Design, Synthesis, and Biological Evaluation of 8-Mercapto-3,7-Dihydro-1H-Purine-2,6-Diones as Potent Inhibitors of SIRT1, SIRT2, SIRT3, and SIRT5

Article abstract of DOI:10.3390/molecules25122755

Sirtuins (SIRT1-7) are a family of NAD+-dependent deacetylases. They regulate many physiological processes and play important roles in inflammation, diabetes, cancers, and neurodegeneration diseases. Sirtuin inhibitors have potential applications in the treatment of neurodegenerative diseases and various cancers. Herein, we identified new sirtuin inhibitors based on the scaffold of 8-mercapto-3,7-dihydro-1H-purine-2,6-dione. To elucidate the inhibitory mechanism, the binding modes of the inhibitors in SIRT3 were established by molecular docking, showing that the inhibitors occupy the acetyl lysine binding site and interact with SIRT3, mainly through hydrophobic interactions. The interactions were validated by site-directed mutagenesis of SIRT3 and structure-activity relationship analysis of the inhibitors. Consistently, enzyme kinetic assays and microscale thermophoresis showed that these compounds are competitive inhibitors to the acetyl substrate, and mix-type inhibitors to NAD+. Furthermore, we demonstrated that the compounds are potent SIRT1/2/3/5 pan-inhibitors. This study provides novel hits for developing more potent sirtuin inhibitors.

Full text of DOI:10.3390/molecules25122755

Article
Design, Synthesis, and Biological Evaluation of
8-Mercapto-3,7-Dihydro-1H-Purine-2,6-Diones as
Potent Inhibitors of SIRT1, SIRT2, SIRT3, and SIRT5
1,2,†
3,†
4
3
3
4,
Haozhen Han , Chunpu Li , Man Li , Lisheng Yang , Sen Zhao , Zhifei Wang *,
3,
1,2,
Hong Liu * and Dongxiang Liu
*
1
Department of Pharmacology III, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu-Chong-Zhi Road, Shanghai 201203, China; hhaozhen@126.com
College of Pharmacy, University of Chinese Academy of Sciences, No.19A Yuquan Road,
Beijing 100049, China
Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu-Chong-Zhi Road, Shanghai 201203, China; lcp1681993@163.com (C.L.); 15250968801@163.com
(L.Y.); zhaosen@shu.edu.cn (S.Z.)
2
3
4
School of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
liman1451098759@163.com
* Correspondence: zfwang4911@163.com (Z.W.); hliu@simm.ac.cn (H.L.); dxl@mail.shcnc.ac.cn (D.L.);
Tel.: +86-21-5080-6600 (H.L. & D.L.); +86-21-5132-2222 (Z.W.); Fax: +86-21-5080-7088 (H.L.)
† These authors contribute equally to this work
Received: 06 May 2020; Accepted: 06 June 2020; Published: 15 June 2020
+
Abstract: Sirtuins (SIRT1-7) are a family of NAD -dependent deacetylases. They regulate many
physiological processes and play important roles in inflammation, diabetes, cancers, and
neurodegeneration diseases. Sirtuin inhibitors have potential applications in the treatment of
neurodegenerative diseases and various cancers. Herein, we identified new sirtuin inhibitors based
on the scaffold of 8-mercapto-3,7-dihydro-1H-purine-2,6-dione. To elucidate the inhibitory
mechanism, the binding modes of the inhibitors in SIRT3 were established by molecular docking,
showing that the inhibitors occupy the acetyl lysine binding site and interact with SIRT3, mainly
through hydrophobic interactions. The interactions were validated by site-directed mutagenesis of
SIRT3 and structure–activity relationship analysis of the inhibitors. Consistently, enzyme kinetic
assays and microscale thermophoresis showed that these compounds are competitive inhibitors to
+
the acetyl substrate, and mix-type inhibitors to NAD . Furthermore, we demonstrated that the
compounds are potent SIRT1/2/3/5 pan-inhibitors. This study provides novel hits for developing
more potent sirtuin inhibitors.
Keywords: sirtuins; inhibitor; structure–activity relationship; deacetylase
1. Introduction
+
Sirtuins are nicotinamide adenine dinucleotide (NAD )-dependent deacylases that catalyze the
+
deacylation of histones and non-histone proteins with the transformation of NAD into nicotinamide
and 2'-O-acyl-adenosine diphosphate (ADP)-ribose [1]. Human sirtuins include seven family
members, SIRT1–7, which have different subcellular localizations and functions [2]. SIRT1 [3], SIRT6
[4], and SIRT7 [5], which are mostly present in the nucleus, are associated with gene homeostasis,
and regulate post-translational modification of proteins. SIRT2 is a cytoplasmic protein that co-
localizes with microtubules and plays important roles in cell differentiation [6]. SIRT3 [7], SIRT4 [8],
and SIRT5 [9], the mitochondrial sirtuins, participate in the regulation of aging and energy
metabolism. Due to the critical roles of sirtuins in neurodegenerative diseases and different cancers
Molecules 2020, 25, 2755; doi:10.3390/molecules25122755
www.mdpi.com/journal/molecules

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[10], sirtuin inhibitors have potential applications in the treatment of Huntington’s disease and
cancers [11]. This has aroused the interest of research groups around the world to develop sirtuin
small molecule inhibitors.
So far, several sirtuin inhibitors have been reported (Figure 1). Among the inhibitors, salermide
[12], sirtinol [13], cambinol [14], suramin [15], and tenovin-6 [16] are SIRT1 and SIRT2 inhibitors and
exhibited antineoplastic, antiproliferative, or antiviral activities. AGK2 [17], AK-1 [18,19], and
SirReal2 [20] are potent SIRT2 inhibitors and prevent the death of dopaminergic cells, hippocampal
neurodegeneration, or induce cell cycle arrest in human colon carcinoma cells. ELT-31 is the most
potent nanomolar SIRT1/2/3 inhibitor, which was discovered by screening the DNA encoded
compound library [21,22]. Ex527, a SIRT1 inhibitor with IC50 of 60–100 nM, has been evaluated in
clinical trials for the treatment of Huntington’s disease [23].
Figure 1. Sirtuin inhibitors.

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Despite the fact that several sirtuin inhibitors have been reported, most of them have limited or
moderate inhibitory activities. Development of more potent inhibitors is needed, which can be used
as tools to elucidate the biological functions of sirtuins, or as clinical interventions for the related
diseases. In this study, novel sirtuin inhibitors with the scaffold of 8-mercapto-3,7-dihydro-1H-
purine-2,6-dione was discovered through a fluorescent assay. The binding modes of the inhibitors in
SIRT3 were established by molecular docking, which were confirmed by site-directed protein
mutagenesis, structure–activity relationship (SAR) analysis, enzyme kinetic assays, and microscale
thermophoresis (MST). In our study, ELT-31 was used as the control. Compared with ELT-31, the
most potent sirtuin inhibitor ever reported [22], one of our inhibitors, 15, exhibited better activities
than ELT-31 for SIRT1 and SIRT5.
2. Results and Discussion
2.1. Discovery of Sirtuin Inhibitors
To discover new sirtuin inhibitors, we screened our in-house compound library (about 1100
compounds) by using a fluorescent assay. An internally quenched fluorescent peptide (sequence:
Abz-GVLKAcAYNO2GV-NH2; Abz: 2-aminobenzoyl; KAc, acetyl lysine; YNO2, 3-nitro-L-tyrosine) was
used as the substrate [24]. After deacetylation by SIRT3, the substrate was hydrolyzed by trypsin to
produce a fluorescent Abz-peptide (sequence: Abz-GVLK), which can be quantified by measuring
the fluorescence of Abz at 420 nm with the excitation wavelength of 320 nm (Figure 2A). As shown
in Figure 2B, compound 1 inhibited SIRT3 activity with an IC50 of 0.79 μM.
(A)
(B)
Figure 2. Discovery of SIRT3 inhibitors. (A) Principle of the enzyme assay. (B) Chemical structure and
IC50 of compound 1.
2.2. Chemistry
To study the structure–activity relationship of the inhibitor, we designed and synthesized the
derivatives (Table 1: 4, 12–20) of compound 1. The synthesis strategy is outlined in Schemes 1 and 2,
which starts from commercially available diaminouracil derivatives 2a and 2b. Treatment of 2a and
2b, with carbon disulfide as one-carbon equivalent, gave access to a series of 8-thiotheophylline
scaffolds 3a and 3b. Transformation of the key intermediates 3a and 3b to compounds 1 and 4 was
achieved under I2-mediated oxidation.
Condensation of commercially available 1-ethylurea 5 with ethyl-2-cyanoacetate 6 in the
presence of NaOEt afforded 6-amino-1-ethyluracil 7 (Scheme 2). Intermediate 8 was prepared by
using dimethyl formamide dimethyl acetal in MeOH. This step was followed by N-1-alkylation of 8
with alkyl bromides and K2CO3. Deprotection of the N,N-dimethylaminomethylene moiety was

