Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.05.080

A series of structurally novel mono-carbonyl curcumin analogues have been synthesized and biologically evaluated to test their inhibitory potencies and the structure-activity relationship (SAR) on human and rat 11β- hydroxysteroid dehydrogenase isoform (11β-HSD1) activities. 11β-HSD1 selective inhibitors have been discovered and compound A10 is discovered as a very potent with an IC₅₀ value of 97 nM without inhibiting 11β-HSD2.

Full text of DOI:10.1016/j.bmcl.2013.05.080

Accepted Manuscript
Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1
inhibitors
Han Lin, Guo-Xin Hu, Jingjing Guo, Yufei Ge, Guang Liang, Qing-Quan Lian,
Yanhui Chu, Xiaohua Yuan, Ping Huang, Ren-Shan Ge
PII:
S0960-894X(13)00675-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.05.080
BMCL 20541
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 January 2013
15 May 2013
24 May 2013
Please cite this article as: Lin, H., Hu, G-X., Guo, J., Ge, Y., Liang, G., Lian, Q-Q., Chu, Y., Yuan, X., Huang, P.,
Ge, R-S., Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1 inhibitors, Bioorganic &
Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.05.080
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Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1 inhibitors
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a
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Han Lin a,c,1, Guo-Xin Hu a,b,1, Jingjing Guo a, Yufei Gec, Guang Liang , Qing-Quan Lian ,
Yanhui Chu d, Xiaohua Yuan d, Ping Huange,*, and Ren-Shan Ge a,c,*
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a The 2nd Affiliated Hospital & Institute of Reproductive Biomedicine, b School of Pharmacy,
Wenzhou Medical College, Wenzhou 325000, China.
c Population Council& Rockefeller University,1230 York Avenue, New York, NY 10065;
d
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Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University,
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Heilongjiang, P. R. China
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e Department of Pharmacy, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, 310007,
China
1Authors contributed equally
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Correspondence should be addressed to R.S.G. (Ren-Shan Ge; The 2nd Affiliated Hospital &
Institute of Reproductive Biomedicine,Wenzhou, China. Tel.: 86-577-88879079; Fax: 86-577-
88879167, E-mail: r_ge@yahoo.com) or huangpwly@sina.com (P. Huang).
Abbreviations
11β-HSD1 11β-hydroxysteroid dehydrogenase isoform
11β-HSDs 11β-hydroxysteroid dehydrogenases
11DHC 11-dehydrocorticosterone
CORT corticosterone
CUR curcumin

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Abstract
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A series of structurally novel mono-carbonyl curcumin analogues have been synthesized and
biologically evaluated to test their inhibitory potencies and the structure–activity relationship
(SAR) on human and rat 11β-hydroxysteroid dehydrogenase isoform (11β-HSD1) activities.
11-HSD1 selective inhibitors have been discovered and compound A10 is discovered as a very
potent with an IC50 value of 97 nM without inhibiting 11-HSD2.
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Keywords: 11β-HSD1; 11β-HSD2; mono-carbonyl curcumin analogues; inhibitors

