Synthesis and activity evaluation of tilorone analogs as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.050

Tilorone is an interferon inducer with anticancer activity. Twenty-two novel tilorone analogs were synthesized by improvements of fluorenone skeleton, side chains and amino groups to screen new anticancer agents. In vitro evaluation showed that ten new co

Full text of DOI:10.1016/j.ejmech.2013.03.050

European Journal of Medicinal Chemistry 64 (2013) 432e441
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and activity evaluation of tilorone analogs as potential
anticancer agents
Dingshan Zhou a, Wei Tuo a, Hao Hu a, Jianrong Xu b, Hongzhuan Chen b, Zhigang Rao a,
,
Yuling Xiao a, Xianming Hu a, Peng Liu a
*
a State Key Laboratory of Virology, Ministry of Education Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of
Pharmaceutical Sciences, Wuhan 430071, China
b Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Tilorone is an interferon inducer with anticancer activity. Twenty-two novel tilorone analogs were
synthesized by improvements of fluorenone skeleton, side chains and amino groups to screen new
anticancer agents. In vitro evaluation showed that ten new compounds had better anticancer activities
Received 23 January 2013
Received in revised form
8 March 2013
Accepted 24 March 2013
Available online 16 April 2013
than tilorone. Among them, 2c (IC50 < 7
m
M against cancer cell lines and IC50 > 35
mM against non-cancer
cell lines) and 5d (IC50 < 10 M against cancer cell lines and IC50 > 53
m
m
M against non-cancer cell lines)
exhibited the best anticancer activities and selectivities. Pharmacophore modeling of highly active
compounds was carried out by Molecular Operating Environment (MOE) to generate a visualized model
for compound design in future study.
Keywords:
Anticancer agent
Tilorone
Ó 2013 Elsevier Masson SAS. All rights reserved.
Fluorenone
Isoflavone
Pharmacophore modeling
1. Introduction
On the basis of these considerations, twenty-two novel tilor-
one analogs containing rotational isoflavone skeleton with anti-
Tilorone (or 2,7-bis[2-diethylaminoethoxy]fluoren-9-one, Fig.1)
is the first synthetic, small molecular weight interferon inducer in
possession of a broad range of therapeutical functions, such as
resistance to systemic candidiasis, antiviral effect and anticancer
activity [1e6]. These activities are mainly achieved by DNA strand
intercalation that alters DNA chemobiological properties and
further activates interferon expression [7e9].
cancer activity [21e24], hydroxyl side chains and non-tertiary
amino groups have been designed, synthesized and assayed for
cytotoxicity and anticancer activity. After in vitro activity evalu-
ation, the highly active compounds were implemented pharma-
cophore modeling by Molecular Operating Environment (MOE)
[25].
Many previous studies showed that the modification of tilor-
one’s fluorenone skeleton and side chain (Fig. 1) enhanced anti-
cancer activity [10e16]. In general, the fluorenone skeleton was
optimized by substitution with differently rigid planar rings
[9,13,17e20], while the modification of the side chain was mostly
derived from length extension, addition of carbonyl group and shift
of linkage site [10,11,14,16] as well as change of N-alkyl groups [10e
20]. It is thus of considerable interest to investigate the necessities
of rigid skeleton and tertiary amino groups as well as the influence
of extra hydroxyl groups.
2. Chemistry
The compounds 1d (Scheme 1) and 6cem (Scheme 2) were
synthesized via a three-step procedure starting from daidzein (D)
and 2,7-dihydroxy-9-fluorenone (F), respectively. This procedure
included hydroxyethylation of phenolic hydroxyls by ethylene
chlorohydrin, further chlorination of alcoholic hydroxyls by thionyl
chloride and final nucleophilic substitution of chlorides by corre-
sponding amines [26]. Note that in the first step, ethylene chloro-
hydrin in excess needed to be used due to its hydrolyzation during
the reaction. The compounds 4c and 4d (Scheme 2) were produced
via a similar procedure to 6cem, where the only difference was
ethylation on one of the two phenolic hydroxyls by diethylsulfate
prior to the three-step procedure. The synthesis of the compounds
* Corresponding author. Tel.: þ86 27 68759887; fax: þ86 27 68759850.
E-mail addresses: zhoudingshan@whu.edu.cn (D. Zhou), liupenghk@gmail.com
(P. Liu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.050

D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
433
Fig. 1. Structures of tilorone and its active derivatives.
Scheme 1. Synthesis of compounds 1d, 2bed, 3b and 3c. Reagents and conditions: (1) NaOH/ethylene chlorohydrin/DMSO, 70 ꢀC; (2) thionyl chloride, 60 ꢀC; (3) potassium
carbonate/diethylamine/CHCl3, reflux; (4) epichlorohydrin/NaOH/DMSO, 70 ꢀC; (5) potassium carbonate/amines/CHCl3, reflux; (6) NaOH/1,3-dibromopropane/DMSO, 70 ꢀC; (7)
potassium carbonate/amines/DMF, 70 ꢀC.
2bed (Scheme 1) and 5bee (Scheme 2) involved epoxypropylation
of starting compounds (D and F) by epichlorohydrin followed by
ring-opening reaction of epoxide using appropriate amines.
The compounds 3b and 3c (Scheme 1) were obtained by bromo-
propylation and nucleophilic substitution. Bromopropylation was
performed with an excess of 1,3-dibromopropane (five equivalents)
in order to reduce by-products. Our attempts indicated that direct
bromoethylation by 1,2-dibromoethane could not be used to pre-
pare 1d and 6cem, resulted in a large amount of polymolecular by-
products.
Scheme 2. Synthesis of compounds 4c, 4d, 5bee and 6cem. Reagents and conditions: (1) NaOH/diethylsulfate/water, R.T.; (2) NaOH/ethylene chlorohydrin/DMSO, 70 ꢀC; (3) thionyl
chloride, 60 ꢀC; (4) potassium carbonate/amines/DMF, 70 ꢀC; (5) epichlorohydrin/NaOH/DMSO, 70 ꢀC; (6) potassium carbonate/amines/DMF, 70 ꢀC; (7) NaOH/ethylene chloro-
hydrin/DMSO, 70 ꢀC; (8) thionyl chloride, 60 ꢀC; (9) potassium carbonate/amines/DMF, 70 ꢀC.

