Novel N-substituted sophoridinol derivatives as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.069

Using sophoridine (1) as the lead compound, a series of new N-substituted sophoridinic acid derivatives were designed, synthesized and evaluated for their cytotoxicity. SAR analysis indicated that introduction of a chlorobenzyl on the 12-nitrogen atom of sophoridinol might significantly enhance the antiproliferative activity. Of the newly synthesized compounds, sophoridinol analogue 9k exhibited a potent effect against six human tumor cell lines (liver, colon, breast, lung, glioma and nasopharyngeal). The mode of action of 9k was to inhibit the DNA topoisomerase I activity, followed by the G0/G1 phase arrest. It also showed a moderate oral bioavailability and good safety in vivo. Therefore, compound 9k has been selected as a novel-scaffold lead for further structural optimizations or as a chemical probe for exploring anticancer pathways of this kinds of compounds.

Full text of DOI:10.1016/j.ejmech.2014.04.069

European Journal of Medicinal Chemistry 81 (2014) 95e105
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel N-substituted sophoridinol derivatives as anticancer agents
Chong-Wen Bi 1, Cai-Xia Zhang 1, Ying-Hong Li, Sheng Tang, Hong-Bin Deng, Wu-Li Zhao,
*
*
Zhen Wang, Rong-Guang Shao , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Using sophoridine (1) as the lead compound, a series of new N-substituted sophoridinic acid derivatives
were designed, synthesized and evaluated for their cytotoxicity. SAR analysis indicated that introduction
of a chlorobenzyl on the 12-nitrogen atom of sophoridinol might significantly enhance the anti-
proliferative activity. Of the newly synthesized compounds, sophoridinol analogue 9k exhibited a potent
effect against six human tumor cell lines (liver, colon, breast, lung, glioma and nasopharyngeal). The
mode of action of 9k was to inhibit the DNA topoisomerase I activity, followed by the G0/G1 phase arrest.
It also showed a moderate oral bioavailability and good safety in vivo. Therefore, compound 9k has been
selected as a novel-scaffold lead for further structural optimizations or as a chemical probe for exploring
anticancer pathways of this kinds of compounds.
Received 19 December 2013
Received in revised form
23 April 2014
Accepted 23 April 2014
Available online 2 May 2014
Keywords:
Sophoridinol
Sophoridinic acid
Structureeactivity relationship
Antiproliferative
Topoisomerase I
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
acid, had an improved antiproliferative activity with IC50 of 15.4
mg/
ml in HepG2 cells, compared to its parent 1 (IC50 > 20 g/ml), with a
m
Sophoridine (1, Fig. 1), a quinolizidine natural product extracted
from Sophora flavescens, was approved by Chinese FDA in 2005 as
an anticancer drug against malignant trophoblastic tumors [1].
Nowadays, it has been widely used to elevate the therapeutic effi-
cacy against gastric cancer, liver cancer, and lung cancer in com-
bination with other anticancer drugs used in clinic including
vinorelbine, cisplatin and docetaxel et al. [2e6]. The mechanism of
action of 1 is to inhibit the DNA topoisomerase I (topo I) activity,
and arrest the cell cycle at the S-phase, then cause apoptotic cell
death [7e10]. Compared to topo I inhibitors in clinical use, the
camptothecin family drugs such as camptothecin and Topotecan
(TPT), compound 1 has couple of advantages, for example, special
scaffold, simple structure, good solubility and considerable oppor-
tunities of formulation for oral administration, suggesting a novel
family of antitumor agents with ideal druggable characteristics.
The structureeactivity relationship (SAR) of 1 has been recently
initiated with the D ring analysis with 1 as the lead compound [11].
The SAR results revealed that sophoridinic acid (2, Fig. 1) with a 3-
ring structure scaffold was more favorable than 1 with a 4-ring
scaffold. Compound 3 (Fig. 1), 12-N-bromoacetyl sophoridinic
mode of action as consistent with that of parent 1. The simple and
unique chemical scaffold and promising antiproliferative activity of
3 provoked our strong interest to further develop structural mod-
ifications and optimizations in an effort to discover a novel class of
anticancer candidates with a 3-ring scaffold. As shown in Fig. 1, in
the present study we would continue to carry out the SAR analysis
on the substituents on the 12-nitrogen atom, carboxyl acid group
and the configuration of the chiral center at position 5, respectively.
Based on this strategy, 47 new sophoridinic acid derivatives were
synthesized and screened for their antiproliferative activity in hu-
man HepG2 cell line.
Herein, we describe the synthesis, in vitro antitumor assess-
ment, SAR analysis, primary mechanism of action as well as phar-
macokinetic and safety evaluation in vivo of the newly synthesized
compounds.
2. Results and discussion
2.1. Chemistry
Forty-seven target compounds were synthesized and grouped
into sophoridinic core (5R-configuration) and matrinic core (5S-
configuration) as described in Schemes 1 and 2, respectively.
Compound 1 was hydrolyzed in 6 N HCl at the refluxing tempera-
ture to give 2, which was then esterified in methanol to provide the
* Corresponding authors.
E-mail addresses: shaor@yahoo.com (R.-G. Shao), songdanqingsdq@hotmail.
com (D.-Q. Song).
1
These authors made equal contribution to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.04.069
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

96
C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
Fig. 1. Structures of sophoridine (1), sophoridinic acid (2), compound 3 and the modification sites of constructed new scaffold (right).
Scheme 1. (a) 6 N HCl, reflux, 4 h; (b) CH3OH, 2 h; (c) R1CHO, triethylamine, 1,2-dichloroethane, reflux, 2 h; (d) sodium triacetoxyborohydride, reflux, 2 h; (e) 3 N HCl, reflux, 3 h; (f)
NH4HCO3/DIBOC, pyridine, CH3CN; (g) LiAlH4, THF; (h) methyl/ethyl benzenesulfonate, THF.
key intermediate 4 with a high yield of 95% [12]. Intermediate 4 was
condensated with the corresponding aldehydes in alkaline condi-
tion to give the Schiff base 5, which was reduced selectively using
sodium triacetoxyborohydride (STB) as a reducing agent to afford
the desired product 6 in 45%e62% yields. As shown in Scheme 1,
product 6 could not be obtained through alkylation of 4 with the
corresponding alkyl halide directly [13]. The product in series 7 was
gained by the hydrolyzation of 6 in 3 N HCl at the refluxing tem-
perature in a good yield. The amide product 8 was gained by
amination of 7 with NH4HCO3/DIBOC as a NH2-donator using pyr-
idine as a catalyst [14]. The alcohol derivative 9 was prepared via
reduction of 6 with LiAlH4 as a reducing agent in THF with over 80%
yields. Another ether product in series 10 was obtained via ether-
ification of 9 with methyl/ethyl benzenesulfonate in THF in yields of
76e87%.
previously [15]. As described in Scheme 2, the product 14 was
readily acquired through alkylation of 13 with the alkyl halide in
70e82% yields; but it could not be obtained by condensation with
the corresponding aldehydes owing to transferring into raw ma-
terial 11 in the amination conditions of 13. The final product 15 was
gained through the reduction of 14 with LiAlH4 as a reducing agent
mentioned above. All crude products were purified with flash
column chromatography on silica gel using CH2Cl2 and MeOH as
gradient eluents.
2.2. SAR analysis for the antiproliferative activity
All of the newly synthesized compounds were initially screened
for their cytotoxicity activities in human HepG2 hepatoma cells
with TPT as a positive reference using MTT assay. Structures of the
47 analogues and their antiproliferative activity at the concentra-
The second synthetic route used commercially available matrine
(11) as the starting material. The key intermediate 13 was gained by
hydrolyzation and esterification with the methods reported
tion of 10 mg/ml were summarized in Table 1.

C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
97
Scheme 2. (a) 3 N NaOH, reflux, 8 h; (b) 3 N HCl, MeOH, 2 h; (c) RCH2X, K2CO3, CH3CN; (d) LiAlH4, THF.
SAR analysis was first focused on the substituents at the 12-
nitrogen atom. The SAR study for a group of N-acyl sophoridinic
acid analogues has been carried out in our previous report [11]. In
this part, a variety of alkyl groups including aliphatic, benzyl, picolyl
were introduced into the nitrogen atom at the 12-position
respectively, by which 10 new N-substituted sophoridinic acids
(7aej) were generated and tested. As indicated in Table 1, all of
them showed a weak activity with inhibition rates less than 26% at
15c and the aimed targets. It was deduced that conformational
alteration into matrinic core might cause decreased activities, and
sophoridine scaffold was probably essential for the activity against
cancer. Among the newly synthesized compounds, sophoridinol 6g
and sophoridinic ester 9k were selected as the representative
compounds for further investigation.
the concentration of 10 mg/ml, regardless of the size of the side-
2.3. Antiproliferative activities of compounds 6g and 9k in six
chains. The results suggested that the improvement of ClogP in
this kind of compounds (ClogP ¼ 0.07e2.5) might be helpful for the
activity. Hence, a series of N-substitued sophoridinic esters (4, 6ae
j) with the improved ClogP value (ClogP ¼ 1.94e5.08) was then
gained, aiming to enhance the activity against cancer. As expected,
a couple of compounds (6d, 6feh) afforded significantly higher
cytotoxicity with inhibition rates ranging from 81.8% to 97.6%, much
higher than TPT. Similarly, among the new family of N-substituted
sophoridinol (9aen), compounds 9c and 9jek showed pleasing
antiproliferative effects with over 85% inhibition rate. The results
indicated that the introduction of a fluorobenzyl or chlorobenzyl on
the 12-nitrogen atom could significantly improve the anticancer
human tumor cell lines
Compounds 6g and 9k were further examined their anti-
proliferative effect in six human tumor cell lines including HepG2,
HCT116 (colon cancer), H1299 (lung cancer), U87 (malignant gli-
oma), MCF-7 (breast cancer) and KB (nasopharyngeal epidermoid
carcinoma). As described in Table 2, both of them showed ideal
cytotoxicities for the six solid tumor lines with IC50 values ranging
from 4
Especially, compounds 6g and 9k had potent activities for the KB
tumor cells with IC50 of 6.1 and 19.7 M respectively, much higher
than that of TPT (IC50 > 200 M). Thus, both of them were chosen to
mM to 20 mM, indicating a broad antiproliferative spectra.
m
m
activity. As indicated in Fig. 2, p-p conjugation between Cl atom and
carry out for further druggable evaluations in vivo studies.
phenyl might be helpful for the binding of compound 9k and target
topo I residues.
