Identification of novel N-acetylcysteine derivatives for the treatment of hepatocellular injury

Article abstract of DOI:10.1039/c7md00409e

New anti-hepatocellular injury drugs with better curative effects and fewer side effects are urgently needed at present. In this study, a series of novel N-acetylcysteine (NAC) derivatives were designed, synthesized and biologically evaluated for their anti-hepatocellular injury activities against two different cell models. In the biological evaluation against hydrogen peroxide (H₂O₂)-induced LO2 hepatocytes, half of the target compounds exhibited moderate to potent activities in improving the model cell viability, and two compounds (6a and 6b) displayed more potent activities in decreasing malondialdehyde (MDA) levels than the positive control NAC. In further 4-acetamidophenol (APAP)-induced LO2 cell experiment, compounds 6a and 6b could not only improve the cell viability but also significantly reduce the secretion of MDA. Additionally, compound 6a displayed excellent Caco-2 permeability and oral bioavailability in rats. All these experimental results suggested that compounds 6a and 6b could serve as potential lead molecules for further development of anti-hepatocellular injury drugs.

Full text of DOI:10.1039/c7md00409e

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1
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Identification of novel N-acetylcysteine derivatives for the treatment of hepatocellular injury
a, §
a, §
a
b
a
a
a
Shourong Liu , Yanmei Zhao , Ruoyu He , Limin Kong , Jianjun Xi , Jingjing Sun , Yidan Shao ,
a
a,
a,
Xuwang Pan , Jiankang Zhang *, Rangxiao Zhuang *
a
Department of pharmaceutical Preparation, Hangzhou Xixi Hospital, Hangzhou 310023, Zhejiang
Province,
China.
Eꢀmail:
zhuangrangxiao@sina.com,
zjk0125@yeah.net;
Tel./Fax:
+
86ꢀ571ꢀ8648ꢀ1960 (Rangxiao Zhuang), +86ꢀ571ꢀ8546ꢀ3955 (Jiankang Zhang)
b
Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Zhejiang University,
Hangzhou 310003, Zhejiang Province, China
§
These authors contributed equally to this work.
Abstract: New antiꢀhepatocellular injury drugs with better curative effects and fewer side effects are
urgently needed at present. In this study, a series of novel Nꢀacetylcysteine (NAC) derivatives were
designed, synthesized and biologically evaluated for their antiꢀhepatocellular injury activities against
2 2
two different cell models. In the biological evaluation against hydrogen peroxide (H O ) induced LO2
hepatocytes, half of the target compounds exhibited moderate to potent activities in improving the
model cell viability, and two compounds (6a and 6b) displayed more potent activities in decreasing
malondialdehyde (MDA) level than that of the positive control NAC. In further 4ꢀacetamidophenol
(APAP) induced LO2 cell experiment, compounds 6a and 6b could not only improve the cell viability
but also significantly reduce the secretion of MDA. Additionally, compound 6a displayed excellent
Cacoꢀ2 permeability and oral bioavailability in rats. All these experimental results suggested that
compounds 6a and 6b could be served as potential lead molecules for further development of
antiꢀhepatocellular injury drugs.

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1
.
Introduction
Liver diseases are highly prevalent all over the world. Regardless of different etiologies,
inflammation and oxidative stress (OS) are the most important pathogenetic events in hepatic
1
ꢀ3
diseases . Chemical drugs, reꢀoxidation after hypoxia and all kinds of acute or chronic
inflammation can cause oxidative damage in hepatocytes, accompanied by the changes of cellular
structure and function, which allows the liver cells to produce excessive reactive oxygen species
4
ꢀ6
(
ROS) . Several studies on hepatic diseases indicated that cells have intrinsic antioxidant
mechanisms, such as peroxidases, catalases and superoxide dismutases to scavenge ROS. Among
these antioxidant systems, the most abundant and outstanding cellular thiol antioxidant glutathione
(GSH) exhibits numerous and versatile functions and therefore protects cells against oxidative
7
stress .
As an antioxidant, Nꢀacetylcysteine (NAC) could directly increase the intracellular GSH,
especially on hepatic tissue, and it has been in clinical practice for several decades with relatively
low toxicity and good efficacy. As a small molecule, NAC has been elucidated to interact with
numerous metabolic pathways including, but not limited to, regulation of cell cycle and apoptosis,
carcinogenesis and tumor progression, mutagenesis, gene expression and signal transduction,
8
ꢀ10
immuneꢀmodulation, cytoskeleton and trafficking and mitochondrial functions . The molecular
mechanisms by which NAC exerts its diverse effects are complex and the insignificant routes
under physiological conditions of NAC is serving as a precursor of cysteine for GSH synthesis
11
which helping in the detoxification of reactive metabolites and scavenging of free radicals . The
second important mechanism of NAC attributes to the antiꢀoxidative activity of its sulfhydryl
•
•
•−
group, which has a fast binding rate with OH, NO
2
, CO
3
and thiyl radicals as well as restitutes
1
2
of impaired targets in vital cellular components . At the meantime, the uniqueness of NAC is
most probably due to efficient reduction of disulfide bonds in proteins thus altering their structures
and disrupting their ligand bonding, competition with larger reducible molecules in sterical less
accessible spaces. Possible chemical and biochemical routes involving NAC are summarized in
1
3
Fig. 1 .

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<
Insert Fig. 1>
At present, administration of NAC has been mostly used as a mucolytic agent. While IV NAC is used
for various types of liver injury and early stage of liver failure on the basis of comprehensive treatment
1
4
in order to reduce bilirubin and increase prothrombin activity . According to the literature, the human
plasma terminal halfꢀlife of NAC after a single intravenous administration is 5.6 h where 30% of the
drug is cleared by renal excretion. NAC's oral bioavailability is less than 5%, which is mainly thought
1
5
to be associated with its Nꢀdeacetylation in the intestinal mucosa and first pass metabolism in the liver .
Researches indicated that NAC is negatively charged under the physiological condition and its neutral,
1
6
membrane permeating form, constitutes as little as 0.001% of the total NAC . Therefore, in order to
achieve a certain well clinical efficacy, higher doses and longer treatment cycles should be performed,
which also bring more drug toxicity risks. The side effects that accompany the use of a high dose of IV
NAC includes rash, pruritus, angioedema and bronchospasm, while oral administration of NAC may be
associated with vomiting and diarrhea along with an unpleasant odor.
Therefore, more effective and safer drugs are urgently required, which would be of great value for the
treatment of various diseases, especially for liver injury. To improve the stability and activity of NAC,
structure optimization was performed at the carboxyl, sulfydryl and acetyl groups in this research (Fig.
2
). Firstly, different carboxylic esters of NAC were designed and synthesized to increase the molecular
permeation rate through the biological membranes by adjusting the molecule PKa. Additionally,
methylꢀsubstituted thiol could not only prevent the selfꢀoxidation and improve the stability of the
1
7
compound, but also play antioxidant effect after deꢀmethylation by demethylase in vivo . Finally, in
order to prevent NAC from Nꢀdeacetylation in the intestinal mucosa and first pass metabolism in the
liver, acetyl was replaced with other more stable acyl, which was expected to prolong the compound
retention time in vivo and improve its bioavailability, thus overcome the adverse side effects of this
drug.
<
Insert Fig. 2>
2
. Synthesis
The synthetic route for all the target compounds was outlined in Scheme 1. Firstly, FmocꢀCys(Me)ꢀOH

