Synthesis of a novel series of (E, E)-4,6-bis(styryl)-2-O-glucopyranosyl- pyrimidines and their potent multidrug resistance (MDR) reversal activity against cancer cells

Article abstract of DOI:10.1080/07328303.2012.689041

A novel series of methoxy or benzyloxy substituted (E,E)-4,6-bis(styryl)-2- O-glucopyranosyl-pyrimidines as curcuminoid analogs were synthesized in four steps with total yields of 21.5% to 33.9%. A549 and HL60 cells were employed for the anticancer activity testing. The results demonstrated that 5a, 5c, and 5e have some inhibitory activity against the HL-60 cell line. Unfortunately, no compound displayed inhibitory activity against A549 except for 5c. MDR reversal activity results demonstrated that compounds 4a (RF = 12.3) and 4b (RF = 18.5) showed strong reversal activity to the P-gp-mediated LCC6MDR cells compared to verapamil (RF = 3.2) and no cytotoxicity to cancer or normal cell lines even at a high concentrations (100M).

Full text of DOI:10.1080/07328303.2012.689041

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Synthesis of a Novel Series of (E,E)-4,6-
bis(styryl)-2-O-Glucopyranosyl-
Pyrimidines and Their Potent Multidrug
Resistance (MDR) Reversal Activity
Against Cancer Cells
Lei Gao a , Qian Liu a , Sumei Ren a , Shengbiao Wan a , Tao Jiang a ,
Iris L. K. Wong b , Larry M. C. Chow b & Shixi Wang c
a Key Laboratory of Marine Drugs, Chinese Ministry of Education,
School of Medicine and Pharmacy , Ocean University of China ,
Qingdao , China
b Department of Applied Biology and Chemical Technology and the
State Key Laboratory for Chirosciences , The Hong Kong Polytechnic
University , Hung Hom , Hong Kong SAR , China
c Department of Chemistry , Jining College , Jining , China
Published online: 31 Oct 2012.
To cite this article: Lei Gao , Qian Liu , Sumei Ren , Shengbiao Wan , Tao Jiang , Iris L. K. Wong ,
Larry M. C. Chow & Shixi Wang (2012) Synthesis of a Novel Series of (E,E)-4,6-bis(styryl)-2-O-
Glucopyranosyl-Pyrimidines and Their Potent Multidrug Resistance (MDR) Reversal Activity Against
Cancer Cells, Journal of Carbohydrate Chemistry, 31:8, 620-633, DOI: 10.1080/07328303.2012.689041
To link to this article: http://dx.doi.org/10.1080/07328303.2012.689041
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Journal of Carbohydrate Chemistry, 31:620–633, 2012
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2012.689041
Synthesis of a Novel Series of
(E,E)-4,6-bis(styryl)-2-O-
Glucopyranosyl-Pyrimidines
and Their Potent Multidrug
Resistance (MDR) Reversal
Activity Against Cancer Cells
Lei Gao,1 Qian Liu,1 Sumei Ren,1 Shengbiao Wan,1 Tao Jiang,1
Iris L. K. Wong,2 Larry M. C. Chow,2 and Shixi Wang3
1Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine
and Pharmacy, Ocean University of China, Qingdao, China
2Department of Applied Biology and Chemical Technology and the State Key
Laboratory for Chirosciences, The Hong Kong Polytechnic University, Hung Hom,
Hong Kong SAR, China
3Department of Chemistry, Jining College, Jining, China
A
novel series of methoxy or benzyloxy substituted (E,E)-4,6-bis(styryl)-2-O-
glucopyranosyl-pyrimidines as curcuminoid analogs were synthesized in four steps with
total yields of 21.5% to 33.9%. A549 and HL60 cells were employed for the anticancer
activity testing. The results demonstrated that 5a, 5c, and 5e have some inhibitory
activity against the HL-60 cell line. Unfortunately, no compound displayed inhibitory
activity against A549 except for 5c. MDR reversal activity results demonstrated that
compounds 4a (RF = 12.3) and 4b (RF = 18.5) showed strong reversal activity to the
P-gp-mediated LCC6MDR cells compared to verapamil (RF = 3.2) and no cytotoxicity
to cancer or normal cell lines even at a high concentrations (100 μM).
Keywords Curcumin; Pyrimidine; Glucosylation; Anticancer; MDR modulator
Received December 15, 2011; accepted April 24, 2012.
Address correspondence to Tao Jiang, Key Laboratory of Marine Drugs, Chinese Min-
istry of Education, School of Medicine and Pharmacy, Ocean University of China Qing-
dao China. E-mail: jiangtao@ouc.edu.cn and Larry M. C. Chow, Department of Applied
Biology and Chemical Technology and the State Key Laboratory for Chirosciences,
The Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, China. E-mail:
bclchow@polyu.edu.hk
620

