Novel sophoridine derivatives bearing phosphoramide mustard moiety exhibit potent antitumor activities in vitro and in vivo

Article abstract of DOI:10.3390/molecules23081960

Novel mustard functionalized sophoridine derivatives were synthesized and evaluated for their cytotoxicity against of a panel of various cancer cell lines. They were shown to be more sensitive to S180 and H22 tumor cells with IC₅₀ values ranging from 1.01–3.65 μM, and distinctly were more cytotoxic to cancer cells than normal cell L929. In addition, compounds 7a, 7c, and 7e displayed moderate tumor suppression without apparent organ toxicity in vivo against mice bearing H22 liver tumors. Furthermore, they arrested tumor cells in the G1 phase and induced cellular apoptosis. Their potential binding modes with DNA-Top I complex have also been investigated.

Full text of DOI:10.3390/molecules23081960

molecules
Article
Novel Sophoridine Derivatives Bearing
Phosphoramide Mustard Moiety Exhibit Potent
Antitumor Activities In Vitro and In Vivo
Dongdong Li *, Linlin Dai, Xiumei Zhao, Shuang Zhi, Hongsheng Shen and Zibo Yang
Tianjin Institute of Medical and Pharmaceutical Sciences, Tianjin 300020, China; abcdll@whu.edu.cn (L.D.);
zxmmlg@163.com (X.Z.); zhishuang0110@126.com (S.Z.); shenhongsheng830@163.com (H.S.);
thesun999@163.com (Z.Y.)
* Correspondence: lidongdong2010@163.com
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 July 2018; Accepted: 3 August 2018; Published: 6 August 2018
Abstract: Novel mustard functionalized sophoridine derivatives were synthesized and evaluated for
their cytotoxicity against of a panel of various cancer cell lines. They were shown to be more sensitive
to S180 and H22 tumor cells with IC50 values ranging from 1.01–3.65
µM, and distinctly were more
cytotoxic to cancer cells than normal cell L929. In addition, compounds 7a
,
7c, and 7e displayed
moderate tumor suppression without apparent organ toxicity in vivo against mice bearing H22 liver
tumors. Furthermore, they arrested tumor cells in the G1 phase and induced cellular apoptosis.
Their potential binding modes with DNA-Top I complex have also been investigated.
Keywords: mustard functionalized sophoridine derivatives; antitumor activity; apoptosis
1. Introduction
Natural phytochemicals have proven to be an extremely helpful tool to aid the discovery of potent
antitumor agents for targets matching or tissue-selective therapy due to their privileged structures
with inherent drug-likeness [
the traditional medicine herb Sophora alopecuroides L. It was approved by China Food and Drug
Administration (CFDA), in 2005, as an anticancer agent against malignant trophoblastic tumors [ ].
1,2]. Sophoridine is a tetracyclic quinolizidine alkaloid extracted from
3
Indeed, Sophoridine has been widely used as adjuvant with other anticancer pharmaceuticals including
vinorelbine, cisplatin, and taxol to improve the therapeutic effect against gastric cancer, liver cancer,
and esophageal cancer in combination therapy over time [4–10]. Sophoridine is also a main chemical
ingredient of the Chinese traditional medicine Fufang Kushen injection, furthermore, it owns many
of drugable advantages, such as high solubility, good safety profiles and a special chemical scaffold,
indicating that it is an ideal lead compound for further investigation.
The mechanism of sophoridine is verified to inhibit the DNA topoiosmerase I activity, which induced
cell cycle arrest in the G0/G1 phase and is accompanied by apoptotic cell death [11
to confirmed safety profiles, sophoridine exerts many favorable features such as potential of T cell
improvement, good solubility, and no myelotoxicity [14 16]. However, the relatively moderate
–13]. In addition
–
potency has generally limited its broader utility in clinic. Hence, considerable attentions have been
devoted to discover sophoridine derivatives with optimized biochemical and/or pharmacological
properties for new antineoplastic drug development. Concerning the unique scaffold of sophoridine,
Xin Li et al. [17] have opened the sophoridine ring D and evaluated the resultant tricyclic sophoridinic
acid. The result is that the sophoridine ring D might not be required for activity. The 12-nitrogen
atom and 40-carboxyl group of tricyclic sophoridinic acid provided modifiable sites for further
diversity-oriented optimizations.
Molecules 2018, 23, 1960; doi:10.3390/molecules23081960
www.mdpi.com/journal/molecules

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Cyclophosphamide is one of the most successful antitumor drugs for the management
of malignancies of various origins from soft tissue tumor to lymphoma [18]. Because of its advantage
of broad spectrum of antitumor and activity against both resting and multiplying cells, it still remains
the drug of choice in routine clinical practices at this time [19]. Under physiological conditions,
cyclophosphamide is metabolized to active phosphoramide mustard A, which is the ultimate cytotoxic
species that cross-links interstrand DNA [20].
