Synthesis and hypoglycemic activity evaluation of rhein amide derivatives

Article abstract of DOI:10.1007/s00044-012-0215-7

In order to investigate the relationship of the structure and hypoglycemic activity, rhein amide derivatives 2a-2e were synthesized and their hypoglycemic activities were evaluated by glucose consumption in HepG2. Their structures were characterized by ¹H-, ¹³C NMR, IR, mass and elemental analysis. All the compounds exhibited strong hypoglycemic activity in improving glucose consumption in HepG2 cell assays in vitro, which was influenced by the diversity of rhein amide derivatives. The compounds 2a-c, 2f, and 2g bearing heterocyclic ring were proved to be more potentially useful in glucose consumption than dimethyldiguanide. Among all the compounds, compound 2f exhibited the strongest activity on glucose consumption, while compound 2d showed the weakest activity.

Full text of DOI:10.1007/s00044-012-0215-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2228–2234
DOI 10.1007/s00044-012-0215-7
ORIGINAL RESEARCH
Synthesis and hypoglycemic activity evaluation of rhein
amide derivatives
•
Xiaokang Zhu Xiaoli Ye Liu Song Yonghuang Luo
•
•
•
•
Qing Tang Yanan Jin Xuegang Li
•
Received: 1 November 2011 / Accepted: 24 August 2012 / Published online: 5 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In order to investigate the relationship of the
structure and hypoglycemic activity, rhein amide derivatives
2a–2e were synthesized and their hypoglycemic activities
were evaluated by glucose consumption in HepG2. Their
Introduction
Diabetes is a metabolic disease, seriously affecting peo-
ple’s health and quality of life. Type II diabetes, the more
prevalent form of diabetes, is characterized by insulin
resistance of internal organs and peripheral tissues that
results in impaired glucose utilization, and consequently, in
abnormally high blood glucose levels between and espe-
cially after meals (Jan-Willem et al., 2011). It has been
estimated that the number of adults affected by diabetes
will grow from 135 million in 1995 to 300 million in 2025
worldwide (King et al., 1998; Benson et al., 2010).
Hyperglycemia resulting from uncontrolled glucose regu-
lation, which cause the disorder of fat and protein metab-
olism and even lead to cardiovascular disease, is widely
recognized as the causal link between diabetes and diabetic
complications (Anabela and Carlos, 2006; Brownlee,
2001). Therefore, it is of great importance to explore more
effective and safer antihyperglycemic drugs, as a pharma-
cological tool to provide important information regarding
treatment for diabetes. Currently, the diabetic drugs are
structurally divided into two major classes, sulfonylureas
and biguanides. Furthermore, other drugs such as a-glu-
cosidase inhibitors are used as an auxiliary type for dia-
betes treatment. The development of more effective and
safe hypoglycemic agents is still a challenge, due to few
species and side effects (McEwen et al., 2009; Chao et al.,
2007). There is a growing trend of using natural products as
an alternative treatment of type II diabetes for the safety
and diversity (Hnatyszyn et al., 2002). China has a long
history of using herbs for the treatment of human diseases
including diabetes. Rheum rhaponticum is one of those
herbs which exhibit many pharmacological activities.
Rhein, as quality control marker of R. rhaponticum, has
good anti-hypoglycemic effect and lipid-lowering activity,
1
structures were characterized by H-, 13C NMR, IR, mass
and elemental analysis. All the compounds exhibited strong
hypoglycemic activity in improving glucose consumption in
HepG2 cell assays in vitro, which was influenced by the
diversity of rhein amide derivatives. The compounds 2a–c,
2f, and 2g bearing heterocyclic ring were proved to be more
potentially useful in glucose consumption than dimethyld-
iguanide. Among all the compounds, compound 2f exhibited
the strongest activity on glucose consumption, while com-
pound 2d showed the weakest activity.
