Discovery of (5-Phenylfuran-2-yl)Methanamine derivatives as new human sirtuin 2 Inhibitors

Article abstract of DOI:10.3390/molecules24152724

Human sirtuin 2 (SIRT2), a member of the sirtuin family, has been considered as a promising drug target in cancer, neurodegenerative diseases, type II diabetes, and bacterial infections. Thus, SIRT2 inhibitors have been involved in effective treatment strategies for related diseases. Using previously established fluorescence-based assays for SIRT2 activity tests, the authors screened their in-house database and identified a compound, 4-(5-((3-(quinolin-5-yl)ureido)methyl) furan-2-yl)benzoic acid (20), which displayed 63 ± 5% and 35 ± 3% inhibition against SIRT2 at 100 μM and 10 μM, respectively. The structure-activity relationship (SAR) analyses of a series of synthesized (5-phenylfuran-2-yl)methanamine derivatives led to the identification of a potent compound 25 with an IC₅₀ value of 2.47 μM, which is more potent than AGK2 (IC₅₀ = 17.75 μM). Meanwhile, 25 likely possesses better water solubility (cLogP = 1.63 and cLogS = ?3.63). Finally, the molecular docking analyses indicated that 25 fitted well with the induced hydrophobic pocket of SIRT2.

Full text of DOI:10.3390/molecules24152724

molecules
Article
Discovery of (5-Phenylfuran-2-yl)methanamine
Derivatives as New Human Sirtuin 2 Inhibitors
Lijiao Wang 1, Chao Li 1, Wei Chen 1, Chen Song 1, Xing Zhang 2, Fan Yang 1, Chen Wang 1,
Yuanyuan Zhang 2, Shan Qian 1, Zhouyu Wang 2,* and Lingling Yang 1,
*
1
College of Food and Bioengineering, Xihua University, Sichuan 610039, China
College of Science, Xihua University, Sichuan 610039, China
2
*
Correspondence: zhouyuwang77@gmail.com (Z.W.); yangll0808@sina.com (L.Y.); Tel.: +86-28-7725898 (L.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 June 2019; Accepted: 20 July 2019; Published: 26 July 2019
Abstract: Human sirtuin 2 (SIRT2), a member of the sirtuin family, has been considered as
a promising drug target in cancer, neurodegenerative diseases, type II diabetes, and bacterial
infections. Thus, SIRT2 inhibitors have been involved in effective treatment strategies for related
diseases. Using previously established fluorescence-based assays for SIRT2 activity tests, the authors
screened their in-house database and identified a compound, 4-(5-((3-(quinolin-5-yl)ureido)methyl)
furan-2-yl)benzoic acid (20), which displayed 63
and 10 M, respectively. The structure-activity relationship (SAR) analyses of a series of synthesized
(5-phenylfuran-2-yl)methanamine derivatives led to the identification of a potent compound 25 with
an IC50 value of 2.47 M, which is more potent than AGK2 (IC50 = 17.75 M). Meanwhile, 25 likely
possesses better water solubility (cLogP = 1.63 and cLogS = 3.63). Finally, the molecular docking
±
5% and 35
±
3% inhibition against SIRT2 at 100 µM
µ
µ
µ
−
analyses indicated that 25 fitted well with the induced hydrophobic pocket of SIRT2.
Keywords: histone deacetylases; sirtuins; SIRT2; SAR studies; molecular docking
1. Introduction
Histone deacetylases (HDACs) are enzymes that catalyze the removal of acyl groups from
ε
-N-acyl-lysine amino groups on histones and non-histone substrates. These have been identified and
grouped into four classes [
]: Classes I, II, and IV HDACs are Zn2+-dependent metalloproteases; class
III HDACs, namely sirtuins (SIRTs), use NAD+ as a cofactor for catalysis [
]. There are seven isotypes
of sirtuins (SIRT1–7), which differ in their catalytic activity and subcellular localization [ ]. The isotype
SIRT2, which is located in both cytoplasm and nucleus [ ], mainly catalyzes deacetylation and
defatty-acylation for a variety of protein substrates, including histones H3 and H4 [ 10], and nonhistone
proteins -tubulin [11], p53 [12], Foxo1 [13], p300 [14], NF B [15], PAR3 and PRLR [16]. Thus,
SIRT2 has been shown to be involved in cell cycle regulation [11 17 18], autophagy [19], peripheral
myelination [20], and immune and inflammatory responses [21 23]. Recently, many studies revealed
that the dysregulation of SIRT2 activity is a key factor contributing to the pathogenesis of cancer [24],
neurodegenerative diseases [25 26], type II diabetes [27], and bacterial infections [21 23], which makes
SIRT2 a promising target for pharmaceutical intervention.
To date, except for some substrate analogues [ ], a number of small molecule inhibitors targeting
SIRT2 have been reported. The representative inhibitors are shown in Figure 1: The moderate
potency or non-specific inhibitors Sirtinol (46 M) [28], EX-527 (46 M) [29 30], AGK2 (3.5 M) [31 32],
AEM1(18.5 M) [33], AK-7 (15.5 M) [34], and AC-93253 (6 M) [35], the highly potent but unselective
inhibitors VII (0.048 M) and VIII (0.001 M) [36], and the potent and highly isotype-selective SIRT2
inhibitor SirReal2 (0.4 M) [23 37]. However, there remains a shortage of novel SIRT2 inhibitors as
1–3
4–6
7
8
9
,
α
κ
,
,
–
,
,
7
µ
µ
,
µ
,
µ
µ
µ
µ
µ
µ
,
lead candidates for drug discovery and development.
Molecules 2019, 24, 2724; doi:10.3390/molecules24152724
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2724
2 of 18
Figure 1. Chemical structures and inhibition potencies of selected examples SIRT2 inhibitors.
The authors previously established a fluorescence-based method for SIRT2 inhibition tests [38
and identified a series of N-(3-(phenoxymethyl)phenyl)acetamide derivatives as highly selective
SIRT2 inhibitors [38 41], some of which showed inhibitory activities against SIRT2 highly-expressed
–40],
,
human breast cancer cells and non-small cell lung cancer cells. Recently, the in-house compound
collection using the fluorescence-based method was screened, and a new compound was identified,
4-(5-((3-(quinolin-5-yl)ureido)methyl)furan-2-yl)benzoic acid (20, Figure 2), which displayed 63
and 35 3% inhibition against SIRT2 at 100 M and 10 M, respectively (Table 1). The scaffold of
compound 20 is novel for SIRT2 inhibitors, and 20 has a relatively low molecular weight (387 Da) with
moderate physicochemical properties (cLogP = 3.05, cLogS = 4.04). Thus, in this study, the authors
±
5%
±
µ
µ
−
used 20 as a starting point for further structural modifications (Linker, A, B, Figure 2) to improve the
inhibitory potency against SIRT2.
Figure 2. Chemical structure of 20 and the focus of structural modifications.
2. Results and Discussion
2.1. Chemistry
This study synthesized a series of (5-phenylfuran-2-yl)methanamine derivatives using the synthetic
routes outlined in Schemes 1–3. Firstly, urea-based compounds 11
condensation reaction between the key intermediate 5a 5i with aromatic-amine compounds
the presence of triphosgene, in 82–93% yields (Scheme 1). The intermediates 5a 5i were obtained
by using Suzuki cross-coupling reaction between commercially available substituted iodobenzenes
–
19 were acquired through the
–
6–10 in
–

Molecules 2019, 24, 2724
3 of 18
1a
–
1i with (5-formylfuran-2-yl)boronic acid (2), respectively. Then, the condensation reaction and
reduction reaction were performed in sequence to produce the intermediates 5a
acid compounds 20 26 were subsequently produced through the hydrolysis reaction from the
corresponding esters.
–5i. The carboxylic
–
Scheme 1. The preparation of target compounds 12
,
17
,
18 and 20
–
26. Reagents and conditions:
HCl, NaOAc, EtOH,
(i) Pd(Pph3)2Cl2, Na2CO3, MeCN/H2O = 1:1, 60 ◦C, 1 h, 80–85% [42]; (ii) NH2OH
·
Ref., 0.5 h, 100% [43]; (iii) Zn, HCl, EtOH, 80 ◦C; 62–83%; (iv) BTC, Et3N, DCM, RT., 0.5 h, 6 h, 82–93%;
(v) NaOH, EtOH/H2O = 1:1, 80 ◦C, 2 h, 86–95%.
Next, the desired target compound 30, a hydroxamic acid derivative, was prepared by a three-step
sequence starting from the synthesized intermediate 4a (Scheme 2). Sodium cyanoborohydride
(NaBH3CN)-mediated reduction reaction was firstly performed to reduce the aldoxime group of
intermediate 4a to the hydroxylamine of intermediate 27 (54% yield), followed by condensation with
2-phenylacetyl chloride in the presence of NaHCO3 to give the compound 29. Further, hydrolysis
of compound 29 using 3.0 equiv NaOH led to the white solid target compound 30. The synthesis of
target compounds 32–37 are also depicted in Scheme 2. The reactions of commercially available amines
(aniline, phenylmethanamine, and pyridin-3-ylmethanamine) or hydrazide (nicotinohydrazide) with
intermediates 3a or 3i in the presence of hantzschester (1.2 equiv), catalytic amount of molecular sieve
and trifluoroacetic acid, resulted in the reductive amination products 31–34. The resulting compounds
31–33 were subsequently hydrolyzed to give the desired compounds 35–37 in high yields.