434
D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
Table 1
Anticancer and cytotoxic activities of synthesized compounds.
a
Compounds
X, Y
IC50 (mM)
Cancer cell lines
Hep3B
Non-cancer cell lines
Vero
HeLa
N. A.
A549
N. A.
293T
N. D.
1c
X ¼ Y ¼
N. A.b
N. D.c
1d
X ¼ Y ¼
35.22
26.39
27.84
5.17
19.43
24.98
4.26
102.41
106.68
52.87
39.65
2b
2c
X ¼ Y ¼
30.36
6.52
60.11
35.48
X ¼ Y ¼
2d
3a
X ¼ Y ¼
12.00
N. A.
5.32
4.34
18.53
N. D.
20.16
N. D.
X ¼ Y ¼
N. A.
N. A.
3b
3c
X ¼ Y ¼
24.37
76.14
22.18
82.37
20.63
73.51
34.92
N. D.
41.89
N. D.
X ¼ Y ¼
4a
4b
4c
4d
X ¼ CH2CH3, Y ¼
X ¼ CH2CH3, Y ¼
X ¼ CH2CH3, Y ¼
X ¼ CH2CH3, Y ¼
N. A.
N. A.
>100
84.75
N. A.
N. A.
N. A.
>100
>100
N. D.
N. D.
N. D.
N. D.
N. D.
N. D.
N. D.
N. D.
N. A.
>100
106.63
5b
5c
X ¼ Y ¼
X ¼ Y ¼
18.31
21.46
11.73
20.23
12.58
16.04
20.95
48.57
28.26
56.38
5d
5e
X ¼ Y ¼
X ¼ Y ¼
9.43
6.76
6.78
1.82
7.42
1.40
57.83
13.01
53.97
17.95
6c
X ¼
, Y ¼
>100
>100
>100
N. D.
N. D.

D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
435
Table 1 (continued )
a
Compounds
X, Y
IC50
(
mM)
Cancer cell lines
Hep3B
Non-cancer cell lines
Vero
HeLa
A549
293T
N. D.
6e
X ¼ Y ¼
>100
>100
>100
N. D.
6f
X ¼ Y ¼
X ¼ Y ¼
>100
>100
>100
>100
>100
>100
N. D.
N. D.
N. D.
N. D.
6g
6h
6i
X ¼ Y ¼
5.17
8.89
2.73
1.72
2.96
2.43
8.47
8.59
X ¼ Y ¼
14.76
13.81
6j
X ¼ Y ¼
3.34
2.21
9.17
2.65
7.86
8.89
10.97
19.82
6k
X ¼ Y ¼
20.08
25.33
6l
X ¼ Y ¼
X ¼ Y ¼
X ¼ Y ¼
N. A.
N. A.
38.78
N. A.
N. A.
39.97
N. A.
N. A.
32.63
N. D.
N. D.
89.53
N. D.
N. D.
76.41
6m
Tilorone
Compounds 1c, 1d, 2bed and 3aec are daidzein skeleton.
Compounds 4aed, 5bee and 6cem are fluorenone skeleton.
a
IC50 is the half maximal (50%) cell growth inhibitory concentration of the compound.
b
c
N. A. means no activity at concentration of 100
N. D. means not determined.
mM or higher.
The structures of the synthesized compounds were character-
The results of anticancer and cytotoxic assay reveal that the
compounds without amino group (1c, 3a, 4a, 4b, 6l and 6m) or
possessing only one amino group (4c, 4d and 6c) were not actively
against cancer cells even though the concentrations were higher
ized by 1H NMR, 13C NMR, elemental analysis and mass spectra. The
data are shown in the Experimental part.
3. Results and discussion
than 100 mM. This result shows double amino groups are critical for
anticancer activities. When the amino groups are substituted by
hydroxyls, halogens, mercapto groups or there was only one amino
group, the compounds lose cytotoxicity. Compounds 6e, 6f and 6g
with two azoles instead of amino groups exhibited low activities
(IC50 > 100 mM). The reason probably is the azole has weaker ability
of forming hydrogen bond than amine and the rigid ring is not
benefit to activity.
3.1. In vitro anticancer activity
The in vitro anticancer activities of the synthesized compounds
were evaluated according to MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) assay by using tilorone as posi-
tive control. Three human cancer cell lines, human hepatoma cell
line (Hep3B), human cervical cancer cell line (HeLa) and human
lung adenocarcinoma epithelial cell line (A549), were used for
screening. The anticancer activities are described by half maximal
inhibitory concentration (IC50) values in Table 1.
The compounds 6hek with two secondary amino groups per-
formed much better activities to cancer cell lines (3.34e20.08
against Hep3B, 1.72e9.17 M against HeLa and 2.43e7.86
m
m
M
M
m
against A549) than tilorone. However, they exhibited high cyto-
toxicities to non-cancer cell lines (8.47e25.33 M against Vero and
8.59e19.82 M against 293T, tilorone was 89.53 M against Vero
and 76.41 M against 293T). The differences of activity between
There are ten new compounds (2c, 2d, 3b, 5bee and 6iek)
which showed strong inhibition (IC50 < 25
lines. The positive control tilorone’s IC50 was 32e39
m
m
M) against cancer cell
m
m
mM to cancer
m
cell lines. These new compounds with 1d, 2b, 6h and tilorone were
then tested against two non-cancer cell lines, African green mon-
key kidney epithelial cells (Vero) and human embryonic kidney
cells (293T), for investigating their cytotoxicities. The IC50 values
are summarized in Table 1.
6hek and tilorone might derive from toxic secondary metabolites
hydroxylamines. Although this class of compounds is effectively
against cancer cells, they have high cytotoxicities.
Compounds 5bee possess all the functional groups as tilorone
with an extra hydroxyl on each side chain. All of them exhibited

436
D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
Fig. 2. Ligand-based pharmacophore model. The pharmacophore features were generated by assembled pharmacophore annotation points. Pharmacophore features: cyan, H-bond
acceptor; pink, H-bond donor and/or acceptor; brown, aromatic centroid; green, hydrophobic centroid. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
better activities than tilorone. 5d (9.43
against HeLa and 7.42 M against A549) and 5e (6.76
Hep3B, 1.82 M against HeLa and 1.40 M against A549) were an
m
M against Hep3B, 6.78
m
M
probable pharmacophores spatial arrangements and can help us to
design compounds in future study.
m
mM against
m
m
order of magnitude more active than tilorone. The inhibition to
4. Conclusions
non-cancer cell lines showed 5e was cytotoxic to tested cell lines
(13.01
exhibited good selectivity between cancer and non-cancer cells
(57.83 M against Vero and 53.97 M against 293T). This class of
compounds indicates that extra hydroxyl on side chain increases
anticancer activity and flexible carbon chain of tertiary amino
group is in favor of selectivity.
The compounds 1d, 2bed, 3b and 3c were synthesized to
determine the necessity of fluorenone skeleton for anticancer ac-
tivity. The isoflavone skeleton has a rotational aromatic ring other
than rigid skeleton. The IC50 values of 1d against cancer cell lines
and non-cancer cell lines were slightly lower than tilorone. 3b and
3c are side chain extension compounds of 1d. There is somewhat
deficiency for their activities. The most active compounds with
isoflavone skeleton are those with hydroxyls on their side chains
such as 2c and 2d. 2c performed excellent anticancer activity
m
M against Vero and 17.95
m
M against 293T), but 5d
Twenty-two novel tilorone analogs were synthesized and
screened as candidates of novel anticancer agents. There are ten
new compounds that performed better anticancer activities than
tilorone. 2c and 5d are the most potent compounds for both anti-
cancer activities and selectivities. A brief sum-up of structureeac-
tivity relationship is outlined as follows: 1. amino groups are
necessary for the anticancer activity; 2. comparing with tertiary
amino groups, secondary amino groups increase the anticancer
activity but decrease the selectivity; 3. the introduced hydroxyls on
side chains improve the activity considerably; 4. the substitution of
fluorenone skeleton by isoflavone skeleton retains the anticancer
activity. Pharmacophore model shows the visualized spatial ar-
rangements of pharmacophores and can be used to direct com-
pound design.
m
m
(6.52
A549) and was good at selectivity (35.48
39.65 M against 293T). This result implies that the totally rigid
m
M against Hep3B, 5.17
m
M against HeLa and 4.26
mM against
5. Experimental protocols
mM against Vero and
m
5.1. Chemistry
skeleton is not necessary for anticancer activity.
