Synthesis and biological evaluation of novel symmetry bis-enediynes

Article abstract of DOI:10.1016/j.ejmech.2008.03.005

A series of acyclic symmetry bis-enediynes have been synthesized successfully and their bioactivities were evaluated. Among them, 1,6-bis(4-((2-(pyridin-2-ylethynyl)phenyl)ethynyl)phenoxy)hexane 8g showed good inhibition activity against the CCRF-CEM (GI<

Full text of DOI:10.1016/j.ejmech.2008.03.005

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 35e41
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel symmetry bis-enediynes
Kuo-Feng Tseng a, Chi-Fong Lin b,1, Yu-Hsiang Lo c, Yi-Ling Hu a, Li-Yi Chen a,
Sheng-Huei Yang a, Shinne-Ren Lin a,e, Long-Sen Chang d,e, Ming-Jung Wu a,e,
*
a Faculty of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
b Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan, Taiwan
c Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan
d Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan
e National Sun Yat-Sen UniversityeKaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan
Received 23 September 2007; received in revised form 3 March 2008; accepted 6 March 2008
Available online 25 March 2008
Abstract
A series of acyclic symmetry bis-enediynes have been synthesized successfully and their bioactivities were evaluated. Among them, 1,6-
bis(4-((2-(pyridin-2-ylethynyl)phenyl)ethynyl)phenoxy)hexane 8g showed good inhibition activity against the CCRF-CEM (GI50 ¼ 0.04 mM)
and HL-60 (GI50 ¼ 0.09 mM) cell lines of human leukemia.. The cell cycle analysis shows that compound 8g arrests cell cycle via inhibiting
Cyclin A and Cyclin B expressions in low concentration and induces a significant apoptosis progress in high concentration.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Bis-enediynes; Cell cycle; Anticancer; Apotosis
1. Introduction
10ꢀ8 to 10ꢀ6 M. Particularly, 2-(6-(2-thienyl)-3(Z )-hexen-
1,5-diynyl)aniline against the MDA-MB-231/ACTT cell line
of human breast cancer was found to be less than 10ꢀ8 M
[6c]. Furthermore, the series of acyclic enediynes performs in-
duced apoptosis phenomenon. It has been known that linking
of two small molecules as a dimer could enhance the effi-
ciency and selectivity of the biological activities. For instance,
the trioxane dimer [7] and bis-acridine [8] have been shown to
enhance the efficiency and selectivity of anticancer activity. In
continuing search for more potent biologically active com-
pounds, bis-enediynes 8ae8g have been designed and synthe-
sized (Fig. 1). The main focus of this study is to modify the
aryl group at the terminal alkyne for the structure and to study
the structureeactivity relationship.
Small molecules can arrest cancer cell cycle machinery in
G2/M phase by the inhibition of Cdk family that led to the
antiproliferation of cancer cell have been reported by many re-
search groups [1e4]. Cyclin A and Cyclin B are critical pro-
teins in G2 and mitosis of cell cycle. Cyclin A can construct
complexes that play critical roles in replication and passage
through G2 with both Cdk1 and Cdk2 during S and G2 phases.
Cyclin B constructs complexes that required for mitosis with
Cdk1 during G2 [5]. The report of controlling cell cycle via
Cdk family is interested in many small molecules. Recently,
we have disclosed novel acyclic enediyne derivatives [6] that
showed remarkable broad spectrum inhibition of the growth
of cancer cell lines and the average GI50 values were from
2. Chemistry
* Corresponding author. Faculty of Medicinal and Applied Chemistry, Kaoh-
siung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan. Tel.:
þ886 7 3121101; fax: þ886 7 3125339.
The synthesis of compounds 8ae8g is summarized in
Scheme 1. Compound 1 [9] was treated with various aryl iodides
2ae2f using Pd(PPh3)4 as the catalyst to obtain compounds
E-mail address: mijuwu@kmu.edu.tw (M.-J. Wu).
1
Equally contributed as the first author.
0223-5234/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ejmech.2008.03.005

36
K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
O
O
n
8a. Ar = 2-anilinyl
n = 5
n = 6
8b. Ar = 2-cyanophenyl
8c. Ar = 2-anilinyl
8d. Ar = 2-nitrophenyl
8e. Ar = 2-anisyl
8f. Ar = 2-trifluoromethylphenyl
8g. Ar = 2-pyridinyl
Ar
Ar
Fig. 1. Chemical structures of bis-enediynes 8ae8g.