Next, we retained the fluorobenzyl or chlorobenzyl at the 12-
position as a pharmacophore for activity, and replaced the car-
boxylic acid group with ether or amide to explore the SAR of the
carboxyl variation. Therefore, several N-substituted sophoridinic
ether (10aef) and amide (8) were made and tested. The results
showed that all of them lost their anticancer activity completely
with less than 7% inhibition rate at the same concentration. The
results revealed that sophoridinic ester/alcohol exhibited potent
antiproliferative effect among this kind of compounds.
In another variation, SAR study was carried out to investigate
the (R)- or (S)-configuration of the 5-chiral center for the activity,
and a group of N-substituted matrinic ester/alcohol (14, 15aec)
were prepared. The results showed that most of them lost the ac-
tivity partly or completely, compared to their corresponding
sophoridinic ester/alcohol. As shown in Fig. 2, the docking analysis
showed that multiple hydrogen bonds formed between 9k and
topo I residues, while there was no obvious interaction between
2.4. In vivo pharmacokinetic and safety assessment of 6g and 9k
The in vivo pharmacokinetic (PK) behaviors of compound 6g and
9k were investigated in rat model. The tested compounds were
administered via oral (ig, 25 mg/kg) and intravenous (iv, 5 mg/kg)
routes to adult male SpragueeDawley (SD) rats. As anticipated,
ester compound 6g was rapidly hydrolyzed into the acid metabolite
7h in vivo (data not shown), which had no anticancer activity
in vitro (Table 1). As indicated in Table 3, the actual oral bioavail-
ability of alcohol 9k in this experiment was moderate (F ¼ 18%) in
the rats.
Single-dose toxicity tests were carried out in mice. After 6g and
9k were given by intragastric administration (i.g) at a dose of 250,
500 or 1000 mg/kg respectively, no death of the mice was observed.
In addition, this treatment with 6g and 9k showed no effect on
body weight of mice as well (data not shown). The results sug-
gested that both of them were considerably safe in vivo.

98
C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
Table 1
indicated in Fig. 3B, different from the parent, compound 6g caused
a major shift of the cell population from G0/G1 to G2/M phase,
indicating a significant change of the action mode after the ester-
ification of carboxyl group. Then, we accessed the inhibitory ac-
tivity of 6g and 9k on DNA Top I. Compound 6g did not show
significant inhibitory activity of Top I at the concentration of 15 mg/
ml (data not shown), while compound 9k significantly inhibited
the activity of Top I at the same concentration as shown in Fig. 4,
which was consistent with that of the parent compound 1 or 2. The
mode of action of 6g is further being investigated in our laboratory.
Structureeactivity relationships of the sophoridinic acid derivatives for their anti-
proliferative activities in HepG2 cells.
3. Conclusion
Taken together, 47 new sophoridinic acid derivatives were
designed, synthesized and evaluated for their antitumor activities.
SAR analysis indicated that (i) R-configuration at the 5-position was
crucial; (ii) introduction of a chlorobenzyl on the 12-nitrogen atom
of the sophoridinol might significantly enhance the activity. Among
them, compound 9k showed a potent anticancer effect in the six
tested tumor cell lines. Its mode of action was to inhibit the activity
of DNA topo I, followed by the G0/G1 phase arrest. In addition, it
displayed a good PK profile and safety in vivo. We consider N-
substituted sophoridinol derivatives to be a novel scaffold of topo I
inhibitors against cancer with good druggable characteristics.
Compound 9k was selected as a novel-scaffold lead for further
structural optimizations or a chemical probe for revealing the
biological pathways against cancer of this kind of compounds.
Compd
5-H
R1
R2
Inhibition ratea
ClogPb
4
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
H
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
33.4 ꢃ 1.5
24.9 ꢃ 0.6
26.8 ꢃ 0.7
60.8 ꢃ 1.0
81.8 ꢃ 1.0
73.1 ꢃ 2.4
84.9 ꢃ 1.9
97.6 ꢃ 2.4
89.6 ꢃ 2.8
29.9 ꢃ 3.6
16.6 ꢃ 5.1
25.3 ꢃ 4.7
10.7 ꢃ 2.4
1.5 ꢃ 1.0
1.6 ꢃ 1.7
25.2 ꢃ 0.8
2.0 ꢃ 2.0
22.8 ꢃ 3.9
3.5 ꢃ 1.9
24.9 ꢃ 0.3
22.3 ꢃ 3.5
1.7 ꢃ 3.2
0.9 ꢃ 0.4
19.7 ꢃ 8.0
95.7 ꢃ 3.3
9.1 ꢃ 2.6
18.7 ꢃ 5.7
17.8 ꢃ 0.7
30.9 ꢃ 3.4
24.3 ꢃ 4.2
35.7 ꢃ 5.8
88.1 ꢃ 1.9
87.7 ꢃ 2.1
1.2 ꢃ 0.3
33.2 ꢃ 2.0
34.6 ꢃ 6.5
4.4 ꢃ 1.6
2.1 ꢃ 2.4
6.8 ꢃ 3.7
5.1 ꢃ 3.2
0.3 ꢃ 0.1
0.2 ꢃ 0.1
0.8 ꢃ 4.4
31.0 ꢃ 4.8
0.1 ꢃ 14.1
4.9 ꢃ 6.8
72.6 ꢃ 2.1
1.94
3.53
3.79
4.36
4.36
4.36
4.93
4.93
5.08
2.72
3.39
0.88
1.28
1.57
1.71
1.71
1.71
2.28
2.28
2.43
0.07
3.63
3.11
3.64
3.91
3.79
3.80
4.30
3.94
3.94
3.94
4.51
4.51
2.30
2.98
2.72
4.51
4.90
4.66
5.04
5.23
5.61
4.36
3.80
3.94
4.51
6a
6b
6c
6d
6e
6f
6g
6h
6i
CH2CH2CH3
CH2CH(CH3)2
CH2PhF-o
CH2PhF-m
CH2PhF-p
CH2PhCl-m
CH2PhCl-p
CH2PhBr-p
4-picolyl
2-furfuryl
CH2CH2CH3
CH2CH(CH3)2
CH2Ph
CH2PhF-o
CH2PhF-m
CH2PhF-p
CH2PhCl-m
CH2PhCl-p
CH2PhBr-p
4-picolyl
CH2PhCl-p
CH2CH2CH3
CH2CH2CH2CH3
CH2C(CH3)3
CH2CH(CH3)2
CH2Ph
6j
7a
7b
7c
7d
7e
7f
7g
7h
7i
4. Experimental section
4.1. Chemistry
7j
8
COOH
CONH2
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OCH3
CH2OCH2CH3
CH2OCH3
CH2OCH2CH3
CH2OCH3
CH2OCH2CH3
COOCH3
CH2OH
CH2OH
CH2OH
Melting points (mp) were obtained with CXM-300 melting
point apparatus. 1H NMR and 13C NMR spectra were recorded on a
Bruker Avance 400 spectrometer using deuterated DMSO or CH3OH
as a solvent and tetramethylsilane (TMS) as an internal standard.
ESI high-resolution mass spectra (HRMS) were recorded on an
AutospecUltima-TOF spectrometer. Flash column chromatography
was performed on Combiflash Rf 200, particle size 0.038 mm. HPLC
9a
9b
9c
9d
9e
9f
9g
9h
9i
CH2PhCH3-p
CH2PhF-o
CH2PhF-m
CH2PhF-p
CH2PhCl-o
CH2PhCl-p
4-picolyl
2-furfuryl
CH2CH2Ph
CH2Ph
analyses were performed on an Agilent 1200 using a 5.0 mm C18
9j
9k
9l
reversed phase column. For purities obtained by HPLC, the major
peak accounted for ꢀ95% of the combined total peak area moni-
tored by a MWD detector at 220 nm.
9m
9n
10a
10b
10c
10d
10e
10f
14
15a
15b
15c
TPT
4.1.1. Methyl sophoridinate dihydrochloride (4)
CH2Ph
Compound 1 (4.96 g, 20 mmol) was evenly dispersed in 6 N
hydrochloric acid solution (50 ml) and heated to reflux for 4 h. Then
the solvent was evaporated under vacuum and anhydrous meth-
anol (50 ml) was added into the residue. The reaction mixture was
stirred at room temperature for 2 h, and then the solvent was
evaporated under vacuum. The resultant solid was purified by flash
chromatography over silica gel to obtain white solid 4 (6.72 g, 95%).
CH2PhF-p
CH2PhF-p
CH2PhCl-p
CH2PhCl-p
CH2PhF-p
CH2Ph
S
S
S
CH2PhF-p
CH2PhCl-p
Mp 112e114 ꢁC; 1H NMR (DMSO-d6)
d: 11.61 (s, 1H), 9.67 (s, 1H),
9.29 (s, 1H), 3.61 (s, 3H), 3.60e3.30 (m, 3H), 3.22e2.93 (m, 3H),
2.86e2.69 (m, 1H), 2.54e2.49 (m, 1H), 2.48e2.31 (m, 4H), 2.12 (m,
a
% of inhibition. Cells were untreated (control) or treated with test compounds
g/ml) for 48 h, respectively.
ClogP value was generated by software Chembiooffice (2010).