Melting points were determined on a XT-4 apparatus (uncor-
rected) and some of the compounds were not determined due to
hygroscopicity or oil like. 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance 400 apparatus in CDCl3 or DMSO-d6
with TMS as internal standard. Chemical shifts were given in
3.2. Pharmacophore modeling
The results of in vitro anticancer activity assay have proposed
some structureeactivity relationships about active compounds.
In this part, we intended to use molecular simulation to build a
model for guiding compound design in the future. Since the
target DNA segment of tilorone has not been ascertained yet
[7,8,27,28], the ligand-based drug design has to be employed in
the near future. As a significant tool of ligand-based drug design,
pharmacophore modeling can generate a visualized spatial model
of active compounds which could be used for directing com-
pound design and virtual screening. Hence, we set up a phar-
macophore model from the highly active tilorone analogs (1d, 2b,
2c, 3b and 5bed) and tilorone (Fig. 2). The procedure included
flexible alignment of the active compounds and pharmacophore
consensus [29].
This pharmacophore model suggests that the compounds
derived from fluorenone and isoflavone share similar pharmaco-
phore features. The carbonyl groups as well as the tertiary amino
groups locate at the same site, while the benzene rings form hy-
drophobic centers. The pharmacophore model also shows that the
pharmacophores of synthesized compounds superpose and fit
tilorone’s pharmacophores well. Thus the model presents the
d
(ppm) and coupling constants (J) were shown in Hz. Mass spectra
of all new compounds were recorded by electrospray ionization
(ESI) method on a LC/MSD Trap SL Plus spectrometer (Agilent
Technologies, Waldbronn, Germany). Optical rotations were
measured on a PerkinElmer Model 341 polarimeter. Elemental
analysis was performed on a VarioEL III instrument. The reactions
were monitored by thin layer chromatography (TLC).
5.1.1. 40,7-Bis(2-hydroxyethoxy)isoflavone (1b)
Daidzein (10 g, 0.0394 mol) was solved in 150 ml DMSO, NaOH
(3.2 g, 0.08 mol) and ethylene chlorohydrin (6.7 ml, 0.1 mol) were
added into the solution. The resulting solution was stirred over-
night at 70 ꢀC, and then the solution was poured into 1 M NaOH
water solution. The precipitate was filtered and washed by 1 M
NaOH till colorless eluate and dried in vacuum drying oven then
recrystallized from 2-propanol to obtain compound 1b. Light yel-
low solid. Yield 51%. M.p.189e191 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
: 8.43 (s, 1H), 8.03 (d, J ¼ 8.9 Hz, 1H), 7.56e7.49 (m, 2H), 7.18 (d,
J ¼ 2.3 Hz, 1H), 7.10 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H),

D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
437
4.15 (t, J ¼ 4.8 Hz, 2H), 4.02 (t, J ¼ 5.0 Hz, 2H), 3.75 (dt, J ¼ 14.6,
4.9 Hz, 4H). 13C NMR (101 MHz, DMSO-d6)
: 174.62, 163.14, 158.42,
157.39, 153.44, 130.05, 126.91, 123.98, 123.33, 117.49, 115.08, 114.13,
101.03, 70.46, 69.48, 59.42, 59.18. Anal. Calcd. for C19H18O6: C 66.66,
H 5.30; Found C 66.58, H 5.31.
5.1.5.1. 40,7-Bis(3-diethylamino-2-hydroxypropoxy)isoflavone (2b).
Light yellow solid. Yield 80%. M.p. 87e88 ꢀC. [ 20 0ꢀ (c 0.5, CH2Cl2).
1H NMR (400 MHz, CDCl3)
: 8.13 (d, J ¼ 8.9 Hz,1H), 7.85 (s,1H), 7.42
d
a]
D
d
(d, J ¼ 8.7 Hz, 2H), 6.98e6.88 (m, 3H), 6.82 (d, J ¼ 2.3 Hz, 1H), 4.05e
3.88 (m, 6H), 2.70e2.50 (m, 12H), 1.02 (td, J ¼ 7.1, 5.0 Hz, 12H). 13
C
NMR (101 MHz, CDCl3) d: 175.85, 163.13, 158.76, 157.84, 152.15,
5.1.2. 40,7-Bis(2-chloroethoxy)isoflavone (1c)
130.13, 127.79, 124.83, 124.51, 118.57, 114.84, 114.59, 100.86, 70.99,
70.41, 65.73, 65.56, 56.14, 55.73, 47.38, 47.28, 11.83, 11.65. Anal.
Calcd. for C29H40N2O6: C 67.94, H 7.86 N 5.46; Found C 67.89, H 7.87,
N 5.45.
1b (2 g, 0.00585 mol) was solved in 40 ml thionyl chloride and
stirred at 60 ꢀC till the reaction completed (monitored by TLC), then
poured the solution into 1 M NaOH carefully. The precipitate was
collected and purified by flash column chromatography to yield
compound 1c. Light yellow solid. Yield 40%. M.p. 166e168 ꢀC. 1H
5.1.5.2. 40,7-Bis(3-dipropylamino-2-hydroxypropoxy)isoflavone (2c).
20
NMR (400 MHz, CDCl3)
d
: 8.15 (d, J ¼ 8.9 Hz,1H), 7.85 (s,1H), 7.43 (d,
Yellow oil. Yield 65%. [
CDCl3)
a
]
0ꢀ (c 0.5, CH2Cl2). 1H NMR (400 MHz,
D
J ¼ 8.7 Hz, 2H), 6.98e6.87 (m, 3H), 6.80 (d, J ¼ 2.2 Hz, 1H), 4.23 (dt,
d
: 8.20 (d, J ¼ 8.9 Hz, 1H), 7.92 (s, 1H), 7.49 (d, J ¼ 8.7 Hz, 2H),
J ¼ 25.2, 5.8 Hz, 4H), 3.78 (dt, J ¼ 17.1, 5.8 Hz, 4H). 13C NMR
7.07e6.96 (m, 3H), 6.89 (d, J ¼ 2.3 Hz, 1H), 4.10e3.97 (m, 6H), 2.64e
(101 MHz, CDCl3)
d
: 175.74, 162.45, 158.21, 157.78, 152.23, 130.26,
2.40 (m, 12H), 1.59e1.41 (m, 8H), 0.90 (td, J ¼ 7.3, 2.5 Hz, 12H). 13
C
128.09, 124.91, 124.83, 118.89, 114.80, 114.63, 101.07, 68.42, 68.10,
41.85, 41.43. Anal. Calcd. for C19H16Cl2O4: C 60.17, H 4.25; Found C
60.20, H 4.25. MS (ESI): m/z [M þ H]þ calcd 379.0, found 378.9.
