Discovery and evaluation of new compounds targeting ribosomal protein S1 in antibiotic-resistant Mycobacterium Tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112317

The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide.

Full text of DOI:10.1016/j.ejmech.2020.112317

Journal Pre-proof
Discovery and evaluation of new compounds targeting ribosomal protein S1 in
antibiotic-resistant Mycobacterium Tuberculosis
Yazhuang Dai, Yinqiu Xu, Chenyun Guo, Xiaowen Xue, Donghai Lin, Kejiang Lin
PII:
S0223-5234(20)30286-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112317
EJMECH 112317
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2019
Revised Date: 11 March 2020
Accepted Date: 6 April 2020
Please cite this article as: Y. Dai, Y. Xu, C. Guo, X. Xue, D. Lin, K. Lin, Discovery and evaluation of new
compounds targeting ribosomal protein S1 in antibiotic-resistant Mycobacterium Tuberculosis, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112317.
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Title Page
Discovery and Evaluation of New Compounds Targeting
Ribosomal Protein S1 in Antibiotic-Resistant Mycobacterium
Tuberculosis
Yazhuang Dai(1,3), Yinqiu Xu(1), Chenyun Guo(2)，Xiaowen Xue(1), Donghai Lin*(2), Kejiang Lin*(1)
(1) Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
(2) The Key Laboratory for Chemical Biology of Fujian Province, MOE Key Laboratory of
Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen 361005, China.
(3) Hangzhou ipharmacare Co., LTD，Hangzhou 310000, China
* Corresponding authors: Kejiang Lin & Donghai Lin
Address: 24 Tongjiaxiang, Nanjing 210009, China (Kejiang Lin);
422, Siming South Road, Xiamen 361005, China (Donghai Lin).
Email address: link@cpu.edu.cn (Kejiang Lin);
dhlin@xmu.edu.cn (Donghai Lin).
Telephone number: +86 25 83271351(Kejiang Lin);
+86 592 2186078(Donghai Lin).
The authors declare no competing financial interest.

Graphical Abstract
Highlights
ò Based on the pyrazinoic acid binding site and an allosteric binding site, molecular
docking was performed to discover potential RpsA antagonists;
ò Derivatives
of
2-((hypoxanthine-2-yl)thio)acetic
acid
and
2-((5-hydroxylflavone-7-yl)oxy)acetamide were synthesized in satisfactory
yields;
ò Six compounds showed good affinity to both RpsA and mutant RpsA;
ò The binding sites of the active compounds are consistent with the molecular
docking sites.

Discovery and Evaluation of New Compounds Targeting
Ribosomal Protein S1 in Antibiotic-Resistant Mycobacterium
Tuberculosis
ABSTRACT
The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections
compels new treatment strategies, of which targeting trans-translation is promising.
During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key
role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows
its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the
structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new
compounds were designed. After being synthesized, the compounds were tested in
vitro with saturation transfer difference (STD), fluorescence quenching titration
(FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new
compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant.
The active compounds provide new choices for targeting trans-translation in Mtb, and
the analysis of the structure-activity relationships will be helpful for further structural
modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and
2-((5-hydroxylflavone-7-yl)oxy)acetamide.
Keywords: anti-tuberculosis agents, pyrazinamide, ribosomal protein S1,
trans-translation

Introduction
Antibiotic resistance is urging new strategies to treat bacterial infections,
of which tuberculosis is the leading cause of death around the world[1].
Recently, the inhibition of the trans-translation process of bacteria
appears to be an effective strategy, and compounds targeting this process
show broad-spectrum antibiotic activity[2]. Compounds affecting the
trans-translation process of Mycobacterium Tuberculosis (Mtb) have also
been discovered[3, 4], showing that the trans-translation process is a
viable target for developing anti-tuberculosis drugs.
In the trans-translation process of Mtb, the ribosomal protein S1 (RpsA)
plays an important role. As shown in Figure 1, Mtb initializes the process
to release the stalled ribosome when stimulated by factors such as stress
and mRNA damage[5]. With other molecules, such as the small protein B
(smpB) and the elongation factor thermo unstable (EF-Tu), RpsA helps
replace mRNA with tmRNA, which contains the information that the
protein being produced is incomplete. After translation, the incomplete
protein has a tmRNA-encoded peptide label, which helps with the
targeted proteolysis of the protein. Without the trans-translation process,
the incomplete protein may be toxic to the Mtb cell. Additionally, this
process also ensures that Mtb can successfully synthesize proteins that are
indispensable for survival in adversity[6].