(10
m
1H), 1.98e1.58 (m, 9H), 1.51e1.27 (m, 2H); 13C NMR
d: 172.8, 56.1,
b
55.4, 51.6, 51.3, 44.1, 41.6, 33.2, 32.8, 26.8, 24.9, 24.8, 21.8, 21.2, 20.8,
17.0; HRMS: calcd for C16H30N2O2Cl2 [Mꢂ2HClþH]þ, 281.2223;
found, 281.2222.
2.5. Primary mechanism of action of the representative compounds
We extended our work to the primary mechanism investigation
of compounds 6g and 9k. Flow cytometric analysis in the HepG2
cells was carried out. As shown in Fig. 3A, compound 9k attested
HepG2 cells at the G0/G1 phase as anticipated, indicating a similar
mechanism of action with its parent compound 1 or 2. However, as
4.1.2. General procedures for the synthesis of the compounds 6aej
To a stirred solution of 4 (2.81 g, 10.0 mmol) and triethylamine
(2.8 ml, 20.0 mmol) in 1,2-dichloroethane (50 ml), aldehyde
(15.0 mmol) was added dropwise. The reaction mixture was
refluxed for 2 h and STB (3.16 g, 15.0 mmol) was added into the

C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
99
Fig. 2. Stereo viewing docked models of 9k (left) and 15f (right) in the active site of DNA topo I complex (from PDB: 1K4T). Molecular figures were generated by Discovery Studio
Client.
reaction mixture slowly. The reaction solution was refluxed till TLC
analysis showed completion of the reaction. After cooling down,
the mixture was separated and washed successively with water
(20 ml), brine (20 ml). The organic layer was evaporated under
vacuum, and the residue was purified by flash chromatography
over silica gel or acidized with 3 N hydrochloride/ether (10 ml) to
obtain the desired product.
4.1.2.2. Methyl 12-N-isobutylsophoridinate dihydrochloride (6b).
The title compound was prepared from 4 and isobutyraldehyde in
the same manner as described above. Yield: 62%; Yellow solid; mp
89e92 ꢁC; 1H NMR (DMSO-d6)
d: 11.69 (s, 1H), 10.09 (s, 1H), 3.63 (s,
3H), 3.41e3.33 (m, 2H), 3.32e3.01 (m, 5H), 3.00e2.77 (m, 3H),
2.71e2.47 (m, 4H), 2.47e2.30 (m, 2H), 1.99e1.50 (m, 10H), 1.46e
1.30 (m, 1H), 1.04e0.83 (m, 6H); 13C NMR
d: 172.8, 60.0, 58.8, 54.9,
52.3, 51.4, 51.3, 44.1, 33.0, 32.7, 25.5, 24.7, 22.9 (2), 21.8, 21.6, 20.9,
20.8, 20.7, 17.2; HRMS: calcd for C20H38N2O2Cl2 [Me2HClþH]þ,
337.2849; found, 337.2852.
4.1.2.1. Methyl 12-N-n-propylsophoridinate dihydrochloride (6a).
The title compound was prepared from 4 and propanal in the same
manner as described above. Yield: 51%; Yellow solid; mp 100e
4.1.2.3. Methyl 12-N-2-fluorobenzylsophoridinate (6c). The title
compound was prepared from 4 and 2-fluorobenzaldehyde in the
same manner as described above. Yield: 57%; Thick yellow solid;
103 ꢁC; 1H NMR (DMSO-d6)
d: 11.59 (s, 1H), 10.72 (s, 1H), 3.61 (s,
3H), 3.43e3.23 (m, 2H), 3.23e2.99 (m, 5H), 2.98e2.74 (m, 3H),
2.69e2.47 (m, 4H), 2.41e2.19 (m, 2H), 1.87e1.49 (m, 10H), 1.37e1.19
mp 52e54 ꢁC; 1H NMR (DMSO-d6)
d: 7.42e7.25 (m, 2H), 7.21e7.06
(m, 2H), 0.97e0.81 (m, 3H); 13C NMR
d
: 172.8, 60.1, 55.1, 54.2, 51.4,
(m, 2H), 3.61 (s, 3H), 2.90e2.73 (m, 3H), 2.63e2.41 (m, 4H), 2.39e
2.26 (m, 3H), 2.26e2.15 (m, 1H), 2.11e1.94 (m, 2H), 1.86e1.29 (m,
51.3, 51.1, 44.1, 33.3, 32.7, 25.5, 24.8, 21.9, 21.6, 21.2, 21.0, 17.0, 16.6,
10.8; HRMS: calcd for C19H36N2O2Cl2 [Me2HClþH]þ, 323.2693;
found, 323.2696.
10H), 1.28e1.18 (m, 1H), 1.10e0.91 (m, 2H); 13C NMR
d: 173.7, 162.3
(d, J ¼ 242 Hz), 130.8, 128.9, 127.1, 124.6, 115.3, 64.2, 58.4, 54.5, 51.6,
Table 2
Antiproliferative activities (IC50
, mM) of the compounds 6g and 9k in six human tumor cell lines.
HepG2
HCT116
H1299
U87
MCF-7
KB
6g
9k
TPT
4.05 ꢃ 0.46
4.59 ꢃ 0.42
10.64 ꢃ 0.22
4.32 ꢃ 2.59
4.06 ꢃ 0.33
5.55 ꢃ 0.31
10.97 ꢃ 0.62
12.60 ꢃ 0.94
4.52 ꢃ 10.04
0.16 ꢃ 0.02
20.02 ꢃ 0.77
5.73 ꢃ 0.48
14.3 ꢃ 1.03
6.11 ꢃ 0.17
19.71 ꢃ 2.02
>200
9.37 ꢃ 0.49
1.66 ꢃ 0.06
HepG2: hepatoma. HCT116: colon cancer. H1299: lung cancer. U87: malignant glioma. MCF-7: breast cancer. KB: nasopharyngeal epidermoid carcinoma.
Table 3
Pharmacokinetic parametersa of 9k in rats after single oral and intravenous dosing (n ¼ 3).
Route
Dosage (mg/kg)
Cmax
(
m
M)
Tmax (h)
AUC
(
m
M$h)
MRT (h)
t1/2 (h)
F (%)
18
0et
iv
ig
5
25
3.43 ꢃ 1.58
5.25 ꢃ 1.66
0.25 ꢃ 0.00
0.66 ꢃ 0.29
20.51 ꢃ 1.28
18.60 ꢃ 2.95
2.39 ꢃ 1.13
3.28 ꢃ 0.37
4.46 ꢃ 1.06
3.87 ꢃ 0.53
a
PK parameters were calculated by non-compartmental analysis using WinNonlin, version 5.3.

100
C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
Fig. 3. Cell cycle analysis of 9k (A) and 6g (B). HepG2 cells were incubated without (control) or with compounds at different concentrations (2.5, 5, 10
then analyzed for their cell cycle distribution using flow cytometry.
mg/ml) for 24 h. Cells were
51.5, 51.3, 45.4, 38.1, 33.8, 29.9, 26.6, 26.1, 25.6, 23.3, 23.0, 19.2;
4.1.2.5. Methyl 12-N-4-fluorobenzylsophoridinate (6e). The title
compound was prepared from 4 and 4-fluorobenzaldehyde in the
same manner as described above. Yield: 59%; Thick yellow solid;
HRMS: calcd for C23H33N2O2F [MþH]þ, 389.2598; found, 389.2593.
mp 59e61 ꢁC; 1H NMR (DMSO-d6)
d: 7.43e7.32 (m, 2H), 7.23e7.03
(m, 2H), 3.63 (s, 3H), 3.17e3.01 (m, 2H), 2.92e2.69 (m, 3H), 2.57e
2.40 (m, 3H), 2.36e2.18 (m, 3H), 2.06e1.86 (m, 3H), 1.80e1.60 (m,
3H), 1.60e1.41 (m, 5H), 1.40e1.31 (m, 1H), 1.31e1.23 (m, 1H), 1.19e
4.1.2.4. Methyl 12-N-3-fluorobenzylsophoridinate dihydrochloride
(6d). The title compound was prepared from
fluorobenzaldehyde in the same manner as described above.
Yield: 51%; White solid; mp 98e101 ꢁC; 1H NMR (DMSO-d6)
: 11.47
4 and 3-
0.94 (m, 2H); 13C NMR
d
: 173.7,160.3 (d, J ¼ 242 Hz),136.6,130.2 (2),
d
115.2 (2), 63.5, 58.6, 57.5, 54.1, 51.6, 49.0, 45.3, 37.8, 33.8, 29.4, 26.9,
25.7, 25.2, 23.2, 22.7, 19.1; HRMS: calcd for C23H33N2O2F [MþH]þ,
389.2598; found, 389.2593.
(s, 1H), 11.11 (s, 1H), 7.90e7.68 (m, 2H), 7.50e7.29 (m, 2H), 4.42e
4.21 (m, 2H), 3.94e3.74 (m, 1H), 3.60 (s, 3H), 3.35e3.01 (m, 6H),
3.01e2.80 (m, 3H), 2.75e2.42 (m, 4H), 1.98e1.62 (m, 5H), 1.60e1.35
(m, 4H), 1.27e1.01 (m, 1H); 13C NMR
d
: 172.6, 162.1 (d, J ¼ 242 Hz),
4.1.2.6. Methyl 12-N-3-chlorobenzylsophoridinate dihydrochloride
131.9, 130.8, 128.8, 117.3, 116.3, 60.9, 55.5, 54.6, 51.5, 51.3, 44.1, 33.4,
32.5, 25.5, 24.7, 21.8, 21.6, 21.0, 20.7, 17.1; HRMS: calcd for
(6f). The title compound was prepared from
chlorobenzaldehyde in the same manner as described above.
