Synthesis, characterization, and biological evaluations of 1,3,5-triazine derivatives of metformin cyclization with berberine and magnolol in the presence of sodium methylate

Article abstract of DOI:10.3390/molecules22101752

The novel target products were synthesized in the formation of a triazine ring from berberine, magnolol, and metformin catalyzed by sodium methylate. The structures of products 1-3 were firstly confirmed by extensive spectroscopic analyses and single-crystal X-ray diffraction. The crystal structures of the target product 2 and the intermediate product 7b were reported for the first time. All target products were evaluated for their anti-inflammatory and antidiabetic activities against INS-1 and RAW264.1 cells in vitro and all products showed excellent anti-inflammatory effects and anti-insulin resistance effects. Our studies indicated that new compounds 1-3 were found to be active against inflammation and insulin resistance.

Full text of DOI:10.3390/molecules22101752

molecules
Article
Synthesis, Characterization, and Biological
Evaluations of 1,3,5-Triazine Derivatives of
Metformin Cyclization with Berberine and Magnolol
in the Presence of Sodium Methylate
1
1
1
1
1
2,
Han Cao , Shili Liao , Wenjing Zhong , Xuerong Xiao , Jiancheng Zhu , Weimin Li *,
1
,
1,
Xia Wu * and Yifan Feng *
1
Central Laboratory of Guangdong, Pharmaceutical University, Guangzhou 510000, China;
caohan19930219@163.com (H.C.); shililiao_03@163.com (S.L.); ZWJzwj112@163.com (W.Z.);
x1784502739@163.com (X.X.); edenss@foxmail.com (J.Z.)
College of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510000, China
2
*
Correspondence: 13925023915@139.com (W.L.); wxiaxia@163.com (X.W.); yffeng@139.com (Y.F.);
Tel.: +86-20-39352522 (Y.F.)
Received: 29 September 2017; Accepted: 16 October 2017; Published: 18 October 2017
Abstract: The novel target products were synthesized in the formation of a triazine ring from
berberine, magnolol, and metformin catalyzed by sodium methylate. The structures of products
1
–3
were firstly confirmed by extensive spectroscopic analyses and single-crystal X-ray diffraction.
The crystal structures of the target product and the intermediate product 7b were reported for the
2
first time. All target products were evaluated for their anti-inflammatory and antidiabetic activities
against INS-1 and RAW264.1 cells in vitro and all products showed excellent anti-inflammatory
effects and anti-insulin resistance effects. Our studies indicated that new compounds
to be active against inflammation and insulin resistance.
1–3 were found
Keywords: cyclization triazine; anti-inflammatory activity; anti-insulin resistance
1
. Introduction
Berberine is an isoquinoline alkaloid originally isolated from the Chinese herb Coptis chinensis
Huang lian) and is one of the main components of R. Coptidis [ ]. Magnolol is a 4-allyl-2-(5-allyl-
-hydroxy-phenyl) phenol, and is present in considerable quantities in the bark of the Houpu magnolia
]. Previous data showed that berberine and magnolol were extensively
(
2
1
(Magnolia officinalis) [
2
employed in traditional Chinese medicine for the treatment of diabetes, cancers, inflammations,
and hyperlipoidemia [3–8].
Metformin has been widely applied in clinics since the 1950s [9]. Over the past decades,
metformin has been recommended as a first-line antidiabetic therapy for the treatment of type 2
diabetes mellitus (T2DM) based on the official guidelines of the American Diabetes Association (ADA),
the European Association for the Study of Diabetes (EASD), and the American Association of Clinical
Endocrinologists (AACE) [10,11]. Moreover, it takes part in the regulation of reduced insulin resistance
and blood glucose concentration [12].
According to a review of recent advances in structural modifications of metformin, a variety
of metformin derivatives have been successfully synthesized. In 2005, Viollet synthesized new
3
d and 4f complex compounds with metformin that were utilized for the treatment of diabetic
patients with non-insulin-dependent diabetes mellitus [13]. Liu synthesized seven derivatives of
-amino-4-dimethylamino-1,3,5-triazine and obtained pure target compounds from silica gel column
chromatography in the same year [14]. Additionally, berberine and magnolol derivatives have
2
Molecules 2017, 22, 1752; doi:10.3390/molecules22101752
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1752
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likewise been synthesized. In 1995, K. Iwsav firstly synthesized berberrubine derivatives [15].
In 2010, Huang designed, synthesized, and evaluated a new series of berberine derivatives as AChE
inhibitors [16]. In 2012, Khaing Zin synthesized clovanemagnolol from the reaction of magnolol and
caryophyllene β-oxide, appending macromolecular groups onto the phenolic hydroxyl groups of
magnolol for the first time [17].
