2-Isocyano glucose used in Ugi four-component reaction: An approach to enhance inhibitory effect against DNA oxidation

Article abstract of DOI:10.1016/j.ejmech.2017.04.041

The Ugi four-component-reaction (Ugi 4CR) allowed synthesizing bisamide from carboxylic acid, aldehyde, amine, and isocyanide in one-pot operation. However, introducing 2-isocyano glucose into the Ugi 4CR and investigating the inhibitory effects of Ugi adducts against radical-induced oxidation of DNA remained technical challenges. We herein applied 2-isocyano glucose (acetylation of hydroxy groups) to perform a catalyst-free Ugi 4CR at room temperature. The gallic, ferulic, caffeic, or p-hydroxybenzoic acids, aniline (or benzylamine and p-aminophenol), and formaldehyde acted as reagents. In the case of inhibiting DNA oxidations induced by 2,2’-azobis(2-amidinopropane hydrochloride) (AAPH), hydroxy radical, and Cu²⁺/glutathione, the Ugi adduct containing glucose moiety exhibited higher antioxidative activities than the structural analog without glucose moiety involved. It was also proved that high antioxidative property was owing to hydroxy groups in glucose moiety. Therefore, sugar-appended Ugi adducts might hold promising inhibitors for DNA oxidation.

Full text of DOI:10.1016/j.ejmech.2017.04.041

Accepted Manuscript
2-isocyano glucose used in Ugi four-component reaction: An approach to enhance
inhibitory effect against DNA oxidation
Peng-Fei Zhao, Zai-Qun Liu
PII:
S0223-5234(17)30307-0
10.1016/j.ejmech.2017.04.041
EJMECH 9392
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2017
Revised Date: 16 April 2017
Accepted Date: 17 April 2017
Please cite this article as: P.-F. Zhao, Z.-Q. Liu, 2-isocyano glucose used in Ugi four-component
reaction: An approach to enhance inhibitory effect against DNA oxidation, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.041.
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ACCEPTED MANUSCRIPT
Graphic Abstract
OAc
O
G NH2
G = Ph, PhCH2,
p-HO-Ph
O
AcO
AcO
CH3OH
+
+
+
OAc
OAc
R
OH
N
R = Ph, p-HO-Ph
HCHO
room
temperature
24 h
C
O
OH
HO
HO
OAc
O
O
deprotection
HO
HO
AcO
AcO
OH
HO
HO
O
O
HN
HN
N
G
N
G
R
R
O
O
HO
OH
Ugi adduct
bisamide

ACCEPTED MANUSCRIPT
2-Isocyano Glucose Used in Ugi Four-Component
Reaction: An Approach to Enhance Inhibitory Effect
against DNA Oxidation
Peng-Fei Zhao, Zai-Qun Liu*
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China.
Email: zaiqun-liu@jlu.edu.cn Tel: +86 431 88499174 Fax: +86 431 88499159
ABSTRACT
The Ugi four-component-reaction (Ugi 4CR) allowed synthesizing bisamide from carboxylic acid,
aldehyde, amine, and isocyanide in one-pot operation. However, introducing 2-isocyano glucose into
the Ugi 4CR and investigating the inhibitory effect of Ugi adducts against radical-induced oxidation of
DNA remained technical challenges. We herein applied 2-isocyano glucose (acetylation of hydroxy
groups) to perform a catalyst-free Ugi 4CR at room temperature. The gallic, ferulic, caffeic, or p-
hydroxybenzoic acids, aniline (or benzylamine and p-aminophenol), and formaldehyde acted as
reagents. In the case of inhibiting DNA oxidations induced by 2,2’-azobis(2-amidinopropane
hydrochloride) (AAPH), hydroxy radical, and Cu2+/glutathione, the Ugi adduct containing glucose
moiety exhibited higher antioxidative activities than the structural analog without glucose moiety
involved. It was also proved that high antioxidative property was owing to hydroxy groups in glucose
moiety. Therefore, sugar-appended Ugi adducts might hold promising inhibitors for DNA oxidation.
KEYWORDS glucose, isocyanide, Ugi 4CR, DNA oxidation.
1

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1. Introduction
A growing body of evidence in antibiotic pathogen expansion [1] and intestinal glucose
metabolism [2] demonstrated that carbohydrate was highly associated with in vivo oxidative stress [3]
via regulating the pathway for glucose tolerance [4]. The glucose was again situated at the convergence
point of designing novel bioactive molecules [5] because glucose-related enzymes were proved to be
able to introduce exogenous molecules into the metabolic process [6]. Hence, some efforts have
recently been directed to ameliorate the sensitivity of glucose-appended anode [7], to specifically
isolate glycoproteins by using glycoform separation column [8], and to design stereoselective catalysts
by introducing glucosyl group [9]. These backgrounds implied that the function of glucose was owing
to the increase of the bioavailability of synthetic molecules [10]. On the other hand, clinic evidences
have addressed the correlation of DNA damage with fatal diseases [11], and therefore, endeavors were
focused on optimizing structures of traditional drugs and improving inhibitory effects against DNA
damage [12]. Of these metrics, some small organic molecules were found to be able to protect DNA by
inhibiting glycosylases [13] or by mimicking biological species undergoing the oxidative damage [14].
2. Chemistry
As an important recognition moiety in the metabolic process [15], attaching glucose moiety to a
molecule became a popular way for increasing the bioavailability of synthetic compounds, and thus, the
glucosylation was usually employed in the design of novel drugs [16]. For example, 1-aminoglucose
and its related isocyanide (hydroxy groups were protected by acetyl group) were applied to perform the
Ugi 4CR in order to prepare carbohydrate-steroid hybrid [17] and divalent neoglycoconjugates [18]. In
addition, 2,3,4-tri-O-pivaloyl-α-D-arabinopyranosylamine was used in the Ugi 4CR because the sugar
moiety could be hydrolyzed under acidic condition to give an amino group. Thus, 1-aminoglucose
2

ACCEPTED MANUSCRIPT
moiety became the precursor of amino group [19]. To sum up the aforementioned results, it was found
that substituents (either amino or isocyano group) for taking part in the reaction located at 1-position,
which was generated by the intramolecular acetalation of glucose. So, substituents at 1-position were so
active that could take part in multicomponent reactions. On the other hand, reports on applications of 2-
aminoglucose (a popularly employed drug) for multicomponent reactions were scanty to-date. This
might be due to the low activity of 2-position in glucose, and therefore, motivated us to explore
applications of 2-aminoglucose and its related isocyanide for the Ugi 4CR.
3. Pharmacology
As part of our continuing program on finding inhibitors against DNA oxidation by using MCRs
[20], presented herein was a study on the Ugi 4CR by using 2-isocyanno glucose along with some
popularly used antioxidants as the carboxylic acid recourses. The obtained glucose-appended Ugi
adducts were applied to inhibit DNA oxidations mediated by 2,2’-azobis(2-amidinopropane
.
hydrochloride) (AAPH, R-N=N-R, R = -CMe2C (=NH)NH2), OH, and Cu2+/glutathione (GSH),
respectively, during which the formation of carbonyl compounds from the oxidation of DNA acted as
the detected targets because carbonyl compounds could be colorized by reacting with thiobarbituric
acid (TBA). Thus, these oxidative products deriving from DNA oxidation were called as thiobarbituric
acid reactive species (TBARS) as well. By the comparison of the amount of TBARS, it was found that
glucose-involved bisamides possessed high abilities to inhibit DNA oxidation.
4. Results and Discussion
4.1. Synthesis of bisamides by Ugi 4CR
3