Yield: 49%; Yellow solid; mp 96e98 ꢁC; 1H NMR (DMSO-d6)
: 11.52
4
and 3-
C
23H35N2O2FCl2 [Me2HClþH]þ, 389.2598; found, 389.2599.
d
(s, 1H), 11.12 (s, 1H), 8.10 (s, 1H), 7.85 (d, J ¼ 7.1 Hz, 1H), 7.56e7.43
(m, 2H), 4.41e4.20 (m, 2H), 3.94e3.74 (m, 1H), 3.60 (s, 3H), 3.31e
3.00 (m, 6H), 2.96e2.71 (m, 3H), 2.58e2.13 (m, 6H), 1.97e1.77 (m,
5H), 1.42e1.13 (m, 3H); 13C NMR
d: 172.6, 133.5, 131.9, 130.6, 130.5,
129.4, 127.3, 60.3, 55.0, 54.5, 51.5, 51.3, 44.1, 40.1, 38.8, 33.5, 32.6,
25.6, 24.7, 21.7, 21.1, 20.8, 17.1; HRMS: calcd for C23H35N2O2Cl3
[Mꢂ2HClþH]þ, 405.2303; found, 405.2309.
4.1.2.7. Methyl 12-N-4-chlorobenzylsophoridinate (6g). The title
compound was prepared from 4 and 4-chlorobenzaldehyde in the
same manner as described above. Yield: 53%; Yellow thick solid;
mp 62e64 ꢁC; 1H NMR (DMSO-d6)
d: 8.16e6.76 (m, 4H), 3.69e3.43
(m, 4H), 3.43e3.22 (m, 2H), 3.22e2.70 (m, 5H), 2.61e2.39 (m, 5H),
2.39e2.17 (m, 2H), 2.19e1.86 (m, 2H), 1.71e1.41 (m, 5H), 1.41e1.12
Fig. 4. DNA Top I inhibitory activity of 9k at different concentrations (0.2, 0.4, 0.6, 0.8
and 1.0 mg/ml).

C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
101
(m, 3H), 1.14e0.92 (m, 1H); 13C NMR
d: 173.7, 139.6, 131.5 (2), 130.3
4.1.3.2. 12-N-n-Butylsophoridinol dihydrochloride (9b). The title
(2), 128.6, 81.8, 63.6, 58.6, 57.5, 53.9, 51.6, 51.3, 49.0, 45.2, 33.8, 27.0,
25.4, 24.9, 23.2, 22.7, 19.0; HRMS: calcd for C23H33N2O2Cl [MþH]þ,
405.2303; found, 405.2307.
compound was prepared in the same manner as described above.
Yield: 83%; Yellow solid; mp 118e121 ꢁC; 1H NMR (DMSO-d6)
d:
11.65 (s, 1H), 10.72 (s, 1H), 4.71e4.35 (m, 2H), 3.78e3.26 (m, 6H),
3.21e2.75 (m, 5H), 2.73e2.08 (m, 5H), 1.69e1.52 (m, 3H), 1.53e1.35
(m, 6H), 1.34e1.10 (m, 4H), 1.00e0.65 (m, 4H); 13C NMR
d: 60.9,
4.1.2.8. Methyl 12-N-4-bromobenzylsophoridinate dihydrochloride
56.4, 53.0, 52.0, 49.0, 44.6, 36.0, 35.5, 33.1, 32.6, 28.0, 25.7, 25.3,
23.7, 22.5, 20.0, 19.0, 17.8, 14.1; HRMS: calcd for C19H38N2OCl2
[Mꢂ2HClþH]þ, 309.2900; found, 309.2901.
(6h). The title compound was prepared from
bromobenzaldehyde in the same manner as described above.
Yield: 45%; White solid; mp 88e91 ꢁC; 1H NMR (DMSO-d6)
: 11.49
4 and 4-
d
(s, 1H), 11.08 (s, 1H), 7.86 (d, J ¼ 8.2 Hz, 2H), 7.65 (d, J ¼ 8.3 Hz, 2H),
4.37e4.18 (m, 2H), 3.93e3.78 (m, 1H), 3.60 (s, 3H), 3.36e3.20 (m,
2H), 3.15e3.00 (m, 2H), 2.99e2.81 (m, 2H), 2.74e2.41 (m, 5H),
2.31e2.14 (m, 2H), 1.88e1.62 (m, 6H), 1.60e1.34 (m, 3H), 1.21e1.09
4.1.3.3. 12-N-Neopentylsophoridinol (9c). The title compound was
prepared in the same manner as described above. Yield: 86%; White
solid; mp 81e83 ꢁC; 1H NMR (DMSO-d6)
d: 3.15e2.97 (m, 4H),
2.63e2.51 (m, 2H), 2.48e2.27 (m, 4H), 2.24e2.01 (m, 4H), 2.02e1.74
(m,1H); 13C NMR
d: 172.7,132.8 (2),131.8,129.0 (2),123.0, 60.1, 55.0,
(m, 4H), 1.74e1.26 (m, 7H), 1.27e0.98 (m, 3H), 0.82 (s, 9H); 13C NMR
54.6, 51.5, 51.3, 48.5, 44.1, 33.4, 32.5, 25.6, 24.7, 21.8, 21.6, 21.0, 20.7,
17.1; HRMS: calcd for C23H35N2O2Cl2Br [Mꢂ2HClþH]þ, 449.1798;
found, 449.1806.
d: 66.0, 65.9, 61.1, 59.1, 53.9, 52.3, 44.8, 35.9, 34.1, 33.1, 28.2, 27.8 (3),
26.5, 24.3, 24.1, 23.4, 22.9, 18.1; HRMS: calcd for C20H38N2O
[MþH]þ, 323.3056; found, 323.3058.
4.1.2.9. Methyl 12-N-4-picolylsophoridinate dihydrochloride (6i).
4.1.3.4. 12-N-Isobutylsophoridinol dihydrochloride (9d). The title
The title compound was prepared from
pyridinecarboxaldehyde in the same manner as described above.
Yield: 56%; White solid; mp 89e91 ꢁC; 1H NMR (DMSO-d6)
: 11.71
4
and 4-
compound was prepared in the same manner as described above.
Yield: 84%; White solid; mp 119e121 ꢁC; 1H NMR (DMSO-d6)
d:
d
11.68 (s, 1H), 10.14 (s, 1H), 3.55e3.22 (m, 7H), 3.23e2.80 (m, 8H),
2.78e2.36 (m, 4H), 2.23e2.09 (m, 2H), 1.26e1.12 (m, 2H), 1.05e0.96
(s, 1H), 9.14e8.92 (m, 2H), 8.75e7.99 (m, 2H), 4.86e4.03 (m, 2H),
3.60 (s, 3H), 3.47e3.35 (m, 2H), 3.24e2.66 (m, 6H), 2.57e2.33 (m,
3H), 2.34e2.05 (m, 3H), 2.05e1.76 (m, 4H), 1.76e1.12 (m, 5H), 1.10e
(m, 3H), 0.96e0.87 (m, 3H), 0.83e0.77 (m, 6H); 13C NMR
d
: 61.1,
60.8, 59.2, 55.4, 44.6, 33.5, 32.5, 26.0, 23.5, 22.5 (2), 22.4, 22.3, 21.4,
0.74 (m, 1H); 13C NMR
d: 173.9, 145.9, 143.0 (2), 125.0 (2), 61.7, 51.5,
21.3, 21.1, 21.0, 20.9, 17.7; HRMS: calcd for
C19H38N2OCl2
[Mꢂ2HClþH]þ, 309.2900; found, 309.2898.
51.3, 48.5, 44.5, 44.2, 33.7, 33.1, 32.8, 25.8, 25.0, 21.9, 21.4, 21.3, 21.0,
17.2; HRMS: calcd for C22H35N3O2Cl2 [Mꢂ2HClþH]þ, 372.2645;
found, 372.2644.
4.1.3.5. 12-N-Benzylsophoridinol dihydrochloride (9e). The title
compound was prepared in the same manner as described above.
Yield: 80%; White solid; mp 104e107 ꢁC; 1H NMR (DMSO-d6)
d:
4.1.2.10. Methyl 12-N-2-furfurylsophoridinate dihydrochloride (6j).
The title compound was prepared from 4 and 2-furaldehyde in the
same manner as described above. Yield: 56%; Yellow solid; mp 98e
11.58 (s, 1H), 11.02 (s, 1H), 7.88e7.51 (m, 2H), 7.43e7.21 (m, 3H),
4.42e4.19 (m, 2H), 3.74e3.42 (m, 1H), 3.37e3.22 (m, 4H), 3.09e2.74
(m, 4H), 2.75e2.53 (m, 2H), 2.54e2.32 (m, 2H), 2.00e1.55 (m, 7H),
111 ꢁC; 1H NMR (DMSO-d6)
d: 11.60 (s, 1H), 11.10 (s, 1H), 7.75 (d,
1.55e1.34 (m,1H), 1.33e1.15 (m, 4H), 0.90e0.66 (m, 1H); 13C NMR
d:
J ¼ 7.1 Hz, 1H), 6.87 (m, 1H), 6.55 (d, J ¼ 7.1 Hz, 1H), 4.62e4.20 (m,
5H), 3.62e3.28 (m, 1H), 3.15 (s, 3H), 3.06e2.84 (m, 3H), 2.78e2.60
(m, 2H), 2.41e2.14 (m, 2H), 1.96e1.69 (m, 5H), 1.65 (m, 4H), 1.43e
131.0 (2), 130.2, 129.8, 129.3 (2), 61.2, 60.6, 55.8, 55.6, 52.1, 52.0,
44.6, 33.8, 32.1, 26.1, 25.2, 22.6, 22.3, 22.2, 21.8, 17.6; HRMS: calcd
for C22H36N2OCl2 [Mꢂ2HClþH]þ, 343.2744; found, 343.2743.
1.30 (m, 3H), 1.28e1.15 (m, 1H); 13C NMR
d: 172.7, 144.6, 143.9,114.7,
111.2, 61.2, 54.9, 51.5, 51.3, 50.5, 48.5, 48.3, 44.1, 33.4, 32.7, 25.7, 24.8,
22.0, 21.8, 21.1, 21.0; HRMS: calcd for C21H34N2O3Cl2 [Mꢂ2HClþH]þ,
361.2485; found, 361.2487.
