Synthesis of novel curcumin analogues for inhibition of 11β-hydroxysteroid dehydrogenase type 1 with anti-diabetic properties

Article abstract of DOI:10.1016/j.ejmech.2014.03.012

In the present study, a series of mono-carbonyl analogues of curcumin were designed and synthesized by deleting the reactive beta-diketone moiety, which is responsible for the pharmacokinetic limitation of curcumin. We demonstrated that 4 of 9 curcumin an

Full text of DOI:10.1016/j.ejmech.2014.03.012

European Journal of Medicinal Chemistry 77 (2014) 223e230
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel curcumin analogues for inhibition of
11 -hydroxysteroid dehydrogenase type 1 with anti-diabetic
properties
b
,
Xiaohuan Yuan a b, Hongzhi Li b, He Bai b, Zhijian Su a, Qi Xiang a, Chaonan Wang b,
,
a,
*
Binghai Zhao b, Yufei Zhang b, Qihao Zhang a, Yanhui Chu b , Yadong Huang
*
a Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China
b Heilongjiang Key Laboratory of Tissue Damage and Repair, Mudanjiang Medical University, Heilongjiang 157011, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a series of mono-carbonyl analogues of curcumin were designed and synthesized by
deleting the reactive beta-diketone moiety, which is responsible for the pharmacokinetic limitation of
curcumin. We demonstrated that 4 of 9 curcumin analogues were selective inhibitors of human and
Received 3 December 2013
Received in revised form
23 February 2014
Accepted 5 March 2014
Available online 6 March 2014
rodent 11
analogues weakly inhibited 11
selective, favoring 11 -HSD1. These 4 curcumin analogues are potential therapeutic agents for type-2
diabetes by targeting 11 -HSD1. The compound 8 displays anti-diabetic properties in diabetic mice
induced by streptozocin and high-fat-diet (STZHFD).
b
-HSD1. The level of this inhibitor was 4e20 times more than that of curcumin. Curcumin
b-HSD2, and further analyses revealed that these compounds were highly
b
b
Keywords:
Curcumin analogues
Stability
Ó 2014 Elsevier Masson SAS. All rights reserved.
Pharmacokinetic
Anti-diabetic activity
1. Introduction
primary reductase in the liver and fat tissues and an NADþ-depen-
dent 11 -HSD2 [4]. 11 -HSD2 acts a unidirectional oxidase to pre-
vent cortisol from stimulating the mineralo-corticoid receptor in the
b
b
Glucocorticoids (mainly cortisol in humans and corticosterone in
rodents) play a fundamental role in controlling physiologic ho-
meostasis. However, when present in excess, they can have a
detrimental impact on glucose control, blood pressure and lipid
levels. Glucocorticoids increase glucose output in the liver, induce fat
accumulation, dampen glucose-dependent insulin sensitivity in the
adipose tissue, thus increasing the risks of metabolic syndrome [1].
Obesity and type-2 diabetes are associated with abnormal regulation
kidneys, and the mutation of human 11b-HSD2 causes severe hy-
pertension and hypokalemia [5,6].
Recent data, mainly from rodents, provide considerable evi-
dence for the causal role of 11 -HSD1 in the development of
visceral obesity and type-2 diabetes. Thus, 11 -HSD1 inhibitors
b
b
have the potential of becoming an interesting novel class of drugs in
the future for the treatment of obesity and type-2 diabetes [7]. It is
of glucocorticoids metabolism. 11
b
-hydroxysteroid dehydrogenase
also important that 11
11 -HSD2 in order to avoid undesirable actions such as sodium
retention, hypokalemia, and hypertension. This led to the industry-
wide search for selective 11 -HSD1 inhibitors.
Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-hepta
diene-3,5-dione] (Fig. 1) is one of the hundreds of components
isolated from the ancient spice turmeric (Curcuma longa). Tumeric
has beenwidely used for centuries as a dietary spice and pigment. In
addition to its unique flavor and color, tumeric has been extensively
used in traditional medicine in several countries including the
United States, India, Japan, Korea, Thailand, China, Turkey, South
Africa, Nepal, and Pakistan [8]. It is being studied intensively today
for its potential therapeutic activity in the treatment of diabetes and
obesity-related metabolic disorders [9,10]. Curcumin has exhibited
b-HSD1 inhibitors not significantly inhibit
type 1 (11 -HSD1) catalyzes functionally inert glucocorticoid pre-
b
b
cursors (cortisone) to active glucocorticoids (cortisol) within the
insulin target tissues, such as adipose tissue, thereby regulating local
b
glucocorticoid action [2,3]. 11
b
-HSD has two known isoforms: an
NADPþ/NADPH dependent 11
b
-HSD1 oxidoreductase that behaves a
Abbreviations: 11
-hydroxysteroid dehydrogenase type 2; IPITT, intraperitoneal insulin tolerance
test; IPGTT, intraperitoneal glucose tolerance test; STZHFD, induced by streptozocin
and high-fat-diet; TG, triglycerides; TC, total cholesterol; LP( ), lipoprotein ; HDL,
b-HSD1, 11b-hydroxysteroid dehydrogenase type 1; 11b-HSD2,
11
b
a
a
high density lipoprotein cholesterol; LDL, low density lipoprotein cholesterol.
* Corresponding authors.
