Structure-activity relationship study and biological evaluation of SAC-Garlic acid conjugates as novel anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2019.06.059

A series of S-allyl-L-cysteine (SAC) with garlic acid conjugates as anti-inflammatory agents were designed and synthesized. Among the 40 tested compounds, SMU-8c exhibited the most potent inhibitory activity to Pam₃CSK₄-induced nitric oxide (NO) in RAW264.7 macrophages with IC₅₀ of 22.54 ± 2.60 μM. The structure-activity relationship (SAR) study suggested that the esterified carboxyl group, carbon chain extension and methoxylation phenol hydroxy could improve the anti-inflammatory efficacy. Preliminary anti-inflammatory mechanism studies showed that SMU-8c significantly down-regulated the levels of Pam₃CSK₄ triggered TNF-α cytokine in human THP-1 cells, mouse RAW 264.7 macrophages, as well as in ex-vivo human peripheral blood mononuclear cells (PBMC) with no influence on cell viability. SMU-8c specifically blocked the Pam₃CSK₄ ignited secreted embryonic alkaline phosphatase (SEAP) signaling with no influence to Poly I:C or LPS triggered TLR3 or TLR4 signaling. Moreover, SMU-8c suppressed TLR2 in HEK-Blue hTLR2 cells and inhibited the formation of TLR1-TLR2, and TLR2-TLR6 complex in human PBMC. In summary, SMU-8c inhibited the TLR2 signaling pathway to down-regulate the inflammation cytokines, such as NO, SEAP and TNF-α, to realize its anti-inflammatory activity.

Full text of DOI:10.1016/j.ejmech.2019.06.059

Accepted Manuscript
Structure-activity relationship study and biological evaluation of SAC-Garlic acid
conjugates as novel anti-inflammatory agents
Jingjie Bi, Wenqing Wang, Junxi Du, Kun Chen, Kui Cheng
PII:
S0223-5234(19)30586-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.059
EJMECH 11462
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 April 2019
Revised Date: 17 June 2019
Accepted Date: 21 June 2019
Please cite this article as: J. Bi, W. Wang, J. Du, K. Chen, K. Cheng, Structure-activity relationship study
and biological evaluation of SAC-Garlic acid conjugates as novel anti-inflammatory agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.059.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-activity relationship study and biological evaluation of SAC-Garlic
Acid conjugates as novel anti-inflammatory agents
Jingjie Bi a, Wenqing Wang a, Junxi Du b, Kun Chen b, and Kui Cheng a,
aGuangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key
Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School
of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515,
China.
bThe Joint Research Center of Guangzhou University and Keele Univeristy for Gene
Interference and Application, School of Life Science, Guangzhou University,
Guangzhou, 510006, China.
Corresponding author. Tel.: +86-20-61647192; fax: +86-20-61648533; e-mail: chengk@smu.edu.cn
Corresponding author. Tel.: +86-20-39366915; fax: +86-20-39366915; e-mail: kchen@gzhu.edu.cn
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Abstract:
A series of S-allyl-L-cysteine (SAC) with garlic acid conjugates as anti-inflammatory
agents were designed and synthesized. Among the 40 tested compounds, SMU-8c
exhibited the most potent inhibitory activity to Pam3CSK4-induced nitric oxide (NO)
in RAW264.7 macrophages with IC50 of 22.54 ± 2.60 µM. The structure-activity
relationship (SAR) study suggested that the esterified carboxyl group, carbon chain
extension and methoxylation phenol hydroxy could improve the anti-inflammatory
efficacy. Preliminary anti-inflammatory mechanism studies showed that SMU-8c
significantly down-regulated the levels of Pam3CSK4 triggered TNF-α cytokine in
human THP-1 cells, mouse RAW 264.7 macrophages, as well as in ex-vivo human
peripheral blood mononuclear cells (PBMC) with no influence on cell viability.
SMU-8c specifically blocked the Pam3CSK4 ignited secreted embryonic alkaline
phosphatase (SEAP) signaling with no influence to Poly I:C or LPS triggered TLR3
or TLR4 signaling. Moreover, SMU-8c suppressed TLR2 in HEK-Blue hTLR2 cells
and inhibited the formation of TLR1-TLR2, and TLR2-TLR6 complex in human
PBMC. In summary, SMU-8c inhibited the TLR2 signaling pathway to down-regulate
the inflammation cytokines, such as NO, SEAP and TNF-α, to realize its
anti-inflammatory activity.
Keywords: SAC-Garlic acid conjugates; Structure-activity relationship;
Anti-inflammatory; Toll-like receptor 2.
1. Introduction
Garlic acid is a type of phenolic acid, and has been widely found in natural plants,
such as Chinese nutgall, rhubarb, lacquer tree, and some fruits [1, 2]. Many drugs
contain a skeleton of methylated garlic acid, such as reserpine, deserpidine, and
trimethoprim [3, 4]. Garlic acid has been shown to have beneficial effects on
anti-inflammation, vascular calcification, and metabolic diseases, as well as scavenges
free radicals and regulates different intracellular signaling pathways [5]. Recent
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researches have demonstrated that garlic acid can down-regulate the expression of
matrix metalloproteinase 2 and matrix metalloproteinase 9 then inhibit tumor growth
[6, 7]. It was also reported as an antagonist of semen amyloid fibrils to attenuate
HIV-1 infection [8], and interacted with a-synuclein, associated with Parkinson's
disease, to prevent the structural change required for its fibril formation [9]. In
addition, garlic acid exhibited that it can suppress the inflammatory reaction by
inhibiting activation of the p65-NF-kB and IL-6/STAT3 pathways [10].
On the other hand, S-allyl-L-cysteine (SAC) and its modifications
S-propargyl-L-cysteine (SPRC) (Fig. 1), or SPRC-Leonurine complex revealed
exciting anti-inflammatory, anti-atherogenic effects by decrease the nitric oxide (NO)
on macrophages and endothelial cells [11], as well as protective effects on acute
myocardial ischaemia through regulating cysthathionine-glyase pathway [12]. SAC is
also a well-known bioactive component of the aqueous garlic, exhibiting NF-κB
inhibition effect in T cells [13]. Thus, the selective inhibition of NO and NF-κB may
contribute to anti-inflammatory effect and prevention of atherosclerosis. SAC or
SPRC-Leonurine complex, a new class of multifunctional antimyocardial ischaemia
agents, displayed potent cardioprotective effect at lower molar concentration [14, 15].
Incorporation of two mutually complementary biological active by conjugating
into one has been widely used in drug design. Recently, researcher attempted to
integrate the chemical features of garlic acid and methyl-L-leucine to yield a novel
garlic acid-L-leucine (GAL) conjugate. The results suggested that GAL conjugate
may serve as a new scaffold compound for the development of new anti-inflammatory
drugs [16]. Garlic acid-β-D-glucose conjugates (BGG), a major component of the
Emblica officinalis medicinal plant, demonstrated specific inhibition to inflammatory
diseases, particularly diabetic eyes diseases [17]. Garlic acid-Quercetin conjugate
increased both the mRNA and protein levels of HO-1 in RAW cells through
MAPK/Nrf2 pathway [18], and Garlic acid-triphenylphosphonium lipophilic cations
showed significantly antiproliferative effect in vivo [19]. Garlic acid and rivastigmine
hybrid GA2 efficiently prevented the self-mediated Aβ aggregation as a new approach
against Alzheimer's disease [20].
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Toll-like receptors (TLRs) is one of the pattern recognition receptors, associated
with variety of immune-related diseases [21-30]. Inspired by above paradigms, as well
as our continued work to develop new modulators of TLRs [31-35], in the present
study, a series new GA-SACs conjugates were developed and their inhibitory activity
to the proinflammatory cytokines were also evaluated, including NO, TNF-α and
SEAP. Next, the preliminary anti-inflammatory mechanisms of the most potent
compound were further explored.