3ae3f. On the other hand, 1,5-dibromopentane (4a) and 1,6-di-
bromohexane (4b) were reacted with 4-iodophenol (5) in DMF
using K2CO3 as the base to give compounds 6a and 6b in 70 and
41% yields, respectively. Compounds 6a and 6b were then cou-
pled with trimethylsilylacetylene under Sonogashira coupling
reaction conditions [10], followed by desilyation gave com-
pounds 7a and 7b in 71 and 78% yields, respectively. Finally,
palladium-catalyzed coupling reaction of 7a and 7b with vari-
ous aryl iodides 3ae3f gave 8ae8g in the yields from 30 to
57%.
Ar
H
a
Ar
I
+
I
I
1
2a. Ar = 2-cyanophenyl
2b. Ar = 2-anilinyl
3a (33%)
3b (23%)
3c (64%)
3d (48%)
3e (25%)
3f (64%)
2c. Ar = 2-nitrophenyl
2d. Ar = 2-anisyl
2e. Ar = 2-trifluoromethylphenyl
2f. Ar = 2-pyridinyl
I
I
Br
b
a, c
O
+
Br
O
n
n
HO
I
4a. n= 5
4b. n= 6
5
6a. n= 5 (70%)
6b. n= 6 (41%)
H
Ar
a
O
O
+
n
I
H
7a. n= 5 (71%)
7b. n= 6 (78%)
3a. Ar = 2-cyanophenyl
3b. Ar = 2-anilinyl
3c. Ar = 2-nitrophenyl
3d. Ar = 2-anisyl
3e. Ar = 2-trifluoromethylphenyl
3f. Ar = 2-pyridinyl
Ar
n = 5
n = 6
8a. Ar = 2-anilinyl (51%)
O
n
O
8b. Ar = 2-cyanophenyl (34%)
8c. Ar = 2-anilinyl (30%)
8d. Ar = 2-nitrophenyl (32%)
8e. Ar = 2-anisyl (57%)
8f. Ar = 2-trifluoromethylphenyl (42%)
8g. Ar = 2-pyridinyl (38%)
Ar
Scheme 1. Reagents and condition: (a) Pd(PPh3)4, CuI, n-BuNH2, ether, rt; (b) K2CO3, DMF, rt; (c) K2CO3, methanol, rt.

K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
37
3. Biological assay result
Table 1
The in vitro testing results of full panel screen of 50 human tumor cell linesa
3.1. Cytotoxicity activities
Panel/cell line
8g
c
GI50b/LC50
Compounds 8ae8g were submitted to the National Can-
cer Institute for testing against a panel of nine cancer cell
lines. Details of this test system have been published by
others [11]. Compounds 8ae8f were found to be inactive
in the pre-screening test. However, compound 8g is found
to be active against 50 tumor cell lines at low concentration,
especially against the CCRF-CEM (GI50 ¼ 0.04 mM) and
HL-60 (GI50 ¼ 0.09 mM) cell lines of human leukemia. The
GI50 and LC50 values of compound 8g are summarized in
Table 1. It was also noted that most of the LC50 values of
compound 8g for these cancer cell lines were 10e1000 times
higher than the GI50 values. This result indicates that com-
pound 8g exhibits very high growth inhibition activity but
not cytotoxic toward human cancer cell lines. This phenom-
enon is meaningful to the advancement of medical therapies
of human cancer diseases, whereas drugs with highly
cytotoxic activities always caused the damage to normal
cells.
Leukemia
CCRF-CEM
HL-60
0.04/>50.0
0.09/2.7
K-562
MOLT-4
1.38/22.8
0.37/28.0
0.62/>50.0
RPMI-8226
Non-Small Cell Lung cancer
A549/ATCC
EKVX
6.41/43.2
4.33/22.9
6.31/38.8
6.53/30.3
7.97/>50.0
1.69/18.7
2.87/38.4
HOP-62
NCI-H226
NCI-H23
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
8.18/30.9
7.21/26.9
8.69/>50.0
2.78/22.0
7.89/27.0
1.38/20.8
4.00/22.7
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
3.2. Cell cycle assay
3.63/22.7
1.24/15.8
2.38/21.3
6.00/26.2
5.13/31.3
5.63/24.1
SF-539
SNB-19
To confirm the mechanism of the biological activity of bis-
enediyne 8g, we examined cell cycle phase distribution by
treating K-562 cell line of leukemia for 24 h by flow cytome-
try. The pattern of cell cycle is shown in Fig. 2. The result of
cell cycle percentage is summarized in Fig. 3(a). On the other
hand, Fig. 3(b) summarized the result of apoptosis percentage.
