1. Synthesis of (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)
designed and synthesized as inhibitors of Sirt-2. A panel with sixty penta-2,4-dienoyl chloride (3). Compounds 2 and 3 were
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prepared according to our previous research work35 and the 141.5, 140.9, 140.1, 136.0, 131.2, 126.2, 125.9, 125.6, 123.9,
experimental details are recorded in Appendix A.
123.6, 121.9, 117.6, 114.9, 113.2, 111.1, 109.0, 106.4, 102.0.
C
4.1.2. Synthesis of hydrazone derivatives (4a–h).49–51 To
20H20N2O6, crystal dimensions 0.12 ꢃ 0.1 ꢃ 0.1 mm3, Mr ¼
a solution of carbonyl compounds (2.5 mmol), hydrazine 384.38, monoclinic, space group P21/c(14) cell: a ¼ 6.6192(6),
ꢂ
ꢂ
˚
monohydrate (158 mL, 3.25 mmol) in THF solution was added b ¼ 19.7039(17), c ¼ 14.2723(13) A, a ¼ 90 , b ¼ 99.079(5) , g ¼
3
ꢂ
˚
and reuxed for 24 h. Then the hydrazone solution was dried by 90 , V ¼ 1841.78(14) A , Z ¼ 4, density (calculated) ¼
200 mg anhydrous Na2SO4 for 0.5 h and then 5 A molecular 1.389 g mꢀ3, m ¼ 0.866 mmꢀ1, F(000) ¼ 808.0, reection
˚
sieves powder (250 mg) for 5–6 h. The obtained hydrazones 4a–h collected/unique ¼ 10 609/2973, renement method ¼ full-
were utilized directly to the next step.
matrix least-squares on F2, Final R indices [I > 2sigma(I)]: R1 ¼
4.1.3. General procedure for synthesis of piperine-based 0.0461, wR2 ¼ 0.1239, R indices (all data): R1 ¼ 0.0507, wR2 ¼
hydrazone derivatives (5a–h). Compound 3 (191 mg, 1.1 mmol) 0.1332, goodness of t on F2 ¼ 1.074. CCDC 1984253 (Fig. 9B).
was dissolved in CH2Cl2 (5 mL) and was added dropwise into
a mixture of the hydrazones (4a–h, 100 mg, 1 mmol) in CH2Cl2 (5
mL) in ice bath. Aer addition of compound 3, the reaction
mixture was stirred for 7 h at room temperature, and then the
solvent was removed under reduced pressure. The crud product
was treated with Na2CO3 solution, then extracted three times by
ethyl acetate. The organic layer was dried over Na2SO4 and the
solvent was evaporated under reduced pressure. The residue was
puried by silica gel column chromatography (CH2Cl2 : MeOH ¼
20 : 1) to give the corresponding compounds 5a–h (69.1–89.2%
yields). Crystals were grown aer slow evaporation of a mixture
solution of methanol and CH2Cl2 (2 : 3) with the appropriate
amount of 5a–f, then le open to the atmosphere at room
temperature, producing yellow sheets and cubic crystals aer 45
days.
4.1.3.1. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-((E,Z)-4-
hydroxybenzylidene)penta-2,4-dienehydrazide (5a). Pale yellow
Fig. 9 The X-ray single crystal structures of 5a (A) and 5b (B).
solid, m.p. 12–127 ꢂC, yield: 83%; IR: 3463, 3104 3050, 2890,
1
1660, 1600, 1500, 1448, 1400, 1384, 1260. H NMR (600 MHz,
DMSO-d6) d 11.40 (s, 0.49H, NH), 11.20 (s, 0.46H, NH), 9.98 (d,
4.1.3.3. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-((E,Z)-4-
0.51H, OH), 9.91 (d, 0.46H, OH), 8.12 (s, 0.51H, 1H, CH]N),
hydroxy-3-methoxybenzylidene)penta-2,4-dienehydrazide
(5c).