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treated with corresponding carboxylic acid or ester at the presence of N, Nꢀdiisopropylethylamine
DIEA) and Nꢀmethylmorpholine (NMM) to give the target compounds 2a-e by using standard
(
Fmocꢀchemistry. The target compounds 2a-e were also important intermediates for the next step, which
were then treated with HCl and MeOH to afford the methyl ester analogues 3a-e. As illustrated in
Scheme 1, treatment of 2a-e with 2ꢀtertꢀbutylꢀ1, 3ꢀdiisopropylꢀisourea provided the tertꢀbutyl ester
compounds 4a-e, while reacted with methylamine hydrochloride, cyclohexylamine and aniline afforded
target compounds 5a-c and 6a-c respectively, with proper reagents and conditions. Treatment of 2a-e
with aniline and HATU afforded the target compounds 7a and 7b with the structure of phenyl amide.
Commercially available Nꢀ(tertꢀbutoxycarbonyl)ꢀSꢀmethylꢀLꢀcysteine 8 was converted to phenyl amide
9
1
in the presence of NMM and IBCF at 15 °C which was then deprotected with TFA to afford the amine
0. Subsequently, condensation reaction of compounds 10 and benzoyl chloride provided the target
compound 7c.
<
Insert Scheme 1>
3
. Pharmacology
. 1 Cell viabilities and MDA measurement on H
3
2
2
O induced LO2 cell injury model
2 2
Human hepatocytes cell line, LO2, was used to build hepatocytes injury model by H O treatment. Cell
Counting Kitꢀ8 (CCKꢀ8) was utilized to detect cell proliferation and determine the optimal damage
condition. All the target compounds were screened for their cell viabilities on this model using NAC as
positive control, and the results are illustrated in Fig. 3.
As the results displayed in Fig. 3, compared with the model group, part of the target compounds (2a,
2
b, 2c, 2d, 5c, 6a, 6b and 7a) exhibited moderate to potent cell protective and repair effects against
H O treated LO2 cells. In Fig. 3 (a), compounds 2a-e had a slight improvement on cell viability when
2
2
1
the carboxyl groups were retained and different substituents were introduced in R position, while
compounds 3a-e with carboxylic methyl ester had slight inhibitory effects on cell proliferation.
Additionally, compound 2a could slightly improve the cell viability and reduce the MDA level, which
indicated that the increase of the antioxidant damage ability at the cell level was limited after the
introduction of methyl at R
2
position compared with NAC. In Fig. 3 (b), the inhibition of cell viability
position. On the contrary, the
increased from compound 4a to 4e with tertꢀbutyl ester substituted at R
3

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inhibitory activity decreased from compound 5a to 5c with formamide at R position, which indicated
3
that the structureꢀactivity relationship of these two series of compounds is not clear and compounds
4
aꢀe have a certain degree of cytotoxicities. Among the formamide derivatives (5aꢀc), compound 5c
with phenyl at R position has a certain recovery effect on cell viability, which displayed more potent
1
cell protective activity than compounds 5a and 5b. Compounds 6aꢀc and 7aꢀc with different
substituents at R
a) exhibited more potent cell protective activities than ethyl substituted compounds (6b and 7b), and
compounds with benzene at this position (6c and 7c) displayed the worst potent activities. These data
also suggested that minor changes at R position could greatly influence the cell viabilities of the target
compounds (4aꢀb, 4d, 6a-c and 7a-c) when R was substituted with tertꢀbutyl, phenyl or cyclohexyl.
1
position, displayed the same regularity, in which methyl replaced analogues (6a and
7
1
3
Among all these derivatives (except 2a and 5a), the ones with methyl substituted at R position
1
displayed more potent activities than other ones. The most potent compound 6a, whose cell protective
and repair activity was superior to model group, deserved further study with regard to its application
potential in the treatment of antiꢀhepatic injury.
<
<
<
Insert Fig. 3 (a)>
Insert Fig. 3 (b)>
Insert Fig. 3 (c)>
Based on the results of cell viabilities of all the target compounds, selected analogues (2aꢀe, 5bꢀc, 6aꢀb
and 7a) were screened for their effects on malondialdehyde (MDA) secretion in H treated LO2 cells
2
O
2
by Lipid oxidation (MDA) Assay Kit using NAC as positive control. The results were shown in Fig. 4.
Except for compound 5c, all the selected compounds could decrease the MDA level in the LO2 cells
2 2
injured by H O compared to that of the model group. In particular, compounds 6a, 6b and 7a exhibited
more potent MDA secretion inhibitory activities than that of the positive control, which indicated that
these active compounds could protect injured liver cells through inhibition of MDA secretion.
<
Insert Fig. 4>

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3
.2 Cell viabilities and MDA measurement on APAP induced LO2 cell injury model
To further confirm the cell protective activities of compounds 6a, 6b and 7a on oxidative injured liver
cells, an additional experiment was performed to determine the effects of the three selected compounds
on APAP induced LO2 injured cells. As displayed in Fig. 5, compared with the model group,
compounds 6a and 6b exhibited moderate improvement on cell viability while 7a have almost no effect.
At the same time, effects of compounds 6a, 6b and 7a on MDA secretion in APAP treated LO2 cells
were studied by using Lipid oxidation (MDA) Assay Kit. As shown in Fig. 6, compared with the model
group, compounds 6a, 6b and 7a could significantly reduce the level of MDA in the APAP treated LO2
cells. However, the recovery abilities of compounds 6a and 6b was no better than that of the positive
control NAC.
<
<
Insert Fig. 5>
Insert Fig. 6>
4
. ADME properties
The preliminary ADME profiles of compound 6a were evaluated by determining its Cacoꢀ2
permeability (Table 1) and pharmacokinetic properties (Table 2). As displayed in Table 1, compound
6
a exhibited a better Cacoꢀ2 permeability compared to that of the positive control NAC, which also
validated the initial structural optimization ideas. In addition, as the pharmacokinetic data depicted in
Table 2, the terminal halfꢀlife of compound 6a after a single oral administration was 5.62 h and its oral
bioavailability could reach 52.8% in SD rats.
<
<
Insert Table 1>
Insert Table 2>
4
. Conclusion

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A novel series of NAC derivatives were designed, synthesized and biologically evaluated for their cell
protective activities on H treated LO2 hepatocytes, and half of the derivatives exhibited moderate to
2
O
2
potent activities. Additionally, selected ten compounds were further screened for their effect on MDA
secretion. What encouraged us was that three compounds (6a, 6b and 7a) exhibiting more potent
activities than that of the positive control NAC were successively obtained. In further APAP treated
LO2 cells study, compounds 6a and 6b could not only improve the cell viability but also significantly
reduce the level of MDA. However, the effect of different substituents on the selectivity and
pharmacological activity of target compounds was not very clear in this study, and the main reason for
the unclear structureꢀactivity relationship was that the target compounds were pharmacological
screened on the cell model. The cell viabilities of various target compounds were affected by both the
electronegativity and size of different substituents and the physicochemical properties of these
compounds. If the test was carried out at a molecular level, the structureꢀactivity relationship would be
more explicit, and we will focus on this issue in subsequent research.
In the ADME studies, compound 6a showed excellent Cacoꢀ2 permeability and good oral
bioavailability in rats. Overall, although we did not get compound significantly superior to the
precursor NAC in pharmacodynamics, the membrane permeability and pharmacokinetic profiles of
compound 6a was significantly better than NAC. All these results suggested that compounds 6a could
be a potential lead molecule for further structural optimization and investigation.
5
5
. Experimental procedures
.1 Chemistry, general methods
Mass spectra (MS) were taken in ESI mode on Agilent 1100 LCꢀMS (Agilent, Palo Alto, CA, U.S.A.).
1
13
H NMR and C NMR spectra were recorded on Bruker 400 MHz spectrometers (Bruker Bioscience,
Billerica, MA, USA) with TMS as an internal standard and CDCl or DMSOꢀd as solvents. Coupling
constants (J) were reported in Hertz (Hz). Splitting patterns were designated as singlet (s), broad singlet
brs), doublet (d), double doublet (dd), triplet (t), quartet (q), and multiplet (m). All materials were
3
6
(
obtained from commercial suppliers and were used without further purification. Reaction time and
purity of the products were monitored by TLC on FLUKA silica gel aluminum cards (0.2 mm thickness)
with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200ꢀ300 mesh) from
Qingdao Ocean Chemicals (Qingdao, Shandong, China). All yields were unoptimized and generally
represent the result of a single experiment.