According to Fig. 3(a), the cell cycle was blocked in G2/M
phase compared with control (17.3%) by compound 8g of
10 mM (35.8%). Furthermore, treatment with 8g in 20 mM
with K-562 cell showed diphasic arrest in S phase (59.7%)
and G2/M (26.6%) that compared with S phase (51.2%) and
G2/M (17.3%) of control. Comparing the percentage of sub-
G1 area (3.13%) of control, compound 8g showed significant
apoptotic activity against K-562 cell line in 50 mM (15.54%).
This experiment indicated that compound 8g can cease cell cy-
cle machinery in G2/M phase in low concentration (10 mM),
diphasic arrest in S phase and G2/M phase (20 mM), and in-
duce significant apoptosis phenomenon in high concentration
(50 mM).
SNB-75
U251
Melanoma
LOX IMVI
M14
0.27/18.4
7.47/41.6
5.73/24.3
2.30/20.5
7.11/31.4
0.47/16.4
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
SK-OV-3
Renal cancer
786-0
2.14/21.3
1.91/23.4
3.56/23.4
7.05/42.0
7.45/>50.0
1.34/19.6
5.42/23.8
7.65/27.9
6.55/32.7
8.51/>50.0
ACHN
CAKI-1
RXF 393
SN12C
UO-31
Prostate cancer
PC-3
DU-145
4.46/29.2
6.14/24.9
3.3. Inhibiting Cyclin A and Cyclin B expressions
Breast cancer
MCF7
1.91/21.8
2.30/>50.0
2.67/23.5
5.41/34.6
1.54/21.7
3.45/22.7
2.35/>50.0
To obtain the understanding of mechanism of cell cycle in
these enediyne analogues, we examined the expression of G2/
M associated proteins, including Cyclin A, Cyclin B, Cdk1,
Cdk2 and Cdk4 expression analyses which are summarized
in Fig. 4. According to the electrophoresis, we found that com-
pound 8g showed significant inhibition of Cyclin A and Cyclin
B expressions in 20 mM. It is indicted that compound 8g af-
fects cancer cell cycle via controlling the expressions of Cy-
clin A and Cyclin B.
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
a
Data obtained from the NCI’s in vitro human tumor cell screen.
The concentration produces 50% reduction in cell growth.
The concentration produces 50% cells death.
b
c

38
K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
Fig. 2. Cell cycle distribution of K-562 cells treated with colchicine (10 mM) and compound 8g for 24 h used at the concentration of 10, 20 and 50 mM by flow
cytometry analysis. Sub G1 ¼ apoptotic area.
4. Conclusion
(15 ml) was added a degassed solution of aryl iodides
(12 mmol) containing Pd(PPh3)4 (0.8 mmol) in ether (20 ml).
The resulting reaction mixture was stirred for 6 h. Methanol
was removed in vacuo, and the residue was quenched with
saturated aqueous NH4Cl solution. The aqueous layer was ex-
tracted with EtOAc (50 ml) and the combined organic extracts
were washed with saturated aqueous Na2CO3 solution (40 ml)
and dried over anhydrous MgSO4. After filtration and removal
of solvent in vacuo, the residue was purified by column chro-
matography on silica gel to yield the desired products.
In conclusion, we have found a new acyclic bis-enediyne
derivative that is potent in growth inhibition on many kinds
of human tumor cancer cells. In this study, several points
were achieved. (i) The structureeactivity relationship of bis-
enediynes showed the distinct cytotoxic activities in 2-pyri-
dinyl of changed aryl group. (ii) The average GI50 values of
compound 8g were from 10ꢀ8 to 10ꢀ6 M, especially for
against the CCRF-CEM (0.04 mM) and HL-60 (0.09 mM)
cell lines of human leukemia. (iii) For 24 h treatment of com-
pound 8g, it can cease cell cycle machinery in G2/M phase
(10 mM), diphasic arrest in S phase and G2/M phase
(20 mM) and induce significant apoptosis phenomenon
(50 mM) (Fig. 3) and (iv) Compound 8g showed significant in-
hibition of Cyclin A and Cyclin B expressions that play criti-
cal roles during cell cycle in low concentration (Fig. 4).
According to the results of this investigation, we found that
there is no biological activity correlation between the substit-
uent on the bis-enediyne derivatives and the mono-enediynes.