E-mail addresses: yanhui_chu@sina.com (Y. Chu), tydhuang@jnu.edu.cn
(Y. Huang).
http://dx.doi.org/10.1016/j.ejmech.2014.03.012
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

224
X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
30 min. Tomren [18] also showed a half-life of 10.5 h when cur-
cumin was kept in a pH 8.0 buffer containing 10% cyclodextrins. It
can be postulated that the hydrolytic degradation starts with an
attack from the nucleophilic OHꢀ ion on the carbonyl carbon in the
keto-enol form of the beta-diketone moiety in curcumin. The main
hydrolytic degradation products and mechanisms have been
identified previously [17]. The curcumin analogues stabilities were
assessed in 0.5% CMCNa solution (pH 7.4) at 37 ꢁC in a sodium
hydroxide and potassium dihydrogen phosphate buffer [19]. Cur-
cumin analogues were analyzed using HPLC (Fig. S1), and the dis-
tribution of the degradation profiles of curcumin and 9 curcumin
analogues were determined by monitoring the buffer solution for
72 h (Fig. 3). In the conditions above, more than 25% of curcumin
was degraded, while 6 of the analogues retained more than 85% of
the original content. Compound 4, 5 retained more than 98% of the
original content after 72 h, and there is no significant difference
between the compound 7 and curcumin. Fig. 3 showed that the
analogues lacking the beta-diketone moiety were much more sta-
ble than the curcumin in the above condition. These results are
similar to some curcumin analogues of the previous reports [19].
Fig. 1. Chemical structure of curcumin.
inhibitory action against human and rat 11
with IC50 values of 2.29 and 5.79 M, respectively, and selectivity
against 11 -HSD2 (IC50, 14.56 and 11.92 M) [11]. At the same time,
b-HSD1 in intact cells,
m
b
m
in the preclinical and clinic studies, curcumin has been shown to
possess several disadvantages in pharmacokinetics due to its poor
bioavailability, rapid metabolism and requirement of repetitive oral
doses, which limit its candidacy as a drug. For instance, it has been
reported that plasma and tissue curcumin levels in human volun-
teers and patients are 11.1 nM and 1.3 mM, respectively. However, it
required oral dose administration of up to 3.6 g/day [12,13]. In the
present study, a series of curcumin analogues with more stable
structures and good pharmacokinetic properties were synthesized.
The analogues that deleting the methylene group and one
carbonyl group of curcumin, we previously synthesized three series
of mono-carbonyl curcumin analogues without the central meth-
2.3. Inhibitory effects of curcumin analogues on 11b-HSD1
ylene functional groups and b-diketone moiety. These compounds
demonstrated better anti-bacterial and anti-inflammatory proper-
ties [14].
In this study, we designed and synthesized a series of mono-
carbonyl analogues of curcumin by deleting the beta-diketone
moiety (Scheme 1). The 9 curcumin analogues were evaluated for
It has been reported that, using microsomes from CHOP cells
transfected with human 11 -HSD1 and adult rat testicular 11
HSD1, curcumin showed inhibitory effects. The inhibitory effects on
11 -HSD1 of the 9 curcumin analogues (shown in Table 1) were
evaluated using the methods described previously [20]. Most of the
curcumin analogues exhibited inhibitory effects on 11 -HSD1.
The IC50 was determined by adding 25 nM substrate, 0.2 mM
cofactor and various concentrations of compound 1e9 in 250
reaction buffer (0.1 mM PBS) containing human or rat microsomal
protein as described previously [20]. As shown in Table 1, all cur-
cumin analogues possessed much higher activities than curcumin.
There was also species-dependent inhibition in which most cur-
cumin analogues exhibited better inhibition with rat testis micro-
somes than with human liver microsomes. For example, the
b
b-
b
b
selective 11b-HSD1 inhibitors activity. We performed a degradation
degree assay in vitro and a pharmacokinetic study in vivo to
examine the eight mono-carbonyl analogues that exhibit enhanced
stability. We found our bis-halo derivatives had more activities
against human HSD1 than the previous report of the analogs with
Single halo derivatives [15]. The compound 8 improved pharma-
cokinetic profiles compared to that of curcumin.
mL
Inhibition of 11
b-HSD1 is being pursued as an approach to the
treatment of type-2 diabetes and visceral obesity [16]. Since 11
b
-
compound 3, 4, 5 and 9 inhibited 11b-HSD1 with IC50 of rat testis
HSD1 primarily modulates levels of cortisol within tissues, these
findings led us to hypothesize that the generated curcumin ana-
logues will display anti-diabetic activity in the mice.
microsomes 1793.8 ꢂ 110.2, 531.6 ꢂ 29.4, 407.4 ꢂ 68.3 and
4767.9 ꢂ 328.9 nM, respectively. However, they showed higher IC50
value with human liver microsomes (>100 mM, data not shown).
The compound 1, 6, 7 and 8 exhibited excellent inhibitory activity
against 11 -HSD1 with both microsomes. Using rat testis micro-
2. Results and discussion
b
somes, IC50 of the four curcumin analogues was 422.5 ꢂ 25.6,
2.1. Chemistry
2240.7 ꢂ 186.5, 3443.0 ꢂ 169.2 and 673.4 ꢂ 86.3 nM, whereas IC50
was 1400.2
ꢂ
369.4, 650.5
ꢂ
101.6, 1100.8
ꢂ
123.6 and
Four series of mono-carbonyl analogues of curcumin, penta-1,4-
dien-3-one (compound 1,5), together with cyclopentanone (com-
pound 2, 4, 8), cyclohexanone (compound 3, 6, 9) and piperidin-4-
one (compound 7) analogues, were designed by displacing beta-
diketone moiety with a single carbonyl group based on previous
reports [14,15]. Different substituents with opposing electronic
properties in the benzene rings were designed to investigate the
structureeactivity relationship. Compounds 1e9 (shown in Fig. 2)
were synthesized by coupling the appropriate aromatic aldehyde
with cyclohexanone, cyclopentanone, acetone or piperidone in an
alkaline medium, respectively.
