Synthesis, cytotoxicity, topoisomerase I inhibition and molecular docking of novel phosphoramide mustard sophoridinic acid analogues

Article abstract of DOI:10.1002/aoc.3565

A series of novel phosphoramide mustard sophoridinic acid analogues, consisting of nitrogen mustard group and sophoridinic acid scaffold, have been designed, synthesized and evaluated for their topoisomerase inhibitory activity as well as cytotoxicity against six tumor cell lines (SMMC-7721, LoVo, MCF-7, K562, S180 and H22) and a normal cell line (L929). Among the compounds tested, five were found to be potent inhibitors and exhibited potent cytotoxicity against S180 and H22 cell lines with IC₅₀ values of 1–4?μM. Further mechanistic studies showed that this class of compounds acted as novel topoisomerase I (Topo I) catalytic inhibitors by preventing the binding of Topo I to DNA and inhibiting the cleavage of DNA, and molecular docking studies revealed that the binding energy for these compounds was comparable to that for classic Topo I inhibitors CPT and HCPT, indicating that the compounds have an interaction with DNA and Topo I.

Full text of DOI:10.1002/aoc.3565

Received: 18 April 2016
DOI 10.1002/aoc.3565
Revised: 26 June 2016
Accepted: 1 July 2016
F U L L PA P E R
Synthesis, cytotoxicity, topoisomerase I inhibition and molecular
docking of novel phosphoramide mustard sophoridinic acid
analogues
2 †
2
2
2
2
Kai Liu1,2† | Dong‐Dong Li
| Xiu‐Mei Zhao | Lin‐Lin Dai | Ting Zhang | Zun‐Wei Tao
*
*
1 School of Graduate, Tianjin Medical University,
Tianjin 300070, China
2 Tianjin Institute of Medical and Pharmaceutical
Sciences, Tianjin 300020, China
A series of novel phosphoramide mustard sophoridinic acid analogues, consisting of
nitrogen mustard group and sophoridinic acid scaffold, have been designed, synthe-
sized and evaluated for their topoisomerase inhibitory activity as well as cytotoxic-
ity against six tumor cell lines (SMMC‐7721, LoVo, MCF‐7, K562, S180 and H22)
and a normal cell line (L929). Among the compounds tested, five were found to be
potent inhibitors and exhibited potent cytotoxicity against S180 and H22 cell lines
with IC50 values of 1–4 μM. Further mechanistic studies showed that this class of
compounds acted as novel topoisomerase I (Topo I) catalytic inhibitors by
preventing the binding of Topo I to DNA and inhibiting the cleavage of DNA,
and molecular docking studies revealed that the binding energy for these com-
pounds was comparable to that for classic Topo I inhibitors CPT and HCPT, indicat-
ing that the compounds have an interaction with DNA and Topo I.
Correspondence
Dong‐Dong Li or Zun‐Wei Tao, Tianjin Institute of
Medical and Pharmaceutical Sciences, Tianjin
300020, China.
Email: lidongdong2010@163.com;
taozunweitj@163.com
KEYWORDS
antitumor activity, molecular docking, phosphoramide mustard sophoridinic acid
analogues, Topo I inhibition
1
|
INTRODUCTION
consequently development of sophoridine derivatives is nec-
essary to discover more effective drug candidates. Bi
et al.[11,12] synthesized a series of N‐substituted sophoridinol
analogues and found that introduction of a chlorobenzyl on
the 12‐nitrogen atom of sophoridinol might significantly
enhance the anti‐proliferative activity. The mode of action
of these compounds was to inhibit DNA Topo I, followed
by G0/G1 phase arrest, and they also showed a moderate oral
bioavailability and good safety in vivo. Li et al.[13] demon-
strated that the D‐ring was not necessary for activity, and
synthesized a family of N‐substituted sophoridinic acid
derivatives, then evaluated their cytotoxicity against HepG‐2
cancer cells. The mechanism of action of these compounds
was to inhibit the activity of DNA Topo I, followed by S‐phase
arrest and then to cause apopototic cell death.
Sophoridine, which was approved by the China Food and
Drug Administration in 2005 for the treatment of cancer
patients, has been widely used to treat liver cancer, gastric
cancer and lung cancer in combination with other anticancer
drugs used in the clinic including cisplatin, vinorelbine,
docetaxel, etc.[1–6] Sophoridine is also a main chemical ingre-
dient of the Chinese traditional medicine Fufang Kushen
injection; what is more, it has many drug‐related advantages
such as high solubility, good safety profiles and a special
chemical scaffold, suggesting that it is an ideal lead com-
pound for further modifications and optimizations. The
mechanism of action of sophoridine is to inhibit DNA topo-
isomerase I (Topo I) activity and induce cell cycle arrest at
the G0/G1 phase, and then cause apoptotic cell death.[7–10]
However, the moderate antitumor activity of sophoridine
limits its use as a drug for clinical applications, and
Nitrogen mustard has been widely used in the clinical
setting as an alkylating agent with the advantages of wide
spectrum of antitumor activity and strong killing ability for
tumor cells, but the lack of selectivity for cells causes serious
side effects. Based on the above suggestion, we hypothesize
† These authors contributed equally to this work
Appl. Organometal. Chem. 2016; 1–7
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
LIU ET AL.
