Cytotoxicities and topoisomerase I inhibitory activities of 2-[2-(2-alkynylphenyl)ethynyl]benzonitriles, 1-aryldec-3-ene-1,5-diynes, and related bis(enediynyl)arene compounds

Article abstract of DOI:10.1002/1522-2675(200208)85:8<2564::AID-HLCA2564>3.0.CO;2-0

The activities of a series of acyclic enediynes, 2-(6-substituted hex-3-ene-1,5-diynyl)benzonitriles (1-5) and their derivatives 7-23 were evaluated against several solid tumor cell lines and topoisomerase I. Compounds 1-5 show selective cytotoxicity with

Full text of DOI:10.1002/1522-2675(200208)85:8<2564::AID-HLCA2564>3.0.CO;2-0

2564
Helvetica Chimica Acta Vol. 85 (2002)
Cytotoxicities and Topoisomerase I Inhibitory Activities of 2-[2-(2-
Alkynylphenyl)ethynyl]benzonitriles,1-Aryldec-3-ene-15, -diynes,and
Related Bis(enediynyl)arene Compounds
by Chi-Fong Lina), Wen-Der Lua), Pei-Chen Hsieha), Yao-Haur Kuob), Huey-Fen Chiuc), Chyi-Jia Wangd),
and Ming-Jung Wu*a)
a) School of Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan (Phone: 886-7-3121101, ext 2220;
fax: 886-7-3125339; e-mail: mijuwu@cc.kmu.edu.tw)
b) National Research Institute of Chinese medicine, Taipei, Taiwan
c) Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
d) School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
The activities of a series of acyclic enediynes, 2-(6-substituted hex-3-ene-1,5-diynyl)benzonitriles (1 5) and
their derivatives 7 23 were evaluated against several solid tumor cell lines and topoisomerase I. Compounds 1
5 show selective cytotoxicity with Hepa cells, and 2-[6-phenylhex-3-ene-1,5-diynyl]benzonitrile (5) reveals the
most-potent activity. Analogues 8 10 and 13 22 also have the same effect with DLD cells; 1-[(Z)-dec-3-ene-
1,5-diynyl)-4-nitrobenzene 21 shows the highest activity among them. Moreover, 1-[(Z)-dec-3-ene-1,5-diynyl]-
2-(trifluoromethyl)benzene (20) exhibits the strongest inhibitory activity with the Hela cell line. Derivatives 9,
10, 18, and 23 display inhibitory activities with topoisomerase I at 87 mm. The cell-cycle analysis of compound 5,
which induces a significant blockage in S phase, indicates that these novel enediynes probably undergo other
biological pathways leading to the cytotoxicity, except the inhibitory activity toward topoisomerase I.
Introduction. Topoisomerases are important enzymes highly associated with the
separation of DNA strands in many cellular metabolic processes that alter the
topological state of duplex DNA. Topoisomerases can be classified into two types based
on the mode of cleavage of duplex DNA [1]; topo I makes a transient nick on a single-
strand of DNA and does not require an energy cofactor [2], while topo II acts by
nicking both strands of the DNA and hydrolyzes ATP during its catalytic cycle [3].
Topoisomerase I can be isolated from some cellular organisms, including nuclear and
mitochondria. Moreover, topo I is present throughout the cell, and its activity varies
less than that of topo II during cell cycle [4], which makes a topo I inhibitor an
attractive target for anticancer, antibacterial, and antiviral drug development.
Although a series of synthetic acyclic enediynes, 2-(6-substituted hex-3-ene-1,5-
diynyl)benzonitriles 2 5, exhibited cytotoxicity toward KB and Hep 2,2,15 cells, and
significant topo I inhibitory properties [5] in low micro-molar concentration ranges
(Scheme 1), the precise relationships between the cytotoxicities and these unique
structures are still under investigation. This prompted us to further study the structure-
activity relationships (SAR) of this new type of enediynes.
In continuation of our interests in the biological activities of 2-(6-substituted hex-3-
ene-1,5-diynyl)benzonitriles, several derivatives thereof, i.e., 2-[2-(2-alkynylphen-
yl)ethynyl]benzonitriles 7 10, 1,4-bis(dec-3-ene-1,5-diynyl)benzene (11), 4,4'-bis-
(dec-3-ene-1,5-diynyl)-1,1'-biphenyl (12), and 1-aryldec-3-ene-1,5-diynes 13 23