1064.3 ꢂ 220.2 nM in the case of human liver microsomes.
2.4. Inhibitory effects of curcumin analogues on 11 -HSD2
b
Curcumin was slightly selective against both rat and human 11
HSD2 with IC50 values of 12.23 and 14.67 M, respectively, this is
consistent with previous reports [11]. However, none of the cur-
b-
m
cumin analogues inhibited human and rat 11
(Table 1).
b-HSD2 at 100 mM
2.5. Pharmacokinetic study for curcumin and the compound 8 in
C57BL/6 mice
The general process for synthesis, synthetic yields, melting
point, 1H NMR analysis, 13C NMR analysis, EIMS analysis and
HREIMS analysis of these compounds are described in the Section 4.
We used the compound 8 and curcumin for the pharmacoki-
netic study in male C57BL/6 mice. The detailed HPLC methodology,
linearity, accuracy and precision are shown in the supplementary
data. The methodological data showed excellent chromatographic
specificity and no interference from plasma by the compound 8
(shown in Fig. S2).
2.2. Stability of analogues in vitro
In a previous report, Wang et al. [17] found that 92% of curcumin
was degraded when added to a 0.1 M phosphate buffer (pH 7.2) for

X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
225
Scheme 1. General synthesis of analogues of curcumin.
The calibration curves of the compound 8 are presented in
compound 8 was to 47.17
mg/mL at 2 h after administration, and its
Fig. S3, with coefficient of correlation R2 ¼ 0.9991 (A-curcumin) and
0.9993 (B- the compound 8). The intra-and inter-day RSD variations
were no more than 6.5% and 10.2% for curcumin and less than 6.0%
and 10.5% for the compound 8 (Table S1). Plasma concentratione
time curves of curcumin, the compound 8 in healthy C57BL/6 mice
(n ¼ 4) are shown in Fig. 4.
half-life period was 18.77 h. Furthermore, there was an apparent
decrease of clearance (CL) when comparing curcumin (11.24 [mg/
(mg/mL/h)]) with the compound 8 (1.22 [mg/(mg/mL/h)]). The dif-
ference in the total amount of compounds in plasma, represented
by the area under the curve (AUC 0-inf) of the compound 8, was
almost 10 times as much as curcumin. The biological utilization of
the compound 8 was 976.4% relative to that of curcumin. In sum-
mary, our results indicate that mono-carbonyl analogues of cur-
cumin, deleted one ketone in beta-diketone moiety, significantly
decreases the degree and speed of metabolism of curcuminoids,
and possess much better pharmacokinetic profiles than curcumin.
The pharmacokinetic parameters are presented in Table 2.
Following the intraperitoneal injection at a dose of 5 mg/kg, the
peak concentration of curcumin reached 4.03 mg/mL at 1.37 h after
administration and had a half-life period was of 26.64 h. The
deletion of beta-diketone enhanced the peak concentration of the
Fig. 2. Structure of analogues of curcumin.

226
X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
Table 2
The pharmacokinetic characteristics of curcumin analogues in the plasma of health
mice (n ¼ 4) that were injected a single dose of 5 mg/kg body weight.
Parameter
Curcumin
Compound 8
t1/2 (h)
Tmax (h)
26.64 ꢂ 7.092
1.375 ꢂ 0.375
4.003 ꢂ 0.486
57.22 ꢂ 8.276
74.39 ꢂ 4.821
0.760 ꢂ 0.071
35.58 ꢂ 8.061
445.5 ꢂ 131.5
11.24 ꢂ 0.757
18.77 ꢂ 2.163
2.00 ꢂ 0.000
47.17 ꢂ 3.009
558.7 ꢂ 44.98
700.1 ꢂ 69.93
0.81 ꢂ 0.0580
28.72 ꢂ 4.470
31.86 ꢂ 1.242
1.220 ꢂ 0.141
Cmax (
AUC 0-t (
AUC 0-inf_obs (
AUC 0-t/0-inf_obs
MRT 0-inf_obs (h)
m
g/mL)
g/mL*h)
g/mL*h)
m
m
Vz/F_obs [mg/(
Cl/F_obs [mg/(
mg/mL)]
m
g/mL/h)]
group. To evaluate the effect of the compound 8 on glucose meta-
bolism, we next conducted an intraperitoneal insulin tolerance test
(IPITT) and intraperitoneal glucose tolerance test (IPGTT). The
compound 8 group showed a significant decrease of plasma glucose
in response to insulin (Fig. 5A) and plasma glucose (Fig. 5B)
compared with the diabetic group mice at 15, 30, 60 and 120 min
after intraperitoneal injection of insulin or glucose. The results
revealed that the compound 8 treatment improved insulin and
glucose tolerance by the compound 8.
Fig. 3. The degradation degree of curcumin and 9 analogues.
2.6. Anti-diabetic effect in mice with diabetes induced by STZHFD
In mice with diabetes induced by streptozocin and high-fat-diet
(STZHFD mice), the compound 8 treatment caused a decrease in
body weight compared with the control group. There is no differ-
ence in food intake with or without the compound 8 treatment in
STZHFD mice (Fig. S4.). At the beginning, the control group mice
gained more body weight, but were stabilized after 21 days, and the
compound 8 group were stabilized (shown in Fig. S5) all along,
indicating that the compound 8 suppressed weight gain in STZHFD
mice without causing appetite loss.