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Some reagents were carefully selected for the Ugi 4CR and provided us with complicated
structures of bisamides. For example, if dicarboxylic acid and diisocyanide reacted with amne and
formaldehyde, non-symmetric cryptands and igloo-shaped macromulticycles could be achieved via two
sequential Ugi 4CRs [21]. In view of reagents used in the Ugi 4CR, changes in R- of R-N+≡C- were not
as abundant as variations in carboxylic acid, aldehyde, and amine [22]. As pointed out in the Chemistry
section, 1-aminoglucose and its related isocyanide were usually used in multicomponent reactions, but
reports on reactions of 2-aminoglucose (a much popularly used drug) were not usually found. This
encouraged us to explore whether 2-aminoglucose and its related isocyanide were capable of reagents
in the Ugi 4CR. Initially, we performed the Ugi 4CR by using 2-aminoglucose to react with aldehyde.
Unfortunately, the produced imine was so stable that could not further react with the carboxylic acid
and isocyanide. This fact implied that the amino group in 2-aminoglucose was not as active as that in 1-
aminoglucose. Hence, we selected glucuronic acid (aqueous solution) to be the carboxylic acid for the
Ugi 4CR, and it was still failure to achieve the target product. So, we attempted to convert 2-
aminoglucose into the isocyanide for carrying out the Ugi 4CR. The isocyanide group (-N+≡C-) was
generally produced by the dehydration of formamide (-NHCHO) [23]. As a water-soluble compound,
2-aminoglucose could not be dissolved in organic media, while isocyanides were lipophilic reagents.
This implied that hydroxy groups in 2-aminoglucose should be acetylized in order to increase
lipophilicity of 2-aminoglucose, and thus, to be dissolved in ethyl formate during the formylation of
amino group. As shown in Figure 1, hydroxy groups in 2-aminoglucose could not be acetylized directly
because the amino group was superior to be acetylized in the presence of acetic anhydride. Thus, the
amino group in 2-aminoglucose firstly reacted with p-anisaldehyde to form the Schiff base,
glucosamine imine, in which hydroxy groups were acetylized. Subsequently, the amino group was
recovered from the hydrolysis of glucosamine imine, and the reaction of acetyl-appended glucosamine
4

ACCEPTED MANUSCRIPT
with ethyl formate allowed to formylate the amino group. Finally, the dehydration of formamide (-
NHCHO) led to the formation of acetyl-appended glucose isocyanide for the usage in the Ugi 4CR.
Figure 1 Synthesis of isocyanides.
O
OH
OAc
O
N
H
O
HO
HO
AcO
AcO
OH
OAc
OH
O
(CH3CO)2O
600C, overnight
p-anisaldehyde
O
N
HO
HO
N
OH
HCl
NaOH (1.2 equiv.) / H2O
NH2
O
O
room temperature, 2 h
glucosamine imine
acetyl-appended glucosamine imine
O
glucosamine hydrochloride
88%
Ac = CH3C-
83%
CH3COCH3
room temperature, 1 h
HCl / CH3OH
OAc
OAc
O
OAc
O
AcO
AcO
O
CH3COONa / H2O
HCOOC2H5
AcO
AcO
AcO
AcO
OAc
HCl
OAc
H
OAc
NH2
room temperature, 1 h
reflux, 3 h
NH2
NH
POCl3
acetyl-appended
glucosamine hydrochloride
acetyl-appended
glucosamine
O
CH2Cl2
00C, 2 h
80%
87%
N
H
OAc
A general method for preparing isocyanide
NH2
NH2
O
RNH2
=
AcO
AcO
OAc
N
HCOOC2H5
H
N
H
POCl3
CH2Cl2
N
H
R
NH2
C
R
reflux, 3 h
R
N
C
acetyl-appended
glucose isocyanide
00C, 2 h
NH2
O
81%
N
H
6 steps, over yield ~40%
As shown in Figure 2, we herein selected p-hydroxybenzoic, gallic, ferulic, and caffeic acids as
the carboxylic acid resources, which were able to provide with antioxidative moieties for the Ugi
adducts. The aniline (or benzylamine and p-aminophenol) and formaldehyde acted as the amine and
aldehyde resources and were mixed with each other to form imine firstly. The hydroxy group in p-
aminophenol did not influence the formation of imine and was not necessary to be acetylized. However,
as outlined in Table 1, the acidity of phenolic hydroxy group in the aforementioned carboxylic acids
5

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hindered the Ugi 4CR, and the phenolic hydroxy group in the carboxylic acids should be protected by
acetyl group before added to the imine system (produced by the reaction of formaldehyde with aniline
in advance). The acetyl-appended glucosyl isocyanide was then added to the imine system at room
temperature, and no catalyst was needed. Subsequently, acetyl groups in 1~4, 7, and 8 were removed
by CH3ONa, and acetyl groups in 9~11 were removed in the methanolic solution of CH3COONH4.
Compounds 5 and 6 were composed of more than three hydroxy groups, the deprotection under basic
condition such as CH3ONa and CH3COONH4 might lead to the formation of phenolic anions, which
were much sensitive towards the ambient oxidation. Hence, the acetyl groups in 5 and 6 were removed
in the methanolic solution of HCl.
Figure 2 Synthesis of bisamides via the Ugi 4CR.
OAc
G NH2
+
O
O
AcO
AcO
CH3OH
+
+
OAc
R
OH
N
HCHO
C
room
temperature
24 h
adding RCOOH
stirring for 15 min
adding isocyanide
stirring for 24 h
stirring for 1 h
.
OH
OAc
O
O
deprotection
HO
HO
AcO
AcO
OH
OAc
O
O
HN
HN
N
N
G
R
R
O
G
O
Ugi adduct
bisamide
6

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Table 1 Structures of bisamides synthesized in this work (HCHO as the aldehyde resource).
Ugi adduct
bisamide
carboxylic
acid
deprotection
agent
isocyanide
amine
(Ugi 4CR yield)
(deprotection yield)
OAc
O
OH
O
AcO
AcO
HO
OAc
O
OAc
OH
HO
O
NH2
O
O
AcO
AcO
HN
HN
OAc
CH3ONa
CH3ONa
OH
N
N
O
N
O
C
1-1 (87%)
1
(58%)
OAc
O
OH
O
AcO
AcO
HO
HO
OAc
OH
OAc
NH2
O
O
O
HN
HN
O
AcO
AcO
OAc
N
N
HO
OH
O
O
N
C
2-1 (81%)
2 (52%)
OH
O
OH
OAc
O
OH
AcO
AcO
OAc
O
O
HO
HO
OAc
OH
HN
O
NH2
O
AcO
AcO
OH
HN
OAc
N
CH3ONa
O
AcO
N
N
O
OAc
C
3-1 (83%)
OH
3
(47%)
OH
O
OAc
O
HO
HO
AcO
AcO
O
OAc
OH
OAc
NH2
O
O
O
HN
HN
AcO
AcO
OH
OAc
OAc
OAc
N
CH3ONa
N
HO
AcO
O
O
N
OAc
OH
C
C
C
4-1 (80%)
4
(43%)
OH
OH
OH
O
OAc
O
OAc
HO
HO
AcO
AcO
O
OH
OAc
O
AcO
AcO
NH2
O
O
AcO
AcO
HN
HN
OH
HCl
OAc
OAc
OH
OH
N
N
N
O
O
OAc
AcO
OH
5-1 (78%)
5
(38%)
OAc
O
OH
O
AcO
AcO
HO
HO
OAc
OH
OAc
NH2
O
O
O
O
AcO
AcO
HN
HN
AcO
AcO
OH
OAc
OAc
OH
OH
N
N
HCl
HO
O
O
N
OAc
AcO
OH
6-1 (72%)
OH
6 (35%)
OH
7