Based on the above suggestion, as depicted in Scheme 1, we have introduced phosphoramide
mustard group to the structure of sophoridine, and designed and synthesized a series of novel
sophoridine derivatives bearing phosphoramide mustard moiety.
Scheme 1. The formula structures of designed targeted compounds.
2. Results
2.1. Chemistry
Recently, we have designed and synthesized series of novel phosphoramide mustard sophoridine
derivatives 7a
subsequent synthesis of the aryloxy-phosphoramidate mustard 6a
Scheme 2, was hydrolyzed in aqueous NaOH, and then acidified with 12 N HCl solution gave
with good yield. Intermediate was afforded via esterification of in thionyl chloride/methanol
solution. was prepared according to the previously reported protocols [21] as a crystalline solid
from diethanolamine. Then we used the appropriate various phenols to react with to obtain
aryloxy-phosphoramidate mustards 6a 6e. Finally, 6a 6e was coupled with in the presence of
triethylamine, giving the target compounds 7a–7e with satisfied yield.
–
7e, the synthetic approach consisted first of preparation of sophoridinic ester
2 and
–6e in parallel. As depicted in
1
2
3
2
5
5
–
–
3

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O
H
H
N
H
N
N
H
COOH
COOCH3
H
a
H
H
b
H
H
H
H
H
H
H
e
N
1
N
2
N
3
Ar
Cl
HCl
HN
OH
Cl
O
HN
O
P
Cl
c
d
O
N
P
Cl
Cl
N
Cl
OH
Cl
4
Cl
Cl
5
6
Ar
H
Cl
O
P
N
H
Ar
H
N
N
O
H
COOCH3
O
H
H
O
f
COOCH3
Cl
P
Cl
N
Cl
H
H
N
H
N
Cl
3
6
7
Ar =
CH3
OCH3
Cl
NO2
a
b
c
d
e
Scheme 2. Synthesis of 7a–7e. Reagents and conditions: (a) 2 N NaOH/H2O, reflux; 12N HCl;
(b) anhydrous CH3OH, SOCl2/CH3OH; (c) SOCl2, CHCl3, ice bath then reflux; (d) excess POCl3,
◦
120 C; (e) substituted phenol, Et3N, anhydrous THF, ice bath; and (f) Et3N, anhydrous CH3CN, r.t.
2.2. In Vitro Activity
7a–7e were evaluated for their antiproliferative activity against S180, H22, K562, MCF-7,
SMMC-7721, and LoVo cells along with non-cancerous cells L929 by means of standard 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Doxorubicin (Dox) was used as
positive control, as illustrated in Table 1, higher activity was observed in some lineages evaluated.
All the newly synthesized sophoridine derivatives were selectively efficacious against S180 and
H22 cells with IC50 values in the range 1.01–3.65
aryl. In S180 cells, compound 7c (IC50, 2.02 M), 7d (IC50, 2.01
considered to be highly active. Another lineage sensitive to the compounds was H22 with three
promising compounds 7c (IC50, 1.01 M), 7d (IC50, 1.80 M), and 7e (IC50, 1.85 M). The targeted
compounds show more potency than the positive control Dox (IC50, 29.20 M for S180, 36.51 M for
H22). Nonselective cytotoxicity is the main effect which hampers the use of optimal doses in most
conventional chemotherapy [22]. Strikingly, the data suggest that derivatives 7a 7e distinctly are more
µM regardless of the type of the substitutions on
µ
µ
M) and 7a (IC50, 2.89 M) were
µ
µ
µ
µ
µ
µ
–
cytotoxic to cancer cells than the tested normal cell L929 according to the selectivity index (SI).
Li et al. have reported a series of 12-N-substituted sophoridinic acid derivatives, structure activity
relationships (SAR) analysis indicated that the substituents introduced on the 12-nitrogen atom might
significantly enhance the anticancer activity of this kind of compounds, especially, compound 6b
(30
µg/mL), possessing a bromoacetly group, afforded the potential antiproliferative activities
with inhibition rate of 88%, 68%, 40%, and 73% against HepG2, HCT1116, A549, and MCF-7 cells,
respectively, much stronger than that of sophoridine. In this work, sophoridine derivatives bearing
phosphoramide mustard moiety 7a–7e have also exhibited much stronger than the lead compound
with IC50 values ranging from 1.01–3.65 µM against S180 and H22 tumor cells.
We also tested the kinetics of growth inhibition of H22 and S180 cell lines for 7c and 7d at the
dosages of 1–20
µM. Seen in Figure S1, they all showed concentration-dependent and time-dependent
on inhibitory effect.