The compound 8 treatment group reduced fasting plasma TG
and plasma TC (Table 3), and significantly reduced fasting blood
glucose, HDL and LDL concentrations compared with the diabetes
group. TG, TC of the compound 8 treatment group was close to
control group, though fasting blood glucose of the compound 8
group was higher than control group, it was lower than diabetes
2.7. Acute toxicity
The compound 8 did not produce any mortality during the 2-
week period at 500 and 100 mg/kg oral doses by oral gavage.
Mice showed no symptoms associated with toxicity, such as
convulsion, ataxia or increased diuresis.
3. Conclusion
Although curcumin is the natural 11
b-HSD1 inhibitor, its poor
bioavailability and rapid metabolism make it less promising for
Table 1
Inhibitory effects on 11
b
-HSD1 and 11
b
-HSD2 profiles of curcumin analogues (IC50, n ¼ 4).
Human liver
Compound
Rat testis
Rat testis
11 -HSD2
Human liver
11 -HSD2
11b-HSD1
11b-HSD1
b
b
1
2
3
4
5
6
7
8
422.5 ꢂ 25.6
1400.2 ꢂ 369.4
11,560.3 ꢂ 2108.8
>100,000
>100,000
>100,000
650.5 ꢂ 101.6
1100.8 ꢂ 123.6
1064.3 ꢂ 220.2
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
12,231 ꢂ 874.2
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
14,672 ꢂ 1136.1
1638.1 ꢂ 384.7
1793.8 ꢂ 110.2
531.6 ꢂ 29.4
407.4 ꢂ 68.3
2240.7 ꢂ 186.5
3443.0 ꢂ 169.2
673.4 ꢂ 86.3
9
4767.9 ꢂ 328.9
11,360.8 ꢂ 1807.9
Curcumin
5236.4 ꢂ 98.6
Fig. 4. Mean plasma concentrationetime of curcumin (A) and the compound 8 (B) in the plasma of healthy C57BL/6 mice (n ¼ 4), which were injected at the dose of 5 mg/kg body
weight.

X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
227
mixture was stirred at a temperature of 50 ꢁC for 50 min. When the
reaction was finished, large amounts of precipitate were generated.
The precipitate was cooled to room temperature and dried under
reduced pressure. The products was washed with water and cold
ethanol, and dried in vacuo. The solid portion was purified through
chromatography over silica gel using CH2Cl2/CH3OH as the eluent.
Table 3
Characteristics of plasma biochemical indexes in mice treated with or without the
compound 8 (n ¼ 5).
C57BL/6
Control
STZHFD mice
Diabetes
Compound 8
Body weight, g
GLU, mg/dL
LP(A), mg/dL
TC, mg/dL
TG, mg/dL
HDL, mg/dL
LDL, mg/dL
30.84 ꢂ 1.84*
113.0 ꢂ 4.01***
0.738 ꢂ 0.05
83.85 ꢂ 0.14
65.14 ꢂ 13.15
55.83 ꢂ 1.50*
12.55 ꢂ 0.38***
38.12 ꢂ 0.41*
372.1 ꢂ 37.38***
1.012 ꢂ 0.08
34.32 ꢂ 0.68
246.4 ꢂ 4.37
0.650 ꢂ 0.15
75.83 ꢂ 2.16
69.47 ꢂ 3.82
43.2 ꢂ 4.30
10.16 ꢂ 0.20
4.1.1. (1E,4E)-1,5-bis(2-bromophenyl)penta-1,4-dien-3-one (1)
Yellow powder, 70.2% yield. mp 111.2e113.5 ꢁC.
153.7 ꢂ 3.82***
101.2 ꢂ 5.04**
97.1 ꢂ 6.51***
16.09 ꢂ 0.63***
1H NMR (500 MHz, DMSO)
d: 10.23 (s, 2H), 7.86 (s, 2H), 7.61 (dd,
J ¼ 8.7, 5.5 Hz, 4H), 7.41e7.30 (m, 4H), 4.45 (d, J ¼ 1.4 Hz, 4H). 13
C
*, p < 0.05, control (or diabetes) vs the compound 8 group; **, p < 0.01, control (or
diabetes) vs the compound 8 group; ***, p < 0.001, control (or diabetes) vs the
compound 8 group.
NMR (75 MHz, DMSO) d: 182.1, 164.2, 160.9, 137.8, 133.0, 132.8,
130.2, 127.6, 116.0, 115.8, 43.7. EIMS m/z:311 (M ꢀ HCl). HREIMS
Calcd for C19H15O1N1F2: 311.1116, found: 311.1116.
clinical applications. In this paper, we designed and synthesized a
series of mono-carbonyl analogues of curcumin. Curcumin ana-
4.1.2. (2E,5E)-2,5-bis(2,6-difluorobenzylidene)cyclopentanone (2)
Yellow powder, 68.5% yield. mp 154.6e156.0 ꢁC.
logues significantly inhibited human and rat 11
b
-HSD1 activities. In
M indicated
1H NMR (400 MHz, CDCl3)
d 7.51 (s, 2H), 7.39e7.29 (m, 2H), 6.95
addition, their IC50s for 11 -HSD2 at the dose of >100
b
m
(t, J ¼ 8.1 Hz, 4H), 2.79e2.71 (m, 2H). 13C NMR (101 MHz, CDCl3)
good primary and secondary selectivities. The curcumin analogues
compound 8 showed anti-diabetic effects, without any associated
toxicity. These compounds may potentially become novel thera-
d
194.41, 162.00 (d, J ¼ 7.3 Hz), 159.49 (d, J ¼ 7.4 Hz), 143.29, 130.69
(t, J ¼ 10.5 Hz), 120.91, 113.34 (t, J ¼ 18.8 Hz), 111.65 (d, J ¼ 6.0 Hz),
111.45 (d, J ¼ 6.0 Hz), 26.36 (t, J ¼ 5.5 Hz). Purity: 98.9% by HPLC.