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efficiently achieved to afford 1,3-disubstituted-6-aminouracils 9a–9h. Reaction of 9a–9h with NaNO2
under acidic conditions yielded the desired 5-nitrosouracil derivatives, which were reduced to the
5,6-diaminouracil 10a–10h with sodium dithionite. Transformation of the key intermediates 10a–10h
to the target compounds 12–19 was achieved by the same method described in scheme 1.
Deprotection of the Boc group of compound 17 under trifluoroacetic acid (TFA) gave the desired
product compound 20. All the target compounds were analyzed by HPLC (see Table S1).
Scheme 1. Reagents and conditions: (a) CS2, NaHCO3, EtOH:H2O, 65 °C, overnight; (b) I2, KOH, EtOH,
r.t. to reflux, overnight.
Scheme 2. Reagents and conditions: (a) NaOEt, EtOH, reflux; (b) N,N-Dimethylformamide dimethyl
acetal (DMF-DMA), MeOH, reflux; (c) R-Br, K2CO3, N,N-dimethylformamide (DMF), reflux; (d)
LiOH.H2O, MeOH:H2O, 55 °C; (e) NaNO2, 50% HAc, 70 °C; (f) Na2S2O4, 14% NH4OH, 70 °C; (g) CS2,
NaHCO3, EtOH:H2O, 65 °C, overnight; (h) I2, KOH, EtOH, r.t. to reflux, overnight; (i) For compound
17, TFA/dichloromethane (DCM) (1:4), rt, overnight.
2.3. Structure–Activity Relationship
We determined the inhibitory activities of derivatives 4, 12–20 for SIRT3. As shown in Table 1,
compound 4 was 1.5-fold more potent than compound 1, indicating that introduction of long-chain
alkyls at the R1 position increases the activity. By comparing the activities of 1, 4, and 12, we can see
that groups with branched chains were not tolerated well at R1, suggesting that there is limited space
around the R1 position in SIRT3. Similarly, introducing (tetrahydro-2H-pyran-4-yl)methyl or
(tetrahydrofuran-2-yl)methyl at the R1 position (compounds 13 and 14) decreased the inhibitory
activity compared to 4. In contrast, introduction of cycloalkanes at R1 increased the hydrophobicity
of the compounds (compounds 13 and 14), leading to an enhanced activity (2.5-fold) compared to
compound 12. These indicated that hydrophobic substituent at R1 with a proper conformation may
strengthen the interaction of the inhibitor with SIRT3. Inspired by this result, we tried to further
improve the activity by introducing phenethyl at R1 (compound 15). Notably, the inhibitory activity
for SIRT3 was dramatically enhanced. Compounds 16–18, and 20, which have a fluoro, Boc-protected
amino, methoxy, or amino at the para position of the phenethyl, respectively, exhibited lower
activities. This indicates that no group was tolerant at the para position of phenethyl. In addition,
replacing the phenethyl with N-(3-(trifluoromethyl)phenyl)propionamide (compound 19)

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remarkably reduced the inhibitory activity (23-fold) compared with 15, suggesting that a larger group
at the R1 position may generate steric hindrance with SIRT3.
Table 1. Inhibitory activities of compound 1 and its derivatives for SIRT1/2/3/5/6.
IC50 (μM)
SIRT2
Cmpd
R1
R2
SIRT3
SIRT1
SIRT5
SIRT6
1
4
12
–CH2CH3
–CH2CH2CH3
–CH2CH(CH3)2
–CH2CH3
0.79 ± 0.06 0.10 ± 0.01 1.17 ± 0.04
0.42 ± 0.01
0.39 ± 0.03
1.51 ± 0.10
116.0 ± 6.2
128.7 ± 16.1
296.6 ± 11.3
–CH2CH2CH3 0.54 ± 0.05 0.12 ± 0.01 1.19 ± 0.06
–CH2CH3
1.77 ± 0.05 0.43 ± 0.03 4.86 ± 0.08
13
14
15
16
–CH2CH3
0.72 ± 0.06 0.17 ± 0.01 2.25 ± 0.12
0.50 ± 0.01
0.54 ± 0.02
0.45 ± 0.01
0.84 ± 0.08
155.8 ± 6.5
126.5 ± 10.2
103.3 ± 6.3
110.9 ± 2.6
–CH2CH3
–CH2CH3
–CH2CH3
0.69 ± 0.12 0.15 ± 0.02 1.62 ± 0.05
0.37 ± 0.05 0.17 ± 0.01 1.35 ± 0.05
0.46 ± 0.04 0.19 ± 0.01 2.03 ± 0.04
17
–CH2CH3
1.15 ± 0.08 0.41 ± 0.04 2.19 ± 0.08
0.61 ± 0.03 0.21 ± 0.02 2.16 ± 0.08
1.00 ± 0.03
0.61 ± 0.02
128.3 ± 4.1
18
19
20
–CH2CH3
–CH2CH3
–CH2CH3
102.3 ± 9.1
452.0 ± 9.8
209.6 ± 5.9
8.52 ± 0.22 1.87 ± 0.04 9.44 ± 0.19 26.12 ± 3.15
0.97 ± 0.06 0.49 ± 0.01 4.61 ± 0.13
0.35 ± 0.02 0.27 ± 0.02 0.12 ± 0.01
0.92 ± 0.04
>500
ELT-31 a
462.0 ± 14.9
a the most potent sirtuin inhibitor ever reported [22].
2.4. Binding Mode of the Inhibitor in SIRT3
Molecular docking is a powerful computational technique that has been widely used for
designing bioactive compounds or studying the interactions of the ligand with its receptor. This
technique is also applicable to the structure-unknown receptor or very large binding site with few
hydrophobic domains such as phosphorylated tau or BACE1 [25–27]. To elucidate the interactions of
our inhibitors, we constructed the binding mode of 15 in SIRT3 by using AutoDock4.2 (Figure 3). As
2+
known, sirtuins have a conserved catalytic core containing a Rossmann fold and a Zn -binding
+
domain [28]. The binding sites of the acetyl substrate and NAD are located in the cleft between the
two domains. According to the binding mode, the inhibitor occupied the acetyl-lysine binding site
(Figure 3A). The carbonyl at the C-2 position of the inhibitor formed a hydrogen-bond with the side-
chain guanidine of Arg158 (Figure 3B,3C). This interacted with SIRT3 mainly through hydrophobic
interactions. Specifically, the purines of the inhibitor were surrounded by hydrophobic residues:
Ala146, Phe157, Phe180, Ile230, Phe251, Ile291, Val292, Phe293, and Phe294. The sulfur atom

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interacted with residues His248 via π–sulfur interaction. The benzene groups at both ends of the
compound were inserted into the hydrophobic pockets constituted by Ile230, Leu199, Ile154 or
Val324, Leu298, Ala247, Pro326, and Phe327, respectively. In addition, 15 interacted with residues
Asp156, Glu177, Asp231, Gln228, and Asn229 through van der Waals interactions.
.
(A)
(B)
(C)
(D)
Figure 3. Interactions of compound 15 with SIRT3. (A) Binding site of 15 (yellow) in SIRT3. (B) 15
(yellow) interacts with the residues composed of the acetyl lysine binding site. Dotted lines in
different colors represent various interactions (hydrogen-bond in green, π–π interaction in purple, π–
cation, and π–sulfur interactions in orange, and CH–π interaction in pink). (C) Interaction details of
15 with SIRT3. (D) Binding modes of 15 or SRT1720 in SIRT3: SIRT3 (green) with 15 (yellow), SIRT3
(cyan)/SRT1720 (black)/carba-NAD+ (magenta) (pdb ID: 4BN5). Images were generated with PyMol.
According to the binding mode, the R1 substituent of 15 is surrounded by hydrophobic residues
of Leu199, Ile230, Ala247, Leu298, Val324, and Pro326. Improvement of the hydrophobicity of R1
would increase the inhibitory activity. Consistently, the IC50 of compounds 1, 4, and 15 were 0.79 μM,
0.54 μM, and 0.37 μM, respectively, indicating that replacement of ethyl at R1 with propyl or
phenethyl can enhance the inhibitory activity (interactions of 1, 4, 12–14, 16–20 with SIRT3 shown in
Figure S1). However, introduction of groups with branched chains at R1 such as isobutyl,
(tetrahydro-2H-pyran-4-yl)methyl or (tetrahydrofuran-2-yl)methyl (compounds 12–14), appears to
collide with Ala146, which decreased the activity down to 1.77 μM, 0.72 μM, and 0.69 μM,
respectively. In addition, the introduction of fluoro, Boc-protected amino, methoxyl, or amino groups
at the para-position to the phenethyl of 15 (compounds 16–18, 20) collided with Leu199, causing the
decline of the inhibitory activity. Compound 19, due to the introduction of a large substituent, N-(3-
(trifluoromethyl)phenyl)propionamide at R1, producing steric hindrance with residues Ile154,