that the introduction of nitrogen mustard group into the struc-
ture of sophoridinic acid might be beneficial for improving
antitumor activity and inducing toxicity; however, research
on the combination of nitrogen mustard and sophoridinic
acid derivatives is rare.
with a Bruker Avance 400 (400 MHz) spectrometer with
CDCl3 as solvent and tetramethylsilane as an internal stan-
dard. Mass spectra were obtained using a Shimadzu LCMS‐
8040 LCMS (ESI). The DigiDoc‐It gel imaging and docu-
mentation system was assessed using LabWorks Imaging
and Analysis Software (UVI, UK). The MTT assay was
coonducted by measuring the absorbance of each well at
570 nm using a Bio‐Rad 680 microplate reader (Bio‐Rad,
USA).
Previously, we have modified the 17‐carboxyl group with
aromatic nitrogen mustard groups to produce target com-
pounds, verifying that the introduction of the nitrogen mustard
group to the 4′‐carboxyl group in the sophoridinic acid scaffold
significantly enhances antitumor activity.[14] The result pro-
voked our strong interest to further develop structural modifica-
tions and optimizations in an effort to discover a novel class of
anticancer candidates with a three‐ring scaffold. As shown in
Scheme 1, in the work reported here, we continued to carry
out the structure–activity relationship (SAR) analysis of the
substituent on the 12‐nitrogen atom. Based on this strategy,
we introduced phosphoramide mustard group to the
sophoridinic acid scaffold to produce target compounds.
2.3 | Preparation of compounds
Compounds 2, 3 and 4 were synthesized and characterized
according to previous reports.[11,15]
2.3.1
| Synthesis of compound 6
To a stirred solution of 2 (3.2 mmol, 1.0 g) and K2CO3
(14.5 mmol, 2.0 g) in anhydrous CH3CN (20 ml) at 80°C
(oil bath), phosphoramidic dichloride (3.9 mmol, 1.0 g) in
10 ml of anhydrous CH3CN was added dropwise. The reac-
tion solution was refluxed until TLC analysis showed com-
pletion of the reaction. This was followed by filtration, the
filtrate being evaporated under vacuum and purified by col-
umn chromatography on silica gel with petroleum ether–
dichloromethane–triethylamine (12:8:1) to afford compound
6 as a yellow oil in 41% yield.
2
| EXPERIMENTAL
2.1 | Materials
Unless otherwise indicated, the chemical reagents used in the
experiments were of analytical grade and obtained from
Tianjin Chemical Reagent Co. Ltd. Methanol, tetrahydrofuran
(THF) and acetonitrile were distilled before use. Calf thymus
DNA, pBR322 plasmid DNA and DNA Topo I were from
Takara Bio Inc.
Anal. Calcd for C20H35Cl3N3O3P (503.0) (%): C, 47.77;
1
H, 7.02; N, 8.36. Found (%): C, 47.75; H, 7.05; N, 8.34. H
NMR (400 MHz, CDCl3, δ, ppm): 3.63 (s, 3H, 18‐H),
3.44–3.24 (m, 7H, 20‐H, 22‐H, 13‐H, 11‐H ), 2.88–2.76
(m, 4H, 19‐H, 21‐H), 2.35–2.20 (m, 6H, 2‐H, 10‐H, 16‐H),
1.63–1.45 (m, 15H, 3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐
H, 15‐H). 13C NMR (δ, ppm): 173.56 (17‐C), 58.83 (2‐C),
58.36 (10‐C), 54.44(19‐C), 51.60 (21‐C), 49.40 (18‐C),
45.61 (11‐C), 45.35 (13‐C), 41.57 (20‐C), 41.42 (22‐C),
38.19 (5‐C), 33.59 (14‐C), 30.80 (15‐C), 29.21 (4‐C), 25.89
(8‐C), 25.78 (3‐C), 24.91 (9‐C), 22.36 (16‐C). ESI‐MS:
C20H35Cl3N3O3P (503.0), [M + H]+ = 504.3. UV–visible
(nm): 209, n–π* transition. IR (ν, cm−1): 3442.54, 2924.20,
1729.76, 1634.69, 1456.00, 1234.84.