5.1.1. Cell cycle analysis
Flow cytometry was used to measure cell cycle profile and
apoptosis. For cell cycle analysis, K-562 cells treated with
compounds 8g for 24 h were harvested by centrifugation. Af-
ter being washed with PBS, the cells were fixed with ice-cold
70% ethanol for 30 min, washed with PBS, and then treated
with 1 ml of 1 mg/ml of RNase A solution at 37 ꢁC for
30 min. Cells were harvested by centrifugation at 1000 rpm
for 5 min and further stained with 250 ml of DNA staining so-
lution (10 mg of propidium iodide [PI], 0.1 mg of trisodium
citrate, and 0.03 ml of Triton X-100 were dissolved in
100 ml H2O) at room temperature for 30 min in the dark. After
loading 500 ml of PBS, the DNA contents of 10,000 cells were
measured by FACScan (Elite ESP, Beckman Coulter, Brea,
CA) and the cell cycle profile was analyzed from the DNA
content histograms with WinCycle software. When cells
were undergoing apoptosis, the containing DNA was digested
5. Experimental
5.1. General procedure for coupling compound 3ae3f,
7ae7b and 8ae8g
To a degassed solution of terminal alkyne (12 mmol) con-
taining CuI (3.2 mmol) and n-BuNH2 (30 mmol) in MeOH
Fig. 3. (a) Cell cycle percentage of K-562 cells treated with compound 8g. The percentages of accumulation of G2/M phase cells of the control and compound 8g
(10, 20 and 50 mM) were 17.3 ꢂ 0.4, 35.8 ꢂ 0.4, 26.6 ꢂ 1.4 and 24.9 ꢂ 2.3%, respectively (n ¼ 3). (b) Apoptosis percentage of K-562 cells treated with compound
8g. The proportions of apoptotic cells for the control and compound 8g (10, 20 and 50 mM) were control (3.13 ꢂ 0.5%), 10 mM (8.57 ꢂ 0.5%), 20 mM
(8.77 ꢂ 0.2%) and 50 mM (15.54 ꢂ 0.7%).

K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
39
107.2, 100.4, 96.5, 90.2. HRMS calcd for C14H10IN,
Mr ¼ 319.1404; found 318.9852.
5.1.5. 1-Iodo-2-((2-nitrophenyl)ethynyl)benzene (3c)
The compound was purified by column chromatography,
eluting with hexane/EA (20:1) to give 64% of yellow oil ac-
cording to general procedure. Mp: 82e83 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 8.12 (dd, 1H, J ¼ 7.2, 1.2 Hz), 7.90
(dd, 1H, J ¼ 8.4, 1.2 Hz), 7.85 (dd, 1H, J ¼ 7.6, 1.6 Hz),
7.65 (td, 2H, J ¼ 7.2, 1.6 Hz), 7.50 (td, 1H, J ¼ 7.6, 1.6 Hz),
7.37 (td, 1H, J ¼ 7.6, 1.2 Hz), 7.09 (td, 1H, J ¼ 7.6,
1.6 Hz); 13C NMR (CDCl3, 100 MHz): d 138.8, 134.8,
133.4, 132.9, 130.3 (2C), 129.0, 128.9, 127.9, 124.7, 118.5,
100.7, 98.8, 88.0. HRMS calcd for C14H8INO2, Mr ¼
349.1233; found 348.9601.
Fig. 4. The western blotting analysis of the G2/M related protein, includes
Cyclin A, Cyclin B, CDK1, CDK2 and CDK4 in 10, 20 and 50 mM.
5.1.6. 1-Iodo-2-((2-methoxyphenyl)ethynyl)benzene (3d)
The compound was purified by column chromatography,
eluting with hexane/EA (20:1) to give 48% of yellow oil ac-
by endonuclease, then the sub G1 pick appeared. The percentage
of sub G1 was analyzed by gating on cell cycle dot blots using
Windows Multiple Document Interface software (WinMDI).
1
cording to general procedure. H NMR (CDCl3, 400 MHz):
d 7.88 (dd, 1H, J ¼ 6.8, 1.2 Hz), 7.58 (td, 2H, J ¼ 6.4,
2.0 Hz), 7.36e7.30 (m, 2H), 7.02e6.91 (m, 3H), 3.93 (s,
3H); 13C NMR (CDCl3, 100 MHz): d 160.0, 138.6, 133.6,
132.5, 130.1(2C), 129.1, 127.6, 120.4, 112.1, 110.8, 100.9,
95.3, 89.6, 55.8. HRMS calcd for C15H11IO, Mr ¼ 334.1517;
found 333.9861.
5.1.2. Western blotting analysis
Cells were washed in PBS, suspended in lysis buffer con-
taining 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA,
10 mM NaCl, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride and placed on ice for 30 min.