However, these reports on berberine, magnolol, and metformin derivatives described individual
structural transformations, while there are no reports on berberine, magnolol, and metformin combined
in the formation of a triazine ring catalyzed by sodium methylate.
Our continuing interest in the chemistry of metformin, berberine, and magnolol and the absence
of any reported synthetic approaches to the structurally distinct frameworks of products
prompted us to begin investigations in this area. We synthesized compounds combined in the
formation of a triazine ring from berberine, magnolol, and metformin (published patents CN 106966997
A and CN 106928246 A) [18 19]. On this basis, we optimized the previous reaction conditions and
identified their structure by UV, IR, H-NMR, C-NMR, 2D-NMR, and HRESIMS. In this study,
the crystal structures of the target product and the intermediate product 7b were never reported in
1–3 (Figure 1)
1–3
,
1
13
2
our cognition. The cytotoxic activities of these derivatives on INS-1 cells and RAW264.1 cells were
established by the MTT method [20]. We also determined that PEG-2 and COX-2 released amounts
of these compounds through ELISA assays, in order to investigate their potential biological and
pharmacological activities.
Figure 1. The structures of compounds 1–3.
2
. Results and Discussion
2
.1. Chemistry
The synthetic strategy for preparing compounds 1 and 2 is outlined in Scheme 1, and the strategy
for compound
of magnolol ( ) and berberrubine (
with metformin (a biguanide) in alkaline medium to afford the final 1,3,5-triazine derivatives (
During the development of compounds and , we started by alkylation; reacting magnolol (
methyl bromoacetate ( ). The reaction proceeded smoothly under mild conditions. Two phenolic OH
groups of magnolol ( ) engaged in alkylation with the bromine group of methyl bromoacetate catalyzed
3
is outlined in Scheme 2. Briefly, it is based on the alkylation of phenolic OH groups
) with methyl bromoacetate and a subsequent ring closure reaction
and ).
) with
5
9
1,
2
5
3
1
2
6
5

Molecules 2017, 22, 1752
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by Na CO . Because the generated HBr was neutralized by Na CO , the alkylation reacting was more
2
3
2
3
rapid and thorough, and compound
dissociated metformin, metformin hydrochloride and sodium methoxide were mixed in a solvent for
0 min at room temperature. In the process of mixing, the displacement reaction occurred between
metformin hydrochloride and sodium methoxide. Because the solution of product could participate in
the subsequent reaction, product was added dropwise to a cyclization reaction with metformin. Before
the cyclization reaction, the ester groups of product were hydrolyzed by sodium methoxide and the
carbonyl groups on product engaged in a ring closure reaction with the two imino groups of metformin
4) in the presence of sodium methylate to afford the final 1,3,5-triazine derivatives (1 and 2).
7 was found to be present in the form of esters. In order to obtain
3
7
7
7
7
(
O
R1
O
O
O
R2
OH
OH
a: R1=OCH3, R2=OH
O
b: R1=OCH3, R2=OCH3
+
Br
Na2CO3
MeOH, 65°C
O
H
+
N
N
NH2
6
h
NH NH
5
6
yield: 75%
4
7
Sodium methylate
solution (5mol/L)
MeOH, 65°C
12h
H2N
N
N
N
N
O
O
O
OH
yield: 17%
1
H2N
N
N
N
N
NH2
N
N
O
N
O
N
yield: 21%
2
Scheme 1. Cyclization of compounds 1 and 2.
Scheme 2. Cyclization of compound 3.
Compared with compounds
analogous process. According to a review of Cao’s and Nechepurenko‘s studies [21
was a pintsch reaction in berberine hydrochloride ( ), which led to the product berberrubine (
the hydroxyl radical of berberrubine ( ) engaged in the nucleophile substitution reaction with the
bromine group of methyl bromoacetate ( ) and gave the intermediate product (10). With the similar
cyclization process of products and , intermediate product 10 was stirred in the metformin solution,
1
and
2
, the ring closure reaction of compound
22], the initial step
). Then,
3 underwent an
,
8
9
9
6
1
2
which consisted of metformin hydrochloride and sodium methoxide that had been mixed in a solvent
for 30 min at room temperature and led to product 3.

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Then, we studied the reaction under various conditions of alkylation reactions (Tables S5 and S6).
Our studies were initiated using dichloromethane (DCM) with Na CO for compound , methanol
7
2
3
(
(
MeOH) for compound 10, and methanol (MeOH) with 8 mL of a sodium methylate (NaOCH ) solution
3
◦
5 mol/L) for compounds
1–
3
at room temperature (25 C). After screening the reaction conditions,
◦
using Na CO as the base catalyst and methanol as the solvent reacted at 65 C for 6 h was determined
to be optimal for the synthesis of product 7 (Table S5, entry 11). DCM as the solvent reacted at 45 C
2
3
◦
for 8 h was determined to be optimal for the synthesis of product 10 in 80% yield (Table S6, entry 9).