HRMS (ESI):calcd for (M þ Na)þ (C19H12F4O):355.0716, found
355.0718.
peutic agents targeting 11
diabetes.
b-HSD1, for the treatment of type-2
4. Experimental section
4.1.3. (2E,6E)-2,6-bis (2,6-fluorobenzylidene) cyclohexanone (3)
Yellow powder, 68.3% yield. mp 135.1e137.1 ꢁC.
4.1. Chemical synthesis
1H NMR (400 MHz, CDCl3)
d 7.56 (s, 2H), 7.36e7.28 (m, 2H), 6.94
(t, J ¼ 7.7 Hz, 4H), 2.68e2.55 (m, 4H), 1.82e1.69 (m, 2H). 13C NMR
Melting points were determined on a OptiMelt MPA-100
apparatus (SRS, USA) and were uncorrected. 1H NMR spectra
were recorded on a INOVA-400 spectrometer (Varian, USA). The
chemical shifts were presented in terms of parts per million, with
TMS as the internal reference. Electron-spray ionization mass
spectra in positive mode (ESI-MS) data were recorded on a Esquire
3000 þ spectrometer (Bruker, USA). Column chromatography pu-
rifications were carried out on Silica Gel 60 (E. Merck, 70e230
mesh).
The general procedure of synthesis of the compound 1e9 was as
follows. Aromatic aldehyde (15.4 mmol) was added to a solution of
7 mmol acetone (for the syntheses of the compound 1, 5), or
cyclopentanone (for the syntheses of the compound 2, 4, 8), or
cyclohexanone (for the syntheses of the compound 3, 6, 9), or
piperidin-4-one (for the syntheses of the compound 7) in ethanol
(10 mL). The solution was stirred at room temperature for 20 min,
followed by drop-wise addition of KOH solution (5%, 2 mL). The
(101 MHz, CDCl3)
d
188.33, 161.63 (d, J ¼ 7.5 Hz), 159.14 (d,
J ¼ 7.4 Hz), 141.20, 130.23 (t, J ¼ 10.3 Hz), 124.26, 113.46 (t,
J ¼ 19.6 Hz), 111.50 (d, J ¼ 6.1 Hz), 111.30 (d, J ¼ 6.1 Hz), 28.91 (t,
J ¼ 3.5 Hz), 22.55.Purity: 98.7% by HPLC. HRMS (ESI):calcd for
(M þ Na)þ (C20H14F4O):369.0873, found 369.0868.
4.1.4. (2E,5E)-2,5-bis(2-bromo-6-fluorobenzylidene)
cyclopentanone (4)
Yellow powder, 64.9% yield. mp 171.2e173.4 ꢁC.
1H NMR (400 MHz, CDCl3)
d 7.50e7.39 (m, 4H), 7.24e7.18 (m,
2H), 7.09 (t, J ¼ 8.8 Hz, 2H), 2.76e2.58 (m, 4H). 13C NMR (101 MHz,
CDCl3)
d
194.29, 159.87 (d, J ¼ 253.7 Hz), 143.11, 130.75 (d,
J ¼ 9.4 Hz), 128.63 (d, J ¼ 3.3 Hz), 126.93, 125.38 (d, J ¼ 4.2 Hz),
124.79 (d, J ¼ 18.3 Hz), 115.10 (d, J ¼ 23.1 Hz), 25.98 (d, J ¼ 8.1 Hz).
Purity: 98.8% by HPLC. HRMS (ESI): calcd for (M
(C19H12Br2F2O): 474.9115, found 474.9089.
þ
Na)þ
Fig. 5. Effect of the compound 8 supplementation on intraperitoneal insulin tolerance test (IPITTA) and intraperitoneal glucose tolerance test (IPGTT B) in C57BL/6 mice and STZHFD
mice (9 week-old). (After a 12 h fast, control (C57BL/6 mice, closed triangle), the compound 8 (STZHFD mice with the compound 8 treatment, closed square) and diabetes (STZHFD
mice without the compound 8, closed circle) were intraperitoneal injected with insulin (1.0 U/kg body weight) or glucose (1.5 g/kg body weight), respectively (n ¼ 5). *, p < 0.05,
diabetes vs the compound 8 group; **, p < 0.01, diabetes vs the compound 8 group.)

228
X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
4.1.5. (1E,4E)-1,5-bis(2-bromo-6-fluorophenyl)penta-1,4-dien-3-
one (5)
4.2. Stability of analogues
Yellow powder, 71.1% yield. mp 161.5e163.7 ꢁC.