After centrifugation at 20,000g for 30 min at 4 ꢁC, the super-
natant was collected. The protein concentration in the superna-
tant was determined with a BCA protein assay kit (Pierce,
Rockford, IL, USA). Whole lysate (50 mg) was resolved by
12% SDS-PAGE, transferred onto PVDF membranes (Roche)
by electroblotting, and probed with, anti-Cdk1, -Cdk2, -Cdk4,
anti-cyclin A, and anti-cyclinB1 (Santa Cruz Biotechnology
Inc., Santa Cruz, CA). The blot was developed by enhanced
chemiluminescence.
5.1.7. 1-Iodo-2-((2-(trifluoromethyl)phenyl)ethynyl)-
benzene (3e)
The compound was purified by column chromatography,
eluting with hexane/EA (3:1) to give 25% of yellow oil ac-
1
cording to general procedure. H NMR (CDCl3, 400 MHz):
d 7.88 (dd, 1H, J ¼ 7.2, 0.8 Hz), 7.78 (d, 1H, J ¼ 7.6 Hz),
7.70 (d, 1H, J ¼ 8.0 Hz), 7.57e7.53 (m, 2H), 7.45 (t, 1H,
J ¼ 7.6 Hz), 7.35 (td, 1H, J ¼ 7.6, 1.2 Hz), 7.05 (td, 1H,
J ¼ 7.6, 1.6 Hz); 13C NMR (CDCl3, 100 MHz): d 138.5,
133.9, 132.9, 131.2, 129.6, 129.1, 128.1, 127.6, 125.6,
124.6, 121.9, 120.9, 100.1, 96.3, 88.4. HRMS calcd for
C15H8F3I, Mr ¼ 372.1237; found 371.9630.
5.1.3. 1-Iodo-2-((2-cyanophenyl)ethynyl)benzene (3a)
The compound was purified by column chromatography,
eluting with hexane/EA (30:1) to give 33% of yellow oil accord-
ing to general procedure. Mp: 90e91 ꢁC. H NMR (CDCl3,
1
400 MHz): d 7.89 (dd, 1H, J ¼ 8.0, 1.2 Hz), 7.73e7.57 (m,
4H), 7.44 (td, 1H, J ¼ 7.6, 1.2 Hz), 7.36 (td, 1H, J ¼ 7.6,
1.2 Hz), 7.07 (td, 1H, J ¼ 7.6, 1.6 Hz); 13C NMR (CDCl3,
100 MHz): d 138.8, 133.4, 132.7, 132.5, 132.3, 132.1, 130.2,
128.6, 127.9, 126.7, 117.5, 114.9, 100.5, 97.3, 88.6. HRMS
calcd for C15H8IN, Mr ¼ 329.1352; found 328.9706.
5.1.8. 1-Iodo-2-((2-pyridinyl)ethynyl)benzene (3f)
The compound was purified by column chromatography,
eluting with hexane/EA (15:1) to give 64% of yellow oil ac-
1
cording to general procedure. . H NMR (CDCl3, 400 MHz):
d 8.64 (dd, 1H, J ¼ 4.8, 0.4 Hz), dd, 7.89 (1H, J ¼ 6.8,
1.2 Hz), 7.70 (td, 1H, J ¼ 7.6, 1.6 Hz), 7.62 (td, 2H, J ¼ 7.2,
1.2 Hz), 7.34 (td, 1H, J ¼ 7.6, 1.2 Hz), 7.28e7.25 (m, 1H),
7.05 (td, 1H, J ¼ 7.2, 1.6 Hz); 13C NMR (CDCl3, 100 MHz):
d 150.0, 143.0, 138.7, 136.2, 133.1, 130.0, 128.8, 127.8,
127.5, 123.0, 101.0, 91.7, 91.1. HRMS calcd for C13H8IN,
Mr ¼ 304.9701; found 304.9701.