Helvetica Chimica Acta Vol. 85 (2002)
2565
Scheme 1
(Scheme 2), were designed and synthesized by modification of the enediyne core and
the aryl group bearing it. The analogues were evaluated for cytotoxic activity against
five human solid tumor cells and the topoisomerase I inhibitory activities, and the cell-
cycle analysis was performed to provide more advanced understanding of the
cytotoxicity.
Results and Discussion. Chemistry. 2-[2-(2-Alkynylphenyl)ethynyl]benzonitriles
7 10 were synthesized from the Pd-catalyzed coupling reaction of 2-(2-substituted
eth-1-ynyl)-1-iodobenzenes 24 27 with 2-ethynylbenzonitrile 28 [6] (Scheme 3). The
yields obtained were 40 98%.
1,4-Bis(dec-3-ene-1,5-diynyl)benzene (11) and 4,4'-bis(dec-3-ene-1,5-diynyl)-1,1'-
biphenyl 12 were prepared from 1-chlorooct-1-ene-3-yne (29) as the starting material
(Scheme 4). Coupling 29 with (trimethylsilyl)acetylene with Pd(PPh3)4 as a catalyst
gave 30 in 53% yield. Treatment of 30 with Bu4NF (TBAF) produced compound 31 in
80% yield. Finally, Pd-catalyzed coupling reaction of dec-3-ene-1,5-diyne (31) with aryl
diiodides 32 and 33 gave bis(enediynyl)arenes 11 and 12 in 65 and 35% yields,
respectively.
Generation of 1-aryldec-3-ene-1,5-diynes 13 23 was accomplished under the same
Pd-catalyzed coupling reactions between dec-3-ene-1,5-diyne (31) and aryl iodides 34
44 in 31 99% yields (Table 1).

2566
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 2. Structures of Enediyne Compounds 1 23
THP ˆ 3,4,5,6-Tetrahydro-2H-pyran-2-yl
Cytotoxicity. The IC50 values of compounds 1 23 with five human solid tumor cells
(KB, Hela, DLD, NCI, and Hepa) are listed in Table 2. Obviously, 2-(6-substituted hex-
3-ene 1,5-diynyl)benzonitriles 1 5 display a tendency to inhibit the growth of Hepa
cells. Among these compounds, 2-(6-phenylhex-3-en-1,5-diynyl)benzonitrile (5) ex-
hibited the highest cytotoxic activity against Hepa cells at 1.09 mg/ml. Moreover, 2-[2-
(2-alkynylphenyl)ethynyl]benzonitriles 8 10 and 1-aryldec-3-ene-1,5-diynes 13 22
also showed the same tendency with DLD cell line, in which 1-[(Z)-dec-3-ene-1,5-
diynyl]-4-nitrobenzene (21) showed selective potency against DLD cells at the low IC50
value of 1.66 mg/ml. 1-[(Z)-dec-3-ene-1,5-diynyl)- 2-(trifluoromethyl)benzene (20) was
more active than the other derivatives against Hela cells (IC50 ˆ 2.15 mg/ml).
Inhibition Tests for Topoisomerase I. On the other hand, the cleavage of supercoiled
DNA by topo I in the presence of 2-[2-(2-alkynylphenyl)ethynyl]benzonitriles 7 190,
1,4-bis(dec-3-ene-1,5-diynyl)benzene (11), 4,4'-bis(dec-3-ene-1,5-diynyl)-1,1'-biphenyl
(12), and 1-aryldec-3-ene-1,5-diynes 13 23 was evaluated by gel electrophoresis, and

Helvetica Chimica Acta Vol. 85 (2002)
2567
Scheme 3. Preparation of 2-[2-(2-Alkynylphenyl)ethynyl)benzonitriles 7 10
THP ˆ 3,4,5,6-Tetrahydro-2H-pyran-2-yl
Scheme 4. Generation of 1,4-Bis(dec-3-ene-1,5-diynyl)benzene (11) and 4,4'-Bis(dec-3-ene-1,5-diynyl)-1,1'-
biphenyl 12