An Agilent 1200 HPLC system, equipped with a Hypersil ODS2
1H NMR (400 MHz, CDCl3)
d
7.89 (d, J ¼ 16.2 Hz, 2H), 7.47 (d,
column (5
mm ꢃ 4.6 mm ꢃ 200 mm), was used in this study. The
J ¼ 7.9 Hz, 2H), 7.24e7.16 (m, 2H), 7.15e7.08 (m, 2H). 13C NMR
hydrolytic stabilities of the curcumin and analogues were investi-
gated in phosphate-buffered 0.3% (w/v) sodium carbox-
ymethycellulose (CMCNa) solutions at pH 7.4. Two milligrams
samples were weighed accurately and dissolved in 1 mL of CMCNa
solution by vortex. After stored in the dark at 37 ꢁC for the indicated
time, the mixture was vortexed and analyzed by HPLC. The peak
areas were recorded and the degradation rate was calculated using
the following formula:
(101 MHz, CDCl3)
d
189.26, 161.81 (d, J ¼ 257.6 Hz), 136.44 (d,
J ¼ 2.6 Hz), 131.88 (d, J ¼ 13.8 Hz), 131.23 (d, J ¼ 10.1 Hz),
129.39 (d, J ¼ 3.4 Hz), 126.70 (d, J ¼ 4.0 Hz), 123.64 (d,
J ¼ 13.4 Hz), 115.62 (d, J ¼ 23.5 Hz). Purity: 98.9% by HPLC.
HRMS (ESI):calcd for (M
found 448.8963.
þ
Na)þ (C17H10Br2F2O):448.8959,
Degradation rate ¼ ðpeak area in 0 h ꢀ peak area in 72 hÞ=peak area in 0 h
4.1.6. (2E,6E)-2,6-bis(2-bromo-6-fluorobenzylidene)cyclohexanone
4.3. Pharmacokinetic study in vivo
(6)
Yellow powder, 66.9% yield. mp 136.7e138.9 ꢁC.
4.3.1. Chromatographic conditions
1H NMR (400 MHz, CDCl3)
d
7.54 (s, 2H), 7.45 (d, J ¼ 7.9 Hz, 2H),
7.25e7.18 (m, 2H), 7.09 (t, J ¼ 8.7 Hz, 2H), 2.60e2.50 (m, 4H), 1.80e
1.70 (m, 2H). 13C NMR (101 MHz, CDCl3)
188.19, 159.54 (d,
An Agilent 1200 HPLC system equipped with a Hypersil ODS2
column (5
m
m ꢃ 4.6 mm ꢃ 200 mm) was used in this study. The
d
mobile phase consisted of a mixture of acetonitrile and water
(containing 4% acetic acid, pH ¼ 2.56) with the ratio 80:20 (v/v). The
HPLC system was operated at a flow rate of 1.0 mL/min and per-
formed at a controlled temperature of 20 ꢁC. UV detection was
performed under a wavelength of 340 nm.
J ¼ 251.9 Hz), 140.28, 130.39 (d, J ¼ 9.2 Hz), 130.29, 128.44 (d,
J ¼ 3.4 Hz), 125.29 (d, J ¼ 18.8 Hz), 124.95 (d, J ¼ 4.0 Hz), 114.84 (d,
J ¼ 23.1 Hz), 28.56 (d, J ¼ 5.0 Hz), 22.30. Purity: 99.6% by HPLC.
HRMS (ESI): calcd for (M þ Na)þ (C20H14Br2F2O): 488.9272, found
488.9302.
4.3.2. Development of HPLC method
The methods used to determine selectivity, standard solution,
linearity, sensitivity, precision, and recovery for the establishment
of HPLC method are shown in the supplemental file.
4.1.7. (3E,5E)-3,5-bis(2-bromo-6-fluorobenzylidene)piperidin-4-
one (7)
Yellow powder, 64.8% yield. mp 165.8e167.5 ꢁC.
1H NMR (400 MHz, CDCl3)
d
7.57 (s, 2H), 7.46 (d, J ¼ 8.0 Hz, 2H),
4.3.3. Pharmacokinetic study
7.26e7.21 (m, 2H), 7.11 (t, J ¼ 8.8 Hz, 2H), 3.78e3.73 (m, 2H). 13C
The pharmacokinetic study of the mice was approved by the
Mudanjiang Medical College Animal Policy and Welfare Committee.
C57BL/6 mice with body weights ranging from 33 to 40 g were used
for the study of pharmacokinetics of curcumin and its analogues
(Vital River Laboratory Animal Technology Co. Ltd, SCXK (Beijing)-
0001). All mice were housed in a temperature-controlled room
with a 12 h light/dark cycle. Eight mice were randomly divided into
two groups, curcumin and the compound 8 solubilized by the aid of
1% CMCNa, was given at a dose of 5 mg/kg with intraperitoneal
injection, respectively. Blood samples (0.2 mL) were obtained from
the fossa orbitalis veniplex at 0, 0.25, 0.5, 1, 2, 3, 5, 9, 15, 24, 36 and
48 h after administration. After collection, each blood sample was
immediately centrifuged at 1500 ꢃ g for 3 min and the plasma was
separated then stored at 20 ꢁC until analysis.
NMR (101 MHz, CDCl3)
d
186.38, 159.59 (d, J ¼ 252.2 Hz), 138.40,
130.92 (d, J ¼ 9.3 Hz), 128.81, 128.63 (d, J ¼ 3.4 Hz), 125.10 (d,
J ¼ 3.8 Hz), 124.23 (d, J ¼ 18.5 Hz), 114.95 (d, J ¼ 23.2 Hz), 48.03 (d,
J ¼ 8.4 Hz). Purity: 99.2% by HPLC. HRMS (ESI): calcd for (M þ Na)þ
(C19H13NBr2F2O): 467.9405, found 467.9406.
4.1.8. (2E,5E)-2,5-bis[2-fluoro-6-(trifluoromethyl)benzylidene]
cyclopentanone (8)
Yellow powder, 66.3% yield. mp 109.2e111.1 ꢁC.