2. Design idea
In view of the operability of the combination and the stability of the forming
compound, we adopted a binding strategy for forming an amide bond between SAC
and garlic acid, and got a new scaffold SGA (Fig. 2). Three positions in SGA were
investigated for the structure-activity relationship (SAR). First, based on the allyl
substitution of SAC (blue color), we varied the length of the carbon chain,
including1-butenyl or 1-pentenyl substitution. Replacing the allyl by a propynyl group
made the structure more linear. Considering the electronic and steric effects, we
replaced the allyl group with a more sterically hindered and more rigid benzyl group.
In the second part (purple color), we methylated the SAC carboxyl group to make the
compound more hydrophobic. In the third part (red color), we investigated the effect
of the hydroxyl groups on the activity according the availability of the substrate. A
series of compounds with hydroxy substitution at the 4th position or the 3rd, 5th
positions, or trimethoxy group substitution at the 3rd, 4th, and 5th positions were
investigated.
3. Results and discussion
These potential anti-inflammatory agents were prepared by following Scheme 1.
All targeted compounds were test for their anti-inflammatory activity by secreting
pro-inflammatory NO in RAW 264.7 cells. To monitor the activation of inflammatory
state, the TLR2 agonist Pam3CSK4 was used as the positive control.
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3.1. Structure-activity relationship study.
The IC50 results of SGA and its derivatives in RAW 264.7 cells were illustrated
in Table 1. Comparison of 1a-f and 2a-f, we knew that SAC and its derivatives had no
nitric oxide inhibitory activity even up to 150 µM. Methylated the carboxyl group
enhanced its anti-inflammatory activity. Compound 2c was better than others (2a to
2f), indicating that the hydrophobic effect was helpful for the activity. Similar result
was also observed between compound 3a-c and 6a. When the -OH substitutions at the
4th position, or the 3rd, 5th positions of the benzene ring were removed, the activity
decreased. Meanwhile, the inhibitory activity was improved when trimethoxy group
substitutions showing up at the 3rd, 4th, and 5th positions. Secondly, among SGA and
its derivatives (8a-f, 10a-r), as we expected, the compound SMU-8c, which was a
combination of 2c and 6a by forming an amide bond, exhibited the best inhibitory
activity with IC50 of 22.54 ± 2.60 µM (Fig. 3A and Table 1). SMU-8c was much better
than SAC (1a, IC50 > 150 µM) and garlic acid (3a, IC50 of 69.25 ± 4.22 µM), as well
as much better than 2a and garlic acid conjugated compound 10a. SAR studies found
that when an allyl substitution was increased to 1-butenyl or 1-pentenyl, the inhibitory
activity was increased follow the carbon chain extension (8a-c, 10a-c, 10g-i, 10m-o).
At the same time, when we replaced the first part of 1-pentenyl with the linear group
propynyl (8d, 10d, 10j, 10p), the activity of the compounds decreased dramatically.
This indicated that the steric hindrance of the substituents greatly influence their
inhibitory activity. This result was further confirmed by replacing propynyl with a
benzyl group or 4-fluorobenzyl group (8e-f, 10e-f, 10k-l, 10q-r), and the inhibitory
activity of these compounds increased. Moreover, the activities of the benzyl
compounds with an electron-withdrawing substitution in the para position (8e, 10e,
10k, 10q) were more potent than that with no substitutions (8f, 10f, 10l, 10r). While
the activity of all these compounds were not superior to that of 1-pentenyl substituted
compounds. From above results we can conclude that the first part (I) of the
substituent favors certain steric hindrance and flexibility chain, while the second (II)
and third (III) part like the hydrophobic substitutions and were necessary for the
inhibitory activity.
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3.2. Target validation of SMU-8c
SMU-8c inhibited the inflammation cytokine nitric oxide on RAW 264.7 cells
with TLR2 agonist Pam3CSK4 as the positive control. To confirm this effect was
associated with TLR2, SMU-8c was test for TLR2 inhibition in HEK-Blue hTLR2
cells. This cell line was obtained by co-transfection of the human TLR2 and SEAP
(secreted embryonic alkaline phosphatase) genes into HEK293 cells. Additionally, the
CD14 co-receptor gene was also transfected into these cells to enhance the TLR2
response. TLR2 specific ligand Pam3CSK4 can activate the NF-kB and AP-1 signaling
pathway to induce the SEAP signaling in HEK-Blue hTLR2 cells. Similar to the
results observed on RAW 264.7 cells, SMU-8c suppressed SEAP signaling in a
concentration dependent manner in HEK-Blue hTLR2 cells, with almost 50 percent
inhibition at 25 µM. SAC as a negative control showed no inhibition (Fig. 3B). So, the
optimized compound SMU-8c was selected out for further assays.
3.3. In vitro cytotoxicity studies
In vitro toxicity of the compound was evaluated by a MTT colorimetric method
in RAW 264.7 cells. From the MTT assay results (Fig. 4), we can confirm that
SMU-8c had no significant cytotoxicity problem as concentration up to 100 µM.
Considering compound SMU-8c having the lowest IC50, high inhibitory activity, low
toxicity, as well as appropriate molecular weight, it might own the higher drug-like
potential ability. Thus, compound SMU-8c was picked out to evaluate its selectivity
and other potential mechanisms.
3.4. Specificity assays
From the previous experiments, we knew that SMU-8c can inhibit the SEAP
signal stimulated by Pam3CSK4 on HEK-Blue hTLR2 cells. But TLR family can
recognize a large range of ligands, so we conducted a selectivity examination to
investigate whether SMU-8c could specifically inhibit TLR2 signaling. The targets
included TLR1/2, TLR2/6, TLR3, and TLR4, and their native ligands were Pam3CSK4,
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Pam2CSK4, Poly I:C, and LPS, respectively. As shown in Fig 5, both TLR1/2 and
TLR2/6 were inhibited (Fig. 5A), which had no influence on TLR3 and TLR4 (Fig.
5B and 5C). These results suggested that SMU-8c was an antagonist of TLR2.
We also evaluated whether SMU-8c influences the TLR2 expression in the
whole-cell environment. HEK-Blue hTLR2 cells, which overexpress the human TLR2,
were stimulated with Pam3CSK4 (50 ng/mL). Cell lysates were subjected to
immunoprecipitation and immuno-detected using TLR2 antibody. As illustrated in Fig.
6A, the TLR2 protein upregulated when the Pam3CSK4 added and this upregulation
could be inhibited by SMU-8c in a dose-dependent manner. TLR2 protein was
suppressed more than 90% at 12.5 µM. A co-immunoprecipitation assay was also
conducted to validate the effect of compound SMU-8c on TLR1–TLR2, TLR2–TLR6
heterodimerization, as well as TLR2 and its adaptor protein MyD88 in cellular level.
Human peripheral blood mononuclear cells (PBMC) pre-treated with compound
SMU-8c were stimulated with Pam3CSK4 or Pam2CSK4 and TLR1–TLR2,
TLR2-TLR6, TLR2-MyD88 complexes were pulled down using protein A agarose
beads coated with anti-TLR2 antibodies. The complexes were then analyzed by
Western blotting using anti-TLR1, anti-TLR2, anti-TLR6, anti-MyD88 antibodies
(Fig. 6B, C). In the absence of Pam3CSK4 (TLR1/2 ligand) or Pam2CSK4 (TLR2/6
ligand), less TLR1, TLR6, MyD88 were detected in the immunoprecipitates, while the
TLR1–TLR2, TLR2–TLR6, and TLR2-MyD88 complexes were detected with the
present of the ligands. Decreased TLR1, TLR6, MyD88 were found in
immunoprecipitates from Pam3CSK4 or Pam2CSK4-treated cells in a dose-dependent
manner,
indicating
that
TLR1–TLR2
heterodimerization,
TLR2–TLR6
heterodimerization, TLR2-MyD88 complexes formation were inhibited and were
pulled down together. These results indicate that SMU-8c, as an antagonist of TLR2,
is capable of inhibit the TLR2 on the cell surface and downregulate Pam3CSK4 or
Pam2CSK4 triggered TLR2 protein and its adaptor protein MyD88.