Figure 1. The role of RpsA in trans-translation. Trans-translation helps
Mtb degrade incomplete proteins which may be toxic. The grey
rectangles in the figure represent information that the protein needs to be
degraded. The grey rectangle in tmRNA is an RNA sequence, and the
grey rectangle in the protein is an amino acid sequence.
Pyrazinamide (PZA), a first-line anti-tuberculosis drug, plays an
important role in the treatment of Mtb infections, and it is also related to
RpsA. Among the first-line anti-tuberculosis drugs, PZA has the
irreplaceable function of shortening the treatment period, even though
there are several new drug candidates in clinical development [7]. These
drug candidates, including the highly potent TMC207, must be used with
PZA, and any combination of drugs without PZA is unsatisfactory[8-11].
PZA acts as pyrazinoic acid (POA), the product of pyrazinamidase

hydrolysis. Research suggests[12] that POA forms a complex with the
C-terminal domain of RpsA (RpsA-CTD) to interfere with
trans-translation, and RpsA-CTD with Ala438 deleted (RpsA-CTD
Δ438A) has weak affinity for POA. Meanwhile, mutants of RpsA are
phenotypically associated with PZA-resistant Mtb[13-15]. By performing
molecular dynamic simulations, researchers found that RpsA mutants
without A438, key amino acids including Phe307, Phe310, Arg355 and
Arg357 are too flexible to bind POA, which could cause the PZA
resistance problem[14, 16]. To discover better drugs for treating Mtb
infections, further drug design not only needs to depend on wild type
RpsA, but also should consider the flexible RpsAA438 deletion mutant,
as well, so that the trans-translation process in both Mtb and
PZA-resistant Mtb can be inhibited effectively.
In our previous work, we found that a barrel of the RpsA-CTD composed
of β-strands shows strong affinity for RNA[17]. It is also the β-strands
that approach POA in the complex structure (PDB code: 4NNI). These
help drug design by showing the potential binding sites of RpsA-CTD
and RpsA-CTD Δ438A. Meanwhile, trans-translation is a unique
physiological mechanism of microorganisms. Therefore, targeting RpsA
for drug design should have good specificity[18]. More importantly, the
PZA-resistance problem is mainly caused by mutations in pncA gene

encoding pyrazinamidase[19]. Although PZA is a prodrug which is
converted to POA by pyrazinamidase, the interaction between POA and
RpsA does not rely on the activity of pyrazinamidase. Design of new
compounds directly interacting with RpsA avoids the conversion process,
which indicates that targeting RpsA could be an effective solution to the
PZA-resistance problem.
This research focuses on the β-strands of RpsA-CTD and RpsA-CTD
Δ438A to design novel inhibitors of RpsA. Molecules were selected from
the docking results and synthesized. Then, saturation transfer difference
(STD) was used to preliminarily evaluate the effects of the compounds,
followed by the determination of dissociation constants with fluorescence
quenching titration (FQT). Finally, chemical shift perturbation (CSP) was
applied to show the binding sites of the active compounds.
Results and discussion
Virtual screening and synthesis
To discover molecules having effects on both RpsA-CTD and RpsA-CTD
Δ438A, both the complex structure of POA with RpsA-CTD (PDB code:
4NNI) and the structure of RpsA-CTD Δ438A (PDB code: 4NNG) were
used for virtual screening. According to the complex structure shown in
4NNI, the basic Arg357 inside the binding site of RpsA-CTD can form an

electrostatic interaction with the acidic POA. This suggests that Arg357
should be protonated before molecular docking. Meanwhile, the scoring
functions used are more reliable than what was used before[4]. The
re-docking results show that the protonated model had a smaller deviation
between the docked POA and the POA in 4NNI (Figure 2a). At the same
time, the protonated model distinguished between POA and PZA more
clearly with the scoring functions shown in Figure 2b, which explains the
fact that PZA is inactive before being hydrolysed. Based on these results,
we screened new compounds with the protonated Arg357 model.
(a)
(b)
Figure 2. Re-docking results of POA. (a) Results from the protonated and
the unprotonated models. POA in the complex structure is in green, while
the docked poses of POA based on the protonated and the unprotonated
models are in cyan and yellow, respectively. (b) Results described with
three functions. The difference between the total binding energy values of
POA and PZA is higher with the Arg357 protonated, and the difference of