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Leu199, Phe302, Pro326, and Phe327, displayed a sharp decline of the inhibitory activity. The binding
mode of 15 may explain the SAR of the derivatives.
2.5. Site-Directed Mutagenesis
To further verify the binding mode of 15, the residues of Phe157, Arg158, Glu177, Phe251, and
Phe294 were selected for single site-directed mutation. The affinity (i.e., Ki) of compound 15 for wild-
type SIRT3 and its mutants was determined by enzyme kinetic assays. The reciprocals of the
maximum reaction velocities were plotted against the concentrations of 15. The Ki is the negative of
the x-intercept of the plot. As shown in Figure 4, the Ki of the mutants were higher than those of the
wild-type SIRT3. These results indicate that these residues play important roles in the interactions of
15.
(A)
(B)
Figure 4. (A) Ki of compound 15 for wild-type SIRT3 and its mutants (E177A, F251A). (B) Ki of 15 for
SIRT3 mutants (F157A, F294L). Ki is the negative of the x-intercept of the plot.
2.6. Mechanism of Inhibition
To identify the mechanism of the inhibition, the inhibition pattern of 15 was determined by
enzyme kinetic analysis. The double-reciprocal plot of the initial reaction velocity (V) versus the
+
concentration of the acetyl peptide or NAD shows that 15 is a competitive inhibitor for the acetyl
+
peptide and a mixed-type inhibitor for NAD (Figure 5A,5B). Increasing the concentration of 15
+
+
reduced Vmax and Km for NAD , which suggested that NAD can promote the binding of 15 to SIRT3.
To confirm this result, we measured the binding affinity of 15 with SIRT3 by microscale
thermophoresis. As shown in Figure 5C, compound 15 exhibited a moderate affinity with Kd of 19.9
± 5.3 μM for SIRT3. Adding 50 μM of the acetyl peptide decreased the affinity of 15 to SIRT3 with Kd
+
> 110 μM, while the addition of 5 mM of NAD increased the affinity of 15 to SIRT3 with Kd of 6.0 ±
1.2 μM. Both enzyme kinetic analysis and MST experiments demonstrated that compound 15
+
competes with the acetyl peptide for binding to SIRT3, whereas NAD enhances the interaction of 15
in SIRT3.
To figure out the mechanism of the inhibition, we compared the binding modes of 15 and
another SIRT3 inhibitor SRT1720. As shown by the superposition of SIRT3/15 structure with that of
+
the SIRT3/SRT1720/carba-NAD complex (pdb ID: 4BN5) [29], several similarities could be observed
between the binding modes of two inhibitors (Figure 3D). Both inhibitors occupied the acetyl-lysine
+
binding site. Additionally, 15 was found in the neighborhood of carba-NAD and may interact with
+
the co-substrate analogue. Thus, binding of NAD can improve the affinity of 15 to SIRT3.

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(A)
(B)
(C)
Figure 5. Inhibition pattern of compound 15. (A) Lineweaver–Burk plot of the velocity of
deacetylation versus the concentration of the acetyl peptide (5.0–100.0 μM) in absence or presence of
0.25 μM, 0.5 μM or 1.0 μM of 15 shows that 15 is a competitive inhibitor for the acetyl peptide. (B)
Lineweaver–Burk plot of the velocity of deacetylation versus the concentration of NAD+ (15.625–500.0
μM) in the absence or presence of 0.125 μM, 0.25 μM, or 0.5 μM of 15 shows that 15 is a mixed-type
inhibitor for NAD+. (C) Microscale thermophoresis to determine the dissociation constant Kd of 15
with SIRT3 in the absence or presence of 5 mM NAD+ or 50 μM acetyl peptide.
2.7. Compounds Are Potent Inhibitors of SIRT1/2/3/5.
In addition to the inhibitory activity of the compounds for SIRT3, we also measured their
inhibitory activities for SIRT1, SIRT2, SIRT5, and SIRT6 (Table 1). In general, these compounds
displayed notable activities for SIRT1, SIRT2, and SIRT5, but not for SIRT6. Compared with other
sirtuins (SIRT1–5) that have a conserved compact acetyl substrate binding pocket, SIRT6 possesses a
larger and wider hydrophobic channel [30], which may contribute to the weak affinities and activities
of the inhibitors for SIRT6.
ELT-31, the most potent sirtuin inhibitor so far, was reported to inhibit SIRT1-3 with an IC50 of
4.3, 1.1, and 7.2 nM, which were determined by measuring the production of 2'-O-acetyl-ADP-ribose

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by mass spectrometry [22]. In our assay, ELT-31 inhibited SIRT1-3 with an IC50 of 0.27, 0.12, and 0.35
μM (Table 1). In contrast, the IC50 of our inhibitor 15 for SIRT1-3 and SIRT5 were 0.17, 1.35, 0.37, and
0.45 μM, respectively, showing that 15 was more potent than ELT-31 for SIRT1 and SIRT5, similar to
ELT-31 for SIRT3.
3. Conclusions
Sirtuins are important deacetylases that regulate metabolism, longevity, DNA repair, apoptosis,
and are involved in diseases such as inflammation, cancer, diabetes, and neurodegeneration. Sirtuin
inhibitors have potential applications in the treatment of neurodegenerative diseases and various
cancers. In this study, we identified novel sirtuin inhibitors based on the scaffold of 8-mercapto-3,7-
dihydro-1H-purine-2,6-dione by a fluorescent assay. To elucidate the inhibitory mechanism, we
established the binding mode of the inhibitor 15 in SIRT3 by molecular docking. The inhibitor
occupied the acetyl-lysine binding site in SIRT3. It interacted with SIRT3 mainly through
hydrophobic interactions and formed a hydrogen-bond with Arg158. The binding mode was verified
by site-directed mutagenesis of SIRT3 and the SAR analysis of the inhibitors. We also determined the
inhibition pattern of 15 by enzyme kinetic analysis, showing that 15 is a competitive inhibitor to the
+
acetyl peptide and a mixed-type inhibitor to NAD . Consistently, MST experiments proved that the
+
acetyl peptide competes 15 for binding to SIRT3 and NAD enhances the affinity of 15 with SIRT3.
Furthermore, we demonstrated that the compounds are potent SIRT1/2/3/5 pan-inhibitors. We also
found that 15 is nontoxic at concentrations up to 20 μM and stable in the assays (see Figures S2 and
S3). This study provides novel hits and structural information for developing potent sirtuin
inhibitors.
4. Experimental Section
4.1. Chemicals and General Methods
Analytical thin layer chromatography (TLC) was HSGF 254 (0.15–0.2 mm thickness). Preparative
thin layer chromatography (PTLC) was HSGF 254 (0.4–0.5 mm thickness). The reagents (chemicals)
were purchased from commercial sources (J&K Scientific Co., Ltd., Beijing, China; TCI Development
Co., Ltd., Shanghai, China; Adamas Reagent, Co., Ltd., Shanghai, China.), and used without further
1
13
purification. All analytical products were characterized by their NMR and MS spectra. H and C-
NMR spectra were recorded on a 400 MHz, 500 MHz, or 600 MHz instrument (Billerica, MA, USA).
Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton
coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), heptet (hept),
multiplet (m), doublet of doublets (dd), and broad (br). Low-resolution mass spectra (LRMS) data
were measured on Agilent 1260 Infinity II (Palo Alto, CA, USA) with electrospray ionization (ESI).
High-resolution mass spectra (HRMS) were measured on Micromass Ultra Q-TOF spectrometer (Palo
Alto, CA, USA).
4.2. Synthesis of Compounds
1,3-Diethyl-8-mercapto-3,7-dihydro-1H-purine-2,6-dione (3a) [31]. To a solution of 2a (0.5 g, 2.52 mmol)
in EtOH/H2O (v/v = 1:2, 12 mL) was added sodium bicarbonate (1.48 g, 17.66 mmol) and CS2 (2 mL,
32.79 mmol). The mixture was stirred overnight under reflux. The mixture was evaporated in
vacuum, and the aqueous solution was acidified to pH 5–6 by using acetic acid. The precipitate
formed was collected by filtration, washed with water, and dried under vacuum to afford compounds
1
3a as a yellow solid (410 mg, 68%). H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) δ 13.44 (br,
1H), 13.00 (br, 1H), 4.04–3.71 (m, 4H), 1.15 (t, J = 7.0 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). LRMS (ESI, m/z):
−
239.0 (M − H) .
8-Mercapto-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione (3b) [32]. Compound 3b was prepared in the
1
same manner as described for compound 3a. H-NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 13.02 (s,