Molecules 2019, 24, 2724
4 of 18
Scheme 2. The preparation of target compounds 30 and 32
–37. Reagents and conditions: (i) NaBH3CN,
HCl, MeOH, 0–60 ◦C, 4 h, 54%; (ii) NaHCO3, diethyl ether, RT, 6 h, 78%; (iii) NaOH, EtOH/H2O = 1:1,
◦
◦
80 C, 2 h, 89%; (iv) hantzschester, TFA, molecular sieve, DCM, 45 C, 6–12 h, 56–95%; (v) NaOH,
EtOH/H2O = 1:1, 80 ◦C, 2 h, 92–96%.
Scheme 3. The preparation of target compounds 39, and 43
–
52
.
Reagents and conditions:
(i) benzenesulfonyl chloride, Et3N, DCM, RT, 2 h, 91%; (ii) NaOH, EtOH/H2O = 1:1, 80 ◦C, 2 h,
90%; (iii) HOBT, EDCI, DIPEA, DCM, RT, 12 h, 73–91%; (iv) NaOH, EtOH/H2O = 1:1, 80 ◦C, 2 h, 90–95%.
Finally, Scheme 3 presents the synthetic routes for compounds 39 and 43–52, which contain a
sulfonamide or amide linker. For sulfonamide linker compound 39, intermediate 5a was used to
react with benzenesulfonyl chloride in the presence of Et3N at room temperature, and the resulting
compound 38 underwent a hydrolysis reaction to give the desired target compound 39, in 80%
yield for two steps. The synthetic access to structurally diverse amide linker compounds 41
achieved using a condensation reaction of carboxylic acid (40) with amine (5a 5c 5f) in the presence
of 1-hydroxybenzotriazole (HOBT), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
–48 was
,
–
(EDCI), and N,N-diisopropylethylamine (DIPEA). The resulting ester-contained compounds 41
and 47 were subjected to hydrolyzation to afford the target compounds 49–52 in good yields.
, 42, 46

Molecules 2019, 24, 2724
5 of 18
2.2. SAR Studies with SIRT2
The enzyme activity assays were performed using a fluorogenic-based method [38–40],
and Ac-Glu-Thr-Asp-Lys(Dec)-AMC, termed p2270, was used as the substrate. The SAR studies with
all of the synthesized (5-phenylfuran-2-yl)methanamine derivatives (Tables 1 and 2) were carried out.
The compounds bearing various linkers or different substituents (A moiety) at 3- or 4-position of the
phenyl of (5-phenylfuran-2-yl)methanamine scaffold (Table 1) were firstly investigated. Compared
with the hit compound 20, compounds 12 and 21, containing a urea as linker, showed comparable
or slightly lower SIRT2 inhibitory activities at 100
compounds 20 and 21, appeared to have better clogP and clogS properties than 12 (with clogP of 5.14
and clogS of 4.43). Compound 22 (23 3%), bearing a thiourea linker, displayed lower inhibitory
activity to SIRT2 than the corresponding compound 21 (33 3%) at 10 M. Further comparison of
the different linkers, including hydroxamic acid (30), secondary amine (35 36), sulfonamide (39) and
amide (49 50) revealed that urea linker derivatives were likely to have more potent SIRT2 inhibition
than other linker derivatives. The additional compounds with the 4-ethyl formate (32), 4-methyl
43), 4-methoxy group (44) replaced the 4-carboxyl of the phenyl of (5-phenylfuran-2-yl)methanamine
scaffold or changed to 3-position substituents (45 47 51, and 52) did not show improved inhibitory
µM or 10 µM;.Carboxyl acid which contained
−
±
±
µ
,
,
(
–
,
activity against SIRT2. These results indicate that the urea linker and 4-carboxyl of the phenyl of
(5-phenylfuran-2-yl)methanamine scaffold may be beneficial to fit with the binding pocket of SIRT2.
Table 1. The inhibitory activities and calculated clogP/clogS values of compounds 12
, 20–22, 30, 32,
35–36, 39, 43–47 and 49–52 against human SIRT2.
Inhibition% 1/SIRT2-p2270
cLogP
cLogS
ID
20
Structure
@ 100 µM
@ 10 µM
63 ± 5
35 ± 3
3.05
−4.04
12
60 ± 3
33 ± 3
5.14
−4.43
21
22
30
35
46 ± 4
44 ± 5
13 ± 2
24 ± 3
33 ± 3
23 ± 3
−2 ± 3
1 ± 2
2.85
3.60
2.99
4.27
−3.98
−4.22
−4.05
−4.13
39
49
38 ± 3
32 ± 3
11 ± 2
8 ± 1
1.85
3.12
−4.13
−4.14

Molecules 2019, 24, 2724
6 of 18
Table 1. Cont.
Inhibition% 1/SIRT2-p2270
cLogP
cLogS
ID
50
Structure
@ 100 µM
@ 10 µM
30 ± 2
5 ± 2
3.34
−4.25
32
36
43
9 ± 2
15 ± 2
3 ± 2
−2 ± 2
3 ± 2
4.17
2.48
4.18
−4.54
−4.29
−4.46
2 ± 3
44
45
20 ± 3
19 ± 2
8 ± 2
5 ± 2
3.79
3.76
−4.36
−4.36
46
51
15 ± 2
3 ± 3
3.46
−4.3
18 ± 3
16 ± 3
13 ± 3
5 ± 3
3.09
3.67
−4.12
−4.4
47
52
18 ± 2
80 ± 6
2 ± 3
3.31
5.65
−4.24
−4.25
AGK2
30 ± 5
1
Each compound was tested in triplicate; the data are presented as the mean ± SD (n = 2).
The authors next synthesized compounds 23
of the phenyl of (5-phenylfuran-2-yl)methanamine. The tested inhibitory activities and calculated
clogP and clogS values are shown in Table 2. Compounds 23 24, and 26 appear to have moderate
physiochemical properties, but compound 25, with the pyridine moiety, likely possesses better water
solubility (cLogP = 1.63 and cLogS = 3.63). Notably, compound 25 (99 2% @ 100 M, 90 3% @
10 M) shows potent inhibition against SIRT2, which is substantially more potent than the structurally
similar compound AGK2 (80 6% @ 100 M, 30 5% @ 10 M). Considering the fact that the
introduction of pyridine at the skeleton has improved SIRT2 inhibition, a series of pyridine-containing
–26, which contain a urea linker and 4-carboxyl
,
−
±
µ
±
µ
±
µ
±
µ
(5-phenylfuran-2-yl)methanamine derivatives (17
Comparing with 25, only compounds 17 (50 4% @ 100
100 M, 23 2% @ 10
whereas compounds 33, 34, 37 and 48 had almost no SIRT2 inhibitory activities (Table 2).
Collectively, the structural optimization and SAR studies led to the discovery of compound 25
,
18
,
33
µ
,
34
M, 37
,
37 and 48) were further synthesized.
3% @ 10 M) and 18 (40 5% @
±
±
µ
±
µ
±
µ
M), which both contained a urea linker, displayed low inhibition to SIRT2,
,
which exhibited high potency against SIRT2, better than the hit compound 20 and positive control
AGK2. Subsequently, the IC50 value of 25 was then measured against SIRT2, and the IC50 curve

Molecules 2019, 24, 2724
7 of 18
has been presented in Figure 3. The study observed that compound 25 inhibited SIRT2 via a dose
dependent manner with an IC50 value of 2.47 M, which is more potent than AGK2 (with an IC50
value of 17.75 M). Molecular docking was then used to investigate the possible binding mode of 25
with SIRT2. The results indicated that 25 appeared to fit well with the induced hydrophobic pocket
(Figure 4) [44 45]. The carboxyl acid group of 25 is likely positioned to make hydrogen-bonding
µ
µ
,
interactions with the main chain of Asp170 and the side chain of Thr171 and Tyr139. The furan
and pyridine moiety likely have hydrophobic contacts with hydrophobic residues Phe119, Phe234,
Phe131, Leu138, and Ile169 (Figure 4). Notably, the pyridine appears to form edge-to-face aromatic
interactions with Phe119, and fits well with the pocket around Phe119, Phe131, and Phe234, suggesting
that introducing substituents on pyridine may result in a clash with these three residues. Together,
these docking results may explain why the replacement of the carboxyl acid group or the introduction
of substituents on pyridine leads to a decrease in SIRT2 inhibition, and indicates the possible inhibition
mode for this series of compounds.
Table 2. The inhibitory activities and calculated clogP/clogS values of compounds 17
37 and 48 against human SIRT2.
–18, 23–26, 33–34,
Inhibition% 1/SIRT2-p2270
cLogP
cLogS
ID
23
Structure
@ 100 µM
@ 10 µM
35 ± 4
10 ± 3
3.15
−3.58
24
25
26
43 ± 2
99 ± 2
9 ± 2
13 ± 2
90 ± 3
−5 ± 3
3.21
1.63
3.05
−3.82
−3.63
−3.83
17
18
33
37
48
50 ± 4
40 ± 5
20 ± 3
30 ± 5
18 ± 2
37 ± 3
23 ± 2
3 ± 2
5 ± 2
0 ± 2
2.85
3.54
3.28
2.56
2.82
−3.83
−4.12
−4.08
−3.83
−4.11
34
3 ± 2
−2 ± 3
30 ± 5
3.19
5.65
−4.12
−4.25
AGK2
80 ± 6
1
Each compound was tested in triplicate; the data are presented as the mean ± SD (n = 2).