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5
5
.2 Synthetic procedures
.2.1 Method for the preparation of compound NꢀacetylꢀSꢀmethylꢀLꢀcysteine (2a)
Compound 2a was synthesized using standard Fmoc chemistry. The mixture of Chlorotrityl Chloride
CTC) Resin (18.2 mmol), FmocꢀCys(Me)ꢀOH (5.2 g, 41.6 mmol, 0.8 eq) and DCM (200.0 mL) were
stirred at 25 °C in vessel for 12 h under N atmosphere and then DIEA (3.2 eq) was added dropwise
(
2
and stirred for another 2 h. Then, MeOH (20.0 mL) was added and stirred for 30 min, and the mixture
was washed with DMF (10×5.0 ml). Subsequently, 20% piperidine/DMF was drained and reacted for
3
0 min, and the mixture was then washed with DMF again (10×5.0 ml). A solution of 85% DMF/10%
Acetyl acetate/5% NMM (200 mL) was added and reacted under N atmosphere for 0.5 h. Finally, 20%
2
piperidine in DMF was used for Fmoc deprotection, and the reaction lasted for 30 min. The coupling
reaction was monitored by ninhydrin test, and the resin was washed with DMF (10×5.0 ml). In peptide
cleavage and purification phase, cleavage buffer (80% DCM/ 20% 1, 1, 1, 3, 3,
3
ꢀhexafluoroꢀ2ꢀpropanol (HFIP)) was added to the flask at room temperature for 0.5 h. Then the
reaction mixture was filtered and the HFIPꢀmixture was concentrated under reduced pressure to remove
2
solvent. The crude peptide was purified by PreHPLC (A: 0.1% TFA in H O, B: acetonitrile) to give the
compound 2a.
1
White solid; Yield: 91%; Purity: 95%; H NMR (400MHz, d ꢀDMSO): δ = 8.23 (d, 1H, J = 8.1 Hz,
6
NH), 4.40 (m, 1H, J = 8.3, 5.0 Hz, CH), 2.77 (ddd, 2H, J = 22.0, 13.6, 6.7 Hz, CH
2
), 2.07 (s, 3H, CH
); C NMR (101MHz, DMSO): δ = 172.78, 169.77, 52.01, 35.54, 22.82, 15.68;
S M + H).
3
) ,
1
3
1
.86 (s, 3H, CH
3
+
HRMS (ES ) m/z 178.0452 (178.0460 Calcd for C
6
H11NO
3
5
.2.2 Preparation of compounds 2bꢀe
Peptides 2bꢀe were also synthesized using standard Fmoc chemistry. The mixture of CTC Resin (18.2
mmol), FmocꢀCys(Me)ꢀOH (5.2 g, 41.6 mmol, 0.8 eq) and DCM (200.0 mL) were stirred at 25 °C in
vessel for 12 h under N atmosphere and then DIEA (3.2 eq) was added dropwise and stirred for 2 h.
2
MeOH (20.0 mL) was added and mixed for 30 min, and the mixture was washed with DMF (10×5.0
ml). After that, 20% piperidine/ DMF was drained and reacted for 30 min, and washed with DMF again
(
10×5.0 ml), and then corresponding acid was added and mixed for 30 s. The activation buffer was
added under N atmosphere for 0.5 h. Finally, 20% piperidine in DMF was used for Fmoc deprotection
for 30 min. The coupling reaction was monitored by ninhydrin test, and the resin was washed with
2

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DMF (10×5.0 ml). In peptide cleavage and purification phase, cleavage buffer (80% DCM/ 20% HFIP)
was added to the flask at room temperature for 0.5 h. Then the reaction mixture was filtered and the
HFIPꢀmixture was concentrated under reduced pressure to remove solvent. The crude peptides were
purified by PreHPLC (A: 0.1% TFA in H O, B: acetonitrile) to give the compounds 2b-e.
2
SꢀmethylꢀNꢀpropionylꢀLꢀcysteine (2b)
1
White solid; Yield: 80%; Purity: 96%; H NMR (400MHz, d
6
ꢀDMSO): δ = 8.13 (d, 1H, J = 8.1 Hz,
), 2.14 (q, 2H,
); C NMR (101MHz, DMSO): δ =
NH), 4.40 (td, 1H, J = 8.3, 5.0 Hz, CH), 2.77 (ddd, 2H, J = 22.2, 13.7, 6.8 Hz, CH
2
J
13
=
7.6 Hz, CH
73.47, 172.84, 51.94, 35.56, 28.64, 15.69, 10.24; HRMS (ES ) m/z 192.0608 (192.0616 Calcd for
S M + H).
2
), 2.07 (s, 3H, CH
3
), 0.99 (t, 3H, J = 7.6 Hz, CH
3
+
1
7 3
C H13NO
Nꢀ(cyclohexanecarbonyl)ꢀSꢀmethylꢀLꢀcysteine (2c)
1
White solid; Yield: 86%; Purity: 95%; H NMR (400MHz, d
6
ꢀDMSO): δ = 7.15 (d, 1H, J = 6.4 Hz,
), 2.11 (t, 1H,
11.1 Hz, CH), 1.97 (s, 3H, CH ), 1.77 – 1.53 (m, 5H, CyclohexaneꢀH), 1.35 – 1.10 (m, 5H,
NH), 3.87 (dd, 1H, J = 10.9, 5.4 Hz, CH), 2.83 (ddd, 2H, J = 18.5, 13.1, 5.0 Hz, CH
2
J
=
3
1
3
CyclohexaneꢀH); C NMR (101MHz, DMSO): δ = 174.38, 173.14, 53.95, 44.66, 37.50, 30.06, 29.64,
+
2
6.03, 25.77, 25.71, 16.16; HRMS (ES ) m/z 246.1090 (246.1086 Calcd for C11
H19NO
3
S M + H).
NꢀbenzoylꢀSꢀmethylꢀLꢀcysteine (2d)
White solid; Yield: 81%; Purity: 95%; H NMR (400MHz, d
NH), 7.84 – 7.72 (m, 2H, ArꢀH), 7.58 – 7.38 (m, 3H, ArꢀH), 4.10 (dd, 1H, J = 10.3, 6.1 Hz, CH), 2.98
1
6
ꢀDMSO): δ = 7.95 (d, 1H, J = 6.4 Hz,
1
3
(ddd, 2H, J = 19.2, 13.2, 5.1 Hz, CH
2
), 2.01 (s, 3H, CH
3
); C NMR (101MHz, DMSO): δ = 173.11,
+
1
65.60, 135.50, 131.43, 128.83, 127.35, 54.57, 37.34, 15.99; HRMS (ES ) m/z 240.0604 (240.0616
3
Calcd for C11H13NO S M + H).
Nꢀ(4ꢀfluorobenzoyl)ꢀSꢀmethylꢀLꢀcysteine (2e)
1
White solid; Yield: 81%; Purity: 95%; H NMR (400MHz, d ꢀDMSO): δ = 7.98 (d, 1H, J = 6.6 Hz,
6
NH), 7.88 (dd, 2H, J = 8.7, 5.6 Hz, ArꢀH), 7.29 (t, 2H, J = 8.9 Hz, ArꢀH), 4.12 (dd, 1H, J = 8.5, 4.5 Hz,
13
CH), 2.96 (ddd, 2H, J = 19.8, 13.2, 5.4 Hz, CH
2
), 2.01 (s, 3H, CH
3
); C NMR (101MHz, DMSO): δ =
+
1
72.96, 164.61, 130.04, 129.96, 115.79, 115.57, 54.64, 37.37, 15.91; HRMS (ES ) m/z 258.0528
(258.0522 Calcd for C11
H12NO
3
S M + H).
5
.2.3 General methods for preparation of the target compounds 3a-e
An appropriate intermediates 2a-2e (1.1 mmol) in HCl/ MeOH (4 M, 5.0 mL) was stirred at 15 °C for 3