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1H), 3.87–3.68 (m, 4H), 1.64–1.46 (m, 4H), 0.85 (dt, J = 14.8, 7.4 Hz, 6H). LRMS (ESI, m/z): 267.0 (M −
−
H) .
8,8’-Disulfanediylbis(1,3-diethyl-3,7-dihydro-1H-purine-2,6-dione) (1). To a solution of 3a (80 mg, 0.33
mmol) in EtOH was added KOH (18 mg, 0.32 mmol), and the mixture was stirred for 0.5 h at room
temperature. Further addition of I2 (88 mg, 0.35 mmol) at room temperature, and reflux overnight
gave a crude product as a pale yellow powder that was filtered off, washed with H2O, and dried
under vacuum. The crude product was purified by silica gel column (DCM/MeOH, 25/1) to afford 1
1
(64 mg, 80%). Mp = 252–254 °C. H-NMR (400 MHz, DMSO-d6) δ 14.28 (br, 2H), 4.03–3.88 (m, 8H),
13
1.19 (t, J = 7.0 Hz, 6H), 1.12 (t, J = 7.0 Hz, 6H). C-NMR (151 MHz, DMSO-d6) δ 153.49, 149.86, 147.46,
+
144.31, 109.95, 39.92, 39.78, 39.64, 39.50, 39.36, 39.22, 39.08, 38.22, 35.85, 13.08. MS (EI, m/z): 478 [M] ;
+
HRMS (EI) cacld for C18H22O4N8S2 ([M] ): 478.1200; found: 478.1201.
8,8'-Disulfanediylbis(1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione) (4). Compound 4 was prepared in
1
the same manner as described for compound 1 (78 mg, 84%). Mp = 241–243 °C. H-NMR (600 MHz,
DMSO-d6) δ 14.25 (br, 2H), 3.92–3.87 (m, 4H), 3.86–3.82 (m, 4H), 1.66–1.59 (m, 4H), 1.59–1.52 (m, 4H),
13
0.86 (t, J = 7.4 Hz, 6H), 0.82 (t, J = 7.4 Hz, 6H). C-NMR (151 MHz, DMSO-d6) δ 153.66, 150.31, 147.76,
144.27, 109.72, 44.52, 42.22, 39.92, 39.78, 39.64, 39.50, 39.36, 39.22, 39.08, 20.75, 20.73, 11.13, 10.85. MS
+
+
(EI, m/z): 534 [M] ; HRMS (EI) cacld for C22H30O4N8S2 ([M] ): 534.1826; found: 534.1819.
6-Amino-1-ethylpyrimidine-2,4(1H,3H)-dione (7) [33]. To a solution of NaOEt (15.4 g, 226 mmol) in dry
EtOH was added 5 (10 g, 113 mmol) and 6 (12.8 g, 113 mmol), and the mixture was stirred under
reflux for 24 h. The solvent was evaporated under vacuum, and the residue was dissolved in water
(50 mL). The aqueous solution was acidified to pH 7 by using concentrated HCl. The solid formed
was collected by filtration, washed with water, and dried under vacuum to afford 8 g of 7 (45% yield).
1
H-NMR (600 MHz, DMSO-d6) δ 10.33 (s, 1H), 6.81 (s, 2H), 4.53 (s, 1H), 3.77 (q, J = 7.0 Hz, 2H), 1.08 (t,
+
J = 7.1 Hz, 3H). MS (ESI, m/z): 156.0 (M + H) .
(E)-N'-(3-Ethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N,N-dimethyl formimidamide (8) [33]. To a
solution of 7 (1 g, 6.45 mmol) in MeOH was added DMF-DMA (922 mg, 7.73 mmol), and the mixture
was stirred at 50 °C for 10 h. The solvent was evaporated under vacuum, and the residue was
1
recrystallized from EtOH to yield 1.1 g of compound 8. H-NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H),
8.06 (s, 1H), 4.96 (d, J = 2.0 Hz, 1H), 3.90 (q, J = 6.9 Hz, 2H), 3.11 (s, 3H), 2.98 (s, 3H), 1.08 (t, J = 7.0 Hz,
+
3H). LRMS (ESI, m/z): 211.0 (M + H) .
6-Amino-1-ethyl-3-phenethylpyrimidine-2,4(1H,3H)-dione (9a). To a solution of 8 (0.3 g, 1.43 mmol) in
dry DMF was added (2-bromoethyl)benzene (396 mg, 2.14 mmol) and K2CO3 (296 mg, 2.14 mmol),
and the mixture was stirred at 100 °C for 24 h. The reaction mixture was diluted with ethyl acetate
(EA), and the organic layer was washed with water for three times, and dried over anhydrous Na2SO4,
and concentrated under reduced pressure to afford the crude product for the next step without
further purification.
.
Crude product in MeOH:H2O (v/v = 9:1) was added LiOH H2O (180 mg, 4.28 mmol), and then
heated to 55 °C for 12 h. The solvent was acidified to pH 7 by using concentrated HCl and evaporated
under vacuum. The residue was purified by silica gel column (DCM/MeOH, 20/1) to afford 9a (210
1
mg, 57% for 2 steps). H-NMR (400 MHz, DMSO-d6) δ 7.31–7.26 (m, 2H), 7.22–7.15 (m, 3H), 6.96 (s,
13
2H), 4.67 (s, 1H), 3.98–3.76 (m, 4H), 2.78–2.67 (m, 2H), 1.07 (t, J = 7.0 Hz, 3H). C-NMR (151 MHz,
DMSO-d6) δ 161.07, 154.23, 151.02, 138.98, 128.59, 128.37, 126.18, 75.03, 41.09, 37.01, 33.65, 13.14. HRMS
−
(ESI) cacld for C14H16N3O2 ([M − H] ): 258.1248; found: 258.1248.
6-Amino-1-ethyl-3-isobutylpyrimidine-2,4(1H,3H)-dione (9b) [33]. Compound 9b was prepared in the
1
same manner as described for compound 9a. H-NMR (400 MHz, CDCl3) δ 5.25 (s, 2H), 4.99 (s, 1H),
3.93 (q, J = 7.1 Hz, 2H), 3.71 (d, J = 7.4 Hz, 2H), 2.17–2.05 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.87 (d, J = 6.7
13
Hz, 6H). C-NMR (151 MHz, CDCl3) δ 163.48, 153.55, 151.76, 78.40, 47.96, 37.91, 27.26, 20.25, 13.44.
+
LRMS (ESI, m/z): 212.0 (M + H) .

