Synthesis and biological evaluation of a novel series of curcumin-peptide derivatives as PepT1-mediated transport drugs

Article abstract of DOI:10.1016/j.bioorg.2019.103163

Curcumin (CUR) is a natural yellow pigment from turmeric with extensive bioactivities. However its relatively poor solubility limited its absorption and bioavailability. In this study, a novel series of CUR-peptide conjugates were designed and synthesized

Full text of DOI:10.1016/j.bioorg.2019.103163

Journal Pre-Proof
Synthesis and Biological Evaluation of a Novel Series of Curcumin-peptide
Derivatives as PepT1-Mediated Transport Drugs
Jiyun Zhang, Hongmei Wen, Fei Shen, Xinzhi Wang, Chen-Xiao Shan, Chuan
Chai, Jian Liu, Wei Li
PII:
S0045-2068(19)30375-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103163
YBIOO 103163
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
8 March 2019
23 July 2019
29 July 2019
Please cite this article as: J. Zhang, H. Wen, F. Shen, X. Wang, C-X. Shan, C. Chai, J. Liu, W. Li, Synthesis and
Biological Evaluation of a Novel Series of Curcumin-peptide Derivatives as PepT1-Mediated Transport Drugs,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103163
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Synthesis and Biological Evaluation of a Novel Series of Curcumin-
peptide Derivatives as PepT1-Mediated Transport Drugs
Jiyun Zhang, Hongmei Wen⁎,Fei Shen, Xinzhi Wang, Chen-Xiao Shan, Chuan Chai,
Jian Liu, Wei Li⁎
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, PR
China
*Corresponding author details: Hongmei Wen (); School of Pharmacy, Nanjing
University of Chinese Medicine, Nanjing, 210023, China. E-mail:
njwenhm@126.com.
Wei Li (); School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing,
210023, China. E-mail: liwaii@126.com.
Abstract
Curcumin (CUR) is a natural yellow pigment from turmeric with extensive
bioactivities. However its relatively poor solubility limited its absorption and
bioavailability. In this study, a novel series of CUR-peptide conjugates were
designed and synthesized as PepT1-mediated transport drugs and their solubility,
cellular uptakes and anti-tumor activities were evaluated. Ten compounds showed
better water solubility than CUR due to the dipeptide moiety. Compared with CUR,
compound 5e exhibited the slightly better activity and 5d showed the similar activity
with CUR. Besides, compounds 5d and 5e performed higher cellular uptakes in Caco-

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2 cell and dose-dependently inhibited by the addition of PepT1 typical substrate
glycylsarcosine (Gly-Sar). Compound 5d and 5e have improved the absorption of
CUR by PepT1-mediated without affected the activity. These new dipeptide
conjugates of CUR may serve as promising lead compounds for future drug
development.
Keywords: CUR-peptide conjugates; Antitumor; PepT1; Caco-2 cells; Celluar
uptake
1. Introduction
Curcumin (CUR, Figure 1) is a phenolic compound isolated from the rhizome of
Curcuma longa L. [1], has a wide range of biological activities chemoprevention [2-7].
CUR is currently being tested in clinical trials for treatment of various types of
cancers, including liver cancer, colon cancer, multiple myeloma etc [8-11]. CUR has
three functional groups including one active methylene group and two phenolic
groups [12]. However, its relatively poor solubility and instability at physiological
condition limited its absorption and bioavailability to a great extent, which restricts
the therapeutic usage of CUR [13,14].
Over the past decade, the human intestinal oligopeptide transporter 1 (PepT1),
has been widely explored as a target for increasing the oral bioavailability of poorly
absorbed drugs [15-19]. PepT1 is a promising and valuable target protein, responsible
for the uptake of dipeptides and tripeptides. Structural information of substrate

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+
identified by PepT1 includes: a strong binding site for an N-terminal NH3 group; a
hydrogen bond to the carbonyl group of the first peptide bond a hydrophobic pocket,
which possesses a strong directional vector as indicated; preference for the
stereochemistry shown (usually L); acarboxylate binding site [20]. Several dipeptide-
drug conjugate (shown in Figure 2) were synthesized, such as Gly-Val-Acyclovir
(Ⅰ), Val-Trp-Acyclovir (II) [21,22], Val-Val-Doxorubicin (III)[23], Pro-Ile-
Zidovudine (IV) [24], Val-Ala-Oleanolic Acid (V) [25], Phe-Gly-Zidovudine (VI)
[26], Gly-Val-Saquinvair (VII) [27] and they appeared to combine both chemical
stability and good affinity for the PepT1 transporter, the peptide carrier strategy
applied to improve oral absorption and bioavailability via targeting the PepT1
transporter. The structure and biochemical characteristics of Caco-2 cells are similar
to those of human intestinal epithelial cells, various transporters and metabolic
enzymes in intestinal epithelial cells could be expressed in Caco-2 cells. Caco-2 cells
were an ideal model for studying absorption and transport of drugs, Caco-2 cell model
is currently used by most laboratories to evaluating PepT1 transport activity [28,29].
In recent years, diester of CUR with valine, glutamic acid and demethylenated
piperic acid have been prepared [30-33], several CUR derivatives have better water
solubility or greater bioavailability, however few of them exhibited more potent
anticancer activity than CUR, the reason for decreased activity was the phenolic
hydroxyl groups of this derivatives were substituted. It had been found that the C4'
phenolic hydroxyl group of CUR has a great influence on the tumor activity, and
when it modified to an ether, an ester or other groups, the antitumor activity was