Molecules 2019, 24, 2724
8 of 18
Figure 3. The chemical structures and IC50 curves against SIRT2 of 25 and AGK2.
Figure 4. The docking pose of the compound 25 in the substrate binding site of SIRT2.
3. Materials and Methods
3.1. Synthesis
As previously reported, proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker
AV-400 (Bruker Company, Billerica, Germany) instrument and are reported in ppm relative to
tetramethylsilane (TMS) and referenced to the solvent in which the spectra were collected. Unless
otherwise noted, all of the commercially available starting materials, reagents, and solvents and
reagents were used without further purification. The analytical thin-layer chromatography (TLC)
was run on Merck silica gel 60 F-254 (Qingdao Haiyang, Qingdao, China). The spots on the
plates were visualized under UV light (λ = 254 nm). Purification was performed on silica gel
chromatography with EtOAc—petroleum ether or CH2Cl2-MeOH solvent systems. The melting points
were measured on an electrothermal melting point apparatus without correction (JIAHANG, Shanghai,
China. ESI-MS was obtained on a Shimadzu-2010EV series liquid chromatograph mass spectrometer
(Shimadzu, Tokyo, Japan). High-resolution mass spectra (HRMS) were determined using a SCIEX

Molecules 2019, 24, 2724
9 of 18
X500 QTOF mass spectrometer (Shanghai Sciex Analytical Instrument Trading Co., Shanghai, China).
All target compounds were purified to >95% purity, as determined by the high-performance liquid
chromatography (HPLC). The HPLC analysis was performed on a Waters 2695 HPLC system equipped
with a Kromasil C18 column (4.6 mm × 250 mm, 5 µm, Waters, Milford, MA, USA).
3.1.1. General Procedure for the Preparation of Key Intermediates 5a–5i
A mixture of substituted iodobenzenes (1a–1i, 15 mmol), (5-formylfuran-2-yl)boronic acid
(
2
, 15 mmol), bis(triphenylphosphine)palladium(II) chloride (Pd(Pph3)2Cl2, 0.6 mmol) an◦d sodium
carbonate (Na2CO3, 30 mmol) in MeCN/H2O (10 mL /10 mL) was stirred for 1 h at 60 C. Upon
completion of the reaction as determined by TLC, MeCN was removed by a rotary evaporator under
reduced pressure, and the residue was acidated with 1M HCl solution (pH 7) and filtered. Next,
the filtrate was partitioned between water (60 mL) and ethyl acetate (3
×
50 mL). The organic layer was
dried over magnesium sulfate anhydrous (MgSO4), filtered and concentrated in vacuo. The crude
products were purified by column chromatography with appropriate eluents to give the coupling
products 3a–3i, in 80–86% yields.
To a solution of the coupling products 3a–3i (12 mmol) in EtOH (25 mL), hydroxylamine
hydrochloride (NH2OH.HCl, 14.4 mmol) and sodium acetate (NaOAc, 14.4 mmol) were added and the
mixture was stirred at reflux for 0.5 h. When TLC indicated that the reaction was finished, the reaction
solution was concentrated and the residue was partitioned between water (50) and ethyl acetate
(3 × 50 mL). The combined organic layer was dried over MgSO4, filtered and concentrated in vacuo
to give the crude products 4a
–4i, which were used without further purification. Subsequently, to a
stirring solution of condensation products, 4a
–4i (12 mmol) in EtOH (25 mL) was added to zinc powder
(Zn, 12 mmol) and 3 M hydrochloric acid (HCl, 8.0 mL) at ambient temperatures. The reaction mixture
was heated to 80 ◦C for further 2 h. After completion (monitored by TLC), the solvent was removed
in vacuo, the crude residue was treated with 100 mL of ice water, and the pH was adjusted to 7–8
with saturated NaHCO3. Then, the mixture was filtered by diatomite and extracted with ethyl acetate
(3 × 80 mL). The combined extracts were dried, concentrated and purified by column chromatography
with appropriate eluents with three ethylamine (Et3N, TEA) to afford the desired intermediates 5a–5i
in high yields.
1-((5-(2,5-Dichlorophenyl)furan-2-yl)methyl)-3-(quinolin-5-yl)urea (12). A solution of quinolin-5-amine
(7, 250 mg, 1.73 mmol) and TEA (200 µL, 2.03 mmol) dissolved in CH2Cl2 (DCM, 15 mL) was
slowly dripped into a stirred solution of triphosgene (BTC, 256 mg, 0.85 mmol) in DCM (10 mL)
by using a constant-pressure dropping funnel. Then, the mixture was stirred for another 0.5 h at
room temperature (RT). After evaporation of the solvent, the residue was taken up in DCM (30 mL),
and (5-(2,5-dichlorophenyl)furan-2-yl)methanamine (5b, 230 mg, 0.95 mmol) was added directly to the
residue. The reaction mixture was stirred at RT for 6 h, and the solvent was subsequently removed in
vacuo. The residue obtained was purified by column chromatography (V(PE):V(EA) = 1:1) to give the
desired target compound 12 (343 mg, 0.84 mmol) in 88% yield. 96.8% HPLC purity. Mp: 245–246 ◦C.
1H-NMR (400 MHz, DMSO-d6)
δ 8.92 (s, 1H), 8.89 (dd, J = 4.0 Hz, J = 4.0 Hz, 1H), 8.54 (d, J = 8.4 Hz,
1H), 8.06–8.04 (m, 1H), 7.86 (d, J = 2.8 Hz, 1H), 7.69 (s, 1H), 7.68 (d, J = 2.8 Hz, 1H), 7.60–7.53 (m, 2H),
7.39 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.20 (d, J = 3.2 Hz, 2H), 6.53 (d, J = 3.2 Hz, 1H), 4.47 (d, J = 5.6 Hz,
2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 155.9, 154.7, 150.7, 148.7, 147.3, 135.8, 133.0, 132.7, 130.8,
130.3, 129.9, 128.6, 127.7, 126.9, 123.8, 121.3, 121.0, 117.4, 113.6, 109.5, 36.90 ppm. HRMS: m/z calcd for
C21H16N3O2 [M + H]+ 412.0577, found 412.0573.
Ethyl 4-(5-((3-(pyridin-3-yl)ureido)methyl)furan-2-yl)benzoate (17). The title compound was prepared from
pyridin-3-amine (9) and ethyl 4-(5-(aminomethyl)furan-2-yl)benzoate (5a) using the same method as
compound 12, purified by column chromatography (V(DCM):V(MeOH) = 30:1). Yield: 82%. HPLC
purity: 98.6%. Mp: 182–184 ◦C. 1H-NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H),
8.12 (dd, J = 4.4, 1.2 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.93–7.88 (m, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.26 (dd,

Molecules 2019, 24, 2724
10 of 18
J = 8.4, J = 4.4 Hz, 1H), 7.18 (s, 1H), 7.10 (d, J = 3.2 Hz, 1H), 6.46 (d, J = 3.2 Hz, 1H), 4.40 (d, J = 5.6 Hz,
2H), 4.32 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H) ppm. 13C-NMR (101 MHz, DMSO-d6)
165.8, 155.6,
δ
155.0, 151.5, 142.6, 139.9, 137.5, 134.8, 130.3, 128.5, 124.8, 124.0, 123.6, 114.6, 109.8, 61.2, 45.8, 31.2 ppm.
HRMS: m/z calcd for C20H20N3O4 [M + H]+ 366.1448 found 366.1444.
1-(Pyridin-3-yl)-3-((5-(4-(trifluoromethyl)phenyl)furan-2-yl)methyl)urea (18). The title compound was
prepared from pyridin-3-amine (9) and ((5-(4-(trifluoromethyl)phenyl)furan-2-yl)methyl)-l2-azane (5h)
using the same method as compound 12, purified by column chromatography (V(PE):V(EA) = 3:1).
Yield: 83%. HPLC purity: 98.0%. Mp: 183–187 ◦C. 1H-NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.58
(s, 1H), 8.14 (s, 1H), 7.91 (d, J = 12.8 Hz, 2H), 7.87 (s, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.27 (dd, J = 12.8 Hz,
J = 2.8 Hz, 1H), 7.11 (d, J = 3.2 Hz, 1H), 6.89 (t, J = 5.6 Hz, 1H), 6.46 (d, J = 3.2 Hz, 1H), 4.41 (d, J = 5.6 Hz,
2H) ppm. 13C-NMR (10 MHz, DMSO-d6)
δ 155.5, 154.9, 154.1, 151.0, 143.6, 142.1, 139.3, 137.9, 134.3,
130.0, 126.4, 126.3, 125.5, 124.0, 109.7, 45.8 ppm. HRMS: m/z calcd for C18H15N3O2 [M + H]+ 362.1061,
found 362.1070.
4-(5-((3-(Quinolin-5-yl)ureido)methyl)furan-2-yl)benzoic Acid (20). A mixture of ethyl 4-(5-((3-(quinolin-
5-yl)ureido)methyl)furan-2-yl)benzoate (11, 415 mg, 1.0 mmol), which was prepared from
quinolin-5-amine (9) and ethyl 4-(5-(aminomethyl)furan-2-yl)benzoate (5a) using the same method
as compound 12, and NaOH (127 mg, 3.0 mmol) reacted for 2 h in the solution of EtOH/H2O (10 mL/10 mL)
at 80 ◦C. After evaporation of the organic solvent, the residue was treated with 50 mL of ice water,
and the PH was adjusted to 6–7 with diluted HCl. Next, the mixture was extracted with ethyl acetate
(3
obtained was purified by column chromatography (V(PE):V(EA) = 2:1) to give the final compound
20 (344 mg) in 89% yield. HPLC purity: 98.2%. Mp: 285–287 ◦C. 1H-NMR (400 MHz, DMSO-d6)
×
50 mL). The combined extracts were washed with brine, dried, and concentrated. The residue
δ
9.84 (s, 1H), 9.01 (d, J = 8.4 Hz, 1H), 8.86 (d, J = 4.0 Hz, 1H), 8.20 (t, J = 5.6 Hz, 1H), 8.14 (q, J = 5.6 Hz
1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.68–7.62 (m, 4H), 7.50 (q, J = 4.0 Hz, 1H), 6.89 (d, J = 3.2 Hz, 1H), 6.45
(d, J = 3.2 Hz, 1H), 4.43 (d, J = 5.2 Hz, 1H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 170.8, 156.2, 153.7,
152.8, 150.5, 148.7, 136.5, 131.6, 130.1, 129.9, 123.0, 122.7, 121.0, 120.7, 116.3, 107.2, 101.7, 99.6, 36.9 ppm.
HRMS: m/z calcd for C22H18N3O4 [M + H]+ 388.1252, found 388.1254.
4-(5-((3-Phenylureido)methyl)furan-2-yl)benzoic Acid (21). The title compound was prepared from
ethyl 4-(5-((3-phenylureido)methyl)furan-2-yl)benzoate (13) using the same method as compound
20, purified by column chromatography (V(PE):V(EA) = 2:1). Yield: 86%. HPLC purity: 97.6%. Mp:
285–288 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
9.28 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz,
2H), 7.85 (d, J = 8.0 Hz, 2H), 7.38 (s, 1H), 7.21 (t, J = 7.6 Hz, 2H), 6.95 (d, J = 3.2 Hz, 1H), 6.88 (t, J = 7.2 Hz,
1H), 6.40 (d, J = 2.8 Hz, 1H), 4.37 (d, J = 5.2 Hz, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
168.9,
δ
155.8, 154.6, 152.0, 141.2, 133.1, 130.3, 129.0, 123.1, 121.3, 118.0, 109.4, 108.5, 36.8 ppm. HRMS: m/z calcd
for C19H16N2O4Na [M + Na]+ 359.0968, found 359.0965.
4-(5-((3-Phenylthioureido)methyl)furan-2-yl)benzoic Acid (22). The title compound was prepared from
ethyl 4-(5-((3-phenylthioureido)methyl)furan-2-yl)benzoate (14) using the same method as compound
20, purified by column chromatography (V(PE):V(EA) = 4:1). Yield: 87%. HPLC purity: 97.6%. Mp:
281–282 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 11.04 (s, 1H), 9.44 (s, 1H), 7.95 (d, J = 8.4 Hz, 3H), 7.70
(d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 4.0 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.96
(d, J = 3.2 Hz, 1H), 6.48 (d, J = 3.2 Hz, 1H), 4.82 (d, J = 5.6 Hz, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ
207.0, 181.4, 169.6 152.9, 152.4, 140.7, 132.7, 130.3, 128.7, 124.0, 123.1, 123.0, 110.1, 108.1, 31.2 ppm.
HRMS: m/z calcd for C19H17N2O3S [M + H]+ 353.0954, found 353.0962.
4-(5-((3-(4-Cyano-3-fluorophenyl)ureido)methyl)furan-2-yl)benzoic Acid (23). The title compound was
prepared from ethyl 4-(5-((3-(4-cyano-3-fluorophenyl)ureido)methyl)furan-2-yl)benzoate (15) using
the same method as compound 20, purified by column chromatography (V(DCM):V(MeOH) = 30:1).
Yield: 92%. HPLC purity: 97.2%. Mp: 192–193 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
10.97 (s, 1H),
8.53 (s, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.83 (dd, J = 12.8 Hz, J = 2.0 Hz 1H), 7.70 (d, J = 8.4 Hz, 3H),

