Synthesis of diosgenyl quaternary ammonium derivatives and their antitumor activity

Article abstract of DOI:10.1016/j.steroids.2020.108774

Giosgenin is a naturally steroidal saponin exhibiting a variety of biological activities including antitumor ones. A series of novel diosgenyl quaternary ammonium derivatives were designed and synthesized to develop potential anti-tumor agents in our research. All novel derivatives were characterized by ¹H NMR, ¹³C NMR and HR-MS, and evaluated for their in vitro anti-proliferative activities using MTT assay. The human cancer cell lines were A549 (Human lung cancer cell), H1975 (Human lung adenocarcinoma cell), A431 (Human skin squamous cell carcinoma), HCT-116 (Human colorectal adenocarcinoma cell), Aspc-1 (Human metastatic pancreatic cancer cell), Ramos (Human B lymphoma cell), HBE (Human bronchial epithelioid cell) and LO2 (Human normal hepatocyte).

Full text of DOI:10.1016/j.steroids.2020.108774

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Synthesis of Diosgenyl Quaternary Ammonium Derivatives and their antitu‐
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Xi Xia, Yu Chen, Lin Wang, Zhi-Gang Yang, Xiao-Dong Ma, Zhi-Gang
Zhao, Hong-Jun Yang
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Please cite this article as: Xia, X., Chen, Y., Wang, L., Yang, Z-G., Ma, X-D., Zhao, Z-G., Yang, H-J., Synthesis
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Synthesis of Diosgenyl Quaternary Ammonium Derivatives
and their antitumor activity
Xi Xia1, Yu Chen1, Lin Wang1, Zhi-Gang Yang1, Xiao-Dong Ma2, Zhi-Gang Zhao1,
Hong-Jun Yang,1，
1 Key Laboratory of Basic Chemistry of the State Ethnic Affairs Commission, School
of Chemistry and Environment, Southwest Minzu University, Chengdu 610041, P. R.
of China
2 College of Pharmacy, Dalian Medical University, Dalian 116044
Graphical Abstract
O
O
O
O
O
O
O
R1
R3
X
R1
R2
X
N
N
R2
O
R3
n
HO
Ⅰ
diosgenin
Ⅱ
Abstract
Giosgenin is a naturally steroidal saponin exhibiting a variety of biological activities
including antitumor ones. A series of novel diosgenyl quaternary ammonium
derivatives were designed and synthesized to develop potential anti-tumor agents in our
research. All novel derivatives were characterized by 1H NMR, 13C NMR and HR-MS,
and evaluated for their in vitro anti-proliferative activities using MTT assay. The human
cancer cell lines were A549 (Human lung cancer cell), H1975 (Human lung
adenocarcinoma cell), A431 (Human skin squamous cell carcinoma), HCT-116
(Human colorectal adenocarcinoma cell), Aspc-1 (Human metastatic pancreatic cancer
cell), Ramos (Human B lymphoma cell), HBE (Human bronchial epithelioid cell) and
LO2 (Human normal hepatocyte).
Key words: Diosgenin; Antitumor activities; Derivatives; Quaternary ammonium
derivatives

Corresponding author at: Key Laboratory of Basic Chemistry of the State Ethnic
Affairs Commission, School of Chemistry and Environment, Southwest Minzu
University, Chengdu, China
E-mail address: yanghj@swun.edu.cn (Hong-Jun Yang).
1