Using these optimized conditions (Table S5, entry 11 and Table S6, entry 9), we examined the
substrate scope of the cyclization reaction (Table S7). The best conditions for the reaction of compounds
◦
1
–3
were determined to be CH ONa as the base catalyst and methanol as the solvent, reacted at 65 C
3
for 12 h (Table S7, entry 12).
2
.2. Characterization of Compounds
Target compound was confirmed to possess a molecular formula of C H N O by
1
26
29
5
4
+
high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z = 476.2322 M ,
calcd = 476.2298) and 13C-NMR data, requiring 13 degrees of unsaturation. Compound
2
was confirmed
+
to possess a molecular formula of C H N O by HRESIMS (m/z = 569.3101 M , calcd = 569.3101)
and 13C-NMR data. The Fourier transform infrared (FT-IR) spectrum of
the presence of a hydroxyl group (3225.36 cm ), a methyl group (2929.34 cm ), a carbonyl group
30
36 10
2
1
(Figure S7) suggested
−
1
−1
−
1
−1
−1
(
1680.66 cm ), and benzene rings (1571.7 cm and 1630.52 cm ). A comparison of the FT-IR data
of and (Figures S15 and S7) suggested that these compounds shared the same methyl group and
benzene rings. The C-NMR and H-NMR spectra of
on the N atom (Tables S8 and S9). A detailed examination of the 2D-NMR spectra of
the existence of two benzene rings—ring-A and ring-B—and the triazine ring-C. The heteronuclear
2
1
13
1
1
clearly showed the two methyl groups located
confirmed
1
0
0
0
0
0
multiple bond correlation (HMBC) of H-5/C-7, C-9, C-8; H-6/C-4, C-9; H-5 /C-7 , C-9 ; and H-8 /C-6 ,
C-5 , C-4 , combined with the H- H correlation spectroscopy (COSY) correlations of H-5/H-6, clearly
showing the locations of the benzene ring-A and -B. The triazine ring-C was established based on
0
0
1
1
0
0
0
0
0
0
the HMBC correlations of C-13 /H-14 , H-15 and H-10 /C-11 , C-12 . The relative configurations of
compounds and were determined by X-ray diffractions using Cu-K radiation with a refinement
parameter of 0.04. The crystal structure of compound is deposited in the CCDC as number 1,547,785
Figure 2), while compound 7b is 1,547,784 (Figure 3). Furthermore, the HMBC and H- H COSY
correlations are shown in Figure 4.
1
7
α
1
1
1
(
Figure 2. X-ray ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of compound 1.

Molecules 2017, 22, 1752
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Figure 3. X-ray ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of coampound 7b.
1
1
Figure 4. Key heteronuclear multiple bond correlation (HMBC) and H- H COSY correlations of
compounds 1–3.
Compound
Table S10) and 2D-NMR spectra of
of . The only difference was the replacement of the carbonyl group in
2
has a completely symmetric structure. A detailed comparison of the 13C-NMR data
and revealed that the structure of was very similar to that
by an additional moiety of
(
2
1
2
1
1
4
in 2, as confirmed by the HMBC of H-10/C-11, H-14/C-13, and H-15/C-13.
+
The molecular formula of compound
3
, C H N O , was established by HRESIMS
25
25
6
4
+
13
(
m/z = 473.1936 M , calcd = 473.1937) and C-NMR data, requiring 13 degrees of unsaturation.
The FT-IR spectrum of compound (Figure S23) suggested the presence of a primary amine group
1
−
1
−1
−1
−1
1
(
1674.87 cm ) and benzene rings (1600.63 cm , 1553.38 cm , and 1491.67 cm ). The H-NMR data
of displayed signals for two methyl groups at H = 2.179 (d) and a methoxyl group at H = 3.10 (s)
Figure S17). The HMBC of H-3/2-C, 17-C, 16-C; 1-H/2-C; and 17H/15-C indicated the existence of
benzene ring-A. Ring-B contained an N atom between C-5 and C-15, as confirmed from the HMBC of
3
δ
δ
(
1
1
5
-H/4-C, 6-C and 6-H/5-C, 15-C, combined with the H- H COSY correlations of H-5/H-6. Meanwhile,
ring-C was deduced by the HMBC of 8H/13C, 7C, 15C, 14C and 14H/8C, 15C, 16C. The HMBC of
1
1
1
1H/9C, 13C, 12C, 10C and 12H/13C, 10C, 11C, combined with the H- H COSY correlations of

Molecules 2017, 22, 1752
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12H/11H, elucidated the structure of ring-D. The HMBC of 20H/21C; 24H/22C, 23C; and 25H/22C,
2
3C suggested the presence of the triazine ring.