2.2 | Instrumentation
All the compounds were characterized with 1H NMR and 13
C
NMR spectroscopy and ESI‐MS. NMR data were recorded
2.3.2
| Synthesis of compounds 5a–5e
To a stirred solution of 4‐methylphenol (10 mmol, 1.08 g)
and phosphoramidic dichloride (12 mmol, 3.10 g) in anhy-
drous THF (30 ml) at 70°C (oil bath), Et3N (24 mmol,
2.44 g) in 10 ml of anhydrous THF was added dropwise.
The reaction solution was refluxed until TLC analysis
showed completion of the reaction. This was followed by
filtration, the filtrate being evaporated under vacuum and
purified by column chromatography on silica gel with petro-
leum ether–dichloromethane–triethylamine (25:5:1) to
afford compound 5a as a yellow oil in 61% yield. Com-
pounds 5b–5e were prepared in a manner similar to that
described above.
SCHEME 1 Structure of sophoridine and the modification sites of newly
constructed scaffold

LIU ET AL.
3
2.3.2.1
|
5a
130.08 (7‐C), 130.10 (9‐C), 126.25 (8‐C), 120.47 (6‐C),
120.42 (10‐C), 49.88 (1‐C), 49.84 (3‐C), 41.46 (2‐C), 41.44
(4‐C). ESI‐MS: C10H13Cl3NO2P (316.5), [M + Na]+ = 340.1.
Yield 61%; yellow oil. Anal. Calcd for C11H15Cl3NO2P
(330.6) (%): C, 39.96; H, 4.57; N, 4.24. Found (%): C,
39.94; H, 4.60; N, 4.22. H NMR (400 MHz, CDCl3, δ,
1
ppm): 7.19 (d, J = 8.6 Hz, 2H, 7‐H, 9‐H), 7.14 (d,
J = 8.6 Hz, 2H, 6‐H, 10‐H), 3.72 (d, J = 5.3 Hz, 8H, 1‐H,
2‐H, 3‐H, 4‐H), 2.35 (s, 3H, 8‐CH3). 13C NMR (δ, ppm):
147.20 (5‐C), 136.10 (7‐C), 136.02 (9‐C), 130.50 (8‐C),
120.15 (6‐C), 120.10 (10‐C), 49.79 (1‐C), 49.31 (3‐C),
41.48 (2‐C), 40.81 (4‐C), 20.83 (8‐CH3). ESI‐MS:
C11H15Cl3NO2P (330.6), [M + Na]+ = 354.0.
2.3.3
| Synthesis of compounds 7a–7e
To a stirred solution of 5a (0.30 mmol, 0.10 g) and 2
(0.56 mmol, 0.20 g) in anhydrous CH3CN (30 ml) at 80°C
(oil bath), Et3N (3.0 mmol, 0.3 g) in 10 ml of anhydrous
CH3CN was added dropwise. The reaction solution was
refluxed until TLC analysis showed completion of the reaction.
This was followed by filtration, the filtrate being evaporated
under vacuum and purified by column chromatography on
silica gel with petroleum ether–dichloromethane–triethylamine
(12:8:1) to afford colorless oil 7a. Compounds 7b–7e were
prepared in a manner similar to that described above.
2.3.2.2
|
5b
Yield 68%; yellow oil. Anal. Calcd for C11H15Cl3NO3P
(346.6) (%): C, 38.12; H, 4.36; N, 4.04. Found (%): C,
1
38.10; H, 4.39; N, 4.02. H NMR (400 MHz, CDCl3, δ,
ppm): 7.19 (dd, J = 9.1, 1.8 Hz, 2H, 7‐H, 9‐H), 6.90 (dd,
J = 9.1, 1.8 Hz, 2H, 6‐H, 10‐H,), 3.82 (s, 3H, 8‐OCH3),
3.78–3.71 (m, 4H, 2‐H, 4‐H ), 3.69–3.57 (m, 4H, 1‐H, 3‐
H). 13C NMR (δ, ppm): 157.55 (8‐C), 143.07 (5‐C), 121.40
(7‐C), 121.35 (9‐C), 114.90 (6‐C), 114.89 (10‐C), 55.64 (8‐
OCH3), 49.87 (1‐C), 49.83 (3‐C), 41.46 (2‐C), 41.44 (4‐C).
ESI‐MS: C11H15Cl3NO3P (346.6), [M + Na]+ = 370.1.