Molecules 2019, 24, 2724
11 of 18
7.41 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 3.2 Hz, 1H), 6.41 (d, J = 3.2 Hz, 1H), 4.40 (d, J = 5.6 Hz, 2H) ppm.
13C-NMR (101 MHz, DMSO-d6)
δ 207.0, 169.6, 165.0, 162.5, 155.2, 153.6, 152.4, 148.45 134.3, 132.7, 130.3,
123.1, 115.4, 114.4, 109.6, 108.0, 45.9 ppm. HRMS: m/z calcd for C20H15FN3O4 [M + H]+ 380.1041, found
380.1040; C20H14FN3O4Na [M + Na]+ 402.0855, found 402.0857.
2-Hydroxy-4-(5-((3-phenylureido)methyl)furan-2-yl)benzoic Acid (24). The title compound was prepared
from ethyl 2-hydroxy-4-(5-((3-phenylureido)methyl)furan-2-yl)benzoate (16) using the same method
as compound 20, purified by column chromatography(V(DCM):V(MeOH) = 30:1). Yield: 86%. HPLC
purity: 96.6%. Mp: 224–227 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
12.05 (s br, 2H), 8.60 (s, 1H), 7.82
(d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 12.8 Hz, 4H), 7.10 (d, J = 3.2 Hz, 1H), 6.91 (s, 1H),
6.68 (s, 1H), 6.43 (d, J = 3.2 Hz, 1H), 4.38 (d, J = 5.6 Hz, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ
172.1, 162.1, 155.5, 155.2, 151.2, 140.8, 137.0, 131.5, 129.2, 121.7, 118.3, 114.6, 111.9, 111.1, 110.2, 109.6,
36.9 ppm. HRMS: m/z calcd for C19H17N2O5 [M + H]+ 353.1132, found 353.1140.
4-(5-((3-(Pyridin-3-yl)ureido)methyl)furan-2-yl)benzoic Acid (25). The title compound was prepared from
ethyl 4-(5-((3-(pyridin-3-yl)ureido)methyl)furan-2-yl)benzoate (17) using the same method ascompound
20, purified by column chromatography (V(PE):V(EA) = 3:1). Yield: 95%. HPLC purity: 98.6%. Mp:
218–220 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.98 (s br, 1H), 8.84 (s, 1H), 8.58 (s, 1H), 8.14 (d, J = 4.0 Hz,
1H), 7.99 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.29 (q, J = 4.8 Hz, 1H), 7.08
(d, J = 3.2 Hz, 1H), 6.87 (t, J = 5.6 Hz, 1H), 6.45 (d, J = 3.2 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H) ppm. 13C-NMR
(101 MHz, DMSO-d6)
δ 167.5, 155.4, 154.7, 151.6, 142.7, 140.1, 137.5, 134.5, 130.5, 129.5, 125.2, 124.1,
123.5, 109.8, 109.5, 31.2 ppm. HRMS: m/z calcd for C18H16N3O4 [M + H]+ 338.1135, found338.1138.
4-(5-((3-(5-Bromo-2-methylpyridin-3-yl)ureido)methyl)furan-2-yl)benzoic acid (26). The title compound was
prepared from ethyl 4-(5-((3-(5-bromo-2-methylpyridin-3-yl)ureido)methyl)furan-2-yl)benzoate (19
)
using the same method as compound 20, purified by column chromatography (V(DCM):V(MeOH)
= 10:1). Yield: 90%. HPLC purity: 97.6%. Mp: 250–254 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.96
(s, 1H), 8.56 (d, J = 2.8 Hz, 1H), 8.35 (s, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.80
(d, J = 8.4 Hz, 2H), 7.58 (s, 1H), 7.09 (d, J = 3.2 Hz, 1H), 6.49 (d, J = 3.2 Hz, 1H), 4.42 (d, J = 5.6 Hz, 2H),
2.40 (s, 3H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 167.4, 155.4, 154.4, 151.8, 146.2, 142.0, 136.2, 134.4,
130.5, 129.6, 127.9, 123.5, 117.5, 110.0, 109.5, 36.8, 21.38 ppm. LCMS m/z: 428.0 [M − H]−.
4-(5-((N-hydroxy-2-phenylacetamido)methyl)furan-2-yl)benzoic Acid Ethyl (30).
To a solution of
(E)-4-(5-((hydroxyimino)methyl)furan-2-yl)benzoate (4a, 610 mg, 2.35 mmol) in methyl alcohol (10 mL),
sodium cyanoborohydride (440 mg, 1.5 mmol) and 12 M hydrochloric acid (780 µL, 9.4 mmol) were
added at 0 ◦C. Then, the mixture was stirred at room temperature for 4 h. When TLC indicated that
the reaction was finished, the reaction solution was concentrated and the residue was basified with
6 N sodium hydroxide solution (pH 8) and extracted several times with ethyl acetate. The combined
organic extracts were dried (Na2SO4), and concentrated under reduced pressure to yield the reduction
product ethyl 4-(5-((hydroxyamino)methyl)furan-2-yl)benzoate (27, 328 mg) in 54% yield. Next,
2-phenylacetyl chloride (28, 195 mg, 1.27 mmol) and NaHCO3 (106 mg, 1.27 mmol) were added to
the solution of 27 (328 mg, 1.27 mmol) in diethyl ether (15 mL) and the reaction was stirred at room
temperature for 6 h. Upon completion of the reaction as determined by TLC, the resulting solution
was concentrated under reduced pressure to dryness and the residue was purified by silica gel column
chromatography to give the light yellow compound 29 in 78% yield. The target compound 30 was
gained from ethyl 4-(5-((N-hydroxy-2-phenylacetamido)methyl)furan-2-yl)benzoate (29) using the
same method as compound 20, purified by column chromatography (V(PE):V(EA) = 3:1). Yield:
89%. HPLC purity: 97.0%. Mp: 162–163 ◦C. 1H-NMR (400 MHz, DMSO-d6)
1H), 7.98 (d, J = 8.8 Hz, 2H) 7.75 (d, J = 8.4 Hz, 2H), 7.32–7.21 (m, 5H), 7.08 (d, J = 3.2 Hz, 1H), 6.49
(d, J = 3.2 Hz, 1H), 4.80 (s, 2H), 3.79 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
171.9, 167.4, 152.0,
δ 12.95 (s, 1H), 10.15 (s,
δ
151.8, 136.1, 134.4, 130.5, 129.9, 129.6, 128.6, 126.8, 123.6, 111.7, 109.4, 45.07, 38.9 ppm. HRMS: m/z calcd
for C20H16NO5 [M − H]− 350.1034, found 350.1066.