1. Introduction
Malignant tumor, known as cancer, is one of the most dangerous diseases
endangering human health [1]. Now, the discover of the antineoplastic drugs form
natural plants has been accredited with a great amount of attention over the years by
medicinal chemists. Besides, restudy on the efficacious components form plants and
structural modification play an important role in field of anti-tumor drugs.
Giosgenin is a naturally steroidal saponin, it can be found in several plants species
including dioscorea, yams, smilax, costus, fenugreek, et al [2, 3]. Giosgenin is widely
used as a precursor to synthetic steroids [4], such as prednisone, norethindrone,
dexamethasone and so on. A large number of studies have shown that diosgenin has a
wide range of anti-tumor activities on a variety of tumor cells [2, 5-9], and it can
effectively plays its role by inhibiting cell proliferation and inducing apoptosis [10],
and blocking cell cycle [11, 12]. In addition, diosgenin exhibits anti-inflammatory [13,
14], anti-photoaging [3], anti-cardiovascular disease [15, 16] and the like [17]. However,
the use of these compounds was always limited by the high toxicity, poor water-soluble
and low bioavailability. A number of studies were attempted by structural modification
to improve utilization of diosgenin, and many successful examples have been reported
[5, 18-22]. Therefore, the design of novel families of giosgenin derivatives to
ameliorate their severe side effect is a pivotal task.
It is found that the introduction of nitrogen atoms into the structure of drugs or
modification of existing nitrogen atoms to make them into quaternary ammonium
derivatives can improve its water-soluble and enhance the biological activity [23-27].
In the reported derivatives of diosgenin, the derivatives modified with triazolium salt
or diazolium salt in C-3 position have good antitumor activity [28-31]. Therefore, in
order to improve the water-soluble of diosgenin and increase its antitumor activity, two
series of quaternary ammonium derivatives were designed and synthesized with
diosgenin as the lead compound, and their antitumor activity was tested in vitro.
2. Results and Discussion
2.1 Synthesis
2.1.1 Synthesis of quaternary ammonium series I derivatives of diosgenin [32, 33]
2

Series I of quaternary ammonium derivatives 2.5a-2.5k were prepared using the
sequences shown in Schemes 1. First, giosgenin as the lead compound was sulfonated
with methylsulfonyl chloride in the presence of trimethylamine to afford compound
2.1, which was reacted with sodium azide to provide compound 2.2. Then, the azidine
of compound 2.2 was reduced to amino by triphenylphosphine to obtain compound 2.3.
Finally, the compound 2.3 obtained through the 3-step reaction was used as the common
intermediate of the C3 derivatives of diosgenin, and substituted with different
halogenated hydrocarbons to afford the corresponding series of quaternary ammonium
derivatives 2.5a-2.5k.
O
O
O
O
O
O
a
b
2.1
2.2
HO
MsO
N3
Diosgenin
O
O
O
O
O
O
c
d
e
2.5b-2.5k
(
)
2.4b-2.4k
(
)
I
R1
R1
N
R2
N
H2N
2.3
2.4
2.5
Me
R2
e 2.5a
(
)
OH
R1=R2=
R1=R2=
R1=R2=
R1=R2=
2.5a
2.4h, 2.5h
2.4i, 2.5i
Me
O
2.4b, 2.5b
2.4c, 2.5c
N
R1=R2=
R1=R2=
O
2.4d, 2.5d
R1=R2=
2.4j, 2.5j
R1=R2=
R1=R2=
R1=R2=
2.4e, 2.5e
2.4f, 2.5f
2.4g, 2.5g
R1=
R1=
R2=
R2=
2.4k
2.5k
H
N
N
O
Me
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, THF, 0 °C, 1 h, yield 93%. (b)
NaN3, DMF, 90 °C, 7 h, yield 51%. (c) Ph3P, THF/H2O, 60 °C, 24 h, yield 60%. (d)
halohydrocarbon, K2CO3, CH3CN or CH3CN/CH2Cl2 (v/v=1:1), 40-80 °C, yield 63-
93%. (e) CH3I, (K2CO3), CH3CN/CH2Cl2 (v/v=1:1), reflux, yield 41-96%.
2.1.2 Synthesis of quaternary ammonium series II derivatives of diosgenin [34, 35]
Series II of quaternary ammonium derivatives 2.7a-2.7k were synthesized as
3