2.3.3.1
|
7a
Yield 39%; colorless oil. Anal. Calcd for C27H42Cl2N3O4P
(574.5) (%): C, 56.44; H, 7.37; N, 7.31. Found (%): C,
56.40; H, 7.40; N, 7.30. H NMR (400 MHz, CDCl3, δ,
1
ppm): 7.15 (d, J = 3.4 Hz, 2H, 25‐H, 27‐H), 7.11 (d,
J = 3.4 Hz, 2H, 24‐H, 28‐H), 3.68 (s, 3H, 18‐H), 3.59 (m,
4H, 20‐H, 22‐H), 3.43 (m, 4H, 19‐H, 21‐H), 3.21 (m, 1H,
11‐H), 2.88 (m, 2H, 13‐H), 2.62–2.44 (m, 2H, 16‐H), 2.33
(s, 3H, 26‐CH3), 2.28–1.96 (m, 4H, 2‐H, 10‐H), 1.81–1.27
(m, 15H, 3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐H, 15‐H).
13C NMR (δ, ppm): 173.46 (17‐C), 148.28 (26‐C), 134.28
(23‐C), 130.17 (25‐C), 130.10 (27‐C), 120.06 (24‐C),
120.01(28‐C), 58.98 (2‐C), 58.54 (10‐C), 51.56 (19‐C),
50.30 (21‐C), 49.97 (18‐C), 45.41 (11‐C), 45.30 (13‐C),
44.32 (7‐C), 42.44 (20‐C), 42.40 (22‐C), 38.58 (5‐C), 33.86
(14‐C), 30.74 (15‐C), 29.16 (4‐C), 26.99 (8‐C), 25.73
(3‐C), 24.74 (9‐C), 22.29 (26‐CH3), 20.80 (16‐C). ESI‐MS:
C27H42Cl2N3O4P (574.5), [M + H]+ = 575.2. UV–visible
(nm): 229, π–π* transition; 263, n–π* transition. IR (ν, cm
−1): 3443.49, 2929.46, 2857.59, 1734.74, 1636.48, 1506.55,
1455.60, 1217.39, 914.49, 823.08, 744.30.
2.3.2.3
|
5c
Yield 62%; yellow oil. Anal. Calcd for C10H12Cl4NO2P
(351.0) (%): C, 34.22; H, 3.44; N, 3.99. Found (%):C,
1
34.20; H, 3.47; N, 3.96. H NMR (400 MHz, CDCl3, δ,
ppm): 7.38 (d, J = 8.5 Hz, 2H, 7‐H, 9‐H), 7.23 (d,
J = 8.5 Hz, 2H, 6‐H, 10‐H), 3.74 (dd, J = 9.7, 2.6 Hz, 4H,
2‐H, 4‐H), 3.65 (dd, J = 9.7, 2.6 Hz, 4H, 1‐H, 3‐H). 13C
NMR (δ, ppm): 148.04 (5‐C), 131.79 (7‐C), 131.76 (9‐C),
130.11 (8‐C), 121.87 (6‐C), 121.81 (10‐C), 49.75 (1‐C),
49.71 (3‐C), 41.38 (2‐C), 41.36 (4‐C). ESI‐MS:
C10H12Cl4NO2P (351.0), [M + Na]+ = 374.0.
2.3.2.4
|
5d
Yield 53%; yellow oil. Anal. Calcd for C10H12Cl3N2O4P
(361.5) (%): C, 33.22; H, 3.34; N, 7.75. Found (%): C,
33.20; H, 3.36; N, 7.73; H NMR (400 MHz, CDCl3, δ,
2.3.3.2
|
7b
1
Yield 41%; colorless oil. Anal. Calcd for C27H42Cl2N3O5P
(590.5) (%): C, 54.91; H, 7.16; N, 7.11. Found (%): C, 54.89;
H, 7.18; N, 7.10. 1H NMR (400 MHz, CDCl3, δ, ppm): 7.15
(dd, J = 25.4, 8.7 Hz, 2H, 25‐H, 27‐H), 6.84 (dd, J = 25.4,
8.7 Hz, 2H, 24‐H, 28‐H), 3.78 (s, 3H, 26‐OCH3), 3.65 (s,
3H, 18‐H), 3.54 (m, 4H, 20‐H, 22‐H), 3.40 (m, 4H, 19‐H, 21‐
H), 3.20–3.12 (m, 1H, 11‐H), 2.85–2.73 (m, 2H, 2‐H,10‐H),
2.62–2.36 (m, 2H, 2‐H, 10‐H), 2.37–2.06 (m, 2H, 16‐H),
1.91–1.28 (m, 15H, 3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐
H, 15‐H). 13C NMR (δ, ppm): 173.62 (17‐C), 156.46 (26‐C),
144.88 (23‐C), 121.13 (25‐C), 120.93 (27‐C), 114.62 (24‐C),
114.59 (28‐C), 58.88 (2‐C), 58.54 (10‐C), 58.40 (26‐OCH3),
55.67 (19‐C), 55.61 (21‐C), 54.55 (18‐C), 50.35 (11‐C),
49.95 (13‐C), 45.31 (7‐C), 42.39 (20‐C), 42.29 (22‐C), 37.88
(5‐C), 33.82 (14‐C), 30.85 (15‐C), 29.31 (4‐C), 26.87 (8‐C),
ppm): 8.31 (d, J = 9.1 Hz, 2H, 7‐H, 9‐H), 7.46 (d,
J = 9.1 Hz, 2H, 6‐H, 10‐H), 3.78–3.60 (m, 8H, 1‐H, 2‐H,
3‐H, 4‐H). 13C NMR (δ, ppm): 154.89 (5‐C), 145.11 (8‐C),
126.17 (7‐C), 125.89 (9‐C), 120.71 (6‐C), 120.65 (10‐C),
49.17 (1‐C), 49.13 (3‐C), 41.57 (2‐C), 41.17 (4‐C). ESI‐
MS: C10H12Cl3N2O4P (361.5), [M + Na]+ = 385.2.