2
.3. Biological Evaluation
In order to evaluate the anti-inflammatory activities and antidiabetic activities of the synthesized
compounds , we chose RAW264.1 cells (mice inflammatory cells) and INS-1 cells (mouse insulinoma
cells). Cell viability was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay. RAW264.7 cell cultures were pretreated with a series of compounds or
vehicle to evaluate the effects of compounds on the release of COX-2/PEG-2. Two target cytokines
were markedly increased in LPS (Lipopolysaccharide)-stimulated RAW264.7 cells; the increase
was dramatically diminished by compounds at 10 Mol/L and compound at 25 Mol/L
concentrations. The level of COX-2 was decreased by 13.06%, 14.24%, and 25.41%, respectively,
by compounds , and the level of PEG-2 was decreased by 27.05%, 32.26%, and 36.41%. Ibuprofen
was used as positive control and the level of COX-2/PEG-2 was decreased by 32.82% and 42.31%
1–3
1–3
1–3
1
,
2
µ
3
µ
1–3
(Figure 5A,B). In general, compounds 1–3 all performed remarkable anti-inflammatory activity.
Figure 5.
The blank group was not stimulated by LPS. The model group and compound
stimulated by LPS. * p < 0.05, ** p < 0.01 compared to the model; ( ) Glucose-mediated (16.7 mM)
insulin secretion by INS-1 cells incubated in the presence of compounds . The blank group was
not stimulated by culture medium containing 40 mmol/L glucose. The model group and compound
groups were stimulated by culture medium containing 40 mmol/L glucose. * p < 0.05, ** p < 0.01
compared to the model.
(
A,
B) The levels of COX-2 and PEG-2 which influenced by compounds
1–
3
and ibuprofen.
1–3 groups were
C
1–3
1–3
Compounds
-cells. All the products had a significant stimulatory effect on the insulin secretion from INS-1 cells.
, an increase of 143% in insulin release was recorded compared to
the model whereas increases of 156% and 137% were recorded in the presence of compound and
compound , respectively (Figure 5C). Metformin was used as positive control and insulin increased
47% compared with the model. The results showed that all compounds had excellent antidiabetic
1–3 were assessed for their effects on the insulin secretion of rat pancreatic INS-1
β
In the presence of compound
3
2
1
1
effects and stimulatory effects on the insulin secretion from INS-1 cells, and the effects of compound
exceeded that of metformin.
2

Molecules 2017, 22, 1752
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3
. Materials and Methods
3
.1. General Experimental Procedures
FT-IR spectra were recorded on a Jasco FT-IR-4600 microscopic spectrometer (Jasco Corporation,
Tokyo, Japan). UV spectra were measured on a Jasco V-550 UV/Vis spectrophotometer (Jasco Corporation,
Tokyo, Japan). Melting points were determined using a Beijing Tech X-6 microscopic melting point
apparatus (Beijing Tech Corporation, Beijing, China). The 1D- and 2D-NMR spectra were taken on
a Bruker-AVANCEIII-010601AM-500 spectrometer (Bruker Corporation, Karlsruhe, Germany) using
TMS (Tetramethylsilane) as an internal standard. ESIMS was measured on a Waters UPLC/Q-TOF
mass spectrometer (Waters Corporation, Milford, MA, USA). The X-ray crystallographic data were
obtained on a Rigaku X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) using Cu-K
HPLC was performed on a Waters 2695 liquid chromatograph (Waters Corporation, Milford, MA,
USA) with DIKMA-Diamonsil-C18 (5 m, 200 4.6 mm). Reactions were monitored by HPLC
DIKMA-Diamonsil-C18, 5 µm, 200 × 4.6 mm).
α radiation.
µ
×
(
3
.2. Synthesis and Purification of Compounds 1–3
All the starting reactants, solvents, and catalysts were commercially available unless otherwise
stated. All solvents, except for the aqueous media, were purified by standard techniques to
exclude moisture.
3
.2.1. Synthesis of Compound 7
Magnolol (2.66 g, 0.01 mol), methyl bromoacetate (2 mL, 0.02 mol), and anhydrous potassium
◦
carbonate (Na CO ) (0.02 mol) in absolute methanol (50 mL) were reacted at 65 C for 6 h. After the
2
3
completion of the reaction, the mixture was filtrated while hot and the methanol was removed by
vacuum distillation to give a solid residue (2.32 g, 86%, containing a mixture of 36% 7a and 46% 7b).
Crystals of pure 7b could be separated manually and were used for characterization.