2568
Helvetica Chimica Acta Vol. 85 (2002)
Table 1. Preparation of 1-Aryldec-3-ene-1,5-diynes 13 23
34 44
13 23 (Yield [%])
34 Arˆ 4-ClC6H4
13 (31)
14 (46)
15 (84)
16 (53)
17 (99)
18 (46)
19 (46)
20 (33)
21 (59)
22 (31)
23 (96)
35 Arˆ 4-CNC6H4
36 Arˆ Pyridin-2-yl
37 Arˆ Thiophen-2-yl
38 Arˆ Pyrazin-2-yl
39 Arˆ 4-CF3C6H4
40 Arˆ 3-CF3C6H4
41 Arˆ 2-CF3C6H4
42 Arˆ 4-NO2C6H4
43 Arˆ 2-CH3CO2C6H4
44 Arˆ 4-CH3COC6H4
the results were shown in Figs. 1 and 2. In Fig. 1,a, compound 10 displayed inhibitory
activity against topo I at 8.7 and 87 mm and no inhibition of topo I at 0.87 mm
concentration. Compound 9 showed inhibition of topo I at 87 mm, and there was no
supercoiled DNA observed at 8.7 or 0.87 mm. Moreover, there was complete untwisting
of DNA revealed at 0.87, 8.7, or 87 mm of 7 and 8 (data not shown). No supercoiled
DNA was observed for 11 and 12 at 8.7 or 87 mm concentration (Fig. 1,b). In Fig. 2,a,
1-aryldec-3-ene-1,5-diynes 13 17 showed no inhibition effects with topo I. 1-[(Z)-dec-
3-ene-1,5-diynyl)]-4-(trifluoromethyl)benzene (18) and 4-[(Z)-dec-3-ene-1,5-diynyl)]-
acetophenone (23) displayed inhibitory activities of topo I on the test concentration as
shown in Fig. 2,b.
Supercoiled pGEM9zf(À) DNA was treated with 0.025 mg/ml topoisomerase I and
compounds 7 101), 11, and 12, then analyzed on a 2% agarose gel. Fig. 1,a, Lane 1:
DNA ‡ topo I; Lane 2: DNA only; Lane 3: DNA ‡ topo I ‡ camptothecin (43.5 mm);
Lanes 4 6: DNA ‡ topo I ‡ compound 10; Lanes 7 9: DNA ‡ topo I ‡ compound 9;
each group of three lanes contained 0.87, 8.7, and 87 mm of the analogs, respectively.
Fig. 1,b, Lane 1: DNA ‡ topo I; Lane 2: DNA only; Lanes 3 and 4: DNA ‡ topo I ‡
compound 11; Lanes 5 and 6: DNA ‡ topo I ‡ compound 12; each group of both lanes
contained 8.7 and 87 mm of the analogs, respectively.
Supercoiled pGEM9zf(À) DNA was treated with 0.025 mg/ml topoisomerase I and
compounds 13 23, then analyzed on a 2% agarose gel. Fig. 2,a, Lane 1: DNA only;
Lane 2: DNA ‡ topo I; Lanes 3 7. DNA ‡ topo I ‡ compounds 13 17 (87 mm).
Fig. 2,b, Lanes 1 6: DNA ‡ topo I ‡ compounds 18 23 (87 mm).
1
)
The unreported results of compounds 7 and 8 display no topo I inhibition at 8.7 and 87 mm.