shown in Scheme 2. We designed to derive diosgenin into a steroidal halogenated
hydrocarbon and then react with different tertiary amines to obtain quaternary
ammonium derivatives. First, diosgenin, the lead compound, was condensed with 5-
bromo- pentanoic acid to provide intermediate 2.6. The compounds 2.7a-2.7k were
synthesized through 2.6 reaction with different tertiary amines.
O
O
O
O
O
O
g
f
O
O
Br
HO
Br
O
Y
O
Diosgenin
2.6
2
.
7
HO
N
N
2.7a
2.7e
2.7i
Y =
Y =
Y =
N
O
OH
HO
2.7b
2.7c
2.7f
2.7j
Y =
Y =
Y =
Y =
Y =
Y =
N
N
N
N
2.7g
2.7k
N
N
O
N
2.7d
2.7h
Y =
Y =
N
Scheme 2. Reagents and conditions: (f) DMAP, EDCHCl, 5-Bromovaleric acid,
CH2Cl2, r.t., 3 h, yield 79%. (g) Tertiary amine or Triphenylphosphine, CH3CN, reflux,
yield 35-90%.
2.2 Water solubility
Water solubility of all derivatives is shown in table 1.
The water solubility of the two series of diosgenin quaternary ammonium salt
derivatives 2.5a-2.5k, 2.7a-2.7l is superior to the lead diosgenin. In the quaternary
ammonium salt series I (2.5a-2.5k), the water solubility of 2.5h and 2.5k is the best,
about 300 times higher than the water solubility of the lead. In the quaternary
ammonium salt derivative series II (2.7a-2.7l), the water solubility of 2.7c is superior
to all synthetic derivatives and is about 3000 times higher than the lead.
Table 1 Water solubility of diosgenyl quaternary ammonium derivatives
Compound
Water-soluble*(mg/mL)
Compound
Water-soluble
4

(mg/mL)
0.0782
0.0999
0.2938
0.0613
0.0758
0.1004
0.0269
0.1992
0.1987
0.0399
0.1276
0.0046
Diosgenin
2.5a
2.5b
2.5c
0.0001
0.0021
0.0116
0.0235
0.0115
0.0170
0.0113
0.0108
0.0351
0.0074
0.0019
0.0351
2.7a
2.7b
2.7c
2.7d
2.7e
2.7f
2.7g
2.7h
2.7i
2.5d
2.5e
2.5f
2.5g
2.5h
2.5i
2.7j
2.7k
2.7l
2.5j
2.5k
* The above results are relative values of water solubility of each derivative.
2.3 Inhibitory activity
All diosgenyl quaternary ammonium derivatives were tested with MTT assay on
five kinds of tumor cells and two kinds of normal human cells, and the results were
shown in table 2 and table 3.
Antiproliferation activity of quaternary ammonium series I derivatives of
diosgenin is showed in table 2.
For tumor cells H1975, Aspc-1, HTC-116 and A549, the antitumor activity of
derivative 2.5a-2.5k was better than the lead compound. Among them, the inhibitory
activity of 2.5g on H1975 (IC50 = 5.09 µM) was comparable to that of Adriamycin
(IC50 < 5 µM). Compounds of 2.5b, 2.5d, 2.5f, 2.5g, 2.5i and 2.5j (IC50 was 4.06, 4.07,
< 5, 5.03, 4.78, < 5 µM, respectively) had comparable inhibitory activity to that of
Adriamycin against Aspc-1 cells (IC50 < 5 µM). The inhibitory activity of 2.5d, 2.5f
and 2.5j on HTC-116 (IC50 = 1.12, 1.03, 0.34 µM, respectively) was better than that
of Adriamycin (IC50 > 1.25 µM). The inhibitory activity of 2.5b, 2.5d, 2.5f, 2.5g and
2.5h on A549 (IC50 = 6.64, 2.69, 2.42, 6.33, 5.35 µM, respectively) was better than
5

