Synthesis, characterization and biological studies of diosgenyl analogues

Article abstract of DOI:10.1016/j.bmcl.2012.10.086

A series of optical amino acid diosgenyl esters and diosgenyl salicylate conjugates were designed and synthesized to develop new anticancer and anti-inflammatory agents. The analogue 9c that contains an 6-aminohexanoic acid residue at C-3 of diosgenin exhibits higher potency against all three tumor cell lines with IC₅₀ values ranging from 4.7 μM in C26 cells to 14.6 μM in Hep G2 cells. In addition, seven of newly synthesized compounds significantly inhibit xylene-induced ear edema and exhibit comparable or better anti-inflammatory activities than those of diosgenin and aspirin. Furthermore, preliminary structure-activity relationship studies demonstrate that diosgenyl salicylate conjugates have stronger anti-inflammatory activities than amino acid diosgenyl esters.

Full text of DOI:10.1016/j.bmcl.2012.10.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7330–7334
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization and biological studies of diosgenyl analogues
⇑
⇑
Baozhan Huang , Dan Du , Rui Zhang, Xiaohua Wu, Zhihua Xing, Yang He , Wen Huang
Institute for Nanobiomedical Technology and Membrane Biology, and Laboratory of Ethnopharmacology, Regenerative Medicine Research Center,
West China Hospital/West China Medical School, Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of optical amino acid diosgenyl esters and diosgenyl salicylate conjugates were designed and
synthesized to develop new anticancer and anti-inflammatory agents. The analogue 9c that contains
an 6-aminohexanoic acid residue at C-3 of diosgenin exhibits higher potency against all three tumor cell
Received 13 August 2012
Revised 14 October 2012
Accepted 17 October 2012
Available online 23 October 2012
lines with IC50 values ranging from 4.7 lM in C26 cells to 14.6 lM in Hep G2 cells. In addition, seven of
newly synthesized compounds significantly inhibit xylene-induced ear edema and exhibit comparable or
better anti-inflammatory activities than those of diosgenin and aspirin. Furthermore, preliminary struc-
ture–activity relationship studies demonstrate that diosgenyl salicylate conjugates have stronger anti-
inflammatory activities than amino acid diosgenyl esters.
Keywords:
Diosgenin-salicylate
Asprin
Ó 2012 Elsevier Ltd. All rights reserved.
Amino acid
Anti-tumor
Anti-inflammatory
Diosgenin (3b-hydroxy-5-spirostene, Fig. 1), which is a major
steroidal sapogenin that is extracted from Dioscorea zingiberensis
C.H. Wright, is a main precursor for the synthesis of sex hormones,
corticosteroids, and other steroid products.1 Recent studies indi-
cate that diosgenin may be an active constituent that contributes
to a variety of biological activities, for example, by exhibiting
anti-inflammatory2 and anti-thrombosis effects,3 inducing megak-
aryocytic differentiation in human erythroleukemia cells,4 enhanc-
ing regulatory T-cell immunity,5 and inducing apoptosis in DU-145
prostate cancer cells6 and HCT-116 human colon carcinoma cells.7
In recent years, diosgenyl esters have received considerable atten-
tion due to their favorable anticancer properties.8 Kvasnica9 de-
signed and synthesized twelve steroidal platinum (II) complexes
with steroidal esters (i.e., cholesterol, diosgenin, and cholestanol)
controls.11 Subsequent studies have shown that long-term aspirin
use aids in the prevention of vascular events and may reduce the
risk of cancer, and it potentially leads to a reduced death rate from
cancer.12–15 Since the relationship between inflammation and
carcinogenesis has been examined in so many case-controlled
studies,16 the synthesis of new complexes containing anti-
inflammatory drugs such as aspirin or salicylic acid is a strategy
that is employed for the development of potential anti-tumor
drugs.17 However, long-term administration of aspirin or high
doses of aspirin could block the production of prostaglandin E2;
this result in the risk of aspirin-induced gastrointestinal bleeding
increases with age and dose.18 A variety of approaches have been
investigated to avoid aspirin-induced gastrointestinal effects
including decreasing the acidity and increasing the pharmacologi-
cal activity of the drugs, which can be achieved by preparing amino
acid salts and ester derivatives of aspirin.19
In recent years, our group has focused on studying the extrac-
tion and applications of Dioscorea zingiberensis C.H. Wright,20,21
the acute toxicity and sub-chronic toxicity of diosgenyl saponins,22
and preliminary structure–activity relationships of steroidal sapo-
nins.23 In addition, we have reported the anti-thrombosis effects of
diosgenin,3 the anticancer activity and anti-angiogenesis of delto-
nin,24,25 the protective effects of diosgenin in human vascular
endothelial cells against hydrogen peroxide-induced apoptosis,26
etc. Clinical studies are underway and preliminary results indicate
that NSAID/aspirin and steroidal sapogenin/diosgenin share some
similar pharmacological activities, including anti-inflammatory
and anti-thrombosis effects as well as anticancer properties.
Herein, we report the synthesis of diosgenyl esters coordinated
to aspirin and diosgenin via a series of amino acids, including
of
significant activities against T-lymphoblastic leukemia CEM cell
line with IC50 values in the range of 14–25 M.
L-histidine and L-methionine; several of the compounds showed
l
Aspirin, which is a synthetic antipyretic, analgesic, and anti-
inflammatory agent, has been known for over 110 years. Today, it
is still one of the most widely consumed nonsteroidal anti-
inflammatory drugs (NSAIDs) in the world. Inflammation is a
symptom of various common diseases, and it can be an early phase
of serious diseases such as cancer.10 In 1988, Kune and his
collaborators reported a 50% reduction in the risk of colorectal
cancer in patients who regularly took aspirin, compared with
⇑
Corresponding authors. Tel.: +86 28 85164076; fax: +86 28 85164073.
E-mail addresses: heyangqx@yahoo.com.cn (Y. He), huangwen@scu.edu.cn
(W. Huang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.086

B. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7330–7334
7331
Scheme 2. Reagents and conditions: (Boc)2O, Et3N, CH3COCH3:H2O (v:v, 1:1), rt,
3 h, 98%.
Figure 1. Structures of diosgenin (1), aspirin (2), and salicylic acid (3).
chloride in the presence of triethylamine afforded 6d, 6e, 7e, 8d
and 9e in good yields. However, the target products 6e, 7e, and
9e by losing of the acetyl group at 2-acetoxybenzoyl part were
respectively obtained after their flash chromatography on silica
gel (Schemes 3–5). The synthesis of diosgenyl salicylate compound
from 8d which features a glycine bridge was unsuccessful. This fact
is very interrogative. The loss of acetyl group was probably due to
mildly basic details made by excess of Et3N and hydrous condition
of post-processing reaction, the phenolic acetyl group was hydro-
lyzed. As steric hindrance of diosgenyl salicylate compounds de-
creased, occurrence of hydrolysis reaction increased. Therefore,
longer amino acid bridging chains might lead to easier lose of acet-
yl group of the 2-acetoxybenzoyl group. The structures of com-
pounds 6e, 7e, and 9e were confirmed by 1H NMR, 13C NMR, and
HR-MS spectral data.
In this work, a series of diosgenyl derivatives were synthesized
and the cytotoxic activities of these compounds were evaluated
through a MTT assay24,25 in the following tumor cell lines: human
breast cancer MDA-MB-231 cells, murine colon carcinoma C26
cells, and human hepatocellular carcinoma Hep G2 cells. The IC50
values of deltonin24,25 were also included in the study for compar-
ison. The data presented in Table 1 show that compound 9c dem-
onstrates significantly higher inhibition against all three cancer cell
lines than either aspirin or diosgenin. Also, 9c demonstrates stron-
ger activity than 6c, 7c, and 8c, and has a better activity than its
salicylate conjugates 9e.
glycine, alanine, glutamic acid, and 6-aminohexanoic acid with the
aim of increasing the anticancer activities and anti-inflammatory
properties of these compounds and eliminating the gastrointesti-
nal side effects of aspirin. These complexes were also investigated
to elucidate how the amino acids affect the cytotoxic activity of the
compounds against human breast cancer MDA-MB-231 cells, mur-
ine colon carcinoma C26 cells, and human hepatocellular carci-
noma Hep G2 cells. Further, preliminary structure–activity
relationships of these compounds were analyzed via their anti-
inflammatory activities using a xylene-induced ear edema model.
In this present study, a series of diosgenyl derivatives, including
four amino acid diosgenyl esters (6c–9c) and six diosgenyl
salicylate compounds (5, 6d, 6e, 7e, 8d and 9e), were synthesized.
The pyridine-promoted esterification of diosgenin and 2-
acetoxybenzoyl chloride27 mainly provided the desired compound
5 (Scheme 1). With increased reaction time or temperature, more
diosgenyl acetate 5a was generated and the yield of compound 5
decreased from 65% to 40%.
N-tert-Butyloxycarbonates are stable toward a variety of exper-
imental conditions and easily convert to the corresponding
primary amines under mildly acidic conditions. In this study, all
N-protected amino acids used were commercially obtained except
for compound 9a, which was synthesized via the reaction of 6-
aminohexanoic acid with (Boc)2O in an acetone–water mixed sol-
vent in the presence of triethylamine (Scheme 2).28 Unfortunately,
the target compound 9a could not be obtained using the method
reported by Chankeshwara without basic catalytic compounds.29
Therefore, the alkalinity provided by triethylamine (Et3N) is impor-
tant for N-tert-butyloxycarbonylation.
Evidence from a wide range of epidemiological studies suggests
that long-term aspirin or related NSAIDs use is associated with a
30–50% reduction in the risk of cancer.12–15 However, aspirin and
the salicylate conjugates (6e, 7e, 8d, and 9e) did not have a signif-
icant effect against any of the three tumor cell lines after 48 h of
Amino acid diosgenyl esters 6c–9c, were prepared by an
esterification reaction involving DCC/DMAP, the corresponding
N-tert-butoxycarbonyl amino acids, and diosgenin in dry dichloro-
methane followed by deprotection of the N-tert-butoxycarbonyl
group using trifluoroacetic acid at room temperature. Meanwhile,
didiosgenyl ester 7c was obtained when N-(tert-butoxycarbonyl)-
incubation (i.e., the IC50 values were greater than 50 lM). Only
amino acid diosgenyl esters 8c and 9c showed significant tumor
cell growth inhibition. It is possible that the introduction of various
amino acid residues to the diosgenyl has a different impact on
cytotoxic activities. 6-Aminohexanoic acid (9), which is an analog
of the amino acid lysine, is used to treat excessive post-operative
bleeding, and block the binding of plasminogen to fibrin and its
activation to plasmin.30,31 Compound 9c, which contains an
D
-glutamic acid was used as the reagent.
The diosgenyl salicylate compounds were synthesized. The cou-
pling of amino acid diosgenyl esters 6c–9c with 2-acetoxybenzoyl
Scheme 1. Reagents and conditions: (a) SOCl2, DMF, 70–75 °C, 3 h; (b) Py, CH2Cl2, 0 °C, 1 h, 65%.