2.3.2.5
|
5e
Yield 65%; yellow oil. Anal. Calcd for C10H13Cl3NO2P (316.5)
(%): C, 37.94; H, 4.14; N, 4.42. Found (%):C, 37.92; H, 4.17;
N, 4.40. 1H NMR (400 MHz, CDCl3, δ, ppm): 7.42 (d,
J = 7.5 Hz, 2H, 7‐H, 9‐H), 7.29 (m, 3H, 6‐H, 8‐H, 10‐H),
3.75 (dd, J = 7.0, 2.1 Hz, 4H, 2‐H, 4‐H), 3.66 (dd, J = 7.0,
2.1 Hz, 4H, 1‐H, 3‐H). 13C NMR (δ, ppm): 150.1 (5‐C),

4
LIU ET AL.
25.98 (3‐C), 24.92 (9‐C), 22.54 (16‐C). ESI‐MS:
C27H42Cl2N3O5P (590.5), [M + H]+ = 592.3. UV–visible
(nm): 232, π–π* transition; 280, n–π* transition. IR (ν, cm
−1): 3464.85, 2930.55, 2310.04, 2056.65, 1732.11, 1504.28,
1454.64, 914.14, 749.58.
(d, J = 6.7 Hz, 2H, 25‐H, 27‐H), 7.32 (d, J = 6.7 Hz, 1H, 24‐
H), 7.26 (d, J = 6.7 Hz, 1H, 28‐H), 7.20 (m, 1H, 26‐H), 3.70
(s, 3H, 18‐H), 3.65–3.56 (m, 4H, 20‐H, 22‐H), 3.47 (m, 4H,
19‐H, 21‐H), 3.23 (m, 1H, 11‐H), 2.90 (m, 4H, 13‐H, 16‐H),
2.66–2.21 (m, 2H, 2‐H), 2.37 (m, 2H, 10‐H), 2.14–0.83 (m,
15H, 3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐H ,15‐H). 13C
NMR (δ, ppm): 173.73 (17‐C), 150.79 (23‐C), 129.72 (25‐
C), 129.65 (27‐C), 124.72 (26‐C), 120.32 (24‐C), 120.08 (28‐
C), 58.94 (2‐C), 58.59 (10‐C), 58.43 (18‐C), 54.56 (19‐C),
51.59 (21‐C), 50.31 (11‐C), 49.96 (13‐C), 45.33 (7‐C), 42.38
(20‐C), 42.28 (22‐C), 33.85 (5‐C), 30.88 (14‐C), 26.98
(15‐C), 25.93 (4‐C), 24.96 (8‐C), 22.57 (3‐C), 22.40 (9‐C),
19.03 (16‐C). ESI‐MS: C26H40Cl2N3O4P (560.5), [M + Na]
+ = 582.2. UV–visible (nm): 221, π–π* transition; 263, n–π*
transition. IR (ν, cm−1): 3443.64, 2931.96, 2858.15, 1733.92,
1636.65, 1594.28, 1491.43, 1455.83, 1440.00, 1265.60,
1208.11, 1162.43, 918.32, 738.23.
2.3.3.3
|
7c
Yield 42%; colorless oil. Anal. Calcd for C26H39Cl3N3O4P
(595.0) (%): C, 52.48; H, 6.61; N, 7.06. Found (%): C,
1
52.45; H, 6.64; N, 7.03. H NMR (400 MHz, CDCl3, δ,
ppm): 7.26 (dd, J = 8.9, 4.4 Hz, 2H, 25‐H, 27‐H), 7.21 (d,
J = 8.9 Hz, 1H, 24‐H), 7.15 (d, J = 4.4 Hz, 1H, 28‐H),
3.63 (s, 3H, 18‐H), 3.58–3.48 (m, 4H, 20‐H, 22‐H), 3.39
(m, 4H, 19‐H, 21‐H), 3.14 (m, 1H, 11‐H), 2.93–2.90 (m,
2H, 13‐H), 2.84–2.24 (m, 6H, 2‐H, 10‐H, 16‐H), 2.60–1.02
(m, 15H, 3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐H ,15‐H).