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OAc
OH
O
AcO
OAc
O
AcO
OAc
HO
HO
O
OH
HN
O
O
O
AcO
AcO
O
HN
NH2
OAc
N
OH
CH3ONa
CH3ONa
O
N
N
AcO
O
OAc
O
7-1 (61%)
C
OH
7
(53%)
O
OAc
O
OH
AcO
AcO
OAc
O
OAc
HO
HO
O
OH
HN
O
O
O
AcO
AcO
AcO
AcO
HN
NH2
OAc
N
OH
O
N
N
O
OAc
OAc
8-1 (65%)
C
OH
8
(46%)
OH
H
N
O
H
N
O
OAc
OH
O
N
N
AcO
AcO
NH2
O
O
OH
C
OAc
N
OH
CH3COONH4
CH3COONH4
AcO
OH
OAc
9-1 (92%)
9
(81%)
H
O
H
O
N
N
N
O
OAc
OAc
OH
OH
N
O
AcO
AcO
NH2
O
OH
N
C
AcO
OH
OAc
10-1 (54%)
10 (76%)
H
O
N
H
O
OAc
OAc
N
N
O
OH
OH
O
N
AcO
AcO
N
NH2
O
H
OH
N
CH3COONH4
OAc
H
OH
C
N
HN
OAc
11-1 (63%)
11 (63%)
4.2. Stoichiometric factor (n) of bisamide in AAPH-induced oxidation of DNA
The decomposition of AAPH at 37°C generated peroxyl radical (ROO.) with a stable rate (Rg), Rg
= (1.4 ± 0.2) × 10-6 [AAPH] s-1 [24], while ROO. abstracted hydrogen atom from the guanine bases,
leading to the oxidative damage of DNA [25]. On the other hand, chemical kinetics demonstrated that
the inhibition period (tinh) resulting from an antioxidant proportionally correlated with the
concentration of the antioxidant as shown as equation (1) [24].
tinh = (n/Ri) [antioxidant]
(1)
8

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The coefficient in the equation (1) consisted of the initiation rate of the radical-induced oxidation (Ri)
and the stoichiometric factor (n, the number of radical-propagation terminated by one molecule of the
antioxidant). Thus, the stoichiometric factor (n) as a quantitative index was capable of characterizing
the antioxidant effectiveness [26]. It was safely assumed that the initiation rate (Ri) of DNA oxidation
was equal to the radical-generation rate, viz., Ri = Rg = 1.4 × 10-6 × 40 mM.s-1 = 3.36 µM.min−1 (the
concentration of AAPH was 40 mM) because ROO. deriving from the decomposition of water-soluble
AAPH attacked DNA sodium salt at the same water phase [27]. Accordingly, a gradient of
concentration of a carboxylic acid was added to inhibit AAPH-induced DNA oxidation for measuring
the inhibition period (tinh). As listed in Table 2, concentrations of these carboxylic acids correlated with
the tinh quantitatively. The stoichiometric factors (n) was the product of the coefficient in the equation
of tinh~[antioxidant] and Ri (3.36 µM.min−1), and n values of gallic, caffeic, ferulic, and p-
hydroxybenzoic acids were obtained as 2.03, 1.86, 1.67, and 0.68, respectively. The aforementioned
data revealed that the inhibitory effects of these carboxylic acids against DNA oxidation depended
upon the number of hydroxy groups.
Table 2 Measurements of tinh~[antioxidant] and stoichiometric factor (n) of carboxylic acids.
antioxidant
tinh = (n/Ri) [antioxidant]
n
O
HO
OH
tinh = 0.61 [gallic acid] + 51.78
2.03
HO
OH
O
HO
HO
OH
OH
tinh = 0.55 [caffeic acid] + 32.55
1.86
1.67
0.68
O
O
tinh = 0.49 [ferulic acid] + 28.61
HO
O
tinh = 0.21 [p-hydroxybenzoic acid] + 23.54
OH
HO
9

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Subsequently, we measured the stoichiometric factors (n) of synthetic bisamides for clarifying the
influence of glucose moiety on the inhibitory effect of the corresponding bisamide against AAPH-
induced oxidation of DNA. As shown in Figure 1S, the addition of 1 did not hinder the increase of the
TBARS when the concentration even increased to 400 ꢀM. Thus, the introduction of glucose moiety
into bisamide was not able to inhibit AAPH-induced oxidation of DNA because of no phenolic
hydroxy group involved. On the other hand, the addition of other bisamides retarded the increase of the
TBARS for a period. As shown in Figure 2S, the inhibitory periods (tinh) were proportionally related to
concentrations of bisamides. The relationships of tinh with concentrations of bisamides were outlined in
Table 1S. The stoichiometric factors (n) of bisamides were also calculated and depicted in Figure 3. It
was found that all of the bisamides (except 1) possessed higher n values than that of the corresponding
carboxylic acid. It was the bisamide skeleton deriving from Ugi 4CR that could actually ameliorate the
antioxidant effect against DNA oxidation.
10

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Figure 3 Stoichiometric factors of bisamides and comparisons with those of carboxylic acids.
6
6
4
2
0
6
4
2
0
5.37
5.12
4.32
3.76
4
2
0
3.43
3.48
3.3
2.41
2.33
2.03
1.64
1.67
1.86
0.68
0
1
gallic
acid
caffeic
acid
p-hydroxy
benzoic
acid
5
6
9
10
11
ferulic
acid
2
3
4
7
8
compound
compound
compound
11

ACCEPTED MANUSCRIPT
Only one hydroxy group was contributed from either p-aminophenol or p-hydroxybenzoic acid.
As a result, stoichiometric factors (n) of 2 and 3 increased to 3.30 and 3.43, respectively, much higher
than that of p-hydroxybenzoic acid (0.68). This result indicated that the single hydroxy group was able
to increase abilities of 2 and 3 to terminate more than 3 radical-propagations, while the same hydroxy
group in p-hydroxybenzoic acid just terminated < 1 radical-propagation. So, the bisamide skeleton
became an efficient structural feature for phenolic hydroxy group to inhibit AAPH-induced DNA
oxidation. Moreover, the aforementioned phenomenon was also found in the middle panel of Figure 3.
After ferulic and caffeic acids performed the Ugi 4CR with benzylamine and formaldehyde, the
produced bisamides 7 and 8 possessed higher stoichiometric factors (n) (3.76 and 3.48, respectively)
than that of ferulic and caffeic acids (1.67 and 1.86, respectively), indicating that inhibitory effects of 7
and 8 were 2~fold higher than that of ferulic and caffeic acids. Furthermore, as shown in the right panel
of Figure 3, the stoichiometric factor (n) of gallic acid (3,4,5-trihydroxybenzoic acid) was 2.03, and
that of 5 increased to 5.12, 2~fold higher than that of gallic acid. On the other hand, 4 consisted of
double hydroxy groups deriving from p-hydroxybenzoic acid and p-aminophenol, respectively. But, the
stoichiometric factor (n) of 4 was 4.32, not much higher than that of 3 (3.43). Thus, double hydroxy
groups allocating at different benzene rings did not markedly increase the inhibitory effect of the
bisamide. This phenomenon was also observed by comparing the stoichiometric factors (n) of 5 (5.13)
with that of 6 (n = 5.37). The hydroxy group deriving from p-aminophenol did not provide much more
antioxidant effect for 6.
Our previous report on the Ugi 4CR revealed that the antioxidant effects of bisamides were mainly
dependent upon the steric hindrance of isocyanide [20]. Thus, we herein selected adamantyl isocyanide
to perform the Ugi 4CR with gallic acid, aniline, and formaldehyde, accessing to 10 in order to
compare the contribution from adamantyl group with that from glucosyl group to the antioxidant effect
(5 possessed the similar structure with 10, see Table 1). But the stoichiometric factor of 10 (2.33) was
12