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enhanced [34-36].
In this work, with the aim to further improve the solubility and oral absorption of
CUR, the structure of parent drugs selected for PepT1-targeted was designed, the
phenolic hydroxyls group of CUR was acylated with the dipeptide group of amino to
form the ether conjugate through a chloroacetic chloride linker and simultaneously the
other phenolic hydroxyl group was retained. Ten novel CUR-dipeptide bioconjugates
5a-j were synthesized (Figure 1) and their solubility, cellular uptakes and anti-tumor
activities were evaluated. The equilibrium solubility was assessed by HPLC in
ammonia water (pH = 8). The MTT assay was used to evaluate their anti-proliferative
activities against HepG2 and SMMC-7721 cells in vitro. PepT1-mediated uptake and
uptake inhibition studies were determined by LC-MS/MS in Caco-2 cells. In vitro
phase I metabolism assay was carried out to determined biotransformation of 5d and
5e.
2. Results and Discussion
2.1 Chemistry
General synthesis procedure for compounds 5a-j
General methods. All reagents involved in the synthesis were used directly
without further purification. The preparation of target compounds 5a-j was described
in Scheme 1. The reaction of N-Boc-amino acids and amino acid methyl esters was

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condensed in chloroform in the presence of dehydrating agents N,N-
dicyclohexylcarbodiimide (DCC) and triethylamine (TEA) as catalysts. The N-Boc
protection of amino group in dipeptides were deprotected with Trifluoroacetate (TFA)
(20%) in dry dichloromethane, the reaction time was 1-2 h. Next free amine group is
acylated by chloroacetyl chloride in dry dichloromethane. Chlorine was replaced by
the phenolic hydroxyl group of CUR, then the methyl ester group is removed under
1
13
alkaline conditions. Compounds 5a-j were characterized by H NMR, C NMR and
HRMS-ESI.
O
R2
Boc
O
R2
R2
H
HN
R1
O
R1
HN
N
O
a
c
O
+
N
OH
Cl
N
H2N
H
H
O
O
R1
O
O
HCl
Boc
1a-j
O
3a-j
Y
X
b
a, X= Val, Y=Val f, X= Val, Y= Gly
b, X= Ala, Y=Val g, X= Val , Y=Ala
c, X= Gly, Y=Val h, X= Ala, Y=Ala
d, X= Phe, Y=Val i, X= Val, Y=Phe
e, X= Ile, Y=Val j, X= Val, Y=Ile
2a-j
d
CUR
O
O
O
O
O
O
O
O
O
O
e
MeO
X
Y
OH
X
OH
Y
O
5a-j
4a-j
O
Scheme 1.Synthesis of target compounds 5a-j. Reagents and condition: (a) DCC, TEA,
chloroform, 0℃ 3h, RT 12h; (b) TFA, anhydrous CH2Cl2; (c) chloroacetyl chloride, TEA,
anhydrous CH2Cl2; (d) K2CO3, KI, acetone; (e) LiOH, MeOH/THF/H2O.
2.2 Solubility of CUR-dipeptide conjugates
The poor aqueous solubility of CUR is the major problem limiting its application.
To investigate whether the aqueous solubility of the derivatives was improved, test
compounds were selected to determine the equilibrium solubility in ammonia water
(pH = 8) according to the Higuchi and Connor's method [37].

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The solubility of all compounds were shown in Table 1. It is shown in Table 1
that 10 novel CUR derivatives have synthesized and their equilibrium solubility in
ammonia water were significantly increased compared with CUR. The effects
revealed that the conjugation of the phenolic group to dipeptide moiety via an ether
linkage improved the water solubility of CUR, The phenolic methoxyl groups of
CUR was linked to dipeptide, solubilities were all enhanced and it as a result of
compounds 5a-j containing carboxylic group. The dipeptide promoiety can improve
the hydrophilicity of CUR. Reasons for different degrees of water-solubility
enhancement of derivatives may be that along with the increase of the carbon number
of the substituent, the liposolubility generally increased and the water solubility
decreased.
2.3 Antitumor activity of CUR and its derivatives in vitro
To explore the antitumor effect of these compounds, we evaluated their anti-
proliferative activities against HepG2 and SMMC-7721 in vitro by MTT assay. The
half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 cells were
described in Table 1 and inhibition of tested compounds on HepG2 cell and SMMC-
7721 was shown in Table S1, S2 (shown in Supplementary material). Cell viability
rate of compounds CUR, 5d and 5e in vitro antitumor activity of SMMC-7721 and
HepG2 was shown in Figure 3.
As indicated in Table 1, Table S1, S2, CUR-dipeptide conjugates have different
degrees of inhibition of cell proliferation on HepG2 and SMMC-7721 cells. Several

