Discovery of 5-(4-methylpiperazin-1-yl)-2-nitroaniline derivatives as a new class of SIRT6 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127215

SIRT6 is a deacetylase of histone H3 and inhibitors of SIRT6 have been thought as potential agents for treatment of diabetes. Herein we report the discovery of a series of new SIRT6 inhibitors containing the skeleton 1-phenylpiperazine. Among them, compound 5-(4-methylpiperazin-1-yl)-2-nitroaniline (6d) is the most potent one, which showed an IC₅₀ value of 4.93 μM against SIRT6 in the Fluor de Lys (FDL) assay. It displayed K_D values of 9.76 μM and 10 μM in surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays, respectively. In selectivity assay, 6d showed no activity against other members of the HDAC family (SIRT1-3 and HDAC1-11) at concentrations up to 200 μM. In a mouse model of type 2 diabetes, 6d could significantly increase the level of glucose transporter GLUT-1, thereby reducing blood glucose. Overall, this study provides a promising lead compound for subsequent drug discovery targeting SIRT6.

Full text of DOI:10.1016/j.bmcl.2020.127215

Journal Pre-proofs
Discovery of 5-(4-Methylpiperazin-1-yl)-2-nitroaniline Derivatives as A New
Class of SIRT6 Inhibitors
Weining Sun, Xiuli Chen, Shenzhen Huang, Wenpei Li, Chenyu Tian,
Shengyong Yang, Linli Li
PII:
S0960-894X(20)30315-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127215
BMCL 127215
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
28 January 2020
21 April 2020
23 April 2020
Please cite this article as: Sun, W., Chen, X., Huang, S., Li, W., Tian, C., Yang, S., Li, L., Discovery of 5-(4-
Methylpiperazin-1-yl)-2-nitroaniline Derivatives as A New Class of SIRT6 Inhibitors, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127215
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Discovery of 5-(4-Methylpiperazin-1-yl)-2-nitroaniline Derivatives as A New
Class of SIRT6 Inhibitors
Weining Suna,c, Xiuli Chenb,c, Shenzhen Huangb, Wenpei Lib, Chenyu Tiana,
Shengyong Yangb, and Linli Lia,*
aKey Laboratory of Drug Targeting and Drug Delivery System of Ministry of
Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan
610041, P.R. China
bState Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University, Chengdu, Sichuan 610041, P.R. China
cThese authors contributed equally to this work.
*To whom correspondence should be addressed. Key Laboratory of Drug Targeting
and Drug Delivery System of Ministry of Education, West China School of
Pharmacy, Sichuan University, Chengdu, Sichuan 610041, P.R. China. E-mail:
ysylilinli@sina.com.cn.
KEYWORDS: SIRT6, small molecule inhibitor, type 2 diabetes
1

ABSTRACT
SIRT6 is a deacetylase of histone H3 and inhibitors of SIRT6 have been thought as
potential agents for treatment of diabetes. Herein we report the discovery of a series
of new SIRT6 inhibitors containing the skeleton 1-phenylpiperazine. Among them,
compound 5-(4-methylpiperazin-1-yl)-2-nitroaniline (6d) is the most potent one,
which showed an IC50 value of 4.93 μM against SIRT6 in the Fluor de Lys (FDL)
assay. It displayed KD values of 9.76 μM and 10 μM in surface plasmon resonance
(SPR) and isothermal titration calorimetry (ITC) assays, respectively. In selectivity
assay, 6d showed no activity against other members of the HDAC family (SIRT1-3
and HDAC1-11) at concentrations up to 200 µM. In a mouse model of type 2
diabetes, 6d could significantly increase the level of glucose transporter GLUT-1,
thereby reducing blood glucose. Overall, this study provides a promising lead
compound for subsequent drug discovery targeting SIRT6.
2