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much lower than that of 5 (5.12), revealing that high steric hindrance of isocyanide did not improve the
antioxidant effect of the bisamide as glucosyl group did. In addition, tryptamino isocyanide was applied
to synthesize 11, in which an indole moiety replaced the glucosyl group. The stoichiometric factor of
11 (2.41) was still lower than that of 5 (5.12), revealing that aromatic ring with abundant π-electron did
not increase the inhibitory effect of the bisamide either. Finally, cyclohexyl isocyanide was employed
to perform the Ugi 4CR because cyclohexyl group possessed the same six-member ring as glucose but
did not contain any substituents. Thus, comparing 9 with 5 was able to reveal the contribution of
hydroxy groups in glucose to the antioxidant effect of 5. It was found that the stoichiometric factor (n)
of 9 (1.64) was much lower than that of 5 (5.12). Therefore, high antioxidant effect of 5 could be
regarded as the contribution from hydroxy groups in glucose because hydroxy groups in glucose might
form hydrogen bond with DNA strand [28], resulting that glucose-appended bisamides protected DNA
more efficiently than those structural analogs.
4.3. Inhibitory effects against .OH- and Cu2+/GSH-induced oxidations of DNA
Alternatively, antioxidant effects of bisamides were tested in other experimental systems, aiming
at confirming the function of glucose moiety for bisamide inhibiting DNA oxidation. A previous report
suggested that glutathione (GSH) was converted into the corresponding radical (GS.) in the presence of
Cu2+, and DNA was then damaged by GS. [29]. Meanwhile, hydroxy radical (.OH) as the main reactive
.
oxygen species (ROS) oxidized bases in DNA [30]. We applied experimental systems of OH- and
Cu2+/GSH-mediated DNA oxidation to test antioxidant effects of synthetic compounds [31]. As shown
in Figure 3S, mixing 3.0 mM tetrachlorohydroquinone (TCHQ) with 1.5 mM H2O2 led to the formation
.
.
of OH via the decomposition of H2O2, and OH degraded DNA strand to produce TBARS [32]. The
amount of TBARS reached a stable value after 90 min. Figure 4S depicted such a similar phenomenon
that mixing 5.0 mM Cu2+ with 3.0 mM GSH initiated DNA oxidation and produced TBARS as well.
13

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However, when 400
ꢀ
M bisamide was added to DNA oxidation systems, the value of TBARS
absorbance was decreased at the balance state (after 90 min), which was ascribed to inhibitory effect of
bisamide against .OH- or Cu2+/GSH-induced oxidation of DNA. With TBARS absorbance in the blank
experiment being assigned to 100%, other TBARS absorbances were compared with it and depicted in
.
Figure 4 (the left columns were TBARS percentages of bisamides against OH-induced oxidation of
DNA, while right ones were those from Cu2+/GSH-induced DNA oxidation. Low TBARS percentage
implied high antioxidant effect of the bisamide). All of the bisamides used herein did not exhibit high
antioxidant effects because the lowest TBARS percentages generated by 5 and 6 were just ~90%. It
.
was found that TBARS percentages decreased from 100% (the addition of 1 to OH-induced DNA
oxidation) and 99.3% (the addition of 1 to Cu2+/GSH-induced DNA oxidation) to 91.8% and 91.0% in
the case of 6 added, and to 92.7% and 90.1% in the case of 5 added. Decreases of TBARS percentages
supported the obtained conclusion that the antioxidant effect depended upon the number of phenolic
hydroxy groups. Moreover, TBARS percentages deriving from additions of 10 and 11 (95.6% and
93.6%; 93.8% and 96.2%, respectively) were higher than that of 5 (92.7% and 90.1%). In addition,
high TBARS percentages of 9 (97.1% and 97.1%) indicated that the glucose moiety still played the key
role in improving antioxidant effect of 5 against .OH- and Cu2+/GSH-induced DNA oxidation.
14

ACCEPTED MANUSCRIPT
Figure 4 TBARS percentages at 90 min in the case of .OH- and Cu2+/GSH-induced DNA oxidation with 400 ꢀM bisamide added.
101
97
100
99.3
left column: hydroxyl radical
right column: copper(II)/GSH
98.3
96.7
97.1
97.1
97.8
96.5
96.2
93.8
95.6
93.6
95
94.6
94.9
94.7
93.7
92.8
92.7
93
91.8
6
91
90.1
89
1
2
3
4
5
7
8
9
10
11
compoud
15

ACCEPTED MANUSCRIPT
5. Conclusion
We herein provided an approach for enhancing antioxidant effect of bisamide by using glucosyl
isocyanide (hydroxy groups were protected by acetyl group) in the Ugi 4CR. After the deprotection of
acetyl groups, the affinity of bisamide towards DNA might be increased via hydrogen bonds produced
between glucose moiety and DNA strand. Therefore, in the continued quest for the antioxidants, the
sugar-appended MCR adducts might offer additional advantage for antioxidative properties.
6. Experimental section
6.1. Materials and instrumentation
AAPH, naked DNA sodium salt, and D-(+)-glucosamine hydrochloride ([α] °, c = 1.0,
20 = +78.1
D
H2O) were products of ACROS Organics (Geel, Belgium) and used as received. Solvents and reagents
used in the synthesis were obtained commercially and used as such unless noted otherwise. All of the
products were identified by 1H and 13C NMR spectroscopy (Bruker Avance III 400 MHz spectrometer)
as shown in the Supporting Information, and the molecular weight was detected by high solution mass
spectra (HRMS) equipped with ESI as the ionization mode (Agilent 1290-micrOTOF Q II).
6.2. Synthesis of bisamides by Ugi 4CR
Bisamides were produced by the Ugi 4CR with amine, formaldehyde, carboxylic acid, and acetyl-
appended glucosyl isocyanide being starting materials. Briefly, to a 5 mL of methanol was added amine
(0.5 mmol) and formaldehyde (40% aqueous solution, 0.5 mmol) and stirred at room temperature for 1
h, followed by adding carboxylic acid (0.5 mmol) and stirred for 15 min. The isocyanide (0.5 mmol,
dissolved in 3 mL of methanol) was added dropwisely within 30 min and stirred for 24 h at room
temperature. After completion of the reaction as indicated by TLC (the spots have been detected by
UV), the methanol was removed under vacuum. The residue was dissolved with 20 mL of CH2Cl2, and
16

ACCEPTED MANUSCRIPT
the CH2Cl2 solution was washed with saturated NaHCO3 aqueous solution and dried over Na2SO4.
After the organic solvent was evaporated under vacuum, the residue was purified by flash column
chromatography with ethyl acetate/petroleum ether being eluent to afford white Ugi adduct as the
precursor of the bisamide.
Compound 1-1: 2-(N-phenylbenzamido)-N-(1,3,4,6-tetra-O-acetylglucopyranosyl)-2-acetamide, Rf =
1
0.45 (ethyl acetate:petroleum ether = 2:1, v:v), yield 250 mg, 87%. H NMR (400 MHz, CDCl3)
δ:
7.34 (d, J = 7.5 Hz, 2H), 7.21 (dd, J = 17.6, 8.1 Hz, 6H), 7.11 (d, J = 7.4 Hz, 2H), 6.66 (d, J = 8.9 Hz,
1H), 5.86 (d, J = 8.7 Hz, 1H), 5.35 (t, J = 9.9 Hz, 1H), 5.14 (t, J = 9.6 Hz, 1H), 4.44 (q, J = 15.2 Hz,
2H), 4.27 (dt, J = 19.3, 6.3 Hz, 2H), 4.14 (d, J = 12.4 Hz, 1H), 3.86 (d, J = 8.9 Hz, 1H), 2.11 (s, 3H),
2.07 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 171.1, 170.9, 170.7, 169.6,
169.4, 169.0, 143.9, 134.6, 130.2, 129.3, 128.9, 127.8, 127.1, 127.0, 92.3, 72.7, 72.2, 68.2, 65.8, 61.7,
54.9, 53.1, 20.9, 20.7, 20.6.
Compound 2-1: 2-[N-(4-hydroxyphenyl)benzamido]-N-(1,3,4,6-tetra-O-acetylglucopyranosyl)-2-
acetamide, Rf = 0.20 (ethyl acetate:petroleum ether = 2:1, v:v), yield 240 mg, 81%. 1H NMR (400 MHz,
DMSO-d6) δ: 9.46 (s, 1H), 8.11 (d, J = 9.1 Hz, 1H), 7.23 (s, 5H), 6.96 (d, J = 6.9 Hz, 2H), 6.57 (d, J =
7.4 Hz, 2H), 5.76 (d, J = 8.5 Hz, 1H), 5.24 (t, J = 10.0 Hz, 1H), 4.89 (t, J = 9.5 Hz, 1H), 4.38-4.14 (m,
3H), 4.09-3.96 (m, 3H), 2.02 (s, 3H), 1.98 (s, 6H), 1.86 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 171.4,
171.0, 170.8, 170.0, 169.6, 169.5, 155.7, 135.4, 134.4, 130.3, 128.9, 128.2, 127.8, 116.1, 92.1, 72.5,
72.2, 68.2, 61.7, 55.2, 53.1, 20.9, 20.7, 20.5.
Compound
3-1:
2-(N-phenyl-4-acetoxybenzamido)-N-(1,3,4,6-tetra-O-acetylglucopyranosyl)-2-
1
acetamide, Rf = 0.39 (ethyl acetate:petroleum ether = 2:1, v:v), yield 260 mg, 83%. H NMR (400
MHz, CDCl3) δ: 7.37 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 7.8 Hz, H), 7.20 (t, J = 7.3 Hz, 1H), 7.11 (d, J =
7.1 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 9.0 Hz, 1H), 5.85 (d, J = 8.8 Hz, 1H), 5.33 (dd, J =
10.6, 9.4 Hz, 1H), 5.15 (t, J = 9.6 Hz, 1H), 4.43 (q, J = 15.2 Hz, 2H), 4.29 (dd, J = 12.5, 4.6 Hz, 1H),
17