Molecules 2019, 24, 2724
12 of 18
3.1.2. Hantzsch-Involved Reductive Amination Used for Compounds 31–34
To a solution of substituted 5-phenylfuran-2-carbaldehydes (3a and 3i, 1.5 mmol), different amines
(1.8 mmol) and diethyl 2,6-dimethyl- 1,4-dihydropyridine-3,5-dicarboxylate (hantzschester, 1.8 mmol)
in DCM (25 mL), catalytic amount of molecular sieve and trifluoroacetic acid were added at room
temperature, and the reaction was warmed to 45 ◦C and reacted for 6–12 h. After completion (monitored
by TLC), the reaction was filtered, and the crude residue was obtained by concentrating the filtrate
in vacuo. Finally, the crude residue was purified by column chromatography to give the desired
compounds 31–34 in high yields.
Ethyl 4-(5-((Benzylamino)methyl)furan-2-yl)benzoate (32). Yield: 56%. HPLC purity: 98.1%. Mp:
250–251 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
7.98 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.37–7.30
(m, 4H), 7.23 (t, J = 8.4 Hz, 1H), 7.08 (d, J = 3.2 Hz, 1H), 6.44 (d, J = 3.2 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H),
3.74 (s, 4H), 2.80 (s br, 1H), 1.33 (t, J = 7.2 Hz, 3H) ppm. 13C-NMR (101 MHz, DMSO-d6)
165.9, 156.4,
δ
151.3, 140.9, 135.0, 130.3, 128.6, 128.5, 128.3, 127.1, 123.5, 110.0, 109.7, 61.2, 52.5, 45.3, 14.67 ppm. LCMS
m/z: 335.2 [M + H]+.
Ethyl (E)-4-(5-((2-nicotinoylhydrazono)methyl)furan-2-yl)benzoate (33). Yield: 72%. HPLC purity: 97.5%.
Mp: 201–204 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.10 (s, 1H), 9.09 (d, J = 1.2 Hz, 1H),8.79 (d, J = 3.6 Hz,
1H), 8.41 (s, 1H), 8.28 (dt, J = 8.0 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 7.62–7.58 (m,
1H), 7.36 (d, J = 3.6 Hz, 1H), 7.16 (d, J =3.6 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H) ppm.
13C-NMR (101 MHz, DMSO-d6)
δ 165.7, 162.2, 154.2, 152.9, 150.4, 149.0, 138.2, 136.0, 133.9, 130.4, 129.5,
129.4, 124.5, 124.1, 117.3, 111.3, 61.3, 14.7 ppm. HRMS: m/z calcd for C20H18N3O4 [M + H]+ 364.1260,
found 364.1264.
1-(5-(4-Bromophenyl)furan-2-yl)-N-(pyridin-3-ylmethyl)methanamine (34). Yield: 95%. HPLC purity:
97.8%. Mp: 146–150 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
8.53 (s, 1H), 8.44 (d, J = 3.6 Hz, 1H), 7.76
(d, J = 7.6 Hz, 1H), 7.64–7.58 (m, 5H), 7.35–7.32 (m, 1H), 6.93 (d, J = 3.2 Hz, 1H), 6.39 (d, J = 3.2 Hz,
1H), 3.75 (s, 2H), 3.72 (s, 2H), 1.23 (s, 1H) ppm. 13C-NMR (101 MHz, DMSO-d6)
155.1, 151.3, 149.9,
δ
148.4, 136.3, 136.2, 132.2, 130.1, 125.6, 123.8, 120.4, 109.8, 107.8, 49.9, 45.4 ppm. HRMS: m/z calcd for
C17H15BrNO2 [M + H]+ 342.0368, found 343.0397 and 345.0387.
4-(5-((phenylamino)methyl)furan-2-yl)benzoic Acid (35). The title compound was prepared from ethyl
4-(5-((phenylamino)methyl)furan-2-yl)benzoate (31) using the same method ascompound 20, purified
by column chromatography (V(PE):V(EA) = 2:1). Yield: 96%. HPLC purity: 98.3%. Mp: 207–210 ◦C.
1H-NMR (400 MHz, DMSO-d6)
δ
12.96 (s, 1H), 7.98 (d, J = 7.6 Hz, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.09 (t,
J = 7.6 Hz, 2H), 7.05 (d, J = 3.2 Hz, 1H), 6.70 (d, J = 7.6 Hz, 2H), 6.57 (t, J = 7.2 Hz, 1H), 6.47 (d, J = 3.2 Hz,
1H), 6.18 (s br, 1H), 4.34 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 167.4, 155.2, 151.5, 148.7, 134.6,
130.5, 129.4, 129.3, 123.4, 116.7, 112.9, 110.1, 109.4, 40.5 ppm. LCMS m/z: 294.1 [M + H]+.
4-(5-((Benzylamino)methyl)furan-2-yl)benzoic Acid (36). The title compound was prepared from ethyl
4-(5-((benzylamino)methyl)furan-2-yl)benzoate (32) using the same method as compound 20, purified
by column chromatography (V(PA):V(EA) = 2:1). Yield: 92%. HPLC purity: 98.0%. Mp: 248–250 ◦C.
1H-NMR (400 MHz, DMSO-d6)
δ
10.96 (s br, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.60
(d, J = 6.4 Hz, 2H), 7.44–7.38 (m, 3H), 7.17 (d, J = 3.2 Hz, 1H), 6.81 (d, J = 3.2 Hz, 1H), 4.27 (s, 2H),
4.19 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 167.4, 153.3, 147.3, 134.0, 132.5, 130.6, 130.5, 130.1,
129.3, 129.1, 124.0, 115.0, 109.5, 50.0, 42.6 ppm. HRMS: m/z calcd for C19H17NO3 [M + H]+ 308.1265,
found 308.1260.
(E)-4-(5-((2-nicotinoylhydrazono)methyl)furan-2-yl)benzoic Acid (37). The title compound was prepared
from ethyl (E)-4-(5-((2-nicotinoylhydrazono)methyl)furan-2-yl)benzoate (33) using the same method
as compound 20, purified by column chromatography (V(PE):V(EA) = 2:1). Yield: 92%. HPLC purity:
97.2%. Mp: 237–240 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
12.10 (s, 1H), 9.09 (s, 1H), 8.79 (d, J = 3.6 Hz,
1H), 8.41 (s, 1H), 8.28 (dt, J = 8.4 Hz, J = 2.0 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz,

Molecules 2019, 24, 2724
13 of 18
2H), 7.62–7.58 (m, 1H), 7.34 (d, J = 3.6 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H) ppm. 13C-NMR (101 MHz,
DMSO-d6) δ 164.7, 162.2, 154.6, 152.8, 150.9, 149.1, 138.4, 137.2, 136.0, 130.5, 129.6, 129.5, 124.2, 124.1,
117.1, 110.7 ppm. HRMS: m/z calcd for C18H14N3O4 [M + H]+ 336.0975, found 336.0952.
4-(5-(Phenylsulfonamidomethyl)furan-2-yl)benzoic Acid (39). The intermediate 5a (100 mg, 0.43 mmol)
reacted with benzenesulfonyl chloride (90 mg, 0.05 mmol,) in the presence of Et3N (179 µL, 1.30 mmol)
at room temperature, in DCM (15 mL). When TLC indicated that the reaction was finished, the reaction
was concentrated in vacuo and the pH was adjusted to 7–8 with saturated NaHCO3. Then, the water
solution was extracted with ethyl acetate (3 ). The combined extracts were concentrated to give brown
×
crude product ethyl 4-(5-(phenylsulfonamidomethyl)furan-2-yl)benzoate (38) in 91% yield, which
was used to synthesize the target compound 39, in 90% yield. HPLC purity: 97.5%. Mp: 222–223 ◦C.
1H-NMR (400 MHz, DMSO-d6)
δ13.00 (s, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.85
(d, J = 6.8 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.62–7.55 (m, 3H), 6.98 (d, J = 3.6 Hz, 1H), 6.38 (d, J = 3.2 Hz,
1H) 4.19 (d, J = 6.0 Hz, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 167.4, 152.1, 152.0, 141.2, 134.2, 132.7,
130.4, 129.5, 129.5, 126.9, 123.6, 111.1, 109.1, 39.9 ppm. HRMS: m/z calcd for C18H16NO5S [M + H]+
358.0671, found 358.0670.
2-Phenyl-N-((5-(p-tolyl)furan-2-yl)methyl)acetamide (43). (5-(p-tolyl)furan-2-yl)methanamine (5c, 378 mg,
1.49 mmol) reacted with 2-phenylacetic acid (200 mg, 1.47 mmol) in the presence of 1-hydroxybenzotriazole
(HOBT, 214 mg, 1.47 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
282 mg, 1.47 mmol), and N,N-diisopropylethylamine (DIPEA, 0.21 mL, 4.3 mmol) in DCM (20 mL).
The mixture was stirred at room temperature for 12 h. Then, the mixture was concentrated and
partitioned between water (60 mL) and ethyl acetate (3
×
60 mL). The organic layer was dried over
MgSO4, filtered, concentrated and purified by column chromatography (V(PE):V(EA) ◦= 6:1) to give
1
the target compound 43 (327 mg) in 73% yield. HPLC purity: 99.2%. Mp: 193–194 C. H-NMR
(400 MHz, DMSO-d6)
δ 8.60 (t, J = 5.6 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 4.4 Hz, 4H), 7.24–7.21
(m, 3H), 6.78 (d, J = 3.2 Hz, 1H), 6.30 (d, J = 3.2 Hz, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.48 (s, 2H), 2.32 (s,
2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ170.5, 152.8, 152.2, 137.1, 136.8, 129.9, 129.4, 128.7, 128.2,
126.8, 123.7, 109.4, 106.1, 42.7, 36.3, 21.3 ppm. HRMS: m/z calcd for C20H19NO2 Na [M + Na]+ 328.1231,
found 328.1228.
N-((5-(4-methoxyphenyl)furan-2-yl)methyl)-2-phenylacetamide (44). The title compound was prepared
from intermediate 5d using the same method as compound 43, purified by column chromatography
(V(PA):V(EA) = 7:1). Yield: 86%. HPLC purity: 98.2%. Mp: 196–197 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
8.59 (t, J = 5.6 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 4.4 Hz, 4H), 7.25–7.21 (m, 1H), 6.99
(d, J = 8.8 Hz, 2H), 6.69 (d, J = 3.2 Hz, 1H), 6.28 (d, J = 3.2 Hz, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.79 (s, 3H),
3.49 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ 170.5, 159.1, 152.8, 151.8, 136.8, 129.4, 128.7, 126.8,
125.2, 123.8, 114.8, 109.4, 105.1, 55.6, 42.7, 36.3 ppm. HRMS: m/z calcd for C20H19NO2 Na [M + Na]+
344.1170, found 344.1175.
N-((5-(3-methoxyphenyl)furan-2-yl)methyl)-2-phenylacetamide (45). The title compound was prepared
from intermediate 5e using the same method as compound 43, purified by column chromatography
(V(PE):V(EA) = 7:1). Yield: 91%. HPLC purity: 97.8%. Mp: 182–184 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
8.61 (t, J = 5.6 Hz, 1H), 7.35–7.29 (m, 5H), 7.25–7.23 (m, 2H), 7.21–7.20 (m, 1H), 6.89 (d, J = 3.2 Hz,
1H), 6.86 (dd, J = 8.0 Hz, J = 3.2 Hz, 1H), 6.32 (d, J = 3.6 Hz, 1H), 4.34 (d, J = 5.6 Hz, 2H), 3.80 (s, 3H),
3.50 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
170.6, 160.1, 152.7, 152.5, 136.8, 132.1, 130.5, 129.4,
δ
128.7, 126.8, 116.2, 113.4, 109.5, 109.1, 107.4, 55.6, 42.7, 36.3 ppm. HRMS: m/z calcd for C20H19NO2 Na
[M + Na]+ 344.1170, found 344.1174.
Methyl 3-(5-(benzamidomethyl)furan-2-yl)benzoate (46). The title compound was prepared from
intermediate 5f and benzoic acid using the same method as compound 43, purified by column
chromatography (V(PE):V(EA) = 8:1). Yield: 90%. HPLC purity: 97.9%. Mp: 197–198 ◦C. 1H-NMR
(400 MHz, DMSO-d6) δ 9.09 (t, J = 6.4 Hz, 1H), 8.22 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 7.6 Hz, 2H),