the -CDocker_energy values is also more obvious.
After virtual screening based on RpsA-CTD and RpsA-CTD Δ438A,
2,500 compounds were retained for the binding site around the β-strands,
which might be new solutions for PZA-resistance. In our previous work,
ZRL15 showed the best affinity[4], and therefore, the PXYC series of
compounds were selected due to their shared
2-((pyrimidine-2-yl)thio)-acetyl moiety (Figure 3). Another reason for
selecting the PXYC series is that their hypoxanthine moiety resembles the
purine of an RNA molecule, which has a high affinity for RpsA. From the
docking results of an allosteric binding site, flavone derivatives were
tentatively selected, which comprise the PXYD series.
Figure 3. Similarities between ZRL15 and the PXYC series. ZRL15 has
dissociation constants of 2.67 μM and 1.74 μM with RpsA-CTD and
RpsA-CTD Δ438A, respectively.
As shown in Scheme 1, the 2-thioxanthine core (PXYC3) was obtained in
five steps in satisfactory yield, and the functionalization of the
2-thioxanthine brought about the PXYC series. The condensation

between thiourea and ethyl cyanoacetate yielded
6-amino-2-mercaptopyrimidin-4(3H)-one (1), in which 5H was further
substituted with a nitroso. The substituted product (2) was then
deoxidized to obtain compound 3. A carbon was introduced to 3 through
the alkanoylation of the reduced amino to obtain 4. The intramolecular
condensation of 4 gave the 2-thioxanthine core.

Scheme 1. Synthesis of the PXYC series of compounds. Reaction
conditions: (a) EtONa, 2 h, 75 °C; (b) NaNO2, H2O, AcOH, 20 h, room
temperature; (c) Na2S2O4, NaHCO3, 7 h, 0 °C; (d) AcOH, pH = 5; (e)
HCOOH, 2 h, 101 °C; (f) formamide, 2 h, 178 °C; (g) ethyl chloroacetate,
H2O, 70 °C; (h) 1 M NaOH, 70 °C; (i) AcOH; (j) Et3N, DCM, 0 °C to
room temperature; (k) Et3N or K2CO3, EtOH, 50 °C or room temperature.
During the functionalization of the 2-mercapto of PXYC3, the direct
introduction of completed side chains had higher yields than stepwise
elongations. PXYC1 was first prepared by the alkylation of the
2-mercapto with ethyl chloroacetate, and the hydrolysis of PXYC1
brought PXYC2. However, direct amination reactions of PXYC1 gave
many side products. Meanwhile, the condensation between PXYC2 and
the corresponding amines under HBTU and HATU catalysis had low
yields. To overcome these obstacles, the completed side chains were
introduced directly, which were obtained by the alkanoylation of the
corresponding amines with chloroacetyl chloride. Then, the alkyl chloride
products (5-13) were used to prepare PXYC5-PXYC13.
PXYD1-PXYD5 have the same side chains as some compounds in the
PXYC series, so some alkyl chloride products mentioned above can also
be used. As shown in Scheme 2, PXYD1-PXYD5 were directly prepared

with 5,7-dihydroxyflavone as the core. An intramolecular hydrogen bond
can form between the 4-one and the 5-hydroxyl, which provides
regioselectivity for the alkylation of the 7-hydroxyl.
Scheme 2. Synthesis of the PXYD series compounds. Reaction conditions:
K2CO3, DMF, 80 °C.
Preliminary tests with STD
The STD experiment is a fast method for identifying hit compounds[20],
which has been widely applied to ligand-based drug design[21-23]. After
the selective irradiation saturation of a receptor, the saturation can be
transferred to its nearby ligand, and their spin system will try to return to
equilibrium. Due to the different relaxation pathways of the system, the
net spin populations belonging to the ligand may increase or decrease,
which is represented as higher or lower ligand peaks on its spectrum.
Based on the principle of STD, the satisfactory results preliminarily
showed that the drug design was successful.