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Excessive glucocorticoids under conditions such as Cushing’s disease cause a spectrum of
clinical features similar to the metabolic syndrome.1 The treatment of the metabolic syndrome in
Cushing’s disease can be achieved by reducing circulating glucocorticoids.2 Glucocorticoids
have been demonstrated to increase the risks of metabolic syndromes. Intracellular levels of
glucocorticoids (cortisol in the human or corticosterone, CORT, in the rat) are regulated by two
11β-hydroxysteroid dehydrogenases (11β-HSDs). 11β-HSD1 is an NADPH-dependent
oxidoreductase that activates glucocorticoid by converting biologically inactive 11keto
glucocorticoids, cortisone (in the human) or 11-dehydrocorticosterone (11DHC, in the rat) into
the active 11-hydroxyl glucocorticoids (cortisol or CORT). In the contrast, 11-HSD2 is an
NAD+-dependent unidirectional enzyme that does the opposite reaction. 11-HSD2 is a critical
enzyme that acts to prevent cortisol from stimulating the mineralocorticoid receptor, thus
preventing from apparent mineralocorticoid excess (Figure 1).3, 4
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Recently, 11β-HSD1 reductase inhibition has gained attention as a potentially effective
method for treating the metabolic syndrome, including type 2 diabetes.5 11β-HSD1 knockout
mice are resistant to diet-induced obesity and glucose intolerance, and the 11β-HSD1 over-
expression in fat and liver tissues causes the metabolic syndrome.6, 7 Selectivity against the 11-
HSD2 is expected to be critical as deficiency of the enzyme leads to apparent mineralocorticoid
excess caused by the occupancy of mineralocorticoid receptor by cortisol.4 Therefore, many
classes of 11-HSD1 inhibitors without any effects on 11-HSD2 have been reported 8-10. In the
present study, we report such a new class of inhibitors.
Curcumin (CUR) was identified as an initial hit by high-throughput screening. CUR had
significant level of potency on the human 11-HSD1 (IC50= 4501 nM, Figure 2), when the
enzyme activity was measured in intact CHOP cells transfected with human 11-HSD1 cDNA
(HSD11B1). CUR showed an equivalent selectivity of inhibiting human liver microsomal 11-
HSD1 (IC50 = 15,983 nM) and human kidney microsomal 11-HSD2 (IC50 = 15,696 nM).
This compound is attractive, because it is a natural product with low toxicity.11 However,
CUR has several disadvantages in pharmacokinetics such as structural instability, poor
bioavailability and fast metabolism, which limit its applications.11 Due to the simplicity of the
scaffold, a large collection of mono-carbonyl curcumin analogues with better pharmacokinetics
profiles and improved structural stability have been synthesized (scheme 1).12-14 These mono-
carbonyl curcumin analogues include cyclopentanones (A), 1,5-diaryl-1,4-pentadiene-3-ones (B)
and cyclohexanones (C). These compounds were synthesized to examine the role of different
substituents on the benzene ring and their influence on inhibitory potencies of 11-HSD1
without affecting 11-HSD2.
The synthesis and spectral properties of compounds A01-A17, B01-B17 and C01-C17 were
reported in our previous papers.12, 15 General procedure for synthesizing compounds A01-C17 is
described in the following section.
An amount of 7.5 mmol acetone (B-class), cyclopentanone (A-class), or cyclohexanone (C-
class) was added to a solution of 15 mmol arylaldehyde in methanol (10 ml). The solution was
stirred at room temperature for 20 min, followed by adding NaOCH3/CH3OH (1.5 ml, 7.5
mmol). The mixture was stirred at room temperature and monitored with thin layer
chromatography. When the reaction was finished, the residue was poured into saturated NH4Cl
solution and filtered. The precipitate was washed with water and cold ethanol, and dried in
vacuum. The solid was purified by chromatography over silica gel using CH2Cl2/CH3OH as the

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eluent to yield compounds. Details of the reaction routes, yields, melting points, NMR and
electrospray ionization mass spectrometry (ESI-MS) analysis were reported as described.12, 15
The synthesized analogues were evaluated for the inhibition of human and rat 11β-HSD1
reductase activities in either microsome (human liver or rat testis) or intact cell (CHOP cells
transfected with human HSD11B1 or intact isolated rat Leydig cell).
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11β-HSD1 reductase assay in human liver and rat testis microsomes was performed as
described.16 Briefly, 11β-HSD1 activity assay tubes contained 25 nM substrate 11DHC (for rat)
or cortisone (for human), spiked with 60,000 dpm of their respective 3H-steriods. 11DHC or
cortisone was used as the substrate to measure 11β-HSD1 activity. 25 nM of steroid substrate
was used because this concentration was within the physiological concentration range. Human
liver (4 µg) or rat testis (10 µg) microsomes were incubated with 11keto steroids, 0.2 mM
NADPH and various concentrations (10-10-10-5 M) of curcumin analogues at 37C for 60-90 mins.
The inhibitory potency of curcumin analogues was measured relative to control (DMSO solvent).
Curcumin analogues were dissolved in DMSO with final concentration of 0.4%, at this
concentration DMSO did not inhibit enzyme activity. At the end of the reaction, the reaction was
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stopped by adding 10 µl of 1 mM glycyrrhentic acid (that terminates the reaction ) and 1 ml
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ice-cold ether. The steroids were extracted by ether, and the organic layer was dried under
nitrogen. 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.16 The percentage
conversion of cortisone to cortisol or 11DHC to CORT was calculated by dividing the
radioactive counts identified as 11-OH-steroids by the total counts. The liver or testis were
selected because of the abundance of expression of 11-HSD1 in these tissues.18
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The 11-HSD1 activity was also measured in intact cells, in which no exogenous NADPH
was added as described.16 Adult rat Leydig cells were isolated from 90-day-old Sprague Dawley
rats as described in our previous study.19 The human 11-HSD1 activity was achieved by
transfecting human HSD11B1 into the CHOP cell line as described.20 0.01 × 106 CHOP cells or
0.025 × 106 rat Leydig cells were used for the cell-based assays.
Human and rat kidney microsomes were used to measure 11β-HSD2 as described.21 In brief,
11-HSD2 activity assay tubes contained 25 nM substrate (cortisol for human and CORT for rat).
Kidney microsome was incubated with substrate and 0.2 mM NAD+ for 30 min. The percentage
conversion of cortisol to cortisone (human) or CORT to 11DHC (rat) was calculated by dividing
the radioactive counts identified as 11keto steroid product by the total counts associated with
both substrate and product.
The inhibitory potencies of chemicals on 11β-HSD1 reductase in intact cell preparations were
included in Table 1, and those values of inhibiting 11β-HSD1 and 11-HSD2 activities of
microsomes were listed in Table 2.
The results indicate that curcumin analogues inhibited 11-HSD1 activity to various degrees
as judged by the results from intact CHOP cells transfected with human HSD11B1. Among these
compounds, A02, A06, A10, B02, B06, B13, B14, C02, C06 and C13 are potent inhibitors
(Table 1). Halogenation of benzene ring such as 4-fluorobenzyl (A02, B02 and C02), 2-
bromobenzyl (A06, B06 and C06) and 3-bromobenzyl (A10) analogues improved 9-46 fold in
potency of inhibiting human 11-HSD1 in intact cells compared to CUR (Table 1).
Hydroxylation of benzene ring at 4’-position for B and C groups such as 4-hydroxybenzyl (B13
and C13) and 3-methoxy-4-hydroxybenzyl (B14) analogues also showed 5-23 fold increases of
potency compared to CUR. Apparently, this hydroxylation at 4’-position for A-group