NMR (101 MHz, CDCl3) d: 175.86, 163.18, 158.86, 157.84, 152.13,
130.10, 127.78, 124.84, 124.43, 118.55, 114.84, 114.60, 100.82, 71.02,
70.55, 65.92, 65.70, 57.23, 56.93, 56.25, 56.22, 20.30, 20.29, 11.80.
Anal. Calcd. for C33H48N2O6: C 69.69, H 8.51 N 4.93; Found C 69.65,
H 8.52, N 4.92. MS (ESI): m/z [M þ H]þ calcd 569.4, found 569.1.
5.1.3. 40,7-Bis(2-diethylaminoethoxy)isoflavone (1d)
To a stirred solution of 1c (0.4 g, 0.00106 mol) in 50 ml CHCl3,
potassium carbonate (0.5 g, 0.0036 mol) and diethylamine (1 ml,
0.01 mol) were added. The reaction was refluxed 24 h and
extracted with brine and CH2Cl2. The organic layer was filtered,
washed with brine and dried. After concentrated in vacuum, the
crude product was separated on flash column to obtain compound
1d. Light yellow solid. Yield 75%. M.p. 80e81 ꢀC. 1H NMR
5.1.5.3. 40,7-Bis(3-piperidino-2-hydroxypropoxy)isoflavone
(2d).
0ꢀ (c 0.5,
20
Light yellow solid. Yield 85%. M.p. 161e163 ꢀC. [
a]
D
CH2Cl2). 1H NMR (400 MHz, CDCl3)
d
: 8.20 (d, J ¼ 8.9 Hz, 1H), 7.92
(s, 1H), 7.49 (d, J ¼ 8.7 Hz, 2H), 7.05e6.94 (m, 3H), 6.89 (d,
J ¼ 2.2 Hz, 1H), 4.16 (dd, J ¼ 12.5, 5.7 Hz, 2H), 4.09e3.96 (m, 4H),
2.73e2.42 (m, 12H), 1.64 (d, J ¼ 4.4 Hz, 8H), 1.48 (t, J ¼ 11.9 Hz, 4H).
(400 MHz, CDCl3)
d
: 8.09 (d, J ¼ 8.9 Hz, 1H), 7.83 (s, 1H), 7.40 (d,
13C NMR (101 MHz, CDCl3)
d: 175.85, 163.09, 158.71, 157.83, 152.17,
J ¼ 8.6 Hz, 2H), 6.89 (t, J ¼ 8.9 Hz, 3H), 6.78 (d, J ¼ 1.9 Hz, 1H), 4.03
(dt, J ¼ 17.6, 6.1 Hz, 4H), 2.83 (q, J ¼ 6.0 Hz, 4H), 2.58 (q, J ¼ 7.1 Hz,
130.14, 127.80, 124.81, 124.53, 118.57, 114.85, 114.60, 100.85, 70.93,
70.34, 65.14, 64.98, 61.31, 60.92, 54.78, 25.88, 25.69, 24.04, 23.94.
Anal. Calcd. for C31H40N2O6: C 69.38, H 7.51 N 5.22; Found C 69.35,
H 7.52, N 5.21. MS (ESI): m/z [M þ H]þ calcd 537.3, found 537.1.
8H), 1.01 (t, J ¼ 7.1 Hz, 12H). 13C NMR (101 MHz, CDCl3)
d: 175.83,
163.23, 158.77, 157.85, 152.11, 130.06, 127.54, 124.71, 124.21, 118.26,
114.96, 114.48, 100.65, 67.36, 66.43, 51.62, 51.41, 47.87, 47.73, 11.81,
11.73. Anal. Calcd. for C27H36N2O4: C 71.65, H 8.02 N 6.19; Found C
71.59, H 8.04, N 6.18.
5.1.6. 40,7-Bis(3-bromopropoxy)isoflavone (3a)
Daidzein (5 g, 0.0197 mol) was solved in 100 ml DMSO, NaOH
(1.6 g, 0.04 mol) and 1,3-dibromopropane (10 ml, 0.1 mol) were
added into the solution. The resulting solution was stirred over-
night at 70 ꢀC. The solution was then poured into 1 M NaOH water
solution. The precipitate was filtered and washed by 1 M NaOH till
colorless eluate and dried in vacuum drying oven to obtain crude
product. The crude product was separated on flash column to
obtain compound 3a. Light yellow solid. Yield 70%. M.p. 138e
5.1.4. 40,7-Bis(2,3-epoxypropoxy)isoflavone (2a)
Daidzein (10 g, 0.0394 mol) was solved in 150 ml DMSO, NaOH
(3.2 g, 0.08 mol) and epichlorohydrin (16 ml, 0.2 mol) were added
into the solution. The solution was stirred overnight at 70 ꢀC. The
resulting solution was then poured into 1 M NaOH water solution.
The precipitate was filtered and washed by 1 M NaOH till colorless
eluate and dried in vacuum drying oven to obtain crude product.
141 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 8.22 (d, J ¼ 8.9 Hz, 1H), 7.92 (d,
The crude product was separated on flash column to get compound
J ¼ 2.8 Hz, 1H), 7.53e7.46 (m, 2H), 7.02e6.95 (m, 3H), 6.88 (d,
20
2a. Light yellow solid. Yield 55%. M.p. 117e118 ꢀC. [
a
]
D
0ꢀ (c 0.5,
J ¼ 2.3 Hz, 1H), 4.19 (dt, J ¼ 29.9, 5.8 Hz, 4H), 3.63 (dd, J ¼ 11.4,
CH2Cl2). 1H NMR (400 MHz, CDCl3)
d
8.15 (d, J ¼ 8.9 Hz, 1H), 7.85 (s,
6.3 Hz, 4H), 2.43e2.29 (m, 4H). 13C NMR (101 MHz, CDCl3)
d: 175.85,
1H), 7.42 (t, J ¼ 5.8 Hz, 2H), 6.95 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.92 (d,
J ¼ 8.8 Hz, 2H), 6.82 (d, J ¼ 2.3 Hz, 1H), 4.33e4.16 (m, 2H), 3.94 (td,
J ¼ 11.1, 5.8 Hz, 2H), 3.37e3.28 (m, 2H), 2.87 (dt, J ¼ 15.0, 4.5 Hz, 2H),
2.72 (ddd, J ¼ 10.6, 4.8, 2.6 Hz, 2H). 13C NMR (101 MHz, CDCl3)
163.00, 158.69, 157.90, 152.12, 130.20, 127.90, 124.89, 124.48, 118.59,
114.76, 114.59, 100.76, 65.94, 65.37, 32.33, 31.98, 30.02, 29.59. Anal.
Calcd. for C21H20Br2O4: C 50.83, H 4.06; Found C 50.78, H 4.05. MS
(ESI): m/z [M ꢁ Br]þ calcd 415.1, found 414.9.
d
175.78, 162.70, 158.49, 157.79, 152.21, 130.19, 127.97, 124.82,
124.75, 118.79, 114.73, 114.68, 101.06, 69.31, 68.81, 50.15, 49.79,
44.76, 44.58. Anal. Calcd. for C21H18O6: C 68.85, H 4.95; Found C
68.81, H 4.96.