ACCEPTED MANUSCRIPT
4.26-4.18 (m, 1H), 4.14 (dd, J = 12.5, 2.1 Hz, 1H), 3.85 (ddd, J = 10.0, 4.6, 2.2 Hz, 1H), 2.27 (s, 3H),
2.11 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ: 170.5, 170.0,
169.7, 169.3, 169.2, 168.6, 151.6, 144.2, 133.6, 130.2, 129.4, 127.7, 127.0, 121.7, 92.1, 72.4, 72.0, 68.6,
61.9, 53.5, 52.5, 21.3, 20.9, 20.8, 20.7.
Compound 4-1: 2-[N-(4-hydroxyphenyl)-4-acetoxybenzamido]-N-(1,3,4,6-tetra-O-acetylglucopyrano
1
syl)-2-acetamide, Rf = 0.19 (ethyl acetate:petroleum ether = 3:1, v:v), yield 270 mg, 80%. H NMR
(400 MHz, DMSO-d6) δ: 9.51 (s, 1H), 8.13 (d, J = 9.2 Hz, 1H), 7.26 (d, J = 7.2 Hz, 2H), 6.98 (t, J =
8.0 Hz, 4H), 6.59 (d, J = 8.1 Hz, 2H), 5.76 (d, J = 8.8 Hz, 1H), 5.24 (t, J = 10.0 Hz, 1H), 4.89 (t, J =
9.7 Hz, 1H), 4.27 (d, J = 7.7 Hz, 2H), 4.20 (dd, J = 12.7, 4.6 Hz, 1H), 4.08-3.96 (m, 3H), 2.23 (s, 3H),
2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.86 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ: 170.5, 170.0,
169.7, 169.3, 169.2, 168.7, 156.4, 151.3, 135.5, 133.9, 130.2, 129.1, 121.6, 115.8, 92.1, 72.4, 72.0,
68.7, 61.9, 53.6, 52.4, 21.3, 21.0, 20.9, 20.8, 20.7.
Compound 5-1: 2-(N-phenyl-3,4,5-triacetoxybenzamido)-N-(1,3,4,6-tetra-O-acetylglucopyranosyl)-2-
acetamide, Rf = 0.23 (ethyl acetate:petroleum ether = 2:1, v:v), yield 280 mg, 78%. 1H NMR (400 MHz,
CDCl3) δ: 7.32 (d, J = 7.1 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 7.12 (s, 2H), 6.51
(d, J = 9.0 Hz, 1H), 5.86 (d, J = 8.8 Hz, 1H), 5.33 (dd, J = 10.5, 9.4 Hz, 1H), 5.14 (t, J = 9.6 Hz, 1H),
4.39 (dd, J = 33.5, 15.2 Hz, 2H), 4.29 (dd, J = 12.5, 4.6 Hz, 1H), 4.22 (dt, J = 10.5, 8.9 Hz, 1H), 4.14
(dd, J = 12.5, 2.1 Hz, 1H), 3.85 (ddd, J = 10.0, 4.6, 2.2 Hz, 1H), 2.25 (s, 3H), 2.22 (s, 6H), 2.11 (s, 3H),
2.07 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 171.2, 170.7, 169.6, 169.4,
168.6, 168.0, 167.2, 166.4, 143.2, 142.8, 136.1, 132.4, 129.6, 127.5, 126.9, 121.8, 92.2, 72.7, 72.1, 68.1,
61.7, 55.0, 53.2, 20.9, 20.7, 20.5, 20.1.
Compound 6-1: 2-[N-(4-hydroxyphenyl)-3,4,5-triacetoxybenzamido]-N-(1,3,4,6-tetra-O-acetylgluco
pyranosyl)-2-acetamide (6), Rf = 0.21 (ethyl acetate:petroleum ether = 3:1, v:v), yield 270 mg, 72%. 1H
NMR (400 MHz, CDCl3)
δ: 7.13 (s, 1H), 6.94 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 9.0 Hz, 1H), 6.63 (d, J =
18

ACCEPTED MANUSCRIPT
8.8 Hz, 2H), 5.87 (d, J = 8.7 Hz, 1H), 5.39-5.31 (m, 1H), 5.13 (t, J = 9.6 Hz, 1H), 4.36 (dd, J = 24.4,
10.5 Hz, 2H), 4.29 (dd, J = 7.9, 4.5 Hz, 1H), 4.21 (dd, J = 19.4, 9.0 Hz, 1H), 4.13 (dd, J = 12.5, 2.1 Hz,
1H), 3.87 (ddd, J = 10.0, 4.5, 2.2 Hz, 1H), 2.25 (s, 3H), 2.23 (s, 6H), 2.10 (s, 3H), 2.07 (s, 3H), 2.05 (s,
3H), 2.01 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 171.2, 170.8, 169.7, 169.5, 169.4, 167.6, 166.6,
155.9, 142.7, 136.1, 132.5, 128.2, 121.9, 116.4, 92.1, 72.6, 72.1, 68.2, 61.7, 55.2, 53.1, 20.9, 20.7, 20.6,
20.5, 20.1.
Compound 7-1: 2-[N-benzyl-(3-methoxyl-4-acetoxystyryl)formamido]-N-(1,3,4,6-tetra-O-acetylgluco
1
pyranosyl)-2-acetamide, Rf = 0.31 (ethyl acetate:petroleum ether = 2:1, v:v), yield 220 mg, 61%. H
NMR (400 MHz, CDCl3) δ: 7.77 (d, J = 15.2 Hz, 1H), 7.44-7.34 (m, 3H), 7.25 (d, J = 7.4 Hz, 2H), 7.11
(d, J = 8.2 Hz, 1H), 7.04 (s, 1H), 7.03 (d, J = 7.3 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 6.79 (d, J = 15.3 Hz,
1H), 5.92 (d, J = 8.6 Hz, 1H), 5.38 (t, J = 9.9 Hz, 1H), 5.13 (t, J = 9.5 Hz, 1H), 4.78 (s, 2H), 4.29 (dd, J
= 12.5, 4.4 Hz, 1H), 4.16-4.07 (m, 2H), 3.97 (dd, J = 40.6, 14.6 Hz, 2H), 3.87 (d, 1H), 3.85 (s, 3H),
2.33 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ:
170.8, 170.6, 169.5, 169.4, 168.7, 167.5, 151.4, 144.1, 141.2, 136.1, 133.7, 129.1, 128.1, 126.7, 123.2,
120.5, 116.3, 112.0, 92.1, 72.6, 72.1, 68.1, 61.7, 55.9, 53.2, 52.7, 51.1, 21.0, 20.8, 20.7, 20.6, 20.5.
Compound 8-1: 2-[N-benzyl-(3,4-diacetoxystyryl)formamido]-N-(1,3,4,6-tetra-O-acetylglucopyrano
1
syl)-2-acetamide, Rf = 0.29 (ethyl acetate:petroleum ether = 2:1, v:v), yield 240 mg, 65%. H NMR
(400 MHz, CDCl3) δ: 7.76 (d, J = 15.3 Hz, 1H), 7.44-7.35 (m, 5H), 7.32 (d, J = 1.9 Hz, 1H), 7.24 (d,
1H), 7.22 (s, 1H), 6.85 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 15.3 Hz, 1H), 5.91 (d, J = 8.7 Hz, 1H), 5.36 (t,
1H), 5.13 (t, J = 9.7 Hz, 1H), 4.76 (s, 2H), 4.30 (dd, J = 12.5, 4.6 Hz, 1H), 4.15-4.05 (m, 2H), 3.95 (dd,
J = 36.5, 14.7 Hz, 2H), 3.85 (ddd, J = 10.0, 4.5, 2.1 Hz, 1H), 2.31 (s, 6H), 2.12 (s, 3H), 2.10 (s, 3H),
2.04 (s, 3H), 2.02 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ: 170.7, 170.6, 169.5, 169.3, 169.2, 168.0,
167.9, 167.3, 143.4, 142.8, 142.4, 135.8, 133.7, 129.1, 128.1, 126.6, 126.3, 123.9, 122.6, 117.2, 92.1,
72.7, 72.0, 68.1, 61.7, 53.3, 52.5, 50.8, 20.9, 20.7, 20.6, 20.5.
19