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h. The solvent was removed under reduced pressure and the residue was purified by preꢀHPLC (acid
condition, TFA) to give desired compounds 3a-e.
methyl NꢀacetylꢀSꢀmethylꢀLꢀcysteinate (3a)
1
White solid; Yield: 78%; Purity: 97%; H NMR (400 MHz, MeOD) : δ = 4.65 (dd, 1H, J = 8.2, 5.2 Hz,
CH), 3.75 (s, 3H, CH
3
), 2.89 (ddd, 2H, J = 22.2, 13.9, 6.8 Hz, CH
2 3
), 2.13 (s, 3H, CH ), 2.01 (s, 3H,
1
3
CH
3
); C NMR (101MHz, DMSO): δ = 171.87, 169.85, 52.48, 51.98, 35.24, 22.73, 15.58; HRMS
+
(
7 3
ES ) m/z 192.0610 (192.0616 Calcd for C H13NO S M + H).
methyl SꢀmethylꢀNꢀpropionylꢀLꢀcysteinate (3b)
1
White solid; Yield: 84%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 4.65 (dd, 1H, J = 8.4, 5.2 Hz,
CH), 3.75 (s, 3H, CH
3
), 2.89 (ddd, 2H, J = 22.3, 13.9, 6.8 Hz, CH
2
), 2.29 (q, 2H, J = 7.6 Hz, , CH
2
),
1
3
2
5
.13 (s, 3H, CH ), 1.15 (t, J = 7.6 Hz, 3H, CH ); C NMR (101MHz, DMSO): δ = 173.56, 171.93,
3
3
+
8 3
2.46, 51.94, 35.25, 28.56, 15.59, 10.17; HRMS (ES ) m/z 206.0787 (206.0773 Calcd for C H15NO S
M + H).
methyl Nꢀ(cyclohexanecarbonyl)ꢀSꢀmethylꢀLꢀcysteinate (3c)
1
White solid; Yield: 86%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 4.62 (dd, 1H, J = 8.6, 5.2 Hz,
CH), 3.75 (s, 3H, CH
3 2
), 2.89 (ddd, 2H, J = 22.5, 13.9, 6.9 Hz, CH ), 2.29 (tt, 1H, J = 11.7, 3.3 Hz, CH),
1
3
1
.88 – 1.67 (m, 5H, CyclohexaneꢀH), 1.53 – 1.21 (m, 5H, CyclohexaneꢀH); C NMR (101MHz,
DMSO): δ = 175.83, 171.96, 52.44, 51.78, 44.00, 35.19, 29.62, 29.46, 25.90, 25.65, 25.16, 15.56;
+
HRMS (ES ) m/z 260.1228 (260.1242 Calcd for C12
H21NO
3
S M + H).
methyl NꢀbenzoylꢀSꢀmethylꢀLꢀcysteinate (3d)
1
White solid; Yield: 81%; Purity: 98%; H NMR (400 MHz, MeOD) : δ = 8.75 (d, 1H, J = 7.0 Hz, NH),
7
.87 (dd, 2H, J = 5.3, 3.3 Hz, ArꢀH), 7.62 – 7.54 (m, 1H, , ArꢀH), 7.54 – 7.43 (m, 2H, , ArꢀH), 4.87 –
.82 (m, 1H, CH), 3.79 (s, 3H, CH ), 3.05 (ddd, 2H, J = 23.0, 13.9, 7.1 Hz, CH ), 2.17 (s, 3H, CH );
4
3
2
3
13
C NMR (101MHz, DMSO): δ = 171.85, 166.91, 134.04, 132.07, 128.82, 127.91, 52.59, 52.57, 34.85,
+
1
5.45; HRMS (ES ) m/z 254.0763 (254.0773 Calcd for C12
H
15NO
3
S M + H).
methyl Nꢀ(4ꢀfluorobenzoyl)ꢀSꢀmethylꢀLꢀcysteinate (3e)
1
White solid; Yield: 79%; Purity: 98%; H NMR (400 MHz, MeOD) : δ = 8.78 (d, 1H, J = 7.2 Hz, NH),
8
.02 – 7.80 (m, 2H, ArꢀH), 7.23 (dd, 2H, J = 9.7, 7.8 Hz, ArꢀH), 4.86 – 4.80 (m, 1H, CH), 3.78 (s, 3H,
13
CH
3
), 3.04 (ddd, 2H, J = 23.1, 13.9, 7.1 Hz, CH
2
), 2.17 (s, 3H, CH
3
); C NMR (101MHz, DMSO): δ =
+
1
71.62, 165.97, 130.66, 130.57, 115.91, 115.69, 52.61, 52.60, 34.82, 15.43; HRMS (ES ) m/z 272.0660