Molecules 2020, 25, 2755
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6-Amino-1-ethyl-3-((tetrahydro-2H-pyran-4-yl)methyl)pyrimidine-2,4(1H, 3H)-dione (9c). Compound 9c
1
was prepared in the same manner as described for compound 9a. H-NMR (500 MHz, DMSO-d6) δ
6.79 (s, 2H), 4.65 (s, 1H), 3.86–3.77 (m, 4H), 3.62 (d, J = 7.2 Hz, 2H), 3.24–3.17 (m, 2H), 1.93–1.82 (m,
13
1H), 1.43–1.36 (m, 2H), 1.24–1.13 (m, 2H), 1.09 (t, J = 7.0 Hz, 3H). C-NMR (126 MHz, DMSO-d6) δ
161.41, 154.09, 151.34, 74.98, 66.65, 44.80, 37.00, 33.61, 30.38, 13.11. HRMS (ESI) cacld for C12H18N3O3
−
([M − H] ): 252.1354; found: 252.1355.
6-Amino-1-ethyl-3-((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione (9d). Compound 9d was
1
prepared in the same manner as described for compound 9a. H-NMR (400 MHz, CDCl3) δ 6.54–6.16
(m, 2H), 4.86 (s, 1H), 4.42–4.32 (m, 1H), 4.22–4.00 (m, 2H), 3.92 (q, J = 7.2 Hz, 1H), 3.81–3.63 (m, 3H),
13
2.12–1.56 (m, 4H), 1.21 (t, J = 7.0 Hz, 3H). C-NMR (101 MHz, CDCl3) δ 163.65, 154.78, 151.93, 76.62,
−
76.55, 67.58, 44.43, 37.68, 29.17, 25.36, 12.73. HRMS (ESI) cacld for C11H16N3O3 ([M − H] ): 238.1197;
found: 238.1191.
3-(4-Amino-3-ethyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)-N-(3-(tri-fluoromethyl)phenyl)propanamide
1
(9e). Compound 9e was prepared in the same manner as described for compound 9a. H-NMR (500
MHz, DMSO-d6) δ 10.34 (s, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.79–7.67 (m, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.37
(dd, J = 7.7, 1.7 Hz, 1H), 6.86 (s, 2H), 4.68 (s, 1H), 4.06–4.00 (m, 2H), 3.82 (q, J = 7.0 Hz, 2H), 2.55 (t, J =
13
8.5, 6.4 Hz, 2H), 1.08 (t, J = 7.0 Hz, 3H). C-NMR (126 MHz, DMSO-d6) δ 169.68, 161.01, 154.21, 151.00,
139.86, 129.81, 129.31 (q, J = 31.5 Hz), 124.10 (q, J = 272.4 Hz), 122.66, 119.47–119.18 (m), 115.38–115.10
+
(m), 75.02, 37.00, 36.39, 35.07, 13.03. HRMS (ESI) cacld for C16H18F3N4O3 ([M + H] ): 371.1326; found:
371.1325.
6-Amino-1-ethyl-3-(4-fluorophenethyl)pyrimidine-2,4(1H,3H)-dione (9f). Compound 9f was prepared in
1
the same manner as described for compound 9a. H-NMR (400 MHz, CDCl3) δ 7.26–7.21 (m, 2H),
7.12–6.86 (m, 2H), 4.97 (s, 1H), 4.35 (s, 2H), 4.15–4.02 (m, 2H), 3.91 (q, J = 7.2 Hz, 2H), 2.88 (q, J = 8.0
13
Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H). C-NMR (126 MHz, DMSO-d6) δ 161.03, 160.84 (d, J = 241.5 Hz),
154.16, 150.98, 135.09 (d, J = 3.2 Hz), 130.36 (d, J = 7.9 Hz), 114.98 (d, J = 21.1 Hz), 75.03, 40.99, 36.97,
−
32.73, 13.07. HRMS (ESI) cacld for C14H15FN3O2 ([M − H] ): 276.1154; found: 276.1161.
6-Amino-1-ethyl-3-(4-methoxyphenethyl)pyrimidine-2,4(1H,3H)-dione (9g). Compound 9g was prepared
1
in the same manner as described for compound 9a. H-NMR (400 MHz, DMSO-d6) δ 7.26–6.73 (m,
13
6H), 4.74–4.59 (m, 1H), 3.99–3.62 (m, 7H), 2.78–2.60 (m, 2H), 1.15–0.99 (m, 3H). C-NMR (151 MHz,
DMSO-d6) δ 161.05, 157.71, 154.12, 150.98, 130.80, 129.79, 129.52, 113.78, 75.07, 54.95, 41.29, 36.98, 32.73,
−
13.12. HRMS (ESI) cacld for C15H18N3O3 ([M − H] ): 288.1354; found: 288.1353.
tert-Butyl(4-(2-(4-Amino-3-ethyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl) ethyl)phenyl)carbamate (9h).
1
Compound 9h was prepared in the same manner as described for compound 9a. H-NMR (400 MHz,
DMSO-d6) δ 9.27 (s, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.12–7.00 (m, 2H), 6.84 (s, 2H), 4.65 (s, 1H), 3.96–3.72
13
(m, 4H), 2.66 (dd, J = 9.2, 6.5 Hz, 2H), 1.46 (s, 9H), 1.08 (t, J = 6.9 Hz, 3H). C-NMR (151 MHz, DMSO-
d6) δ 161.01, 154.10, 152.77, 150.97, 137.67, 132.41, 128.69, 118.14, 78.83, 75.04, 41.14, 36.96, 32.91, 28.14,
−
13.12. HRMS (ESI) cacld for C19H25N4O4 ([M − H] ): 373.1881; found: 373.1879.
5,6-Diamino-1-ethyl-3-phenethylpyrimidine-2,4(1H,3H)-dione (10a). To a solution of 9a (150 mg, 0.58
mmol) in 50% acetic acid was added NaNO2 (120 mg, 1.74 mmol), and the reaction mixture was
stirred at 70 °C for 1.5 h. The mixture was evaporated in vacuum. The precipitate formed was
collected by filtration, washed with water, and dried under vacuum to afford the crude product for
the next step without further purification.
Crude product in 14% aqueous ammonium hydroxide was added to Na2S2O4 (302 mg, 1.74
mmol), and then heated to 70 °C for 1.5 h. The mixture was cooled to room temperature, and then
evaporated under vacuum. The residue was purified by silica gel column (DCM/MeOH, 20/1) to
1
afford 10a (110 mg, 65% for 2 steps). H-NMR (400 MHz, CDCl3) δ 7.35–7.27 (m, 4H), 7.25–7.17 (m,
1H), 5.14–4.91 (m, 2H), 4.25–4.05 (m, 2H), 3.93 (q, J = 7.2 Hz, 2H), 3.00–2.83 (m, 2H), 2.47–2.13 (m, 3H),
13
1.29 (td, J = 7.2, 2.6 Hz, 3H). C-NMR (101 MHz, CDCl3) δ 161.55, 150.07, 148.80, 139.00, 129.12, 128.52,

Molecules 2020, 25, 2755
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−
126.43, 95.59, 42.99, 38.40, 34.29, 13.63. HRMS (ESI) cacld for C14H17N4O2 ([M − H] ): 273.1357; found:
273.1349.
5,6-Diamino-1-ethyl-3-isobutylpyrimidine-2,4(1H,3H)-dione (10b) [33]. Compound 10b was prepared in
1
the same manner as described for compound 10a. H-NMR (400 MHz, CDCl3) δ 4.98 (s, 2H), 3.94 (q,
J = 7.2 Hz, 2H), 3.76 (d, J = 7.4 Hz, 2H), 2.34 (s, 2H), 2.11 (dp, J = 13.9, 7.0 Hz, 1H), 1.30 (t, J = 7.2 Hz,
13
3H), 0.89 (d, J = 6.7 Hz, 6H). C-NMR (101 MHz, CDCl3) δ 162.01, 150.45, 148.54, 95.62, 48.41, 38.40,
+
27.29, 20.26, 13.64. MS (ESI, m/z): 227.0 (M + H) .
5,6-Diamino-1-ethyl-3-((tetrahydro-2H-pyran-4-yl)methyl)pyrimidine-2,4 (1H,3H)-dione (10c). Compound
1
10c was prepared in the same manner as described for compound 10a. H-NMR (400 MHz, CDCl3) δ
5.07 (s, 2H), 4.01–3.88 (m, 4H), 3.83 (d, J = 7.1 Hz, 2H), 3.31 (td, J = 11.7, 2.3 Hz, 2H), 2.32 (s, 2H), 2.01
(ddq, J = 14.7, 7.2, 3.6 Hz, 1H), 1.56–1.48 (m, 2H), 1.40 (dtd, J = 13.3, 11.6, 4.4 Hz, 2H), 1.29 (t, J = 7.2
13
Hz, 3H). C-NMR (101 MHz, CDCl3) δ 161.88, 150.42, 148.77, 95.40, 67.74, 46.64, 38.45, 34.20, 30.79,
−
13.59. HRMS (ESI) cacld for C12H19N4O3 ([M − H] ): 267.1463; found: 267.1456.
5,6-Diamino-1-ethyl-3-((tetrahydrofuran-2-yl)methyl)pyrimidine-2,4(1H, 3H)-dione (10d). Compound 10d
1
was prepared in the same manner as described for compound 10a. H-NMR (400 MHz, CDCl3) δ 5.23–
5.11 (m, 2H), 4.34 (dq, J = 10.8, 5.5 Hz, 1H), 4.18 (dd, J = 13.1, 8.9 Hz, 1H), 4.04–3.67 (m, 5H), 2.64 (s,
13
2H), 2.07–1.78 (m, 3H), 1.66 (ddt, J = 10.4, 8.3, 4.3 Hz, 1H), 1.34–1.21 (m, 3H). C-NMR (101 MHz,
CDCl3) δ 161.63, 150.42, 148.82, 76.18, 67.88, 44.81, 38.42, 29.27, 25.37, 13.51. HRMS (ESI) cacld for
−
C11H17N4O3 ([M − H] ): 253.1306; found: 253.1303.
3-(4,5-Diamino-3-ethyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)-N-(3
(trifluoromethyl)phenyl)propanamide (10e). Compound 10e was prepared in the same manner as
1
described for compound 10a. H-NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.06 (d, J = 2.5 Hz, 1H),
7.79–7.69 (m, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 6.23 (s, 2H), 4.10 (t, J = 7.4 Hz, 2H),
13
3.88 (q, J = 7.0 Hz, 2H), 2.93 (s, 2H), 2.57 (t, J = 7.5 Hz, 2H), 1.09 (t, J = 6.9 Hz, 3H). C-NMR (101 MHz,
DMSO-d6) δ 170.09, 158.99, 149.58, 144.58, 140.28, 130.27, 129.78 (q, J = 31.5 Hz), 124.55 (q, J = 273.1
Hz), 123.12, 119.94–119.64 (m), 115.88–115.56 (m), 96.37, 37.85, 37.40, 35.49, 13.61. HRMS (ESI) cacld
+
for C16H19F3N5O3 ([M + H] ): 386.1435; found: 386.144.
5,6-Diamino-1-ethyl-3-(4-fluorophenethyl)pyrimidine-2,4(1H,3H)-dione (10f). Compound 10f was
1
prepared in the same manner as described for compound 10a. H-NMR (400 MHz, CDCl3) δ 7.25–7.19
(m, 2H), 7.01–6.91 (m, 2H), 5.00 (s, 2H), 4.16–4.05 (m, 2H), 3.92 (q, J = 7.2 Hz, 2H), 2.91–2.82 (m, 2H),
13
2.26 (s, 2H), 1.29 (t, J = 7.2 Hz, 3H). C-NMR (101 MHz, CDCl3) δ 161.68 (d, J = 243.9 Hz), 161.52,
150.06, 148.83, 134.64 (d, J = 3.0 Hz), 130.52 (d, J = 7.9 Hz), 115.26 (d, J = 21.1 Hz), 95.59, 42.90, 38.41,
−
33.43, 13.61. HRMS (ESI) cacld for C14H16FN4O2 ([M − H] ): 291.1263; found: 291.1259.
5,6-Diamino-1-ethyl-3-(4-methoxyphenethyl)pyrimidine-2,4(1H,3H)-dione (10g). Compound 10g was
1
prepared in the same manner as described for compound 10a. H-NMR (400 MHz, DMSO-d6) δ 7.13–
7.05 (m, 2H), 6.93–6.73 (m, 2H), 6.23 (s, 2H), 3.99–3.81 (m, 4H), 3.71 (s, 3H), 3.00 (s, 2H), 2.77–2.64 (m,
13
2H), 1.09 (t, J = 6.9 Hz, 3H). C-NMR (101 MHz, DMSO-d6) δ 158.62, 157.75, 149.13, 144.11, 130.79,
−
129.56, 113.84, 95.95, 54.99, 41.93, 37.42, 32.80, 13.32. HRMS (ESI) cacld for C15H19N4O3 ([M − H] ):
303.1463; found: 303.1456.
tert-Butyl(4-(2-(4,5-Diamino-3-ethyl-2,6-dioxo-3,6-dihydropyrimidin-1
(10h). Compound 10h was prepared in the same manner as described for compound 10a. H-NMR
(500 MHz, CDCl3) δ 7.27 (d, J = 7.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 6.56 (s, 1H), 4.99 (s, 2H), 4.15−4.03
(2H)-yl)ethyl)phenyl)carbamate
1
13
(m, 2H), 3.92 (q, J = 7.3 Hz, 2H), 2.90−2.77 (m, 2H), 2.19 (s, 2H), 1.50 (s, 9H), 1.29 (t, J = 7.2 Hz, 3H). C-
NMR (126 MHz, CDCl3) δ 161.63, 153.04, 150.10, 148.84, 136.76, 133.73, 129.63, 118.94, 95.68, 80.51,
+
43.03, 38.43, 33.59, 28.49, 13.62. HRMS (ESI) cacld for C19H28N5O4 ([M + H] ): 390.2136; found: 390.2131.
4-Ethyl-8-mercapto-1-phenethyl-3,7-dihydro-1H-purine-2,6-dione (11a). Compound 11a was prepared in
1
the same manner as described for compound 3a. H-NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H),
7.43−7.08 (m, 5H), 4.08−3.98 (m, 2H), 3.90 (q, J = 7.0 Hz, 2H), 2.91−2.71 (m, 2H), 1.13 (t, J = 7.0 Hz, 3H).