Molecules 2019, 24, 2724
14 of 18
7.85 (d, J = 7.6 Hz, 1H), 7.59–7.53 (m, 2H), 7.50–7.46 (m, 2H), 7.03 (d, J = 3.2 Hz, 1H), 6.44 (d, J = 3.2 Hz,
1H), 4.57 (q, J = 5.6 Hz, 2H), 3.89 (s, 3H) ppm. 13C-NMR (101 MHz, DMSO-d6)
166.7, 166.5, 153.6, 151.4,
δ
134.6, 131.8, 131.3, 130.8, 130.0, 128.8, 128.3, 128.2, 127.8, 123.8, 109.9, 108.3, 52.8, 36.8 ppm. HRMS:
m/z calcd for C20H17NO4 [M + H]+ 336.1210, found 336.1211; C20H16NO4Na [M + Na]+ 358.1010,
found 358.1015.
Methyl 3-(5-((2-Phenylacetamido)methyl)furan-2-yl)benzoate (47). The title compound was prepared from
intermediate 5f and phenylacetic acid using the same method as compound 43, purified by column
chromatography (V(PE):V(EA) = 8:1). Yield: 90%. HPLC purity: 97.2%. Mp: 183–184 ◦C. 1H-NMR
(400 MHz, DMSO-d6)
δ 8.66 (t, J = 5.6 Hz, 1H), 8.21 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz,
1H), 7.57 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 4.4 Hz, 4H), 7.25–7.20 (m, 1H), 7.01 (d, J = 3.2 Hz, 1H), 6.36
(d, J = 3.2 Hz, 1H), 4.37 (d, J = 5.6 Hz, 2H), 3.89 (s, 3H), 3.49 (s, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ
170.6, 166.5, 153.4, 151.5, 136.8, 131.2, 130.8, 129.9, 129.4, 128.7, 128.3, 126.8, 123.8, 109.8, 108.2, 52.8,
42.7, 36.2 ppm. HRMS: m/z calcd for C21H19NO4Na [M + Na]+ 372.1171, found 372.1177.
Ethyl 4-(5-(Nicotinamidomethyl)furan-2-yl)benzoate (48). The title compound was prepared from
intermediate 5a and nicotinic acid using the same method as compound 43, purified by column
chromatography (V(PE):V(EA) = 6:1). Yield: 78%. HPLC purity: 97.8%. Mp: 182–183 ◦C. 1H-NMR
(400 MHz, DMSO-d6) δ 9.30 (t, J = 2.0 Hz, 1H), 9.07 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 6.0 Hz, 1H), 8.25 (t,
J = 8.0 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 6.4 Hz, 1H), 7.11 (d, J = 3.6 Hz,
1H), 6.52 (d, J = 3.2 Hz, 1H), 4.61 (d, J = 5.6 Hz, 2H), 4.31 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H) ppm.
13C-NMR (101 MHz, DMSO-d6)
δ 165.8, 165.4, 153.9, 152.5, 151.6, 149.0, 135.6, 134.8, 130.3, 130.1, 128.6,
124.0, 123.6, 110.3, 109.8, 61.2, 36.8, 14.7 ppm. LCMS m/z: 351.1 [M + H]+.
4-(5-(Benzamidomethyl)furan-2-yl)benzoic Acid (49). Using the intermediate 5a and benzoic acid, the title
compound 49 was synthesized via condensation reaction (87% yield), and hydrolysis reaction (92%
yield) in turn. HPLC purity: 97.0%. Mp: 183–186 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 9.09 (t, J = 5.6 Hz,
1H), 7.98 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 1.6 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.77–7.49 (m, 3H), 7.08
(d, J = 2.4 Hz, 1H), 6.47 (d, J = 3.2 Hz, 1H), 4.58 (d, J = 5.6 Hz, 2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ
167.5, 166.7, 154.2, 151.6, 134.5, 134.5, 131.9, 130.5, 129.5, 128.8, 127.8, 123.5, 110.1, 109.5, 36.8 ppm.
LCMS m/z: 320.1 [M − H]−.
4-(5-((2-Phenylacetamido)methyl)furan-2-yl)benzoic Acid (50). Using the intermediate 5a and phenylacetic
acid, the title compound 49 was synthesized via condensation reaction (82% yield), and hydrolysis
reaction (95% yield) in turn. HPLC purity: 97.1%. Mp: 228–230 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ
12.98 (s, 1H), 8.65 (t, J = 5.4 Hz, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 4.4 Hz,
4H), 7.26–7.22 (m, 1H), 7.05 (d, J = 3.2 Hz, 1H), 6.38, (d, J = 3.2 Hz, 1H), 4.37 (d, J = 5.6 Hz, 2H), 3.49 (s,
2H) ppm. 13C-NMR (101 MHz, DMSO-d6)
δ170.6, 167.4, 154.0, 151.7, 136.8, 134.5, 130.5, 129.5, 129.5,
128.7, 126.9, 123.5, 109.9, 109.4, 42.7, 36.3 ppm. HRMS: m/z calcd for C20H17NO4Na [M + Na]+ 358.1026,
found 358.1015.
3-(5-(Benzamidomethyl)furan-2-yl)benzoic Acid (51). The title compound was prepared from compound
46 via hydrolysis reaction (90% yield), purified by column chromatography (V(PE):V(EA) = 3:1). HPLC
purity: 97.0%. Mp: 190–191 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 13.15 (s br, 1H), 9.20 (t, J = 6.4 Hz,
1H), 8.22 (s, 1H), 7.94–7.91 (m, 3H), 7.83 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 8.0 Hz, 2H), 7.47 (t, J = 8.0 Hz,
2H), 7.00 (d, J = 3.2 Hz, 1H), 6.42 (d, J = 3.2 Hz, 1H), 4.55 (d, J = 5.6 Hz, 2H) ppm. 13C-NMR (101 MHz,
DMSO-d6) δ 167.5, 166.7, 153.5, 151.5, 134.5, 132.0, 131.8, 131.1, 129.8, 128.8, 128.4, 127.8, 124.1, 109.8,
108.1, 36.7 ppm. HRMS: m/z calcd for C19H15NO4 [M + Na]+ 344.0852, found 344.0855.
3-(5-((2-Phenylacetamido)methyl)furan-2-yl)benzoic Acid (52). The title compound was prepared from
compound 47 via hydrolysis reaction (91% yield), purified by column chromatography (V(PE):V(EA)
= 3:1). HPLC purity: 97.2%. Mp: 220–221 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 8.85 (t, J = 5.6 Hz, 1H),
8.27 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.58 (t, J = 8.0 Hz, 1H), 7.36–7.34 (m, 4H), 7.29–7.25 (m, 1H), 7.02
(d, J = 3.2 Hz, 1H), 6.40 (d, J = 3.2 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H), 3.55 (s, 2H) ppm. 13C-NMR (101 MHz,