5.1.4. 1-Iodo-2-((2-anilinyl)ethynyl)benzene (3b)
The compound was purified by column chromatography,
eluting with hexane/EA (15:1) to give 23% of yellow oil ac-
cording to general procedure. Mp: 67e68 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.87 (dd, 1H, J ¼ 6.8, 1.2 Hz), 7.55
(dd, 1H, J ¼ 6.0, 2.0 Hz), 7.41 (dd, 1H, J ¼ 5.6, 2.0 Hz),
7.34 (td, 1H, J ¼ 7.2, 1.2 Hz), 7.17 (td, 1H, J ¼ 7.6, 1.2 Hz),
7.01 (td, 1H, J ¼ 8.0, 2.0 Hz), 6.74e6.71 (m, 2H), 4.58e
4.48 (bs, 2H); 13C NMR (CDCl3, 100 MHz): d 148.4, 138.5,
132.3, 132.1, 130.1, 129.9, 129.1, 127.9, 117.7, 114.3,
5.1.9. 1,5-Bis(4-iodophenoxy)pentane (6a)
To a stirred solution of 1,5-dibromopentane (10 mmol) and
4-iodophenol (10 mmol) in DMF (20 ml) was added K2CO3
(50 mmol) under nitrogen at room temperature. The resulting
solution was stirred for 24 h. The aqueous layer was extracted

40
K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
with EtOAc (50 ml) and the combined organic extracts were
washed with saturated aqueous NH4Cl solution (40 ml) and
dried over anhydrous MgSO4. After filtration and removal of
solvent in vacuo, the residue was purified by column chroma-
tography on silica gel to yield the desired products. The com-
pound was purified by column chromatography, eluting with
hexane/EA (50:1) to give 70% of white solid according to gen-
eral procedure. Mp: 106e107 ꢁC. 1H NMR (CDCl3,
400 MHz): d 7.54 (dd, 4H, J ¼ 4.4, 2.4 Hz), 6.66 (dd, 4H,
J ¼ 4.4, 2.4 Hz), 3.94 (t, 4H, J ¼ 6.4 Hz), 1.84 (quin, 4H,
J ¼ 6.4 Hz), 1.64e1.60 (m, 2H); 13C NMR (CDCl3,
100 MHz): d 158.8, 138.1, 116.8, 82.5, 67.7, 28.81, 22.6.
HRMS calcd for C17H18I2O2, Mr ¼ 508.1326; found
507.9395.
83.716, 67.843, 29.077, 25.803. HRMS calcd for C22H22O2,
Mr ¼ 318.4089; found 318.1625.
5.1.13. 1,6-Bis(4-((2-((2-anilinyl)ethynyl)phenyl)ethynyl)-
phenoxy)pentane (8a)
According to general procedure, we can obtain compound
8a. The compound was purified by column chromatography,
eluting with hexane/EA (5:1) to give 51% of yellow oil ac-
1
cording to general procedure. H NMR (CDCl3, 400 MHz):
d 7.59e7.49 (m, 8H), 7.41 (dd, 2H, J ¼ 8.0, 1.6 Hz), 7.33e
7.26 (m, 4H), 7.13 (td, 2H, J ¼ 8.0, 1.2 Hz), 6.87 (dt, 4H,
J ¼ 8.8, 2.8 Hz), 6.73e6.66 (m, 4H), 4.60e6.20 (bs, 2H),
3.99 (t, 4H, J ¼ 6.4 Hz), 1.85 (quin, 4H, J ¼ 8.0 Hz), 1.65
(quin, 2H, J ¼ 7.2 Hz); 13C NMR (CDCl3, 100 MHz):
d 159.2, 148.1, 133.2, 131.9, 131.8, 131.2, 129.8, 127.7,
127.6, 125.5, 125.2, 117.4, 114.7, 114.4, 113.9, 107.4, 93.7,
93.1, 90.2, 87.4, 67.7, 28.8, 22.6. HRMS (FABþ) calcd for
C49H39 N2O2 (M þ H)þ, Mr ¼ 687.3012; found 687.3008.