Helvetica Chimica Acta Vol. 85 (2002)
2569
Table 2. IC50 Values [mg/ml] of Cytotoxicities of Compound 1 23a)
Compound
Cells
KB
Hela
DLD
8.61
NCI
Hepa
1
2
3
4
5
n.d.
n.d.
n.d.
n.d.
n.d.
14.43
9.97
10.62
14.26
14.66
12.49
5.03
6.38
7.28
7.01
7.35
1.09
6
7
8
9
11.65
8.98
10
11
12
13
14
15
16
17
18
19
20
21
22
23
16.25
9.22
8.00
5.13
5.57
9.83
5.22
11.93
5.15
6.75
3.89
5.71
7.05
8.91
1.66
7.27
5.93
4.32
6.74
5.30
5.86
9.43
11.15
7.73
12.34
17.08
14.39
12.24
19.65
2.15
5.02
11.19
13.13
a
)
: IC50 > 20 mg/ml; the standard of cytotoxicity test is doxorubicin with ED50 0.1 mg/ml. KB: human oral
epidermoid carcinoma, Hela: human cervix epithelial carcinoma, DLD (DLD-1): human colon adenocarci-
noma, NCI (NCI-H661): human lung large-cell carcinoma, Hepa: human hepatoma.
Cell Cycle Assay of Compound 5. To obtain more information about the differences
of these novel enediynes in affecting whole cells, human hepatoma Hep G2 cell was
used, and the growth characteristic of cells following treatment with compounds 5 was
measured. As shown in Fig. 3, cells were exposed to the vehicle solvent (DMSO) as
control, 1 and 10 mm 5 was added to the cells, and, after exposure to the compounds for
72 h, attached cells were analyzed by flow cytometry. The majority (71.5%) of control
cells exposed to DMSO were in the G0 G1 phase of cell cycle and only a few cells
were detected in either the S phase (22.7%) or G2/M (5.8%) phase. After treatment
of compound 5 (1 and 10 mm) for 72 h, cells progressed to the S phase, and the majority
of cell population was almost arrested at the S phase. Consistent with this cell-cycle
arrest, only 36.6% (1 mm) and 46.9% (10 mm) of the cells were found at the G0 G1
phase, 63.2% (1 mm) and 53.0% (10 mm) in S phase, and 0.2% (1 mm) and 0.1% (10 mm)
in G2/M phase. However, the significant blockage of the Hep G2 cell cycle in S phase
was observed and induced by both concentrations (1 mm and 10 mm) of compound
5.
Conclusions. There are several results arising from this study (Scheme 5), and the
conclusions are summarized in two parts:

2570
Helvetica Chimica Acta Vol. 85 (2002)
Fig. 1. Cleavage of supercoiled pGEM9zf(À) DNA by topoisomerase I in the presence of 2-[2-(2-alkynylphen-
yl)ethynyl)benzonitriles 7 10,1,4-bis(dec-3-ene-1,5-diynyl)benzene (11), and 4,4'-bis(dec-3-ene-1,5-diynyl)-1,1'-
biphenyl (12)
Fig. 2. Cleavage of supercoiled pGEM9zf(À) DNA by topoisomerase I in the presence of 1-aryldec-3-ene-1,5-
diynes 13 23
I) For the cytotoxic assay: 1) New enediynes 1 5 have a tendency to exhibit
cytotoxic activity with Hepa cells, and the longer alkyl chain, the higher the cytotoxic
activity observed. When the substituent at C(6) is a Ph group, it provides the strongest
activity against the Hepa cell line. 2) The series of enediynes 13 22 induce the growth
inhibition of DLD cells. Among these, compound 21 shows the highest biological
activity. Comparison of the results for 15 (Arˆ pyridin-2-yl) and 17 (Arˆ pyrazin-2-yl)
shown in Table 2 indicates that the increasing number of N-atoms in the aromatic ring
leads to a lower IC50 value and a wider spectrum of cytotoxicity. Compound 6 and its
dimer 12 have similar structures and remain selectively toxic against KB cells.
II) The inhibitory activity of topo I: 1) There are two essential features that facilitate
the inhibition of topo I by these enediyne derivatives, the enediyne core and CN group.
The series of new enediynes 2 5 reveal inhibition of topo I [5]. Replacement of the
enediyne core with 1,2-diethynylbenzene lowers the inhibition abilities of topo I, which