13C NMR (δ, ppm): 173.60 (17‐C), 149.90 (23‐C), 129.84
(26‐C), 129.65 (25‐C), 129.61 (27‐C), 121.60 (24‐C),
121.38 (28‐C), 58.80 (2‐C), 58.65 (10‐C), 58.46 (18‐C),
54.50 (19‐C), 51.53 (21‐C), 50.22 (11‐C), 49.83 (13‐C),
46.17 (7‐C), 42.36 (20‐C), 42.21 (22‐C), 30.80 (5‐C), 29.27
(14‐C), 26.87 (15‐C), 25.89 (4‐C), 24.98 (8‐C), 22.48
(3‐C), 22.32 (9‐C), 18.97 (16‐C). ESI‐MS: C26H39Cl3N3O4P
(595.0), [M + H]+ = 596.2. UV–visible (nm): 233, π–π*
transition; 267, n–π* transition. IR (ν, cm−1): 3446.24,
2928.96, 2855.76, 1735.79, 1487.07, 1441.15, 1234.35,
1212.86, 1162.63, 1089.96, 1060.88, 914.49.
2.4 | Anti‐proliferation assay
The anti‐proliferative activity of the compounds against
tumor cell lines was evaluated. Tumor cell lines were grown
to the log phase in an RPMI‐1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 × 104 cells
ml−1 with the complete medium, 100 μl of the obtained cell
suspension was added to each well of 96‐well culture plates.
The subsequent incubation was performed for 48 h at 37°C
under a 5% CO2 atmosphere prior to the cytotoxicity assess-
ments. The tested samples at preset concentrations were
added to six wells. After a 48 h exposure period, 40 μl of
phosphate‐buffered saline containing 2.5 mg ml−1 MTT was
added to each well. After 4 h, 100 μl of extraction solution
(10% sodium dodecylsulfate, 5% isobutyl alcohol, 0.01 M
HCl) was added. After overnight incubation at 37°C, the opti-
cal density was measured at 570 nm with an enzyme‐linked
immunosorbent assay microplate reader. Three replicate
wells were used for each concentration in all experiments.
Each assay was performed at least three times. The concentra-
tion required for 50% reduction in cell survival (IC50) of test
substances was calculated using the inverse Hill equation.
2.3.3.4
|
7d
Yield 31%; light yellow oil. Anal. Calcd for C26H39Cl2N4O6P
(605.5) (%): C, 51.57; H, 6.49; N, 9.25. Found (%): C, 51.54;
H, 6.51; N, 9.23. 1H NMR (400 MHz, CDCl3, δ, ppm): 8.17
(d, J = 6.9 Hz, 2H, 25‐H, 27‐H), 7.38 (d, J = 6.9 Hz, 2H,
24‐H, 28‐H), 3.59 (s, 3H, 18‐H), 3.55–3.36 (m, 8H, 19‐H,
20‐H, 21‐H, 22‐H), 3.17 (m, 1H, 11‐H), 2.87 (m, 4H, 13‐H,
16‐H ), 2.54–2.26 (m, 4H, 2‐H,10‐H ), 1.76–1.18 (m, 15H,
3‐H, 4‐H, 5‐H, 6‐H, 7‐H, 8‐H, 9‐H, 14‐H, 15‐H). 13C NMR
(δ, ppm): 172.56 (17‐C), 155.15 (23‐C), 143.28 (26‐C),
124.68 (25‐C), 124.66 (27‐C), 119.63 (24‐C), 119.44
(28‐C), 57.82 (2‐C), 57.63 (10‐C), 57.45 (18‐C), 53.39
(19‐C), 50.61 (21‐C), 49.09 (11‐C), 48.75 (13‐C), 44.34
(7‐C), 41.36 (20‐C), 41.18 (22‐C), 37.51 (5‐C), 32.48
(14‐C), 28.09 (15‐C), 26.07 (4‐C), 24.65 (8‐C), 23.83
(3‐C), 21.31 (9‐C), 17.89 (16‐C). ESI‐MS: C26H39Cl2N4O6P
(605.5), [M + H]+ = 606.3. UV–visible (nm): 226, π–π*
transition; 252, n–π* transition. IR (ν, cm−1): 3448.89,
2926.04, 2855.28, 1735.21, 1611.49, 1591.33, 1521.97,
1440.67, 1383.69, 1346.23, 1120.51, 1065.05, 947.95,
899.58, 862.28, 745.20.
2.5 | Molecular docking
Molecular docking studies were performed using the Glide
model from Schrödinger software. The structure of the com-
pounds was sketched using ChemDraw and saved as mol for-
mat. The structure of human DNA–Topo I complex (PBD ID:
1T8I) was downloaded from the Protein Data Bank.