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DOI: 10.1039/C7MD00409E
(
272.0678 Calcd for C H NO S M + H).
12 14 3
5
.2.4 General procedure for the preparation of 4a-e
A solution of appropriate intermediates 2a-e, 2ꢀtertꢀbutylꢀ1, 3ꢀdiisopropylꢀisourea (507.8 mg, 2.5 mmol)
in DCM (5.0 mL) was stirred at 80°C for 12h. Then 2ꢀtertꢀbutylꢀ1, 3ꢀdiisopropylꢀisourea (507.0 mg,
2
.5 mmol) was added and the resulting mixture was stirred at 80 °C for another 12 h. The mixture was
cooled to room temperature, filtered, and the filtrate was concentrated in vacum. The residue was
purified by preꢀHPLC (acid condition, TFA) to get desired compounds 4a-e.
tertꢀbutyl NꢀacetylꢀSꢀmethylꢀLꢀcysteinate (4a)
1
Colorless oil; Yield: 65%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 4.51 (dd, 1H, J = 8.2, 5.3 Hz,
CH), 2.85 (ddd, 2H, J = 21.9, 13.8, 6.8 Hz, CH
2
), 2.14 (s, 3H, CH
3 3
), 2.01 (s, 3H, CH ), 1.50 (s, 9H,
13
C H ); C NMR (101MHz, DMSO): δ = 170.49, 169.78, 81.45, 52.73, 35.49, 28.08, 22.77, 15.68;
4
9
+
6 3 4 9
HRMS (ES ) m/z 178.0456 (178.0460 Calcd for C H11NO S M + HꢀC H ).
tertꢀbutyl SꢀmethylꢀNꢀpropionylꢀLꢀcysteinate (4b)
1
Yellow oil; Yield: 72%; Purity: 98%; H NMR (400 MHz, MeOD) : δ = 4.57 (s, 1H, CH), 3.08 – 2.76
2 2 3 4 9
(m, 2H, CH ), 2.43 – 2.28 (m, 2H, CH ), 2.21 (d, J = 2.0 Hz, 3H, CH ), 1.56 (s, 9H, C H ), 1.30 –
1
3
1.15 (m, 3H, CH
3
); C NMR (101MHz, DMSO): δ = 173.50, 170.50, 81.38, 52.69, 35.46, 28.64,
+
2
8.08, 15.69, 10.28; HRMS (ES ) m/z 192.0602 (192.0616 Calcd for C
7
H13NO
3
S M + HꢀC
4
H
9
).
tertꢀbutyl Nꢀ(cyclohexanecarbonyl)ꢀSꢀmethylꢀLꢀcysteinate (4c)
1
Yellow oil; Yield: 71%; Purity: 95%; H NMR (400 MHz, MeOD) : δ = 8.13 (d, 1H, J = 6.8 Hz, NH),
4
.47 (td, 1H, J = 8.2, 5.2 Hz, CH), 2.86 (ddd, 2H, J = 22.2, 13.7, 6.8 Hz, CH
2
), 2.29 (tt, 1H, J = 11.8,
3
.3 Hz, CH), 2.13 (s, 1H, CH ), 1.77 (dd, 5H, J = 40.8, 9.4 Hz, CyclohexaneꢀH), 1.57 – 1.16 (m, 14H,
3
13
CyclohexaneꢀH+ C
4
H
9
); C NMR (101MHz, DMSO): δ = 175.68, 170.51, 81.25, 52.59, 44.01, 35.31,
+
2
9.61, 29.52, 28.08, 25.91, 25.66, 25.63, 15.65; HRMS (ES ) m/z 246.1094 (246.1086 Calcd for
C H NO S M + HꢀC H ).
11
19
3
4
9
tertꢀbutyl NꢀbenzoylꢀSꢀmethylꢀLꢀcysteinate (4d)
1
Yellow oil; Yield: 61%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 8.64 (d, 1H, J = 7.0 Hz, NH),
7
1
1
.92 – 7.79 (m, 2H, ArꢀH), 7.65 – 7.43 (m, 3H, ArꢀH), 4.76 – 4.64 (m, 1H, CH), 3.02 (ddd, 2H, J = 22.8,
13
3.8, 7.1 Hz, CH
2
), 2.17 (s, 3H, CH
3
), 1.51 (s, 9H, C
4
H
9
); C NMR (101MHz, DMSO): δ = 170.44,
+
66.95, 134.33, 131.95, 128.79, 127.86, 81.52, 53.33, 35.01, 28.11, 15.50; HRMS (ES ) m/z 240.060
3 4 9
(240.0616 Calcd for C11H13NO S M + HꢀC H ).

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DOI: 10.1039/C7MD00409E
tertꢀbutyl Nꢀ(4ꢀfluorobenzoyl)ꢀSꢀmethylꢀLꢀcysteinate (4e)
1
Yellow oil; Yield: 75%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 8.67 (d, 1H, J = 7.4 Hz, NH),
8
.00 – 7.81 (m, 2H, ArꢀH), 7.33 – 7.12 (m, 2H, ArꢀH), 4.69 (ddd, 1H, J = 9.0, 8.0, 5.1 Hz， CH), 3.02
1
3
(
tt, 2H, J = 22.9, 6.8 Hz, CH ), 2.17 (s, 3H, CH ), 1.51 (s, 9H, C H ); C NMR (101MHz, DMSO): δ =
2 3 4 9
+
1
2
5
70.39, 165.89, 130.60, 130.51, 115.86, 115.64, 81.57, 53.37, 35.01, 28.10, 15.49; HRMS (ES ) m/z
58.0536 (258.0522 Calcd for C11 S M + HꢀC ).
.2.5 Preparation of compounds 5a, 5b and 5c
H12NO
3
4 9
H
To a mixture of appropriate intermediate (2b, 2c or 2e, 1.2 mmol), EDCl (189.2 mg, 1.5 mmol), DIEA
315.4 mg, 2.4 mmol), HOBt (197.8 mg, 1.5 mmol) in DMF (5.0 mL) was added methanamine
(
hydrochloride (90.6 mg, 1.3 mmol) at 15 °C. Then the mixture was stirred at 15 °C for 16 h, diluted
with DCM (100.0 mL), washed with 1 M HCl (1×20.0 mL), H O (1×20.0 mL), brine (1×20.0 mL),
2
dried over anhydrous Na
2 4
SO , and concentrated under reduced pressure. The residue was purified by
preꢀHPLC (acid condition, TFA) to get desired compounds 5a, 5b and 5c as white solids.
(
R)ꢀNꢀmethylꢀ3ꢀ(methylthio)ꢀ2ꢀpropionamidopropanamide (5a)
1
White solid; Yield: 82%; Purity: 97%; H NMR (400 MHz, MeOD) : δ = 8.08 (s, 1H, NH), 4.50 (ddd,
1
3
3
2 3 2
H, J = 8.1, 7.0, 4.3 Hz, CH), 2.96 – 2.65 (m, 5H, CH +CH ), 2.30 (q, J = 7.6 Hz, 2H, CH ), 2.12 (s,
1
3
H, CH
3
), 1.15 (t, J = 7.6 Hz, 3H, CH
3
); C NMR (101MHz, DMSO): δ = 173.55, 171.22, 52.24,
+
6.30, 28.70, 26.07, 15.52, 10.20; HRMS (ES ) m/z 205.0516 (205.0932 Calcd for C
8
H
16
N
2
O
2
S M +
H).
(
R)ꢀNꢀ(1ꢀ(methylamino)ꢀ3ꢀ(methylthio)ꢀ1ꢀoxopropanꢀ2ꢀyl)cyclohexanecarboxamide (5b)
1
White solid; Yield: 79%; Purity: 98%; H NMR (400 MHz, MeOD) : δ = 7.96 (d, 1H, J = 7.8 Hz, NH),
4
.55 – 4.40 (m, 1H, CH), 2.96 – 2.65 (m, 5H, CH
2 3
+CH ), 2.29 (tt, 1H, J = 11.7, 3.4 Hz, CH), 2.12 (s,
1
3
3
H, CH ), 1.91ꢀ1.65 (m, 5H, CyclohexaneꢀH), 1.53ꢀ1.18 (m, 5H, CyclohexaneꢀH); C NMR (101MHz,
3
DMSO): δ = 175.67, 171.28, 52.01, 44.15, 36.34, 29.94, 29.32, 26.08, 25.94, 25.77, 25.63, 15.48;
+
HRMS (ES ) m/z 259.1419 (259.1402 Calcd for C12
H
22
N
2
O
2
S M + H).
(
R)ꢀNꢀ(1ꢀ(methylamino)ꢀ3ꢀ(methylthio)ꢀ1ꢀoxopropanꢀ2ꢀyl)benzamide (5c)
1
White solid; Yield: 89%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 7.89 (dd, 2H, J = 5.3, 3.3 Hz,
ArꢀH), 7.63 – 7.43 (m, 3H, ArꢀH), 4.74 (dd, 1H, J = 8.6, 5.8 Hz, CH), 2.97 (ddd, 2H, J = 22.5, 13.9, 7.2
1
3
Hz, CH
2
), 2.79 (s, 3H, CH
3
), 2.17 (s, 3H, CH
3
); C NMR (101MHz, DMSO): δ = 171.21, 166.82,
+
1
34.47, 131.83, 128.65, 128.03, 53.07, 36.02, 26.18, 15.42; HRMS (ES ) m/z 253.0912 (253.0932