Sirtuins are NAD+-dependent lysine deacetylases widespread in prokaryotes and
eukaryotes1,2. It contains 7 members (SIRT1-SIRT7) in mammals, which have
different subcellular localization and biological functions3. SIRT6, as a member of the
sirtuin family, is closely related to many physiological processes such as
developmental retardation4, DNA repair5-7, genomic stability6 and life span8,9.
Importantly, SIRT6 has been demonstrated to be a corepressor of the transcription
factor Hif1α. And knockdown of SIRT6 gene could upregulate glycolysis-related
genes, such as GLUT-1, PDK1, ALDOC, and LDH, which are modulated by Hif1α10.
SIRT6 is thus thought as a potential intervening target for metabolic diseases such as
diabetes and obesity.
To date, a number of SIRT6 inhibitors have been reported. Representative
compounds are shown in Figure 1, including 5-Cl-PZA11, BMC-1212, EJMC-313,
SYN1773930314, and POA11, JMC-914, and JMC-1614. Unfortunately, potencies of
these compounds are poor (see Figure 1). In addition, most of the SIRT6 inhibitors are
multi-target compounds. Therefore, it is of great significance to find SIRT6 inhibitors
with potent activity and selectivity at present.
H
O
O
S
N
R1
R2
OH
O
O
HN
O
O
N
N
NH2
N
O
N
H
N
H
COOH
Cl
O
R2=
R1=
N
N
O
R1, EJMC-3, IC50=37 M
R2, SYN17739303, IC50=106M
BMC-12
5-Cl-PZA

IC50=20 M
IC50=33.2 M


O
O
N
O
O
H
N
N
NH
N
N
OH
OH
OH
N
H
H
N
N
N
O
O
N
JMC-16, IC50=1339 M

JMC-9, IC50=89 M
POA, IC50=926.8 M


Figure 1. Reported synthetic SIRT6 inhibitors.
To discover new SIRT6 inhibitors, we performed a screening study against our
in-house chemical library containing about 2000 compounds by Fluor de Lys (FDL)
3

assay. Through this screening, a hit compound 1-(4-nitrophenyl)piperazine (Hit01,
Figure 2A) was obtained, which showed an IC50 value of 35 μM (Figure 2B). Further
structural optimization was then performed on this compound.
Figure 2. (A) Chemical structure of Hit01. (B) Dose-dependent activities of SIRT6
deacetylation
of
Hit01,
determined
with
acetyl
substrate
peptide
Ac-RYQK(Ac)-AMC. Data are presented as the meanꢀ±ꢀs.d., n=ꢀ3 wells, from three
independent experiments.
The structural optimization of Hit01 was focused on 1-position piperazine
(region I), 3-position (region II) and 4-position (region Ш) of the phenylpiperazine
(Figure 3). Structure-activity relationship (SAR) analyses are based on inhibitory
activities of compounds against SIRT6, which were determined by FDL assay.
Inhibitory activities of compounds were measured at a single concentration of 300 μM
in advance. For compounds with higher potency at 300μM, further IC50 values were
tested.
Hit compound
Region III
R3
R2
NO2
Region II
N
N
HN
N
R1
Hit01
Region I
Figure 3. Regions for structural optimization and SAR analyses.
4

Region I was firstly optimized. To this end, we installed various substituents on
the 1-H of piperazine and fixed region II (-NO2) and III (-H) as their original groups.
Six compounds (PC01-06) were purchased from the commercial chemical provider
Specs. Chemical structures and bioactivities of compounds PC01-06 are shown in
Table 1. All the compounds except PC06 showed decreased bioactivity against
SIRT6. The only compound with activity improved, PC06, contains a methyl group at
R1, implying that the methyl group at R1 is the best choice.
Table 1. Chemical Structures and SIRT6 Inhibitory Activities of Compounds
PC01-06.
H
NO2
N
N
R1
Inhibition (%) @ 300 μM
ID
R1
(IC50/μM)
81.79
Hit01
H
(35.0)
O
PC01
PC02
13.07
61.70
O
O
O
PC03
50.69
Cl
OH
PC04
PC05
32.01
69.67
-CH2CH3
5

112.70
(21.91)
PC06
-CH3
Region II (R2) was then optimized. For this purpose, region I and III were fixed
as –CH3 and –H, respectively, and region II (R2, -NO2) was replaced by various
substituents. We purchased nine compounds (PC07-15) from Specs. Chemical
structures and bioactivities are presented in Table 2. Although various substituents at
R2, bioactivities of the new compounds are still less than that of PC06, indicating that
a nitro group at R2 position is suitable.
Table 2. Chemical Structures and SIRT6 Inhibitory Activities of Compounds
PC06-15.
H
R2
N
N
Inhibition (%) @ 300μM
ID
R2
(IC50/μM)
112.70
(21.91)
35.77
PC06
-NO2
PC07
PC08
PC09
H
F
24.31
-CF3
45.67
O
O
PC10
41.64
N
H
6