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Table 1. Antitumor effect of 7a–7e in vitro (48 h).
Cell Lines (IC50, µM)
Compound
S180
H22
K562
MCF-7
SMMC-7721
LoVo
L929
SI *
SI **
5
7a
7b
7c
7d
7e
Dox
1
12.13 ± 0.55
2.89 ± 0.23
3.50 ± 0.35
2.02 ± 0.17
2.01 ± 0.14
3.65 ± 0.25
29.20 ± 2.07
>100
11.70 ± 1.32
2.17 ± 0.13
2.65 ± 0.25
1.01 ± 0.08
1.80 ± 0.12
1.85 ± 0.16
36.51 ± 2.56
>100
48.76 ± 2.01
27.07 ± 2.51
54.50 ± 2.32
18.07 ± 1.34
20.31 ± 2.05
32.73 ± 2.56
17.23 ± 1.78
>100
111.37 ± 3.95
61.20 ± 2.21
38.86 ± 2.56
31.92 ± 2.45
34.62 ± 2.21
86.57 ± 2.67
0.44 ± 0.08
>100
48.76 ± 2.74
60.80 ± 2.34
65.16 ± 2.67
31.73 ± 1.21
36.51 ± 2.45
77.89 ± 2.78
1.22 ± 0.05
>100
83.98 ± 2.36
62.47 ± 2.32
46.17 ± 2.32
33.43 ± 2.45
32.25 ± 2.21
69.95 ± 2.78
0.15 ± 0.02
>100
52.84 ± 2.80
180.66 ± 4.56
104.30 ± 3.20
162.41 ± 4.12
105.32 ± 3.10
138.54 ± 3.35
0.23 ± 0.08
>100
4.36
62.51
29.80
80.40
52.40
37.96
-
4.52
83.10
39.36
160.80
58.51
74.89
-
IC50 values were expressed as mean values. * Indicates a selectivity index (SI) of S180 against L929. ** Indicates a
selectivity index (SI) of H22 against L929.
2.3. In Vivo Activity
Based on the above suggestion, 7a
,
7c, and 7e were selected for evaluating the antitumor activity
in H22 tumor bearing mice models. Twenty-four hours after implantation, the mice were randomly
divided into eight experimental groups. Normal saline (NS) and cyclophosphamide (CP) were utilized
as a negative control and positive control, respectively. For the tested compounds, the cytotoxicity
against H22 indicated 7c (IC50: 1.01 µM) > 7e (IC50: 1.85 µM) > 7a (IC50: 2.17 µM), and the tumor
inhibition rate showed 7c (54.83%) > 7e (47.78%) > 7a (46.95%) at the dose of 50 mg/kg. This suggested
that the in vivo antitumor potency correlated well with their in vitro antiproliferative activities.
Compared with CP groups, though their activities were weaker than that of positive control CP
(72.22%), 7a
even at the dose of 50 mg/kg. Compared with NS group, 7a
effect at the indicated dosage, there was no obvious different between 7c
7a and 7c containing electron-donating groups did not enhance binding affinity with corresponding
receptor in vivo compared with 7e. For 7a 7c, and 7e treated groups, the tumor inhibitory effects of
,
7c, and 7e did not significantly cause body weight change comparing with the NS group
7c and 7e treated groups showed similar
7a, and 7e, this indicated that
,
,
,
high dose were superior to that of low dose in all treated groups, and the reduction of tumor growth
depends on dosage. Seen in Figure 1.
Figure 1. The effect of different dosage of 7a, 7c, and 7e on the in vivo tumor growth of H22 mice.
Normal saline (NS) and cyclophosphamide (CP) were utilized as a negative control and positive control,
respectively. * p < 0.05, * p < 0.01, *** p < 0.001 compared with untreated control group.
To evaluate the toxicity of 7a, 7c, and 7e, necropsy of the dead mice was carried out. Organ-to-body
weights ratios were compared between treated (50 and 30 mg/kg dose) and vehicle groups of animals,
the data were listed in Figure 2. The thymus, brain, lung, heart, liver, kidney, and spleen indexes
of tested compounds were comparable to that of NS, and did not show any significant difference.

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Compared with NS, CP was found to significantly increase the liver and spleen indexes, suggesting that
CP could cause change in liver (p < 0.05) and spleen (p < 0.01) function of H22 mice. The results
indicated that 7a, 7c, and 7e induced low toxicity to the mice in vivo.
Figure 2. Effects of different dosage of 7a, 7c, and 7e on organ index in H22 bearing mice. Normal saline
(NS) and cyclophosphamide (CP) were utilized as a negative control and positive control, respectively.
p < 0.05, p < 0.01 compared with untreated control group.