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DOI: 10.1039/C7MD00409E
Calcd for C H N O S M + H).
1
2
16
2
2
5
.2.6 General procedure for the synthesis of 6a, 6b and 6c
To a solution of appropriate intermediate (2a, 2b or 2e, 1.1 mmol), NMM (137.2 mg, 1.4 mmol) in
THF (4.0 mL) was added isobutyl chloroformate (IBCF) (169.8 mg, 1.2 mmol) dropwise at 0 °C. After
addition, the mixture was stirred at 0 °C for 1 h and cyclohexanamine (123.3 mg, 1.2 mmol) was added
at the same temperature. The resulting mixture was stirred at 0 °C for another 1 h, which was then
diluted with DCM (50.0 mL), washed with 1 M HCl (1×20.0 mL), brine (1×20.0 mL), dried over
anhydrous Na
2
SO
4
, and concentrated under reduced pressure. The residue was purified by preꢀHPLC
(
(
acid condition, TFA) to obtain the desired compounds 6a, 6b and 6c as white solid.
R)ꢀ2ꢀacetamidoꢀNꢀcyclohexylꢀ3ꢀ(methylthio)propanamide (6a)
1
White solid; Yield: 68%; Purity: 92%; H NMR (400 MHz, MeOD) : δ = 8.12 (dd, 1H, J = 55.0, 7.6 Hz,
NH), 4.50 (td, 1H, J = 7.9, 4.7 Hz, CH), 3.65 (tdt, 1H, J = 11.6, 7.9, 3.9 Hz, CH), 2.78 (ddd, 2H, J =
2
1
3
2 3 3
1.6, 13.7, 7.1 Hz, CH ), 2.14 (s, 3H, CH ), 2.01 (s, 3H, CH ), 1.92 – 1.59 (m, 5H, CyclohexaneꢀH),
1
3
.44 – 1.14 (m, 5H, CyclohexaneꢀH); C NMR (101MHz, DMSO): δ = 169.86, 169.52, 52.28, 48.08,
+
6.62, 32.77, 32.62, 25.66, 24.99, 24.93, 22.97, 15.60; HRMS (ES ) m/z 259.1416 (259.1402 Calcd for
12 22 2 2
C H N O S M + H).
(
R)ꢀNꢀcyclohexylꢀ3ꢀ(methylthio)ꢀ2ꢀpropionamidopropanamide (6b)
1
White solid; Yield: 63%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 4.50 (dd, 1H, J = 7.7, 6.6 Hz,
CH), 3.66 (td, 1H, J = 10.7, 5.6 Hz, CH), 2.79 (ddd, 2H, J = 21.6, 13.7, 7.2 Hz, CH
2
), 2.28 (q, J = 7.6
Hz, 2H, CH ), 2.14 (s, 3H, CH ), 1.93ꢀ1.60 (m, 5H, CyclohexaneꢀH), 1.45ꢀ1.19 (m, 5H,
2
3
1
3
CyclohexaneꢀH), 1.15 (t, 3H, J = 7.6 Hz, CH ); C NMR (101MHz, DMSO): δ = 173.27, 169.74,
3
+
5
2.23, 48.06, 36.62, 32.77, 32.66, 28.71, 25.66, 24.96, 24.90, 15.62, 10.30; HRMS (ES ) m/z 273.1546
24 2 2
(273.1558 Calcd for C13H N O S M + H).
(
R)ꢀNꢀ(1ꢀ(cyclohexylamino)ꢀ3ꢀ(methylthio)ꢀ1ꢀoxopropanꢀ2ꢀyl)benzamide (6c)
1
White solid; Yield: 73%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 8.42 (d, 1H, J = 7.8 Hz, NH),
8
.15 (d, 1H, J = 8.2 Hz, NH), 7.93ꢀ7.76 (m, 2H, ArꢀH), 7.53 (dt, 3H, J = 14.9, 7.3 Hz, ArꢀH), 4.80ꢀ 4.65
m, 1H, CH), 3.77ꢀ3.60 (m, 1H, CH), 2.94 (ddd, 2H, J = 22.1, 13.8, 7.2 Hz, CH ), 2.18 (s, 1H, CH ),
); C NMR (101MHz, DMSO): δ = 169.77, 166.69,
(
2
3
13
1
1
5 5
.95ꢀ1.62 (m, 5H, CH ), 1.48ꢀ1.12 (m, 5H, CH
34.54, 131.81, 128.69, 127.95, 53.08, 48.19, 36.29, 32.78, 32.69, 25.66, 24.99, 24.95, 15.51; HRMS
+
(
ES ) m/z 321.1552 (321.1558 Calcd for C17
H
24
N
2
O
2
S M + H).

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5
.2.7 General procedure for the preparation of 7aꢀb
(
R)ꢀ2ꢀacetamidoꢀ3ꢀ(methylthio)ꢀNꢀphenylpropanamide (7a)
A solution of compound 2a (200.0 mg, 1.1 mmol), aniline (160.8 mg, 1.7 mmol), HATU (515.6 mg, 1.4
mmol) in DMF (3.0 mL) was stirred at 15 °C for 16 h. Then DMF was removed under reduced pressure.
The residue was diluted with DCM (100.0 mL), washed with 1 M HCl (1×30.0 mL), H
2
O (1×30.0 mL),
brine (1×30.0 mL), dried over anhydrous Na SO , and concentrated under reduced pressure to get
2 4
compound 7a as a white solid.
1
White solid; Yield: 61%; Purity: 99%; H NMR (400 MHz, MeOD) : δ = 10.02 (s, 1H, NH), 7.66ꢀ7.51
(m, 2H, ArꢀH), 7.33 (dd, 2H, J = 10.8, 5.2 Hz, ArꢀH), 7.13 (t, 1H, J = 7.4 Hz, ArꢀH), 4.76ꢀ4.59 (m, 1H,
1
3
2 3 3
CH), 2.90 (ddd, 2H, J = 21.7, 13.8, 7.2 Hz, CH ), 2.17 (s, 3H, CH ), 2.04 (s, 3H, CH ); C NMR
(101MHz, DMSO): δ = 169.85, 169.77, 139.25, 129.18, 123.96, 119.82, 53.36, 36.14, 22.91, 15.67;
+
HRMS (ES ) m/z 253.0922 (253.0932 Calcd for C12
16 2 2
H N O S M + H).
(
R)ꢀ3ꢀ(methylthio)ꢀNꢀphenylꢀ2ꢀpropionamidopropanamide (7b)
A solution of compound 2b (300.0 mg, 1.6 mmol), aniline (160.8 mg, 1.7 mmol), EDCI (451.5 mg, 2.4
mmol) in pyridine (5.0 mL) was stirred at 15 °C for 16 h. The solvent was removed under reduced
pressure. The residue was diluted with DCM (100.0 mL), washed with 1 M HCl (1×30.0 mL), H
2
O
(
1×30.0 mL), brine (1×30.0 mL), dried over anhydrous Na SO , and concentrated under reduced
2
4
pressure. The residue was purified by preꢀHPLC (acid condition, TFA) to get compound 7b as a white
solid.
1
White solid; Yield: 55%; Purity: 97%; H NMR (400 MHz, MeOD) : δ = 10.02 (s, 1H, NH), 7.64 –
7
.52 (m, 2H, ArꢀH), 7.33 (dd, J = 10.8, 5.1 Hz, 2H, ArꢀH), 7.13 (t, J = 7.4 Hz, 1H, ArꢀH), 4.77 – 4.62
(
m, 1H, CH), 2.90 (ddd, J = 21.8, 13.8, 7.2 Hz, 2H, CH ), 2.32 (q, J = 7.6 Hz, 2H, CH ), 2.17 (s, 3H,
2
2
1
3
CH
3
), 1.17 (t, J = 7.6 Hz, 3H, CH
3
); C NMR (101MHz, DMSO): δ = 173.49, 169.90, 139.27, 129.18,
+
1
23.95, 119.81, 53.31, 36.18, 28.65, 15.68, 10.26; HRMS (ES ) m/z 267.1095 (267.1089 Calcd for
C
13
H
18
N
2
O
2
S M + H).
5
.2.8 Preparation of the target compound 7c
tertꢀbutyl (R)ꢀ(3ꢀ(methylthio)ꢀ1ꢀoxoꢀ1ꢀ(phenylamino)propanꢀ2ꢀyl)carbamate (9)
To a solution of compound 8 (700 mg, 2.97 mmol), NMM (360.50 mg, 3.56 mmol) in THF (20 mL)
was added IBCF (446.21 mg, 3.27 mmol) dropwised at 0 °C. Then the mixture was stirred at 15 °C for
1
h. Then the mixture was added to aniline (331.92 mg, 3.56 mmol) at 0 °C. The mixture was stirred at