5.1.7. General procedure for the synthesis of compounds 3b and 3c
To a stirred solution of 3a (0.5 g, 0.001 mol) in 40 ml dime-
thylformamide, potassium carbonate (0.5 g, 0.0036 mol) and
dimethylamine solution (33%, 3.1 ml, 0.02 mol) were added. The
reaction was taken 24 h at 70 ꢀC and then extracted with brine and
CH2Cl2. The organic layer was filtered, washed with brine and dried.
After concentrated in vacuum, the crude product was separated on
flash column to obtain compound 3b. 3c was prepared by instead of
dimethylamine solution with diethylamine in the same condition.
5.1.5. General procedure for the synthesis of compounds 2bed
To a stirred solution of 2a (0.4 g, 0.00109 mol) in 50 ml CHCl3,
potassium carbonate (0.5 g, 0.0036 mol) and diethylamine (1 ml,
0.01 mol) were added. The reaction was refluxed 24 h and extracted
with brine and CH2Cl2. The organic layer was filtered, washed with
brine and dried. After concentrated, the crude product was sepa-
rated on flash column to obtain compound 2b. Compounds 2c and
2d were produced by reacted with dipropylamine and piperidine
respectively in the same method.
5.1.7.1. 40,7-Bis(3-dimethylaminopropoxy)isoflavone (3b). Light yel-
low solid. Yield 75%. Hygroscopicity. 1H NMR (400 MHz, CDCl3)
d:
8.12 (d, J ¼ 8.9 Hz, 1H), 7.84 (s, 1H), 7.44e7.37 (m, 2H), 6.88 (d,

438
D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
J ¼ 8.6 Hz, 3H), 6.78 (d, J ¼ 2.3 Hz,1H), 4.05 (t, J ¼ 6.3 Hz, 2H), 3.98 (t,
J ¼ 6.3 Hz, 2H), 2.54 (t, J ¼ 7.5 Hz, 2H), 2.49 (t, J ¼ 7.5 Hz, 2H), 2.29 (s,
6H), 2.26 (s, 6H), 1.97 (dd, J ¼ 13.7, 6.8 Hz, 4H). 13C NMR (101 MHz,
flash column to obtain compound 4c. 4d was prepared by instead of
dimethylamine solution with diethylamine in the same condition.
CDCl3)
d: 175.88, 163.31, 158.85, 157.93, 152.10, 130.13, 127.70,
5.1.10.1. 2-(2-Dimethylaminoethoxy)-7-ethoxyfluoren-9-one
(4c).
124.81, 124.28, 118.33, 114.86, 114.49, 100.62, 66.72, 66.01, 56.13,
55.97, 45.18, 44.93, 26.92. Anal. Calcd. for C25H32N2O4: C 70.73, H
7.60, N 6.60; Found C 70.70, H 7.61, N 6.58. MS (ESI): m/z [M þ H]þ
calcd 425.2, found 425.0.
Orange solid. Yield 85%. M.p. 80e81.6 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 7.19 (d, J ¼ 8.1 Hz, 2H), 7.06 (d, J ¼ 7.0 Hz, 2H), 6.89 (d, J ¼ 8.0 Hz,
1H), 6.84 (d, J ¼ 8.0 Hz,1H), 4.03 (t, J ¼ 5.4 Hz, 2H), 3.98 (dd, J ¼ 13.8,
6.9 Hz, 2H), 2.70 (t, J ¼ 5.3 Hz, 2H), 2.30 (s, 6H), 1.34 (t, J ¼ 6.9 Hz,
3H). 13C NMR (101 MHz, CDCl3)
d: 193.77, 159.35, 159.08, 137.72,
5.1.7.2. 40,7-Bis(3-diethylaminopropoxy)isoflavone (3c). Light yel-
137.31, 135.95, 135.93, 120.99, 120.85, 120.56, 120.49, 110.19, 110.14,
66.31, 63.98, 58.11, 45.77, 14.76. Anal. Calcd. for C19H21NO3: C 73.29,
H 6.80 N 4.50; Found C 73.24, H 6.82, N 4.51. MS (ESI): m/z [M þ H]þ
calcd 312.2, found 312.0.
low solid. Yield 70%. M.p. 82e84 ꢀC. 1H NMR (400 MHz, CDCl3)
d:
8.19 (d, J ¼ 8.9 Hz, 1H), 7.92 (s, 1H), 7.49 (d, J ¼ 8.6 Hz, 2H), 6.97 (dd,
J ¼ 15.5, 5.4 Hz, 3H), 6.86 (d, J ¼ 2.0 Hz, 1H), 4.12 (dt, J ¼ 26.9, 5.9 Hz,
4H), 2.95e2.74 (m, 12H), 2.25e2.08 (m, 4H), 1.25 (t, J ¼ 7.2 Hz, 6H),
1.17 (t, J ¼ 7.2 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d
: 175.87, 163.18,
5.1.10.2. 2-(2-Diethylaminoethoxy)-7-ethoxyfluoren-9-one
(4d).
158.60, 157.93, 152.16, 130.20, 127.75, 124.79, 124.53, 118.41, 114.85,
114.47, 100.65, 66.57, 65.63, 49.32, 49.18, 46.93, 46.86, 30.95, 25.89,
25.36, 10.77, 10.13. Anal. Calcd. for C29H40N2O4: C 72.47, H 8.39, N
5.83; Found C 72.45, H 8.40, N 5.83. MS (ESI): m/z [M þ H]þ calcd
481.3, found 481.1.
Orange solid. Yield 88%. M.p. 60e61 ꢀC. 1H NMR (400 MHz, CDCl3)
d:
7.21 (s, 1H), 7.18 (s, 1H), 7.07 (t, J ¼ 2.4 Hz, 2H), 6.86 (dd, J ¼ 7.9 Hz,
2H), 4.05 (t, J ¼ 5.9 Hz, 2H), 3.98 (q, J ¼ 6.9 Hz, 2H), 2.89 (t,
J ¼ 5.8 Hz, 2H), 2.66 (q, J ¼ 7.1 Hz, 4H), 1.35 (t, J ¼ 7.0 Hz, 3H), 1.05 (t,
J ¼ 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d: 193.77, 159.36, 159.00,
137.68, 137.33, 135.98, 135.96, 120.91, 120.60, 120.58, 120.49, 110.49,
110.14, 66.56, 63.99, 51.48, 47.78, 14.76, 11.41. Anal. Calcd. for
5.1.8. 2-(2-Hydroxyethoxy)-7-ethoxyfluoren-9-one (4a)
2,7-Dihydroxy-fluoren-9-one (10 g, 0.047 mol) was added into
200 ml water with NaOH (4 g, 0.1 mol), then stirred in room tem-
perature till completely solved. Diethylsulfate (9.2 ml, 0.07 mol)
was added dropwise into the system. The mixture was stirred 5 h
and the precipitate was filtered out and dried. The precipitate was
then solved in 80 ml DMSO, NaOH (2 g, 0.05 mol) and ethylene
chlorohydrin (4.1 ml, 0.06 mol) were added. The reaction was car-
ried out overnight at 70 ꢀC, then the solution was poured into 1 M
NaOH and precipitate was collected by filtration. After dried in
vacuum oven, the mixture was stirred in 50 ml CH2Cl2 for half an
hour, then filtered the insoluble substance and washed with CH2Cl2
then recrystallized from 2-propanol to get 4a. Orange solid. Yield
C21H25NO3: C 74.31, H 7.42 N 4.13; Found C 74.26, H 7.43, N 4.12.