O
N
PC11
PC12
PC13
26.24
46.68
46.86
H
O
O
N
H
Cl
O
O
N
H
O
PC14
PC15
39.13
22.83
N
H
O
O
OH
Region III (R3) was finally optimized. To this end, we synthesized thirteen new
1-methyl-4-(4-nitrophenyl)piperazine derivatives (6a-m), in which region I and II
were fixed as the methyl group and the nitro group, respectively, and region III was
changed with various substituent groups. Scheme 1 illustrates synthetic routes for
compounds 6a-m. Condensation reactions between 2,4-difluoro-1-nitrobenzene (3) or
5-fluoro-2-nitrobenzoic acid (7) and various substituted amino (4a-c, 8a-f) in DMF
solution gave compounds 5a-c or 9a-f as intermediates. Final products 6a-m were
then obtained from reactions of 2a-d, 5a-c, and 9a-f with commercially available
1-methylpiperazine (1).
Scheme 1. Synthesis of compounds 6a-m.a
7

NO2
R3
NH
+
N
F
F
1
2a-d
NO2
R3
NO2
F
b
NO2
R3
R4-NH2
4a-c
+
a
F
N
3
5a-c
N
NH
6a-m
N
1
NO2
NO2
R3
c
R5-NH2
8a-f
+
OH
F
F
9a-f
O
7
2a, 6a: R3= OH
2b, 6b: R3= CF3
2c, 6c: R3= OCH3
2d, 6d:R3= NH2
O
O
O
3
H
9a, 6h: R3 =
9d, 6k: R3 =
9e, 6l: R3 =
9f, 6m: R3 =
5a, 6e: R =
N
N
N
N
H
H
H
O
O
5b, 6f: R3 =
5c, 6g: R3 =
9b, 6i: R3 =
9c, 6j: R3 =
N
N
N
N
H
H
O
O
O
O
N
O
H
N
O
N
H
Cl
a Reagents and conditions: a) Pd(OAc)2, Xant-phos, 1,4-dioxane,Cs2CO3, 100 °C, 12
h; b): R4-NH2, K2CO3, DMF, 50 C, overnight; c) i: SOCl2, 50 °C, 4-5 h; ii: R5-NH2,
DMAP, DMF, RT, overnight.
Bioactivities of compounds 6a-m are shown in Table 3. Compounds 6a-d, which
contain smaller subgroups at R3 (-OH, -CF3, -OCH3, -NH2, respectively), displayed
enhanced activity compared with PC06. Among them, compound 6d is the most
potent one with an IC50 value of 4.93 μM, which bears an amino group at R3 (Figure
4A). Compounds 6e-g, having a substituted amino group at R3, also showed activity
but weaker potency compared with 6d. Compound 6h-m, which harbor relatively
larger substituents, again displayed considerable activity. But their potencies did not
exceed that of 6d. Overall, among all the purchased and synthesized compounds, 6d is
8

the most potent one. Further studies were conducted on this compound.
Table 3. Chemical Structures and SIRT6 Inhibitory Activities of Compounds 6a-m.
R3
NO2
N
N
Inhibition (%)
@ 300 μM
(IC50/μM)
112.70
Inhibition (%)
@ 300 μM
(IC50/μM)
119.1
ID
R3
ID
R3
PC0
6
H
6a
6c
6e
6g
-OH
(21.91)
(11.04)
88.3
107.8
6b
6d
6f
-CF3
-NH2
-OCH3
(21.13)
(9.09)
107.20
O
H
63.70
N
N
(4.93)
O
N
96.00
97.08
H
H
N
N
(12.49)
(12.37)
Cl
97.10
O
O
6h
85.10
6i
N
H
N
H
(16.82)
O
87.50
108.00
(17.53)
100.90
(9.46)
O
O
6j
6l
6k
N
H
N
H
(20.94)
O
O
N
H
N
82.30
6m
O
O
Several other methods including differential scanning fluorimetry (DSF) assays,
9