0
0
0
0
◦
Dimethyl 2,2 -{[5,5 -diallyl-(1,1 -biphenyl)-2,2 -diyl]bis(oxy)}diacetate (7b): White powder; m.p. 209–211 C;
−
1
Yield: 75%; ESI-MS m/z 409.17 M ; H-NMR (500 MHz, DMSO-d )
δ
ppm: 3.31 (4H, d, J = 7.0 Hz, H-1,
6
0
0
0
0
H-1 ), 3.65 (6H, s, H-12, H-12 ), 4.67 (4H, s, H-10, H-10 ), 5.01–5.11 (2H, m, H-3, H-3 ), 5.91–5.99 (2H,
0
0
0
0
m, H-2, H-2 ), 6.89 (2H, d, H-9, H-9 ), 7.04 (2H, d, J = 2.5 Hz, H-6, H-6 ), 7.04–7.10 (2H, m, H-5. H-5 );
1
3
0
0
0
0
C-NMR (125 MHz, DMS0-d )
12.5 (C, C-9, C-9 ), 115.5 (C, C-3, C-3 ), 127.3 (C, C-8, C-8 ), 128.2 (C, C-5, C-5 ), 131.3 (C, C-6, C-6 ),
32.1 (C, C-4, C-4 ), 137.8 (C, C-2, C-2 ), 153.5 (C, C-7, C-7 ), 169.3 (C, C-11, C-11 ).
δ
ppm: 38.5 (C, C-1, C-1 ), 51.6 (C, C-12, C-12 ), 65.4 (C, C-10, C-10 ),
6
0
0
0
0
0
0
1
1
0
0
3
.2.2. Synthesis of Compounds 1 and 2
Metformin hydrochloride (6.6 g, 0.04 mol), a sodium methylate solution (5 mol/L, 8 mL),
and absolute methanol (20 mL) were added to a flask and mixed at room temperature for 30 min.
◦
A methanol solution of compound
7
(4.4 g, 0.01 mol, 50 mL) was added dropwise to the flask at 65 C,
and the mixture was reacted for 12 h. After the reaction, the mixture was filtrated and the precipitate
was isolated (5.33 g,
1
: 47.85%,
2
: 29.79%). Methanol (70 mL) and the precipitate were mixed at
◦
60 C for 6 h and then filtered to obtain a mixture (3.34 g,
1
: 56.89%, : 33.58%). Then, the mixture
2
◦
and 70 mL DCM were mixed at 40 C for 4 h and then filtered to obtain dichloromethane solution,
which was vacuum distilled to eliminate the DCM. The white powder was washed with water to
obtain compound
2
(1 g, 0.0017 mol, 97%). Then, absolute methanol solution (26 mL) of the filter
◦
cake was added into formic acid (1 mL) and then precipitated in 4 C for 12 h. Then, the mixture was
filtrated and the precipitated was isolated (1, 0.99 g, 0.0021 mol, 97%).
0 0 0
-{{5,5 -Diallyl-2 -{[4-amino-6-(dimethylamino)-1,3,5-triazin-2-yl]methoxy}-(1,1 -biphenyl)-2-yl}oxy}acetic
2
◦
+ 1
acid (
1): White powder; m.p. 277–280 C; Yield: 17%; ESI-MS m/z 476.2322 M ; H-NMR (500 MHz,

Molecules 2017, 22, 1752
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0 0
δ ppm: 3.30 (4H, t, J = 7.5 Hz, H-1, H-1 ), 5.88–5.99 (2H, m, H-2, H-2 ), 4.98–5.04 (2H, m,
DMSO-d6)
H-3), 5.04–5.10 (2H, m, H-3 ), 6.90 (1H, d, J = 8.4 Hz, H-5), 7.02–7.11 (4H, m, H-5 , H-6, H-8 , H-9), 6.86
0
0
0
0
0
0
0
(
1H, d, J = 8.4 Hz, H-9 ), 4.69 (2H, s, H-10), 4.55 (2H, s, H-10 ), 2.99 (6H, s, H-14 , H-15 ), 6.80 (2H, s,
0
13
0
0
H-16 ); C-NMR (125 MHz, DMSO-d ) δ ppm: 39.6 (C, C-1, C-1 ), 137.9 (C, C-2, C-2 ), 115.4 (C, C-3,
6
0
0
0
0
C-3 ), 131.6 (C, C-4), 131.5 (C, C-4 ), 112.8 (C, C-5), 131.3 (C, C-5 ), 127.4 (C, C-6), 127.1 (C, C-6 ), 154.4
0
0
0
(
(
C, C-7), 153.8 (C, C-7 ), 127.1 (C, C-8), 127.4 (C, C-8 ), 131.2 (C, C-9), 112.5 (C, C-9 ), 70.4 (C, C-10), 65.6
0
0
0
0
0
C, C-10 ), 170.5 (C, C-11 ), 172.8 (C, C-12), 166.5 (C, C-12 ), 165.1 (C, C-13 ), 35.6 (C, C-14 ).