5.1.11. 2,7-Bis(2,3-epoxypropoxy)fluoren-9-one (5a)
2,7-Dihydroxy-fluoren-9-one (10 g, 0.047 mol) was solved in
150 ml DMSO, NaOH (4 g, 0.1 mol) and epichlorohydrin (16 ml,
0.2 mol) were added into the solution. The solution was stirred
overnight at 70 ꢀC. The resulting solution was then poured into 1 M
NaOH water solution. The precipitate was collected and washed by
1 M NaOH till colorless eluate and dried in vacuum drying oven to
obtain crude product. The crude product was separated on flash
column to get compound 5a. Orange solid. Yield 73%. M.p. 93e
20
95 ꢀC. [
a]
0ꢀ (c 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3)
d: 7.31 (d,
D
30%. M.p. 158e160 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 7.27 (s, 1H), 7.25
J ¼ 8.2 Hz, 2H), 7.16 (d, J ¼ 2.2 Hz, 2H), 6.99 (dd, J ¼ 8.2, 2.3 Hz, 2H),
4.29 (dd, J ¼ 11.0, 2.8 Hz, 2H), 3.96 (dd, J ¼ 11.0, 5.8 Hz, 2H), 3.37 (d,
J ¼ 2.8 Hz, 2H), 2.93 (t, J ¼ 4.5 Hz, 2H), 2.78 (dd, J ¼ 4.7, 2.7 Hz, 2H).
(s, 1H), 7.13 (dd, J ¼ 4.4, 2.4 Hz, 2H), 6.93 (ddd, J ¼ 10.4, 8.2, 2.4 Hz,
2H), 4.12e4.08 (m, 2H), 4.05 (q, J ¼ 7.0 Hz, 2H), 3.98 (s, 2H), 1.42 (t,
J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
: 193.73, 159.41, 158.94,
13C NMR (101 MHz, CDCl3)
d: 193.37, 158.93, 137.87, 135.97, 121.08,
137.97, 137.22, 135.99, 135.92, 120.93, 120.81, 120.63, 120.55, 110.30,
110.18, 69.71, 64.00, 61.35, 14.76. Anal. Calcd. for C17H16O4: C 71.82,
H 5.67; Found C 71.79, H 5.69. MS (ESI): m/z [M þ H]þ calcd 285.1,
found 285.0.
120.72, 110.33, 69.21, 49.99, 44.57. Anal. Calcd. for C19H16O5: C
70.36, H 4.97; Found C 70.33, H 5.00.
5.1.12. General procedure for the synthesis of compounds 5bee
To a stirred solution of 5a (0.4 g, 0.00123 mol) in 50 ml dime-
thylformamide, potassium carbonate (0.5 g, 0.0036 mol) and
diethylamine (1 ml, 0.01 mol) were added. The reaction was pro-
cessed 24 h at 70 ꢀC and extracted with brine and CH2Cl2. The
organic layer was filtered, washed with brine and dried. After
concentrated in vacuum, the crude product was separated on flash
column to obtain compound 5c. Compounds 5b, 5d and 5e were
produced by reacted with dimethylamine solution, dipropylamine
and piperidine respectively in the same method.
5.1.9. 2-(2-Chloroethoxy)-7-ethoxyfluoren-9-one (4b)
4a (2 g, 0.007 mol) was solved in 40 ml thionyl chloride and
stirred at 60 ꢀC till the reaction completed (monitored by TLC), and
then poured the solution into 1 M NaOH carefully. The precipitate
was collected and purified by flash column chromatography to
yield compound 4b. Orange solid. Yield 90%. M.p.146.6e147.4 ꢀC. 1H
NMR (400 MHz, CDCl3)
d
: 7.23 (d, J ¼ 2.3 Hz, 1H), 7.21 (d, J ¼ 2.3 Hz,
1H), 7.08 (d, J ¼ 1.9 Hz, 2H), 6.88 (ddd, J ¼ 14.3, 8.1, 2.5 Hz, 2H), 4.19
(t, J ¼ 5.8 Hz, 2H), 3.99 (q, J ¼ 7.0 Hz, 2H), 3.75 (t, J ¼ 5.8 Hz, 2H), 1.35
(t, J ¼ 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d: 196.44, 159.50,
5.1.12.1. 2,7-Bis(3-dimethylamino-2-hydroxypropoxy)fluoren-9-one
20
158.50, 138.30, 137.14, 136.03, 135.95, 121.19, 120.97, 120.70, 120.62,
110.32, 110.19, 68.48, 64.02, 41.76, 14.76. Anal. Calcd. for C17H15ClO3:
C 67.44, H 4.99; Found C 67.41, H 5.01. MS (ESI): m/z [M ꢁ Cl]þ calcd
267.4, found 267.3.
(5b). Orange solid. Yield 75%. M.p. 95e97 ꢀC. [
a
]
0ꢀ (c 0.5,
D
CH2Cl2). 1H NMR (400 MHz, CDCl3)
d
: 7.18 (d, J ¼ 8.1 Hz, 2H), 7.07 (d,
J ¼ 2.3 Hz, 2H), 6.89 (dd, J ¼ 8.2, 2.4 Hz, 2H), 4.03e3.96 (m, 2H), 3.91
(qd, J ¼ 9.6, 4.8 Hz, 4H), 2.48 (dd, J ¼ 12.1, 9.9 Hz, 2H), 2.29 (dd,
J ¼ 12.3, 3.7 Hz, 2H), 2.25 (s, 12H). 13C NMR (101 MHz, CDCl3)
d:
5.1.10. General procedure for the synthesis of compounds 4c and 4d
To a stirred solution of 4b (0.4 g, 0.0013 mol) in 40 ml dime-
thylformamide, potassium carbonate (0.5 g, 0.0036 mol) and
dimethylamine solution (33%, 1.5 ml, 0.01 mol) were added. The
reaction was carried out in 24 h at 70 ꢀC and then extracted with
brine and CH2Cl2. The organic layer was filtered, washed with brine
and dried. After concentrated, the crude product was separated on
192.57, 158.14, 136.62, 134.87, 119.85, 119.56, 109.26, 69.90, 65.14,
60.63, 44.58. Anal. Calcd. for C23H30N2O5: C 66.65, H 7.30, N 6.76;
Found C 66.63, H 7.32, N 6.75. MS (ESI): m/z [M þ H]þ calcd 415.2,
found 415.0.