7.93 (s, 0.46H, 1H, CH]N), 7.54 (d, J ¼ 8.6 Hz, 2H), 7.41–7.26
(m, 2H), 7.20–7.07 (m, 1H), 7.02 (dd, J ¼ 15.7, 6.3 Hz, 1H), 6.99–
6.95 (m, 1H), 6.93 (dd, J ¼ 8.0, 4.3 Hz, 1H), 6.83 (d, J ¼ 8.6 Hz,
2H), 6.15 (d, J ¼ 14.9 Hz, 1H), 6.06 (d, J ¼ 2.8 Hz, 2H, –OCH2Oꢀ).
13C NMR (150 MHz, DMSO-d6) d 166.47, 161.91, 159.81, 159.57,
148.45, 148.37, 147.12, 131.31, 131.25, 129.34, 129.21, 128.93,
128.79, 125.82, 123.33, 116.23, 116.04, 109.02, 106.23, 106.07,
101.79. C20H20N2O5, crystal dimensions 0.12 ꢃ 0.1 ꢃ 0.1 mm3,
Mr ¼ 368.38, monoclinic, space group P21/n (14) cell: a ¼
Pale yellow solid, m.p. 210–213 ꢂC, yield: 76.4%; IR: 3401, 3231,
1
3029, 2909, 2831, 1624, 1606, 1507, 1445, 1375, 1337, 1258. H
NMR (600 MHz, DMSO-d6) d 11.44 (s, 0.55H, NH), 11.25 (s,
0.43H, NH), 9.54 (s, 0.53H, OH), 9.50 (s, 0.39H, OH), 8.13 (s,
0.51H, 1H, CH]N), 7.93 (s, 0.40H, 1H, CH]N), 7.50–7.23 (m,
3H), 7.19–7.06 (m, 2H), 7.03 (dd, J ¼ 10.3, 7.8 Hz, 1H), 6.99 (s,
1H), 6.93 (d, J ¼ 13.2 Hz, 1H), 6.85 (s, 1H), 6.18 (d, J ¼ 14.9 Hz,
1H), 6.06 (d, J ¼ 1.7 Hz, 2H, –OCH2O–), 3.85 (d, J ¼ 18.9 Hz, 3H,
OCH3). 13C NMR (150 MHz, DMSO-d6) d 166.46, 161.95, 149.39,
149.11, 148.48, 148.44, 148.37, 147.38, 143.74, 142.86, 141.50,
139.63, 139.35, 131.25, 126.26, 126.22, 125.68, 123.41, 123.01,
122.51, 121.41, 120.34, 116.06, 115.89, 110.25, 109.47, 108.94,
106.16, 101.79, 56.18. C20H18N2O5, crystal dimensions 0.12 ꢃ
0.1 ꢃ 0.1 mm3, Mr ¼ 366.36, monoclinic, space group P21/c (14)
ꢂ
˚
10.9249(5), b ¼ 6.5740(3), c ¼ 25.7341(11) A, a ¼ 90 , b ¼
3
ꢂ
ꢂ
˚
94.790(3) , g ¼ 90 , V ¼ 1841.78(14) A , Z ¼ 4, density (calcu-
lated) ¼ 1.329 g mꢀ3, m ¼ 0.799 mmꢀ1, F(000) ¼ 776.0, reection
collected/unique ¼ 10 352/3035, renement method ¼ full-
matrix least-squares on F2, Final R indices [I > 2sigma(I)]: R1
¼ 0.0925, wR2 ¼ 0.2368, R indices (all data): R1 ¼ 0.1011, wR2 ¼
0.2530, goodness of t on F2 ¼ 1.026. CCDC 1984251 (Fig. 9A).