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
621
INTRODUCTION
Curcuminoids as dietary polyphenolic compounds are of interest since
tumeric plants are often used as herbal medicines.[1] Curcumin (curcumin I),
demethoxycurcumin (curcumin II), and bis-demethoxycurcumin (curcumin III)
are the major forms of curcuminoids (Fig. 1) found in turmeric powder, which
exhibit anticancer, antioxidant, and anti-inflammatory activities.[2] Evidence
has also been found that curcumin has hepatoprotective and nephroprotective
activities, can suppress thrombosis and protect against myocardial infarction,
and has hypoglycemic and antirheumatic properties. Most importantly, cur-
cumin has been shown in various animal models and human studies to be
extremely safe even at very high doses.[3] Owing to their pharmacological ef-
fects and excellent safety profile, curcuminoids have been investigated as lead
compounds for treatment of human diseases.[1]
However, the utility of curcumin as a therapeutic agent is limited by its low
water solubility and bioavailability, poor stability, and fast metabolism that re-
sults in rapid systematic elimination. It is therefore necessary to administer
a high dose to achieve a significant intracellular concentration.[4] The pheno-
lic group and the 1,3-dicarbonyl moiety can be degraded via oxidative and hy-
drolytic pathways both in vitro and in vivo.[5,6] Many strategies have been used
to improve the bioavailability of curcumin, such as protection of the phenolic
groups and substitution of the 1,3-dicarbonyl moiety, which have been shown to
enhance the curcuminoids’ stability and result in increased bioavailability.[6–8]
Figure 1: Structures of major curcuminoids.

L. Gao et al.
622
The nitrogen element plays an important role in many drugs, as it is easy
to form water-soluble salts with acids and improve the water solubility of the
drugs. Therefore, it is of great significance to introduce nitrogen into the struc-
ture when designing the structure modification of natural products. It has been
found that an increased cytotoxicity can be provided by incorporating an α,β-
unsaturated keto group into a nitrogen heterocyclic ring.[9]
Glucose as a ubiquitous fuel in biology is used as an energy source
in most organisms, from bacteria to humans. Glycosylation allows water-
insoluble and unstable organic compounds to be converted into the corre-
sponding water-soluble and stable compounds, which could probably improve
their bioavailability and pharmacological properties. Furthermore, glycosides
of physiologically active compound (i.e., vitamin glycosides) have been reported
to be useful antiallegic agents.[10] Many sugar-modified curcumin derivatives
have been synthesized by different methods, including enzymatic glycosyla-
tion[11] and phase-transfer catalyst glycosylation.[6] While these glycosylation
reactions mainly occurred on the hydroxyl of the curcumin aromatic ring,
there is no report on the glycosylation of the pyrimidine ring. We herein re-
port the preparation of a series of novel curcumin analogs by incorporating the
1,3-dicarbonyl into 2-hydroxyl pyrimidine, followed by glycosylation of the hy-
droxyl. The resulting methoxy- or benzyloxy-substituted (E,E)-4,6-bis(styryl)-
2-O-glucopyranosyl-pyrimidines and (E,E)-4,6-bis-(3,4- dimethoxystyryl)-2-(2-
O-glucopyranosyl)ethyloxgyl-pyrimidines were evaluated in vitro using HL-60
and A549 cell lines.
Chemotherapy plays an important role in the treatment of cancer, but the
emergence of multidrug resistance (MDR) has made many of the currently
available chemotherapeutic agents ineffective.[12] To continue our preliminary
research on development of new MDR modulators from natural products,[13]
the synthetic compounds were assessed for MDR-reversing activity mediated
by P-gp, BCRP, and MRP1 in three different multidrug resistance cancer cell
lines. Here we report the synthesis of this novel series of curcumin analogs
and their potent multidrug resistance reversal activity against cancer cell
lines.
RESULTS AND DISCUSSION
Syntheses of Target Compounds 5a–e
The synthesis of the target compounds 5a–e is outlined in Scheme 1. First,
2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1a) as a glycosyl donor
was synthesized in one-pot as previously reported.[14] 1-O-Bromoethy-2,3,4,6,-
tetra-O-acetyl-β-D-glucopyranose (1b) was synthesized as described in the
Experimental section. 4,6-Dimethyl-2-hydroxyl-pyrimidine hydrochloride (2a)

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
623
Scheme 1: Synthetic route of compounds 4a-4e, 5a-5e.
was prepared by a reported procedure, in which 2,4-pentanedione and urea
were refluxed in anhydrous ethyl alcohol and catalyzed by HCl.[15] Mixtures of
2a and differently substituted benzaldehydes were then refluxed in anhydrous
ethyl alcohol and toluene in the presence of concentrated HCl as catalyst to
give 3a–d with yields of 65% to 70%. To improve the yield, the water generated
during the reaction was removed by using a Dean-Stark trap. It is worth noting
that 4-(benzyloxy) benzaldehyde was prepared from 4-hydroxybenzaldehyde
and benzyl bromide following the reported method.[16] Subsequently, interme-
diates 4a–e were synthesized by using tetrabutyl ammonium bromide as the
phase-transfer catalyst, which can facilitate the migration of 3a–d from the