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of the CUR-dipeptide products exhibited increased antitumor activity toward HepG2
and SMMC-7721 cells compared to that of the parent molecule (CUR), while others
showed decreased activity. It could be found in Figure 3 that CUR, 5d and 5e have
obvious antitumor activities. The IC50 values of CUR, 5d and 5e against HepG2 cells
were 35.8μM, 40.2μM and 23.4μM; the IC50 values of CUR, 5d and 5e against
SMMC-7721 cells were 22.9μM, 35.4μM and 19.7μM. It is also evident from Table 1
that 5e exhibited increased cellular inhibition toward HepG2 and SMMC-7721
compared to that of the CUR, 5d performed almost the resemble activity as the parent
drug. Besides, when X was Ala, anti-tumor activities of 5b and 5h almost disappeared.
While the benzyl substituent (5d) was replaced with 2-methyl butyl group (5e), the
activity was becoming decreased. The 5c demonstrated the best solubility, however it
has relatively weak activity, which showed that great anti-tumor activity requires
appropriate liposolubility. The antitumor activity of 5d (X = Phe, Y = Val) was better
than that of 5i (X = Val, Y = Phe), which explained that X = Phe plays a great
important role in anti-tumor activity. This observation encouraged us to prepare the
CUR derivatives modifying with rational substituents to improve its solubility and
inhibition on human hepatoma cell line.
2.4 Uptake and PepT1-mediated inhibition studies
Compounds were carried out uptake studies using caco-2 cell and LC-MS/MS
method. The inhibition study was evaluated whether PepT1 was involved in 5d and 5e
cellular uptake. The LC-MS/MS method was established to determine the cellular
uptake of CUR, 5d and 5e. Moreover, the conditions of chromatography and mass

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spectrometry were optimized, selectivity and linear range were investigated (shown in
Table S3). Effect of pH, uptake time, concentration on uptake were explored in Caco-
2 cells (shown in Figure 4). The uptake inhibition studies was displayed in Figure 5.
PepT1 employs a proton gradient as the driving force, and its activity is pH
dependent. Therefore, two drug solutions with different pH (6.0 and 7.4) were used in
the uptake studies. From Figure 4A, cellular uptake was significantly enhanced in pH
6.0 compared to that in pH 7.4, respectively. pH 6.0 was selected in the subsequent
experiments. The time-dependent curves (Figure 4A) indicated that as the uptake
time increased, the uptake of the drug in Caco-2 cells also increased, and the uptake
rate was faster at 60 min, thus the appropriate uptake time was selected 60 min in the
subsequent experiments. The celluar uptakes of CUR, 5d and 5e were increased in a
dose-dependent manner (Figure 4B), however, compared to CUR, compounds 5d
and 5e all showed obvious higher celluar uptakes at the same concentration,
especially at 100 μM. These findings suggested that PepT1 might be involved in the
uptake of 5d and 5e. To prove the assumption that the increase in the uptake of the 5d
and 5e was possibly due to the recognition of the 5d and 5e by the PepT1, the uptake
inhibition test with Gly-Sar (a specific of PepT1 substrate transporters) was
performed. The Caco-2 monolayers were incubated with CUR, 5d and 5e (100 μM)
in the absence or in the presence of Gly-sar. As shown in Figure 5A, there was no
difference in the uptake of CUR with or without the presence of Gly-Sar, reflecting
its poor affinity for the PepT1 transporter in Caco-2 cells, but 5d and 5e were
significantly reduced at in the presence of Gly-sar, indicating that 5d and 5e have an

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affinity for the PepT1 transporter. To further evaluate the affinity of the 5d and 5e to
PepT1, 5d and 5e were incubated with Gly-Sar at various concentrations (100, 200,
400, 800μM) in Caco-2 cells. The uptakes of both 5d (IC50 = 148.5 μM) and 5e (IC50
= 145.6μM) by Caco-2 cells were significantly and dose-dependently inhibited by the
addition of PepT1 substrate for 60 min (shown in Figure 5B).These results
demonstrated that 5d and 5e have high affinity to PepT1. The dipeptide structure of
promoiety 5d and 5e may be recognized by the PepT1 and PepT1 might be involved
in the uptake of 5d and 5e.
2.5 In vitro phase I metabolism assay
To investigate the metabolites of 5d and 5e, 5d and 5e were incubated with rat
liver microsomes determined by LC-QTOF MS method. The high resolution MS/MS
fragment ions were listed in Table 2, MS/MS spectra was shown in Figure S22-30.
The mass spectra of M1 and M4 showed an [M-H]- ion at m/z 367.1191 (C21H20O6),
as shown in Table 2 and Figure S22, S24, S28, the MS/MS fragment ions of M1 and
M4 resembled that of CUR. Consequently, M1 and M4 were assigned as CUR, it was
suggested that 5d and 5e could be metabolized into CUR, 5d and 5e were the prodrug
of CUR. Besides the tetrahydrogen reduction products and hexahydro reduction
product of 5d and 5e were detected in liver microsome samples.
3. Conclusions
In this study, a novel series of CUR-peptide conjugates were designed and

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synthesized as PepT1-mediated transport drugs and their solubility, cellular uptakes
and anti-tumor activities were evaluated. The efficient and easy synthetic procedure
has been outlined for the synthesis of ten novel dipeptide conjugates of CUR. All of
the compounds showed higher water solubility than CUR due to the conjugation of
the phenolic group to dipeptide moiety via an ether linkage. These synthesized
compounds were evaluated for anti-HepG2 and anti-SMMC-7721 activity by MTT
assay. Compared with CUR, compound 5e exhibited slightly stronger inhibitory
activities against two liver cells and compound 5d showed the similar activity.
Besides, compound 5d and 5e showed obvious higher cellular uptakes than CUR in
Caco-2 cell model. The cellular uptakes of 5d and 5e were dose-dependently inhibited
by PepT1 typical substrate Gly-Sar and attributed to PepT1-mediated uptake.
Furthermore metabolites of compound 5d and 5e were detected and found CUR in rat
liver microsomes by LC-QTOF MS. Overall, these results suggested that compound
5d and 5e have improved the absorption of CUR by PepT1-mediated without affected
the activity. These new dipeptide conjugates of CUR may serve as promising lead
compounds for future drug development.
4. Experimental
4.1. Materials and methods
The reagents used in the synthesis process are all purchased from Shanghai
Macklin Biochemical Co., Ltd. All reactions were monitored by thin-layer