ACCEPTED MANUSCRIPT
Compound 9-1: 2-(N-phenyl-3,4,5-triacetoxybenzamido)-N-cyclohexylacetamide, Rf = 0.19 (ethyl
acetate:petroleum ether = 1:1, v:v), yield 230 mg, 92%. 1H NMR (400 MHz, CDCl3)
δ: 7.32 (d, J = 7.5
Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 7.7 Hz, 2H), 7.13 (s, 2H), 6.24 (d, J = 7.7 Hz, 1H), 4.48
(s, 2H), 3.80 (dd, J = 18.4, 10.0 Hz, 1H), 2.25 (s, 3H), 2.22 (s, 6H), 1.92 (d, J = 9.5 Hz, 2H), 1.72 (dd, J
= 9.6, 3.7 Hz, 2H), 1.62 (d, J = 12.8 Hz, 1H), 1.38 (dd, J = 24.4, 12.1 Hz, 2H), 1.20 (dd, J = 20.5, 9.7
Hz, 3H). 13C NMR (100 MHz, CDCl3)
δ: 168.3, 167.3, 167.2, 166.4, 143.0, 142.8, 136.1, 132.6, 129.6,
127.5, 126.9, 121.7, 55.2, 48.4, 32.9, 25.4, 24.7, 20.5, 20.1.
Compound 10-1: 2-(N-phenyl-3,4,5-triacetoxybenzamido)-N-adamantylacetamide, Rf = 0.24 (ethyl
acetate:petroleum ether = 1:1, v:v), yield 160 mg, 54%. 1H NMR (400 MHz, CDCl3)
δ: 7.32 (d, J = 7.6
Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 7.13 (s, 2H), 5.96 (s, 1H), 4.39 (s, 2H), 2.25
(s, 3H), 2.22 (s, 6H), 2.09 (s, 3H), 2.03 (s, 6H), 1.74-1.66 (m, 6H). 13C NMR (100 MHz, CDCl3)
δ:
168.1, 167.2, 166.4, 143.1, 142.8, 136.1, 129.6, 127.4, 126.9, 121.7, 56.1, 52.2, 41.5, 36.3, 29.4, 20.5,
20.1.
Compound 11-1: 2-(N-phenyl-3,4,5-triacetoxybenzamido)-N-2-(3-indolyl)ethylacetamide, Rf = 0.26
1
(ethyl acetate:petroleum ether = 2:1, v:v), yield 180 mg, 63%. H NMR (400 MHz, CDCl3)
δ: 8.56 (s,
1H), 7.58 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 7.11 (q, J = 7.1 Hz,
2H), 7.06 (s, 2H), 7.01 (m, J = 12.9, 5.1 Hz, 3H), 6.60 (d, J = 7.7 Hz, 2H), 6.35 (t, 1H), 4.46 (s, 2H),
3.65 (dd, J = 12.0, 5.7 Hz, 2H), 3.03 (t, J = 6.2 Hz, 2H), 2.31 (s, 3H), 2.26 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ: 167.9, 167.8, 167.5, 166.7, 142.7, 142.4, 136.7, 136.2, 131.9, 129.7, 127.6, 126.8, 126.5,
122.9, 122.2, 122.0, 119.2, 118.6, 111.7, 111.5, 55.5, 38.6, 24.8, 20.6, 20.1.
6.3. Deprotection for Ugi adducts
The deprotection for hydroxy groups in Ugi adducts was performed by CH3ONa, CH3COONH4,
and HCl, respectively. The acetyl groups in Ugi adducts of 1~4, 7, and 8 were removed by CH3ONa,
20

ACCEPTED MANUSCRIPT
which was dissolved in 5 mL of CH3OH (the equivalence was equal to the molar mass of hydroxy
groups in the corresponding bisamide). To a 15 mL of methanol (dried by magnesium chips in advance)
was dissolved the corresponding bisamide (0.2 mmol), and methanolic solution of CH3ONa was added
dropwisely at 0°C and stirred for 2 h at the room temperature. After completion of the reaction as
indicated by TLC, the pH value of the mixture was adjusted with formic acid to 5~7. After the solvent
was evaporated, the residue was purified by silica gel chromatography with methanol and
dichloromethane being eluent to afford white solid.
2-( C.
N-Phenylbenzamido)-N-glucopyranosyl-2-acetamide (1), yield 48 mg, 58%. m.p. 149-150
°
[
α
]
D
20 = +19.6 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ: 7.72 (d, J = 6.4 Hz, 1H), 7.33 (d, J =
7.6 Hz, 2H), 7.23 (m, J = 18.0, 13.6, 7.4 Hz, 6H), 7.11 (d, J = 7.8 Hz, 2H), 6.53 (d, J = 9.0 Hz, 1H),
4.92 (s, 1H), 4.81 (s, 1H), 4.70 (t, J = 6.8 Hz, 1H), 4.55 (s, 1H), 4.43 (q, J = 15.2 Hz, 2H), 3.73 (d, J =
11.7 Hz, 1H), 3.49 (d, 1H), 3.43 (t, J = 9.0 Hz, 1H), 3.27-3.21 (m, 1H), 3.14 (t, J = 9.1 Hz, 1H), 2.79 (t,
J = 8.5 Hz, 1H). HRMS (ESI): m/z calcd for [M+H+] of C21H25N2O7 417.1662; found 417.1644.
2-[N-(4-Hydroxyphenyl)benzamido]-N-glucopyranosyl-2-acetamide (2), yield 45 mg, 52%.
m.p. 177-178
°C. [α
]
D
20 = +25.7 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ: 9.26 (s, 1H), 7.92 (d,
J = 9.3 Hz, 1H), 7.69 (d, J = 7.4 Hz, 2H), 7.51 (t, J = 8.3 Hz, 2H), 7.45 (d, J = 6.6 Hz, 1H), 7.37 (d, J =
7.2 Hz, 1H), 6.99 (d, J = 6.9 Hz, 2H), 6.22 (d, J = 9.4 Hz, 1H), 5.10 (s, 1H), 5.02 (s, 1H), 4.70 (t, J =
8.6 Hz, 1H), 4.38 (s, 1H), 4.14 (dd, J = 15.6, 4.8 Hz, 1H), 3.84 (t, J = 8.0 Hz, 1H), 3.51 (s, 1H), 3.43 (t,
J = 9.0 Hz, 1H), 3.24 (t, J = 8.5 Hz, 1H), 3.16 (t, J = 9.8 Hz, 1H), 2.80 (t, J = 8.2 Hz, 1H). HRMS (ESI):
m/z calcd for [M+H+] of C21H25N2O8: 433.1611; found 433.1597.
2-(N-Phenyl-4-hydroxybenzamido)-
N-glucopyranosyl-2-acetamide (3), yield 41 mg, 47%. m.p.
162-163
°C. [
α
]
20 = +28.8 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ: 10.19 (s, 1H), 8.06 (d, J
D
= 9.2 Hz, 1H), 7.37 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 7.3 Hz, 1H), 7.11 (d, J =
7.1 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.53 (d, J = 9.0 Hz, 1H), 5.03 (d, J = 8.8 Hz, 1H), 4.92 (d, J =
21