2.4. Apoptosis Analysis
We use an annexin V-FITC/PI apoptosis detection kit to evaluate the efficacy of 7a
tumor cell line. The result is shown in Figure 3. Compared with the negative control, 7a 7e increased
the apoptosis at the concentration of 3 M for 48 h, the order of the ability to induce apoptosis is
–7e on H22
–
µ
7a > 7b > 7e > 7c > 7d. The results demonstrated that the antitumor activity of targeted compounds
was due to the induction of apoptosis in H22 tumor cell line.
Figure 3. Effects of 7a
–7e on cell apoptosis of H22 for 48 h: (1) control; (2) 7a, 3 µM; (3) 7b, 3 µM; (4) 7c,
3 µM; (5) 7d, 3 µM; and (6) 7e, 3 µM.

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2.5. Cell Cycle Analysis
To determine whether the suppression of cell growth by the compounds was caused by a cell-cycle
effect, we used a cell cycle analysis kit to detect the DNA content of cell nuclei. As shown in Figure 4,
when H22 cells were treated with 7a–7e at the concentration of 3 µM for 48 h, the percentage of
G1-phase cells increased from 15.32% to 32.02% associated with a percentage decrease of S-phase cells
(72.42% to 52.83%) for 7a, the percentage of G1-phase cells increased from 15.32% to 15.44% associated
with a percentage decrease of S-phase cells (72.42% to 71.86%) for 7b, the percentage of G1-phase cells
increased from 15.32% to 51.22% associated with a percentage decrease of S-phase cells (72.42% to
42.48%) for 7c, the percentage of G1-phase cells increased from 15.32% to 16.10% associated with a
percentage decrease of S-phase cells (72.42% to 68.24%) for 7d, and a percentage increase of G1-phase
cells (15.32% to 17.21%) associated with a percentage decrease of S-phase cells (72.42% to 71.04%)
for 7e. Cell cycle analysis showed that the compounds all induced G1 cell cycle arrest in H22 cells,
especially for 7a and 7c.
Figure 4. Cell cycle analysis by flow cytometry of H22 cells treated with 7a
(2) 7a, 3 µM; (3) 7b, 3 µM; (4) 7c, 3 µM; (5) 7d, 3 µM; and (6) 7e, 3 µM.
–7e for 48 h: (1) control;
2.6. Molecule Docking
Previously, Top I was found to be the antitumor target of sophoridine derivatives [23,24].
Typical Top I inhibitor camptothecin acts by stabilizing a covalent Top I-DNA complex called the
cleavable complex. Molecular docking studies were performed to find out the possible binding mode of
the compounds with DNA-Top I complex (Protein Data Bank (PDB) ID: 1T8I). 7a–7e was chosen for the
docking analysis. As seen in Figure 5, alkyl ester chain and parts of tricycle scaffold of the compound
is buried in the hydrophobic pocket by ILE427, TYR426, LEU429, ALA351, PRO431, and MET428
residues. Base-stacking interactions (
DNA base pair TGP11. The carbonyl oxygen atom of 7a
MET428, TYR426, ASN352, and ASN352, respectively. Moreover, the protonated-cationic nitrogen at
the bottom of the tricycle scaffold for 7a 7b, and 7e is involved in a hydrogen bond with the GLU365
π
–π
) are formed between the substituted aryl and the surrounding
–
7e formed a hydrogen bond with TYR426,
,
or LEU356 residue. The halogen bond for 7c and 7d is also observed. The docking statistics was seen
in Table S2. Camptothecin is the positive control, the docking results show that the stability between
7a–7e and DNA-Top I is more potent than that of CPT, indicating that the compounds have interaction
with DNA-Top I [25], the result suggested that the chemical modification retains the mode of action of
its parent.

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Figure 5. Schematic representation of the proposed binding mode for 7a–7e with Top I-DNA (Protein
Data Bank (PDB) code: 1T8I).
3. Materials and Methods
3.1. Instrumentation
All the compounds were characterized with 1H-NMR, 13C-NMR and ESI-MS. Nuclear magnetic
resonance data were recorded on spectrometer (Bruker Avance 400, Billerica, MA, USA) with CDCl3
as solvent, tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained from an
¯
LCMS-8040 (SHIMADZU, Kyoto, Japan). The optical density was measured at the 570 nm wavelength
on a multimode reader (Tecan Infinite M200, Zürich, Switzerland). The fluorescence intensity was
measured with a Flow Cytometer (BD FACSCanto II, East Rutherford, NJ, USA).