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chromatography (TLC) on GF-254 silica gel plates (Tsingdao Silica Gel Corp.,
Tsingdao, China). A silica gel column was used for flash column chromatography
(200–300 mesh, Tsingdao Silica Gel Corp., Tsingdao, China). Visualisation was
1
13
achieved using UV light (254 nm). H and C NMR spectra were recorded with a
13
Bruker NMR spectrometer (1H NMR: 500 MHz, C NMR: 125 MHz) (Bruker Co.,
Germany). The deuterated solvent used was DMSO with tetramethylsilane (TMS) as
the internal standard. High-resolution electrospray ionisation mass spectrometry
(HRMS-ESI) was measured using a TripleTM TOF 5600 system (AB SCIEX CO.,
Foster City, CA) tested in negative ionisation mode.
4.2. Synthesis of CUR-dipeptides conjugates
4.2.1 Synthesis of dipeptides (1a-j)
Boc-amino acids and amino acid methyl esters were prepared according to
literature procedure [38]. Boc-amino acids (10 mmol) was dissolved in chloroform
(20 mL) and cooled to 0℃. To this, N,N’-dicyclohexyl carbodiimide (DCC) (2.25g,
11 mmol) was added with stirring , the amino acid methyl ester hydrochloride (10
mmol) was added followed by Triethylamine (TEA, 10 mmol). Stirring was continued
at this temperature for 3 h, then at room temperature for 12 h. The completion of the
reaction was assessed by precipitation of dicyclohexylurea (DCU). DCU was filtered
off and the filtrate was washed with 1M HCl (3 x 20mL), 0.5M Na2CO3 (3 x 20mL)
and water (2 x 10mL). The organic layer thus collected was dried over anhydrous
sodium sulphate and evaporated in vacuum. The residue was dissolved in acetonitrile

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(20 mL), the undissolved dicyclohexylurea (DCU) present in the solution was filtered.
The filterate was evaporated in vacuum, which yielded a solid mass.
4.2.2 General method for deprotection of dipeptides (2a-j)
1a-j (1 mmol) were dissolved in 4 ml anhydrous dichlormethane at 0℃. TFA (1
mL) was added dropwise to the stirring solution. After this time, the mixture was
allowed to warm to room temperature and stirred for 3.5 h. The solvent was removed
under reduced pressure to a small volume, the crude product was not purified and
used in next step reaction.
4.2.3 N-Chloroacetyl- dipeptides (3a-j)
The TEA was added dropwise with stirring to a solution of compounds 2a-j (l
mmol) in anhydrous dichloromethane at 0℃ until pH=7, which was observed by
extensive pH indicator paper. Chloroacetyl chloride (l mmol) in 2 mL of anhydrous
dichloromethane was introduced dropwise to the reaction vessel, and with stirring at
0℃. Next, the addition is completed, the reaction was stirred 4h at room temperature.
The reaction mixture was washed with water three times. The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated. After removing the solvent
by reduced pressure distillation, the solid is obtained finally.
4.2.4 Amino acid derivatives of CUR (4a-j)
To the solution of CUR (1 mmol) in acetone (6 mL), and stirred for a few
minutes to get a clear solution, Further, potassium carbonate (1.2 mmol) was added,

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the whole solution stirred for 15min.Then a solution of compound 3a-j (1 mmol) in
acetone (12 mL) was added, and the mixture was stirred for 10 h . After completion of
reaction, reaction mixture was filtered and concentrated in vacuum. The residue was
diluted with ethyl acetate and followed by washing with water and brine (100 mL)
The organic layer thus collected was dried over anhydrous sodium sulphate and
evaporated to dryness under vacuum to give a crude product, which was purified by
silica gel column chromatography using mixture of ethyl acetate and petroleum ether
as eluent.
4.2.5 Deprotection of amino acid derivatives of CUR (5a-j)
4a-j (1 mmol) was dissoved in methanol (MeOH), tetrahydrofuran (THF), H2O
(v/v/v = 1:3:1, 10mL), lithium hydroxide (LiOH, 4 mmol) was added subsequently,
The mixture was stirred at room temperature until the completion of the reaction (2–4
h), as indicated by TLC, where the disappearance of 4a-j was observed. The reaction
mixture evaporated to a small volume, which removed the MeOH and THF, then
carefully acidified (1M HCl aq) to pH 3. Yellow solids precipitated from the aqueous
solution, thereafter the precipitate was collected by filtration and washed with water
to give the final product 5a-j.
4.3 Characterization result of 10 target compounds.1H NMR (Figure S1-S10)and
13C NMR (Figure S11-S20) Spectra of all the final compounds were shown in
Supplementary material.
CUR-Val-Val (5a)