7332
B. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7330–7334
Scheme 3. Reagents and conditions: (a) DMAP, TsOH, DCC, CH2Cl2, rt, 94%; (b) CF3COOH, CH2Cl2, rt, 90%; (c) Et3N, CH2Cl2, 0 °C, flash chromatography, 57% for 6d and 17% for
6e.
Scheme 4. Reagents and conditions: (a) DMAP, TsOH, DCC, CH2Cl2, rt, 92%; (b) CF3COOH, CH2Cl2, rt, 86%; (c) Et3N, CH2Cl2, 0 °C, flash chromatography, 93%.
Scheme 5. Reagents and conditions: (a) DMAP, TsOH, DCC, CH2Cl2, rt; (b) CF3COOH, CH2Cl2, rt; (c) Et3N, CH2Cl2, 0 °C, flash chromatography, 73% for 8d and 82% for 9e.
aminocaproic acid residue, shows a strong activity against the pro-
liferation of MDA-MB-231 (IC50 7.6 M) and C26 (IC50 4.7 M). This
result suggests that the aminohexanoic residue is optimal for the
anti-tumor effect. In summary, 6-aminohexanoic acid diosgenyl
ester (9c) is a potential new anticancer drug.
The anti-inflammatory activities of the diosgenyl esters were
evaluated using a xylene-induced ear edema model assay.32–34
The results shown in Table 2 reveal that diosgenin (at a dose of
100 mg/kg, inhibition 32.2%) has an anti-inflammatory activity
that is comparable to that of the standard reference drug, aspirin
l
l

B. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7330–7334
7333
Table 1
Cytotoxic activity (IC50
However, no significant effects were found for diosgenyl salicylate
conjugates against any of the three tumor cell lines investigated.
Meanwhile, structure–activity relationship studies indicate that
compound 9c and all salicylate conjugates exhibit desirable anti-
inflammatory activity compared to aspirin and diosgenin. The
incorporation of an aminocaproic residue with diosgenin is optimal
for the anti-tumor and anti-inflammatory activities.
Further investigations on the synthesis and biological behavior
of diosgenyl esters with long, straight amino acid chains to eluci-
date their anticancer effects and anti-inflammatory properties are
forthcoming.
,
l
M) of synthetic diosgenyl estersa,b
Cancer cell line
Compound
MDA-MB-231
C26
Hep G2
Aspirin
Diosgenin
5
6c
6e
7c
7e
8c
8d
9c
9e
Deltonin
1510
>50
>50
>50
>50
>50
>50
48.2
>50
7.6
1740
>50
>50
>50
>50
>50
>50
>50
>50
4.7
3841
>50
>50
>50
>50
>50
>50
>50
>50
14.6
>50
7.66
Acknowledgments
>50
>50
1.5824
1.2225
This research was supported by grants from the China National
‘12.5’ Foundation (No. 2011BAJ07B04), National Natural Science
Foundation of China (No. 20972105, No.30900493) and Sichuan
Province Foundation (No. 2008SZ0024).
a
IC50 is the concentration in
lM that inhibits cell growth by 50% in 48 h com-
pared to cells that remained untreated.
b
Means obtained from a minimum of n = 3 independent MTT assays for each
compound.
Supplementary data
Table 2
Anti-inflammatory activities of diosgenyl esters against xylene-induced ear edema in
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.086. These data include MOL files and InChiKeys of the most
important compounds described in this article.
micea,b
Agents
Edema weight (X SD mg)
10.73 3.01
Inhibition (%)
CMC
Aspirin
Diosgenin
5
6c
6e
7c
7e
8c
8d
9c
9e
d
7.22 2.50
7.18 2.57
5.67 2.23
7.57 2.75
6.81 2.53
8.58 2.18
31.9
32.2
46.5
28.6
35.8
19.1
30.1
30.6
39.1
38.6
42.6
c
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d
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