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1
5 °C for 1 h and then was diluted with EA (50 mL), washed with 1M HCl (20 mL), H O (20 mL),
2
brine (20 mL), dried over anhydrous Na
as a white solid.
2
SO
4
, concentrated under reduced pressure to get compound 9
+
White solid; Yield: 79%; Purity: 95%; ESIꢀMS: m/z = 254.8[M+HꢀC H ] .
4
9
(
R)ꢀ2ꢀaminoꢀ3ꢀ(methylthio)ꢀNꢀphenylpropanamide (10)
A solution of compound 9 (700.00 mg, 2.26 mmol) in DCM (10.00 mL) was added TFA (67.53 mmol,
.00 mL), which was stirred at 15 °C for 1 h. The solvent was removed under reduced pressure to get
5
the crude product as brown oil. The product was put into next step directly without further purification.
Yield: 91%.
Preparation of (R)ꢀNꢀ(3ꢀ(methylthio)ꢀ1ꢀoxoꢀ1ꢀ(phenylamino)propanꢀ2ꢀyl)benzamide (7c)
To a mixture of compound 10 (700.00 mg, 2.16 mmol), NaHCO (725.30 mg, 8.64 mmol) in THF (10
3
2
mL) and H O (5 mL) was added benzoyl chloride (425.08 mg, 3.02 mmol) at 0 °C. Then the mixture
was stirred at 0 °C for 2 h. The solvent was removed under reduced pressure. The residue was diluted
with DCM (50 mL), washed with 1 M HCl (20 mL), H O (20 mL), brine (20 mL), dried over
2
anhydrous Na
2 4
SO , concentrated under reduced pressure. The residue was purified by silicagel column
(PE : EA = 5:1) to get compound 7c as a white solid.
1
White solid; Yield: 61%; Purity: 96%; H NMR (400 MHz, MeOD) : δ = 10.13 (s, 1H, NH), 8.63 (d, J
7.5 Hz, 1H, NH), 8.00 – 7.82 (m, 2H, ArꢀH), 7.56 (ddd, 5H, J = 31.8, 11.9, 4.2 Hz, ArꢀH), 7.34 (t, 2H,
J = 8.0 Hz, ArꢀH), 7.13 (t, 1H, J = 7.4 Hz, ArꢀH), 3.06 (ddd, 2H, J = 22.1, 13.8, 7.3 Hz, CH ), 2.22 (s,
=
2
1
3
1
1
H, CH
3
); C NMR (101MHz, DMSO): δ = 169.41, 166.46, 138.82, 133.84, 131.43, 128.69, 128.22,
+
27.54, 123.45, 119.35, 53.73, 35.29, 15.12; HRMS (ES ) m/z 315.1064 (315.1089 Calcd for
17 18 2 2
C H N O S M + H).
5
5
.3 Pharmacology
.3.1 Cell proliferation assay against hepatocellular injury model induced by H O
2
2
Human hepatocytes cell line, LO2 (provided by Shanghai Institutes for Biological Sciences, SIBS),
was used to build hepatocytes injury model induced by H or APAP. The liver cell protective
2
activities of target compounds were evaluated against hepatocellular injury model induced by H O ,
2
O
2
2
and Cell Counting Kitꢀ8 (CCKꢀ8, Beyotime, C0038) kit was utilized to detect the cell proliferation
6
rates. Approximate 2×10 LO2 cells were suspended in 10 ml fresh medium and cultured in the relative
2
saturation humidity incubator in 5% CO at 37 °C for 24 h. Then the culture liquid was removed and

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3
6
00 ꢁL H O fresh culture liquid with no fetal bovine serum (FBS) was added at the same condition for
2 2
h. The culture fluid was removed again, washed with phosphate buffered saline (PBS) for three times,
and proceeded for trypsin digestion and suspending. Subsequently, the culture liquid was centrifuged
with the supematant abandoned, and then 1 ml PBS was added and the cells were suspended and
counted. At last, the cells were transferred to 1.5 ml EP tube and centrifuged (4000 r × 10 min). The
supernatant was discarded and 150 ꢁL PBS was added for cell suspension, and then the cells were
storaged at ꢀ20 °C. All of the following procedures were carried out strictly accordance with the
instruction of the CCKꢀ8 kit. The absorbance was measured at 450 nm using a microplate reader. All
the target compounds were screened against this cell model and the processing of the model group was
similar to that of the compound test group, except that the tested compounds were not included in the
hepatocellular injury cells induced by H O when the CCKꢀ8 kit was used. The results were calculated
2
2
by using the GraphPad Prism 5.
.3.2 Cell proliferation assay against hepatocellular injury model induced by APAP
5
The liver cell protective activities of selected compounds were evaluated against hepatocellular injury
model induced by APAP, and CCKꢀ8 kit was used to detect the cell proliferation rates. Similar to the
6
injury model induced by H
in 5% CO
with no FBS was added for incubation with 5% CO
to the experimental procedures illustrated in paragraph 5.3.1.
.3.3 In vitro MDA secretion assay
In this experiment, human normal LO2 cells were cracked directly and measured to determine the
MDA level of normal group. The ꢀ20 °C storaged cells (hepatocellular injury cells induced by H or
2
O
2
, approximate 2×10 cells were suspended in fresh medium and cultured
2
at 37 °C for 24 h, then the culture liquid was removed and 1% DMSO plus 40 mmol APAP
at 37 °C for 3 h. The following operation referred
2
5
2
O
2
APAP) were cracked with repeated freezing and thawing method, and the following assay was
determined according to the instruction of the MDA kit (Beyotime, S0131). 0.37% thiobarbituric acid
(
TBA) storage liquid was prepared and the MDA detection work fluid was diluted according to the
number of samples. 100 ꢁl (100 mmol) of various target compounds was added and treated with 200
M of MDA test solution. The processing of the model group was similar to that of the compound test
group, except that the tested compounds were not included in the hepatocellular injury cells induced by
or APAP in the above experimental step. The solution was mixed at 100 °C for 15 min, cooled to
room temperature and centrifuged (1000 g) for 10 min. Finally, the supernatant (200 ꢁl) was preloaded
ꢁ
2 2
H O