3.5. Downstream signaling evaluation
We performed ELISA to analyze the effect of compound SMU-8c on
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downstream signaling. In our experiments, we tested the inflammatory cytokine
TNF-α to characterize the compound’s inflammatory inhibition ability. The data in
RAW 264.7 cells showed that SMU-8c can dose dependently decrease the
Pam3CSK4-triggered TNF-α signaling, with 25 µM almost inhibiting 50% TNF-α
signaling (Fig. 7A), which was similar to results observed in human THP-1 monocyte
cells (Fig. 7B). In ex-vivo human peripheral blood mononuclear cells (PBMC)
experiment, the inhibition rate of SMU-8c at 12.5 µM has exceeded 95%, which
showed the compound maybe more potent in the ex-vivo model (Fig. 7C).
3.6. Molecular docking studies of SMU-8c
To examine possible explanations for the significantly TLR2 inhibition potency
in human cells, we explored the predicted binding mode(s) of compound SMU-8c
with the TLR1/TLR2 complex by conducting molecular docking experiments in the
human protein crystal complex (PDB 2Z7X). We selected the most favorable energy
configuration of SMU-8c (shown in orange) and compared the binding of Pam3CSK4
(shown in purple) bound to the complex (Fig. 8). Of particular interest is that SMU-8c
is well binding to the interface of TLR1 (A) and TLR2 (B), tightly fits into the
binding site of Pam3CSK4 and TLR1 and TLR2, and shows significant interaction
with the important residues, such as Gly313, Gln316 on TLR1 and Phe349 on TLR2
(Fig. 8). The long chain of olefins binds well to the hydrophobic cavity in which
Pam3CSK4 binds to TLR1, which probably explains why carbon chain elongation has
a certain increase in activity. The methoxy group has a hydrophobic interaction with
the active site of TLR2 which probably explain that the activity is significantly
reduced when the methoxy groups changed into hydroxyl groups. The top three active
compounds (SMU-8c, 8b and 8f) also showed similar binding mode to the
protein-protein interaction domain of TLR1/2 (Fig. S1).
4. Conclusion
In conclusion, a series of SGA or SGA derivatives have been designed and
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synthesized based on our continued work to develop new anti-inflammatory reagents.
SAR studies of a series compounds yield a new SAC and garlic acid conjugate
SMU-8c with the most anti-inflammatory potential. Specificity experiments
confirmed that SMU-8c had low toxicity and selectivity to TLR2, with no influence to
TLR3 and TLR4. Immunoprecipitation assays confirmed that SMU-8c could inhibit
the TLR2 protein, and influence the formation of TLR1-TLR2, TLR2-TLR6 as well
as TLR2-MyD88 complex to downregulate the downstream signaling. Following
ELISA result showed that SMU-8c can not only inhibit the TNF-α in the mouse
macrophages, but also inhibit the TNF-α in human THP-1 monocytes, as well as in
human PBMC cells. In summary, our study has provided a novel, drug-like and potent
nature product conjugate small molecular as anti-inflammatory reagents by inhibit
TLR2, which might be further explored involving some diseases associated with
inflammation or tumor.
5. Experimental section
5.1. Chemistry
Commercially available chemicals were purchased from Macklin or
Sigma-Aldrich and without further purification. 1H NMR and 13C NMR spectra of all
compounds were obtained with a Bruker Advance 400 spectrometer at 400 MHz in a
1
solution of CDCl3, DMSO-d6 or D2O. Data for H NMR spectra were reported as
follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), integration.
Multiplicities were reported as follows: s=singlet, d=doublet, t=triplet, q=quartet,
m=multiplet. All compounds were routinely checked by thin-layer chromatography
(TLC). UV light was used to examine the spots. Low-resolution mass spectra ESI-MS
were recorded on a Waters ZQ 4000 apparatus. Evaporation was performed in vacuum
(rotating evaporator). Sodium sulfate was always used as the drying agent.
The general synthetic procedures followed for the synthesis of final compounds.
Briefly, the brominated unsaturated alkane or aromatic hydrocarbon were reacted with
L-cysteine in NH3·H2O to obtain compounds 1a-f. Then, in a methanol solution, the
methyl esterified products 2a-f were obtained under the catalysis of SOCl2. The
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reaction of a hydroxy-substituted benzoic acid compounds 3a-c with acetic anhydride
gived a hydroxyacetylated protective products 4a-c. Compounds 8a-f or 9a-r were
prepared by the reaction of different substituted benzoyl chloride compounds 5a-c or
7a，freshly prepared by treatment of SOCl2, and the amine-derivatives. Finally,
deacetylation protection in NaHCO3 (1M), yielded the final compounds 10a-r.
General procedure for the synthesis of compounds 1a-f
3-bromoprop-1-ene (3271 mg, 20.490 mmol) was added dropwise to a solution
of L-cysteine (3000 mg, 24.789 mmol) in NH3·H2O (8 ml), pre-cooled for 15 minutes
in ice water. The reaction mixture was stirred for 12h, after which the solvent was
removed using a rotary evaporator under 40 oC, and any residual 3-bromoprop-1-ene
was removed by washing with ethanol. The crude product was recrystallized in a
mixed solution of water:ethanol = 2:3. After filtration, the pure product was obtained
o
and dried in an oven under 40 C. Yielding 65% compound 1a (2595 mg) as a white
solid. Compounds 1b-f were synthesized following the procedure of preparation 1a.
General procedure for the synthesis of compounds 2a-f
SOCl2 (2038 mg, 17.132 mmol) was added dropwise to a solution of 1a (1000
mg, 6.209 mmol) in CH3OH (3167 mg, 98.845 mmol), pre-cooled for 15 minutes in
ice water. The reaction mixture was stirred for 12h. The solvent was removed under
reduced pressure. The crude product was recrystallized in CH2Cl2 and dried under
vacuum. Yielding 93% compound 2a (1011 mg) as a white solid. Compounds 2b-f
were synthesized following the procedure of preparation 2a.
General procedure for the synthesis of compounds 4a-c
Garlic acid (2000 mg, 11.763 mmol) was dissolved in acetic anhydride (7205 mg,
o
70.578 mmol). The solution was then heated under reflux at 130 C for 4 h, after
which the deionized water (4 ml) was add to the solution to remove the unreacted
acetic anhydride. The solvent was removed using a rotary evaporator under reduced
pressure to yield white solid. The product was recrystallized in CH2Cl2 and dried
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under vacuum. Yielding 95% compound 4a (3308 mg) as a white solid. Compounds
4b-c were synthesized following the procedure of preparation 4a.
General procedure for the synthesis of compounds 5a-c, 7a
3,4,5-triacetoxybenzoic acid 4a ( 2000 mg, 6.756 mmol) was dissolved in SOCl2
(8000 mg, 67.244 mmol). The solution was then heated under reflux at 78 oC for 4 h.
The solvent was removed using a rotary evaporator under reduced pressure to yield
white solid. Yielding 99% compound 5a (2100 mg) as a white solid. Compounds 5b-c
and 7a were synthesized following the procedure of preparation 5a.
General procedure for the synthesis of compounds 8a-f, 9a-r
To a solution of methyl (S)-2-aminopent-4-enoate 2a (600 mg, 4.648 mmol) in
CH2Cl2 (4 mL) was added triethylamine (1500 mg, 14.823 mmol). The mixture was
allowed to stir 30 min. 3,4,5-trimethoxybenzoyl chloride 7a (1069 mg, 4.648 mmol)
in CH2Cl2 (2 mL) was added dropwise at 0 oC. The reaction mixture was stirred for 12
h. The reaction was then concentrated and the residue was purified on a silica gel
column eluting with 50% ethyl acetate in petroleum. Yielding 76% compound 8a
(1141 mg) as a white solid. Compounds 8b-c and 9a-r were synthesized following the
procedure of preparation 8a.