Keywords Rhein derivatives Á Glucose consumption Á
Hypoglycemic activity
X. Zhu Á Y. Luo Á Q. Tang Á Y. Jin Á X. Li (&)
College of Pharmaceutical Sciences, Southwest University,
Chongqing 400716, People’s Republic of China
e-mail: xuegangli2000@yahoo.com.cn
X. Zhu
e-mail: xiaokangzhu1982@yahoo.com.cn
X. Ye
School of Life Sciences, Southwest University,
Chongqing 400715, People’s Republic of China
L. Song
School of Chemistry and Chemical Engineering, Southwest
University, Chongqing 400715, People’s Republic of China
123

Med Chem Res (2013) 22:2228–2234
2229
as well as low cytotoxic property (Bettag et al., 2008). It has
been reported rhein exerts anti-hypoglycemic effect through
agitating PPAR-c (Mu et al., 2008). The clinical application
of rhein, however, has been limited because of its poor
bioavailability and stimulating gastrointestinal disorder.
Thus, there is continued interest in the synthesis of new
anthraquinone derivatives, either as promising biologically
active compounds or as valuable synthons for the prepara-
tion of photodynamically active hypericin derivatives.
The modification of rhein had been mainly concentrated
on 1,8-hydroxyl and 3-carboxyl derivative of rhein (Owton
et al., 1995; Baxter and Walmsley, 2005; Pietrangelo
and Travagli, 2005). The introduction of acetyl in the
1,8-hydroxy can increase the effect of inhibition of bone
and joint inflammation. The introduction of a series of
long-chain ester in the 1,8-hydroxy, fluorine atoms on
anthraquinone ring, and carboxylic acid in the 3-carboxyl
can get better antihypoglycemic or anti-inflammation
activity (Kalyoncu et al., 2009; Owton et al., 1995). Our
previous studies have shown that the hypoglycemic activity
of rhein derivatives was significantly influenced by the
aliphatic chain length. It was found that their activity
increased with the aliphatic chain length and then gradually
decreased when the number of carbon atoms was higher
than eight (Zhu et al., 2012). The result suggests that the
changes of hydrophobicity or solubility of rhein affect their
pharmacological activities. Based on our previous studies,
we designed and synthesized a series of rhein homologs by
introducing various groups on the carboxylic acid and/or
hydroxyl of C4 and C5, and these rhein derivatives were
assayed for the activity of promoting glucose consumption
in HepG2 cells.
Results and discussion
Chemistry
Our approach to the synthesis of 2-carboxamide-rhein is
depicted in Scheme 1. The 4,5-unprotected rhein amides
2a–2b was obtained by the reaction of substituted amines
and the highly reactive acyl chloride (1a, 2-carbonyl chlo-
ride rhein), which was generated by the treatment of SOCl2
with rhein. The approach to 4,5-diprotected rhein amides
2c–2g was achieved by the reaction of substituted amines
or alcohols with the anhydride, which was obtained by
treatment of compound 1b–1c with oxalyl chloride. The
resulting compounds were purified by silica gel chroma-
tography and confirmed by IR, H-, 13C NMR, mass spec-
1
tra (MS), and elemental analysis spectroscopy.
For synthesizing the target compounds 2a–2b, SOCl2 was
used to activate the reactions, by generating the active acyl
chloride intermediates. On the contrary, the commonly used
condensation reagents EDC and dicyclohexyl carbodiimide
(DCC) did not efficientlypromote theamidation reactions. In
the reactions with EDC as the promoter, the TLC indicated
that there were many by-products produced. In contrast, the
DCC generated compound 3a as the major product. The
possible reaction mechanism of the formation of 3a is as
follows (Scheme 2): at the first stage, rhein reacted with
DCC to form the unstable carbodiimide intermediate (4),
which could be re-arranged to more stable urea (3a, Path b).