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compounds even lowered the inhibitory potency (Table 1). For example, 4-hydroxybenzyl (A13)
and 3-methoxy-4-hydroxybenzyl (A14) at maximum concentration (100 µM) only inhibited
human 11-HSD1 of intact cells by 49% and 12%, respectively (Table 1). Interestingly, the
halogenated curcumin analogues showed greater selectivity for 11-HSD1 because they almost
did not inhibit human and rat 11-HSD2 activities at 100 µM (Table 2). When B06 was selected
for the mode of inhibition of 11-HSD1, it was found that B06 was a competitive inhibitor
against substrate cortisone (Figure 3).
Although the synthesis of several analogues has been reported previously,12, 15, 22, 23 the
inhibition on 11-HSD1 and 11-HSD2 have not been explored. Interestingly, some of these
analogues have been examined for the inhibition on another hydroxysteroid dehydrogenase, 17-
hydroxysteroid dehydrogenase isoform 3 and data showed that there were completely different
structure activity responses with potent inhibition of this enzyme by C03 (IC50 = 100 nM), which
did not inhibit human 11-HSD1 and 11-HSD2 activities at all.23 Furthermore, these
compounds have been showed to have different anti-inflammatory properties with SAR
difference from the inhibition of 11β-HSD1 (Table 3).12, 14, 15, 22, 23 Liang et al. has reported that
some these compounds showed cytotoxic effects on several cell lines including HeLa cell (Table
3).14 According to the described method by Liang et al14, we examined the cytotoxic effects of
compound B06 and C06 on CHOP cells and rat Leydig cells, and we did not find any cytotoxic
effects of these compounds (data not shown). This indicates that these curcumin analogues
showed selectivity of inhibition on 11-HSD1 activity.
In summary, three series of mono-carbonyl analogues of curcumin were synthesized. Their
structures were identified by NMR analysis and the inhibitory potencies of curcumin analogues
on human and rat 11-HSD1 in intact cells and microsomes were examined. The selectivity
against 11-HSD2 was compared. SAR studies of these chemicals have resulted in the
identification of novel potent 11β-HSD1 inhibitors A02, A06, A10, B02, B06, B13, B14, C02,
C06 and C13 with IC50 values between 97-830 nM. These compounds displayed selectivity for
11β-HSD1 compared to human and rat 11β-HSD2.
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Acknowledgment
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We thank Dr. White PC from Baylor Department of Pediatrics, University of Texas
Southwestern Medical Center, Dallas, Texas provided us with human HSD11B1 vector. This
work was supported in part by the Wenzhou Science & Technology Funding (Y20090003 to
R.S.G.; Y20090009 to G.L.), NSFC (81070329 to Y.H.C.).
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F.; Li, Y.; Zhou, H. Bioorg Med Chem Lett 2008, 18, 1525.
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Legends
Figure 1. The catalysis of 11-HSD1 and 11-HSD2 in the human.
Figure 2. Initial hit curcumin discovered by high-throughput screening ( HTS).
Scheme 1. Reagents and conditions: cyclopentanone (A), acetone (B), or cyclohexanone (C),
NaOH/EtOH, rt.
Figure3. The mode of B06 on 11-HSD1 in human liver microsome. Lineweaver–Burk plotting
in presence of substrate cortisone shows the competitive inhibition.