L. Gao et al.
624
aqueous phase into the organic phase containing 1a or 1b in chloroform. The
use of a phase-transfer catalyst in heterogeneous reactions can result in faster
reactions and higher conversions or yields. Thus, the yields of this reaction
increased from about 20% to about 50% as compared to the common method.
For the last step, compounds 4a–e were deacetylated by a catalytic amount of
CH3ONa in methanol to obtain target compounds 5a–e with yields of 80% to
85% after recrystallization in methanol. All the compounds were characterized
by 1H NMR, 13C NMR, and HRMS spectrometry.
Cytotoxicity Evaluation
The anticancer activity of compounds 5a–e against A549 and HL-60 cell
lines in vitro was determined by the MTT assay and compared with those of the
parent compounds curcumin I and curcumin III. Preliminary in vitro results
demonstrated that most of the synthesized curcumin analogs showed low or
moderate inhibition of A549 and HL-60 cells at the concentration of 10 μg/mL
as shown in Table 1. Among the analogs, 5a, 5c, and 5e showed slightly higher
inhibitory activity against the HL-60 cell line (11.02%, 12.51%, and 16.75%,
respectively) when compared with curcumin I and III (8.39% and −6.84%, re-
spectively). However, no inhibition was observed with any of the compounds
except for 5c (8.31%) toward A549.
Evaluating the MDR Reversal Activity of Compound 4a–e
We employed three drug resistance cell lines in this study: (1) a P-
glycoprotein (P-gp) overexpressing human breast cancer cell line, LCC6MDR,
which displayed 70.5-fold resistance to paclitaxel (IC50 = 183.2
6.3 nM)
compared with parental LCC6 cells (IC50 = 2.6
0.5 nM);[13] (2) a BCRP-
transfected human embryonic kidney cell line, HEK293/R2, which was 25.2-
fold more resistant to topotecan (IC50 = 563.4 41.1 nM) than the parental
HEK293/pcDNA3.1 cell line (IC50 = 22.4
2.4 nM);[17] and (3) an MRP1-
transfected ovarian cancer cell line, 2008/MRP1, which was 9.2-fold more resis-
tant to DOX (IC50 = 577.1 39.4 nM) than parental 2008/P cells (IC50 = 63.5
Table 1: Inhibition effect of target compounds 5a–e at 10 μg/mL toward A549 and
HL-60 cell lines in vitro
Compounds
Inhibition of A549 (%)
Inhibition of HL-60 (%)
Curcumin I
−24.91
−21.94
−30.83
−14.00
8.31
8.39
−6.84
11.02
4.29
12.51
−1.67
16.75
Curcumin III
5a
5b
5c
5d
5e
−29.18
−14.68

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
625
Table 2: MDR reversal activity of 4a–e
LCC6MDR
HEK293/R2
+2008MRP1
Paclitaxel
IC50 (nM)
Topotecan
IC50 (nM)
DOX
IC50 (nM)
Cpds 1 µM
RF
RF
RF
Control
Verapamil
Ko143
Curcumin I
Curcumin III
4a
140.0
43.8
1.0
3.2
612.5
1.0
395.8
73.3
1.0
5.4
30.5
417.2
610.5
82.4
20.3
1.3
107.7
107.2
11.4
1.3
1.3
439.8
494.8
234.6
201.2
362.0
441.2
365.0
0.9
0.8
1.7
2.0
1.1
0.9
1.1
1.0
12.3
18.5
1.3
7.4
4b
7.6
56.0
10.9
4.5
4c
106.2
195.6
91.8
135.1
403.4
117.6
4d
0.7
1.5
1.5
4e
5.2
a
IC50 values were determined for paclitaxel, topotecan, and DOX with 1.0 µM of curcumin
analogs using LCC6MDR, HEK293/R2, and 2008/MRP1 cells. The Relative Fold (RF) represents
the fold change of drug sensitivity in different cell lines. RF-(IC50 without modulator)/(IC50 with
modulator) N- 3 independent experiments were performed, and averaged IC50 values are
presented. No modulator was used in LCC6MDR, HEK293/R2, and 2008/MRP1 cells and the
IC50 value from this control is used for normalization (RF = 1.0). Verapamil was used at 20 µM
for testing MRP1-modulating activity.
5.1 nM).[18] Verapamil is a well-known P-gp and MRP1 inhibitor, and Ko143
is a BCRP-specific modulator. Here, we employed these two positive controls in
the cell proliferation assay. The MDR reversal activity of synthetic curcumin
analogs is listed in Table 2.
Since the preliminary research results showed that introduction of a non-
polar and hydrophobic group into the molecule enhanced the potency of MDR
reversal activity,[13,17] a relative low and safe concentration of compounds 4a–e
(1 μM) was used in the assay. All of the compounds displayed no cytotoxicity to-
ward LCC6, LCC6MDR, and normal mouse connective tissue fibroblast (L929)
even at the concentration of 100 μM. (Table 3).
The reversal MDR activities of curcumin analogs were compared by mea-
suring the relative fold (RF), defined as a ratio of IC50 without modulator to
IC50 with modulator. Table 2 demonstrates the reversal activities of P-gp-,
BCRP-, and MRP1-mediated MDR in three different cell lines. Worth noting,
to the P-gp-mediated LCC6MDR cell lines, compound 4b displayed the high-
est modulating activity with an RF of 18.5 (Table 2). Besides compound 4b, 4a
also demonstrated strong modulating activity with an RF of 12.3. Verapamil
as the well-known P-gp modulator displayed a modulate activity with an RF
of 3.2 (Table 2), and curcumin I and curcumin III gave relative low modulating
activity with an RF of 1.3 (Table 2). To the BCRP-mediated HEK293/R2 cell
lines, the same results were obtained. Compound 4b (RF = 10.9) and 4a (RF
= 7.4) demonstrated promising modulating activity, though it is lower than