that of the control group Tae226 (IC50 = 9.67 µM). For tumor cell Ramos, except 2.5f
and 2.5h, other derivatives showed better levels of inhibitory effects on the anti-tumor
than lead compound. For tumor cells A431, we observed that most derivatives exhibit
better anti-tumor activity than the lead compound, among which, the inhibitory
activity of 2.5j (IC50 < 2.5 µM) was similar to that of the control group Adriamycin
(IC50 < 2.5 µM).
Results from biological activity studies of compounds 2.5a-2.5k indicate that all
derivatives are more toxic than their lead compounds. However, compared with
control group, the cytotoxicity of all the derivatives except compound 2.5j (IC50 < 5
µM) was less than Adriamycin (IC50 < 5 µM) on HBE. For the LO2, all the derivatives
except compound 2.5g 、 2.5j (IC50 = 7.44, 2.58 µM) exhibit weaker toxic than
Adriamycin (IC50 = 7.88 µM), even compound 2.5c 、 2.5f 、 2.5k (IC50 = 18.88,
21.00, 26.77 µM) showed frailer toxic than Tae226 (IC50 = 18.14 µM).
Comparison of the observed IC50 values suggest that derivatives 2.5a-2.5k had
stronger anti-tumor activity against tumor cells H1975, Aspc-1 and HCT-116 than
Ramos on the whole, and stronger against HCT-116 than H1975. Overall, 2.5h and
2.5k showed less antitumor activity than the rest of the derivatives. While, derivative
2.5d demonstrated excellent anti-tumor activity against tumor cells H1975, Aspc-1,
HCT-116, Ramos and A549 (IC50 = 5.32, 4.07, 1.12, 7.52, 2.69 µM, respectively).
Compound 2.5j had better antitumor activity against tumor cells H1975, Aspc-1,
HCT-116 and A431 (IC50 was 5.72, <5, 0.34, <2.5 µM), especially against HCT-116
and A431. The anti-tumor activity of diosgenin can be improved by improving water
solubility and introducing hydrophobic groups, which may be one of the reasons why
compound 2.5j has better anti-tumor activity.
Table 2 Antiproliferation activity of quaternary ammonium series I derivatives of diosgenin
Antiproliferation activity (IC50, µM)
Compound
2.5a
A549
A431
H1975
HTC-
116
Aspc-1 Ramos
HBE
LO2
15.23
6.38
18.96
9.67
9.44
39.06
28.67
14.43
6

2.5b
2.5c
6.64
12.67
2.69
4.01
14.53
4.71
10.36
3.87
2.85
3.26
4.33
<2.5
8.25
4.75
<2.5
\
5.96
5.92
5.32
11.17
6.39
5.09
21.70
9.17
5.73
20.59
50.59
<5
<5
4.71
1.12
3.94
1.03
>1,<5
11.45
3.23
0.34
9.74
31.41
>1.25
\
4.06
4.41
4.07
5.54
<5
7.18
9.56
7.52
22.96
>40
7.17
8.99
6.84
13.00
10.11
9.67
19.73
9.94
<5
10.99
18.44
8.07
2.5d
2.5e
10.48
2.42
12.54
21.00
7.44
2.5f
2.5g
6.33
5.03
17.77
4.78
<5
32.57
>40
2.5h
5.35
11.89
8.70
2.5i
10.72
9.86
11.05
15.47
38.35
>40
2.5j
2.85
2.5k
27.53
71.23
0.78
19.32
63.11
<5
18.89
46.56
<5
26.77
>40
Diosgenin
Adriamycin
Tae226
<2.5
\
7.88
9.67
\
\
\
18.14
Note: The symbol "\ " indicates that the test was not performed.
Anti-proliferation activity of quaternary ammonium series II derivatives of diosgenin
is presented as table 3.
The anti-proliferative activity of all the derivatives were superior to the lead
compound diosgenin for tumor cells A549, H1975 and aspc-1. And for A549, all the
derivatives had better effect on anti-proliferative than Tae226. For H1975, all the
derivatives were more excellent than Adriamycin (IC50 < 5) except 2.7d and 2.7k (IC50
=5.08, 5.45 µM). Compounds 2.7a-d, 2.7f and 2.7i (IC50 =1.23, 2.27, 2.15, 2.83, 4.18,
4.46 µM) showed better anti-proliferative than Adriamycin (IC50 < 5 µM) on Aspc-1.
All the derivatives had better effect than diosgenin (IC50 =4.75 µM) on anti-proliferative
for A431 except compounds 2.7e, 2.7i, and 2.7k (IC50 = 6.21, 6.12, 5.88 µM).
Compound 2.7f (IC50 = 2.80 µM) had comparable anti-proliferative activity to that of
Adriamycin (IC50 < 2.5 µM) on Ramos, and the rest of derivatives was similar levels
effect on the anti-proliferative activity to diosgenin against Ramos.
As is evident from data, 2.7a-2.7k are more toxic than their lead compounds.
Compounds 2.7a, 2.7c, 2.7d, 2.7h and 2.7j (IC50 = 5.61, 7.65, 6.06, 6.42, 7.10 µM) had
7