General procedure for the synthesis of compounds 10a-r
To
a
solution
of
(S)-5-((1-methoxy-1-oxopent-4-en-2-yl)carbamoyl)
benzene-1,2,3-triyl triacetate 9a (600 mg, 1.474 mmol) in a solvent of 1:1 ratio of
tetrahydrofuran to methanol (6 ml) was added dropwise NaHCO3 (1M, 3ml). The
reaction was allowed to 3h at rt. The mixture was treated with an aqueous solution of
HCl (1M) to reach pH=7. The solvent was removed using a rotary evaporator under
reduced pressure. Afterwards deionized water was added and the phases were
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extracted with ethyl acetate in three times. The organic layer was washed with
deionized water in three times and was dried over Na2SO4. The solvent was removed
under reduced pressure. Yielding 82% compound 10a (339 mg) as a light yellow oil.
Compounds 10b-r were synthesized following the procedure of preparation 10a.
S-allyl-L-cysteine (1a)
1
White solid, yield: 65%. H NMR (400 MHz, DMSO) δ 7.61 (s, 2H), 5.76 (m, ,
1H), 5.13 (m, 2H), 3.30 (m, 1H), 3.17 (m, 2H), 2.95 (m, 1H), 2.67 (m, 1H). 13C NMR
(101 MHz, D2O) δ 172.77, 133.45, 118.31, 53.43, 33.84, 30.65. ESI-MS: m/z calcd
for C6H11NO2S (M + H+) 162.3, found 162.3.
S-(prop-2-yn-1-yl)-L-cysteine (1d)
1
Light yellow solid, yield: 76%. H NMR (400 MHz, D2O) δ 3.92 (m, 1H), 3.31
(d, 2H), 3.22 (m, 1H), 3.09 (m, 1H), 2.61 (t, 1H). 13C NMR (101 MHz, D2O) δ 172.57,
79.90, 72.54, 53.22, 31.73, 18.67. ESI-MS: m/z calcd for C6H9NO2S (M + H+) 160.5,
found 160.6.
Methyl S-allyl-L-cysteinate (2a)
White solid, yield: 93%. 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 2H), 5.88 – 5.73
(m, 1H), 5.21 (m, 2H), 4.45 (d, 1H), 3.87 (s, 3H), 3.39 – 3.05 (m, 4H). 13C NMR (101
MHz, CDCl3) δ 168.52, 133.21, 118.53, 53.51, 52.62, 34.93, 30.28. ESI-MS: m/z
calcd for C7H13NO2S (M + H+) 176.7, found 176.8.
Methyl S-(prop-2-yn-1-yl)-L-cysteinate (2d)
Brown solid, yield: 90%.1H NMR (400 MHz, DMSO) δ 8.88 (s, 2H), 4.32 (t,
1H), 3.76 (s, 3H), 3.55 – 3.40 (m, 2H), 3.28 (t, 1H), 3.23 – 3.11 (m, 2H). 13C NMR
(101 MHz, DMSO) δ 169.00, 80.16, 74.82, 53.38, 51.81, 31.18, 19.39. ESI-MS: m/z
calcd for C7H11NO2S (M + H+) 174.5, found 174.5.
Methyl S-benzyl-L-cysteinate (2e)
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White solid, yield: 78%. 1H NMR (400 MHz, DMSO) δ 8.85 (s, 2H), 7.52 – 7.16
13
(m, 5H), 4.29 (s, 1H), 3.83 (d, 2H), 3.75 (s, 3H), 2.97 (m, 2H). C NMR (101 MHz,
DMSO) δ 169.03, 138.16, 129.39, 128.86, 127.49, 53.34, 52.12, 35.81, 31.21.
ESI-MS: m/z calcd for C11H15NO2S (M + H+) 226.2, found 226.3.
3,4,5-triacetoxybenzoic acid (4a)
White solid, yield: 95%. 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 2H), 2.34 (s, 3H),
13
2.34 (s, 6H). C NMR (101 MHz, CDCl3) δ 169.53, 167.59, 166.37, 143.44, 139.28,
127.37, 122.77, 20.51, 20.12. ESI-MS: m/z calcd for C13H12O8 (M + H+) 297.3, found
297.4.
3,5-diacetoxybenzoic acid (4b)
White solid, yield: 91%. 1H NMR (400 MHz, DMSO) δ 13.34 (s, 1H), 7.59 (d, J
= 2.2 Hz, 2H), 7.28 (t, J = 2.2 Hz, 1H), 2.29 (s, 6H). 13C NMR (101 MHz, DMSO) δ
169.33, 166.13, 151.30, 133.33, 120.77, 120.57, 21.19. ESI-MS: m/z calcd for
C11H10O6 (M + H+) 239.3, found 239.3.
4-acetoxybenzoic acid (4c)
1
White solid, yield: 97%. H NMR (400 MHz, CDCl3) δ 10.85 (s, 1H), 8.18 (d, J
= 8.3 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 2.36 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
171.32, 168.75, 154.95, 131.81, 126.76, 121.70, 21.10. ESI-MS: m/z calcd for
C9H8O4 (M + H+) 181.7, found 181.8.
Methyl S-allyl-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (8a)
White solid, yield: 76%. 1H NMR (400 MHz, CDCl3) δ 7.06 (s, 2H), 6.91 (d, 1H),
5.83 – 5.70 (m, 1H), 5.14 (m, 1H), 5.12 (m, 1H), 4.99 (m, 1H), 3.91 (d, 9H), 3.83 (s,
3H), 3.16 (m, 3H), 3.13 – 2.98 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 171.45,
166.61, 153.19, 141.31, 133.45, 128.96, 117.96, 104.54, 60.85, 56.30, 52.69, 52.12,
35.10, 32.60. ESI-MS: m/z calcd for C17H23NO6S (M + H+) 370.4, found 370.4.
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ACCEPTED MANUSCRIPT
Methyl S-(but-3-en-1-yl)-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (8b)
White solid, yield: 78%. 1H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 1.7 Hz, 2H),
6.97 (d, J = 7.2 Hz, 1H), 5.78 (m, 1H), 5.09 – 4.96 (m, 3H), 3.89 (d, 9H), 3.81 (s, 3H),
3.20 – 3.07 (m, 2H), 2.61 (m, 2H), 2.33 (q, 2H). 13C NMR (101 MHz, CDCl3) δ
171.40, 166.66, 153.17, 141.32, 136.07, 128.95, 116.21, 104.57, 60.82, 56.28, 52.68,
52.32, 34.12, 33.66, 31.95. ESI-MS: m/z calcd for C18H25NO6S (M + H+) 384.6,
found 384.5.
Methyl S-(pent-4-en-1-yl)-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (SMU-8c)
White solid, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 1.7 Hz, 2H),
6.94 (d, J = 7.3 Hz, 1H), 5.75 (m, 1H), 5.04 – 4.95 (m, 3H), 3.91 (d, 9H), 3.82 (s, 3H),
3.19 – 3.06 (m, 2H), 2.55 (t, 2H), 2.13 (q, 2H), 1.66 (q, 2H). 13C NMR (101 MHz,
CDCl3) δ 171.42, 166.62, 153.19, 141.34, 137.32, 128.97, 115.34, 104.57, 60.83,
56.30, 52.67, 52.31, 34.11, 32.52, 32.01, 28.58. ESI-MS: m/z calcd for C19H27NO6S
(M + H+) 398.9, found 398.9.
Methyl S-(prop-2-yn-1-yl)-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (8d)
White solid, yield: 74%. 1H NMR (400 MHz, CDCl3) δ 7.07 (s, 2H), 6.94 (m, J =
7.3, 3.3 Hz, 1H), 5.06 (m, 1H), 3.91 (d, 9H), 3.84 (s, 3H), 3.41 – 3.32 (m, 2H), 3.32 –
3.20 (m, 2H), 2.30 (t, 1H). 13C NMR (101 MHz, CDCl3) δ 171.34, 166.75, 153.18,
141.32, 128.90, 104.57, 79.12, 72.08, 60.85, 56.31, 52.78, 52.10, 33.50, 19.78.