We optimized the reaction conditions for the synthesis of
the target compounds 2c–2g by using ethyl oxalyl chloride to
react with compound 1b–1c, to form highly reactive anhy-
dride intermediates, which was easier to react with amines or
Scheme 1 The synthetic routes
of rhein amide or rhein ester
derivatives; I SOCl2, DMF,
reflux; II Ac2O, PyH, 80 °C or
TBS, Et3N, CH2Cl2, CH3CN;
III RNH2, CH2Cl2, Et3N, room
temperature; IV RXH,
ClCOOEt, Et3N, room
temperature
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Med Chem Res (2013) 22:2228–2234
Scheme 2 The possible
reaction mechanism of the
formation of 3a
Table 1 The glucose uptake of different rhein derivatives and dimethyldiguanide in HepG2 cell
Group
C (mg L-1
)
GC (mmol L-1
)
MTT/A490
GC/MTT
CLgP
–
Control
10
10
10
10
10
10
10
10
10
1.972 0.071
0.574 0.019
0.560 0.042
0.565 0.023
0.591 0.021
0.577 0.017
0.573 0.011
0.565 0.026
0.563 0.017
0.574 0.013
3.438 0.139
2a
2.485 0.083*
2.315 0.076*
2.541 0.100*
2.256 0.098*
2.268 0.071*
2.578 0.072*
2.529 0.090*
2.250 0.078*
4.449 0.240**
4.116 0.289**
4.304 0.316**
3.916 0.230*
3.958 0.218*
4.563 0.123**
4.499 0.276**
3.923 0.171*
2.623
2b
2.949
1.532
2.743
3.941
8.924
9.938
-1.633
2c
2d
2e
2f
2g
Dimethyldiguanide
Data were expressed as mean SD (n = 6), analyzed by ANOVA
* P \ 0.05 compared with control group; ** P \ 0.01 compared with control group
alcohols and gave the desired amides or esters, respectively.
The spectral data and other characteristic parameters of these
compounds were listed in ‘‘Experimental protocols’’ section.
According to the experimental data, the introduction of
acetyl ester showed moderate activity on glucose con-
sumption. However, the N-substituent amides at C1 position
did not significantly affect the hypoglycemic activity, when
compared with that induced by O-substituents at C1 position.
O-acetyl group at C4 and C5 position of rhein amide (2c)
increased glucose consumption, compared with the rhein
amide (2b). Among all the compounds, compound 2f and 2g
exhibited the strongest activity on glucose consumption,
while compound 2d showed the weakest activity. The
introduction of TBS group of target molecule 2f and 2g to
rhein derivatives increased CLgP value of the compounds,
indicating that TBS group increased their hydrophobicity
(Table 1). Furthermore, O-TBS at C4, C5 position of rhein
amide (2f and 2g) showed higher hypoglycemic activity
compared with O-acetyl at C4, C5 position (2d and 2e).
Hypoglycemic activity in vitro
The glucose consumption of the target compounds 2a–2g in
HepG2 cells in vitro was evaluated to investigate the possible
antihyperglycemic activity and the preliminary structure–
activity relationship. HepG2 cells were used in this study due
to their common physiological function to lipid or glucose
metabolism with normal hepatic cells (Xu et al., 2003).
The glucose consumption of HepG2 cell assay (Table 1)
showed that the glucose consumption of all the tested
compounds have significant difference compared with vehi-
cle control (P \ 0.05). All of the 2-substitutional amide-
rhein possessed higher glucose consumption ability than
the positive control (dimethyldiguanide) at the same con-
centration, except compound 2d and 2e. The result indi-
cated that glucose consumption of 2a–2f has a very
significant difference compared to control group (P \
0.01). In Table 2, we can find that the glucose consumption
in HepG2 cell increased with the concentration of 2-sub-
stitutional amide-rhein.
Conclusion
In summary, we established an efficient method for the
synthesis of rhein amides using SOCl2 as activator, which
afforded the corresponding amides in higher yield than
that by the use of condensation reagents DCC and EDC.