Helvetica Chimica Acta Vol. 85 (2002)
2571
Fig. 3. Effect of compound 5 on cell-cycle distribution [%] of human hepatoma Hep G2 cells for 72 h. Cell-cycle
distribution of human hepatoma Hep G2 cells were measured by flow cytometry. Values are expressed as mean
for triplicate samples. (Significantly different from control, p < 0.05).
may be due to the alteration of conformation of the original enediyne core, or because
the ability of 1,2-diethynylbenzene to form a complex with topo I is lower than that of
original enediyne core. 2) All unwinding DNA detected in Fig. 1,b, indicates that
compounds 11 and 12 are not topo I inhibitors. The results clearly show that increasing
the quantities of enediyne core cannot enhance the activity against topo I. 3) Moreover,
only compounds 18 and 23 among 1-aryldec-3-ene-1,5-diynes 13 23 display topo I
inhibitory activity; hence, it is considered that topoisomerase I inhibitors reveal the
highly structural selectivity. The observation that 6 and its dimer 12 are not topo I
inhibitors but show activities against KB cells indicates that the structures of 12 and 6
interfere with an unknown biological path to cause the cytotoxic activity. In summary,
we have synthesized a series of novel enediynes that display selective growth inhibition
of various human tumor cell lines, and some of them revealed inhibitory effects with
topoisomerase I, although there seems to be no significant relationship between the
cytotoxicity and topo I.
On the other hand, it was considered that whole cancer cells had more-complete
biological functions than enzymes. Other compensatory pathways would be active
when topoisomerase I was inhibited during the periods of DNA replication. The data
shown in Fig. 3 demonstrated that compound 5 induced blockage of the cell-growth
cycle in S phase. It was assumed that only inhibiting topoisomerase I would not
completely arrest the cell cycle in S phase. Therefore, the results indicate that
compound 5 not only targets topo I but also interferes with topo II. Moreover,
generally, the cells progressed to a final accrual at G2/M or later phase. The observed
small amount of G2/M cells also implied that 5 probably inhibits mitosis or tubulin
polymerization, though more evidence is necessary to support this conclusion.
However, the significant blockage of the Hep G2 cell cycle in S phase suggested that
these novel enediynes probably undergo metabolism by other biological pathways to
exhibit cytotoxicity, aside from the inhibitory activity toward topoisomerase I.
Although the picture concerning the cytotoxicities of the compounds is still
incomplete, this study has provided several new lead compounds for enediynes, which
exhibit activities against various human solid tumors and topo I, and the cell-cycle test
of compound 5 indicates that these compounds block the replication of DNA. The
above results are helpful in the elucidation of undiscovered biological modes related to
the anticancer properties of enediynes.

2572
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 5. Results of Biological Activities of Three Series of Nonradical Enediynes
Experimental Part
General Procedure of the Coupling Reaction of 1-Alkynyl-2-iodobenzene with 2-Ethynylbenzonitrile. A
degassed soln. of 1-alkynyl-2-iodobenzene (12 mmol) in dry Et2O (30 ml) containing Pd(PPh3)4 (0.8mmol) and
CuI (3.2 mmol) was added to a soln. of 2-ethynylbenzonitrile (24 mmol) containing BuNH2 (34 mmol). The
resulting soln. was stirred for 6 h at 258, quenched with sat. aq. NH4Cl and Na2CO3 solns., and extracted with
AcOEt. The org. layer was separated and dried (MgSO4). After filtration, the solvent was evaporated in vacuo.
The residue was purified by flash chromatography (FC) to give the products.
(Z)-1-Chlorooct-1-ene-3-yne (29). Yield 65%. Oil. 1H-NMR (400 MHz, CDCl3): 6.28( dd, J ˆ 7.3, 0.4, 1 H);
5.84 (dt, J ˆ 7.3, 2.2, 1 H); 2.38( td, J ˆ 7.0, 2.2, 2 H); 1.59 1.42 (m, 4 H); 0.92 (t, J ˆ 7.3, 3 H). EI-MS: 142 (32,
‡
M ), 86 (53), 49 (56), 35 (25). HR-EI-MS: 142.0550 (C8H11Cl; calc. 142.0548).
1-(Trimethylsilyl)dec-3-ene-1,5-diyne (30). Yield 53%. Oil. 1H-NMR (200 MHz, CDCl3): 8.81 8.71
(m, 2 H); 2.41 (t, J ˆ 6.8, 2 H); 1.56 1.44 (m, 4 H); 0.92 (t, J ˆ 6.8, 3 H); 0.19 (s, 9 H). 13C-NMR (50 MHz,
‡
CDCl3): 121.5; 118.1; 102.2; 101.7; 99.5; 78.1; 30.7; 21.9; 19.4; 13.6; À0.10. EI-MS: 204 (91, M ), 189 (100), 145
‡
(32), 131 (28). HR-EI-MS: 204.1335 (C13H20Si ; calc. 204.1331).
Dec-3-ene-1,5-diyne (31). Compound 30 (3.02 g, 14.80 mmol) was dissolved in dry MeOH (10 ml), and the
soln. was stirred with K2CO3 (1.0 g) at r.t. for 1.5 h. After the evaporation of MeOH in vacuo, the reaction was