L. Gao et al.
626
Table 3: In vitro cytotoxicity of 4a–e to LCC6, LCC6MDR, and L929 cell linesa
IC50 (µM)
Compounds
LCC6
LCC6MDR
L929
Curcumin I
19.8 3.4
>100
18.0 2.2
>100
25.0 2.0
>100
Curcumin III
4a
4b
4c
4d
4e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
aThe IC50 values were determined after exposure to a series of concentrations of synthetic
curcumin analogs using LCC6, LCC6MDR, and L929. L929 is a mouse connective tissue fibroblast
cell line. Each experiment has been repeated one to three times with the data presented as
the mean standard error of mean.
Ko143. All the results indicate that the presence of a methoxy substituent is
needed for inhibiting the functionality of P-gp and BCRP. Moreover, the num-
ber of methoxy substituents on the phenyl group is also an important factor for
determining the P-gp- and BCRP-modulating activity. Meanwhile, the length
of the linker also plays an important role in modulating P-gp and BCRP ac-
tivity. The compound 4a with a shorter linker at the 2-position of pyrimidine
displayed higher modulating activity than 4e. Unfortunately, compounds 4a–e
displayed no activity in modulating MRP1-mediated MDR cell lines except for
4b with an RF of 2.0. At a high concentration (20 μM), verapamil completely
reversed DOX resistance in 2008/MRP1 (RF = 5.4 in Table 2). This indicates
that the MRP1 modulator maybe has its own special pharmacophore. It is well
known that P-gp is thought to transport neutral or positively charged com-
pounds, whereas MRP1 is a transporter of neutral and anionic compounds such
as glutathione-conjugated drugs, sulphates, and glucuronidates.[19] This wide
difference in substrate specificity of various ABC transporters may explain
why our curcumin pyrimidine derivatives markedly exhibit different potency
against P-gp, BCRP, and MRP1.
EXPERIMENTAL
General
All reagents used were commercially available. Solvents were treated us-
ing standard techniques. Thin-layer chromatography (TLC) was performed on
precoated E. Merck silica-gel 60 F254 plates. Column chromatography was

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
627
performed on silica gel (200–300 mesh Qingdao China). Melting points were
1
determined on a Mitamura-Riken micro-hot stage and were not corrected. H
NMR and 13C NMR spectra were obtained on a Jeol JNM-ECP 600 spectrom-
eter with tetramethylsilane (Me4Si) as the internal standard, and chemical
shifts were recorded in δ values. Mass spectra were recorded on a Q-TOF
Global mass spectrometer.
Synthesis of 1-O-Bromoethy-2,3,4,6,-tetra-O-acetyl–β-D-Glucose
(1b)
Compound 1 (6.0 g, 15.5 mmol) and 2-bromoethanol (1.35 mL 19 mmol)
were dissolved in dry CH2Cl2 (27 mL), stirring at 0◦C under N2 atmosphere.
Then boron trifluoride ethyl ether (10 mL) was added dropwise to the solution
at 0◦C in the darkness over a period of 20 min. The reaction mixture was al-
lowed to stir for 1.5 h at 0◦C and for a further 20 h at rt. After completion
of the reaction, the mixture was poured into ice water (45 mL) and extracted
with CH2Cl2 (45 mL × 2). The organic lay was combined and washed with satu-
rated NaHCO3 (45 mL × 2) and H2O (45 mL × 2) and dried over NaSO4 for 4 h.
The solvent was removed under reduced pressure and the residue was purified
through column chromatography on silica gel (eluent ethyl acetate/petroleum
1
ether, 3/7) to give 1b as a white powder. Yield 54%; m.p. 105–107◦C, H NMR
(CDCl3, 600 MHz): δ 5.24–5.21 (1H, t, J = 9.6 Hz), 5.11–5.07 (1H, t, J = 9.6 Hz),
5.04–5.01 (1H, t, J = 9.6 Hz), 4.58–4.57 (1H, d, J = 7.8 Hz), 4.28–4.25 (1H, dd,
J = 12.4, 5.0 Hz), 4.19–4.16 (2H, m), 2.09 (3H, s), 2.08 (s, 3H), 2.03 (s, 3H),
2.01 (s, 3H); 13C NMR (CDCl3,150 MHz): δ 170.8, 170.5, 169.6, 169.5, 101.1,
72.7, 72.0, 71.1, 69.9, 68.4, 61.9, 30.0, 20.9, 20.8, 20.7, 20.6; ESI-MS 477.1
(M + Na)+.
General Method for the Synthesis of Methoxy or Benzyloxy
Substituted (E,E)-4,6-bis(styryl)-2-Hydroxyl-Pyrimidines (3a–3d)
Compound 2a (2.18 g, 15 mmol) and different substituted benzaldehyde
(45 mmol) were dissolved in anhydrous ethyl alcohol (150 mL) in a round-
bottomed flask (500 mL). Then 3 mL concentrated hydrochloric acid was added
and the mixture refluxed at 100◦C for 36 h. After completion of the reaction,
the solvent was evaporated under reduced pressure. The saturated NaHCO3
solution (250 mL) was added into the brown oil residue for desalting. After
stirring at rt for 5 h, the resulting precipitate was filtered and washed with
water and ether to obtain crude product and then purified by chromatography
on silica gel (eluent, dichloromethane/methanol) to give 3a–d as brown powder.
(E,E)-4,6-bis(3,4-Dimethoxystyryl)-2-hydroxyl-pyrimidine (3a) yield 70%;
1
m.p. 255–257◦C; H NMR (DMSO-d6, 600M): δ 11.55 (1H, s), 7.80 (1H, s),
7.77 (1H, s), 7.29 (2H, s), 7.20 (2H, d, J = 7.74), 7.04–7.02 (2H, d, J = 7.92),