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2231
Table 2 The glucose uptake of different concentration of rhein derivatives in HepG2 cells
Group
GC (mmol L-1
)
0
1 mg L-1
10 mg L-1
100 mg L-1
–
Control
1.972 0.071
–
–
2a
–
–
–
–
–
–
–
–
2.444 0.106**
2.379 0.052**
2.476 0.068**
2.151 0.103*
2.198 0.042**
2.369 0.088**
2.576 0.080**
–
2.485 0.083**
2.315 0.076**
2.541 0.100**
2.256 0.098**
2.268 0.098**
2.578 0.072**
2.529 0.090**
2.250 0.078**
2.598 0.100**
2.262 0.009**
2.567 0.148**
2.390 0.064**
2.292 0.089**
2.733 0.010**
2.506 0.043**
–
2b
2c
2d
2e
2f
2g
Dimethyldiguanide
Data were expressed as mean SD (n = 6), analyzed by ANOVA
– Mean not detected
* P \ 0.05 compared with control group; ** P \ 0.01 compared with the control group
The novel rhein derivatives exhibited strong ability in
improving glucose uptake in HepG2 cells. The introduction
of TBS groups in the dihydroxyl groups at anthraquinone ring
greatly increased the glucose uptake ability. Compounds
2a–c, 2f, and 2g showed higher activity than dimethyldigu-
anide in glucose uptakein vitro. However, compounds 2d and
2e showed similar abilities as dimethyldiguanide.
and dried under reduced pressure, and recrystallized from ethyl
acetate to obtain pure 1a as yellow solid, yield 93 %, mp
1
217.2–218.0 °C, H NMR (CDCl3, 300 MHZ, ppm) d: 12.2
(s, 1H, COOH), 8.14 (s, 1H, ArH), 7.85–7.79 (d, J = 7.4, 1H,
ArH), 7.74–7.72 (d, J = 7.8, 2H, ArH), 7.41–7.39 (d, J = 7.4,
1H, ArH), 2.50 (s, 6H, CH3); which is accord with reference.
Procedure for preparation of 4,5-bis (tert-
butyldimethylsilyloxy)-9,10-dioxo-9,10-
dihydroanthracene-2-carboxylic acid (1c)
Experimental protocols
Chemistry
A solution of rhein (0.568 g, 2 mmol) in dry CH2Cl2 (30 mL)
and 2.78 mL triethylamine was treated with tert-butyl-
chlorodimethylsilane (3.0146 g, 20 mmol) at 0 °C and stirred
at reflux temperature for 24 h under nitrogen. After comple-
tion of the reaction, the reaction was quenched with 50 mL
distilled water and extracted with dichloromethane (3 9
1.5 dm3) and the combined extracts were washed with water,
dried (MgSO4), filtered, and evaporated to dryness under
reduced pressure to give crude product 1c. The crude product
was purified by silica gel column purification using 2:1 (v/v,
petroleum ether:ethyl acetate) to obtain pure solid product 1c
as orange solid, yield 82 %, mp [300 °C, 1H NMR (CDCl3,
300 MHZ, ppm) d: 8.56 (s, 1H, ArH), 7.89 (s, 2H, ArH),
7.56–7.54 (t, 1H, ArH), 7.24–7.21 (d, J = 8.1, 1H, ArH,),
1.09–1.08 (d, J = 3.9, 18, CH3), 0.36–0.32 (d, J = 9.6, 12H,
CH3); IR (KBr) m: 3422, 2967, 2923, 1678, 1630, 1489, 1432,
1405, 1380, 1286, 1200, 1192 cm-1; MS (ESI, m/z): 514
(M ? 1).
Rhein (purity of more than 98 %) was purchased from
Xi’an Natural Product Company (Shanxi, China). Other
reagents were AR grade and purchased from Chongqing
Chemical Reagent Company (Chongqing, China). DMF,
CH2Cl2, Et3N, and CH3CN were redistilled and dried using
molecular sieves.
Melting points were recorded on a Buchi apparatus
and were not corrected. IR spectra and KBr pellets of
400–4,000 cm-1 were recorded on a Perkin Elmer 16PC-FT
spectrophotometer. 1H- and 13C NMR spectra were recorded
on a Brucker ARX-300 instrument (chemical shifts are
expressed as d values relative to TMS as internal standard).