ESI-MS: m/z calcd for C17H21NO6S (M + H+) 368.3, found 368.4.
Methyl S-benzyl-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (8e)
White solid, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 4.4 Hz, 4H),
7.28 – 7.22 (m, 1H), 7.05 (s, 2H), 6.85 (d, J = 7.3 Hz, 1H), 5.04 – 4.98 (m, 1H), 3.93
(s, 6H), 3.92 (s, 3H), 3.81 (s, 3H), 3.76 (s, 2H), 3.10 (m, 1H), 2.99 (m, 1H). 13C NMR
(101 MHz, CDCl3) δ 171.42, 166.67, 153.20, 141.34, 137.48, 128.97, 128.80, 128.54,
127.23, 104.58, 60.86, 56.32, 52.71, 52.08, 36.64, 33.43. ESI-MS: m/z calcd for
C21H25NO6S (M + H+) 420.9, found 420.9.
14

ACCEPTED MANUSCRIPT
Methyl S-(4-fluorobenzyl)-N-(3,4,5-trimethoxybenzoyl)-L-cysteinate (8f)
White solid, yield: 83%. 1H NMR (400 MHz, CDCl3) δ 7.28 – 7.22 (m, 2H), 7.04
(s, 2H), 6.97 (m, 2H), 6.94 – 6.82 (m, 1H), 5.02 – 4.95 (m, 1H), 3.94 – 3.88 (m, 9H),
3.79 (d, 3H), 3.72 (s, 2H), 3.06 (m, 1H), 2.95 (m, 1H). 13C NMR (101 MHz, CDCl3) δ
171.38, 166.64, 163.13, 160.69, 153.19, 141.40, 133.24, 133.21, 130.41, 130.33,
128.85, 115.49, 115.27, 104.60, 60.83, 56.30, 52.71, 52.06, 35.80, 33.34. ESI-MS:
m/z calcd for C21H24FNO6S (M + H+) 420.4, found 420.5.
(R)-5-((3-(but-3-en-1-ylthio)-1-methoxy-1-oxopropan-2-yl)carbamoyl)benzene-1,
2,3-triyl triacetate (9b)
Clear oil, yield: 73%. 1H NMR (400 MHz, CDCl3) δ 7.60 (s, 2H), 6.94 (d, J = 7.4
Hz, 1H), 5.81 (m, 1H), 5.11 – 4.97 (m, 4H), 3.82 (s, 3H), 3.17 – 3.07 (m, 2H), 2.64 –
2.59 (m, 2H), 2.35 (m, 2H), 2.32 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.00,
167.50, 166.38, 164.52, 143.57, 137.62, 136.09, 131.82, 119.81, 116.27, 52.76, 52.33,
34.08, 33.61, 31.97, 20.52, 20.10. ESI-MS: m/z calcd for C21H25NO9S (M + H+) 468.9,
found 468.9.
(R)-5-((3-(benzylthio)-1-methoxy-1-oxopropan-2-yl)carbamoyl)benzene-1,2,3-triy
l triacetate (9e)
Clear oil, yield: 79%. 1H NMR (400 MHz, CDCl3)δ 7.56 (s, 2H), 7.29 (d, J = 4.4
Hz, 4H), 7.26 – 7.18 (m, 1H), 6.88 (d, J = 7.5 Hz, 1H), 4.99 – 4.91 (m, 1H), 3.78 (s,
3H), 3.72 (s, 2H), 3.00 (m, 2H), 2.32 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.00,
167.55, 166.42, 164.58, 143.52, 137.60, 137.47, 131.80, 128.81, 128.57, 127.24,
119.86, 52.73, 52.08, 36.62, 33.24, 20.52, 20.10. ESI-MS: m/z calcd for C24H25NO9S
(M + H+) 504.6, found 504.7.
(R)-5-((3-(allylthio)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,3-phenylene
diacetate (9g)
15

ACCEPTED MANUSCRIPT
Clear oil, yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.52 – 7.42 (m, 2H), 7.15 (t,
J = 1.9 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 5.82 – 5.71 (m, 1H), 5.17 (m, 1H) , 5.11 (m,
1H), 4.98 (q, 1H), 3.83 (s, 3H), 3.15 (d, J = 7.2 Hz, 2H), 3.12 – 2.98 (m, 2H), 2.33 (s,
6H). 13C NMR (101 MHz, CDCl3) δ 171.09, 168.65, 164.99, 151.11, 135.76, 133.42,
119.00, 118.03, 117.87, 52.74, 52.15, 35.15, 32.54, 20.97. ESI-MS: m/z calcd for
C18H21NO7S (M + H+) 396.3, found 396.3.
(R)-5-((1-methoxy-1-oxo-3-(prop-2-yn-1-ylthio)propan-2-yl)carbamoyl)-1,3-phen
ylene diacetate (9j)
Clear oil, yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 2.1 Hz, 2H),
7.14 (t, J = 2.1 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 5.06 (m, 1H), 3.83 (s, 3H), 3.36 –
3.30 (m, 2H), 3.29 – 3.24 (m, 2H), 2.33 (s, 6H), 2.31 (t, 1H). 13C NMR (101 MHz,
CDCl3) δ 170.97, 168.66, 165.16, 151.10, 135.76, 118.99, 117.94, 79.15, 72.21, 52.81,
52.11, 33.58, 20.97, 19.91. ESI-MS: m/z calcd for C18H19NO7S (M + H+) 394.1,
found 394.1.
Methyl N-(4-acetoxybenzoyl)-S-allyl-L-cysteinate (9m)
White solid, White solid, yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.91 – 7.84
(m, 2H), 7.25 – 7.16 (m, 2H), 6.96 (d, J = 7.4 Hz, 1H), 5.84 – 5.69 (m, 1H), 5.16 (m,
1H), 5.09 (m, 1H), 5.04 – 4.97 (m, 1H), 3.82 (s, 3H), 3.15 (m, 2H), 3.12 – 2.97 (m,
2H), 2.34 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.27, 168.86, 166.00, 153.36,
133.44, 131.18, 128.57, 121.80, 117.97, 52.69, 52.07, 35.12, 32.60, 21.06. ESI-MS:
m/z calcd for C16H19NO5S (M + H+) 338.9, found 338.8.
Methyl N-(4-acetoxybenzoyl)-S-(but-3-en-1-yl)-L-cysteinate (9n)
White solid, yield: 83%. 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.3 Hz, 2H),
7.21 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 7.4 Hz, 1H), 5.80 (m, 1H), 5.10 – 5.00 (m, 3H),
3.82 (s, 3H), 3.21 – 3.10 (m, 2H), 2.62 (t, 2H), 2.34 (m, 5H). 13C NMR (101 MHz,
CDCl3) δ 171.23, 168.83, 166.04, 153.38, 136.08, 131.19, 128.57, 121.79, 116.24,
52.70, 52.24, 34.19, 33.66, 31.99, 21.05. ESI-MS: m/z calcd for C17H21NO6S (M + H+)
352.3, found 352.3.
16

ACCEPTED MANUSCRIPT
Methyl S-allyl-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10a)
Clear oil, yield: 83%. 1H NMR (400 MHz, DMSO) δ 9.13 (s, 2H), 8.73 (s, 1H),
8.44 (d, J = 7.7 Hz, 1H), 6.87 (s, 2H), 5.76 (m, 1H), 5.18 – 5.04 (m, 2H), 4.53 (m, 1H),
3.64 (s, 3H), 3.17 (m, 2H), 2.96 – 2.81 (m, 2H). 13C NMR (101 MHz, DMSO) δ
171.93, 166.97, 145.82, 137.05, 134.58, 124.24, 117.81, 107.41, 52.67, 52.43, 34.30,
31.45. ESI-MS: m/z calcd for C14H17NO6S (M + H+) 328.5, found 328.6.
Methyl S-(but-3-en-1-yl)-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10b)
1
Clear oil, yield: 81%. H NMR (400 MHz, DMSO) δ 9.09 (s, 2H), 8.73 (s, 1H),
8.43 (d, J = 7.7 Hz, 1H), 6.87 (s, 2H), 5.83 – 5.75 (m, 1H), 5.09 – 4.99 (m, 2H), 4.53
(m, 1H), 3.65 (s, 3H), 3.01 – 2.90 (m, 2H), 2.60 (t, 2H), 2.29 (m, 2H). 13C NMR (101
MHz, DMSO) δ 171.97, 166.90, 145.81, 137.27, 137.01, 124.20, 116.44, 107.37,
53.03, 52.43, 33.70, 32.61, 31.04. ESI-MS: m/z calcd for C15H19NO6S (M + H+) 342.8,
found 342.9.