4.1.3.2. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-((E)-2,4-
ꢂ
˚
cell: a ¼ 18.1988(7), b ¼ 4.8105(2), c ¼ 22.1672(8) A, a ¼ 90 , b ¼
3
ꢂ
ꢂ
˚
112.874(3) , g ¼ 90 , V ¼ 1788.03(13) A , Z ¼ 4, density (calcu-
lated) ¼ 1.361 g mꢀ3, m ¼ 0.822 mmꢀ1, F(000) ¼ 768.0, reection
collected/unique ¼ 14 927/3069, renement method ¼ full-
matrix least-squares on F2, nal R indices [I > 2sigma(I)]: R1 ¼
0.0490, wR2 ¼ 0.1431, R indices (all data): R1 ¼ 0.0687, wR2 ¼
0.1575, goodness of t on F2 ¼ 1.052. CCDC 2009516.
dihydroxybenzylidene)penta-2,4-dienehydrazide
(5b). Yellow
ꢂ
solid, m.p. 153–156 C, yield: 80%; IR: 3432, 3300, 3030, 2902,
1650, 1602, 1515, 1439, 1367, 1252. 1H NMR (600 MHz, DMSO-
d6) d 11.64 (s, 1H, NH), 11.38 (s, 1H, OH), 9.95 (s, 1H, OH), 8.24
(d, J ¼ 4.8 Hz, 1H, CH]N), 7.38–7.31 (m, 1H), 7.29 (d, J ¼ 7.8 Hz,
2H), 7.07–6.96 (m, 3H), 6.93 (t, J ¼ 8.2 Hz, 1H), 6.37–6.28 (m,
2H), 6.14 (d, J ¼ 14.9 Hz, 1H), 6.06 (s, 2H, –OCH2Oꢀ). 13C NMR
(151 MHz, DMSO-d6) d 166.4, 164.1, 161.9, 150.3, 148.5, 142.5,
4.1.3.4. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-((E/Z)-4-
(dimethylamino)-benzylidene)penta-2,4-dienehydrazide (5d). Pale
yellow solid, m.p. 203–207 ꢂC, yield: 69.1%; IR: 3136, 3053, 2932,
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1
2853, 1640, 1610, 1497, 1447, 1372, 1340, 1260. H NMR (600 (600 MHz, DMSO-d6) d 10.60 (s, 1H, OH), 7.77 (d, J ¼ 7.6 Hz,
MHz, DMSO-d6) d 11.31 (s, 0.54H, NH), 11.12 (s, 0.42H, NH), 2H), 7.62 (d, J ¼ 8.5 Hz, 2H), 7.38 (dd, J ¼ 27.5, 13.6 Hz, 2H), 7.14
8.08 (s, 0.56H, CH), 7.93 (s, 0.44H, CH), 7.52 (dd, J ¼ 8.9, 2.7 Hz, (d, J ¼ 14.1 Hz, 1H), 7.01 (dd, J ¼ 29.7, 6.8 Hz, 2H), 6.93 (d, J ¼
2H), 7.43–7.24 (m, 2H), 7.22–6.89 (m, 4H), 6.74 (dd, J ¼ 8.8, 8.0 Hz, 1H), 6.48 (d, J ¼ 14.9 Hz, 1H), 6.06 (s, 2H, –OCH2O-), 2.28
4.2 Hz, 2H), 6.15 (d, J ¼ 14.9 Hz, 1H), 6.06 (d, J ¼ 2.4 Hz, 2H, (d, J ¼ 16.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) d 166.56,
–OCH2O–), 2.97 (s, 6H, N(CH3)2). 13C NMR (150 MHz, DMSO-d6) 148.46, 146.69, 143.03, 141.61, 139.65, 139.41, 131.24, 129.11,
d 166.26, 151.92, 151.75, 148.44, 148.39, 148.33, 147.63, 141.22, 128.73, 127.39, 126.26, 125.68, 123.44, 122.94, 120.27, 114.77,
131.33, 131.29, 128.93, 128.79, 128.52, 128.38, 123.42, 122.91, 108.95, 106.16, 101.79, 28.41.