MS and ESI (positive) were recorded on a Thermo Fisher-
Ltq-Orbitra spectrometer. Satisfactory micro-analysis was
obtained on FlashEA1112 CHN analyzer.
Procedure for preparation of 4,5-dihydroxy-9,10-dioxo-
9,10-dihydro anthracene-2-carboxylic acid (1b)
Procedure for preparation of 4,5-dihydroxy-N,N-dimethyl-
9,10-dihydro anthracene-2-carboxamide (2a)
For 1b preparation, a mixture of 5 mmol rhein and 60 mL
Pyridine was added dropwise to 60 mL acetic anhydride under
stirring. The resulting mixture was refluxed at 120 °C for 6 h.
The resulting yellow solution was poured into ice water, filtered
Dry SOCl2 (1 mL) was added to a solution of rhein
(0.568 g, 2 mmol) in dry DMF (5 mL). The reaction
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Med Chem Res (2013) 22:2228–2234
mixture was heated under reflux for 45 min under nitrogen.
After completion of the reaction, the reaction mixture was
cooled to room temperature and diluted with 50 mL water.
2.5 % Na2CO3 was added to achieve a pH of 8–9. The
aqueous solution was extracted with dichloromethane
(3 9 1.5 dm3) and the combined extracts were washed with
water, dried (MgSO4), filtered, and evaporated to dryness
under reduced pressure to give crude product 2a. The crude
product was purified by silica gel column purification using
20:1 (v/v, petroleum ether:ethyl acetate) to obtain pure
solid product 2a as brown crystal, yield 64.9 %, mp
219.2–220.0 °C, 1H NMR (CDCl3, 300 MHZ, ppm) d:
11.99 (s, 1H, OH), 11.93 (s, 1H, OH), 7.82–7.87 (t, 1H,
ArH), 7.73–7.76 (d, J = 7.8, 1H, ArH), 7.64 (s, 1H, ArH),
7.44 (s, 1H, ArH), 7.40–7.42 (d, J = 7.1, 1H, ArH), 3.02 (s,
3H, CH3), 2.92 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
191.5, 180.8, 168.7, 162.4, 162.1, 142.4, 136.3, 133.6,
132.1, 124.7, 122.5, 120.1, 118.0, 116.0, 115.4, 38.9, 38.6.
Anal. calcd. for 2a: C17H13NO5, C, 65.59; H, 4.21; N, 4.50.
Found: C, 65.25; H, 4.60; N, 4.28; IR (KBr) : 3421, 2956,
2923, 2852, 2360, 1673, 1630, 1480, 1452, 1404, 1350,
1274, 1208, 1182 cm-1; MS (ESI, m/z): 312 (M ? 1).
122.5, 120.0, 118.0, 116.0, 115.4, 49.1, 46.2, 26.2, 24.2.
Anal. calcd. for 2b: C19H15NO5, C, 67.65; H, 4.48; N, 4.15.
Found: C, 67.21; H, 4.82; N, 3.88; IR (KBr) m: 3422, 2971,
2881, 2362, 1669, 1630, 1476, 1452, 1381, 1351, 1288,
1267, 1202, 1158 cm-1; MS (ESI, m/z): 338 (M ? 1).
Procedure for preparation of 2c–2e
For 2c–2e preparation, a solution of 1b (0.368 g, 1 mmol) in
dry CH2Cl2 (20 mL) and 0.42 mL triethylamine was treated
with ethyl oxalyl chloride (0.29 mL, 3 mmol) at 0 °C, stirred
for 30 min at 0 °C, and then dropwised substitution amine
(2 mmol) for 45 min under nitrogen. After completion of the
reaction, it was quenched with 30 mL distilled water and
extracted with ethyl acetate (3 9 1.5 dm3) and the combined
extracts were washed with water, dried (MgSO4), filtered, and
evaporated to dryness under reduced pressure to give crude
product 2c–2e. The crude product was purified by silica gel
column purification using 10:1 (v/v, CH2Cl2:CH3OH) to
obtain pure solid product 2c–2e as yellow solid.