Table 1. Structure and the inhibition of compounds (Com.) on 11β-HSD1 in intact cells.
R2 group
Rat
Human
% inhibition a
Com.
CUR
A01
% inhibition a
IC50 (nM) b
IC50 (nM)b
-
-
3,921
-
-
4501
-
4-ethanoxy
27%
14%
4-fluoro
-
18%
13%
-
22%
22%
-
3,469
-
-
1,448
-
-
1,147
-
4,171
-
-
-
-
9945
229
-
-
129
17,239
52,476
986
292
-
-
-
1,111
-
-
148
-
-
310
-
-
144
-
-
97
-
-
-
-
-
-
-
507
-
-
115
-
-
832
257
-
-
A02
A03
A04
A06
A07
A08
A10
A11
A12
A13
A14
A16
A17
B01
B02
B03
B04
B06
B07
B11
B13
B14
B16
B17
C01
C02
C03
C04
C06
C07
C11
C13
C16
C17
3,4,5-trimethoxy
4-dimethylamino
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
2-fluoro-3-trifluoromethyl
3-bromo
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-3-dimethyl aminopropoxy
4-hydroxy
4-hydroxy-3-methoxy
4-allyloxy
4-allyloxy-3-methoxy
4-ethanoxy
4-fluoro
3,4,5-trimethoxy
4-dimethylamino
31%
16%
-
9%
24%
-
26%
49%
12%
17%
23%
31%
9%
-
40%
29%
-
54%
28%
-
18%
-
21%
19%
10%
17%
-
-
5%
10%
-
-
-
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-hydroxy
4-hydroxy-3-methoxy
4-allyloxy
4-allyloxy-3-methoxy
4-ethanoxy
4-fluoro
3,4,5-trimethoxy
4-dimethylamino
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-hydroxy
-
-
-
22%
18%
26%
-
42%
37%
-
28%
8%
-
8%
6%
4%
1%
16%
-
14%
26%
-
28%
40%
-
18%
16%
-
263
-
-
346
-
-
195
-
-
633
4-allyloxy
4-allyloxy-3-methoxy
-
-
-
a% inhibition at the maximum concentration of each compound used; IC50 value for inhibitory potency; All
b
potency data are reported as the mean of 2-3 determinations.

Table 2. Structure and the inhibition of compounds (Com.) on 11β-HSD1 and 11-HSD2 in
microsomes.
R2 group
11-HSD1 at 100µM
11-HSD2 at 100µM
Rat Human
%inhibition %inhibition
Rat
Human
Com
%inhibitiona
IC50
(nM) b
4,125
%inhibitiona
IC50
(nM) b
15983
-
-
0
-
(14,939) c
10%
CUR
A01
(15,696) c
4-ethanoxy
-
14%
-
6%
4-fluoro
3,4,5-trimethoxy
-
21,987
-
17,393
9%
9%
1%
22%
13%
5%
26%
5%
0
36%
0
0
23%
0
24%
0
13%
0
10%
8%
26%
1%
0
53%
13%
0
44%
39%
2%
13%
11%
24%
13%
21%
33%
17%
6%
0
A02
A03
A04
A06
A07
A08
A10
A11
A12
A13
A14
A16
A17
B01
B02
B03
B04
B06
B07
B11
B13
B14
B16
B17
C01
C02
C03
C04
C06
C07
C11
C13
C16
C17
12%
0
-
11%
2%
-
13%
2%
32%
52%
9%
14%
7%
2%
0
-
-
16%
15%
-
30%
23%
-
26%
15%
39%
49%
28%
13%
36%
37%
16%
24%
-
-
-
4-dimethylamino
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
2-fluoro-3-trifluoromethyl
3-bromo
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-3-dimethyl aminopropoxy
4-hydroxy
4-hydroxy-3-methoxy
4-allyloxy
4-allyloxy-3-methoxy
4-ethanoxy
4-fluoro
3,4,5-trimethoxy
4-dimethylamino
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-hydroxy
4-hydroxy-3-methoxy
4-allyloxy
4-allyloxy-3-methoxy
4-ethanoxy
4-fluoro
3,4,5-trimethoxy
4-dimethylamino
2-bromo
4-tetrahydro-2H-pyran-2-yloxy
3-methoxy-4-tetrahydro-2H-pyran-2-yloxy
4-hydroxy
2,882
-
-
599
-
-
-
-
-
-
-
-
-
-
6,109
-
-
3,512
-
-
-
-
-
-
-
-
-
3%
11%
12%
17%
34%
15%
0
27%
22%
11%
13%
39%
24%
16%
7%
35%
16%
19%
14%
16%
23%
20%
39%
48%
14%
24%
23%
8%
0
-
13%
19%
41%
-
-
529
-
288
-
-
-
36%
28%
-
-
4,423
70,901
16,077
-
0
-
-
-
35%
14%
17%
-
21%
14%
9%
17%
-
35%
16%
-
45%
-
63%
19%
15%
-
13,502
2,973
-
-
1,111
-
-
1,944
-
-
-
-
218
-
14%
37%
-
18%
7%
65,430
-
-
-
4-allyloxy
4-allyloxy-3-methoxy
6%
6%
a% inhibition at the maximum concentration of each compound used; bIC50 value for inhibitory potency; c() shows
the IC50 values (nM) for inhibitory potency of curcumin on 11-HSD2; All potency data are reported as the mean of
at least two determinations.