5.1.12.2. 2,7-Bis(3-diethylamino-2-hydroxypropoxy)fluoren-9-one
20
(5c). Orange solid. Yield 80%. M.p. 103e105 ꢀC. [
a]
0ꢀ (c 0.5,
D

D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
439
CH2Cl2). 1H NMR (400 MHz, CDCl3)
d
: 7.31 (d, J ¼ 8.2 Hz, 1H), 7.14 (d,
5.1.15.1. 2-(2-Chloroethoxy)-7-(2-diethylaminoethoxy)fluoren-9-one
(6c). Orange solid. Yield 40%. M.p. 75e76.3 ꢀC. 1H NMR (400 MHz,
J ¼ 2.3 Hz, 1H), 6.99 (dd, J ¼ 8.2, 2.4 Hz, 1H), 4.19 (dd, J ¼ 9.0, 4.4 Hz,
1H), 4.04 (dt, J ¼ 9.6, 4.5 Hz, 2H), 2.93e2.77 (m, 6H), 1.18 (t,
CDCl3)
d
: 7.19 (dd, J ¼ 8.1, 1.8 Hz, 2H), 7.06 (dd, J ¼ 5.1, 2.4 Hz, 2H),
J ¼ 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d: 193.53, 159.01, 137.53,
6.87 (ddd, J ¼ 8.0, 5.4, 2.4 Hz, 2H), 4.17 (t, J ¼ 5.8 Hz, 2H), 4.02 (t,
135.75, 120.70, 120.66, 110.43, 70.71, 65.29, 55.18, 47.51, 11.06. Anal.
Calcd. for C27H38N2O5: C 68.91, H 8.14, N 5.95; Found C 68.93, H 8.16,
N 5.94. MS (ESI): m/z [M þ H]þ calcd 471.3, found 471.1.
J ¼ 6.0 Hz, 2H), 3.74 (t, J ¼ 5.8 Hz, 2H), 2.85 (t, J ¼ 6.0 Hz, 2H), 2.62 (q,
J ¼ 7.1 Hz, 4H), 1.03 (t, J ¼ 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d:
193.45, 159.28, 158.52, 138.17, 137.29, 135.99, 135.91, 121.14, 120.72,
120.67, 110.48, 110.31, 68.46, 66.79, 51.52, 47.79, 41.77, 11.57. Anal.
Calcd. for C21H24ClNO3: C 67.46, H 6.47, N 3.75; Found C 67.44, H
6.46, N 3.75. MS (ESI): m/z [M þ H]þ calcd 374.1, found 374.0.
5.1.12.3. 2,7-Bis(3-dipropylamino-2-hydroxypropoxy)fluoren-9-one
(5d). Orange oil. Yield 80%. [
(400 MHz, CDCl3)
a]
D
0ꢀ (c 0.5, CH2Cl2). 1H NMR
20
d
: 7.15 (d, J ¼ 8.1 Hz, 2H), 7.03 (d, J ¼ 2.0 Hz, 2H),
6.86 (dd, J ¼ 8.1, 2.1 Hz, 2H), 4.00 (td, J ¼ 9.5, 4.8 Hz, 2H), 3.91 (d,
5.1.15.2. 2,7-Bis[2-(1H-1,2,4-triazol-1-yl)ethoxy]fluoren-9-one (6e).
Orange solid. Yield 55%. M.p. 209e212 ꢀC. 1H NMR (400 MHz,
J ¼ 4.8 Hz, 2H), 2.60e2.39 (m, 12H), 1.54e1.37 (m, 8H), 0.83 (t,
J ¼ 7.3 Hz, 12H). 13C NMR (101 MHz, CDCl3)
d: 193.54, 159.11, 137.58,
CDCl3)
d
: 8.22 (s, 2H), 7.97 (s, 2H), 7.28 (d, J ¼ 8.2 Hz, 2H), 7.11 (d,
135.84,120.69,120.58,110.35, 70.78, 65.73, 56.96, 56.14,19.82,11.70.
Anal. Calcd. for C31H46N2O5: C 70.69, H 8.80, N 5.32; Found C 70.64,
H 8.81, N 5.33. MS (ESI): m/z [M þ H]þ calcd 527.3, found 527.1.
J ¼ 2.4 Hz, 2H), 6.89 (dd, J ¼ 8.2, 2.5 Hz, 2H), 4.59 (t, J ¼ 5.0 Hz, 4H),
4.36 (t, J ¼ 5.0 Hz, 4H). 13C NMR (101 MHz, CDCl3)
d: 192.99, 160.26,
158.33, 152.24, 144.01, 138.05, 135.97, 120.91, 110.28, 66.08, 49.12.
Anal. Calcd. for C21H18N6O3: C 62.68, H 4.51, N 20.88; Found C 62.67,
H 4.53, N 20.87. MS (ESI): m/z [M þ H]þ calcd 403.1, found 403.0.
5.1.12.4. 2,7-Bis(3-piperidino-2-hydroxypropoxy)fluoren-9-one (5e).
Orange solid. Yield 85%. M.p. 126e128 ꢀC. [ 20 0ꢀ (c 0.5, CH2Cl2). 1H
a]
D
NMR (400 MHz, CDCl3)
d
: 7.22 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 2.3 Hz,
5.1.15.3. 2,7-Bis[2-(1H-imidazol-1-yl)ethoxy]fluoren-9-one
Orange solid. Yield 50%. M.p. 128e130 ꢀC. 1H NMR (400 MHz,
CDCl3)
: 7.63 (s, 2H), 7.28 (d, J ¼ 1.2 Hz, 2H), 7.09 (m, 6H), 6.91
(dd, J ¼ 8.2, 2.4 Hz, 2H), 4.36 (t, J ¼ 4.9 Hz, 4H), 4.24 (t, J ¼ 5.0 Hz,
4H). 13C NMR (101 MHz, CDCl3)
: 193.14, 158.42, 138.01, 137.56,
135.93, 129.60, 121.00, 120.92, 119.38, 110.14, 67.68, 45.79. Anal.
Calcd. for C23H20N4O3: C 68.99, H 5.03, N 13.99; Found C 68.98, H
5.05, N 13.96. MS (ESI): m/z [M þ H]þ calcd 401.2, found 401.0.
(6f).
2H), 6.94 (dd, J ¼ 8.2, 2.4 Hz, 2H), 4.08 (td, J ¼ 9.3, 4.7 Hz, 2H), 4.02e
3.92 (m, 4H), 2.60 (d, J ¼ 4.3 Hz, 4H), 2.52e2.32 (m, 8H), 1.67e1.52
d
(m, 8H), 1.44 (dd, J ¼ 12.9, 6.1 Hz, 4H). 13C NMR (101 MHz, CDCl3)
d:
193.52, 159.23, 137.60, 135.89, 120.86, 120.53, 110.35, 71.08, 65.37,
61.02, 54.75, 26.05, 24.20. Anal. Calcd. for C29H38N2O5: C 70.42, H
7.74, N 5.66; Found C 70.38, H 7.75, N 5.68. MS (ESI): m/z [M þ H]þ
calcd 495.3, found 495.1.
d
5.1.13. 2,7-Bis(2-hydroxyethoxy)fluoren-9-one (6a)
5.1.15.4. 2,7-Bis[2-(1H-1,2,3-triazol-1-yl)ethoxy]fluoren-9-one (6g).