less toxic than Adriamycin (IC50 < 5) on HBE. All the derivatives had more toxic than
Tae226 (IC50 = 18.14 µM) on LO2, while compound 2.7e (IC50 =8.233 µM) showed less
toxic than Adriamycin (IC50 = 7.88 µM).
In general, 2.7a-2.7k had stronger anti-proliferative activity against tumor cells
H1975, Aspc-1 and HCT-116 than Ramos overall. Compounds 2.7b-c, 2.7f-g, 2.7j had
excellent anti-proliferative activity on the tumor cells tested. Compound 2.7k exerted
less anti-proliferative activity compared to the impact of rest of the investigated
compounds.
Table 3 Anti-proliferation activity of quaternary ammonium series II derivatives of
diosgenin
Anti-proliferation activity(IC50, µM)b
Compound
A549
>40.0
1.84
2.81
4.47
5.83
12.6
6.63
5.30
2.52
4.07
4.68
5.83
71.23
A431
>10.0
2.55
3.11
4.05
3.03
6.21
3.52
1.85
3.46
6.12
4.14
5.88
4.75
<2.5
\
H1975
>40.0
0.788
3.68
3.44
5.08
3.36
4.09
1.51
3.23
2.88
1.68
5.45
50.59
<5
Aspc-1
>40.0
1.23
2.27
2.15
2.83
11.1
4.18
7.12
6.56
4.46
6.07
8.48
63.11
<5
Ramos
>40.0
>40.0
36.5
HBE
>40.0
5.61
4.29
7.65
6.06
3.39
4.32
4.19
6.42
3.88
7.10
4.26
46.56
<5
LO2
>40.0
4.57
5.54
6.26
6.30
8.233
5.79
7.12
5.72
6.70
5.75
7.21
>40
2.6
2.7a
2.7b
2.7c
31.9
2.7d
2.7e
>40.0
~ 39.1
2.80
2.7f
2.7g
2.7h
2.7i
35.4
>40.0
33.6
2.7j
37.0
2.7k
Diosgenin
>40.0
>40
Adriamycin 0.78
Tae226 9.67
<2.5
\
7.88
18.14
\
\
\
Note: The symbol "\ " indicates that the test was not performed.
In the investigated two series of compounds, series II had better anti-proliferative
activity than series I on A549, A431, H1975 and Aspc-1. However, series II was also
more toxic. Besides, the anti-proliferation activity of the two series derivatives against
Ramos was worse than that of other tumor cells tested, but the anti-proliferation activity
of series I was better than that of series II.
Analyzing water solubility and anti-tumor activity of two series of diosgenyl
quaternary ammonium derivatives, we observed that the water-soluble of two series of
8