For both RpsA-CTD (285-476) and RpsA-CTD Δ438A (285-476), STD
experiments were performed to test if they interact with the compounds.
As shown in the difference spectra (Figure 4), PXYC1, PXYC2, PXYC12,
PXYC13, PXYD3 and PXYD4 have positive signals with both
RpsA-CTD and RpsA-CTD Δ438A.
(a)
(b)
(c)
(d)
(e)
(f)

Figure 4. Results of the STD experiments. The hydrogens showing
signals on the difference spectra are labelled in red. Diff. and Ref. in the
figure represent difference spectra and reference spectra, respectively. Wt
and Mut stand for RpsA-CTD and RpsA-CTD Δ438A, respectively. (a-f)
Spectra of PXYC1, PXYC2, PXYC12, PXYC13, PXYD3 and PXYD4,
respectively.
Determination of dissociation constants with FQT
FQT experiments can be used to conveniently evaluate the interactions
between biomacromolecules and small molecules[24, 25]. The quenching
rate constants (Kq) and dissociation constants (Kd) of the active
compounds screened by STD are shown in Table 1. According to the
Stern-Volmer equation[26], all the Kq values of the compounds are
greater than 2 × 1010 M-1 sec-1, which show that their quenching process is
static rather than dynamic. Namely, the compounds have specificity for
their targets. According to the number of binding ligands fitted by the

double logarithm regression curve[27], each active molecule combines
with one target, which also supports the specificity of the interactions
between the compounds and their targets. The Kd values fitted by the
double logarithm regression curve show that all the active compounds
have a stronger affinity to RpsA-CTD than POA. Notably, the Kd values
of PXYC1 with the both targets are less than 1 μM, which shows that
PXYC1 has the best affinity among the compounds reported in this work
and our previous work[4]. In our previous work[4], compounds with
moderate affinity to RpsA-CTD and RpsA-CTD Δ438A show satisfying
results in drug susceptibility assays. Therefore, the performance of
PXYC1 in biological tests is worth expecting. Additionally, six
compounds show comparable affinity to RpsA-CTD Δ438A, which
indicates their potential in antibiotic-resistance Mtb infections.
Table 1. Results of the FQT experiments
Hill
Stern–Volmer
equation Adj. R2
Compound
Target
Kq (M-1 sec-1)
equation
Adj. R2
0.997
0.997
0.986
0.997
0.991
0.998
0.956
0.994
0.943
0.986
0.992
Kd (μM)
RpsA-CTD
RpsA-CTD Δ438A
RpsA-CTD
0.989
0.976
0.993
0.990
0.976
0.995
0.977
0.998
0.992
0.994
0.997
3.37 × 1012
3.40 × 1012
1.79 × 1012
1.91 × 1012
3.75 × 1012
2.54 × 1012
4.09 × 1012
3.68 × 1012
1.94 × 1012
2.33 × 1012
6.45 × 1012
0.81
0.31
6.35
5.11
2.67
4.67
7.61
8.50
5.66
6.91
3.24
PXYC1
PXYC2
PXYC12
PXYC13
RpsA-CTD Δ438A
RpsA-CTD
RpsA-CTD Δ438A
RpsA-CTD
RpsA-CTD Δ438A
RpsA-CTD
PXYD3
PXYD4
RpsA-CTD Δ438A
RpsA-CTD

RpsA-CTD Δ438A
0.989
0.994
6.64 × 1012
8.22 × 1011
0.996
0.995
1.64
9.65
POA
RpsA-CTD
Analysis of Structure-activity relationships based on STD and FQT
STD has shown its advantages in fragment-based drug design[23].
Because the saturation of proteins can only propagate to fragments within
a certain distance, fragments that are close to the protein show signals.
These fragments can be important scaffolds for further structural
modifications. Combined with the Kd values given by FQT, SAR can be
analysed for the active compounds.
For the PXYC series, appropriate side chains attached to the 2-mercapto
moiety are important. Most PXYC series difference spectra show signals
belonging to methyl and methylene groups, indicating the importance of
the side chains. Similarly, PXYC3 shows no activity without a side chain.
An acetic acid chain brings moderate activity, whereas its ethyl
esterification product shows the best affinity. It seems that both
hydrophilic and hydrophobic chains can bring moderate affinity. Because
the binding site for which the PXYC series was designed is a surface
rather than a cavity, the hydrophobicity of the side chain may not be
important. Because the ester or acylamide derivatives of PXYC3 show
activity, hydrogen bond acceptors are indispensable. For the acylamide
derivatives, the distance between the amide and an aryl group has the