9,10-Dioxo-3-(pyrrolidine-1-carbonyl)-9,10-dihydroan-
thracene-1,8-diyl diacetate (2c) Deep yellow solid, yield
1
Procedure for preparation of N-cyclopentyl-4,
5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
carboxamide (2b)
56.9 %, mp 190.5–192.2 °C, H NMR (CDCl3, 300 MHZ,
ppm) d: 8.33 (s, 1H, ArH), 8.22–8.24 (d, J = 7.5, 1H, ArH),
7.76–7.81 (t, 1H, ArH), 7.59 (s, 1H, ArH), 7.42–7.45
(d, J = 8.1, 1H, ArH), 3.48 (s, 2H, CH2), 3.68 (s, 2H, CH2),
2.26 (m, 6H, CH3), 1. 89–2.05 (m, 4H, CH2); 13C NMR
(75 MHz, CDCl3): 185.1, 182.4, 169.0, 168.5, 166.0, 156.3,
154.1, 138.5, 135.2, 134.8, 133.5, 130.0, 128.6, 127.6, 121.5,
119.3, 117.4, 49.1, 46.2, 26.2, 24.2, 21.3. Anal. calcd. for 2c:
C23H19NO7, C, 65.55; H, 4.54; N, 3.32. Found: C, 65.01; H,
4.85; N, 3.02; IR (KBr) m: 3421, 2971, 2875, 2364, 1868,
1766, 1678, 1626, 1592, 1474, 1370, 1342, 1251, 1204,
1188 cm-1; MS (ESI, m/z): 422 (M ? 1).
Dry DMF (0.1 mL) was added to a solution of rhein
(0.568 g, 2 mmol) in dry SOCl2 (5 mL). The reaction
mixture was heated under reflux for 45 min under nitrogen.
After completion of the reaction, the reaction mixture was
heated to distill the excrescent SOCl2, cooled to room
temperature, and the intermediate product 1a was obtained
as brown liquid. The solution of 1a and dry dichlorometh-
ane (10 mL) was dropwised into a dry solution of triethyl-
amine (4 mL), pyrrolidine (4 mL), and dichloromethane
(10 mL) at 0 °C under nitrogen and then stirred at room
temperature for 1 h. After completion of the reaction, the
reaction mixture was diluted with 50 mL water, and 2.5 %
Na2CO3 was added to achieve a pH of 8–9. The aqueous
solution was extracted with dichloromethane (3 9 1.5 dm3)
and the combined extracts were washed with water, dried
(MgSO4), filtered, and evaporated to dryness under reduced
pressure to give crude product 2b. The crude product was
purified by silica gel column purification using 4:1 (v/v,
petroleum ether:ethyl acetate) to obtain pure solid product
3-(Cyclopentylcarbamoyl)-9,10-dihydroanthracene-1,
8-diyl diacetate (2d) Light yellow crystal, yield 53.0 %,
1
mp 263.5–265.0 °C, H NMR (CDCl3, 300 MHZ, ppm) d:
8.40 (s, 1H, ArH), 8.25–8.23 (d, J = 7.5, 1H, ArH), 7.91
(s, 1H, ArH), 7.82–7.70 (t, 1H, ArH), 7.45–7.43 (d, J = 7.8,
1H, ArH), 6.31–6.29 (d, J = 6.3, 1H, NH), 4.46–4.39 (m,
1H, CH), 2.46 (s, 6H, CH3), 1.75–1.53 (t, 8H,CH2); 13C
NMR (75 MHz, CDCl3): 185.1, 182.4, 168.9, 168.6, 165.9,
156.3, 154.2, 138.6, 135.1, 134.9, 133.6, 129.7, 128.5, 127.5,
121.3, 119.3, 117.4, 53.0, 24.1, 21.2, 18.7. Anal. calcd. for
2d: C24H21NO7, C, 66.20; H, 4.86; N, 3.22. Found: C, 65.91;
H, 5.01; N, 3.02; IR (KBr) m: 3421, 2958, 2873, 2360, 1771,
1677, 1632, 1594, 1543, 1448, 1377, 1349, 1298, 1261, 1184,
1116 cm-1; MS (ESI, m/z): 436 (M ? 1).