Molecules 2020, 25, 2755
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13
C-NMR (101 MHz, DMSO-d6) δ 150.98, 149.49, 138.70, 128.66, 128.45, 126.32, 41.82, 39.03, 33.46, 13.20.
−
HRMS (ESI) cacld for C15H15N4O2S ([M − H] ): 315.0921; found: 315.0919.
3-Ethyl-1-isobutyl-8-mercapto-3,7-dihydro-1H-purine-2,6-dione (11b). Compound 11b was prepared in
1
the same manner as described for compound 3a. H-NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 3.92
(q, J = 7.0 Hz, 2H), 3.66 (d, J = 7.4 Hz, 2H), 2.00 (hept, J = 6.9 Hz, 1H), 1.15 (t, J = 7.0 Hz, 3H), 0.82 (d, J
13
= 6.7 Hz, 6H). C-NMR (126 MHz, DMSO-d6) δ 151.88, 149.71, 47.31, 39.20, 26.60, 19.91, 13.10. HRMS
−
(ESI) cacld for C11H15N4O2S ([M − H] ): 267.0921; found: 267.0922.
3-Ethyl-8-mercapto-1-((tetrahydro-2H-pyran-4-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione
Compound 11c was prepared in the same manner as described for compound 3a. H-NMR (400 MHz,
(11c).
1
DMSO-d6) δ 13.40 (s, 1H), 12.40 (s, 1H), 3.91 (q, J = 7.0 Hz, 2H), 3.85–3.65 (m, 4H), 3.26–3.11 (m, 2H),
13
1.97−1.90 (m, 1H), 1.50−1.34 (m, 2H), 1.22 (td, J = 12.2, 4.2 Hz, 2H), 1.15 (t, J = 7.0 Hz, 3H). C-NMR
(101 MHz, DMSO-d6) δ 149.97, 66.69, 45.49, 38.82, 33.61, 30.34, 13.22. HRMS (ESI) cacld for
−
C13H17N4O2S ([M − H] ): 309.1027; found: 309.1028.
3-Ethyl-8-mercapto-1-((tetrahydrofuran-2-yl)methyl)-3,7-dihydro-1H- purine-2,6-dione (11d). Compound
1
11d was prepared in the same manner as described for compound 3a. H-NMR (400 MHz, DMSO-d6)
δ 13.44 (s, 1H), 13.03 (s, 1H), 4.10 (dd, J = 9.4, 4.0 Hz, 1H), 4.02–3.84 (m, 3H), 3.79–3.66 (m, 2H), 3.66–
13
3.55 (m, 1H), 1.94–1.71 (m, 3H), 1.67–1.52 (m, 1H), 1.15 (t, J = 7.0 Hz, 3H). C-NMR (101 MHz, DMSO-
d6) δ 164.13, 151.43, 149.51, 138.78, 103.77, 75.01, 66.82, 43.89, 39.57, 28.67, 24.79, 13.18. HRMS (ESI)
−
cacld for C12H15N4O3S ([M − H] ): 295.087; found: 295.0871.
3-(3-Ethyl-8-mercapto-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)-N-(3-
(trifluoromethyl)phenyl)propanamide (11e). Compound 11e was prepared in the same manner as
1
described for compound 3a. H-NMR (600 MHz, DMSO-d6) δ 13.46 (s, 1H), 12.99 (s, 1H), 10.31 (s, 1H),
8.05 (s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 4.15 (t, J = 7.3 Hz,
13
2H), 3.90 (q, J = 7.1 Hz, 2H), 2.60 (t, J = 7.4 Hz, 2H), 1.12 (t, J = 7.0 Hz, 3H). C-NMR (151 MHz, DMSO-
d6) δ 169.51, 149.38, 139.80, 129.87, 129.34 (q, J = 31.5 Hz), 124.10 (q, J = 272.2 Hz), 122.68, 119.61–119.26
+
(m), 115.54–114.81 (m), 39.31, 37.32, 34.82, 13.02. HRMS (ESI) cacld for C17H17F3N5O3S ([M + H] ):
428.0999; found: 428.1007.
3-Ethyl-1-(4-fluorophenethyl)-8-mercapto-3,7-dihydro-1H-purine-2,6-dione (11f). Compound 11f was
1
prepared in the same manner as described for compound 3a. H-NMR (400 MHz, DMSO-d6) δ 13.44
(s, 1H), 13.03 (s, 1H), 4.10 (dd, J = 9.4, 4.0 Hz, 1H), 4.02–3.84 (m, 3H), 3.79–3.66 (m, 2H), 3.66–3.55 (m,
13
1H), 1.94–1.71 (m, 3H), 1.67–1.52 (m, 1H), 1.15 (t, J = 7.0 Hz, 3H). C-NMR (101 MHz, DMSO-d6) δ
164.13, 151.43, 149.51, 138.78, 103.77, 75.01, 66.82, 43.89, 39.57, 28.67, 24.79, 13.18. HRMS (ESI) cacld
−
for C12H15N4O3S ([M − H] ): 295.087; found: 295.0871.
3-Ethyl-8-mercapto-1-(4-methoxyphenethyl)-3,7-dihydro-1H-purine-2,6-dione (11g). Compound 11g was
1
prepared similarly as described for compound 3a. H-NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 13.02
(s, 1H), 7.13–7.05 (m, 2H), 6.90–6.75 (m, 2H), 4.04–3.82 (m, 4H), 3.70 (s, 3H), 2.73 (t, J = 7.7 Hz, 2H),
13
1.13 (t, J = 7.0 Hz, 3H). C-NMR (101 MHz, DMSO) δ 164.16, 157.82, 151.13, 149.21, 138.88, 130.39,
−
129.63, 113.85, 103.79, 54.99, 42.17, 39.45, 32.45, 13.16. HRMS (ESI) cacld for C16H17N4O3S ([M − H] ):
345.1027; found: 345.1022.
tert-Butyl(4-(2-(3-Ethyl-8-mercapto-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)ethyl)phenyl)carbamate
1
(11h). Compound 11h was prepared similarly as described for compound 3a. H-NMR (500 MHz,
DMSO-d6) δ 12.98 (s, 1H), 9.22 (s, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 4.02–3.95 (m,
13
2H), 3.91 (q, J = 7.0 Hz, 2H), 2.77–2.67 (m, 2H), 1.46 (s, 9H), 1.14 (t, J = 7.0 Hz, 3H). C-NMR (126 MHz,
DMSO-d6) δ 164.13, 152.74, 151.09, 149.17, 138.77, 137.77, 131.98, 128.72, 118.18, 103.75, 78.83, 41.98,
+
39.40, 32.60, 28.11, 13.08. HRMS (ESI) cacld for C20H26N5O4S ([M + H] ): 432.17; found: 432.1708.
8,8'-Disulfanediylbis(3-ethyl-1-isobutyl-3,7-dihydro-1H-purine-2,6-dione) (12). Compound 12 was
1
prepared similarly as described for compound 1 (30 mg, 46%). Mp = 240–242 °C. H-NMR (400 MHz,
DMSO-d6) δ 14.29 (br, 2H), 4.01 (q, J = 7.0 Hz, 4H), 3.76 (d, J = 7.3 Hz, 4H), 2.14–2.00 (m, 2H), 1.24–1.18
13
(m, 6H), 0.90–0.86 (m, 12H). C-NMR (151 MHz, CDCl3) δ 155.27, 150.68, 148.25, 146.70, 110.06, 77.37,