3.2. Materials
Sophoridine (98%) was obtained from Shanghai PureOne Biotechnology Co., Ltd. (Shanghai,
China). All the other chemicals reagents used in the experiments were of analytical grade and obtained
from Tianjin Chemical Reagent Co., Ltd. (Tianjin, China). Ethyl acetate, methanol, and petroleum
ether were distilled before use. Chloroform, acetonitrile and triethylamine were distilled from
CaH2. Tetrahydrofuran was purified by distillation under Ar atmosphere from Na/benzophenone
immediately prior to use.
3.3. Synthesis of Compounds
3.3.1. Synthesis of 2
Sophoridine 1 (0.03 mol, 7.44 g) was dissolved in 2 N sodium hydroxide solution and heated to
reflux for several days, after cooling down, the pH of the mixture was adjusted to 5–6 with 20% H2SO4
and then filtered. The solution was evaporated under vacuum and the resultant solid was crystallized
in methanol, the yellow solid was obtained and dried under vacuum. Yield (85%), m.p. 154~155 ◦C.
C15H26N2O2 (266.4): MS(ESI) m/z: [M + H]+ = 267.2, [M − H]− = 265.2.

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3.3.2. Synthesis of 3
To a stirred anhydrous methanol solution of
2
(0.02 mol, 5.33 g), the anhydrous methanol solution
of SOCl2 was added dropwise under ice bath. The mixture was stirred for 4 h and evaporated under
vacuum and light yellow solid was obtained, and then recrystallized in methanol-acetone, white solid
1
was obtained and dried under vacuum. Yield (63%). H-NMR (400 MHz, CDCl3)
δ: 11.55 (s, 1H),
9.43 (d, J = 38.0 Hz, 2H), 3.88 (s, 1H), 3.73 (d, J = 28.2 Hz, 4H), 3.53–3.34 (m, 3H), 3.21 (d, J = 46.1 Hz,
2H), 2.82 (d, J = 60.2 Hz, 3H), 2.35 (d, J = 65.8 Hz, 4H), 1.97 (d, J = 45.2 Hz, 9H), 1.70 (d, J = 11.0 Hz, 1H),
1.49 (s, 1H); 13C-NMR
δ: 173.11, 57.98, 56.84, 54.93, 52.82, 51.76, 45.41, 42.88, 33.82, 33.13, 27.70, 25.60,
22.35, 22.16, 21.56, 17.92; C16H28N2O2 (280.4) MS(ESI) m/z: [M + H]+ = 281.2.
3.3.3. Synthesis of 4
To a stirred chloroform solution of diethanol amine (0.20 mol, 21.01 g), the chloroform solution of
SOCl2 was added dropwise under ice-bath. The mixture was stirred for 6 h and evaporated under
vacuum and white solid was obtained, and then recrystallized in absolute alcohol, white needle solid
was obtained and dried under vacuum. Yield (50%). m.p. 215~216 ◦C. C4H10NCl3 (178.5), MS(ESI)
m/z: [M − HCl + H]+ = 142.0.
3.3.4. Synthesis of 5
Complex
4 (0.056 mol, 10.00 g) was resolved in 26 mL POCl3, and heated under reflux until
complete solution resulted. The excess POCl3 was removed by distillation and the residue distilled at
reduced pressure. The oil was crystallized as a solid mass on cooling, dried under vacuum. Yield (74%).
◦
m.p. 53~54 C. 1H-NMR (400 MHz, CDCl3)
δ
3.74 (dd, J = 9.9, 4.8 Hz, 4H), 3.68 (dd, J = 14.4, 8.6 Hz, 4H).
3.3.5. Synthesis of 6a–6e
To a stirred solution of 4-methyl phenol (10 mmol, 1.08 g) and
5
(12 mmol, 3.10 g) in anhydrous THF
◦
(30 mL) at 70 C oil bath, Et3N was added dropwise. The reaction solution was refluxed till TLC analysis
showed completion of the reaction and filtered, the filtrate was evaporated under vacuum and purified
by column chromatography on the silica gel with petroleum ether-dichloromethane/triethylamine as
eluents to give compound 6a as yellow oil in 61% yield.
Compound 6b–6e was prepared in the same manner as described above.
6a: Yield: 61%; yellow oil; 1H-NMR (400 MHz, CDCl3)
2H), 3.72 (d, J = 5.3 Hz, 8H), 2.35 (s, 3H); 13C-NMR
: 147.20, 136.10, 136.02, 130.50, 120.15, 120.10,
49.79, 49.31, 41.48, 40.81, 20.83; ESI-MS: C11H15Cl3NO2P (330.6), [M + Na]+ = 354.0.
δ: 7.19 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.6 Hz,
δ
6b: Yield: 68%; yellow oil; 1H-NMR (400 MHz, CDCl3)
6.90 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.78–3.71 (m, 4H), 3.69–3.57 (m, 4H); 13C-NMR
δ
: 7.19 (dd, J = 9.1, 1.8 Hz, 2H),
δ: 157.55, 143.07,
121.40, 121.35, 114.90, 114.89, 55.64, 49.87, 49.83, 41.46, 41.44; ESI-MS: C11H15Cl3NO3P (346.6),
[M + Na]+ = 370.1.