L. Gao et al.
628
6.94–6.92 (2H, d, J = 15.42), 6.85 (1H, s), 3.84 (6H, s), 3.81 (6H, s); 13C NMR
(DMSO-d6, 150M): δ 151.09, 149.60, 138.78, 128.63, 122.66, 112.31, 110.39,
56.13 ppm; ESI-MS 421.2 (M + H)+.
(E,E)-4,6-bis(3, 4, 5-Trimethoxystyryl)-2-hydroxyl-pyrimidine (3b) yield
65%; m.p. 222–224◦C, 1H NMR (DMSO-d6, 600M): δ 11.71 (1H, s,), 7.79 (2H, d,
J = 16.5Hz), 6.99 (7H, m), 3.85 (12H, s), 3.71 (6H, s) ppm; 13C NMR (DMSO-d6,
150M): δ 153.7, 139.6,139.0, 131.4, 107.6, 105.7, 67.2, 60.7, 56.5 ppm; ESI-MS
481.2 (M + H)+.
(E,E)-4,6-bis(4-Methoxystyryl)-2-hydroxyl-pyrimidine (3c) yield 69%; m.p.
1
210–212◦C, H NMR (DMSO-d6, 600M): δ 7.81 (1H, s), 7.79 (1H, s), 7.63–7.62
(4H, d, J = 8.22), 7.03–7.02 (4H, d, J = 8.28), 6.89 (1H, s), 6.86 (2H, s), 3.81 (6H,
s) ppm; 13C NMR (DMSO-d6, 150M): δ 161.26, 138.53, 130.02, 128.38, 115.12,
55.90 ppm; ESI-MS 361.2 (M + H)+.
(E,E)-4,6-bis(4-(Benzyloxy)styryl)-2-hydroxyl-pyrimidine (3d) yield 66%;
m.p. 215–217◦C, 1H NMR (DMSO-d6, 600M), δ 11.62 (1H, s), 7.80 (1H, s), 7.78
(1H, s), 7.62–7.61 (4H, d, J = 7.68), 7.47–7.46 (4H, d, J = 7.68), 7.42–7.39 (4H,
t, J = 7.44), 7.35–7.34 (2H, d, J = 7.14), 7.11–7.09 (4H, d, J = 8.28), 6.89 (1H,
s), 6.87 (1H, s), 6.84 (1H, s), 5.17 (4H, s) ppm; 13C NMR (DMSO-d6, 150M):
δ 160.31, 137.30, 129.99, 129.04, 128.52, 128.34, 115.94, 69.93 ppm; ESI-MS
513.2 (M + H)+.
General Method for the Synthesis of Alkoxy Substituted
(E,E)-4,6-bis(styryl)-2-O-(2,3,4,6,-tetra-O-
acetyl)Glucopyranosyl-Pyrimidines (4a-4d) and
(E,E)-4,6-bis(3,4-dimethoxystyryl)-2-(2-O-(2,3,4,6-tetra-O-
acetyl)glucopyranosyl)Ethyloxgyl-Pyrimidine
(4e)
A mixture of 10 mL H2O and 10 mL CHCl3 was stirred at 40◦C and tetra-
butyl ammonium bromide (484 mg 1.5 mmol) was added. Then a solution of
K2CO3 (622 mg 4.5 mmol) and compounds 3a–d (1.5 mmol) in 25 mL H2O and
10 mL DMF was added to the mixture. After that, solution of compound 1a or
1b (2.0 mmol) in CHCl3 (25 mL) was added dropwise at 40◦C over a period of
30 min to the solution. The reaction mixture was allowed to stir overnight at
40◦C. After completion of the reaction, the mixture was extracted with CH2Cl2
(50 mL × 3). The organic layer was combined and washed with H2O (50 mL
× 3) and dried over MgSO4 for 4 h. Then the solution was removed under
reduced pressure and purified by chromatography on silica gel (eluent, ethyl
acetate/petroleum ether) to give product 4a–e as yellow solid.
(E,E)-4,6-bis(3,4-Dimethoxystyryl)-2-O-(2,3,4,6,-tetra-O-acetyl)glucopyra-
1
nosyl-pyrimidine (4a) yield 65.5%; m.p. 93–95◦C; H NMR (CDCl3, 600M): δ
7.84 (1H, s), 7.82 (1H, s), 7.19 (1H, d, J = 2.22), 7.17 (1H, d, J = 1.62), 7.15