Methyl S-(pent-4-en-1-yl)-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10c)
1
Clear oil, yield: 83%. H NMR (400 MHz, DMSO) δ 9.09 (s, 2H), 8.82 – 8.63
(m, 1H), 8.43 (d, J = 7.7 Hz, 1H), 6.87 (s, 2H), 5.84 – 5.74 (m, 1H), 5.05 – 4.95 (m,
2H), 4.52 (m, 1H), 3.65 (s, 3H), 3.00 – 2.90 (m, 2H), 2.54 (d, 2H), 2.09 (m, 2H), 1.59
(q, 2H). 13C NMR (101 MHz, DMSO) δ 171.98, 166.90, 145.81, 138.26, 137.01,
124.21, 115.72, 107.37, 53.07, 52.42, 32.56, 31.13, 28.55. ESI-MS: m/z calcd for
C16H21NO6S (M + H+) 356.2, found 356.3.
Methyl S-(prop-2-yn-1-yl)-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10d)
1
Clear oil, yield: 79%. H NMR (400 MHz, DMSO) δ 9.08 (s, 2H), 8.71 (s, 1H),
8.46 (d, J = 7.8 Hz, 1H), 6.86 (s, 2H), 4.59 (m, 1H), 3.66 (s, 3H), 3.40 (t, 2H), 3.21 –
13
3.13 (m, 2H), 3.02 (m, 1H). C NMR (101 MHz, DMSO) δ 171.84, 166.94, 145.82,
137.05, 124.19, 107.39, 80.54, 74.32, 52.48, 52.28, 32.37, 18.96. ESI-MS: m/z calcd
for C14H15NO6S (M + H+) 326.4, found 326.5.
17

ACCEPTED MANUSCRIPT
Methyl S-benzyl-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10e)
1
Clear oil, yield: 74%. H NMR (400 MHz, DMSO) δ 9.08 (s, 2H), 8.71 (s, 1H),
8.45 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 5.2 Hz, 4H), 7.27 – 7.22 (m, 1H), 6.88 (s, 2H),
4.61 – 4.55 (m, 1H), 3.77 (s, 2H), 3.64 (s, 3H), 2.90 – 2.82 (m, 2H). 13C NMR (101
MHz, DMSO) δ 171.92, 166.95, 145.82, 138.57, 137.03, 129.28, 128.79, 127.28,
124.25, 107.40, 52.59, 52.42, 35.55, 32.19. ESI-MS: m/z calcd for C18H19NO6S (M +
H+) 378.2, found 378.3.
Methyl S-(4-fluorobenzyl)-N-(3,4,5-trihydroxybenzoyl)-L-cysteinate (10f)
1
Clear oil, yield: 71%. H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.74 (s, 1H),
8.47 (d, J = 7.7 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.17 – 7.12 (m, 2H), 6.87 (s, 2H), 4.56
(m, 1H), 3.77 (s, 2H), 3.64 (s, 3H), 2.88 – 2.80 (m, 2H). 13C NMR (101 MHz, DMSO)
δ 171.91, 166.95, 162.74, 145.82, 137.03, 134.88, 131.21, 131.13, 124.21, 115.65,
115.44, 107.38, 52.58, 52.44, 34.66, 32.12. ESI-MS: m/z calcd for C18H18FNO6S (M
+ H+) 396.4, found 396.5.
Methyl S-allyl-N-(3,5-dihydroxybenzoyl)-L-cysteinate (10g)
1
Clear oil, yield: 70%. H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 8.63 (d, J =
7.7 Hz, 1H), 6.71 (d, J = 2.1 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 5.77 (m, 1H), 5.17 –
5.09 (m, 2H), 4.55 (m, 1H), 3.66 (s, 3H), 3.20 – 3.17 (m, 2H), 2.94 (m, 1H), 2.86 (m,
1H). 13C NMR (101 MHz, DMSO) δ 171.70, 167.07, 158.66, 136.19, 134.63, 117.82,
106.05, 105.84, 52.65, 52.49, 34.27, 31.39. ESI-MS: m/z calcd for C14H17NO5S (M +
H+) 312.5, found 312.6.
Methyl S-(but-3-en-1-yl)-N-(3,5-dihydroxybenzoyl)-L-cysteinate (10h)
Clear oil, yield: 72%. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 2H), 8.63 (d, J =
7.7 Hz, 1H), 6.71 (d, J = 2.1 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 5.80 (m, 1H), 5.11 (m,
1H), 4.98 (m, 1H), 4.55 (m, 1H), 3.66 (s, 3H), 3.04 – 2.89 (m, 2H), 2.61 (t, 2H), 2.28
(m, 2H). 13C NMR (101 MHz, DMSO) δ 171.74, 167.05, 158.66, 137.25, 136.19,
18

ACCEPTED MANUSCRIPT
116.45, 106.03, 105.80, 53.02, 52.49, 33.69, 32.54, 31.02. ESI-MS: m/z calcd for
C15H19NO5S (M + H+) 326.8, found 326.8.
Methyl N-(3,5-dihydroxybenzoyl)-S-(pent-4-en-1-yl)-L-cysteinate (10i)
1
Clear oil, yield: 76%. H NMR (400 MHz, DMSO) δ 9.50 (s, 2H), 8.62 (d, J =
7.7 Hz, 1H), 6.71 (d, J = 2.1 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 5.79 (m, 1H), 5.05 (m,
1H), 4.95 (m, 1H), 4.54 (m 1H), 3.66 (s, 3H), 2.95 (m, 2H), 2.55 (d, 2H), 2.13 – 2.05
13
(m, 2H), 1.60 (m, 2H). C NMR (101 MHz, DMSO) δ 171.75, 167.04, 158.66,
138.25, 136.19, 115.72, 106.03, 105.79, 53.06, 52.48, 32.56, 32.50, 31.11, 28.55.
ESI-MS: m/z calcd for C16H21NO5S (M + H+) 340.2, found 340.3.
Methyl N-(3,5-dihydroxybenzoyl)-S-(prop-2-yn-1-yl)-L-cysteinate (10j)
Clear oil, yield: 78%. 1H NMR (400 MHz, DMSO) δ 9.48 (s, 2H), 8.65 (d, J =
7.7 Hz, 1H), 6.71 (d, J = 2.2 Hz, 2H), 6.38 (s, 1H), 4.61 (m, 1H), 3.67 (s, 3H), 3.46 –
13
3.36 (m, 2H), 3.22 – 3.13 (m, 2H), 3.02 (m, 1H). C NMR (101 MHz, DMSO) δ
171.61, 167.07, 158.67, 136.17, 106.04, 105.83, 80.52, 74.46, 52.63, 52.31, 32.31,
18.97. ESI-MS: m/z calcd for C14H15NO5S (M + H+) 340.2, found 340.3.
Methyl S-benzyl-N-(3,5-dihydroxybenzoyl)-L-cysteinate (10k)
1
Clear oil, yield: 76%. H NMR (400 MHz, DMSO) δ 9.49 (d, J = 1.8 Hz, 2H),
8.64 (d, J = 7.7 Hz, 1H), 7.35 – 7.21 (m, 5H), 6.72 (d, J = 2.2 Hz, 2H), 6.38 (t, J = 1.9
Hz, 1H), 4.60 (q, 1H), 3.78 (s, 2H), 3.65 (s, 3H), 2.92 – 2.81 (m, 2H). 13C NMR (101
MHz, DMSO) δ 171.68, 167.09, 158.67, 138.54, 136.23, 129.27, 128.79, 127.29,
106.06, 105.83, 52.58, 52.48, 35.55, 32.16. ESI-MS: m/z calcd for C18H19NO5S (M +
H+) 362.3, found 362.3.