1H NMR (400 MHz, CDCl3) (
d 7.61e7.50 (m, 1H), 7.50e7.42 (m,
1H), 7.33 (t, J ¼ 8.8 Hz, 1H), 2.66e2.55 (m, 4H). 13C NMR (101 MHz,
CDCl3)
d 193.46, 160.92, 158.43, 144.14, 131.06 (m), 130.00 (d,
J ¼ 9.1 Hz), 123.38, 122.61 (m), 122.19 (m), 119.77 (d, J ¼ 23.9 Hz),
25.51 (d, J ¼ 7.0 Hz). Purity: 99.6% by HPLC. HRMS (ESI): calcd for
(M þ Na)þ (C22H114F8O): 455.0653, found 455.0664.
4.3.4. Sample preparation
Samples were determined after extraction by organic solvent. To
every 1 mL extractant of ethyl acetate, 100 mL of plasma was added.
The mixture was vortexed for 2 min and centrifuged at 5000 g for
5 min and then the organic phase was then transferred to a new vial
and dried with N2. The dried residue was reconstituted with 100 mL
4.1.9. (2E,6E)-2,6-bis[2-fluoro-6-(trifluoromethyl)benzylidene]
cyclohexanone (9)
Yellow powder, 69.1% yield. mp 119.8e122.1 ꢁC.
of acetonitrile and vortexed for 1 min. The volume of 10
natant was injected into the HPLC system.
mL super-
1H NMR (400 MHz, CDCl3)
d
7.63 (s, 2H), 7.53 (d, J ¼ 7.8 Hz, 2H),
7.44 (dd, J ¼ 13.4, 7.9 Hz, 2H), 7.31 (t, J ¼ 8.7 Hz, 2H), 2.50e2.43 (m,
4H), 1.74e1.64 (m, 2H). 13C NMR (101 MHz, CDCl3)
187.60, 160.48,
d
4.3.5. Data analysis
158.01, 141.36 (s, 4H), 130.90 (m), 129.67 (d, J ¼ 8.8 Hz), 126.86,
123.18 (m), 121.81 (m), 119.49 (d, J ¼ 23.2 Hz), 28.25 (d, J ¼ 3.9 Hz),
22.07. Purity: 99.1% by HPLC. HRMS (ESI): calcd for (M þ Na)þ
(C22H14F8O): 469.0809, found 469.0800.
A non-compartmental pharmacokinetic analysis with PKSolver
software was used to analyze plasma concentrationetime data [21].
The pharmacokinetic parameters such as area under the concen-
trationetime curve (AUC), mean residence time (MRT), total

X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
229
clearance (CL/F) and biological half-life (t1/2) for each compound
were assessed.
Graph Pad Software Inc., San Diego, CA) for IC50. A Lineweavere
Burk plot was used for the analysis of the mode of inhibition.
ANOVA was used to determine differences. A post hoc test using
Tukey’s analysis was used to determine the differences between the
two groups. All data are expressed as means ꢂ SEM. Differences
were regarded as significant at P < 0.05.
4.4. Determination of half maximum inhibitory concentrations
(IC50)
4.4.1. 11
Six male adult rat testes were used for preparation of rat testis
microsomes to measure 11 -HSD1 activity, as they contained a
large amount of 11 -HSD1 enzyme [22]. The preparation of mi-
b-HSD1 assay in rat and human microsomes
4.5. Anti-diabetic animal experiments
b
b
4.5.1. Animals
crosomes was performed as described previously [20]. In brief, rat
testes were homogenized in 0.01 mM PBS buffer containing 0.25 M
sucrose. The nuclei and large cell debris were removed by centri-
fugation at 1500 ꢃ g for 10 min. The post-nuclear supernatants
were centrifuged twice at 105,000 ꢃ g. The resultant microsomal
pellets were resuspended. Protein contents were measured by
NanoDrop 2000 (Thermo Scientific). The microsomes (20 mg/mL)
were used for measurement of 11b-HSD1 or 11b-HSD2 activities.
Human liver and kidney microsomes were purchased from Gentest
(Cat# 452156, Woburn, MA), which were prepared from 50 pooled
Five-week-old, male C57BL/6 mice were purchased from Vital
River Laboratory Animal Technology Co. Ltd. All experimental
procedures were performed according to the “Guidelines for the
Care and Use of Laboratory Animals” approved by the Mudanjiang
Medical University Committee for Animal Experiment. The mice
were housed two to three per cage, with free access to food and
water. The cages were kept in standard light (12 h light/dark),
temperature (22 ꢂ 2 ꢁC) and humidity (40 ꢂ 10%) conditions.
4.5.2. Generation of mice with diabetes induced by STZHFD
One week after purchase, the C57BL/6 mice were injected
intraperitoneally once every other day for 2 times with vehicle or
livers and stored with final concentrations of 20 mg/mL. 11b-HSD1
activity assay tubes contained 25 nM substrate cortisone (for hu-
man) or 11DHC (for rat), spiked with 30,000 cpm of their respective
[3H] cortisol or [3H] CORT. Cortisone or 11DHC was used as the
low-dose STZ (70 mg/g body weight in 10 mmol/L sodium citrate
buffer, pH 4.0, SigmaeAldrich) after 4 h fasting. The vehicle-
injected mice (control) were placed on SD diet consisting of 18.0%
protein, 4.0% fat, 5.0% fiber, 8.0% ash, 7.0% water, 7.0% amino acid
mix, 1.0% vitamin mix, and 1% salt mix (SLRC, SHANGHAI), and STZ-
injected mice were placed on HFD diet consisting of 18.0% protein,
35.0% fat, 18.0% carbohydrate, 5.0% fiber, 8.0% ash, 7.0% water, 7.0%
amino acid mix, 1.0% vitamin mix, and 1% salt mix (SLRC,
SHANGHAI). After 4 weeks, Blood glucose of STZHFD mice were up
to 14.2 mmol/L. Then the STZHFD mice with similar degrees of
hyperglycaemia and body weight were also randomly divided
diabetes and the compound 8 group (n ¼ 5). Then the three groups
(n ¼ 5) were placed on SD. The diabetes and the control group were
gavaged with 1% CMC, and the compound 8 group was gavaged
with a dose of 5 mg/kg body weight of the compound 8 in 1% CMC.