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in 96ꢀwell plate and the absorbance was determined by enzymeꢀmeter 532.
5
.4 ADME assay
5
.4.1 Cacoꢀ2 permeability assay
Cacoꢀ2 cells (human colorectal adenocarcinoma line) were cultivated in MEM medium supplemented
with 10% FBS, 1% nonꢀessential amino acids solution and 0.1% penicillinstreptomycin in humidified
4
atmosphere at 37 °C in 5% CO
2
and then were seeded at 4.8×10 cells/well on 24ꢀwell semipermeable
insert plates (Millicell # PSHT 010 R5). The medium was changed every two days. After 21 days of
cell growth the integrity of differentiated Cacoꢀ2 monolayers was verified by transepithelial electrical
resistance (TEER) measurements using MilicellꢀERS Voltohmmeter (Millipore # MERS 000 01).
Cacoꢀ2 cell monolayers were considered acceptable for transport studies if the final values of TEER
2
were greater than 250 Ω/cm . For the permeability studies, 24ꢀwell insert plate was removed from its
feeder plate and placed in a new sterile 24ꢀwell receiver plate. The cell layer was washed twice with
HBSS (# 14025ꢀ092). In AꢀB direction, aliquots (400 ꢁL) of the test compound solution (in duplicates,
at 10 ꢁM, in HBSS with 5.56 mM glucose buffered with 25 mM HEPES, pH 7.4) were added into the
apical compartments of the transꢀwell insert and 1000 ꢁL of the same buffer was added to the
basolateral compartments; in BꢀA direction, aliquots (1000 ꢁL) of the test compound solution (in
duplicates, at 10 ꢁM, in HBSS with 5.56 mM glucose buffered with 25 mM HEPES, pH 7.4) were
added into the basolateral compartments of the transꢀwell insert and 400 ꢁL of the same buffer was
added to the apical compartments. The plates were then incubated for 1.5 h at 37 °C. High, low and
Pgp substrate permeability controls (Atenolol, Metoprolol, Erythromycin) were run with every
experimental batch to verify assay validity. The concentrations of the compounds tested in the
permeability assay were determined using LCꢀMSꢀMS method. The formula for calculating Papp
ꢀ
6
(
expressed in 10 cm/s) was as follows:
Papp = (VA / (Area × time)) × ([drug]accepter /(([drug]initial, donor) ×Dilution Factor)
VA ꢀ the volume in the acceptor well, Area ꢀ the surface area of the membrane , time ꢀ the total
transport time in seconds, [drug]accepter ꢀ concentration of test compound in the acceptor well,
[
drug]initial,donor ꢀ initial concentration of test compound in the donor well.
.4.2 Pharmacokinetic studies
5
The protocol of rat pharmacokinetics study was reviewed and approved by the Institutional Animal
Care and Use committee, Shanghai Chempartner (protocol number: A998HL0002) prior to being

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instituted, and the experiments were performed in accordance with the Guidelines for the Care and Use
of Laboratory Animals at Shanghai ChemPartner Co., Ltd. The compounds were dissolved in 5%
DMSO + 5% Solutol HS 15 + 90% (20% HPꢀβꢀCD in water) as a vehicle and were administered
intravenously (i.v.) or orally (p.o.) to SD rats at 10 or 20 mg/kg. Ten male animals (bodyweight:
2
4
20ꢀ240 g) were used for i.v. or p.o. group, and blood was collected via tail vein at 5, 15, 30 min, 1, 2,
, 8 and 24 h or 15, 30 min, 1, 2, 4, 8 and 24 h for i.v. or p.o administration, respectively. All animals
had hasted overnight and fed 4 h post dosing and free access to water. EDTAꢀK2 was used as an
anticoagulant, and blood sample was centrifuged at 4 °C (2000 g, 5 min) to obtain plasma within 15
min after sample collection. Plasma samples were analyzed after protein precipitation with ACN and
centrifugation at 5800 rpm for 10 min using liquid chromatography coupled to mass spectrometry
(
LCꢀMSꢀMS) using appropriate calibration curves and an internal standard (Diclofenac) and
bioanalytical quality controls. Pharmacokinetic parameters were calculated using Phoenix Winnonlin
.4 software package (Pharsight) using nonꢀcompartmental analysis. Area under the plasma
6
concentration versus time curve (AUC) was calculated by Linear Trapezoidal Linear Interpolation rule
and bioavailability (BA, %) was calculated by (AUCINFꢀPO×DOSEIV) / (AUCINFꢀIV×DOSEPO)*100%.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Acknowledgments
This study was supported by Grants from the Major science and technology innovation program of
Hangzhou (Grant 20142013A60 and Grant 20152013A03), the Health care and key specialist scientific
research project of Hangzhou Administration of Science and Technology (Grants 20150733Q49 and
2
0140733Q42),
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Figure captions:
Figure 1. Metabolic pathways affected by NAC and its molecular mechanisms
Figure 2. Design of novel NAC derivatives
Figure 3. Effect of NAC and target compounds on cell proliferation in H
Viabilities of H treated LO2 cell line cultured with compounds 2a-e and 3a-e. (b) Viabilities of
treated LO2 cell line cultured with compounds 4a-e and 5a-c. (c) Viabilities of H treated LO2
cell line cultured with compounds 6a-c and 7a-c. **, significantly different from the model group
2 2
O treated LO2 cells. (a)
2
O
2
H
2
O
2
2 2
O

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with p < 0.01; ***, significantly different from the model group with p < 0.001.
Figure 4. Effect of NAC and selected compounds on MDA secretion on H
.3 Cell viabilities and MDA secretion inhibitory activities on 4ꢀAcetamidophenol (APAP) induced
LO2 cell injury model
2 2
O treated LO2 cells.
2
Figure 5. Effect of NAC and selected compounds on cell viability in APAP induced LO2 cells. *,
significantly different from the model group with p < 0.05.
Figure 6. Effect of NAC and selected compounds on MDA secretion in APAP treated LO2 cells. ***,
significantly different from the model group with p < 0.001.
Scheme 1. Synthesis of target compounds 2a-e, 3a-e, 4a-e, 5a-c, 6a-c and 7a-c. Reagents and
conditions: (I) DIEA, FmocꢀCys(Me)ꢀOH, NMM, DMF, DCM, rt; (II) HCl, MeOH, 15 °C; (III)
2
ꢀtertꢀbutylꢀ1, 3ꢀdiisopropylꢀisourea, DCM, 80 °C to rt; (IV) methylamine hydrochloride, EDCI, HOBt,
DMF, DIEA, 15 °C; (V) NMM, IBCF, THF, 0°C; (VI) aniline, HATU, DMF, 15 °C or aniline, EDCI,
pyridine, 15 °C; (VIII) TFA, DCM,15 °C; (IX) benzoyl chloride, NaHCO , THF, H O, 0 °C.
3
2

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