Methyl N-(3,5-dihydroxybenzoyl)-S-(4-fluorobenzyl)-L-cysteinate (10l)
1
Clear oil, yield: 74%. H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 8.64 (d, J =
7.7 Hz, 1H), 7.38 – 7.34 (m, 2H), 7.17 – 7.12 (m, 2H), 6.71 (d, J = 2.2 Hz, 2H), 6.38
(t, J = 2.2 Hz, 1H), 4.58 (m, 1H), 3.78 (s, 2H), 3.65 (s, 3H), 2.89 (m, 1H), 2.83 (d, 1H).
19

ACCEPTED MANUSCRIPT
13C NMR (101 MHz, DMSO) δ 171.66, 167.09, 158.67, 131.20, 131.12, 115.65,
115.44, 106.05, 52.49, 34.68. ESI-MS: m/z calcd for C18H18FNO5S (M + H+) 380.5,
found 380.6.
Methyl S-allyl-N-(4-hydroxybenzoyl)-L-cysteinate (10m)
White solid, yield: 77%. 1H NMR (400 MHz, DMSO) δ 10.03 (s, 1H), 8.60 (d, J
= 7.7 Hz, 1H), 7.76 (d, J = 8.3 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H), 5.83 – 5.71 (m, 1H),
5.12 (m, 2H), 4.58 (m, 1H), 3.66 (s, 3H), 3.19 (d, 2H), 2.98 – 2.82 (m, 2H). 13C NMR
(101 MHz, DMSO) δ 171.91, 166.43, 160.85, 134.65, 129.84, 124.63, 117.81, 115.23,
52.68, 52.45, 34.30, 31.51. ESI-MS: m/z calcd for C14H17NO4S (M + H+) 296.6,
found 296.5.
Methyl S-(but-3-en-1-yl)-N-(4-hydroxybenzoyl)-L-cysteinate (10n)
White solid, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.77 – 7.69 (m, 2H), 7.01
– 6.96 (m, 1H), 6.93 – 6.86 (m, 2H), 5.79 (m, 1H), 5.10 – 4.99 (m, 3H), 3.82 (s, 3H),
3.20 – 3.10 (m, 2H), 2.62 (t, 2H), 2.37 – 2.30 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
171.50, 167.14, 159.75, 136.07, 129.17, 125.12, 116.27, 115.51, 52.76, 52.27, 34.21,
33.64, 31.98. ESI-MS: m/z calcd for C15H19NO4S (M + H+) 296.5, found 296.5.
Methyl N-(4-hydroxybenzoyl)-S-(pent-4-en-1-yl)-L-cysteinate (10o)
White solid, yield: 81%. 1H NMR (400 MHz, CDCl3) δ 7.74 – 7.70 (m, 2H), 6.99
(d, J = 7.4 Hz, 1H), 6.91 – 6.87 (m, 2H), 5.75 (m, 1H), 5.00 (m, 3H), 3.82 (s, 3H),
3.19 – 3.08 (m, 2H), 2.60 – 2.50 (m, 2H), 2.17 – 2.09 (m, 2H), 1.67 (m, 2H). 13C
NMR (101 MHz, CDCl3) δ 171.52, 167.18, 159.84, 137.35, 129.16, 125.04, 115.52,
115.36, 52.75, 52.30, 34.16, 32.52, 32.03, 28.54. ESI-MS: m/z calcd for C16H21NO4S
(M + H+) 325.2, found 325.3.
Methyl N-(4-hydroxybenzoyl)-S-(prop-2-yn-1-yl)-L-cysteinate (10p)
White solid, yield: 83%. 1H NMR (400 MHz, DMSO) δ 10.03 (s, 1H), 8.62 (d, J
= 7.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 4.65 (m, 1H), 3.67 (s,
20

ACCEPTED MANUSCRIPT
3H), 3.41 (t, 2H), 3.29 – 3.11 (m, 2H), 3.03 (m, 1H). 13C NMR (101 MHz, DMSO) δ
171.82, 166.47, 160.87, 129.84, 124.59, 115.24, 80.53, 74.34, 52.51, 52.34, 32.42,
19.00. ESI-MS: m/z calcd for C14H15NO4S (M + H+) 294.4, found 294.5.
Methyl S-benzyl-N-(4-hydroxybenzoyl)-L-cysteinate (10q)
White solid, yield: 85%. 1H NMR (400 MHz, DMSO) δ 10.03 (s, 1H), 8.61 (d, J
= 7.7 Hz, 1H), 7.80 – 7.74 (m, 2H), 7.34 – 7.23 (m, 5H), 6.86 – 6.80 (m, 2H), 4.68 –
4.60 (m, 1H), 3.78 (s, 2H), 3.65 (s, 3H), 2.93 – 2.82 (m, 2H). 13C NMR (101 MHz,
DMSO) δ 171.89, 166.46, 160.85, 138.55, 129.85, 129.27, 128.79, 127.29, 124.63,
115.23, 52.61, 52.46, 35.57, 32.25. ESI-MS: m/z calcd for C18H19NO4S (M + H+)
346.1, found 346.2.
Methyl S-(4-fluorobenzyl)-N-(4-hydroxybenzoyl)-L-cysteinate (10r)
White solid, yield: 83%. 1H NMR (400 MHz, DMSO) δ 10.03 (d, J = 1.2 Hz,
1H), 8.60 (d, J = 7.6 Hz, 1H), 7.76 (m, J = 8.7, 2.1 Hz, 2H), 7.37 – 7.33 (m, 2H), 7.17
– 7.12 (m, 2H), 6.83 (m, J = 8.7, 1.8 Hz, 2H), 4.62 (m, 1H), 3.78 (s, 2H), 3.65 (s, 3H),
2.93 – 2.81 (m, 2H). 13C NMR (101 MHz, DMSO) δ 171.86, 171.64, 166.47, 162.75,
160.86, 160.34, 134.86, 134.82, 131.19, 131.11, 129.85, 124.63, 115.65, 115.44,
115.24, 52.62, 52.45, 34.73, 32.22. ESI-MS: m/z calcd for C18H18FNO4S (M + H+)
364.2, found 364.2.
5.2. Biology assays
5.2.1. NO activation in Raw 264.7 cells
The free nitrite was monitored by using a modification of the colorimetric Griess
assay. Briefly, dissolving 85% strong phosphoric acid (3 mL) and sulfanilic acid (0.5
g) in 40 mL water to make 50 mL obtained substrate solution A, then dissolving
naphthyl ethylenediamine dihydrochloride (0.05 g) in 50 mL water to make substrate
solution B. RAW 264.7 cells (mouse leukemic monocyte macrophage cell line) were
cultured in medium of DMEM supplemented with 10% FBS, penicillin (100 U/ml),
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and streptomycin (100 mg/ml), seeded in 96-well plates at 80,000 cells of density per
well, and pre-incubated for 24 hours at 37°C in a 5% CO2 humidified incubator.
Removed nonadherent cells away and replaced with fresh unsupplemented DMEM
medium after 24 hours incubation. The adherent macrophages were treated with 200
ng/mL Pam3CSK4 (InvivoGen) and different concentrations of compounds. Cells were
then incubated for an additional 24 hours. After incubation, 50 µL of medium was
collected to a transparent 96-well plate, then added 50 µL substrate solution A and
incubated for 10 minutes in dark. After that, another 50 µL substrate solution B was
added and incubated in 37 °C incubator for 10 min. Finally, absorbance was detected
at 560 nm (OD560) by a microplate reader (Thermo Scientific) and afforded the
content of nitric oxide by Origin 9.0 software.