Helvetica Chimica Acta Vol. 85 (2002)
2573
quenched with sat. aq. NaHCO3 solns. and the soln. was extracted with AcOEt. The org. layer was separated and
dried (MgSO4). After filtration, the solvent was evaporated in vacuo. The residue was purified by FC to give 31
(80% yield). Oil. 1H-NMR (200 MHz, CDCl3): 5.88 5.75 (m, 2 H); 3.28 (s, 1 H); 2.41 (t, J ˆ 7.0, 2 H); 1.58 1.40
(m, 4 H); 0.92 (t, Jˆ 7.0, 3 H ) . 13C-NMR (50 MHz, CDCl3): 122.4; 116.9; 99.6; 83.6; 80.9; 77.8; 30.5; 21.8; 19.4; 13.5.
2-(2-{2-[2-(3,4,5,6-Tetrahydro-2H-pyran-2-yl)ethynyl]phenyl}ethynyl)benzonitrile (7). Yield 98%. Oil.
1H-NMR (200 MHz, CDCl3): 7.84 7.37 (m, 8H); 5.08 5.04 ( m, 1 H); 4.70 (d, J ˆ 2.2, 2 H); 4.21 3.93
(m, 1 H); 3.69 3.61 (m, 1 H); 1.93 1.58( m, 6 H). 13C-NMR (50 MHz, CDCl3): 132.7; 132.6; 132.5; 132.3;
132.2; 128.9; 128.4; 128.2; 127.1; 125.6; 124.9; 117.4; 115.1; 96.8; 94.4; 89.1; 84.0; 61.9; 54.9; 30.3; 25.4; 19.0. HR-
MS (EI) calc. for C23H19NO2 341.1414, found 341.1452.
2-(2-{2-[5-(3,4,5,6-Tetrahydro-2H-pyran-2-yl)pent-1-ynyl]phenyl}ethynyl)benzonitrile (8). Yield 89%. Oil.
1H-NMR (200 MHz, CDCl3): 7.70 7.51 (m, 4 H); 7.47 7.34 (m, 2 H); 7.31 7.28( m, 2 H); 4.59 4.56 (m, 1 H);
3.95 3.81 (m, 1 H); 3.62 3.47 (m, 1 H); 2.64 (t, J ˆ 7.4, 2 H); 1.97 1.46 (m, 10 H). 13C-NMR (50 MHz,
CDCl3): 132.7; 132.6; 132.5; 132.3; 132.1; 132.0; 128.9; 128.2; 127.4; 127.3; 126.4; 124.3; 117.5; 115.2; 98.8; 94.9;
‡
88.6; 79.3; 66.1; 62.2; 30.7; 28.9; 25.5; 19.5; 16.6. HR-EI-MS: 369.1729 (C25H23NO2 ; calc. 369.1725).
1
2-{2-[2-(Hex-1-ynyl)phenyl]ethynyl}benzonitrile (9). Yield 62%. Oil. H-NMR (200 MHz, CDCl3): 7.71
7.52 (m, 4 H); 7.47 7.37 (m, 2 H); 7.33 7.25 (m, 2 H); 2.52 (t, J ˆ 7.6, 2 H); 1.66 1.43 (m, 4 H); 0.89 (t, J ˆ 7.6,
‡
3 H). HR-EI-MS: 283.1326 (C21H17N ; calc. 283.1326).
2-{2-[2-(Hept-1-ynyl)phenyl]ethynyl}benzonitrile (10). Yield 40%. Oil. 1H-NMR (200 MHz, CDCl3): 7.70
7.53 (m, 4 H); 7.47 7.34 (m, 2 H); 7.30 7.25 (m, 2 H); 2.51 (t, J ˆ 7.6, 2 H); 1.69 1.25 (m, 6 H); 0.85 (t, J ˆ 7.6,
‡
3 H). HR-EI-MS: 297.1518(C 21H17N ; calc. 297.1515).
1,4-Bis[(Z)-dec-3-ene-1,5-diynyl]benzene (11). Yield 78%. Oil. 1H-NMR (200 MHz, CDCl3): 7.41 (s, 2 H);
6.02 5.58( m, 4 H); 2.45 (t, J ˆ 7.2, 2 H); 1.70 1.47 (m, 8H); 0.90 ( t, J ˆ 7.2, 6 H). 13C-NMR (50 MHz, CDCl3):
‡