122.14, 112.39, 112.13, 109.02, 108.87, 106.21, 106.06, 101.78,
40.40. C21H24.75N3O4.42, crystal dimensions 0.12 ꢃ 0.04 ꢃ 0.03
4.2. Biology
mm3, Mr ¼ 389.87, monoclinic, space group P21/n (14) cell: a ¼
4.2.1. Screening of cytotoxic activity. The methodology of
the NCI anticancer screening has been described in detail
elsewhere (https://dtp.cancer.gov/).52
4.2.2. Sirtuin inhibitory assay. Sirt-1 and Sirt-2 inhibitory
assay was performed to evaluate the inhibitory potency of 5a–h
against Sirt-1 and Sirt-2.53 See ESI.†
4.2.2.1. Cell apoptosis assay. Apoptosis was determined by
ow cytometry based on the Annexin-V-uoresce in iso-
thiocyanate (FITC) and propidium iodide (PI) staining kit (BD
Pharmingen, San Diego, USA).54,55 See ESI.†
ꢂ
˚
13.6957(16), b ¼ 6.3687(7), c ¼ 24.058(3) A, a ¼ 90 , b ¼
3
ꢂ
ꢂ
˚
104.090(6) , g ¼ 90 , V¼2035.3(4) A , Z ¼ 4, density (calculated)
¼ 1.272 g mꢀ3, m ¼ 0.740 mmꢀ1, F(000) ¼ 828.0, reection
collected/unique ¼ 9153/2692, renement method ¼ full-matrix
least-squares on F2, nal R indices [I > 2sigma(I)]: R1 ¼ 0.0706,
wR2 ¼ 0.1880, R indices (all data): R1 ¼ 0.1081, wR2 ¼ 0.2150,
goodness of t on F2 ¼ 1.029. CCDC 1984254.
4.1.3.5. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-((E)-4-
(diethylamino)-2-hydroxy benzylidene)penta-2,4-dienehydrazide
(5e). The spectroscopic analysis matched with those previ-
ously our work published35 and included in appendix A.
4.1.3.6. (2E,4E)-5-(Benzo[d][1,3]dioxol-5-yl)-N0-(bis(4-
Conflicts of interest
uorophenyl)methylene)penta-2,4-dienehydrazide
(5f).
Deep
The authors declare no competing nancial interest.
yellow solid, m.p. 190–193 ꢂC, yield: 71.7%; IR: 3276, 3052, 2989,
2895, 1650, 1623, 1595, 1486, 1384, 1339, 1255. 1H NMR (600 MHz,
DMSO-d6) d 10.24 (s, 0.59H, NH), 9.61 (s, 0.44H, NH), 7.52 (d, J ¼
2.9 Hz, 2H), 7.41 (s, 2H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.31 (s, 1H), 7.24
(s, 3H), 6.92 (t, J ¼ 4.6 Hz, 4H), 6.36 (d, J ¼ 14.7 Hz, 1H), 6.05 (s, 2H,
–OCH2O-). 13C NMR (151 MHz, DMSO-d6) d 164.02, 162.39, 148.44,
143.81, 141.82, 139.57, 131.69, 131.17, 129.97, 126.24, 125.51,
123.41, 117.00, 115.92, 108.98, 106.16, 101.81. 19F NMR (565 MHz,
DMSO-d6) d ꢀ106.46. C25H18F2N2O3, crystal dimensions 0.20 ꢃ
0.12 ꢃ 0.10 mm3, Mr ¼ 432.41, monoclinic, space group P21/n (14)
Acknowledgements
This work was supported by the National Key Research and
Development Program of China (2017YFE0113900) and Huaz-
hong Agriculture University, Talent Young Scientist Program
(Grant No. 42000481-7).
References
ꢂ
˚
cell: a ¼ 5.329(3), b ¼ 10.708(5), c ¼ 15.496(7)A, a ¼ 83.878(9) , b ¼
3
ꢂ
ꢂ
˚
87.608(9) , g ¼ 82.240(9) , V¼870.8(7) A , Z ¼ 4, density (calcu-
lated) ¼ 1.649 g mꢀ3, F(000) ¼ 448, reection collected/unique ¼
6085/3045, renement method ¼ full-matrix least-squares on F2,
nal R indices [I > 2sigma(I)]: R1 ¼ 0.0623, wR2 ¼ 0.1690, R indices
(all data): R1 ¼ 0.0965, wR2 ¼ 0.1941, goodness of t on F2 ¼ 1.029.
CCDC 1984255.
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25750 | RSC Adv., 2021, 11, 25738–25751
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© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 25738–25751 | 25751