1
2b as orange solid, yield 62.9 %, mp 198.5–201.2 °C, H
NMR (dimethyl sulphoxide [DMSO], 300 MHZ, ppm) d:
11.99 (s, 1H, OH), 11.94 (s, 1H, OH), 7.78 (s, 1H, ArH),
7.76 (s, 1H, ArH), 7.49–7.50 (m, 2H, ArH), 7.43–7.46
(d, J = 8.1, 1H, ArH), 3.49–3.54 (m, 2H, CH2), 3.35–3.43
(t, 6H, CH2); 13C NMR (75 MHz, CDCl3): 192.2, 180.7,
166.5, 162.4, 162.3, 145.4, 137.3, 133.6, 133.1, 124.7,
3-((4-Nitrobenzyloxy)carbonyl)-9,10-dioxo-9,10-dihydro-
anthracene-1,8-diyl diacetate (2e) Deep yellow crystal,
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Med Chem Res (2013) 22:2228–2234
2233
1
yield 78.3 %, mp 225.2–227.5 °C, H NMR (CDCl3, 300
MHZ, ppm) d: 8.86 (s, 1H, ArH), 8.30–8.27 (t, 3H, ArH),
8.06 (s, 1H. ArH), 7.84–7.79 (t, 1H, ArH), 7.65–7.63 (d,
J = 8.1, 2H, ArH), 7.47–7.44 (d, J = 7.8, 1H, ArH), 5.51
(s, 2H, CH2), 2.46 (s, 6H, CH3); 13C NMR (75 MHz,
CDCl3): 185.2, 181.6, 168.9, 168.7, 165.9, 156.1, 155.9,
148.1, 142.5, 135.3, 135.0, 133.9, 129.2, 128.6, 128.4,
128.2, 125.8, 124.2, 124.1, 121.0, 120.3, 66.0, 21.1. Anal.
calcd. for 2e: C26H17NO10, C, 62.03; H, 3.40; N, 2.78.
Found: C, 61.82; H, 3.81; N, 2.46; IR (KBr) m: 3428, 3082,
2813, 2724, 1777, 1756, 1723, 1681, 1668, 1631, 1593,
1522, 1447, 1372, 1353, 1331, 1281, 1255, 1220,
1156 cm-1; MS (ESI, m/z): 502 (M - 1).
(d, J = 7.2, 2H, ArH), 7.56–7.54 (d, J = 6.9, 1H, ArH),
5.50 (s, 2H, CH2), 1.36–1.26 (m, 6H, CH3), 1.07 (s, 9H,
CH3), 0.85 (s, 9H, CH3), 0.33–0.32 (d, J = 5.1, 6H, CH3);
13C NMR (75 MHz, CDCl3): 183.5, 181.4, 164.6, 156.1,
155.9, 147.9, 142.8, 135.4, 134.9, 133.8, 129.2, 128.6,
128.5, 128.2, 125.8, 124.1, 123.9, 120.7, 120.3, 65.9, 30.9,
26.0. Anal. calcd. for 2g: C34H41NO8Si2, C, 63.03; H, 6.38;
N, 2.16; O, 19.76; Si, 8.67. Found: C, 62.76; H, 6.00; N,
1.98; IR (KBr) m: 3422, 2958, 2928, 2856, 1733, 1679,
1631, 1603, 1528, 1478, 1453, 1417, 1381, 1345, 1279,
1238, 1164 cm-1; MS (ESI, m/z): 649 (M ? 1).