Body weights and food intake were recorded every day. Plasma
glucose (tail prick) was measured every 7 days using a blood
glucose meter (qx046701, ROCHE). On day 42 of gavaging IPITT and
IPGTT were conducted. Then the mice were sacrificed.
substrate to measure 11
b
-HSD1 activity. The rat testes microsomes
g) microsomes were incubated with
(10 g) or human liver (4
m
m
substrate, 0.2 mM NADPH and 0.5 mM glucose-6-phosphate (G6P)
and various concentrations of the compound 1e9 at 37 ꢁC for 60e
90 min. The inhibitory potency of curcumin analogues was
measured relative to control (only DMSO). The compound 1e9 was
dissolved in DMSO with final DMSO concentration of 0.4%, at which
DMSO did not inhibit this enzyme activity. The reaction was
stopped by adding 1 mL of ice-cold ether. The steroids were
extracted, and the organic layer was dried with N2. The steroids
were separated chromatographically on the thin layer plate in
chloroform and methanol (90:10, v/v), and the radioactivity was
measured using a scanning radiometer (System AR2000, Bioscan
Inc., Washington, DC) as described previously [23]. The percentage
conversion of 11DHC to CORT, or cortisone to cortisol was calcu-
lated by dividing the radioactive counts identified as 11-OH-ste-
roids by the total counts.
4.4.2. 11
Six male adult rat kidneys were used for preparation of rat
kidney microsomes to measure 11 -HSD2 as high levels of 11b-
b-HSD2 assay in rat and human kidney microsomes
4.6. The compound 8 acute toxicity experiment
b
HSD2 expression and activity a represented in the kidney [24].
Preparation of kidney microsomes was carried out as previously
Adult C57BL/6 mice of either sex, weighing between 25 and 30 g,
were used for toxicity study. They were randomly divided to 3
groups. The compound 8 was administered orally at 500 and
100 mg/kg doses (100 and 20 times the optimal effective dose of
5 mg/kg) every day. At the same time, the control group that
received distilled water was used as a negative control. The volume
administered by gavage in the mice was approximately 0.5 mL per
animal. Water and food were freely available to the animals. Sub-
sequent to the compound 8 administration, the animals were
observed closely for the first three hours for any toxicity such as
increased motor activity, salivation, convulsion, coma, piloerection,
diarrhea, altered muscle tone, hypnosis, hyperexcitability, writhing
(abdominal constrictions), and death. The animals were fully
monitored up to a period of 2 weeks.
All of the animals were sacrificed after 2 weeks. The kidneys,
hearts, lungs, spleens and livers were removed, weighed, and
evaluated for macroscopic abnormalities. If the changes were
observed in the autopsies, the microscopic examination of the or-
gans was performed following paraffin embedding and
Hematoxylin-Eosin staining for further study.
described [20]. 11b-HSD2 activity assay tubes contained 25 nM
(within the range of physiological levels of CORT). [3H] cortisol and
[3H] CORT were used as substrates to measure either human or rat
11b-HSD2 oxidase activity. Kidney microsomes were incubated
with substrates, NADþ. The reactions were stopped by adding 1 mL
of ice-cold ether. The steroids were extracted, and the organic layer
was dried with nitrogen. The steroids were separated chromato-
graphically on thin layer plates in chloroform and methanol
(90:10), and the radioactivity was measured using a scanning
radiometer (System AR2000, Bioscan Inc., Washington, DC). The
percentage conversion of CORT to 11DHC and cortisol to cortisone
was calculated by dividing the radioactive counts identified as
11DHC (or cortisone) by the total counts associated with both
substrate and product.
4.4.3. Statistics
All experiments were repeated at least four times. Data were
subjected to nonlinear regression analysis by Graph Pad (Version 5,

230
X. Yuan et al. / European Journal of Medicinal Chemistry 77 (2014) 223e230
[9] M.R. Maradana, R. Thomas, B.J. O’Sullivan, Targeted delivery of curcumin for
Acknowledgments
treating type 2 diabetes, Molecular Nutrition & Food Research 57 (2013)
1550e1556.
This work was supported in part by the National Natural Science
Funding of China (81070329 to Y.H.C and 81070477 to Y.D.H). The
project was supported in part by the Guangdong Province Higher
Vocational Colleges & Schools Pearl River Scholar Funded Scheme
(2012), by the National Natural Science Funding of Heilongjiang
Province (H201378 to X.H.Y), by the Foundation of Heilongjiang
Educational Committee (13530865 to X.H.Y) and supported by the
Open Project Program of the Key Laboratory of Tissue Injury and
Repair (zdsys2013-01 to X.H.Y). We also thank SubrataChakrabarti
(Western University) for reviewing this manuscript.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.012. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
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