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1H NMR (500 MHz, DMSO-d6) δ: 12.57 (s, 1H), 9.70 (s, 1H), 8.17 (d, J = 8.5 Hz,
1H), 7.87 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 15.7 Hz, 2H), 7.40 (s, 1H), 7.34 (s, 1H),
7.24 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.97 (m, 1H), 6.85 (m, 2H), 6.78 (d,
J = 15.7 Hz, 1H), 6.09 (s, 1H), 4.65 (s, 2H), 4.43 (m, 1H), 4.11 (m, 1H), 3.87(s, 3H),
13
3.84 (s, 3H), 2.09 - 1.97 (m, 2H), 0.92 - 0.84 (m, 9H), 0.81 (m, 3H); C NMR (125
MHz, DMSO-d6) δ: 183.91, 182.40, 172.66, 170.80, 167.14, 149.42, 149.29, 149.18,
147.97, 141.02, 139.79, 128.77, 126.26, 123.16, 122.60, 122.38, 121.10, 115.69,
114.00, 111.43 , 111.06, 100.93, 67.74, 57.31, 56.49, 55.81, 55.69, 31.11, 29.55,
19.06, 19.01, 18.04, 17.62; HRMS-ESI calcd for C33H40N2O10 [M-H]- 623.2610,
found 623.2611.
CUR-Ala-Val (5b)
1H NMR (500 MHz, DMSO-d6) δ: 9.72 (s, 1H), 8.12 (d, J = 8 Hz,1H), 8.08 (d, J = 8
Hz, 1H), 7.57 (d, J = 15.8 Hz, 2H), 7.39 (s, 1H), 7.33 (s, 1H), 7.23 (d, J = 8.2 Hz, 1H),
7.16 (d, J = 8.2 Hz, 1H), 6.97 (m, 1H), 6.84 (m, 2H), 6.77 (d, J = 15.8 Hz, 1H), 6.09
(s, 1H), 4.60 (s, 2H), 4.52 (m, 1H), 4.16 (m, 1H), 3.87(s, 3H), 3.84 (s, 3H), 2.06 (m,
13
1H), 1.26 (m,3H), 0.88 (m, 6H); C NMR (125 MHz, DMSO-d6) δ: 184.39, 182.90,
173.27, 172.57, 167.40, 149.87, 149.72, 149.67, 148.44, 141.53, 140.33, 129.18,
126.74, 123.67, 123.02, 122.93, 121.55, 116.16, 114.41, 111.80, 111.39, 101.52,
68.15, 57.63, 56.20, 56.13, 48.05, 30.27, 19.55, 19.16, 18.39; HRMS-ESI calcd for
C31H36N2O10 [M-H]- 595.2297, found 595.2286.
CUR-Gly-Val (5c)

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1H NMR (500 MHz, DMSO-d6) δ: 9.71 (s, 1H), 8.12 (m, 2H), 7.58 (d, J = 15.8 Hz,
2H), 7.39 (s, 1H), 7.33 (s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 6.99
(m, 1H), 6.85 (m, 2H), 6.78 (d, J = 15.8 Hz, 1H), 6.09 (s, 1H), 4.61 (s, 2H), 4.28 -
4.13 (m, 1H), 4.39- 4.10 (m, 1H), 3.92 - 3.88 (m, 2H), 3.87(s, 3H), 3.84 (s, 3H), 2.05
(m, 1H), 0.94 - 0.80 (m, 6H); 13C NMR (125 MHz, DMSO-d6) δ: 184.38, 182.91,
173.33, 169.04, 168.17, 149.88, 149.74, 149.66, 148.44, 141.52, 140.34, 129.18,
126.74, 123.67, 123.01, 122.92, 121.55, 116.16, 114.34, 111.80, 111.42, 101.51,
68.20, 57.61, 56.19, 56.13, 41.92, 30.41, 19.56, 18.38; HRMS-ESI calcd for
C30H34N2O10 [M-H]- 581.2142, found 581.2133.
CUR-Phe-Val (5d)
1H NMR (500 MHz, DMSO-d6) δ: 12.74 (s, 1H), 9.71 (s,1H), 8.29 (d, J = 8.4 Hz, 1H),
8.07 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 15.5 Hz, 2H), 7.36 (d, J = 15.5 Hz, 2H), 7.23 (s,
5H), 7.16 (m, 2H), 6.86 (m, 2H), 6.76 (m,2H), 6.11 (s, 1H), 4.79 (m, 1H), 4.52 (s, 2H),
4.20 (m, 2H), 3.84 (s,3H), 3.82 (s,3H), 3.07 (m, 1H), 2.85 (m, 1H), 2.09 (m, 1H), 0.91
(m, 6H); 13C NMR (125 MHz, DMSO-d6) δ: 184.37, 182.95, 173.29, 171.42, 167.58,
149.89, 149.80, 149.66, 148.45, 141.52, 141.16, 140.37, 137.73, 129.76, 129.08,
128.47, 126.79, 126.74, 123.68, 123.58, 122.98, 122.93, 121.57, 116.17, 114.19,
111.82, 111.34, 101.51, 67.98, 57.70, 56.16, 56.14, 53.46, 38.27, 30.35, 19.57, 18.48;
HRMS-ESI calcd for C37H40N2O10 [M-H]- 671.2610, found 671.2611.
CUR-Ile-Val (5e)
1H NMR (500 MHz, DMSO-d6) δ: 8.13 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H),