5.1.10. 1,6-Bis(4-iodophenoxy)hexane (6b)
To a stirred solution of 1,6-dibromohexane (10 mmol) and
4-iodophenol (10 mmol) in DMF (20 ml) was added K2CO3
(50 mmol) under nitrogen at room temperature. The resulting
solution was stirred for 24 h. The aqueous layer was extracted
with EtOAc (50 ml) and the combined organic extracts were
washed with saturated aqueous NH4Cl solution (40 ml) and
dried over anhydrous MgSO4. After filtration and removal of
solvent in vacuo, the residue was purified by column chroma-
tography on silica gel to yield the desired products. The com-
pound was purified by column chromatography, eluting with
hexane/EA (50:1) to give 41% of yellow solid according to
general procedure. Mp: 104e108 ꢁC. 1H NMR (CDCl3,
400 MHz): d 7.539 (dt, 4H, J ¼ 8.8, 3.2 Hz), 6.665 (dt, 4H,
J ¼ 8.8, 3.2 Hz), 3.923 (t, 4H, J ¼ 6.4 Hz), 1.799 (t, 4H,
J ¼ 6.4 Hz), 1.575e1.499 (m, 4H); 13C NMR (CDCl3,
100 MHz): d 158.905, 138.150, 116.872, 82.473, 67.858,
5.1.14. 1,6-Bis(4-((2-((2-cyanophenyl)ethynyl)phenyl)-
ethynyl)phenoxy)hexane (8b)
According to general procedure, we can obtain compound
8b. The compound was purified by column chromatography,
eluting with hexane/EA (5:1) to give 34% of yellow solid ac-
cording to general procedure. Mp: 147e148 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.69e7.63 (m, 6H), 7.57e7.52 (m,
8H), 7.51e7.29 (m, 6H), 6.87 (dt, 4H, J ¼ 9.2, 2.8 Hz), 3.99
(t, 4H, J ¼ 6.8 Hz), 1.83 (t, 4H, J ¼ 6.4 Hz), 1.55 (quin, 4H,
J ¼ 4.0 Hz); 13C NMR (CDCl3, 100 MHz): d 159.3, 133.2,
132.7, 132.6, 132.4, 132.3, 131.7, 128.9, 128.3, 127.7,
127.2, 126.3, 124.7, 117.4, 115.1, 115.0, 114.5, 94.8, 94.1,
89.1, 86.6, 67.8, 29.6, 25.8. HRMS calcd for C52H36N2O2,
Mr ¼ 720.2777; found 720.2766.
29.039,
25.772.
HRMS
calcd
for
C18H20I2O2,
Mr ¼ 522.1591; found 521.9548.
5.1.15. 1,6-Bis(4-((2-((2-anilinyl)ethynyl)phenyl)ethynyl)-
phenoxy)hexane (8c)
5.1.11. 1,5-Bis(4-ethynylphenoxy)pentane (7a)
According to general procedure, we can obtain compound
8c. The compound was purified by column chromatography,
eluting with hexane/EA (5:1) to give 30% of yellow solid ac-
cording to general procedure. Mp: 124e125 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.59e7.51 (m, 8H), 7.41 (dd, 2H,
J ¼ 7.6, 1.6 Hz), 7.32e7.29 (m, 4H), 7.14 (td, 2H, J ¼ 7.2,
1.6 Hz), 6.91e6.87 (m, 4H), 6.73e6.67 (m, 4H), 4.60e4.20
(br, 4H), 4.00 (t, 4H, J ¼ 6.4 Hz), 1.85 (t, 4H, J ¼ 6.4 Hz),
1.56 (quin, 4H, J ¼ 4.0 Hz); 13C NMR (CDCl3, 100 MHz):
d 159.3, 148.1, 133.3, 131.9, 131.8, 131.2, 129.8, 127.8,
127.6, 125.5, 125.2, 117.4, 114.7, 114.4, 113.9, 107.4, 93.8,
93.1, 90.2, 87.4, 67.8, 29.0, 25.8. HRMS (FABþ) calcd for
C50H41N2O2 (M þ H)þ, Mr ¼ 701.3168; found 701.3173.
According to general procedure and followed by desilyla-
tion, we can obtain compound 7a. The compound was purified
by column chromatography, eluting with hexane/EA (50:1) to
give 71% of yellow solid according to general procedure. Mp:
86e88 ꢁC. 1H NMR (CDCl3, 400 MHz): d 7.41 (dt, 4H,
J ¼ 8.8, 2 Hz), 6.823 (dt, 4H, J ¼ 8.8, 2 Hz), 3.99 (t, 4H,
J ¼ 6.4 Hz), 2.991 (s, 2H), 1.89e1.82 (m, 4H), 1.68e1.63
(m, 2H); 13C NMR (CDCl3, 100 MHz): d 133.568, 114.404,
113.979, 83.684, 68.725, 28.881, 22.668. HRMS calcd for
C21H20O2, Mr ¼ 304.3823; found 304.1462.