6c: Yield: 62%; yellow oil; 1H-NMR (400 MHz, CDCl3)
2H), 3.74 (dd, J = 9.7, 2.6 Hz, 4H), 3.65 (dd, J = 14.4, 7.1 Hz, 4H); 13C-NMR
130.11, 121.87, 121.81, 49.75, 49.71, 41.38, 41.36. ESI-MS: C10H12Cl4NO2P (351.0), [M + Na]+ = 374.0.
δ
: 7.38 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 7.3 Hz
,
δ: 148.04, 131.79, 131.76,
6d: Yield: 53%; yellow oil; 1H-NMR (400 MHz, CDCl3)
2H), 3.78–3.60 (m, 8H); 13C-NMR
41.57. ESI-MS: C10H12Cl3N2O4P (361.5), [M + Na]+ = 385.2.
δ: 8.31 (d, J = 9.1 Hz, 2H), 7.46 (d, J = 8.9 Hz
: 154.89, 145.11, 142.40, 126.17, 125.89, 120.71, 120.65, 49.17, 49.13,
,
δ

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6e: Yield: 65%; yellow oil; 1H-NMR (400 MHz, CDCl3)
δ: 7.42 (t, J = 7.5 Hz, 2H), 7.29 (d, J = 1.6 Hz,
2H), 3.75 (dd, J = 7.0, 2.1 Hz, 4H), 3.66 (dd, J = 19.1, 11.3 Hz, 4H); 13C-NMR
δ: 150.1, 130.08, 130.10,
126.25, 120.47, 120.42, 49.88, 49.84, 41.46, 41.44. ESI-MS: C10H13Cl3NO2P (316.5), [M + Na]+ = 340.1.
3.3.6. Synthesis of 7a–7e
To a stirred solution of 6a (0.30 mmol, 0.10 g) and
3 (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 anhydrous CH3CN was added dropwise.
The reaction solution was refluxed till TLC analysis showed completion of the reaction and filtered,
the filtrate was evaporated under vacuum and purified by column chromatography on the silica gel to
obtain colorless oil 7a.
7b–7e was prepared in the same manner as described above.
7a: Yield: 39%; colorless oil; 1H-NMR (400 MHz, CDCl3)
2H), 3.68 (d, J = 2.4 Hz, 2H), 3.57 (d, J = 6.5 Hz, 2H), 3.43 (d, J = 7.0 Hz, 2H), 3.05–2.80 (m, 4H),
2.62–2.44 (m, 4H), 2.44–2.09 (m, 8H), 2.08–1.98 (m, 2H), 1.81 (dd, J = 8.9, 4.1 Hz, 2H), 1.68 (dt, J = 11.4
6.3 Hz, 6H), 1.49 (t, J = 7.3 Hz, 2H), 1.45–1.27 (m, 4H); 13C-NMR
: 173.46, 130.17, 130.10, 120.03, 119.98,
δ: 7.15 (d, J = 3.4 Hz, 2H), 7.11 (d, J = 6.2 Hz,
,
δ
119.81, 119.76, 58.98, 58.54, 54.45, 51.56, 50.30, 49.97, 45.41, 44.32, 42.44, 38.58, 33.86, 33.68, 30.74, 29.16,
26.99, 25.73, 24.74, 22.29, 20.80, 18.97; ESI-MS: C27H42Cl2N3O4P (574.5), [M + H]+ = 575.2.
7b: Yield: 41%; colorless oil; 1H-NMR (400 MHz, CDCl3)
δ: 7.15 (dd, J = 25.4, 8.7 Hz, 2H), 6.84 (dd,
J = 9.1, 3.4 Hz, 2H), 3.78 (s, 3H), 3.65 (s, 3H), 3.54 (dd, J = 8.8, 2.9 Hz, 2H), 3.40 (dd, J = 6.9, 4.8 Hz,
2H), 3.20–3.12 (m, 2H), 3.04–2.86 (m, 3H), 2.85–2.73 (m, 2H), 2.62–2.36 (m, 3H), 2.37–2.06 (m, 3H),
1.91–1.70 (m, 5H), 1.70–1.57 (m, 6H), 1.56–1.39 (m, 2H), 1.28 (dd, J = 21.8, 13.5 Hz, 2H). 13C-NMR
δ:
173.62, 156.46, 144.88, 121.13, 120.93, 114.62, 114.59, 58.88, 58.54, 58.40, 55.67, 54.55, 51.66, 50.35, 49.95,
45.31, 42.29, 38.71, 37.88, 33.82, 30.85, 29.31, 26.87, 25.98, 24.92, 22.54, 19.02. ESI-MS: C27H42Cl2N3O5P
(590.5), [M + H]+ = 592.3.