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7.57 (d, J = 15.7 Hz, 2H), 7.39 (s, 1H), 7.33 (s, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.16 (m,
1H), 6.96 (d, J = 8.3 Hz, 1H), 6.85 (m, 2H), 6.80 - 6.74 (m, 1H), 6.09 (s, 1H), 4.64 (s,
2H), 4.46 - 4.40 (m, 1H), 4.12 (m, 1H), 3.87(s, 3H), 3.84 (s, 3H), 2.13 - 2.00 (m, 1H),
1.82 - 1.71 (m, 1H), 1.46 - 1.39 (m, 1H), 1.08 - 0.98 (m, 1H), 0.91 - 0.77 (m, 12H);
13C NMR (125 MHz, DMSO-d6) δ: 184.40, 182.88, 173.35, 171.39, 167.58, 149.92,
149.75, 149.63, 148.46, 141.52, 140.30, 129.24, 126.74, 123.66, 123.57, 122.88,
121.59, 116.18, 114.39, 111.82, 111.41, 101.53, 68.16, 57.92, 56.48, 56.25, 56.14,
37.75, 30.15, 24.50, 19.56, 18.57, 15.71, 11.4; HRMS-ESI calcd for C34H42N2O10 [M-
H]- 637.2767, found 637.2761
CUR-Val-Gly (5f)
1H NMR (500 MHz, DMSO-d6) δ: 12.61 (s, 1H), 9.69 (s, 1H), 8.46 (d, J = 8.5 Hz,1H),
7.89 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 15.8 Hz, 2H), 7.39 (s, 1H), 7.25 (s, 1H), 7.17 (d,
J = 8.2 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 6.92 – 6.82 (m, 2H), 6.77 (d, J = 15.8 Hz,
1H), 6.09 (s, 1H), 4.65 (s, 2H), 4.31 (m, 1H), 3.87(s, 3H), 3.84 (s, 3H), 3.80 (d, J =
5.8 Hz, 1H), 3.77 (d, J = 5.8 Hz, 1H), 2.03 (m, 1H), 0.86 (m, 6H); 13C NMR (125
MHz, DMSO-d6) δ: 184.40, 182.90, 171.51, 171.35, 167.75, 149.89, 149.73, 149.69,
148.45, 141.53, 140.32, 129.19, 126.74, 123.67, 123.05, 122.91, 121.57, 116.17,
114.36, 111.85, 111.46, 101.49, 68.11, 57.28, 56.27, 56.15, 41.03, 31.41, 19.56, 18.08;
HRMS-ESI calcd for C30H34N2O10 [M-H]- 581.2142, found 581.2140.
CUR-Val-Ala (5g)
1H NMR (500 MHz, DMSO-d6) δ: 8.40 (d, J = 8.6 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H),

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7.60 (d, J = 15.8 Hz, 2H), 7.42 (s, 1H), 7.35 (s, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.19 (d,
J = 8.0 Hz, 1H), 7.02 (m, 1H), 6.91 - 6.84 (m, 2H), 6.79 (d, J = 15.8 Hz, 1H), 6.12 (s,
1H), 4.67 (s, 2H), 4.39 - 4.33 (m, 1H), 4.23 (m, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 2.03
(m,1H), 1.31 (m, 3H), 0.88 (m, 6H); 13C NMR (125 MHz, DMSO-d6) δ: 184.40,
182.88, 174.35, 170.66, 167.64, 149.92, 149.81, 149.68, 148.48, 141.51, 140.27,
129.29, 126.76, 123.64, 123.12, 122.86, 121.61, 116.20, 114.54, 111.93, 111.58,
101.44, 68.25, 57.01, 56.31, 56.19, 47.99, 31.64, 19.50, 18.13, 17.46; HRMS-ESI
calcd for C31H36N2O10 [M-H]- 595.2297, found 595.2283.
CUR-Ala-Ala (5h)
1H NMR (500 MHz, DMSO-d6) δ: 8.29 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H),
7.60 (d, J = 15.8 Hz, 2H), 7.41 (s, 1H), 7.35 (s, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.18 (d,
J = 8.1 Hz, 1H), 7.01 (m, 1H), 6.89 - 6.84 (m, 2H), 6.79 (d, J = 15.8 Hz,1H), 6.12
(s,1H), 4.62 (s, 2H), 4.45 (m, 1H), 4.25 (m, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 1.30 (m,
6H); 13C NMR (125 MHz, DMSO-d6) δ: 184.37, 182.88, 174.39, 171.93, 167.35,
149.93, 149.80, 149.73, 148.48, 141.48, 140.27, 129.26, 126.76, 123.63, 123.11,
122.88, 121.61, 116.21, 114.57, 111.94, 111.57, 101.44, 68.27, 56.27, 56.19, 48.02,
47.98, 19.13, 17.63; HRMS-ESI calcd for C29H32N2O10 [M-H]- 567.1984, found
567.1980.
CUR-Val-Phe (5i)
1H NMR (500 MHz, DMSO-d6) δ: 8.36 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H),
7.57 (d, J = 15.8 Hz, 2H), 7.40 (s, 1H), 7.33 (s, 1H), 7.28 - 7.21 (m, 5H), 7.20- 7.15