Molecules 2019, 24, 2724
15 of 18
DMSO-d6) δ 170.7, 167.8, 153.2, 151.8, 136.8, 131.0, 129.6, 129.5, 128.7, 128.4, 127.6, 126.8, 124.1, 109.7,
107.8, 42.7, 36.2 ppm. HRMS: m/z calcd for C20H17NO4Na [M + Na]+ 358.1011, found 358.105.
3.2. Inhibition Assays
This study tested the inhibitory activities of the synthesized compounds against recombinant
human SIRT2 proteins using a fluorogenic substrate p2270(Ac-Glu-Thr-Asp-Lys(Dec)-AMC)-coupled
trypsin assay. The assay buffer is 25 mM Tris–HCl pH 8.0, 150 mM NaCl, and 10% glycerol. The test
compounds were added to 60
compound was prepared in a 3-fold dilution series (300
< 1%. After incubation at 25 ◦C for 30 min, the reaction started by the addition of the substrate p2270
(10 mM) and NAD+(400 mM) at 25 ◦C. After 2 h, 50
L 3~4 U/ L trypsin and 4 mM nicotinamide were
added to terminate the reaction, followed by further incubation for 30 min at 25 ◦C. The fluorescence
intensity was measured using a microplate reader ( ex = 380 nm, em = 460 nm). All determinations
µL of reaction mixture containing SIRT2 enzymes (0.2
µM), and each
µM–15 nM) with the final DMSO concentration
µ
µ
λ
λ
were performed in triplicate. The IC50 values were obtained using GraphPad Prism software as
described previously.
3.3. Molecular Docking Assays
All the docking simulations were performed using AutoDock Vina. The crystal structure of
SIRT2 complexed with an N-(3-(phenoxymethyl)phenyl)acetamide derivative (24a) (PDB ID: 5YQO)
and was used as the docking template. All the water and solvant molecules, as well as 24a were
removed, and clean protein structure coordinates were obtained. AutoDockTools was used to assign
Gasteiger-Marsili charges to the protein structure model, and merge non-polar hydrogens onto their
respective heavy atoms of the protein structure (saved as pdbqt format). The 3D coordinates of the
compound structures were prepared using the Discovery Studio viewer, followed by assigning atom
types and partial charges using AutoDockTools (saved as pdbqt format). The binding site was defined
as a rectangular grid, with the grid center coordinates of [x, y, z =
−
13.5,
−
10.1, 18.4] and the grid
−
size of [25, 25, 25], to encompass the entire binding site. The number of possible docking poses were
set as 10, and the other docking parameters were set as default. The docking results were inspected
using PyMOL.
4. Conclusions
In this study, a series of (5-phenylfuran-2-yl)methanamine derivatives were synthesized. The SAR
analyses of these compounds with SIRT2 led to the identification of compound 25 with 99
100 M and 90 3 % @ 10 M inhibition against SIRT2. Meanwhile, 25 likely possesses better water
solubility (cLogP = 1.63 and cLogS = 3.63). The IC50 measurements revealed that 25 had considerable
potency against SIRT2 with an IC50 value of 2.47 M, which is more potent than AGK2. The molecular
±
2% @
µ
±
µ
−
µ
docking analyses indicated that 25 fits well with the induced hydrophobic pocket of SIRT2. This study
will aid future investigations to discover new potent and selective SIRT2 inhibitors to provide potential
treatments for relevant diseases.
Author Contributions: L.W., C.L., C.S. and X.Z. designed and synthesized the target compounds. W.C., F.Y., C.W.
and Y.Z. performed the biological evaluation. S.Q. performed the molecular docking. Z.W. and L.Y. interpreted
the data and wrote the paper. All authors have approved the final manuscript.
Funding: This research was funded by Science and Technology Benefiting People National Natural Science
Foundation (No. 81703355), Project of Chengdu (No. 2015-HM01-00335-SF), Sichuan Education Department
(No. 18TD0023), Science and Technology Department of Sichuan Province (No. 2016HH0075), Chun-Hui Project
from Ministry of Education of China (No.172507), Open research Subject of Key Laboratory of Food Biotechnology of
Sichuan province of China (No. szj2016-021), and the Center of Comprehensive Health Management (No. szj2017-043).
The APC was funded by Young Scholars Reserve Talents program of Xihua University (No. 0220170305).
Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2019, 24, 2724
16 of 18
References
1.
2.
3.
Bhalla, K.N. Epigenetic and Chromatin Modifiers as Targeted Therapy of Hematologic Malignancies. J. Clin.
Oncol. 2005, 23, 3971–3993. [CrossRef] [PubMed]
Blander, G.; Guarente, L. The Sir2 Family of Protein Deacetylases. Annu. Rev. Biochem. 2004, 73, 417–435.
[CrossRef]
Liu, S.; Ji, S.; Yu, Z.-J.; Wang, H.-L.; Cheng, X.; Li, W.-J.; Jing, L.; Yu, Y.; Chen, Q.; Yang, L.-L.; et al.
Structure-based discovery of new selective small-molecule sirtuin 5 inhibitors. Chem. Biol. Drug Des. 2018
,
91, 257–268. [CrossRef] [PubMed]
4.
Hirschey, M.D. Old Enzymes, New Tricks: Sirtuins Are NAD+-Dependent De-acylases. Cell Metab. 2011, 14,
718–719. [CrossRef]
5.
6.
Jing, H.; Lin, H. Sirtuins in Epigenetic Regulation. Chem. Rev. 2015, 115, 2350–2375. [CrossRef]
Yang, L.; Ma, X.; He, Y.; Yuan, C.; Chen, Q.; Li, G.; Chen, X. Sirtuin 5: A review of structure, known inhibitors
and clues for developing new inhibitors. Sci. China Life Sci. 2016, 60, 249–256. [CrossRef] [PubMed]
Yamagata, K.; Goto, Y.; Nishimasu, H.; Morimoto, J.; Ishitani, R.; Dohmae, N.; Takeda, N.; Nagai, R.;
Komuro, I.; Suga, H.; et al. Structural basis for potent inhibition of SIRT2 deacetylase by a macrocyclic
peptide inducing dynamic structural change. Structure 2014, 22, 345–352. [CrossRef] [PubMed]
Wang, Y.; He, J.; Liao, M.; Hu, M.; Li, W.; Ouyang, H.; Wang, X.; Ye, T.; Zhang, Y.; Ouyang, L. An overview of
Sirtuins as potential therapeutic target: Structure, function and modulators. Eur. J. Med. Chem. 2019, 161,
48–77. [CrossRef]
7.
8.
9.
Das, C.; Lucia, M.S.; Hansen, K.C.; Tyler, J.K. CBP/p300-mediated acetylation of histone H3 on lysine 56.
Nature 2009, 459, 113–117. [CrossRef]
10. Vaquero, A.; Scher, M.B.; Lee, D.H.; Sutton, A.; Cheng, H.L.; Alt, F.W.; Serrano, L.; Sternglanz, R.; Reinberg, D.
SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis. Genes Dev. 2006, 20,
1256–1261. [CrossRef]
11. North, B.J.; Marshall, B.L.; Borra, M.T.; Denu, J.M.; Verdin, E. The Human Sir2 Ortholog, SIRT2, Is an
NAD+-Dependent Tubulin Deacetylase. Mol. Cell 2003, 11, 437–444. [CrossRef]
12. Peck, B.; Chen, C.-Y.; Ho, K.-K.; Di Fruscia, P.; Myatt, S.S.; Coombes, R.C.; Fuchter, M.J.; Hsiao, C.-D.;
Lam, E.W.-F. SIRT Inhibitors Induce Cell Death and p53 Acetylation through Targeting Both SIRT1 and SIRT2.
Mol. Cancer Ther. 2010, 9, 844–855. [CrossRef] [PubMed]
13. Jing,E.; Gesta,S.; Kahn,C.R.SIRT2RegulatesAdipocyteDifferentiationthroughFoxO1Acetylation/Deacetylation.
Cell Metab. 2007, 6, 105–114. [CrossRef] [PubMed]
14. Li, Y.; Matsumori, H.; Nakayama, Y.; Osaki, M.; Kojima, H.; Kurimasa, A.; Ito, H.; Mori, S.; Katoh, M.;
Oshimura, M.; et al. SIRT2 down-regulation in HeLa can induce p53 accumulation via p38 MAPK
activation-dependent p300 decrease, eventually leading to apoptosis. Genes Cells 2011, 16, 34–45. [CrossRef]
[PubMed]
15. Rothgiesser, K.M.; Erener, S.; Waibel, S.; Luscher, B.; Hottiger, M.O. Correction: SIRT2 regulates
NF-kappaB-dependent gene expression through deacetylation of p65 Lys310. J. Cell Sci. 2019, 132,
4251–4258. [CrossRef] [PubMed]
16. Huang, S.; Song, C.; Wang, X.; Zhang, G.; Wang, Y.; Jiang, X.; Sun, Q.; Huang, L.; Xiang, R.; Hu, Y.; et al.
Discovery of New SIRT2 Inhibitors by Utilizing a Consensus Docking/Scoring Strategy and Structure–Activity
Relationship Analysis. J. Chem. Inf. Model. 2017, 57, 669–679. [CrossRef] [PubMed]
17. Dryden, S.C.; Nahhas, F.A.; Nowak, J.E.; Goustin, A.-S.; Tainsky, M.A. Role for Human SIRT2 NAD-Dependent
Deacetylase Activity in Control of Mitotic Exit in the Cell Cycle. Mol. Cell. Biol. 2003, 23, 3173–3185.
[CrossRef] [PubMed]
18. Inoue, T.; Hiratsuka, M.; Osaki, M.; Oshimura, M. The Molecular Biology of Mammalian SIRT Proteins:
SIRT2 Functions on Cell Cycle Regulation. Cell Cycle 2007, 6, 1011–1018. [CrossRef]
19. Machado de Oliveira, R.; Sarkander, J.; Kazantsev, A.; Outeiro, T. SIRT2 as a Therapeutic Target for
Age-Related Disorders. Front. Pharmacol. 2012, 3, 1–9. [CrossRef]
20. Beirowski, B.; Gustin, J.; Armour, S.M.; Yamamoto, H.; Viader, A.; North, B.J.; Michán, S.; Baloh, R.H.;
Golden, J.P.; Schmidt, R.E.; et al. Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through
polarity protein Par-3/atypical protein kinase C (aPKC) signaling. Proc. Natl. Acad. Sci. USA 2011, 108,
E952–E961. [CrossRef]