Orange solid. Yield 50%. M.p.186e189 ꢀC. 1H NMR (400 MHz, CDCl3)
2,7-Dihydroxy-fluoren-9-one (20 g, 0.094 mol) was solved in
200 ml DMSO, NaOH (8 g, 0.2 mol) and ethylene chlorohydrin
(13.4 ml, 0.2 mol) were added into the solution. The resulting so-
lution was stirred overnight at 70 ꢀC, and then poured into 1 M
NaOH water solution. The precipitate was washed by 1 M NaOH till
colorless eluate and dried in vacuum drying oven then recrystal-
lized from 2-propanol to obtain compound 6a. Orange solid. Yield
d
: 7.87 (dd, J ¼ 6.6, 3.0 Hz, 2H), 7.39 (dd, J ¼ 6.6, 3.0 Hz, 2H), 7.22 (d,
J ¼ 8.2 Hz, 2H), 7.13 (d, J ¼ 2.2 Hz, 2H), 6.90 (dd, J ¼ 8.2, 2.3 Hz, 2H),
5.11 (t, J ¼ 5.5 Hz, 4H), 4.68 (t, J ¼ 5.5 Hz, 4H). 13C NMR (101 MHz,
CDCl3) d: 193.11,158.50,137.97,135.90,126.57,121.22,120.71,118.09,
110.45, 66.43, 55.49. Anal. Calcd. for C21H18N6O3: C 62.68, H 4.51, N
20.88; Found C 62.65, H 4.53, N 20.89. MS (ESI): m/z [M þ H]þ calcd
403.1, found 402.9.
55%. M.p. 166e168 ꢀC. 1H NMR (400 MHz, DMSO)
7.08 (s, 4H), 4.05 (s, 4H), 3.72 (s, 4H). 13C NMR (101 MHz, DMSO)
d: 7.54 (s, 2H),
d:
192.81, 159.09, 136.72, 135.07, 121.41, 120.82, 110.06, 70.08, 59.45.
Anal. Calcd. for C17H16O5: C 67.99, H 5.37; Found C 68.01, H 5.37.
5.1.15.5. 2,7-Bis(2-propylaminoethoxy)fluoren-9-one (6h). Orange solid.
Yield 80%. Hygroscopicity. 1H NMR (400 MHz, CDCl3)
d: 7.26 (d,
J ¼ 8.1 Hz, 1H), 7.14 (d, J ¼ 2.4 Hz, 1H), 6.93 (dd, J ¼ 8.2, 2.5 Hz, 1H), 4.09
(t, J ¼ 5.2 Hz, 2H), 3.01 (t, J ¼ 5.1 Hz, 2H), 2.68e2.62 (m, 2H), 2.34 (s,1H),
1.55 (dd, J ¼ 14.7, 7.4 Hz, 2H), 0.94 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz,
5.1.14. 2,7-Bis(2-chloroethoxy)fluoren-9-one (6b)
6a (10 g, 0.033 mol) was solved in 80 ml thionyl chloride and
stirred at 60 ꢀC till the reaction completed (monitored by TLC), then
poured the solution into 1 M NaOH carefully. The precipitate was
collected and purified by flash column chromatography to yield
compound 6b. Orange solid. Yield 90%. M.p. 142e144 ꢀC. 1H NMR
CDCl3) d: 193.65, 159.25, 137.53, 135.93, 120.66, 120.54, 110.33, 67.96,
51.76, 48.64, 23.21, 11.77. Anal. Calcd. for C23H30N2O3: C 72.22, H 7.91, N
7.32; Found C 72.20, H 7.93, N 7.32.
(400 MHz, CDCl3)
d
: 7.29 (d, J ¼ 8.1 Hz, 2H), 7.14 (d, J ¼ 2.3 Hz, 2H),
5.1.15.6. 2,7-Bis(2-isopropylaminoethoxy)fluoren-9-one
Orange solid. Yield 82%. Hygroscopicity. 1H NMR (400 MHz,
CDCl3)
(6i).
6.97 (dd, J ¼ 8.1, 2.4 Hz, 2H), 4.25 (t, J ¼ 5.8 Hz, 4H), 3.82 (t,
J ¼ 5.8 Hz, 4H). 13C NMR (101 MHz, CDCl3)
d
: 193.25, 158.68, 137.98,
d
: 7.18 (d, J ¼ 8.1 Hz, 1H), 7.06 (s, 1H), 6.86 (d, J ¼ 7.9 Hz,
136.00, 121.23, 120.81, 110.39, 68.49, 41.75. Anal. Calcd. for
1H), 4.01 (t, J ¼ 4.8 Hz, 2H), 2.92 (t, J ¼ 4.8 Hz, 2H), 2.80 (dt,
C
17H14Cl2O3: C 60.55, H 4.18; Found C 60.53, H 4.20.
J ¼ 12.3, 6.1 Hz, 1H), 1.61 (s, 1H), 1.03 (d, J ¼ 6.2 Hz, 6H). 13C
NMR (101 MHz, CDCl3) d: 192.61, 158.22, 136.51, 134.91, 119.61,
5.1.15. General procedure for the synthesis of compounds 6cem
To a stirred solution of 6b (0.4 g, 0.0012 mol) in 40 ml dime-
thylformamide, potassium carbonate (0.5 g, 0.0036 mol) and
diethylamine (1 ml, 0.01 mol) were added. The reaction was taken
24 h at 70 ꢀC and then extracted with brine and CH2Cl2. The organic
layer was filtered, washed with brine and dried. After concentrated
in vacuum, the crude product was separated on flash column to
obtain compound 6c. Compounds 6eek were prepared by instead
of diethylamine by according amines in the same condition. 6l and
6m required NaH as base and anhydrous dimethylformamide with
isopropyl mercaptan and t-butyl mercaptan, respectively.
119.50, 109.35, 67.21, 47.52, 45.23, 21.92. Anal. Calcd. for
C
23H30N2O3: C 72.22, H 7.91, N 7.32; Found C 72.18, H 7.94, N
7.30. MS (ESI): m/z [M þ H]þ calcd 383.2, found 383.1.
5.1.15.7. 2,7-Bis(2-butylaminoethoxy)fluoren-9-one (6j). Orange solid.
Yield 75%. Hygroscopicity. 1H NMR (400 MHz, CDCl3)
d: 7.19 (d,
J ¼ 8.1 Hz,1H), 7.07 (d, J ¼ 2.2 Hz,1H), 6.86 (dd, J ¼ 8.1, 2.4 Hz,1H), 4.02
(t, J ¼ 5.1 Hz, 2H), 2.93 (t, J ¼ 5.1 Hz, 2H), 2.64e2.57 (m, 2H), 1.70 (s,
1H), 1.46e1.39 (m, 2H), 1.34e1.24 (m, 2H), 0.86 (t, J ¼ 7.3 Hz, 3H). 13
C
NMR (101 MHz, CDCl3) d: 193.69, 159.25, 137.54, 135.93, 120.67,
120.55, 110.33, 67.94, 49.62, 48.72, 32.24, 20.47, 14.04. Anal. Calcd. for

440
D. Zhou et al. / European Journal of Medicinal Chemistry 64 (2013) 432e441
C25H34N2O3: C 73.14, H 8.35, N 6.82; Found C 73.11, H 8.37, N 6.81. MS
References
(ESI): m/z [M þ H]þ calcd 411.3, found 411.1.
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d
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Eight compounds (1d, 2b, 2c, 3b, 5bed and tilorone) were
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