4.1.3.6. 12-N-4-Methylbenzylsophoridinol
dihydrochloride
(9f).
The title compound was prepared in the same manner as described
above. Yield: 89%; White solid; mp 104e107 ꢁC; 1H NMR (DMSO-d6)
d
: 11.50 (s, 1H), 10.90 (s, 1H), 7.73 (d, J ¼ 7.7 Hz, 2H), 7.22 (d,
4.1.3. General procedures for the synthesis of compounds (9aen)
After the suspension of LiAH4 (0.5 g, 12.0 mmol) in anhydrous
tetrahydrofuran, Compound 6 (4.0 mmol) in anhydrous tetrahy-
drofuran was added dropwise under strict moistureless conditions.
Then the reaction solution was stirred at room temperature till TLC
analysis showed completion of the reaction. The reaction system
was deactivated successively with water (0.5 ml), 3 N NaOH
aqueous solution (0.5 ml) under ice bath condition. The reaction
mixture was filtered and the filtrate was evaporated to afford crude
target compound, which was purified by flash chromatography
over silica gel or acidized with 3 N hydrochloride/ether (10 ml) to
obtain desired compounds.
J ¼ 7.7 Hz, 2H), 4.43e4.24 (m, 1H), 4.13e3.71 (m, 1H), 3.43e3.26 (m,
3H), 2.98e2.75 (m, 2H), 2.67e2.40 (m, 3H), 2.39e2.03 (m, 4H),
2.00e1.53 (m, 7H), 1.53e1.14 (m, 8H), 0.90e0.68 (m, 2H); 13C NMR
d: 139.2, 131.0, 129.8 (2), 127.1 (2), 61.1, 60.6, 55.6, 52.0, 49.0, 48.1,
44.6, 33.8, 32.2, 27.9, 26.1, 25.2, 22.6, 22.3, 21.8, 21.3, 17.6; HRMS:
calcd for C23H38N2OCl2 [Mꢂ2HClþH]þ, 357.2900; found, 357.2904.
4.1.3.7. 12-N-2-Fluorobenzylsophoridinol
dihydrochloride
(9g).
The title compound was prepared in the same manner as described
above. Yield: 80%; White solid; mp 107e110 ꢁC; 1H NMR (DMSO-d6)
d: 11.57 (s, 1H), 11.14 (s, 1H), 7.93e7.68 (m, 2H), 7.49e7.29 (m, 2H),
4.42e4.21 (m, 3H), 3.74e3.58 (m, 1H), 3.42e3.18 (m, 4H), 3.10e2.74
(m, 4H), 2.75e2.54 (m, 2H), 2.55e2.31 (m, 2H), 2.05e1.58 (m, 6H),
4.1.3.1. 12-N-n-Propylsophoridinol dihydrochloride (9a). The title
1.54e1.13 (m, 5H), 0.99e0.51 (m, 1H); 13C NMR
d: 163.7 (d,
compound was prepared in the same manner as described above.
J ¼ 242 Hz), 132.8, 131.4, 127.3, 117.8, 117.0, 61.3, 60.6, 55.5, 55.0,
52.4, 52.0, 49.0, 44.6, 33.8, 32.1, 26.0, 25.1, 22.6, 22.3, 21.8, 17.6;
HRMS: calcd for C22H35N2OFCl2 [Mꢂ2HClþH]þ, 361.2649; found,
361.2654.
Yield: 81%; Yellow solid; mp 123e125 ꢁC; 1H NMR (DMSO-d6)
d:
11.55 (s, 1H), 10.63 (s, 1H), 3.71e3.39 (m, 7H), 3.23e2.74 (m, 6H),
2.52e2.20 (m, 3H), 1.97e1.20 (m, 14H), 0.97e0.75 (m, 3H); 13C NMR
d: 61.2, 60.8, 55.6, 54.7, 52.0, 51.4, 44.6, 33.8, 32.5, 32.4, 26.0, 25.2,
22.7, 22.4, 21.8, 17.5, 17.2, 11.3; HRMS: calcd for C18H36N2OCl2
4.1.3.8. 12-N-3-Fluorobenzylsophoridinol
The title compound was prepared in the same manner as described
dihydrochloride
(9h).
[Mꢂ2HClþH]þ, 295.2743; found, 295.2745.

102
C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
above. Yield: 82%; Yellow solid; mp 101e104 ꢁC; 1H NMR (DMSO-
d6) : 11.53 (s, 1H), 11.14 (s, 1H), 7.93e7.70 (m, 1H), 7.68e7.50 (m,
2H), 7.29e7.15 (m, 1H), 4.48e4.35 (m, 1H), 4.31e4.16 (m, 2H), 3.81e
3.68 (m, 1H), 3.38e3.02 (m, 6H), 3.02e2.79 (m, 2H), 2.75e2.41 (m,
4H), 2.01e1.58 (m, 6H), 1.57e1.17 (m, 5H), 0.94e0.71 (m, 1H); 13C
above. Yield: 83%; Yellow solid; mp 110e112 ꢁC; 1H NMR (DMSO-
d6) : 11.45 (s, 1H), 10.98 (s, 1H), 7.60e6.93 (m, 5H), 3.67e3.23 (m,
8H), 3.22e2.92 (m, 5H), 2.87e2.53 (m, 4H), 1.90e1.80 (m, 4H),
1.77e1.59 (m, 4H), 1.59e0.99 (m, 5H); 13C NMR
: 137.5, 129.2 (2),
129.0 (2),127.2, 61.6, 60.8, 55.5, 54.2, 51.9, 51.3, 49.0, 44.6, 33.9, 32.4,
29.9, 26.1, 22.7, 22.5, 22.4, 21.9, 17.5; HRMS: calcd for C23H38N2OCl2
[Mꢂ2HClþH]þ, 357.2900; found, 357.2901.
d
d
d
NMR
d
: 163.2 (d, J ¼ 242 Hz), 132.3, 130.9, 126.8, 117.5, 116.3, 60.9,
60.1, 55.0, 54.5, 51.5, 48.5, 44.1, 33.4, 31.7, 25.5, 24.6, 23.5, 22.1, 21.8,
21.3, 17.1; HRMS: calcd for
361.2649; found, 361.2652.
C
22H35N2OFCl2 [Mꢂ2HClþH]þ,
4.1.4. General procedures for the synthesis of sophoridinic acid
(7aej)
4.1.3.9. 12-N-4-Fluorobenzylsophoridinol
dihydrochloride
(9i).
Compound 6 (4.0 mmol) was dissolved in 3 N HCl (15 ml) and
heated to reflux for 3 h. Then the pH of reaction solution was
adjusted to 8e9 by addition of NH4OH. The reaction solvent was
removed under reduced pressure. Then the residue was dissolved
with MeOH and filtered to remove the inorganic salts. The solution
was concentrated to obtain crude targeted compound, which was
further separated by flash column chromatography on silica gel to
afford purified title compounds.
The title compound was prepared in the same manner as described
above. Yield: 83%; White solid; mp 102e104 ꢁC; 1H NMR (DMSO-
d6) d: 11.56 (s, 1H), 10.91 (s, 1H), 8.21e7.77 (m, 2H), 7.14e7.07 (m,
2H), 3.64e3.45 (m, 3H), 3.21e2.90 (m, 5H), 2.70e2.23 (m, 2H),
2.22e2.02 (m, 4H), 1.98e1.56 (m, 6H), 1.55e1.02 (m, 7H), 1.02e0.71
(m, 1H); 13C NMR
63.3, 61.0, 59.1, 57.3, 52.2, 50.3, 44.7, 35.6, 33.1, 28.0, 26.5, 23.8, 23.1,
22.8, 22.4, 18.1; HRMS: calcd for C22H35N2OFCl2 [Mꢂ2HClþH]þ,
361.2649; found, 361.2647.
d
: 159.2 (d, J ¼ 242 Hz), 133.5 (2), 130.2, 116.7 (2),
4.1.4.1. 12-N-n-Propylsophoridinic acid (7a). The title compound
was prepared in the same manner as described above. Yield: 81%;
4.1.3.10. 12-N-2-Chlorobenzylsophoridinol
dihydrochloride (9j).
White solid; mp 127e130 ꢁC; 1H NMR (DMSO-d6)
d: 3.60e3.40 (m,
The title compound was prepared in the same manner as described
2H), 3.31e3.02 (m, 3H), 2.76e2.35 (m, 5H), 2.35e2.23 (m, 3H),
2.22e2.05 (m, 2H), 1.98e1.95 (m, 2H), 1.93e1.82 (m, 1H), 1.80e1.59
(m, 3H), 1.59e1.39 (m, 6H), 1.37e1.20 (m, 1H), 1.16e0.91 (m, 3H);
above. Yield: 88%; Thick yellow solid; mp 107e110 ꢁC; 1H NMR
(DMSO-d6) d: 11.60 (s, 1H), 11.06 (s, 1H), 8.77e8.10 (m, 1H), 7.79e
6.98 (m, 3H), 4.56e4.31 (m, 1H), 3.70e3.54 (m 1H), 3.40e2.97 (m,
7H), 2.88e2.34 (m, 6H), 2.03e1.77 (m, 4H),1.79e1.58 (m, 2H), 1.58e
13C NMR
d: 180.1, 64.1, 61.1, 56.8, 52.4, 46.3, 37.3, 37.0, 29.2, 27.7,
24.7, 24.3, 24.0, 23.0, 21.8, 21.6, 19.2, 11.9; HRMS: calcd for
1.12 (m, 6H), 0.94e0.71 (m, 1H); 13C NMR
d
: 133.9, 132.7, 131.7,
C
18H32N2O2 [MþH]þ, 309.2536; found, 309.2538.
130.6, 128.3, 127.9, 62.1, 60.6, 55.5, 52.6, 52.2, 52.0, 44.6, 33.9, 32.2,
28.2, 26.0, 25.2, 22.8, 22.3, 21.7, 17.6; HRMS: calcd for C22H35N2OCl3
[Mꢂ2HClþH]þ, 377.2354; found, 377.2358.