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
629
(2H, d, J = 1.62), 6.99 (1H, s), 6.91–6.89 (3H, d, J = 6.6), 6.88 (1H, s), 6.36–6.35
(1H, d, J = 8.22), 5.45–5.42 (1H, t, J = 9.36), 5.39–5.36 (1H, t, J = 8.79),
5.23–5.20 (1H, t, J = 9.6), 4.21–4.20 (2H, t, J = 3.75), 4.05–4.02 (1H, m), 3.96
(6H, s), 3.93 (6H, s), 2.07 (3H, s), 2.05 (3H, s), 2.03 (3H, s), 1.94 (3H, s) ppm; 13
C
NMR (CDCl3, 150M): δ 170.8, 170.5, 169.5, 169.3, 165.4, 163.5, 150.6, 149.3,
137.4, 128.7, 123.3, 121.9, 112.1, 111.2, 109.7, 73.4, 72.4, 71.1, 68.6, 62.2, 56.0,
20.7 ppm HRMS (ESI) M/Z calcd. for C38H43N2O14 [M+H+]: 751.2709, found
751.2708.
(E,E)-4,6-bis(3,4,5-Trimethoxystyryl)-2-O-(2,3,4,6,-tetra-O-acetyl)glucopy-
ranosyl-pyrimidine (4b) Yield 56.3%; m.p. 108–110◦C, 1H NMR (CDCl3, 600M):
δ 7.83 (1H, s), 7.80 (1H, s), 7.03 (1H, s), 6.94 (1H, s), 6.92 (1H, s), 6.85 (4H, s),
6.37–6.36 (1H, d, J = 8.22), 5.46–5.43 (1H, t, J = 9.36), 5.39–5.36 (1H, t, J =
8.28), 5.23–5.20 (1H, t, J = 9.9), 4.21–4.20 (2H, t, J = 2.22), 4.06–4.03 (1H, m),
3.94 (12H, s), 3.90 (6H, s), 2.07 (3H, s), 2.06 (3H, s), 2.03 (3H, s), 1.96 (3H, s)
ppm; 13C NMR (CDCl3, 150M): δ 170.8, 170.6, 169.5, 169.3, 165.3, 163.6, 153.6,
139.7, 137.7, 131.2, 124.7, 112.5, 105.0, 94.4, 73.3, 72.3, 71.0, 68.6, 62.2, 61.1,
56.3, 20.7 ppm; HRMS (ESI) M/Z calcd. for C40H47N2O16 [M+H+]: 811.2920,
found 811.2918.
(E,E)-4,6-bis(4-Methoxystyryl)-2-O-(2,3,4,6,-tetra-O-acetyl)glucopyranos-
yl-pyrimidine (4c) yield 50.2%; m.p. 225–227◦C, 1H NMR (CDCl3, 600M): δ 7.86
(1H, s), 7.83 (1H, s), 7.56–7.54 (4H, d, J = 8.82), 6.94–6.93 (5H, t, J = 4.41),
6.90 (1H, s), 6.87 (1H, s), 6.35–6.33 (1H, d, J = 8.28), 5.44–5.41 (1H, t, J =
9.36), 5.39–5.36 (1H, t, J = 8.79), 5.22–5.19 (1H, t, J = 9.63), 4.23–4.19 (2H,
m), 4.03–4.00 (1H, m), 3.85 (6H, s), 2.07 (3H, s), 2.05 (3H, s), 2.02 (3H, s), 1.93
(3H, s) ppm; 13C NMR (CDCl3, 150M): δ 170.8, 170.5, 169.5, 169.3, 165.5, 163.5,
160.9, 137.2, 129.3, 128.4, 123.1, 114.4, 112.3, 94.4, 73.6, 72.5, 71.0, 68.6, 62.2,
55.5, 20.7 ppm; HRMS (ESI) M/Z calcd. for C36H39N2O12 [M+H+]: 691.2498,
found 691.2495.
(E,E)-4,6-bis(4-(Benzyloxy)styryl)-2-O-(2,3,4,6,-tetra-O-acetyl)glucopyran-
1
osyl-pyrimidine (4d) yield 47.6%; m.p. 173–175◦C, H NMR (CDCl3, 600M): δ
7.85 (1H, s), 7.83 (1H, s), 7.55–7.54 (4H, d, J = 8.82), 7.44–7.43 (4H, d, J =
7.14), 7.41–7.38 (4H, t, J = 7.68), 7.35–7.33 (4H, t, J = 7.56), 7.01–6.99 (4H, d,
J = 8.82), 6.92 (1H, s), 6.89 (1H, s), 6.87 (1H, s), 6.34–6.33 (1H, d, J = 8.28),
5.44–5.41 (1H, t, J = 9.33), 5.39–5.6 (1H, t, J = 8.82), 5.22–5.19 (1H, t, J =
9.63), 5.10 (4H, s), 4.21–4.16 (2H, m), 4.02–3.99 (1H, m), 2.07 (3H, s), 2.05
(3H, s), 2.07 (3H, s), 1.93 (3H, s) ppm; 13C NMR (CDCl3, 150M): δ 170.8, 170.5,
169.5, 169.4, 165.5, 163.5, 160.1, 136.6, 129.3, 128.7, 128.7, 128.2, 127.5, 123.2,
115.3, 112.4, 73.6, 72.5, 71.0, 70.2, 62.2, 20.7 ppm; HRMS (ESI) M/Z calcd. for
C48H47N2O12 [M+H+]: 843.3124, found 843.3132.
(E,E)-4,6-bis(3,4-Dimethoxystyryl)-2-(2-O-(2,3,4,6-tetra-O-acetyl)glucopy-
ranosyl)ethyloxgyl-pyrimidine (4e) yield 40.7%; m.p. 90–92◦C, 1H NMR
(CDCl3, 600M): δ 7.85 (1H, s), 7.82 (1H, s), 7.18–7.16 (2H, dd, J = 2.19, 8.22),
7.14 (2H, d, J = 1.68), 6.92 (1H, s), 6.90 (1H, s), 6.88 (2H, d, J = 3.3), 6.87