2.6 | Topo I inhibition assay
DNA Topo I was purchased from Takara Bio Inc. One unit of
enzyme was defined as the amount that completely relaxes
0.5 μg of supercoiled pBR322 DNA in 30 min at 37°C. The
reaction mixture (20 μl) contained 40 mM tri‐HCl (pH = 7.5),
100 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol,
2.3.3.5
|
7e
Yield 39%; colorless oil. Anal. Calcd for C26H40Cl2N3O4P
(560.5) (%): C, 55.71; H, 7.19; N, 7.50. Found (%): C, 55.69;
H, 7.22; N, 7.48. H NMR (400 MHz, CDCl3, δ, ppm): 7.37
1

LIU ET AL.
5
0.1 mg ml−1 bovine serum albumin, 0.2 μg of pBR322 DNA,
2 units of Topo I and test compounds. The reaction mixtures
were incubated at 37°C for 30 min, and the final reaction was
terminated by the addition of 4 μl of stop solution consisting
of 0.25% bromophenol blue, 0.5% sodium dodecylsulfate and
15% glycerol. The samples were electrophoresed on 1% agarose
inTBE buffer (89 mM Tris–boric acid, 2 mM EDTA, pH = 7.5)
at 30 V for 8 h. The gel was stained with 5 μg ml−1 ethidium
bromide and photographed under UV light. Hydroxycamp-
tothecine was used as positive control.
dehydrating agent, but we failed. Even if we strictly controlled
the anhydrous and oxygen‐free conditions and increased the
reaction time, we still could not obtain the target compounds.
A possible reason may be due to large steric hindrance of 6,
which leads to difficult reaction conversion with substituted
phenol, so we tried to develop another method to prepare the
target compounds.
In order to produce target compounds 7a–7e, we initially
introduced equivalent substituted phenol to 4 to produce
intermediates 5a–5e, which were obtained by substitution
reaction of equivalent substituted phenol with equivalent 4
using triethylamine as the deacid reagent in anhydrous THF
(Scheme 3). In the reaction, two substituted products would
be formed if the amount of substituted phenol was used in
excess, so the amount of phenol must be controlled strictly.
Compound 5b was prepared much easier than 5a, 5c, 5d
and 5e, which may be due to the electron‐donating effect of
4‐methoxyphenol. Compounds 7a–7e were obtained by sub-
stitution reaction of equivalent compounds 5a–5e with equiv-
alent 2 using triethylamine as the deacid reagent in anhydrous
CH3CN. Among the five target compounds, 7d was difficult
to obtain, which was attributed to the electron‐absorption
effect of 4‐nitrophenol.
3
| RESULTS AND DISCUSSION
3.1 | Synthesis of compounds
Firstly, we introduced phosphoramidic dichloride group as
substituent to the nitrogen atom at the 12‐positition of 2 to syn-
thesize compound 6 (Scheme 2). Sophoridine was hydrolyzed
with sodium hydroxide to produce sophoridinic acid (1),
which was transformed to acyl chloride using sulfoxide chlo-
ride and then reacted with methanol to produce intermediate
2. Compound 6 was prepared by the reaction of 2 with
phosphoramidic dichloride (4) and anhydrous potassium car-
bonate in anhydrous CH3CN. In order to change the lipid–
water partition coefficient, which might be beneficial for
penetrating cells, and improving antitumor activity, we tried
to obtain compounds 7a–7e through the etherification reaction
of 6 with the corresponding phenol in the presence of
3.2 | Cytotoxicity
All the desired compounds were evaluated for their cytotoxic
activities against six tumor cell lines, namely SMMC‐7721
(human hepatoma), LoVo (human pancreatic cancer), MCF‐
7 (human breast cancer), K562 (human leukemia), S180
(mouse sarcoma) and H22 (mouse hepatoma), along with nor-
mal cell line L929 (fibroblast) using MTT assay. As evident
from Table 1, compound 4 exhibits cytotoxic activity to the
six tumor cells and normal cells, whereas compound 6 shows
less cytotoxic activity to normal cells, indicating that the
SCHEME 2 Synthesis of phosphoramide mustard sophoridinic acid ana-
logue 6. Reagents and conditions: (a) 2 M NaOH–H2O, 100°C, 20%
H2SO4; (b) CH3OH, SOCl2, 0°C; (c) anhydrous CH3CN, potassium
carbonate, 80°C
SCHEME 3 Synthesis of phosphoramide mustard sophoridinic acid ana-
logues 7a–7e. Reagents and conditions: (a) thionyl chloride, CH2Cl2, 0°C,
5 h; (b) POCl3, 120°C, 48 h; (c) substituted phenol, anhydrous THF, Et3N,
70°C; (d) anhydrous CH3CN, Et3N, 80°C

6
LIU ET AL.