0 0 0
,6-{[5,5 -Diallyl-(1,1 -biphenyl)-2,2 -diyl]bis(methylene)}bis(N2,N2-dimethyl-1,3,5-triazine-2,4-diamine) (2):
6
◦
+ 1
White powder; m.p. 211–213 C; Yield: 21%; ESI-MS m/z 569.3101 M ; H-NMR (500 MHz, DMSO-d )
6
0
0
δ
ppm: 3.28 (4H, t, J = 6.7 Hz, H-1, H-1 ), 5.90 (2H, ddt, J = 16.8, 10.0, 6.8 Hz, H-2, H-2 ), 5.05 (2H, s,
0
0
0
H-3), 4.96 (2H, s, H-3 ), 7.01, 7.02 (2H, dd, J = 8.4, 2.3 Hz, H-5, H-5 ), 7.11 (2H, d, J = 2.3 Hz, H-6, H-8 ),
0
0
0
0
6
6
.92 (2H, d, J = 8.5 Hz, H-9, H-9 ), 4.71 (4H, S, H-10, H-10 ), 2.99 (12H, s, H-15, H-16, H-15 , H-16 ),
0
2
0
0
0
.8 (4H, s, H-NH , NH ), 4.69 (2H, s, H-10), 4.55 (2H, s, H-10 ), 2.99 (6H, s, H-14 , H-15 ), 6.80 (2H, s,
ppm: 38.6 (C, C-1, C-1 ), 137.9 (C, C-2, C-2 ), 115.3 (C, C-3,
C-3 ), 131.4 (C, C-4, C-4 ), 127.2 (C, C-5, C-5 ), 131.1 (C, C-6, C-6 ), 154.4 (C, C-7, C-7 ), 127.9 (C, C-8,
2
0
13
0
0
H-16 ); C-NMR (125 MHz, DMSO-d )
δ
6
0
0
0
0
0
0
0
0
0
0
C-8 ), 112.9 (C, C-9, C-9 ), 70.5 (C, C-10, C-10 ), 172.8 (C, C-11, C-11 ), 166.5 (C, C-12, C-12 ), 165.1 (C,
0
0
0
C-13, C-13 ), 35.6 (C, C-14, C-14 , C-15, C15 ).
3
.2.3. Synthesis of Compound 9
Berberine (3.71 g, 0.01 mol) in DMF (20 mL) was reacted at 160 C for 2 h. After the reaction,
◦
the mixture was then poured into cold water and a red precipitate was generated. The red solid was
filtrated (2.78 g, 94%).
1
0-DiMethoxy-5,6-dihydro-(1,3)dioxolo(4,5-g)isoquinolino(3,2-a)isoquinolin-7-ium chloride hydrate (
9
δ
): Red
ppm:
.07 (s, 1H, H-5), 7.97 (s, 1H, H-14), 7.60 (s, 1H, H-17), 7.20 (d, J = 8 Hz, 1H), 6.94 (s, 1H, H-3), 6.34
d, J = 8.5 Hz, 1H, H-12), 6.08 (s, 2H, H-1), 4.47 (s, 2H, H-6), 3.72 (s, 3H), 3.03 (d, 2H, H-5); 13C-NMR
125 MHz, DMSO-d6) ppm: 167.1, 149.6, 148.3, 147.2, 145.7, 133.2, 132.0, 129.2, 121.8, 121.31, 119.9,
17.1, 108.2, 104.6, 101.5, 104.6, 101.5, 100.8, 55.7, 52.3, 27.4.
◦
+ 1
crystal; m.p. 245 C; Yield: 75%; ESI-MS m/z 322.1002 M ; H-NMR (500 MHz, DMSO-d )
9
(
(
1
6
δ
3
.2.4. Synthesis of Compound 10
Berberrubine (3.57 g, 0.01 mol) and methyl bromoacetate (4 mL, 0.04 mol) were reacted in DCM
◦
(
20 mL) at 45 C for 8 h. After the reaction, the mixture was filtered, and the precipitate was isolated.
◦
Ethanol (95%) was added, and the solution was stirred and refluxed for 4 h at 70 C to obtain compound
1
0 (3.15 g, 0.008 mol, 4%).