ACCEPTED MANUSCRIPT
10.6 Hz, 1H), 4.79 (t, J = 6.6 Hz, 1H), 4.65 (t, J = 13.2 Hz, 2H), 4.42 (dd, J = 12.1, 4.2 Hz, 1H), 4.85-
4.76 (m, 2H), 3.70 (t, J = 12.5 Hz, 1H), 3.26 (d, J = 10.1 Hz, 1H), 3.06 (t, J = 9.4 Hz, 1H), 2.81 (t, J =
8.8 Hz, 1H). HRMS (ESI): m/z calcd for [M+H+] of C21H25N2O8 433.1611; found: 433.1594.
2-[N-(4-Hydroxyphenyl)-4-hydroxybenzamido]-
N-glucopyranosyl-2-acetamide (4), yield 39
mg, 43%. m.p. 173-175
°
C. [
α
]
D
20 = +27.4 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ: 10.13 (s,
1H), 9.43 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.33 (d, J = 7.2 Hz, 2H), 7.05 (t, J = 5.1 Hz, 3H), 6.57 (d, J
= 3.5 Hz, 2H), 6.51 (d, J = 4.8 Hz, 1H), 4.96 (s, 1H), 4.91 (d, J = 5.4 Hz, 1H), 4.67 (d, J = 5.3 Hz, 1H),
4.46-4.31 (m, 3H), 3.62 (m, J = 11.4, 6.7 Hz, 2H), 3.53-3.46 (m, 2H), 3.18 (d, J = 5.2 Hz, 1H), 3.15-
3.07 (m, 1H). HRMS (ESI): m/z calcd for [M+H+]of C21H25N2O9 449.1560; found: 449.1537.
2-[
N
-Benzyl-(3-methoxyl-4-hydroxystyryl)formamido]-
N
-glucopranosyl-2-acetamide
(7),
yield 53 mg, 53%. m.p. 159-160
°C. [
α
]
20 = +18.1 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ:
D
9.58 (s, 1H), 8.21 (d, J = 9.5 Hz, 1H), 7.77 (d, J = 17.8 Hz, 1H), 7.45-7.27 (m, 4H), 7.25 (d, J = 7.3 Hz,
1H), 7.07-7.01 (m, 2H), 6.79 (d, J = 18.1 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H), 4.98 (s, 1H), 4.83 (s, 1H),
4.79 (s, 2H), 4.70 (t, J = 8.9 Hz, 1H), 4.50 (s, 1H), 4.30 (dd, J = 15.3 Hz, 1H), 3.97 (dd, J = 41.2, 13.7
Hz, 2H), 3.88-3.81 (m, 4H), 3.51 (m, 2H), 3.27 (t, J = 9.1 Hz, 1H), 2.95 (t, J = 8.3 Hz, 1H). HRMS
(ESI): m/z calcd for [M+H+] of C25H30N2O9 503.2023; found 503.1998.
2-[N-Benzyl-(3,4-dihydroxystyryl)formamido]-
N-glucopranoyl-2-acetamide (8), yield 45
1
mg, 46%. m.p. 144-146
°C. [
α
]
D
20 = +21.6 (c = 1.0, H2O). H NMR (400 MHz, DMSO-d6)
δ: 9.54 (s,
1H), 9.13 (s, 1H), 8.23 (d, J = 9.5 Hz, 1H), 7.42 (d, J = 15.3 Hz, 1H), 7.05-6.98 (m, 5H), 6.96 (d, J =
9.0 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.52 (d, J = 8.9 Hz, 1H), 6.17 (d, J = 15.6 Hz, 1H), 4.97 (t, J =
9.6 Hz, 1H), 4.80 (t, J = 9.3 Hz, 1H), 4.65 (s, 2H), 4.50 (t, J = 7.8 Hz, 1H), 4.34 (s, 1H), 4.15 (dd, J =
38.5, 11.3 Hz, 2H), 3.74 (d, J = 15.3 Hz, 1H), 3.52-3.40 (m, 2H), 3.24 (t, J = 7.8 Hz, 1H), 3.17 (d, J =
9.0 Hz, 1H), 2.80 (t, J = 8.6 Hz, 1H). HRMS (ESI): m/z calcd for [M+H+] of C24H28N2O9 489.1873;
found 489.1845.
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The acetyl groups in Ugi adducts of 5 and 6 were removed by HCl. To a 15 mL of methanol (dried
by magnesium chips in advance) was dissolved the corresponding bisamide (0.2 mmol), and HCl (10
mmol, dissolved in 3 mL of methanol) was added dropwisely at 0°C under stirring. The temperature
was allowed to reach the room temperature, and the mixture was stirred for 12 h. The solvent was
evaporated under vacuum, and the residue was purified by silica gel chromatography with methanol
and dichloromethane being eluent to afford white solid.
2-(N-Phenyl-3,4,5-trihydroxybenzamido)-
N-glucopyranosyl-2-acetamide (5), yield 35
1
mg, 38%. m.p. 166-168
°C. [
α
]
D
20 = +22.4 (c = 1.0, H2O). H NMR (400 MHz, DMSO-d6)
δ: 9.22 (s,
2H), 8.75 (s, 1H), 8.05 (d, J = 9.2 Hz, 1H), 7.45-7.34 (m, 4H), 7.28 (d, J = 7.7 Hz, 1H), 6.69 (s, 2H),
6.36 (d, J = 8.9 Hz, 1H), 4.99 (s, 1H), 4.89 (s, 1H), 4.78 (s, 2H), 4.59 (t, J = 9.6 Hz, 1H), 4.43 (s, 1H),
4.28 (d, J = 10.2 Hz, 1H), 4.17-4.10 (m, 2H), 4.02 (d, J = 10.8 Hz, 1H), 3.92 (d, J = 9.5 Hz, 1H), 3.16 (t,
J = 9.2 Hz, 1H), 2.85 (t, J = 8.6 Hz, 1H). HRMS (ESI): m/z calcd for [M+H+] of C21H25N2O10 465.1509;
found 465.1483.
2-[N-(4-Hydroxyphenyl)-3,4,5-trihydroxybenzamido]-
N-glucopranosyl-2-acetamide
(6),
yield 34 mg, 35%. m.p. 157-158
°C. [
α
]
20 = +28.3 (c = 1.0, H2O). 1H NMR (400 MHz, DMSO-d6)
δ:
D
9.31 (s, 2H), 8.88 (s, 1H), 8.46 (s, 1H), 7.76 (d, J = 10.6 Hz, 1H), 6.89 (d, J = 9.2 Hz, 1H), 6.70 (d, J =
9.4 Hz, 1H), 6.56 (s, 2H), 6.43 (d, J = 8.0 Hz, 1H), 5.13 (t, J = 7.2 Hz, 1H), 5.04 (s, 1H), 4.97 (s, 1H),
4.63 (s, 1H), 4.46 (m, 2H), 4.18-4.09 (m, 2H), 3.94 (d, J = 12.8 Hz, 1H), 3.86 (m, 1H), 3.20 (t, J = 8.2
Hz, 1H), 2.93 (t, J = 7.8 Hz, 1H). HRMS (ESI): m/z calcd for [M+H+] of C21H25N2O11 481.1458; found
481.1452.
The acetyl groups in Ugi adducts of 9~11 were removed by CH3COONH4. To a 15 mL of
methanol (dried by magnesium chips in advance) was dissolved the corresponding bisamide (0.2
mmol), and CH3COONH4 was added and stirred for 6 h at room temperature. The solvent was removed
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under vacuum, and the residue was purified by silica gel chromatography with methanol and
dichloromethane being eluent to afford white solid.
2-(N-Phenyl-3,4,5-trihydroxybenzamido)-
N-cyclohexylacetamide (9), yield 62 mg, 81%. m.p.
175-176
°
C. 1H NMR (400 MHz, DMSO-d6)
δ: 8.88 (s, 2H), 8.41 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.24
(t, J = 7.3 Hz, 1H), 7.12 (t, J = 8.4 Hz, 2H), 6.25 (s, 1H), 4.31 (s, 1H), 3.52 (dd, J = 17.2, 9.4 Hz, 1H),
1.69 (t, J = 14.6 Hz, 3H), 1.33-1.22 (m, 1H), 1.20-1.08 (m, 3H). HRMS (ESI): m/z calcd for [M+H+] of
C21H24N2O5 385.1763; found 385.1743.
2-(
N
-Phenyl-3,4,5-trihydroxybenzamido)- -adamantylacetamide (10), yield 66 mg, yield 76%.
N
m.p. 195-196
°
C. 1H NMR (400 MHz, DMSO-d6)
δ: 8.74 (s, 1H), 8.17 (s, 1H) 7.71 (s, 1H), 7.31 (t, J =
7.6 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 6.77 (s, 2H), 4.39 (s, 2H), 2.00 (s, 3H),
1.66-1.48 (m, 12H). HRMS (ESI): m/z calcd for [M+H+] of C25H28N2O5 437.2076; found 437.2061.
2-(N-Phenyl-3,4,5-trihydroxybenzamido)-
N-2-(3-indolyl)ethylacetamide (11), yield 56
mg, 63%. m.p. 283-285
°
C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.23 (s, 1H), 8.96 (s, 1H), 8.63 (s, 1H),
7.87 (t, J = 8.9 Hz, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.26 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.1 Hz, 1H),
7.18-7.00 (m, 5H), 6.79 (s, 2H), 6.48 (d, J = 8.6 Hz, 2H), 4.46 (s, 2H), 3.65 (dd, J = 12.0, 5.7 Hz, 2H),
3.05 (t, J = 6.2 Hz, 2H). HRMS (ESI): m/z calcd for [M+H+]of C25H23N3O5 446.1716; found 446.1697.
6.4. AAPH-induced oxidation of DNA
DNA was dissolved in phosphate buffered solution (PBS1: 8.1 mM Na2HPO4, 1.9 mM NaH2PO4,
10.0 µM EDTA, pH = 7.4) to reach 2.24 mg/mL as the stock solution, and AAPH was also dissolved in
PBS1 to reach 400 mM as the stock solution. Solutions of DNA (13.4 mL), AAPH (1.5 mL), and 0.1
mL of bisamide solution (compounds 1~8 were dissolved in water as the stock solution; others were
dissolved in dimethyl sulfoxide as the stock solution) were mixed and aliquoted into test tubes, with
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each containing 2.0 mL (the final concentrations of DNA and AAPH were 2.00 mg/mL and 40.0 mM,
respectively, and the final concentration of bisamide was shown in the corresponding chart in the
Supporting Information). The test tubes were incubated at 37°C for initiating the oxidation of DNA,
and three of them were taken out every 2 hours and cooled immediately in ice water. Then, 1.0 mL of
PBS1 solution of thiobarbituric acid (TBA, 1.00 g of TBA and 0.40 g of NaOH were dissolved in 100
mL of PBS1) and 1.0 mL of trichloroacetic acid (3.0% aqueous solution) were added, and the mixture
was heated in boiling water for 15 min. After the test tubes were cooled in cold water for 5 min, 1.5 mL
of n-butanol was added and shaken vigorously to extract thiobarbituric acid reactive species (TBARS),
whose absorbance was measured at 535 nm and plotted vs the reaction period.
6.5. •OH- and Cu2+/GSH-induced oxidations of DNA
DNA was dissolved in phosphate buffered solution (PBS2: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4)
to reach 2.24 mg/mL as the stock solution, and CuSO4 and glutathione (GSH) were also dissolved in
PBS2 to reach 75.0 and 90.0 mM, respectively, as the stock solutions. To a 13.4 mL of DNA solution
was added 1.0 mL of CuSO4 and 0.5 mL of GSH PBS2 solution, and 0.1 mL of bisamide solution (the
stock solution was prepared as in the AAPH-induced oxidation of DNA test) was added. The
aforementioned mixture was aliquoted into test tubes, with each containing 2.0 mL (the final
concentrations of DNA, Cu2+, and GSH were 2.00 mg/mL, 5.0 mM, and 3.0 mM, respectively, and the
final concentration of bisamide was 400 ꢀM). The test tubes were incubated at 37°C for initiating the
oxidation of DNA, and three of them were taken out at every 15 min. A 1.0 mL of PBS2 solution of
30.0 mM EDTA was added to chelate Cu2+, and then, 1.0 mL of PBS2 solution of TBA (1.00 g of TBA
and 0.40 g of NaOH were dissolved in 100 mL of PBS2) and 1.0 mL of trichloroacetic acid (3.0%
aqueous solution) were added and heated in boiling water for 30 min. After the test tubes were cooled
25