L. Gao et al.
630
(1H, s), 5.25–5.22 (1H, t, J = 9.37), 5.12–5.09 (1H, t, J = 9.6), 5.05–5.02 (1H,
t, J = 8.79), 4.73–4.72 (1H,d, J = 8.28), 4.71–4.67 (1H, m), 4.61–4.58 (1H, m),
4.28–4.24 (2H, m), 4.13–4.10 (1H, dd, J = 2.22, 12.12), 4.07–4.03 (1H, m), 3.96
(6H, s), 3.93 (6H, s), 3.77–3.74 (1H, m), 2.07 (3H, s), 2.02 (3H, s), 2.01 (3H, s),
1.99 (3H, s) ppm; 13C NMR (CDCl3, 150M): δ 170.8, 170.4, 169.6, 165.2, 150.4,
149.3, 136.9, 128.8, 123.7, 121.8, 111.2, 109.5, 101.2, 72.9, 71.9, 71.3, 68.4, 68.2,
66.0, 62.0, 56.0, 20.8 ppm; HRMS (ESI) M/Z calcd. for C40H47N2O15 [M+H+]:
795.2971, found 795.2980.
General Method for Synthesis of Methoxy or Benzyloxy Substituted
(E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines (5a–5d)
and (E,E)-4,6-bis(3,4-dimethoxystyryl)-2-(2-O-glucopyranosyl)
Ethyloxgyl-Pyrimidine (5e)
To a round-bottomed flask (100 mL), compound 4a–e (0.5 mmol) was dis-
solved in 30 mL anhydrous CH3OH and then 2 mL 0.5 M CH3ONa/CH3OH was
added to the solution. The mixture was allowed to stir on at rt for 3 h. When
the reaction was completed (monitored by TLC), 5g 732-type cation exchange
resin was added to the reaction solution, and the mixture was stirred at rt for
5 min. Then the solvent was filtered and removed under reduced pressure. The
crude products were recrystallized from cold methanol to give product 5a–e as
yellow solids.
(E,E)-4,6-bis(3,4-Dimethoxystyryl)-2-O-glucopyranosyl-pyrimidine
(5a)
yield 82.3%; m.p. 207–209◦C, H NMR (DMSO-d6, 600M): δ 7.86 (1H, s), 7.83
(1H, s), 7.37 (2H, d, J = 1.68), 7.28 (1H, s), 7.26–7.24 (2H, dd, J = 1.68, 8.82),
7.16 (1H, s), 7.13 (1H, s), 7.02 (2H, d, J = 8.28), 5.93 (1H, d, J = 7.68), 5.42
(1H, d, J = 5.46), 5.16 (1H, d, J = 4.38), 5.07 (1H, d, J = 5.52), 4.60–4.58
(1H, t, J = 5.79), 3.85 (6H, s), 3.81 (6H, s), 3.70–3.68 (1H, m), 3.52–3.48
(1H, m), 3.40–3.72 (2H, m), 3.34–3.31 (1H, m), 3.24–3.21 (1H, m) ppm; 13C
NMR (DMSO-d6, 150M): δ 165.62, 164.56, 150.82, 149.58, 137.45, 128.92,
124.16, 122.71, 112.24, 112.01, 110.53, 97.45, 78.22, 77.42, 73.43, 70.34, 61.24,
56.13 ppm; HRMS (ESI) M/Z calcd. for C30H35N2O10 [M+H+]: 583.2286, found
583.2282.
1
(E,E)-4,6-bis(3,4,5-Trimethoxystyryl)-2-O-glucopyranosyl-pyrimidine (5b)
yield 84.5%; m.p. 145–147◦C, 1H NMR (DMSO-d6, 600M): δ 7.87 (1H, s), 7.84
(1H, s), 7.35 (1H, s), 7.25 (1H, s), 7.22 (1H, s), 7.07 (4H, s), 5.94–5.83 (1H, d, J
= 7.68), 5.42–5.41 (1H, d, J = 4.98), 5.17–5.16 (1H, d, J = 4.8), 5.09–5.08 (1H,
d, J = 5.46), 4.61–4.59 (1H, t, J = 5.76), 3.86 (12H, s), 3.70 (6H, s), 3.69–3.67
(1H, m), 3.51–3.47 (1H, m), 3.41–3.37 (2H, m), 3.33–3.30 (1H, m), 3.23–3.20
(1H, m) ppm; 13C NMR (DMSO-d6, 150M): δ 165.56, 164.63, 153.70, 139.36,
137.65, 131.69, 125.82, 112.23, 105.89, 97.48, 78.21, 77.41, 73.47, 70.36, 60.70,
56.58 ppm; HRMS (ESI) M/Z calcd. for C32H39N2O12 [M+H+]: 643.2498, found
643.2495.