greatest influence on activity, from which it can be inferred that around
the binding site there may be an amino acid containing an aryl group.
Within the distance of one carbon, a π-π interaction may form. However,
this interaction looks unnecessary as the affinity of the acylamide
derivatives did not exceed PXYC1, which may be due to the flexibility of
the amino acid aryl group. This one carbon distance also affects the
activity of the PXYD series, but the hydrophobicity of the aryl groups
might be responsible for maintaining the affinities of PXYD3 and
PXYD4, as PXYD5 shows no signal with STD, which has an ester
extension rather than an aryl group.
Validation of binding sites with CSP
CSP has been a successful method in fragment-based drug design since
the proposal of “SAR by NMR”[23, 28]. The binding of ligands changes
the environment of nearby amino acids, which results in the change in
peak shifts belonging to these amino acids. Based on the assignment of
RpsA 280-438 which has been reported[29], the binding sites of the
active compounds on RpsA-CTD can be found.
The change of peaks on 15N-1H heteronuclear single quantum coherence
(HSQC) spectra of RpsA (280-438) and its ligands is concluded in Figure
5, and Figure 5a shows the spectrum of RpsA (280-438) and PXYC1. As

shown in Figure 5b-5 g, for each compound, some peaks move and some
peaks become broader. In solution, proteins and ligands form an
exchanging system, where ligands bind to or unbind from proteins. When
ligands show low affinity, the peaks from the nearby amino acids move
due to the fast unbinding of the ligand. For ligands with high affinity, the
peaks get broader because the back reaction of binding is slower than the
frequency difference between the bound and free proteins[30]. The
broader peaks shown in Figure 5 mean that the compounds have a high
affinity for RpsA (280-438), which is in consistent with the Kd values
given by FQT. More importantly, considering moving and broader peaks,
the visualized binding sites of the active compounds correspond to the
sites for in silico design (Figure 6). The PXYC1, PXYC2, PXYC12 and
PXYC13 sites are around the β-strand barrel that binds RNA. These four
strong compounds may compete with RNA. The PXYD3 and PXYD4
sites are between an α-helix and the β-strand barrel, so they may be
successful allosteric inhibitors. Though the idea of allosteric inhibitors
needs further validation based on biological tests, their high affinity
makes them chemical tools for further evaluation. Based on these facts,
compounds with high affinities were successfully designed.

(a)
(b)
(c)
(e)
(g)
(d)
(f)

Figure 5. Results of the CSP experiments. (a) Superimposed spectrum of
PXYC1. The peaks in red, green and blue represent compound/protein
ratios of 1, 5 and 15, respectively. (b-g) Quantitative descriptions for
changes in the spectra belonging to PXYC1, PXYC2, PXYC12, PXYC13,
PXYD3 and PXYD4, respectively. Hfree/Hcomplex describes the changes in
intensity, and chemical shift perturbation describes the movement of
peaks. In each figure, the lower horizontal line represents the mean value
of the corresponding changes, and the upper horizontal line represents the
mean value plus the corresponding standard deviation. Change values
larger than the upper horizontal line indicate significant changes.

(a)
(b)
Figure 6. Binding sites. (a) Predefined binding sites for molecular
docking. The two spheres are binding sites. The site near the blue
β-strands was used to design the PXYC series, and the other site was used
to design the PXYD series. (b) Binding sites determined by the CSP
experiments. Amino acids with shifts in the spectra are in yellow, while
amino acids with broader peaks are in red.
Conclusion
Based on the complex structure of POA and RpsA-CTD, 17 compounds
were screened and synthesized. With STD experiments, six compounds
were found to have effects on both RpsA-CTD and RpsA-CTD Δ438A,
and these interactions were further proven by FQT. The FQT experiments
also helped to determine the Kd values between each protein and the
active compounds, of which PXYC1 showed the highest affinity. The