TABLE 1 Structure–activity relationships of phosphoramide mustard sophoridinic acid analogues for their anti‐proliferative activities in vitro (48 h)
IC50 (μM)
Clog
Compound
R
SMMC
94.10
77.92
60.80
65.16
31.73
36.51
77.89
LoVo
83.98
77.52
62.47
46.17
33.43
32.25
69.95
MCF‐7
111.37
75.98
61.20
38.86
31.92
34.62
86.57
K562
48.76
34.33
27.07
54.50
18.07
20.31
32.73
S180
11.70
10.06
2.89
H22
12.13
11.7
2.17
2.65
1.01
1.80
1.85
L929
52.84
Pa
4
0.38
3.14
5.33
4.92
5.69
4.88
4.83
6
150.52
180.66
104.30
162.41
105.32
138.54
7a
7b
7c
7d
7e
CH3
OCH3
Cl
3.50
2.02
NO2
H
2.01
3.65
aLipohilicity of the compounds is expressed in terms of their Clog P values which were calculated using ChemBioDraw Ultra 12.0 software.
introduction of sophoridinic acid scaffold induces cytotoxic-
ity to normal cells.
In order to change the lipid–water partition coefficient,
which might be beneficial for penetrating cells, and improv-
ing antitumor activity, we introduced equivalent substituted
phenol to 6 to obtain the compounds 7a–7e. Compared with
compound 6, they display higher antitumor activity against
the tumor cells, especially for S180 and H22 tumor cells
(IC50 = 1–4 μM). It seems that the improved antitumor activ-
ities of 7a‐7e are consistent with their relatively higher Clog
P values (>4) calculated using ChemBioDraw 12.0 software.
Among these compounds, the antitumor activities of 7c and
7d are higher than those of the other compounds, which
may be due to the substituent phenyl ring in the structure.
FIGURE 1 Molecular docking models of 7a–7d in the active site of DNA–
Topo I complex (PDB ID: 1T8I)
3.3 | Molecular docking
hydrogen‐bonded to LYS436 in protein, while hydrogen‐
bonded to MET428 in protein for 7c and 7d. Moreover, the
phenyl ring of 7a–7d approaches towards the DNA cleavage
site in the DNA–Topo I complex, forming a stable complex
through π–π stacking interaction with purine ring of DNA.
In addition, the tertiary amine in compounds 7c and 7d is also
hydrogen‐bonded to purine ring of DNA in protein and
increases the interaction between compounds and protein.
The result is consistent with that of the evaluation of
antitumor activity.
Previous literature[13] reported that sophoridinic acid deriva-
tives could strongly inhibit the activity of DNA–Topo I and
cause apoptotic cell death. Moreover, the target of nitrogen
mustard was DNA. So, in order to better understand the
potency of nitrogen mustard sophoridinic acid derivatives
and to guide further SAR study, molecular docking studies
with human DNA–Topo I complex (PBD ID: 1T8I) were
performed. The X‐ray crystallographic structure of human
DNA–Topo I complex was retrieved from the Protein Data
Bank. The arrangements of target compounds into the active
site of protein were generated based on the scores.
Compounds 7a–7d were analyzed and compared with
typical topoisomerase inhibitors CPT and HCPT. As seen in
Figure 1, the rings in the compounds have hydrophobic inter-
action with amino acids in the protein around the molecule.
The carbonyl oxygen atoms of compounds 7a and 7b are
The resulting docking model gives minimum relative
binding energy for 7a–7d as −46.69, −58.31, −57.92 and
−65.25 kcal mol−1
,
comparable to that of CPT
(−51.13 kcal mol−1) and HCPT (−53.10 kcal mol−1), indi-
cating that the compounds have interaction with DNA–Topo
I. The binding modes of these compounds are similar to
those of CPT and HCPT in DNA–Topo I active sites.[16,17]

LIU ET AL.
7
ACKNOWLEDGMENTS
This work was supported by the Science and Technology
Foundation of Tianjin Health Bureau (2014KY38) and the
National Natural Science Foundation of China (21501134).
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A series of phosphoramide mustard sophoridinic acid ana-
logues have been successfully synthesized and characterized.
SAR analysis revealed the introduction of phosphoramide
mustard and substituted phenyl ring groups on the 12‐nitro-
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How to cite this article: Liu, K., Li, D.‐D., Zhao, X.‐
M., Dai, L.‐L., Zhang, T., and Tao, Z.‐W. (2016), Syn-
thesis, cytotoxicity, topoisomerase I inhibition and
molecular docking of novel phosphoramide mustard
sophoridinic acid analogues, Appl. Organometal.
Chem., doi: 10.1002/aoc.3565