1
0-Methoxy-9-(2-oxoethoxy)-(1,3) dioxolo (4,5-g) isoquinolino (3,2-a) isoquinolin-7-ium chloride (10): Yellow
◦
+ 1
powder; m.p. 212–215 C; Yield: 80%; ESI-MS m/z 395.11 M ; H-NMR (500 MHz,DMSO-d )
δ
ppm:
.9 (s, 1H), 8.9 (s, 1H), 8.20 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18
s, 2H), 5.07 (s, 2H), 4.94 (t, J = 6.3 Hz, 2H), 4.03 (s, 3H, -OCH ), 3.72 (s, 3H, -OCH ), 3.23–3.15 (m, 2H);
6
9
(
3
3
1
3
C-NMR (125 MHz, DMSO-d6)
δ ppm: 169.3, 149.8, 149.2, 147.6, 145.7, 141.4, 137.5, 132.9, 130.6, 126.7,
1
23.5, 121.1, 120.4, 120.1, 108.4, 105.4, 102.1, 69.2, 57.1, 55.4, 51.8, 26.3.
3
.2.5. Synthesis of Compound 3
Metformin hydrochloride (4.125 g, 0.025 mol), a sodium methylate solution (5 mol/L, 8 mL),
and absolute methanol (60 mL) were added to a flask and mixed at room temperature for 30 min.
The absolute methanol (15 mL) solution of compound 10 (1.67 g, 0.004 mol, 94%) was added dropwise
◦
to the flask at 65 C, and the resulting mixture was reacted for 12 h. After the reaction, the mixture
was filtered, and the precipitate was isolated (1.78 g, 36.25%). Methanol (100 mL) and the precipitate
◦
were mixed at 60 C until the filtrate become colorless, and then the mixture was filtrated again (1.25 g,

Molecules 2017, 22, 1752
9 of 11
6
8.17%). Compound
3
was obtained from the precipitate (0.62 g, 0.0013 mol, 97%) after crystallization
in ethanol (3.6 mL) and concentrated hydrochloric acid (22 µL).
9
-[4-Amino-6-(dimethylamino)-1,3,5-triazin-2-yl]-10-methoxy-(4,5-g)isoquinolino(3,2-a)isoquinolin-7-ium
◦
+ 1
chloride (
3
): Red solid; m.p. 215–217 C; Yield: 33%; ESI-MS m/z 473.1936 M ; H-NMR (500 MHz,
ppm: 7.17 (2H, s, H-1), 5.84 (1H, s, H-3), 2.17 (2H, s, H-5), 3.83–4.02 (2H, m, H-6), 6.41
1H, s, H-8), 6.74 (1H, d, J = 8.5 Hz, H-11), 6.94 (1H, d, J =8 .5 Hz, H-12), 5.48 (1H, s, H-14), 5.48 (1H, s,
H-17), 3.10 (3H, s, H19), 5.02 (2H, s, H-20), 2.21 (4H, s, H-24, H-25); 13C-NMR (125 MHz, DCOOD-d2)
ppm: 166.5 (C, C-1), 121.2 (C, C-2), 102.4 (C, C-3), 159.7 (C, C-4), 27.1 (C, C-5), 53.7 (C, C-6), 130.9
C, C-7), 106.4 (C, C-8), 123.9 (C, C-9), 142.1 (C, C-10), 120.5 (C, C-11), 132.7 (C, C-12), 131.1 (C, C-13),
08.9 (C, C-14), 143.0 (C, C-15), 151.3 (C, C-16), 147.7 (C, C-17), 161.4 (C, C-18), 58.3 (C, C-19), 103.0 (C,
C-20), 148.9 (C, C-21), 156.4 (C, C-22), 156.0 (C, C-23), 38.1 (C, C-24), 37.9 (C, C-25).
DCOOD-d2)
(
δ
δ
(
1
3
.3. HPLC Analysis of the Products
The yields of products were determined by HPLC analysis on the crude reaction mixture.
The analytical HPLC system consisted of a Waters 2695 liquid chromatograph, a Waters 2996-visible
detector, and Empower chromatography data station software. All samples were analyzed by HPLC
using a reverse-phase DIKMA-Diamonsil-C18 (5
rate of 1.0 mL/min. For products and , the mobile phases consisted of solvent A (0.2% phosphoric
acid in water, v/v) and solvent B (acetonitrile). The gradient elution was as follows: 45% B and 55% A.
The flow rate was set at 1.0 mL/min. The sample injection volume was 10 L. The UV detector was set
at 277 nm. For product , the gradient elution was as follows: initially, 0–50 min, 10–50% B; 50–55 min,
0–10% B; 55–60 min, 10% B. The flow rate was set at 1.0 mL/min. The sample injection volume was
0 µL. The UV detector was set at 254 nm.
µm, 200
×
4.6 mm) at room temperature with a flow
1
2
µ
3
5
1
3
.4. Cellular Proliferation Assay
3
.4.1. Testing the Cytotoxicities of 1–3 against Mouse Macrophages and Mouse Insulinoma
Compounds 1–3 were tested for their cytotoxicities against RAW264.7 mouse macrophages and
INS-1 cell lines. RAW264.7 cells were cultured in DMEM, and INS-1 cells were cultured in RMPI 1640.