SAR analysis of the PXYC and PXYD series can help design compounds
with higher affinity, and thus, better lead compounds for treating
tuberculosis may be further developed. Finally, the CSP experiments
validate the binding sites of the six compounds. By targeting the
trans-translation process of Mtb, PXYC1, PXYC2, PXYC12 and
PXYC13 provide new leads for treating Mtb infections, and their effects
on both RpsA-CTD and RpsA-CTD Δ438A may limit antibiotic
resistance. In addition, PXYD3 and PXYD4 can be chemical tools to
further study the feasibility of allosteric inhibitors of RpsA.
Experimental section
Molecular docking
Based on the complex structure of RpsA-CTD and POA (PDB code:
4NNI) and the structure of RpsA-CTD Δ438A (PDB code: 4NNG)[12],
molecular docking was performed by CDocker[30], and CDocker energy
and CDocker interaction energy were used as the scoring functions to
screen the compound libraries of Antibacterial_5460 and
Antivriral_77260 in the ChemDiv commercial library, Drugbank and
Zinc. In 4NNI with Arg357 protonated, a coordinate (x=12.681, y=3.955,
z=22.145) was set as the centre of a sphere with a radius of 7.0 Å to
perform the first round of virtual screening. Then, docking was performed
in 4NNG with Arg357 protonated and a coordinate (x=6.065, y=16.396,

z=1.71) and a radius (7.7 Å) were set. Then, the same settings in 4NNI
without the protonated Arg357 were used for the next round of screening,
followed by screening based on the unprotonated 4NNG, with the
following settings: x=-2.291, y=12.954, z=-11.134 and radius=7.0 Å. The
compounds having scores better than POA (protonated model:
-CDocker_energy > 20, -CDocker_interaction_energy > 30; unprotonated
model: -CDocker_energy > 8, -CDocker_interaction_energy > 22) were
selected. For the allosteric site, the coordinates (x=-6.553 y= -11.292
z=27.764) and (x=-8.378 y= -5.605 z=1.888) were set for 4NNI and
4NNG, respectively, and radii of 8.1 Å and 8.2 Å, respectively, were set
tentatively. Antibacterial_5460 and Antivriral_77260 in the ChemDiv
commercial library and Drugbank were screened, and the compounds
with higher -CDocker energy and -CDocker interaction energy were
preferred.
Synthesis
Ethyl cyanoacetate was purchased from Energy Chemical (Shanghai,
China). Chloroacetyl chloride and ethyl chloroacetate were purchased
from Shaoyuan Technology (Shanghai, China). Reagents commonly used
in the laboratory were all chemically pure. The 1H and 13C NMR spectra
were collected with Bruker Avance 300 or 850 MHz spectrometers.
High-resolution mass spectra (HRMS) were collected on an Agilent

ESI-TOF mass spectrometer.
Synthesis of the PXYC series
Synthesis of 6-amino-2-mercaptopyrimidin-4(3H)-one (compound 1). The
reaction was carried out in a three-necked round bottom flask, using the
two side necks for refluxing and purging nitrogen with a dry tube. To the
flask, 37.5 mL of absolute ethanol was added, and a sodium block (1.13 g,
49.2 mmol) was slowly added with ice-cooling and magnetic stirring.
After the sodium block disappeared, thiourea (3.923 g, 51.5 mmol) was
added and the system was purged with nitrogen. Ethyl cyanoacetate (5
mL, 46.9 mmol) was added slowly under the nitrogen atmosphere.
Initially, a yellow solid started to precipitate and finally the solution
clarified. Then, a large amount of white solid appeared, and the reaction
stirred at 75 °C for 2 h. The white solid was obtained and then dissolved
in water (35 mL). The pH of the solution was adjusted to 4 with acetic
acid, and the white precipitate was separated by filtration and washed
with ethyl acetate and petroleum ether to obtain a white solid (5.84 g,
87%). 1H NMR (300 MHz, DMSO-d6) δ 11.64 (s, 1H), 11.54 (s, 1H),
6.38 (s, 2H), 4.69 (s, 1H).
Synthesis of 6-amino-2-mercapto-5-nitrosopyrimidin-4(3H)-one
(compound 2). Compound 1 (0.5 g, 3.5 mmol) was dissolved in a solution
of 8.93 mL water and 1.8 mL acetic acid. At the same time, 0.446 g (6.5