5.1.12. 1,6-Bis(4-ethynylphenoxy)hexane (7b)
After general procedure and desilyation, we can obtain com-
pound 7b. The compound was purified by column chromato-
graphy, eluting with hexane/EA (50:1) to give 78% of yellow
solid according to general procedure. Mp: 94e96 ꢁC. 1H
NMR (CDCl3, 400 MHz): d 7.41 (dt, 4H, J ¼ 8.0, 2.8 Hz),
6.827 (dt, 4H, J ¼ 7.8, 2 Hz), 3.968 (t, 2H, J ¼ 6.4 Hz), 2.991
(s, 1H), 1.816 (t, 4H, J ¼ 6.4 Hz), 1.563e1.515 (m, 4H); 13C
NMR (CDCl3, 100 MHz): d 133.564, 114.439, 113.969,
5.1.16. 1,6-Bis(4-((2-((2-nitrophenyl)ethynyl)phenyl)-
ethynyl)phenoxy)hexane (8d)
According to general procedure, we can obtain compound
8d. The compound was purified by column chromatography,
eluting with hexane/EA (4:1) to give 32% of yellow solid ac-
cording to general procedure. Mp: 150e151 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 8.09 (dd, 2H, J ¼ 8.0, 1.2 Hz), 7.75

K.-F. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 35e41
41
(dd, 2H, J ¼ 7.6, 1.2 Hz), 7.62 (dt, 2H, J ¼ 7.2, 1.2 Hz), 7.57e
7.43 (m, 10H), 7.37e7.29 (m, 4H), 6.87 (dt, 4H, J ¼ 9.6,
2.8 Hz), 3.98 (t, 4H, J ¼ 6.4 Hz), 1.83 (t, 4H, J ¼ 6.4 Hz),
1.55 (quin, 4H, J ¼ 4.0 Hz); 13C NMR (CDCl3, 100 MHz):
d 159.3, 149.3, 134.9, 133.2, 132.7, 132.6, 131.8, 128.9,
128.6, 127.7, 126.5, 124.7, 124.4, 118.9, 115.0, 114.5, 96.2,
94.0, 88.4, 86.6, 67.8, 29.0, 25.8. HRMS (FABþ) calcd for
C50H37N2O6 (M þ H)þ, Mr ¼ 761.2652; found 761.2657.
eluting with hexane/EA (3:1) to give 38% of yellow solid ac-
1
cording to general procedure. H NMR (CDCl3, 400 MHz):
d 8.63 (d, 2H, J ¼ 4.4 Hz), 7.65e7.60 (m, 4H), 7.55e7.52
(m, 8H), 7.44e7.20 (m, 6H), 6.86e6.80 (m, 4H), 3.97 (td,
4H, J ¼ 6.4, 3.6 Hz), 1.82 (t, 4H, J ¼ 6.4 Hz), 1.52 (quin,
4H, J ¼ 3.2 Hz); 13C NMR (CDCl3, 100 MHz): d 159.2,
149.9, 143.4, 136.0, 133.9, 133.1, 132.1, 131.4, 128.6,
127.5, 127.2, 126.6, 124.4, 122.7, 115.0, 114.5, 114.4, 94.1,
92.3, 88.1, 86.7, 67.8, 29.0, 25.7. HRMS (FABþ) calcd for
C48H37N2O2 (M þ H)þ, Mr ¼ 672.2777; found 673.2857.
5.1.17. 1,6-Bis(4-((2-((2-methoxyphenyl)ethynyl)phenyl)-
ethynyl)phenoxy)hexane (8e)
According to general procedure, we can obtain compound
8e. The compound was purified by column chromatography,
eluting with hexane/EA (5:1) to give 57% of yellow solid ac-
cording to general procedure. Mp: 105e106 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.55e7.45 (m, 10H), 7.28e7.21 (m,
6H), 6.90e6.78 (m, 8H), 3.92 (t, 4H, J ¼ 6.4 Hz), 1.76 (t,
4H, J ¼ 6.4 Hz), 1.48 (quin, 4H, J ¼ 3.6 Hz); 13C NMR
(CDCl3, 100 MHz): d 159.8, 159.0, 133.6, 133.0, 131.7,
131.3, 129.7, 127.3, 125.8, 125.7, 120.2, 115.2, 114.2,
112.4, 110.5, 93.5, 92.2, 89.7, 86.9, 67.6, 55.5, 28.9, 25.6.
HRMS calcd for C52H42O4, Mr ¼ 730.3083; found 730.3088.
Acknowledgements
We would like to thank the National Science Council of the
Republic of China and National Sun Yat-Sen University-
Kaohsiung Medical University Joint Research Center for their
financial support.
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5.1.18. 1,6-Bis(4-((2-((2-trifluoromethylphenyl)ethynyl)-
phenyl)ethynyl)phenoxy)hexane (8f)
According to general procedure, we can obtain compound
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eluting with hexane/EA (3:1) to give 42% of yellow solid ac-
cording to general procedure. Mp: 147e148 ꢁC. 1H NMR
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5.1.19. 1,6-Bis(4-((2-(pyridin-2-ylethynyl)phenyl)ethynyl)-
phenoxy)hexane (8g)
According to general procedure, we can obtain compound
8g. The compound was purified by column chromatography,
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