4.1.4.2. 12-N-Isobutylsophoridinic acid (7b). The title compound
was prepared in the same manner as described above. Yield: 84%;
White solid; mp 133e135 ꢁC; 1H NMR (DMSO-d6)
d: 3.47e3.29 (m,
4.1.3.11. 12-N-4-Chlorobenzylsophoridinol (9k). The title compound
1H), 3.27e3.13 (m, 1H), 3.13e2.83 (m, 3H), 2.62e2.45 (m, 3H),
2.45e2.36 (m, 1H), 2.33e2.05 (m, 6H), 2.07e1.91 (m, 2H), 1.91e1.70
(m, 3H), 1.69e1.48 (m, 3H), 1.48e1.28 (m, 3H), 1.18e0.83 (m, 7H);
was prepared in the same manner as described above. Yield: 86%;
Yellow solid; mp 69e71 ꢁC; 1H NMR (DMSO-d6)
d: 7.61e7.19 (m,
4H), 3.60e3.20 (m, 5H), 3.17e2.99 (m, 2H), 2.91e2.42 (m, 3H),
2.40e1.98 (m, 5H), 1.89e1.67 (m, 4H), 1.64e1.27 (m, 7H), 1.26e1.04
13C NMR
d: 180.1, 64.1, 61.1, 56.8, 52.4, 46.3, 37.3, 37.0, 29.2, 27.7,
25.1, 24.7, 24.3, 24.0, 23.0, 21.8 (2), 19.2, 11.9; HRMS: calcd for
(m, 2H); 13C NMR
d: 139.3, 131.6, 130.3 (2), 128.6 (2), 63.5, 61.0, 59.0,
C
19H34N2O2 [MþH]þ, 323.2693; found, 323.2696.
57.3, 52.4, 50.5, 44.7, 35.8, 33.1, 27.8, 26.8, 23.9, 23.3, 23.1, 22.6, 18.2;
HRMS: calcd for C22H33N2OCl [MþH]þ, 377.2354; found, 377.2358.
4.1.4.3. 12-N-Benzylsophoridinic acid (7c). The title compound was
prepared in the same manner as described above. Yield: 84%; White
4.1.3.12. 12-N-4-Picolylsophoridinol dihydrochloride (9l). The title
solid; mp 139e142 ꢁC; 1H NMR (DMSO-d6)
d: 7.47e7.36 (m, 2H),
compound was prepared in the same manner as described above.
7.33e7.17 (m, 3H), 3.59e3.43 (m, 3H), 3.28e2.79 (m, 2H), 2.78e2.52
(m, 2H), 2.47e2.04 (m, 6H), 2.01e1.53 (m, 9H), 1.53e1.35 (m, 2H),
Yield: 82%; Yellow solid; mp 117e119 ꢁC; 1H NMR (DMSO-d6)
d:
11.62 (s, 2H), 11.06 (s, 1H), 8.99 (d, J ¼ 6.8 Hz, 2H), 8.52 (d, J ¼ 6.8 Hz,
2H), 4.81e3.55 (m, 4H), 3.48e3.23 (m, 3H), 3.23e2.68 (m, 4H),
2.49e2.28 (m, 3H), 2.00e1.75 (m, 4H), 1.75e1.53 (m, 3H), 1.53e1.10
1.35e1.09 (m, 2H); 13C NMR
d: 172.3, 139.4, 135.8 (2), 129.4 (2),
126.5, 62.7, 60.0, 57.9, 52.6, 52.5, 50.3, 44.9, 36.1, 28.0, 26.4, 23.9,
22.8, 22.7, 21.7, 17.8; HRMS: calcd for
357.2536; found, 357.2539.
C
22H32N2O2 [MþH]þ,
(m, 5H), 1.08e0.75 (m, 1H); 13C NMR
d: 149.0 (2), 148.3, 143.4 (2),
62.8, 61.8, 56.0, 55.6, 55.5, 55.4, 55.3, 54.9, 54.3, 53.6, 52.0, 37.9,
33.3, 32.1, 26.2, 25.5; HRMS: calcd for C21H35N3OCl2 [Mꢂ2HClþH]þ,
344.2696; found, 344.2696.
4.1.4.4. 12-N-2-Fluorobenzylsophoridinic acid (7d). The title com-
pound was prepared in the same manner as described above. Yield:
86%; White solid; mp 127e130 ꢁC; 1H NMR (DMSO-d6)
d: 7.43e7.25
4.1.3.13. 12-N-2-Furfurylsophoridinol
dihydrochloride
(9m).
(m, 2H), 7.16e6.99 (m, 2H), 3.75e3.43 (m, 4H), 3.37e3.21 (m, 3H),
3.03e2.54 (m, 3H), 2.43e2.04 (m, 5H), 2.03e1.82 (m, 3H),1.78e1.56
The title compound was prepared in the same manner as described
above. Yield: 85%; Yellow solid; mp 121e124 ꢁC; 1H NMR (DMSO-
(m, 5H), 1.46e1.20 (m, 3H); 13C NMR
d
: 180.8, 162.3 (d, J ¼ 242 Hz),
d6)
d
: 11.51 (s, 1H), 11.01 (s, 1H), 7.78 (d, J ¼ 7.4 Hz, 1H), 6.88e6.56
132.2, 129.8, 127.6, 125.1, 117.1, 64.6, 61.2, 53.8, 52.1, 51.6, 46.2, 37.6,
37.3, 29.4, 27.8, 24.9, 24.1, 24.0, 23.3, 19.2; HRMS: calcd for
(m, 2H), 3.70e3.42 (m, 2H), 3.36e3.25 (m, 1H), 3.23e3.09 (m, 3H),
3.0e2.85 (m, 2H), 2.75e2.58 (m, 2H), 2.55e2.04 (m, 4H), 1.99e1.75
C
22H31N2O2F [MþH]þ, 375.2442; found, 375.2450.
(m, 5H),1.75e1.58 (m, 2H),1.52e1.21 (m, 5H),1.18e1.08 (m, 2H); 13
C
NMR
d
: 144.6, 144.0, 114.6, 111.3, 61.8, 60.2, 54.9, 51.5, 50.5, 48.2,
4.1.4.5. 12-N-3-Fluorobenzylsophoridinic acid (7e). The title com-
44.1, 33.4, 31.9, 25.7, 24.7, 22.3, 22.2, 21.8, 21.3, 17.0; HRMS: calcd for
C
pound was prepared in the same manner as described above. Yield:
20H34N2O2Cl2 [Mꢂ2HClþH]þ, 333.2464; found, 333.2538.
81%; Yellow solid; mp 121e133 ꢁC; 1H NMR (DMSO-d6)
d: 7.81e7.51
(m, 1H), 7.43e7.23 (m, 1H), 7.23e6.95 (m, 2H), 3.75e3.42 (m, 4H),
3.35e3.06 (m, 3H), 2.68e2.35 (m, 3H), 2.36e2.16 (m, 4H), 2.14e1.80
4.1.3.14. 12-N-2-Phenethylsophoridinol
dihydrochloride
(9n).
The title compound was prepared in the same manner as described
(m, 4H), 1.78e1.36 (m, 7H), 1.34e1.20 (m, 1H); 13C NMR
d: 180.8,

C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
103
165.7 (d, J ¼ 242 Hz), 144.0, 131.0, 126.2, 118.7, 115.8, 66.9, 61.4, 58.8,
54.0, 51.6, 46.3, 37.6, 36.9, 29.5, 27.7, 24.6, 24.2, 24.0, 23.3, 19.2;
HRMS: calcd for C22H31N2O2F [MþH]þ, 375.2442; found, 375.2449.
deactivated with methanol and evaporated to afford crude product,
which was purified by flash chromatography over silica gel.
4.1.5.1. 12-N-Benzylsophoridinic methyl ether (10a). The title com-
4.1.4.6. 12-N-4-Fluorobenzylsophoridinic acid (7f). The title com-
pound was prepared in the same manner as described above. Yield:
pound was prepared in the same manner as described above. Yield:
87%; Yellow thick solid; mp 56e59 ꢁC; 1H NMR (DMSO-d6)
d: 7.62e
82%; White solid; mp 133e135 ꢁC; 1H NMR (DMSO-d6)
d: 7.45e7.24
7.49 (m, 3H), 7.30e7.11 (m, 2H), 4.44e4.26 (m, 2H), 3.60e3.39 (m,
3H), 3.39e3.07 (m, 6H), 3.07e2.75 (m, 3H), 2.75e2.42 (m, 5H),
(m, 2H), 7.05e6.95 (m, 2H), 3.56e3.45 (m, 3H), 3.41e3.33 (m, 1H),
3.26e3.04 (m, 2H), 2.71e2.59 (m, 1H), 2.54e2.34 (m, 2H), 2.33e
2.13 (m, 4H), 2.13e1.82 (m, 4H), 1.73e1.52 (m, 5H), 1.51e1.33 (m,
2.14e1.88 (m, 2H), 1.87e1.50 (m, 5H), 1.40e1.13 (m, 5H); 13C NMR
d:
137.9, 128.6 (2), 128.4 (2), 127.5, 74.6, 67.5, 62.0, 60.8, 57.8, 54.8,
53.2, 40.8, 37.9, 30.9, 30.5, 28.2, 27.4, 26.2, 24.1, 23.4, 23.2; HRMS:
calcd for C23H36N2O [MþH]þ, 357.2900; found, 357.2904.
2H), 1.34e1.20 (m, 2H); 13C NMR
d
: 179.7, 163.1 (d, J ¼ 242 Hz), 135.3
(2), 129.8, 114.5 (2), 62.6, 59.8, 57.0, 52.3, 50.2, 47.8, 36.4, 35.8, 28.0,
26.4, 23.5, 22.6, 21.6, 21.1, 17.8; HRMS: calcd for C22H31N2O2F
[MþH]þ, 375.2442; found, 375.2447.