mmol) of sodium nitrite was dissolved in 1.4 mL of water. In an ice bath,
the aqueous sodium nitrite solution was slowly added to the compound 1
solution with stirring. During the addition, the solution gradually turned
brick red. After 16 h of stirring at room temperature, the mixture was
filtered and the filter cake was washed with water and ethanol to give a
brick red solid (0.45 g, 76%). As the compound has only active
hydrogens, we have listed only its 13C NMR spectrum. 13C NMR (75
MHz, DMSO-d6) δ 140.38, 142.60, 159.38, 176.43.
Synthesis of 5,6-diamino-2-mercaptopyrimidin-4(3H)-one (compound 3).
Compound 2 (0.45 g, 2.6 mmol) was dissolved in 10.56 mL aqueous
saturated sodium bicarbonate. Sodium dithionite (1.366 g, 7.8 mmol) was
slowly added to the reaction solution in an ice bath. The reaction stirred
for 7 h, during which the solution turned from blood red to pale yellow
and then white. After the reaction was complete, the pH of the solution
was adjusted to 5 with acetic acid, and the solution was placed in a
refrigerator for a few minutes. The solution was filtered and the filter
cake was washed with water and ethanol to give a white solid (0.369 g,
89%). 1H NMR (300 MHz, DMSO-d6) δ 5.68 (br, 6H); 13C NMR (75
MHz, DMSO-d6) δ 102.43, 104.56, 157.86, 167.45.
Synthesis of N-(4-amino-2-mercapto-6-oxo-1,6-dihydropyrimidin-5-yl)

formamide (compound 4). Compound 3 (2.85 g, 18 mmol) was dissolved
in 90% formic acid and the solution stirred at 101 °C for 2 h. During the
reaction, a dry tube was added to the reflux device, and formic acid was
added to the reaction flask to avoid solidification. After the reaction, the
mixture was cooled to 0 °C. The mixture was then filtered and the filter
cake was washed and dried to obtain a pale yellow solid (3.16 g, crude
yield: 94%).
Synthesis of 2-thioxanthine (compound PXYC3). Compound 4 (3.16 g, 17
mmol) was dissolved in 8 mL of formamide solution, and the solution
was allowed to stir at 178 °C. A large amount of bubbles were generated
and there was only a small amount of liquid left in the reaction flask at
the end of the reaction. After cooling to room temperature, a filter cake
was obtained by filtration, which was then dissolved in 1 M sodium
hydroxide, producing a yellow liquid. The solution was decolorized with
activated carbon, and the filtrate was retained after filtration. The solution
was adjusted to acidic pH with acetic acid, and a yellow solid precipitated.
After filtration, the filter cake was washed with ethyl acetate and
petroleum ether to give a pale yellow solid (2.36 g). 1H NMR (300 MHz,
DMSO-d6) δ 8.05 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 110.84,
141.97, 149.32, 153.90, 173.82; HRMS (ESI) m/z [M+H]+: 169.0175.
Synthesis of ethyl 2-((6-oxo-6,7-dihydro-1H-purin-2-yl)thio)acetate