Novel Series of (E,E)-4,6-bis(styryl)-2-O-Glucopyranosyl-Pyrimidines
631
(E,E)-4,6-bis(4-Methoxystyryl)-2-O-glucopyranosyl-pyrimidine (5c) yield
1
82.7%; m.p. 176–178◦C, H NMR (DMSO-d6, 600M): δ 7.88 (1H, s), 7.85 (1H,
s), 7.69–7.68 (4H, d, J = 8.82), 7.25 (1H, s), 7.11 (1H, s), 7.09 (1H, s), 7.02–7.00
(4H, d, J = 8.82), 5.91–5.89 (1H, d, J = 7.68), 5.42–5.41 (1H, d, J = 5.52), 5.16
(1H, d, J = 4.92), 5.07 (1H, d, J = 5.52), 4.59–4.57 (1H, t, J = 6.06), 3.81 (6H,
s), 3.70–3.67 (1H, m), 3.51–3.48 (1H, m), 3.41–3.37 (2H, m), 3.34–3.32 (1H, m),
3.24–3.21 (1H, m) ppm; 13C NMR (DMSO-d6, 150M): δ 165.58, 164.50, 160.95,
137.01, 130.0, 128.68, 123.93, 114.98, 112.23, 97.51, 78.23, 77.43, 73.37, 70.33,
61.20, 55.86 ppm; HRMS (ESI) M/Z calcd. for C28H31N2O8 [M+H+]: 523.2075,
found 523.2062.
(E,E)-4,6-bis(4-(Benzyloxy)styryl)-2-O-glucopyranosyl-pyrimidine
(5d)
yield 84.0%; m.p. 212–214◦C, H NMR (DMSO-d6, 600M): δ 7.87 (1H, s), 7.84
(1H, s), 7.69–7.68 (4H, d, J = 8.76), 7.48–7.47 (4H, d, J = 7.14), 7.42–7.39 (4H,
t, J = 7.44), 7.35–7.33 (2H, t, J = 7.44), 7.24 (1H, s), 7.12 (1H, s), 7.09–7.08
(5H, d, J = 8.76), 5.90–5.89 (1H, d, J = 7.68), 5.42 (1H, d, J = 4.98), 5.17 (4H,
s), 5.16 (1H, s), 5.07 (1H, d, J = 4.98), 4.59–4.57 (1H, t, J = 6.06), 3.70–3.67
(1H, m), 3.51–3.47 (1H, m), 3.40–3.37 (2H, m), 3.34–3.31 (1H, m), 3.24–3.20
(1H, m) ppm; 13C NMR (DMSO-d6, 150M): δ 165.56, 164.50, 160.04, 137.37,
136.96, 130.02, 129.04, 128.89, 128.49, 128.34, 124.05, 115.82, 112.31, 97.52,
78.25, 77.44, 73.37, 70.33, 69.90, 61.20 ppm; HRMS (ESI) M/Z calcd. for
C40H39N2O8 [M+H+]: 675.2701, found 675.2691.
1
(E,E)-4,6-bis(3,4-Dimethoxystyryl)-2-(2-O-glucopyranosyl)ethyloxgyl-
pyrimidine (5e) yield 83.7%; m.p. 74–76◦C, 1H NMR (DMSO-d6, 600M): δ
7.86 (1H, s), 7.84 (1H, s), 7.36 (2H, d, J = 1.68), 7.27–7.25 (2H, dd, J = 2.16,
8.76), 7.23 (1H, s), 7.14 (1H, s), 7.11 (1H, s), 7.02–7.01 (2H, d, J = 8.28), 5.10
(1H, d, J = 5.1), 5.00 (1H, d, J = 4.92), 4.96 (1H, d, J = 4.98), 4.57–4.55
(2H, t, J = 6.03), 4.27–4.26 (1H, d, J = 7.68), 4.18–4.14 (1H, m), 3.85 (6H, s),
3.80 (6H, s), 3.71–3.68 (1H, m), 3.64–3.63 (1H, d, J = 5.46), 3.49–3.43 (2H,
m), 3.17–3.14 (2H, m), 3.09–3.07 (1H, m), 3.03–3.00 (1H, m) ppm; 13C NMR
(DMSO-d6, 150M): δ 165.51, 165.21, 150.73, 149.55, 137.17, 128.93, 124.29,
122.40, 112.28, 110.66, 103.58, 77.53, 77.32, 74.00, 70.60, 67.46, 66.45, 61.68,
56.12 ppm; HRMS (ESI) M/Z calcd. for C32H39N2O11 [M+H+]: 627.2548, found
627.2540.
Cytotoxicity Evaluation
The anticancer activity of synthesized compounds (5a–e) was evaluated in
A549 and HL-60 cell lines in the Molecular Pharmacology Laboratory of our
school. Cytotoxicity was determined using an MTT microplate assay and the
results were expressed as the inhibition values of cell growth at the 10 μg/mL
concentration of the compounds.

L. Gao et al.
632
MDR Reversal Activity Evaluation
The method of evaluating the MDR reversal activity of the compounds,
such as cell culture, cell proliferation assay, cytotoxicity assay, and DOX accu-
mulation assay, was carried out according to the reported procedures.[17]
CONCLUSIONS
In conclusion, five new compounds including five methoxy- or benzyloxy- sub-
stituted (E,E)-4,6-bis(styryl)-2-O-glucopyranosyl-pyrimidines and one (E,E)-4,
6-bis(3,4-dimethoxystyryl)-2-(2-O-glucopyranosyl)ethyloxgyl-pyrimidine were
synthesized in four steps with total yields of 21.5% to 33.9%. The preliminary
in vitro study demonstrated that most new compounds showed some inhibi-
tion effect against A549 and HL-60 cell lines at the concentration of 10 μg/mL.
Meanwhile, the compounds 4a–e were evaluated for their MDR reversal activ-
ity, and the results demonstrated compound 4a and 4b displayed high P-gp-
and BCRP-modulating activity. The preliminary structure-activity relation-
ship could be deduced and is of great significance for the finding of safe and
effective MDR reversal agents.
In this paper, we have proposed a new strategy for the modification of cur-
cuminoids with carbohydrate with no increase of the toxicity and found a novel
type of MDR reversal agents that is very safe and effective. Further modifica-
tion and evaluation of curcumin-carbohydrate conjugates are still needed to
study their therapeutic efficacy and potential medicinal use.
ACKNOWLEDGMENTS
This research was supported by the Natural Science Foundation of China
(Grant No. 21171154) and Special Fund for Marine Scientific Research in the
Public Interest (01005024).
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