Molecules 2019, 24, 2724
17 of 18
21. Eskandarian, H.A.; Impens, F.; Nahori, M.-A.; Soubigou, G.; Copp
é
e, J.-Y.; Cossart, P.; Hamon, M.A. A
Role for SIRT2-Dependent Histone H3K18 Deacetylation in Bacterial Infection. Science 2013, 341, 1238858.
[CrossRef] [PubMed]
22. Pais, T.F.; Szego˝, É.M.; Marques, O.; Miller-Fleming, L.; Antas, P.; Guerreiro, P.; de Oliveira, R.M.; Kasapoglu, B.;
Outeiro, T.F. The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain
inflammation. EMBO J. 2013, 32, 2603–2616. [CrossRef] [PubMed]
23. Zhao, T.; Alam, H.B.; Liu, B.; Bronson, R.T.; Nikolian, V.C.; Wu, E.; Chong, W.; Li, Y. Selective Inhibition of
SIRT2 Improves Outcomes in a Lethal Septic Model. Curr. Mol. Med. 2015, 15, 634–641. [CrossRef] [PubMed]
24. Kim, H.-S.; Vassilopoulos, A.; Wang, R.-H.; Lahusen, T.; Xiao, Z.; Xu, X.; Li, C.; Veenstra, T.D.; Li, B.; Yu, H.;
et al. SIRT2 Maintains Genome Integrity and Suppresses Tumorigenesis through Regulating APC/C Activity.
Cancer Cell 2011, 20, 487–499. [CrossRef] [PubMed]
25. Donmez, G.; Outeiro, T.F. SIRT1 and SIRT2: Emerging targets in neurodegeneration. EMBO Mol. Med. 2013
,
5, 344–352. [CrossRef] [PubMed]
26. Luthi-Carter, R.; Taylor, D.M.; Pallos, J.; Lambert, E.; Amore, A.; Parker, A.; Moffitt, H.; Smith, D.L.; Runne, H.;
Gokce, O.; et al. SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis. Proc. Natl.
Acad. Sci. USA 2010, 107, 7927–7932. [CrossRef] [PubMed]
27. Park, S.H.; Zhu, Y.M.; Ozden, O.; Kim, H.S.; Jiang, H.Y.; Deng, C.X.; Gius, D.; Vassilopoulos, A. SIRT2 is a
tumor suppressor that connects aging, acetylome, cell cycle signaling, and carcinogenesis. Transl. Cancer Res.
2012, 1, 15–21. [PubMed]
28. Grozinger, C.M.; Chao, E.D.; Blackwell, H.E.; Moazed, D.; Schreiber, S.L. Identification of a class of small
molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J. Biol.
Chem. 2001, 276, 38837–38843. [CrossRef] [PubMed]
29. Gertz, M.; Fischer, F.; Nguyen, G.T.; Lakshminarasimhan, M.; Schutkowski, M.; Weyand, M.; Steegborn, C.
Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism. Proc. Natl.
Acad. Sci. USA 2013, 110, E2772–E2781. [CrossRef]
30. Zhou, Y.; Cui, H.; Yu, X.; Peng, T.; Wang, G.; Wen, X.; Sun, Y.; Liu, S.; Zhang, S.; Hu, L.; et al. Synthesis
and Evaluation of Novel Benzofuran Derivatives as Selective SIRT2 Inhibitors. Molecules 2017, 22, 1348.
[CrossRef]
31. Outeiro, T.F.; Kontopoulos, E.; Altmann, S.M.; Kufareva, I.; Strathearn, K.E.; Amore, A.M.; Volk, C.B.;
Maxwell, M.M.; Rochet, J.C.; McLean, P.J.; et al. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity
in models of Parkinson’s disease. Science 2007, 317, 516–519. [CrossRef] [PubMed]
32. Pirrie, L.; McCarthy, A.R.; Major, L.L.; Morkunaite, V.; Zubriene, A.; Matulis, D.; Lain, S.; Lebl, T.;
¯ ˙ ˙
Westwood, N.J. Discovery and Validation of SIRT2 Inhibitors Based on Tenovin-6: Use of a 1H-NMR Method
to Assess Deacetylase Activity. Molecules 2012, 17, 12206–12224. [CrossRef] [PubMed]
33. Hoffmann, G.; Breitenbucher, F.; Schuler, M.; Ehrenhofer-Murray, A.E. A Novel Sirtuin 2 (SIRT2) Inhibitor
with p53-dependent Pro-apoptotic Activity in Non-small Cell Lung Cancer. J. Biol. Chem. 2014, 289,
5208–5216. [CrossRef] [PubMed]
34. Taylor, D.M.; Balabadra, U.; Xiang, Z.; Woodman, B.; Meade, S.; Amore, A.; Maxwell, M.M.; Reeves, S.;
Bates, G.P.; Luthi-Carter, R.; et al. A Brain-Permeable Small Molecule Reduces Neuronal Cholesterol by
Inhibiting Activity of Sirtuin 2 Deacetylase. ACS Chem. Biol. 2011, 6, 540–546. [CrossRef] [PubMed]
35. Liu, P.Y.; Xu, N.; Malyukova, A.; Scarlett, C.J.; Sun, Y.T.; Zhang, X.D.; Ling, D.; Su, S.P.; Nelson, C.;
Chang, D.K.; et al. The histone deacetylase SIRT2 stabilizes Myc oncoproteins. Cell Death Differ. 2012, 20,
503–514. [CrossRef] [PubMed]
36. Cui, H.; Kamal, Z.; Ai, T.; Xu, Y.; More, S.S.; Wilson, D.J.; Chen, L. Discovery of Potent and Selective Sirtuin
2 (SIRT2) Inhibitors Using a Fragment-Based Approach. J. Med. Chem. 2014, 57, 8340–8357. [CrossRef]
[PubMed]
37. Schiedel, M.; Rumpf, T.; Karaman, B.; Lehotzky, A.; Gerhardt, S.; Ovádi, J.; Sippl, W.; Einsle, O.; Jung, M.
Structure-Based Development of an Affinity Probe for Sirtuin2. Angew. Chem. Int. Ed. 2016, 55, 2252–2256.
[CrossRef]
38. Yang, L.-L.; Wang, H.-L.; Zhong, L.; Yuan, C.; Liu, S.-Y.; Yu, Z.-J.; Liu, S.; Yan, Y.-H.; Wu, C.; Wang, Y.; et al.
X-ray crystal structure guided discovery of new selective, substrate-mimicking sirtuin 2 inhibitors that
exhibit activities against non-small cell lung cancer cells. Eur. J. Med. Chem. 2018, 155, 806–823. [CrossRef]

Molecules 2019, 24, 2724
18 of 18
39. Yang, L.-L.; Xu, W.; Yan, J.; Su, H.-L.; Yuan, C.; Li, C.; Zhang, X.; Yu, Z.-J.; Yan, Y.-H.; Yu, Y.; et al.
Crystallographic and SAR analyses reveal the high requirements needed to selectively and potently inhibit
SIRT2 deacetylase and decanoylase. MedChemComm 2019, 10, 164–168. [CrossRef]
40. Galleano, I.; Schiedel, M.; Jung, M.; Madsen, A.; Olsen, C. A Continuous, Fluorogenic Sirtuin 2 Deacylase
Assay: Substrate Screening and Inhibitor Evaluation. J. Med. Chem. 2016, 59, 1021–1031. [CrossRef]
41. Yang, L.; Ma, X.; Yuan, C.; He, Y.; Li, L.; Fang, S.; Xia, W.; He, T.; Qian, S.; Xu, Z.; et al. Discovery of
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as new potent and selective human
sirtuin 2 inhibitors. Eur. J. Med. Chem. 2017, 134, 230–241. [CrossRef] [PubMed]
42. Liang, D.; Robinson, E.; Hom, K.; Yu, W.; Nguyen, N.; Li, Y.; Zong, Q.; Wilks, A.; Xue, F. Structure-based
design and biological evaluation of inhibitors of the pseudomonas aeruginosa heme oxygenase (pa-HemO).
Bioorg. Med. Chem. Lett. 2018, 28, 1024–1029. [CrossRef] [PubMed]
43. Yano, J.K.; Denton, T.T.; Cerny, M.A.; Zhang, X.; Johnson, E.F.; Cashman, J.R. Synthetic Inhibitors of
Cytochrome P-450 2A6: Inhibitory Activity, Difference Spectra, Mechanism of Inhibition, and Protein
Cocrystallization. Eur. J. Med. Chem. 2006, 49, 6987–7001. [CrossRef] [PubMed]
44. Finnin, M.S.; Donigian, J.R.; Pavletich, N.P. Structure of the histone deacetylase SIRT2. Nat. Struct. Biol. 2001
,
8, 621–625. [CrossRef] [PubMed]
45. Rumpf, T.; Schiedel, M.; Karaman, B.; Roessler, C.; North, B.J.; Lehotzky, A.; Oláh, J.; Ladwein, K.I.;
Schmidtkunz, K.; Gajer, M.; et al. Selective Sirt2 inhibition by ligand-induced rearrangement of the active
site. Nat. Commun. 2015, 6, 6263. [CrossRef] [PubMed]
Sample Availability: Samples of all the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).