(compound PXYC1). To 25 mL of water, PXYC3 (0.44 g, 2.6 mmol) was
added and the solution was heated to 70 °C, followed by the addition of
2.5 mL of 1 M sodium hydroxide with stirring. Acetic acid was added to
disperse PXYC3 as white granules, and then 560 μl (0.6418 g, 5.2 mmol)
of ethyl chloroacetate was added. The reaction was monitored via TLC.
After the reaction was complete, the pH of the solution was adjusted to 5
and the solution was filtered. The filter cake was washed with ethanol and
ethyl acetate and finally dried in vacuo to yield a white solid (0.42 g). 1H
NMR (300 MHz, DMSO-d6) δ 1.19 (t, J = 7.1 Hz, 3H), 4.07 (s, 2H), 4.13
13
(q, J = 7.1 Hz, 2H), 8.02 (s, 1H), 12.64 (s, 1H), 13.08 (s, 2H); C NMR
(213MHz, DMSO-d6) δ 14.47, 32.84, 61.66, 141.88, 149.38, 153.99,
168.84, 173.83; HRMS (ESI) m/z [M+H]+: 255.0520.
Synthesis of 2-((6-oxo-6,7-dihydro-1H-purin-2-yl)thio)acetic acid
(compound PXYC2). PXYC1 (0.42 g, 1.65 mmol) was dissolved in 6 mL
of 1 M aqueous sodium hydroxide. The reaction stirred at 70 °C for 2 h,
during which the solution became pale yellow. The reaction was
monitored via TLC, and the mixture was cooled to room temperature
after the reaction was complete. Acetic acid was added to the reaction
mixture to precipitate the product as a white solid. The solid was obtained
by filtration and dried to give a white solid powder (0.3 g). 1H NMR (300
13
MHz, DMSO-d6) δ 7.99 (s, 1H), 3.80 (s, 2H); C NMR (75 MHz,

DMSO-d6) δ 34.62, 116.65, 139.58, 153.02, 156.03, 155.56, 170.62;
HRMS (ESI) m/z [M+H]+: 227.0226.
General procedure for the synthesis of compounds 5-14. The
corresponding amine was dissolved in anhydrous dichloromethane at
0 °C, and then triethylamine (1.1 eq) was added under a nitrogen
atmosphere. Chloroacetyl chloride (1.1 eq) diluted with anhydrous
dichloromethane was slowly added to the reaction mixture in an ice bath.
The reaction stirred at 0 °C for 30 min, after which the reaction slowly
warmed to room temperature overnight. TLC was used to monitor the
reaction. After the reaction was complete, it was quenched with water and
extracted with dichloromethane. The organic phase was retained and
washed with an aqueous hydrochloric acid solution, an aqueous saturated
sodium hydrogen carbonate solution, and brine three times. Anhydrous
sodium sulfate was used to dry the organic phase, after which the solvent
was removed under reduced pressure with a rotary evaporator to obtain
crude product. The crude product was dissolved in ethyl acetate and
recrystalized with petroleum ether to afford the final product. Compounds
5-14 were in 58-86% yield.
1
2-chloro-N-phenylacetamide (compound 5). H NMR (300 MHz,
DMSO-d6) δ 4.26 (s, 2H), 7.10 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 8.0 Hz,

2H), 7.59 (d, J = 7.9 Hz, 2H), 10.30 (s, 1H).
1
Methyl 4-(2-chloroacetamido)benzoate (compound 6). H NMR (300
MHz, DMSO-d6) δ 3.83 (s, 3H), 4.31 (s, 2H), 7.74 (d, J = 8.4 Hz, 2H),
7.95 (d, J = 8.4 Hz, 2H), 10.65 (s, 1H).
1
2-chloro-N-(3-methoxyphenyl)acetamide (compound 7). H NMR (300
MHz, CDCl3) δ 2.34 (s, 3H), 4.16 (s, 2H), 6.98 (d, J = 7.5 Hz, 1H), 7.23
(t, J = 7.8 Hz, 1H), 7.41-7.29 (m, 2H), 8.24 (s, 1H).
1
2-chloro-N-(thiazol-2-yl)acetamide (compound 8). H NMR (300 MHz,
CDCl3) δ 5.13 (s, 2H), 7.91 (d, J = 3.5 Hz, 1H), 8.10 (s, 1H), 8.36 (d, J =
3.6 Hz, 1H).
1
2-chloro-N-phenethylacetamide (compound 9) H NMR (300 MHz,
CDCl3) δ 2.87 (t, J = 7.0 Hz, 2H), 3.59 (q, J = 6.9 Hz, 2H), 4.04 (s, 2H),
6.65 (s, 1H), 7.41-7.28 (m, 2H), 7.33-7.18 (m, 3H).
1
N-benzyl-2-chloroacetamide (compound 10). H NMR (300 MHz,
DMSO-d6) δ 4.12 (s, 2H), 4.51 (d, J = 5.8 Hz, 2H), 6.89 (s, 1H), 7.28 (d,
J = 6.0 Hz, 1H), 7.32 (s, 2H), 7.44-7.30 (m, 2H).