surface plasmon resonance (SPR) assays and isothermal titration calorimetry (ITC)
assays were used to validate the bioactivity of compound 6d. In the DSF assay, 6d
showed an ability to shift the melting temperature (Tm) with △Tm = 1.88 °C at a
concentration of 40 µM (Figure 4B). In the SPR assay, 6d displayed a KD value of
9.76 μM (Figure 4C and 4D). In the ITC assay, 6d showed an KD value of 10.00 μM
(Figure 4E). All of these reuslts verified the bioactivity of 6d.
Figure 4. Bioactivity verification of compound 6d by FDL, DSF, SPR, and ITC
assays. (A) Dose-dependent activities of SIRT6 deacetylation of 6d, determined with
acetyl substrate peptide Ac-RYQK(Ac)-AMC. Data are presented as the meanꢀ±ꢀs.d.,
10

n=ꢀ3 wells, from three independent experiments. (B) The effect of 6d on the thermal
stability of SIRT6 was detected using DSF. (C, D) The binding curve and steady-state
evaluation of 6d binding to SIRT6 using SPR. (E) ITC binding curves for SIRT6 and
6d.
To examine the selectivity of 6d, we measured the bioactivity of 6d against 14
other deacetylases including SIRT1-3 and HDAC1-11 by FDL assays. The results
showed that 6d did not exhibit inhibitory activity against the tested deacetylases at
concentrations up to 200 μM, implying a good selectivity (Table 4).
Table 4. Target selectivity of 6d among HDAC family enzymes a
Protein
SIRT1
IC50 (μM)
>200
>200
>200
4.93
Protein
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
IC50 (μM)
>200
SIRT2
>200
SIRT3
>200
SIRT6
>200
HDAC1
HDAC2
HDAC3
HDAC4
>200
>200
>200
>200
>200
>200
>200
aThe activity of 6d against SIRT6 was determined by the FDL assay with acetyl
substrate peptide Ac-RYQK(Ac)-AMC.
To evaluate the inhibition effect of 6d on SIRT6 deacetylation in intact cells, we
monitored the acetylation status of H3K9 and H3K18 in BxPC-3 cells treated with 6d
11

at different concentrations; H3K9 and H3K18 were selected because they are known
targets of SIRT6. It was found that 6d increased the level of both H3K9ac and
H3K18ac in a dose-dependent manner, suggesting that 6d is active in cultured cells
(Figure 5A).
Finally, western blot assay was performed to examine the effect of 6d on the
level of glucose transporter GLUT-1. In this assay, BxPC-3 cells were used again. As
shown in Figure 5B, 6d could increase the GLUT-1 expression in a dose-dependent
manner. In a mouse model of type 2 diabetes (T2D, in high-fat-diet-fed animals), 6d
could significantly reduce the blood glucose content, and has no effect on the body
weight of mice (Figure 5C and 5D). In normal mice, 6d had no impact on blood
glucose or body weight (Figure S1 A, B).
Figure 5. (A) Western blot analysis of H3K9ac and H3K18ac protein expression in
BxPC-3 cells treated with 0.78, 1.56, 3.13, 6.25, 12.5, and 25 μM 6d for 48ꢀh. β-actin,
loading control. (B)Western blot analysis of GLUT1 protein expression in BxPC-3
cells treated with 1.25, 2.5, 5, and 10 μM 6d for 72ꢀh. α-tubulin, loading control. (C)
The changes of blood glucose were observed in a mouse model of T2DM after
intraperitoneal injection of 6d. (D) The changes of body weight were observed in a
12

mouse model of T2DM after intraperitoneal injection of 6d.
In summary, we discovered a number of new SIRT6 inhibitors containing the
skeleton 1-phenylpiperazine. Compound 6d is the most potent one, which showed an
IC50 value of 4.93 μM in FDL assays, and KD values of 9.76 μM and 10 μM in SPR
and ITC assays, respectively. In BxPC-3 cells, 6d could increase both H3K9ac and
H3K18ac levels in a concentration-dependent manner, and upregulate the expression
of glucose transporter GLUT1. In vivo, 6d also showed efficacy in a mouse model of
T2D. Collectively, 6d could be a potential lead compound for further drug discovery
targeting SIRT6.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(21772130),
National
Science
and
Technology
Major
Project
(2018ZX09711002-011-019, 2018ZX09201018, and 2019ZX09301-135), and 1.3.5
project for disciplines of excellence, West China Hospital, Sichuan University.
13

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Graphical abstracts
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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