Table 3. Structure and various activities of compounds (Com.)
Com Inhibition of TNF-
Cytotoxic to
HeLa
Inhibition on human enzyme at 100µM
α Formation at 10
17β-HSD3
11-HSD1
11-HSD2
μM
Ref.12
Ref.14
This paper
Ref.23
(67.3µM) c
NIa
<50%
NI
NIa
(17.5µM)c
(16.0µM) c
<50% b
(17.4µM)
<50%
(15.7µM) c
<50% b
<50%
<50%
NI
CUR
A01
<50%b
NI
NI
NI
(24.0 µM)
(1.2 µM)
A02
A03
A04
A06
A07
A08
A10
A11
A12
A13
A14
A16
A17
B01
B02
B03
B04
B06
B07
B11
B13
B14
B16
B17
C01
C02
C03
C04
C06
C07
C11
C13
C16
C17
-
-
<50%
NI
-
NI
-
-
NI
-
-
-
-
-
-
NI
-
(6.1µM)
<50%
<50%
(3.5µM)
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
(0.5µM)
<50%
<50%
(4.4µM)
(70.9µM)
<50%
<50%
<50%
(3.0µM)
<50%
<50%
(1.1µM)
<50%
<50%
(1.9µM)
<50%
<50%
<50%
NI
NI
<50%
NI
<50%
NI
<50%
NI
NI
<50%
<50%
<50%
NI
<50%
<50%
NI
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
NI
<50%
NI
NI
NI
NI
<50%
<50%
-
(39.1µM)
-
-
-
-
-
-
-
-
-
-
-
NI
(95.0µM)
<50%
<50%
NI
(2.4µM)
(2.7µM)
NI
<50%
<50%
-
NI
NI
-
>50% b
(34.2µM)
NI
-
-
-
-
-
-
NI
-
-
(217.0µM)
NI
-
-
NI
-
-
NI
-
<50%
(0.1µM)
<50%
<50%
NI
<50%
<50%
-
NI
(27.5µM)
NI
-
NI
-
-
>50% b
NI
-
-
-
-
-
(27.5µM)
-
<50%
<50%
-
NI=no inhibition; b% inhibition at the maximum concentration of each compound used;
c() shows the IC50 values for inhibitory potency.

Figure 1

Figure 2
O
O
H O
O H
O
O
IC50 = 4501 nM

Scheme 1
A
B
Acetone
C

Figure 3
1.0
0.8
0.6
0.4
0.2
B6 0 μM
B6 1 μM
B6 5 μM
B6 10 μM
0
10
20
30
40
1/[Cortisone]

Legends
Figure 1. The catalysis of 11 -HSD1 and 11 -HSD2 in the human.
Figure 2. Initial hit curcumin discovered by HTS.
Scheme 1. Reagents and conditions: cyclopentanone (A), acetone (B), or cyclohexanone (C),
NaOH/EtOH, rt.
Figure3. The mode of B06 on 11 -HSD1 in human liver microsome. Lineweaver–Burk plotting
in presence of substrate cortisone shows the competitive inhibition.

+ Cyclopentanone
A10
IC50=97 nM 11β-HSD1
IC50>100 µM 11β-HSD2