Pharmacology
Procedure for preparation of 2f–2g
HepG2 cells were provided by the Cell Bank of the Insti-
tute of Fundamental Medicine, Chinese Academy of Medi-
cal Science (Beijing, China). Fetal bovine serum (FBS),
RPMI 1640 medium, and blood glucose diagnosis kits were
obtained from Nanjing Jiancheng Bioengineering Institute
(Nanjing, China). MTT and trypsin were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Dimethyld-
iguanide hydrochloride was supplied by Nanjin Langze
Institute (Nanjin, China). Final concentrations of DMSO
for dissolving drugs in medium were below 0.1 % (v/v).
MTT and rhein derivatives were diluted with PBS buffer
and filtered for sterilization. Cells with 10 % fetal calf
serum were cultured in RPMI 1640 medium at 37 °C in a
humidified incubator in 5 % CO2 atmosphere.
For 2f–2g preparation, a solution of 1c (0.512 g, 1 mmol)
in dry THF (20 mL) and 0.42 mL triethylamine was treated
with ethyl oxalyl chloride (0.29 mL, 3 mmol) at 0 °C,
stirred for 30 min at 0 °C, and then dropwised substitution
amine (2 mmol) for 45 min at room temperature under
nitrogen. After completion of the reaction, it was quenched
with 30 mL distilled water and extracted with ethyl acetate
(3 9 1.5 dm3) and the combined extracts were washed
with water, dried (MgSO4), filtered, and evaporated to
dryness under reduced pressure to give crude product
2f–2g. The crude product was purified by silica gel column
purification using 2:1 (v/v, petroleum ether:ethyl acetate)
to obtain pure solid product 2f–2g as yellow solid.
Glucose consumption activity was analyzed by mea-
suring the consumption glucose in HepG2 cell. The HepG2
cell in 48-well plates was pre-incubated with RPMI 1640
medium containing 1, 10, 100 mg/L 2a–2g and 10 mg/L
dimethyldiguanide 250 lL at 37 °C for 24 h, each repeated
three times. Glucose consumption was measured at 505 nm
using a glucose enzymatic kit on an automatic biochemical
analyzer. The media was ended to MTT for 4 h. The
absorbance (OD) retained by the cell lysates was deter-
mined at 490 nm by Multiskan Spectrum.
4,5-Bis(tert-butyldimethylsilyloxy)-N-cyclopentyl-9,10-
dioxo-9,10-dihydroanthracene-2-carboxamide (2f) Yellow
crystal, yield 48.5 %, mp 202.0–204.5 °C, 1H NMR
(CDCl3, 300 MHZ, ppm) d: 8.00 (s, 1H, ArH), 7.89–7.86
(d, J = 7.5, 1H, ArH), 7.79 (s, 1H, ArH), 7.58–7.53 (t, 1H,
ArH), 7.24–7.21 (d, J = 8.1, 1H, ArH), 6.32–6.30 (d,
J = 6.3, 1H, NH), 4.50–4.33 (m, 1H, CH), 1.62 (s, 8H,
CH2), 1.08 (s, 18H, CH3), 0.340–0.314 (d, 12H, CH3); 13
C
NMR (75 MHz, CDCl3): 184.1, 181.4, 164.9, 156.5, 155.9,
138.9, 135.1, 134.9, 133.6, 128.4, 127.7, 127.2, 120.3,
119.2, 116.4, 52.2, 33.3, 26.1, 25.9, 24.1, 18.8. Anal. calcd.
for 2f: C32H45NO5Si2, C, 66.28; H, 7.82; N, 2.42. Found:
C, 65.92; H, 7.65; N, 2.11; IR (KBr) m: 3421, 3306, 2954,
2930, 2657, 2361, 1677, 1632, 1588, 1558, 1454, 1417,
1386, 1347, 1315, 1284, 1256, 1227, 1123 cm-1; MS (ESI,
m/z): 581 (M ? 1).
Acknowledgments This work was supported by Scientific Research
Foundation for PhD of Southwest University (SWU111064, 111072),
the Fundamental Research Funds for the Central Universities (XDJK
2011C050), and Chongqing Engineering Technology Research Center
of Veterinary Drug (CSTC, 2009CB1010).
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