Molecules 2020, 25, 2755
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+
77.16, 76.95, 48.76, 39.69, 27.46, 20.30, 13.54. MS (EI, m/z): 534 [M] ; HRMS (EI) cacld for C22H30O4N8S2
+
([M] ): 534.1826; found: 534.1816.
8,8'-Disulfanediylbis(3-ethyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-3,7- dihydro-1H-purine-2,6-dione) (13).
Compound 13 was prepared similarly as described for compound 1 (36 mg, 50%). Mp = 229–231 °C.
1
H-NMR (500 MHz, DMSO-d6) δ 3.98 (q, J = 7.0 Hz, 4H), 3.84–3.74 (m, 8H), 3.21 (td, J = 11.7, 2.2 Hz,
13
4H), 1.97–1.88 (m, 2H), 1.51–1.40 (m, 4H), 1.30–1.13 (m, 10H). C-NMR (151 MHz, CDCl3) δ 155.21,
150.67, 148.30, 146.95, 109.98, 77.37, 77.16, 76.95, 67.71, 47.01, 39.76, 34.34, 30.84, 13.58. MS (ESI, m/z):
−
−
617.4 [M−H] ; HRMS (ESI) cacld for C26H33O6N8S2 ([M − H] ): 617.197; found: 617.1958.
8,8'-Disulfanediylbis(3-ethyl-1-((tetrahydrofuran-2-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione)
(14).
Compound 14 was prepared similarly as described for compound 1 (21 mg, 53%). Mp = 166–168 °C.
1
H-NMR (400 MHz, DMSO-d6) δ 4.19–4.10 (m, 2H), 4.07–3.96 (m, 5H), 3.82–3.70 (m, 5H), 3.64–3.53 (m,
13
4H), 1.93–1.83 (m, 4H), 1.82–1.74 (m, 2H), 1.69–1.57 (m, 2H), 1.24–1.14 (m, 6H). C-NMR (151 MHz,
CDCl3) δ 154.87, 150.62, 148.12, 146.57, 109.99, 77.37, 77.16, 76.95, 76.16, 67.95, 45.02, 39.69, 29.37, 25.35,
+
+
13.52. MS (EI, m/z): 590 [M] ; HRMS (EI) cacld for C24H30O6N8S2 ([M] ): 590.1724; found: 590.1733.
8,8'-Disulfanediylbis(3-ethyl-1-phenethyl-3,7-dihydro-1H-purine-2,6-dione) (15). Compound 15 was
1
prepared similarly as described for compound 1 (45 mg, 53%). Mp = 225–227 °C. H-NMR (600 MHz,
DMSO-d6) δ 14.26 (br, 2H), 7.35–7.09 (m, 10H), 4.20–3.88 (m, 8H), 2.93–2.71 (m, 4H), 1.22–1.12 (m, 6H).
13
C-NMR (126 MHz, DMSO-d6) δ 153.55, 149.95, 147.31, 144.48, 138.53, 128.56, 128.36, 126.26, 110.00,
+
+
41.92, 38.27, 33.42, 12.97. MS (EI, m/z): 630 [M] ; HRMS (EI) cacld for C30H30O4N8S2 ([M] ): 630.1826;
found: 630.1821.
8,8'-Disulfanediylbis(3-ethyl-1-(4-fluorophenethyl)-3,7-dihydro-1H- purine-2,6-dione) (16). Compound 16
1
was prepared similarly as described for compound 1 (20 mg, 33%). Mp = 216–218 °C. H-NMR (400
MHz, DMSO-d6) δ 14.28 (br, 2H), 7.28–7.19 (m, 4H), 7.15–7.06 (m, 4H), 4.12–4.05 (m, 4H), 3.98 (q, J =
13
6.9 Hz, 4H), 2.84 (t, J = 7.6 Hz, 4H), 1.17 (t, J = 7.0 Hz, 6H). C-NMR (151 MHz, DMSO-d6) δ 160.90 (d,
J = 241.7 Hz), 153.58, 149.97, 147.45, 144.65, 134.74 (d, J = 3.1 Hz), 130.45 (d, J = 8.1 Hz), 115.08 (d, J =
-
21.1 Hz), 109.99, 41.92, 38.29, 32.59, 12.99. MS (ESI, m/z): 665.4 [M-H] ; HRMS (ESI) cacld for
−
C30H27F2N8O4S2 ([M − H] ): 665.157; found: 665.1561.
di-tert-Butyl(((Disulfanediylbis(3-ethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purine-8,1-diyl))bis(ethane-2,1-
diyl))bis(4,1-phenylene))dicarbamate (17). Compound 17 was prepared similarly as described for
1
compound 1 (29 mg, 29%). Mp = 168–171 °C. H-NMR (400 MHz, DMSO-d6) δ 9.26 (s, 2H), 7.36 (d, J =
8.2 Hz, 4H), 7.09 (d, J = 8.2 Hz, 4H), 4.15–3.92 (m, 8H), 2.76 (t, J = 7.8 Hz, 4H), 1.46 (s, 18H), 1.18 (t, J =
13
7.0 Hz, 6H). C-NMR (126 MHz, DMSO-d6) δ 154.42, 153.25, 150.55, 147.95, 145.92, 138.27, 132.60,
−
129.23, 118.69, 111.41, 79.33, 55.38, 42.46, 38.81, 33.31, 28.61, 13.53. MS (ESI, m/z): 859.5 [M − H] ; HRMS
−
(ESI) cacld for C40H47N10O8S2 ([M − H] ): 859.3025; found: 859.3033.
8,8'-Disulfanediylbis(3-ethyl-1-(4-methoxyphenethyl)-3,7-dihydro-1H- purine-2,6-dione) (18). Compound
1
18 was prepared similarly as described for compound 1 (55 mg, 66%). Mp = 216–219 °C. H-NMR (500
MHz, DMSO-d6) δ 7.13 (d, J = 8.6 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 4.09–3.93 (m, 8H), 3.71 (s, 6H), 2.81–
13
2.70 (m, 4H), 1.18 (t, J = 7.1 Hz, 6H). C-NMR (126 MHz, DMSO-d6) δ 157.76, 154.47, 150.19, 147.60,
−
130.62, 129.58, 113.90, 113.82, 54.95, 42.01, 38.37, 32.72, 13.13. MS (ESI, m/z): 689.4 [M − H] ; HRMS
−
(ESI) cacld for C32H33N8O6S2 ([M − H] ): 689.197; found: 689.1963.
3,3'-(Disulfanediylbis(3-ethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purine-8,1-diyl))bis(N-(3-
(trifluoromethyl)phenyl)propanamide) (19). Compound 19 was prepared in the same manner as
1
described for compound 1 (31 mg, 40%). Mp = 174–175 °C. H-NMR (500 MHz, DMSO-d6) δ 10.32 (s,
2H), 8.06 (d, J = 2.0 Hz, 2H), 7.76–7.71 (m, 2H), 7.57–7.51 (m, 2H), 7.38 (d, J = 7.7 Hz, 2H), 4.23 (t, J = 7.3
13
Hz, 4H), 4.01 (q, J = 7.4 Hz, 4H), 2.65 (t, J = 7.3 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H). C-NMR (126 MHz,
DMSO-d6) δ 169.57, 154.07, 150.15, 148.48, 147.55, 139.82, 129.84, 129.34 (q, J = 31.6 Hz), 124.10 (q, J =
−
272.2 Hz), 122.71, 119.41, 115.29, 111.16, 38.35, 37.34, 35.02, 12.98. MS (ESI, m/z): 851.4 [M − H] ; HRMS
−
(ESI) cacld for C34H29F6N10O6S2 ([M − H] ): 851.1623; found: 851.1622.