7c: Yield: 42%; colorless oil; 1H-NMR (400 MHz, CDCl3)
δ: 7.26 (dd, J = 8.9, 4.4 Hz, 2H),
7.21 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 3.63 (s, 3H), 3.47 (dddd, J = 26.8, 20.3, 14.8, 7.3 Hz, 9H),
3.14 (td, J = 9.5, 4.8 Hz, 1H), 2.96–2.75 (m, 4H), 2.60–2.21 (m, 4H), 1.99–1.21 (m, 13H), 1.02 (t, J = 7.2 Hz,
1H); 13C-NMR
δ: 173.60, 149.90, 129.65, 121.60, 121.38, 77.44, 77.12, 76.80, 58.80, 58.65, 58.46, 54.50,
51.53, 51.51, 50.26, 50.22, 49.87, 49.83, 46.17, 45.35, 45.28, 44.95, 44.40, 44.35, 42.36, 42.21, 38.66, 37.88,
37.83, 33.71, 33.57, 30.80, 30.73, 30.68, 29.30, 29.27, 26.87, 25.89, 24.98, 24.91, 22.48, 22.32, 18.97. ESI-MS:
C26H39Cl3N3O4P (595.0), [M + H]+ = 596.2.
7d: Yield: 31%; light yellow oil; 1H-NMR (400 MHz, CDCl3)
8.9 Hz, 2H), 3.59 (d, J = 4.0 Hz, 3H), 3.48–3.35 (m, 5H), 3.17 (d, J = 9.9 Hz, 1H), 2.87 (dt, J = 32.4, 14.3 Hz,
4H), 2.70–2.05 (m, 6H), 1.97–1.85 (m, 1H), 1.79–1.15 (m, 15H); 13C-NMR
: 172.56, 155.15, 143.28, 124.68,
δ: 8.17 (d, J = 6.9 Hz, 2H), 7.38 (dd, J = 17.7,
δ
124.66, 119.63, 119.44, 57.82, 57.45, 53.42, 50.61, 50.57, 49.13, 49.09, 48.75, 48.70, 44.34, 41.36, 41.18, 37.51,
32.48, 28.09, 26.07, 24.65, 23.83, 21.31. ESI-MS: C26H39Cl2N4O6P (605.5), [M + H]+ = 606.3.
7e: Yield: 39%; colorless oil; 1H-NMR (400 MHz, CDCl3)
δ: 7.37 (d, J = 6.7 Hz, 2H), 7.32 (d, J = 5.8 Hz,
1H), 7.26 (d, J = 7.7 Hz, 1H), 7.20 (t, J = 7.0 Hz, 1H), 3.70 (s, 3H), 3.60 (qd, J = 17.8, 10.8 Hz, 5H),
3.47 (d, J = 6.3 Hz, 4H), 3.23 (t, J = 11.6 Hz, 1H), 2.90 (ddd, J = 26.2, 24.5, 12.3 Hz, 4H), 2.66–2.21 (m,
5H), 2.14–0.83 (m, 13H). 13C-NMR
δ: 173.73, 150.79, 129.75, 129.65, 124.63, 120.37, 120.08, 58.94, 58.43,
58.43, 54.56, 51.59, 50.31, 49.96, 44.45, 42.38, 38.69, 37.89, 33.69, 30.88, 29.29, 26.98, 25.93, 24.96, 22.57,
19.03. ESI-MS: C26H40Cl2N3O4P (560.5), [M + Na]+ = 582.2.

Molecules 2018, 23, 1960
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3.4. Anti-Proliferation Assay
The ability of anti-proliferation of compounds was evaluated by MTT assay. Targeted tumor cell
lines were grown in an RPMI-1640 medium supplemented with 10% fetal bovine serum. The cells
were incubated at 37 ◦C under a 5% CO2 atmosphere for 24 h prior to the addition of the compounds.
All the targeted compounds were dissolved in DMSO as stock solution. After a 48 h exposure
period, PBS containing MTT was added to each well for 4 h, the media with MTT were removed,
and DMSO was added, and then measured by UV spectrometer at 570 nm using a multimode reader
(Tecan infinite M200, Zürich, Switzerland), all experiments were performed in triplicate.