5.2.2 Secreted embryonic alkaline phosphatase (SEAP) and specificity
experiments.
HEK-blue hTLR2 or 3 or 4 cells (InvivoGen) were cultured in 96-well plates (4
× 104 cells per well) (Thermo Scientific) at 37°C with condition of 5% CO2 for 24
hours on the first day in 200 µL DMEM, supplemented with 10% FBS and 1%
penicillin/streptomycin. After 24 hours, nonadherent cells and medium were removed
and replaced with unsupplemented fresh DMEM medium. The cells were treated with
indicated concentrations of compounds and 20 ng/ml Pam3CSK4 (InvivoGen) in 200
µL DMEM totally. After added compounds, cells were cultured for another 24 hours.
On the third day, a sample buffer (50 µL) was collected and transferred from each
well of the cell culture supernatants to a transparent 96-well plate. Each well was
treated with 50 µL of QUANTI-Blue (InvivoGen) buffer and incubated at 37°C in
dark place. Then measure the purple color by using a plate reader at an absorbance of
620 nm (OD620) at 15-30 minutes. Finally, analyze the data by using Origin 9.0
software.
5.2.3. Cell toxicity assays
In a 96-well plate, 80,000 RAW 264.7 cells were seeded in 200 µL of growth
22

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medium [DMEM supplemented with 10% FBS, penicillin (100 U/ml), and
streptomycin (100 mg/ml)]. The plates were incubated at 37°C, 5% CO2 for 24 hours.
In the next 24 hours of treatment, the medium was changed to DMEM only and added
positive control Pam3CSK4 (final conc. 200 ng/ml), as well as indicated concentration
compounds to 200 µL totally, incubated for 24 h. Then, 20 µL (5 mg/ml in PBS) of
MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide] solution was
added to each well and incubated (37°C, 5% CO2) for another 4 hours to allow the
MTT to be metabolized. The medium was removed and the plate was dried on paper
towels to remove any residue. Then, 150 µL of DMSO was added in each well and
shaken continuously for 40 min. When all the MTT metabolic products were
dissolved, results were read by spectrophotometer at 450 nm (OD450). Optical
density should be directly correlated with cell quantity.
5.2.4. Cytokine ELISA assays
Raw 264.7 cells or human PBMC cells were seeded in twelve-well plates at a
density of 1×106 cells per well with 0.5 mL of medium DMEM medium supplemented
with 10% FBS, and 1% penicillin/streptomycin. The cells were treated with indicated
concentrations of SMU-8c and 50 ng/ml Pam3CSK4 (Invivogen) as positive control
o
and incubated for 24 h at 37 C in a 5% CO2 humidified incubator. The cell culture
supernatants were collected and frozen at -80 oC until measurement. THP-1 cells at a
density of 2.5 × 105 cells/mL were differentiated by treatment with 100 nM PMA
(Sigma) in RPMI cell culture medium, containing 10% FBS, 1% penicillin and
streptomycin for 24 h at 37 °C in a 5% CO2 humidified incubator. After that, cells
were washed with PBS and cultured in fresh RPMI cell culture medium (containing
10% FBS, 1% pen/strep) for 24 h. Then, change the medium to RPMI only and treat
with 50 ng/ml Pam3CSK4 and indicated concentration of compound for 4 hours. The
cell culture supernatants were collected and froze at -80 °C until measurement. The
level of cytokine TNF-α was determined using recombinant human-cytokine
standards, cytokine-specific capture antibodies and detection antibodies according to
the commercially available ELISA kit (BD Biosciences) with each sample for
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triplicate.
5.2.5. Immunoblot analysis
In the first day, HEK-Blue hTLR2 cells were seeded in 6-well plate (Thermo
Scientific) at density of 1.5 × 106 per well in 3 mL DMEM (supplemented with 10%
FBS, 10 × penicillin/streptomycin, and 10 × Lglutamine) and incubated for 24 hours.
In the second day, removed and replaced the medium with DMEM medium only to 3
mL totally and treated the cells with different concentration of SMU-8c and incubated
for another 24 hours. In the third day, the cells were harvested and lysed with 150 µL
cell lysis buffers (PIPA mixed with PMSF at ratio of 50:1 before use, Boster). Cell
lysates of equal amount were denatured, separated by sodium dodecyl sulfate
polycrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose
membrane. Then use PBS (containing 5% nonfat milk) to block the membrane for 2 h ,
and then incubated with the primary antibody of TLR2 overnight at 4 °C, followed
with incubating by a horseradish peroxidase conjugated secondary antibody. The
immunoblots were visualized by enhanced exposure machine. During the operation,
the following primary and secondary antibodies were employed: mouse-GAPDH
(1:2000, Solarbio, M1000110), rabbit-TLR2 (1:2000, Cell Signaling, 12276),
HRP-goat-anti-mouse-IgG (1:2500, Boster, BA1050) and HRP-goat-anti-rabbit-IgG
(1:1000, Boster, BA1054).
5.2.6. Immunoprecipitation assay
Human peripheral blood mononuclear cells (PBMC) with cells density of
1,000,000 cells/mL were treated with vehicle, Pam3CSK4 (50 ng/mL) or Pam2CSK4
(50 ng/mL) and indicated concentrations SMU-8c in a 6-well plate for 24 h. Cells
were lysed with 200 µL lysis buffer (containing protease and phosphatase inhibitors).
Then, 50 µL of the cell lysates were mixed with 1 µL of anti-TLR2 antibody and
incubated overnight at 4 °C under constant rotation. To recover immunoprecipitated
complexes, 10 µL of protein A agarose beads (EarthOx, nf1501) were added to the
lysates and incubated for additional 4 h of incubation at 4 °C under constant rotation.
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The beads were harvested by centrifugation, and the supernatant was discarded. The
beads were washed with cell lysis buffers (PIPA mixed with PMSF at ratio of 50:1
before use, Boster) for three times to remove nonspecific binding protein. The beads
with bound proteins were eluted by boiling in 5X SDS loading buffer at 95 °C for 5
min. The eluted proteins were subjected to electrophoresis SDS-PAGE [36]. The
signals of bands were detected by probing with anti-TLR1, anti-TLR2 antibodies,
anti-MyD88 antibodies, anti-TLR6 antibodies. During the operation, the following
primary and secondary antibodies were employed: rabbit-TLR1 (1:2000, Cell
Signaling, 2209), rabbit-TLR2 (1:2000, Cell Signaling, 12276), rabbit-TLR6 (1:1000,
Proteintech, 22240-1-AP), rabbit-MyD88 (1:2000, Proteintech, 23230-1-AP) and
HRP-goat-anti-rabbit-IgG (1:1000, Boster, BA1054).
5.2.7. Molecular docking modeling
Compound SMU-8c was docked into the TLR1 and TLR2 binding domain (PDB:
2Z7X) using Glide 7.4. The molecule is created, as appropriate, with multiple
protonation and tautomeric states. The TLR1/2 conformations were prepared using
standard Glide protocols. This includes addition of hydrogens, restrained
energy-minimizations of the protein structure with the Optimized Potentials for
Liquid Simulations-All Atom (OPLS-AA) force field, and finally setting up the Glide
grids using the Protein and Ligand Preparation Module [37].
Acknowledgments
This work was supported by National Natural Science Foundation of China (No.
81773558 (Cheng, K), 31171281 (Chen, K)), Youth Pearl River Scholar Program of
Guangdong Province (No. C1034007), Natural Science Foundation of Guangdong
Province (2018B030312010), Guangdong Provincial Key Laboratory of New Drug
Screening Open Fund (No. GDKLNDS-2018OF001), Major International
Cooperation Projects in Universities in Guangdong Province (2015KGJHZ020), and
Science and Technology Program of Guangzhou, China (201904010380).
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Appendix A. Supplementary data
Supporting Information Available: Supplementary data for the representative
NMR spectra associated with this article can be found in the online version at http://
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