ACCEPTED MANUSCRIPT
in cold water for 5 min, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS,
whose absorbance was measured at 535 nm and plotted vs the reaction period.
DNA and bisamide stock solutions were prepared following the procedure in AAPH-induced
oxidation. Tetrachlorohydroquinone (TCHQ) was dissolved in DMSO to reach 120 mM as the stock
solution, and H2O2 was diluted in PBS1 to reach 30.0 mM as the stock solution. To a 13.4 mL of DNA
solution was added 1.0 mL of H2O2, 0.5 mL of TCHQ solution, and 0.1 mL bisamide solution (the
stock solution was prepared as in the AAPH-induced oxidation of DNA test). The aforementioned
mixture was aliquoted into test tubes, with each containing 2.0 mL (the final concentrations of DNA,
H2O2, and TCHQ were 2.00 mg/mL, 1.5 mM, and 3.0 mM, respectively, and the final concentration of
bisamide was 400 ꢀM). The test tubes were incubated at 37°C for initiating the oxidation of DNA, and
three of them were taken out at every 15 min, and 1.0 mL of PBS2 solution of TBA and 1.0 mL of
trichloroacetic acid (3.0% aqueous solution) were added and heated in boiling water for 15 min. After
the test tubes were cooled in cold water for 5 min, 1.5 mL of n-butanol was added and shaken
vigorously to extract TBARS, whose absorbance was measured at 535 nm and plotted vs the reaction
period.
6.6. Statistical analysis
All of the data were the average values from at least three independent measurements with the
experimental error within 10 %. The equations were analyzed by one-way ANOVA in Origin 7.0
professional Software, and p < 0.001 indicated a significance difference.
Acknowledgment
Financial support from the Jilin Provincial Science and Technology Department, China, is
acknowledged gratefully (20160101317JC).
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Supporting Information
NMR and MS spectra of the synthetic compounds, the variation of TBARS absorbance via the reaction
period, the relationship between the inhibition period (tinh) and the concentration of antioxidant. This
material is available free of charge via the Internet at the Journal.
Author Information
Corresponding Author
*E-mail: zaiqun-liu@jlu.edu.cn
Notes
The authors declare no competing financial interest.
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