All cell lines were supplemented with 10% fetal bovine serum (FBS, Gibco Laboratories, Grand Island,
◦
NY, USA), 100 IU/mL penicillin, and 100
µ
g/mL streptomycin at 37 C with 5% CO . The media were
2
changed every day. The viabilities of cells were determined by an MTT colorimetric assay system.
For the toxicity assay, RAW264.7 and INS-1 cells were trypsinized and seeded onto 96-well plates at
4
a density of 1
1
×
10 cells per well. Then, the cells were treated with various concentrations (5, 10, 25, 50,
00, 200, 400, 800, and 1600
µ
M) of compounds
L of MTT (5 mg/mL) was added to each well, and the plates were incubated for 4 h at
L/well) was added. The optical density of lysate at 490 nm was measured by
1–3 with a volume of 100 µL in sextuplicates. After 48 h
of treatment, 20
µ
◦
3
7 C. Then, DMSO (100
µ
an enzyme immunoassay instrument (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA, USA).
3
.4.2. Testing the Anti-Inflammatory Activities of 1–3 against a Mouse Insulinoma Cell Line
For the anti-inflammatory activity assay, RAW264.7 cells were trypsinized and seeded onto 96-well
4
plates at a density of 1
×
10 cells per well. Then, according to the results of the cytotoxicities of
1–3
against mouse macrophages and mouse insulinoma, we chose cells whose survival rate was between
0–90% and the cells were treated with the target compounds (10 M for compounds and , 25 M for
compound ) with a volume of 100 L in sextuplicates, and ibuprofen (Sigma Inc., Shenzhen, China)
was used as a positive control. After 1 h of treatment, the model group and positive control group were
given LPS (Sigma Inc., Shenzhen, China), and the final concentrations were adjusted to 0.5 g/mL.
8
µ
1
2
µ
3
µ
µ
After 48 h, the results of PEG-2 and COX-2 were determined by ELISA kits (Nanjing Jiancheng
Bioengineering Insitute, Nanjing, China).

Molecules 2017, 22, 1752
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3
.4.3. Testing the Anti-Insulin Resistance Activities of 1–3 against a Mouse Insulinoma Cell Line
For the anti-insulin resistance activity assay, INS-1 cells were trypsinized and seeded onto 96-well
4
plates at a density of 1
8
×
10 cells per well. Then, we chose cells whose survival rate was between
0–90% and the cells were treated with the target compounds (50 µM for compound 1, 5 µM for
compound 2, 25 µM for compound 3) in 100 µL of culture medium containing 40 mmol/L glucose in
sextuplicates, and metformin was used (Sigma Inc., Shenzhen, China) as a positive control. After 48 h
of treatment, the supernatant was removed, and 100 µL of sugar-free culture medium was seeded and
settled for 30 min. Then, each group was seeded in 100
glucose and incubated for 1 h at 37 C. The results were determined by Rat-Insulin ELISA kits
µ
L of culture medium containing 16.7 mM
◦
(Nanjing Jiancheng Bioengineering Insitute, Nanjing, China).
4
. Conclusions
In summary, we have synthesized three novel target products,
1–3, which combined
two pharmacophores from berberine, magnolol, and metformin. All the products were synthesized
and their anti-inflammatory activities and antidiabetic activities against INS-1 cells and RAW264.1
cells were evaluated in vitro for the first time. It is noteworthy that the crystal structures of the target
product
an inexpensive, safe, and simple way to synthesize 1,3,5-triazine derivatives. Compounds
showed remarkable anti-inflammatory and antidiabetic effects in INS-1 cells and RAW264.1 cells
compared with ibuprofen and metformin. The results demonstrated that compounds were able to
relieve the COX-2/PEG-2 destruction and stimulate an insulin secretion increment. Summarizing the
obtained results in our study, we believe that compounds have great potential as anti-inflammatory
2
and the intermediate product 7b were reported for the first time, and this method provided
1–3
all
1–3
1–3
and antidiabetic agents. Moreover, various pharmacophoric groups were well-tolerated under the
optimized reaction conditions; thus, they may serve as drug leads in pharmaceutical chemistry.
Supplementary Materials: The following are available online.
Acknowledgments: The authors gratefully acknowledge finanial support from the National Natural Science
Foundation of China (NSFC, Grant No. 21275036 and No. 81202429).
Author Contributions: Yifan Feng, Han Cao and Wenjing Zhong designed the research; Han Cao, Wenjing Zhong,
and Shili Liao performed experiments; Han Cao and Xia Wu analyzed the data, Han Cao, Xuerong Xiao,
Jiancheng Zhu, and Weimin Li wrote or contributed to the writing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).