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8,8'-Disulfanediylbis(1-(4-aminophenethyl)-3-ethyl-3,7-dihydro-1H-purine-2,6-dione) (20). To a solution of
17 (20 mg, 0.33 mmol) in DCM (4 mL) was added TFA (1 mL), and the mixture was stirred for 12 h at
room temperature. After completion, the reaction mixture was diluted with saturated Na2CO3
solution, extracted with DCM, dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude mixture was purified by silica gel column (DCM/MeOH, 25/1) to afford 20 (9 mg,
1
59%). Mp = 179–182 °C. H-NMR (400 MHz, DMSO-d6) δ 7.12 (d, J = 7.8 Hz, 4H), 6.92 (d, J = 7.7 Hz,
13
4H), 4.13–3.93 (m, 8H), 2.88–2.64 (m, 4H), 1.17 (t, J = 7.0 Hz, 6H). C-NMR (101 MHz, DMSO-d6) δ
153.64, 150.01, 147.36, 144.37, 137.83, 132.65, 129.58, 119.05, 110.12, 42.13, 38.36, 32.83, 13.06. MS (ESI,
−
−
m/z): 659 [M − H] ; HRMS (ESI) cacld for C30H31N10O4S2 ([M − H] ): 659.1977; found: 659.1967.
4.3. Protein Expression and Purification
Human SIRT3 (118-399aa) with a tobacco etch virus (TEV) cleavage site and an N-terminal
hexahistidine (His6) tag was expressed in E. coli BL21 cells. Cells were grown in LB medium
containing 100 μg/mL ampicillin at 37 °C until the A600 reached 0.6–0.8. A total of 100 μM isopropyl
1-β-D-galactopyranoside (IPTG) was then added to the culture and the protein expression was
induced at 16 °C overnight. Cells were harvested by centrifugation and the pellet was lysed in lysis
buffer (25 mM Hepes, pH 7.5, 200 mM NaCl, 5% (v/v) glycerol) and loaded with Ni–NTA. The resin
was washed with 40 mM imidazole and then eluted with lysis buffer containing 250 mM imidazole.
Then, the eluted protein was digested with TEV protease at 4 °C and reloaded onto the Ni–NTA
column to remove the His tag. The untagged SIRT3 protein was loaded to Superdex 75 (GE
Healthcare) in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl. The SIRT3 protein was
collected for further use. The mutants were prepared the same as the wild-type SIRT3.
Human SIRT1 (183-505-(GGGS)2-641-665) was expressed in E. coli BL21 cells with a small
ubiquitin-related modifier (sumo)-cleavage site and an N-terminal hexahistidine (His6) tag [34]. Cells
were grown in LB medium containing 50 μg/mL kanamycin at 37 °C until the A600 reached 0.6–0.8.
Then, 300 μM IPTG was added to the cell culture and the protein was induced at 16 °C overnight.
Cells were harvested by centrifugation and lysed in lysis buffer (20 mM Tris (pH 8.0), 500 mM NaCl,
5% (v/v) glycerol) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and loaded with Ni–
NTA. The resin was washed with 20 mM imidazole and then the protein was eluted with 250 mM
imidazole. The purity of the elution was confirmed by SDS−PAGE analysis, then the protein was
dialyzed in a buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, and 5% (v/v) glycerol.
Human SIRT2/5/6 proteins were prepared as previously reported. [35–37]
4.4. Molecular Modeling
Docking of inhibitor 15 in SIRT3 was performed by using AutoDock4.2. The structures of 15
were optimized by energy minimization with a total of 200 steps until the energy difference was less
than 0.1. The PDBQT format files of 15 and SIRT3 were prepared with AutoDock Tools 1.5.6. The grid
box of 40 × 40 × 40 grid size with a spacing of 0.375 Å was centered at the acetyl-lysine binding pocket.
The Lamarckian genetic algorithm procedure was employed while the maximum number of
evaluations was set to 2,500,000, and the maximum number of generations was set to 27,000. The rate
of gene mutation and crossover were set to 0.02 and 0.80, respectively. The other parameters were set
as default values. The solutions were clustered into groups with root-mean-square deviation (RMSD)
lower than 0.5 Å . AutoDock results were analyzed based on the lowest binding energy of the
conformations [38]. The structure with a low RMSD and low energy was selected for further
verification according to the structure–activity relationship.
4.5. Screening for Sirtuin Inhibitor
All compounds were dissolved in 10 mM stock solution in DMSO. Reactions were performed in
the assay buffer (25 mM Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2) containing 1.0
+
μM SIRT3, 10 μM Abz-GVLK(Ac)AY(NO2)GV-NH2, and 500 μM NAD . The reactions were
performed at 37 °C for 30 mins, and then terminated by adding 10 mM nicotinamide, and developed

Molecules 2020, 25, 2755
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with 0.01 mg/mL trypsin for 15 mins. The fluorescence was measured using a microplate reader
(SpectraMax M5, Molecular Devices, San Jose, CA, USA) with excitation at 320 nm and emission at
420 nm. All reactions were done in triplicate.
IC50 were determined for other sirtuins using the same assay as for SIRT3. The concentrations of
SIRT1, SIRT2, SIRT5, and SIRT6 were set at 0.8 μM, 1.4 μM, 0.75 μM, and 1.3 μM to attain the same
+
+
enzymatic activity as SIRT3. The concentrations of NAD were set at ~32.5-fold of Km for NAD
against sirtuins (SIRT1, 1.2 mM; SIRT2, 450 μM; SIRT5, 710 μM; SIRT6, 500 μM), and the
concentrations of the peptide substrates were set to ~2.5-fold of Km for peptide substrates against
sirtuins (SIRT1, 10 μM acetyl-peptide; SIRT2, 8.5 μM acetyl-peptide; SIRT5, 2 μM succinyl-peptide;
SIRT6, 50 μM acetyl-peptide).
4.6. Determination of the Inhibition Pattern of 15
To identify the mechanism of the inhibition, the inhibition pattern of 15 was determined by
+
enzyme kinetic analysis. The assay was carried out using 0.5 μM SIRT3, 500 μM NAD , and 0.01
mg/mL trypsin with variable peptide concentrations (5.0, 10.0, 20.0, 50.0, 75.0, 100.0 μM peptide
sequence: Abz-GVLK(Ac)AY(NO2)GV-NH2). The concentrations of compound 15 was set at 1.0, 0.5,
0.25, and 0 μM. The double-reciprocal plot of V versus [peptide] (where V is the initial reaction
velocity and [peptide] is the concentration of the acetyl peptide) was analyzed. The inhibition type of
+
NAD by compound 15 was examined in a similar manner. The concentrations of SIRT3 and peptide
+
were set at 0.5 μM and 30 μM, respectively. The concentrations of NAD were set at 15.625, 31.25,
62.5, 125, 250, and 500 μM, and the concentrations of compound 15 were set at 0.5, 0.25, 0.125, and 0
+
+
+
μM. The double-reciprocal plot of V vs. [NAD ] ([NAD ] is the concentration of NAD ) was analyzed.
4.7. Microscale Thermophoresis
Microscale thermophoresis was performed on a Monolith NT.115 instrument (NanoTemper
Technologies) according to the protocol [39]. SIRT3 was labeled by incubating with a 10-fold molar
excess of Cy5 (GE Healthcare) in buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05% (v/v)
Tween-20) for 12–16 hours at 4 °C. Free dye was removed by dialysis and size-exclusion column
(Superdex 75, GE Healthcare). The labeled SIRT3 was then supplemented with Bio-Beads SM-2
Adsorbent (catalog no. 152-3920, Bio-Rad Laboratories Inc., Hercules, CA, USA) to further remove
the free dye. Cy5-labeled SIRT3 was mixed well with serial concentrations of compound 15 and
+
thermophoresis was detected in the presence or absence of 50 μM acetyl peptide or 5 mM NAD .
Experiment settings: before MST time 3 s, MST-On time 20 s, after MST time 1 s. All measurements
were done independently at least twice.
Supplementary Materials: The following are available online, Table S1: HPLC analyses of all the target
compounds, Figure S1: 2D schematic representation of the interactions of the compounds with SIRT3, Figure S2:
MTT assays to evaluate the toxicity of compounds 4 and 15, Figure S3: The stability of the compounds.
Author Contributions: Manuscript writing, H.H., C.L., and D.L.; Biological work, H.H. and M.L.; Chemical
work, C.L., L.Y., and S.Z.; Design and supervise the study, D.L., H.L., and Z.W. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Fund of China (Grant 21672233, 81973239,
81620108027, 21632008, 91229204, 81220108025, and 81602975).
Acknowledgments: MST experiments were done by using the Monolith NT.115 instrument at the National
Facility for Protein Science in Shanghai.
Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2020, 25, 2755
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Abbreviations
+
NAD
ADP
nicotinamide adenine dinucleotide
adenosine diphosphate
dimethyl sulfoxide
dichloromethane
N,N-dimethylformamide
dimethyl acetamide
DMSO
DCM
DMF
DMA
DMF-DMA N,N-Dimethylformamide dimethyl acetal
EA
ethyl acetate
TFA
trifluoroacetic acid
TEV
tobacco etch virus
PMSF
sumo
RMSD
phenylmethanesulfonyl fluoride
small ubiquitin-related modifier
root-mean-square deviation
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