4.1.5.2. 12-N-Benzylsophoridinic ethyl ether (10b). The title com-
pound was prepared in the same manner as described above. Yield:
81%; Yellow thick solid; mp 62e65 ꢁC; mp 127e130 ꢁC; 1H NMR
4.1.4.7. 12-N-3-Chlorobenzylsophoridinic acid (7g). The title com-
pound was prepared in the same manner as described above. Yield:
(DMSO-d6) d: 7.65e7.58 (m, 3H), 7.37e7.32 (m, 2H), 4.44e4.25 (m,
79%; White solid; mp 130e133 ꢁC; 1H NMR (DMSO-d6):
d
7.42 (s,
2H), 4.05e3.71 (m, 2H), 3.69e3.40 (m, 3H), 3.40e3.13 (m, 5H),
3.13e2.63 (m, 5H), 2.53e2.45 (m, 2H), 2.14e1.87 (m, 2H), 1.70 (m,
1H), 7.39e7.25 (m, 2H), 7.25e7.11 (m, 1H), 3.68e3.42 (m, 2H), 3.30e
3.14 (m, 1H), 3.15e2.84 (m, 4H), 2.69e2.30 (m, 3H), 2.25e2.07 (m,
4H), 2.05e1.94 (m, 1H), 1.88e1.56 (m, 4H), 1.56e1.44 (m, 4H), 1.42e
5H), 1.37e1.12 (m, 7H); 13C NMR
d: 137.9, 128. 7 (2), 128.5 (2), 127.6,
71.9, 67.6, 66.8, 62.0, 60.8, 54.8, 53.2, 40.9, 37.9, 30.9, 30.4, 28.3,
26.3, 25.5, 24.2, 23.4, 23.3, 14.8; HRMS: calcd for C24H38N2O
[MþH]þ, 371.3057; found, 371.3056.
1.21 (m, 3H); 13C NMR:
d 175.6, 133.4, 132.9, 130.4, 130.0, 128.2,
127.1, 63.6, 58.1, 57.6, 52.7, 51.1, 44.6, 36.4, 35.3, 28.2, 27.3, 24.2, 24.1,
23.4, 22.6, 18.6; HRMS: calcd for C22H31N2O2Cl [MþH]þ, 391.2147;
found, 391.2152.
4.1.5.3. 12-N-4-Fluorobenzylsophoridinic
methyl
ether
(10c).
The title compound was prepared in the same manner as described
4.1.4.8. 12-N-4-Chlorobenzylsophoridinic acid (7h). The title com-
above. Yield: 85%; Yellow thick solid; mp 60e62 ꢁC; 1H NMR
pound was prepared in the same manner as described above. Yield:
(DMSO-d6)
2H), 3.60e3.10 (m, 10H), 3.09e2.75 (m, 3H), 2.66e2.42 (m, 5H),
1.89e1.12 (m,11H); 13C NMR
115.3 (2), 74.6, 67.5, 62.0, 60.1, 57.8, 54.8, 53.1, 40.8, 37.9, 35.4, 30.9,
30.5, 28.2, 26.2, 24.1, 23.4, 23.2; HRMS: calcd for C23H35N2OF
[MþH]þ, 375.2806; found, 375.2802.
d: 7.61e7.58 (m, 2H), 7.30e7.20 (m, 2H), 4.48e4.25 (m,
82%; White solid; mp 127e130 ꢁC; 1H NMR (DMSO-d6)
d: 7.39 (d,
J ¼ 7.5, 2H), 7.30 (d, J ¼ 7.5, 2H), 3.52e3.42 (m, 2H), 3.33e3.26 (m,
1H), 3.26e3.04 (m, 2H), 2.64e2.49 (m, 2H), 2.44e2.26 (m, 2H),
2.27e2.13 (m, 3H), 2.11e2.02 (m, 3H), 2.02e1.80 (m, 4H), 1.67e1.50
d
: 163.6 (d, J ¼ 242 Hz),132.3,130.8 (2),
(m, 4H), 1.50e1.35 (m, 2H), 1.33e1.18 (m, 1H); 13C NMR
d: 179.2,
138.3,132.2,130.5 (2),127.9 (2), 62.7, 59.7, 57.1, 52.4, 50.3, 44.8, 36.0,
35.9, 28.0, 26.4, 23.4, 22.7, 21.7, 17.8; HRMS: calcd for C22H31N2O2Cl
[MþH]þ, 391.2147; found, 391.2152.
4.1.5.4. 12-N-4-Fluorobenzylsophoridinic ethyl ether (10d). The title
compound was prepared in the same manner as described above.
Yield: 76%; Yellow thick solid; mp 55e57 ꢁC; 1H NMR (DMSO-d6)
d:
4.1.4.9. 12-N-4-Bromobenzylsophoridinic acid (7i). The title com-
7.62e7.57 (m, 2H), 7.28e7.15 (m, 2H), 4.47e4.21 (m, 2H), 3.99e3.41
(m, 5H), 3.40e3.11 (m, 4H), 3.09e2.80 (m, 3H), 2.75e2.26 (m, 5H),
pound was prepared in the same manner as described above. Yield:
72%; White solid; mp 137e139 ꢁC; 1H NMR (DMSO-d6)
d: 7.44 (d,
2.14e1.54 (m, 6H), 1.55e0.69 (m, 8H); 13C NMR
d: 163.5 (d,
J ¼ 8.1 Hz, 2H), 7.27 (d, J ¼ 8.1 Hz, 2H), 3.68e3.44 (m, 4H), 3.40e3.33
(m, 1H), 3.29e3.19 (m, 2H), 3.06e2.66 (m, 2H), 2.57e2.35 (m, 2H),
2.35e2.15 (m, 4H), 2.11e1.70 (m, 5H), 1.70e1.36 (m, 5H), 1.28 (d,
J ¼ 242 Hz), 132.2, 130.7 (2), 115.2 (2), 71.9, 67.5, 66.8, 62.0, 60.8,
54.8, 53.2, 40.8, 37.9, 30.9, 30.4, 28.2, 26.2, 25.1, 24.1, 23.4, 23.2,14.8;
HRMS: calcd for C24H37N2OF [MþH]þ, 389.2962; found, 389.2958.
J ¼ 3.9 Hz, 1H); 13C NMR
d: 170.1, 130.7, 122.9 (2), 122.0 (2), 112.1,
57.4, 54.7, 52.0, 49.1, 44.6, 42.2, 36.9, 28.2, 27.1, 20.0, 18.2, 15.0, 14.8,
13.7; HRMS: calcd for C22H31N2O2Br [MþH]þ, 435.1659; found,
435.1658.
4.1.5.5. 12-N-4-Chlororobenzylsophoridinic methyl ether (10e).
The title compound was prepared in the same manner as described
above. Yield: 81%; Yellow thick solid; mp 62e65 ꢁC; 1H NMR
(DMSO-d6) d: 7.69e7.57 (m, 2H), 7.41e7.29 (m, 2H), 4.48e4.18 (m,
4.1.4.10. 12-N-4-Picolylsophoridinic acid (7j). The title compound
2H), 4.03e3.71 (m, 2H), 3.71e3.42 (m, 3H), 3.42e3.12 (m, 5H),
3.12e2.83 (m, 3H), 2.78e2.55 (m, 3H), 1.89e1.56 (m, 5H), 1.54e1.11
was prepared in the same manner as described above. Yield: 80%;
White solid; mp 151e154 ꢁC; 1H NMR (DMSO-d6)
d: 8.72e8.37 (m,
(m, 8H); 13C NMR
d: 136.4,132.0,129.3 (2), 128.2 (2), 74.0, 67.5, 62.0,
2H), 7.66e7.46 (m, 2H), 3.81e3.69 (m, 3H), 3.57e3.35 (m, 3H),
3.18e2.73 (m, 2H), 2.59e2.24 (m, 6H), 2.01e1.81 (m, 5H), 1.73e1.41
60.8, 57.8, 54.4, 53.2, 40.8, 37.9, 30.9, 30.5, 28.2, 26.2, 25.8, 24.1, 23.4,
23.2; HRMS: calcd for C23H35N2OCl [MþH]þ, 389.2510; found,
391.2509.
(m, 5H), 1.39e1.21 (m, 2H); 13C NMR
d: 177.4, 153.5, 150.6 (2), 125.4
(2), 64.9, 61.5, 58.2, 54.3, 51.7, 46.5, 38.0, 34.7, 29.4, 27.5, 24.3, 24.0,
23.9, 23.8, 19.2; HRMS: calcd for C21H31N3O2 [MþH]þ, 358.2489;
found, 358.2494.
4.1.5.6. 12-N-4-Chlororobenzylsophoridinic methyl ether (10f).
The title compound was prepared in the same manner as described
above. Yield: 82%; Yellow thick solid; mp 60e62 ꢁC; 1H NMR
4.1.5. General procedures for the synthesis of compounds (10aef)
To a stirred solution of compound 9 (5.0 mmol) in anhydrous
tetrahydrofuran, methyl/ethyl benzenesulfonate (20.0 mmol) was
added dropwise under strict moistureless conditions. Then the
reaction solution was stirred at room temperature till TLC analysis
showed completion of the reaction. The reaction mixture was
(DMSO-d6) d: 7.69e7.58 (m, 2H), 7.39e7.28 (m, 2H), 4.47e4.21 (m,
2H), 3.99e3.41 (m, 5H), 3.40e3.11 (m, 4H), 3.09e2.80 (m, 3H),
2.75e2.26 (m, 5H), 2.14e1.54 (m, 6H), 1.55e0.69 (m, 8H); 13C NMR
d: 136.9, 132.2, 129.5 (2), 128.2 (2), 71.9, 67.5, 66.8, 62.2, 60.1, 54.4,
53.1, 40.7, 37.5, 30.3, 30.4, 28.9, 26.8, 25.2, 24.7, 23.4, 23.5, 14.4;
HRMS: calcd for C24H37N2OCl [MþH]þ, 405.2666; found, 405.2667.