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(m, 2H), 6.97 (m,1H), 6.85 (m, 2H), 6.78 (d, J = 15.8 Hz, 1H), 6.09 (s,1H), 4.63 (s,
2H), 4.44 (m, 1H), 4.30 (m, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.08 (dd, J = 13.9, 5.1 Hz,
1H), 2.90 (dd, J = 13.9, 5.1 Hz,1H), 2.05 – 1.94 (m, 1H), 0.80 (m,6H); 13C NMR (125
MHz, DMSO-d6) δ: 184.40, 182.87, 173.34, 170.88, 167.58, 149.93, 149.75, 149.64,
148.46, 141.57, 141.53, 140.29, 138.09, 129.53, 129.24, 128.57, 126.80, 126.73,
123.67, 123.09, 122.92, 122.88, 121.57, 116.18, 114.42, 111.84, 111.48, 101.51,
68.15, 57.11, 56.27, 56.15, 54.05, 37.13, 31.53, 19.60, 18.02; HRMS-ESI calcd for
C37H40N2O10 [M-H]- 671.2610, found 671.2610.
CUR-Val-Ile (5j)
1H NMR (500 MHz, DMSO-d6) δ 8.16 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H),
7.57 (d, J = 15.8 Hz, 2H), 7.40 (s, 1H), 7.33 (s, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.16 (d,
J = 8.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.86 (m, 1H), 6.77 (d, J = 15.8 Hz,2H), 6.09
(s, 1H), 4.65 (s, 2H), 4.42 (m, 1H), 4.16 (m, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 2.05 -
1.97 (m, 1H), 1.78 (m, 1H), 1.47-1.37 (m, 1H), 1.25 - 1.15 (m, 1H), 0.85 (m, 12H);
13C NMR (125 MHz, DMSO-d6) δ: 184.39, 182.88, 173.25, 171.15, 167.63, 149.91,
149.74, 149.64, 148.45, 141.52, 140.29, 129.22, 126.73, 123.66, 123.06, 122.88,
121.57, 116.17, 114.38, 111.84, 111.46, 101.50, 68.15, 56.99, 56.96, 56.26, 56.15,
36.62, 31.64, 25.22, 19.56, 18.14, 15.99, 11.77; HRMS-ESI calcd for C34H42N2O10
[M-H]- 637.2767, found 637.2767.
4.4 Solubility of CUR-dipeptide conjugates
4.4.1 UPLC (Acquity UPLC, Waters Co., USA ) condition

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Chromatographic separation of CUR and its derivative was achieved using a
Inertsil ODS-SP C18 (5 µm, 4.6 × 150 mm, Shimadzu, Tokyo) column at 30 °C.
The injection volume was 10 μL. The mobile phase consisted of 0.1% formic acid in
ultra-pure water - methanol (20:80, v/v) at a flow rate of 1mL/min. The method gave
baseline separation of the analytes within a run time of 10 min, and reproducible peak
shapes and retention times.
4.4.2 Sample preparation
Excess amounts (∼ 5 mg) of samples were suspended 1ml ammonia water in
1.5mL centrifuge tube. These vials were kept in a thermostated oscillator (SHZ-C,
Shanghai Boxun Industrial Co., Ltd., China) maintained at 37°C. After 1 h, the
samples were centrifuged at 13000rpm for 10 min with a microcentrifuge (MicroCL
21, Thermo Scientific Co., USA), and the supernatant was filtered through a 0.22 μm
organic membrane and detected by UPLC (Acquity UPLC, Waters Co., USA ). All
experiments were performed in triplicate.
4.5. Antitumor activity evaluation
4.5.1 Cell culture
The human Hepatocellular carcinoma (HCC) line, HepG2 and SMMC-7721
were cultured in RPMI-1640 medium (Gibco, New York, USA) supplemented with
10% fetal bovine serum (FBS, ScienCell, Carlsbad, CA, USA), 100 U/mL penicillin,
100 µg/mL streptomycin; grown in an atmosphere of 5% CO2 and 90% humidity at
37°C. The medium was replaced every 2-3 days after incubation.

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4.5.2 Antitumor Assays.
The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721
cells were determined by the MTT assay. Briefly, cells in logarithmic growth were
seeded at 1×105 cells/ml and were treated with CUR and derivatives 5(a-j) at various
concentrations for 3 days. After 72 h of incubation, 20 μL of 5 mg/mL MTT solution
was added to each well and the cells were further incubated for 4 h at 37°C. Then, the
media were removed and 150 μL DMSO was added to each well. The optical density
(OD) of each well was measured using a microplate reader at 570nm (Spark 10M,
Tecan, Switzerland). The IC50 values of 11 compounds against SMMC-7721 and
HepG2 were calculated by GraphPad Prism 7.0 statistical software.
4.6 Uptake and PepT1-mediated experiments
4.6.1 Caco-2 cells culture
Caco-2 cells were cultured in DMEM medium with 10% FBS, 100 U/mL
penicillin, 100 µg/mL streptomycin, 1% non-essential amino acid. Culture medium
was changed every other day, when the cells reached confluence, they were plated on
24-well dishes at a density of 5×104 cells/well. The cell monolayers were given fresh
medium every other day and were used on the 15th day for uptake experiments.
4.6.2 LC-MS/MS condition
A Nexera UHPLC 30A LC system (Shimadzu Corporation, Kyoto, Japan)
coupled with a QTRAP TM4500 MS system (AB Sciex Corporation., Foster City, CA)