3.5. Measurement of Apoptosis by Annexin V Analysis
H22 Cells were seeded in a 6-well culture plate for 12 h at 37 ◦C and 5% CO2, and 7a
added to each well respectively at 3 M and incubated for 48 h. The H22 cells were then harvested by
trypainization and washed twice with cold PBS, centrifugated, resuspended in binding buffer which
was then added to 5 L of annexin V-FITC and incubated at room temperature for 15 min. After adding
10 L of PI, the cells were incubated for another 15 min in dark. The stained cells were analyzed by
–7e were
µ
µ
µ
a Flow Cytometer and the average percentage of Annexin V-positive cells was used as a measure
of apoptosis.
3.6. Cell Cycle Analysis
◦
H22 cells were treated with 7a
–
7e at 3
µ
M at 37 C for 48 h and then cells were harvested,
centrifuged, resuspended and fixed in 70% EtOH overnight at 4 ◦C. The fixed cells were digested by
RNase A (0.25 mg
mL−1) at 37 ◦C for 30 min, and stained with PI (50 mL−1) in the dark at room
temperature for 30 min. The distribution of the cell cycle was analyzed by Flow Cytometer.
·
µg
·
3.7. Molecular Docking
The chemical structures of 7a–7e were drawn with chemoffice, protonated using the protonate
3D protocol and subjected to an energy minimization with Ligand Preparation of Schrödinger using
OPLS_2005 force field. Then, all of the conformations of the studied compounds were generated
for further docking. The crystal structure of DNA-Top I in complex with camptothecin (PBD ID:
1T8I) was obtained from Protein Data Bank. Water molecules in the 1T8I.pdb file were deleted,
bond orders were assigned, and hydrogen atoms were added to the protein. And then the protein was
prepared using protein preparation and refinement tool. For the active site, a grid box centered at the
ligand camptothecin (EHD) was used to accommodate a maximum ligand length of 15 Å. Docking all
conformations of ligands flexibly with extra precision (XP) mode of Glide of default parameters,
and writing out a maximum of two poses per ligand, and also enabling the post-docking minimization
of the ligands. Glide score and glide energy are used as scoring functions.
3.8. Antitumor Activity In Vivo
Tumor-bearing mice were prepared by inoculating a suspension of H22 cells (1.2
×
106 cells per
mouse) subcutaneously into the right armpit of KM mousse, five days inoculation, according to body
weight, 40 mice were assigned to eight equal groups: i.v. Cyclophosphamide group (positive control,
50 mg/kg), 7a (50 mg/kg), 7a (30 mg/kg), 7c (50 mg/kg), 7c (30 mg/kg), 7e (50 mg/kg), 7e (30 mg/kg),
and isotonic saline group (negative control). All treatments were administered for 6 days. One day
after, mice were sacrificed, the body, neoplasm and organ weight were measured. The inhibition was
calculated as follows: inhibition (%) = [1
of control mice] × 100.
−
(mean tumor weight of tested mice)/(mean tumor weight
All in vivo assays were performed in the Central Laboratory of the Tianjin Institute of Medical
and Pharmaceutical Sciences, and approved by the Committee on Ethics of the Tianjin Institute of
Medical and Pharmaceutical Sciences. The ethical approval code is IMPS-EAEP-Z-17JCQNJC13600.

Molecules 2018, 23, 1960
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4. Conclusions
In conclusion, a series of novel phosphoramide mustard functionalized sophoridine derivatives
were evaluated for their cytotoxic potential against a panel of various cancer cell lines. All targeted
compounds bearing this type of scaffold are more sensitive to S180 and H22 cells with IC50 values
ranging from 1.01–3.65
difference in activity is modest and within the same order of magnitude. All targeted derivatives
distinctly are more cytotoxic to cancer cells than the tested non-cancerous cell L929. Compound 7a 7c
µM. Substitution on the aryl affects the activity of the compounds, while this
,
,
and 7e were selected for further antitumor therapeutic efficacy in vivo against mice bearing H22 liver
cancer. Compared with vehicle group, the selected compounds showed moderate tumor suppression
and are of low organ toxicity. Moreover, they could induce apoptosis and lead to G1 cell cycle arrest
in H22 tumor cells, molecular docking studies revealed that these compounds have interaction with
DNA-Top I, so they could be considered as the potent targeted antitumor agents to further develop.
Supplementary Materials: The following are available online. The synthesis and characterization of
and 6a 6e, time-dependent growth inhibition of 7c and 7d on H22 and S180 cell lines, cell cycle inhibition in H22
cell line by treating with 7a–7e, and docking statistics of 7a–7e against DNA-Top I.
2, 3, 4, 5,
–
Author Contributions: L.D. and Z.Y. synthesized and characterized the compounds; S.Z. performed the docking;
X.Z. and H.S. performed the activity assay experiments; D.L. prepared the manuscript and designed the study.
Funding: This work was supported by the Tianjin Natural Science Foundation (17JCQNJC13600).
Conflicts of Interest: The authors declare no potential conflicts of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).