derivatives was better than diosgenin and the water-soluble and anti-proliferation activity of
series II were preferable to series I. Therefore, we deduced that increasing water-soluble
could improve the anti-tumor of diosgenyl quaternary ammonium derivatives.
Particularly, compounds 2.7c and 2.7f had fantastic water-soluble, both of which
showed excellent anti-proliferation activities against tumor cells A549, A431, H1975,
Aspc-1 and Ramos. Besides, we also found an interesting phenomenon that compounds
2.5h, 2.5k, 2.7h and 2.7k, which were more water-soluble, had weaker antitumor
activity than other derivatives, while 2.5d, 2.5j, 2.7a, 2.7g and 2.7j showed stronger
anti-proliferation activity in most tumor cells with relatively small water solubility. This
research indicates that not only to improve the water solubility, but also to further study
the structure-activity relationship of the drug, so as to design the diosgenin derivative
with better antitumor activity.
We carried out other experiments on the best anti-proliferative activity of 2.7a in
the two series of compounds to further verify the drug activity, including cell scratch
test, natural light imaging, dual AO/EB flurorescence staining and DAPI staining. In
these four groups, Gefitinib and Osimertinib (AZD9291) were used as the control group
to carry out in vitro anti-proliferative activity experiments on H1975 with a
concentration of 0, 100, 500, and 2500 nM, respectively. In the scratch test (Fig. 1), the
repair ability of cancer cells decreased with the increase of 2.7a concentration,
indicating that the higher the concentration, the stronger the anti-proliferation activity.
Following 72h of treatment with different concentration, as the concentration increases,
the number of cancer cells is significantly reduced in natural light imaging (Fig. 2). In
the AO/EB test (Fig. 3), we can observe that the normal cancer cells represented by
green decreased significantly with the increase of drug concentration, and the anti-
proliferation activity of 2.7a compound was similar to that of Gefitinib and Osimertinib.
In the DAPI test (Fig. 4), blue indicates normal cancer cells, as shown in the figure, the
number of cancer cells decreases as the concentration of 2.7a increases. It can be
concluded that 2.7a shows good anti-proliferation activity, and the anti-proliferation
activity increases with the increase of concentration.
9

Fig. 1 The scratch test of H1975
10

Fig. 2 Natural light imaging of H1975
Fig. 3 The AO/EB test of H1975
11

Fig. 4 The DAPI test of H1975
3 Conclusion
22 new diosgenyl quaternary ammonium derivatives were synthesized from 11 new
diosgenin amines and ester derivatives with diosgenin as a precursor. The water
solubility of diosgenyl quaternary ammonium derivatives and their anti-proliferation
activity against 8 kinds of human cells were investigated. The results showed that the
water solubility of all diosgenyl quaternary ammonium derivatives was higher than that
of the precursors. The anti-tumor activity of most derivatives was better than that of
diosgenin, and the anti-tumor activity of some derivatives was similar to that of
Adriamycin and Tae226. In summary, improving the water solubility of diosgenyl
derivatives is beneficial to improve its antitumor activity. The results of this study have
certain theoretical significance and potential application value for the structural repair
of diosgenin.
12

Financial & interests disclosure
This work was supported by the Key Laboratory of Basic Chemistry, the State Ethnic
Affairs Commission and the Fundamental Research Funds for the Central Universities,
Southwest Minzu University (NO. 2020PTJS20004); and the National Natural Science
Foundation of China (Grant No. 21602183).
Acknowledgements
Supplementary data
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15

16

Highlights:
1.Two series of novel diosgenyl quaternary ammonium derivatives were designed and
synthesized.
1
13
2.The structures of all novel derivatives were confirmed by H NMR, C NMR and
HR-MS, and evaluated for their in vitro anti-proliferative activities using MTT assay.
3. The experimental results show that improving the water solubility of diosgenyl
derivatives is beneficial to improve its antitumor activity.
17