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was used. Chromatographic separation of CUR, 5d, 5e and internal standard
(Honokiol) was achieved using a Inertsil ODS-SP C18 (5 µm, 4.6 × 150 mm,
Shimadzu, Tokyo) column at 40 °C. The injection volume was 2 μl. The mobile phase
consisted of 0.1% formic acid in ultra-pure water - methanol (22:78, v/v) at a flow
rate of 1ml/min. Mass spectrometric condition: ionization was achieved using
electrospray ionization (ESI) with multi-ion monitoring (MRM) in the negative mode.
The optimized quantitative analysis of ion pairs and parameters were described in
Table S3 (shown in Supplementary material).
4.6.3 Selectivity
The LC-MS/MS method has high selectivity because only ions derived from the
analytes of interest were monitored. The typical MRM chromatograms of a blank cell
lysate were shown in Figure S21 (shown in Supplementary material). No interfering
peak was observed in blank cell lysates.
4.6.4 Cell lysate sample preparation
Aliquots of cell lysate (50 μL) were mixed with 50 μl internal standard solution
(Honokiol, 200 ng/mL) and 400 μL methanol and vortex-mixed for 3 min. After
centrifugation for 10 min at 12,000 rpm at 4 °C. 2 μL of these samples was injected
into the LC-MS/MS system for analysis.
The typical equation of calibration curves and linearity ranges for the three
analytes as follows: y = 0.0818x - 0.513 (R2 = 0.9973) at 1–500 ng/mL of CUR, y =
0.0036x - 0.0085 (R2 = 0.9984) at 5–500 ng/mL of compound 5d, y = 0.0066x +

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0.0466 (R2 = 0.9955) at 2–500 ng/mL of compound 5e.
4.6.3 Uptake and uptake inhibition studies
The Caco-2 cells were cultured for 15 days, the culture medium was removed on
the day of experiment, and the Caco-2 cell monolayers were washed twice with HBSS
(Hyclone, USA), after washing, the cells were preincubated with HBSS for 30 min at
37°C. The medium was removed after the preincubation period and uptake was
initiated by adding 1 mL of the preincubated drug solution. Caco-2 cells were
incubated with the drug solution at the appropriate time. The drug solution was
aspirated and the cells were then quickly rinsed three times with ice-cold PBS (pH 7.4,
Hyclone, USA) to terminate uptake. Then the cells were lysed with 0.3 mL of RIPA
lysate, after the cell lysis was completed, the cell lysate was collected, centrifuged at
4°C, 12000 rpm for 20 min and a part of the supernatant was used to determine the
cellular protein by BCA Protein Assay Kit, the LC-MS/MS was utilized to detect the
drug concentration. All experiments were conducted in three independence tests. For
time and pH dependence of uptake experiments, the cell monolayers were incubated
treated with the drug solution (pH = 6.0 or 7.4) at different time (0, 15, 30, 60, 90
min). For the effect of concentration to cellular uptake of CUR, 5d and 5e studies, the
Caco-2 cells were incubated with uptake buffer (pH = 6.0) containing drug solution at
various concentrations (50, 100, 200, 400, 800 μM) for 60 min. In order to evaluate
the in vitro PepT1 involved in the uptake of 5d and 5e, the uptake inhibition test with
Gly-Sar was carried out. The Caco-2 monolayers were incubated with CUR, 5d and
5e (100 μM) in the absence or in the presence of Gly-sar for 60 min at pH = 6.0. To

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explore the inhibition rate of gly-sar on compounds 5d and 5e, Caco-2 monolayers
were incubated with CUR, 5d and 5e at various concentrations ( 100, 200, 400, 800
μM) in the presence of Gly-sar for 60 min at pH = 6.0.
4.7 In vitro phase I metabolism assay
4.7.1 Incubations with rat liver microsomes for 5d and 5e Metabolism
The rat liver microsomes was thawed carefully on ice before the experiment.
CUR, 5d and 5e (20 μM) was incubated with liver microsomes in 200 μL of
phosphate-buffered saline (PBS, 100 mM, pH 7.4), containing magnesium chloride (5
mM). Reactions were initiated by the addition of NADPH (1.3 mM). After 2 h of
incubation, the reaction was terminated by the addition of 1 mL of ice-cold methanol.
Mixtures were vortexed and centrifuged at 12,000g for 10 min at 4 °C to precipitate
proteins. Supernatants were transferred to fresh tubes and evaporated to dryness. The
residues were dissolved in methanol/water (50:50, v/v) for analysis by UFLC-
QTOF/MS. All experiments were performed in duplicate. Control incubations
were performed without the cofactor NADPH.
4.7.2 LC-QTOF MS condition
A UFLC 20ADXR LC system in-line (Shimadzu Corporation, Kyoto, Japan)
coupled with a hybrid quadrupole time-of fight mass spectrometer QTOF MS (Triple
TOF™ 5600 MS system, AB Sciex Corporation., Foster City, CA) was used.
Chromatographic separation of CUR, 5d and 5e and its metabolites was achieved
using a a Inertsil ODS-SP C18 (5 µm, 4.6 × 150 mm, Shimadzu, Tokyo) column at

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40 °C. The injection volume was 2 μL. The mobile phase consisted of 0.1% formic
acid in ultra-pure water - methanol (22:78, v/v) at a flow rate of 1ml/min. Mass
spectrometric condition: MS and MS/MS data were acquired by Analyst® software
using a TripleTOF™ 5600 mass spectrometer with MS scan and information
dependent acquisition (IDA) MS/MS scans. Ionization was achieved using
electrospray ionization (ESI) in the negative mode from m/z 50 to 1200 at a source
temperature of 500 °C. Nitrogen gas was used as ion source gases and collision gas.
The parameters of MS mass spectrometric analysis were as follows: ion Source Gases
1 and 2 (GS1 and GS2), 50 psi; declustering potential, -60 eV; and collision energy
(CE) -5eV.
Acknowledgements
This work was supported by the National Science Foundation of China (No
81573304), and Research Fund for Jiangsu Graduate Research Innovation Program
(KYCX18_1595). Thank you for help in instrumental analysis provided by Center for
Instrumental Analysis of Nanjing University of Chinese Medicine.
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