104
C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
4.1.6. 12-N-4-chlorobenzylsophoridinic amide dihydrochloride (8)
To a solution of the mixture 7h (160 mg, 0.41 mmol) and
NH4HCO3 (40 mg, 0.49 mmol) in acetonitrile (10 ml), di-tert-butyl
4.1.7.3. 12-N-4-Fluorobenzylmatrinol
The title compound was prepared in the same manner as described
above. Yield: 79%; mp 99e101 ꢁC; 1H NMR (DMSO-d6)
: 11.51 (s,
dihydrochloride
(15b).
d
dicarbonate (115
m
L, 0.53 mmol) and pyridine (20
m
L, 0.25 mmol)
1H), 11.08 (s, 1H), 7.75e7.58 (m, 2H), 7.36e7.19 (m, 2H), 4.28e4.10
(m, 2H), 4.07e3.76 (m, 6H), 3.67e3.52 (m, 1H), 3.32e3.17 (m, 2H),
3.10e2.78 (m, 3H), 2.73e2.53 (m, 3H), 1.97e1.64 (m, 5H), 1.64e1.38
were added. Then the reaction solution was stirred at room tem-
perature till TLC analysis showed completion of the reaction. The
reaction mixture was filtered and the filtrate was evaporated to
afford crude compound 8, which was further purified with flash
column chromatography and acidized with 3 N hydrochloride/
ether. Yield: 82%; White solid; mp 127e130 ꢁC; 1H NMR (DMSO-d6)
(m, 6H); 13C NMR
d
: 164.2 (d, J ¼ 242 Hz), 134.3 (2), 126.6, 116.2 (2),
61.0, 60.6, 59.0, 57.0, 54.6, 54.6, 49.0, 36.5, 32.5, 30.4, 28.7, 24.4,
24.0, 23.4, 23.2, 18.3; HRMS: calcd for
C22H35N2OFCl2
[Mꢂ2HClþH]þ, 361.2649; found, 361.2647.
d
: 11.54 (s, 1H), 11.05 (s, 1H), 7.95 (d, J ¼ 8.4 Hz, 2H), 7.52 (d,
J ¼ 8.4 Hz, 2H), 4.38e4.19 (m, 2H), 3.32e3.19 (m, 3H), 3.14e2.76 (m,
4.1.7.4. 12-N-4-Chlorobenzylmatrinol
The title compound was prepared in the same manner as described
above. Yield: 85%; mp 96e99 ꢁC; 1H NMR (DMSO-d6)
: 11.63 (s,
dihydrochloride
(15c).
5H), 2.70e2.36 (m, 5H), 2.07e1.94 (m, 2H), 1.94e1.59 (m, 7H), 1.56e
1.30 (m, 3H), 1.18e1.07 (m, 1H); 13C NMR
d: 179.2, 138.3,132.2,130.5
d
(2), 127.9 (2), 62.7, 59.7, 57.1, 52.4, 50.3, 44.8, 36.0, 35.9, 28.0, 26.4,
23.4, 22.7, 21.7, 17.8; HRMS: calcd for C22H34N3OCl3 [Mꢂ2HClþH]þ,
390.2307; found, 390.2306.
1H), 11.05 (s, 1H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.53 (d, J ¼ 8.4 Hz, 2H),
4.24e4.10 (m,1H), 4.04e3.83 (m, 2H), 3.71e3.53 (m,1H), 3.48e3.34
(m, 8H), 3.09e2.83 (m, 3H), 2.75e2.54 (m, 2H), 2.54e2.39 (m, 2H),
1.64e1.43 (m, 9H); 13C NMR
d: 133.0, 131.5, 125.4 (2), 124.8 (2), 64.9,
61.5, 58.2, 54.3, 51.7, 46.5, 38.0, 34.7, 29.4, 28.4, 27.5, 24.3, 24.0, 23.9,
23.8, 19.2; HRMS: calcd for C22H35N2OCl3 [Mꢂ2HClþH]þ, 377.2354;
found, 377.2352.
4.1.7. General procedures for the synthesis of compounds 14 and
15aec
Compound 11 (4.96 g, 20 mmol) was evenly dispersed in 3 N
NaOH solution (50 ml) and heated to reflux for 8 h. Then the solvent
was evaporated under vacuum and anhydrous methanol (50 ml)
and 10 ml 12 N HCl solution were added into the residue. The re-
action mixture was stirred at room temperature for 2 h and the
solvent was evaporated under vacuum. To a solution of residue in
CH3CN (30 ml), anhydrous K2CO3 (3.5 eq) and benzyl chloride
(1.5 eq) were added and then the reaction solution was stirred at
room temperature till TLC analysis showed completion of the re-
action. The reaction mixture was filtered and the filtrate was
evaporated to afford crude compound 14, which was further puri-
fied via flash column chromatography on silica gel with dichloro-
methane/methanol as eluents.
After the suspension of LiAH4 (0.5 g, 12.0 mmol) in anhydrous
THF, 14 (4.0 mmol) in anhydrous THF was added dropwise under
strict moistureless conditions. Then the reaction solution was stir-
red at room temperature till TLC analysis showed completion of the
reaction. The reaction system was deactivated successively with
water (0.5 ml), 3 N NaOH aqueous solution (0.5 ml) under ice bath
condition. The reaction mixture was filtered and the filtrate was
evaporated to afford crude product, which was purified by flash
chromatography over silica gel or acidized with 3 N hydrochloride/
ether to obtain 15aec.
4.2. Tumor cell lines
The human tumor cell lines HepG2, HCT116, H1299, U87, MCF-7
and KB were obtained from American Type Culture Collection. Cells
were routinely cultured in the MEM-EBSS medium (Hyclone, UT)
with 10% FBS (Gibco, USA) and 1% penicillinestreptomycin and
incubated at 37 ꢁC with 5% CO2.
4.3. Cell growth inhibition assay
Cells were counted and plated at a density of 4000 cells per well
in 96-well plates. After 24 h, cells were treated with different
compounds at different concentrations for 48 h. The effect on cell
growth inhibition was determined by MTT assay as previous
described [16]. The growth inhibition rate (%) was calculated at
each concentration and IC50 value was calculated with Sigmaplot
software [17,18]. Results were obtained from triplicate de-
terminations and shown as mean SD.
4.4. Flow cytometric analysis
Flow cytometric analysis was performed as previously described
[19]. HepG2 cells were treated with 6g and 9k at different con-
4.1.7.1. Methyl 12-N-4-fluorobenzylmatrinate dihydrochloride (14).
centrations (0, 2.5, 5 and 10 mg/ml) for 24 h and then cells were
The title compound was prepared in the same manner as described
harvested. Cells were fixed with 70% ethanol and stored at ꢂ20 ꢁC
above. Yield: 82%; mp 99e101 ꢁC; 1H NMR (DMSO-d6)
d: 11.59 (s,
overnight. The fixed cells were incubated with 200 mg/ml RNase at
1H), 11.08 (s, 1H), 7.80e7.58 (m, 2H), 7.38e7.25 (m, 2H), 4.25e4.15
(m, 2H), 4.05e3.84 (m, 2H), 3.60e3.43 (m, 3H), 3.31e3.10 (m, 3H),
3.05e2.78 (m, 3H), 2.63e2.53 (m, 2H), 2.17e1.90 (m, 3H), 1.85e1.65
37 ꢁC for 30 min and then stained with 50
mg/ml propidium iodide
in the dark for 30 min. Cell cycle distribution was then analyzed by
flow cytometry using FACS analysis (BD FACSCalibur, USA).
(m, 7H), 1.63e1.30 (m, 4H); 13C NMR
d
: 173.7, 164.2 (d, J ¼ 242 Hz),
134.2 (2), 126.6, 116.0 (2), 60.7, 56.8, 54.6, 52.0, 51.8, 49.0, 36.3, 32.8,
30.4, 27.9, 26.6, 24.4, 23.9, 21.7, 18.2; HRMS: calcd for C22H35N2O2F
[Mꢂ2HClþH]þ, 389.2958; found, 389.2564.
4.5. TOP I enzyme activity
A gel assay as previously described [20], was used to determine
if 9k inhibited Top I enzyme activity by measuring the decreased
mobility of the relaxed isomers of supercoiled pBR322 DNA. Human
Top I and pBR322 were obtained from Beyotime (Haimen, Jiangsu,
China). One unit of human Top I and the different concentrations
compound were mixed and incubated at 37 ꢁC for 20 min, and then
4.1.7.2. 12-N-Benzylmatrinol dihydrochloride (15a). The title com-
pound was prepared in the same manner as described above. Yield:
82%; mp 92e95 ꢁC; 1H NMR (MeOD)
d: 7.71e7.54 (m, 2H), 7.53e7.36
(m, 3H), 4.28e3.87 (m, 6H), 3.67e3.50 (m, 1H), 3.37e3.15 (m, 2H),
3.07e2.80 (m, 2H), 2.75e2.36 (m, 5H), 2.01e1.62 (m, 6H), 1.63e1.33
0.5
incubate at 37 ꢁC for 30 min. The reaction was terminated by adding
0.5% SDS, 0.25 g/ml bromophenol blue, and 15% glycerol and the
mg pBR322 DNA was added and the mixture was continued to
(m, 6H); 13C NMR
d: 132.6, 131.1 (2), 130.0 (2), 129.4, 61.9, 61.4, 61.3,
58.7, 55.4, 55.3, 49.9, 37.2, 33.0, 31.2, 30.4, 29.5, 25.2, 24.8, 24.0,19.1;
HRMS: calcd for C22H34N2O [Mꢂ2HClþH]þ, 389.2958; found,
389.2958.
m
reaction products were separated on a horizontal 1% agarose gel in
tris-acetate/EDTA buffer at 4 V/cm for 40 min at room temperature.

C.-W. Bi et al. / European Journal of Medicinal Chemistry 81 (2014) 95e105
105
The gel was stained with GelRed I and imaging was conducted by
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.069.