131.5; 122.2; 120.8; 117.9; 99.9; 95.7; 89.2; 78.4; 30.7; 21.9; 19.6; 13.6. HR-EI-MS: 338.2017 (C26H26 ; calc. 338.2035).
1
4,4'-Bis[(Z)-dec-3-ene-1,5-diynyl]-1,1'-biphenyl (12). Yield 51%. Oil. H-NMR (200 MHz, CDCl3): 7.58
7.51 (m, 8H); 5.98 5.86 ( m, 4 H); 2.45 (td, J ˆ 7.2, 2.0, 4 H); 1.65 1.48( m, 8H); 0.91 ( t, J ˆ 7.4, 6 H). 13C-NMR
(50 MHz, CDCl3): 140.1; 132.2; 126.85; 122.6; 120.4; 116.1; 99.6; 95.9; 88.3; 78.5; 31.5; 30.7; 21.9; 14.2. HR-EI-
‡
MS: 414.2346 (C32H30 ; calc. 414.2349).
1-Chloro-4-[(Z)-dec-3-ene-1,5-diynyl]benzene (13). Yield 31%. Oil. 1H-NMR (200 MHz, CDCl3): 7.36
(d, J ˆ 8.8, 2 H); 7.31 (d, J ˆ 8.6, 2 H); 5.97 5.84 (m, 2 H); 2.45 (t, J ˆ 6.8, 2 H); 1.61 1.45 (m, 4 H); 0.9 (t, J ˆ
7.0, 3 H). 13C-NMR (50 MHz, CDCl3): 134.4; 132.8; 128.6; 121.7; 120.7; 117.7; 99.7; 94.7; 88.1; 78.3; 30.7; 21.8;
‡
19.5; 13.5. EI-MS: 242 (100, M ), 201 (16), 199 (40), 192 (78), 165 (63), 164 (45), 163 (52). HR-EI-MS: 242.0863
‡
(C16H15Cl ; calc. 242.0859).
4-[(Z)-Dec-3-ene-1,5-diynyl]benzonitrile (14). Yield 46%. Oil. 1H-NMR (200 MHz, CDCl3): 7.61 (d, J ˆ
6.4, 2 H); 7.53 (d, J ˆ 8.4, 2 H); 5.95 (d, J ˆ 1.6, 2 H); 2.44 (t, J ˆ 6.8, 2 H); 1.59 1.44 (m, 4 H); 0.9 (t, J ˆ 7.2,
3 H). 13C-NMR (50 MHz, CDCl3): 132.0; 131.9; 128.1; 122.2; 118.4; 117.1; 111.5; 100.6; 93.9; 91.3; 78.2; 30.5;
‡
21.8; 19.4; 13.5. EI-MS: 233 (51, M ), 203 (51), 190 (100), 177 (35), 164 (46), 140 (28). HR-EI-MS: 233.1205
‡
(C17H15N ; calc. 233.1206).
1
2-[(Z)-Dec-3-ene-1,5-diynyl]pyridine (15). Yield 84%. Oil. H-NMR (200 MHz, CDCl3): 8.58 (dt, J ˆ 5.0,
1.0, 1 H); 7.63 (td, J ˆ 7.6, 1.8, 1 H); 7.42 (td, J ˆ 7.6, 1.0, 1 H); 7.23 7.16 (m, 2 H); 5.99 5.93 (m, 2 H); 2.43
(td, J ˆ 7.0, 1.6, 2 H); 1.92 1.43 (m, 4 H); 0.8 7 (t, J ˆ 7.0, 3 H). 13C-NMR (50 MHz, CDCl3): 150.5; 143.3; 135.9;
‡
127.2; 122.7; 122.1; 117.3; 100.3; 94.7; 86.8; 78.2; 30.6; 21.8; 19.5; 13.5. EI-MS: 209 (19, M ), 180 (100), 78 (18), 51
‡
(18). HR-EI-MS: 209.1205 (C15H15N ; calc. 209.1209).
2-[(Z)-Dec-3-ene-1,5-diynyl]thiophene (16). Yield 53%. Oil. 1H-NMR (200 MHz, CDCl3): 7.29 (d, J ˆ 5.2,
1 H); 7.22 (d, J ˆ 2.6, 1 H); 7.00 (t, J ˆ 5.2, 1 H); 5.94 (d, J ˆ 10.4, 1 H); 5.86 (d, J ˆ 10.8, 1 H); 2.45 (d, J ˆ 6.8,
2 H); 1.63 1.47 (m, 4 H); 0.92 (t, J ˆ 6.8, 3 H). 13C-NMR (50 MHz, CDCl3): 132.1; 127.7; 127.2; 123.3; 120.1;
‡
117.7; 99.8; 91.2; 89.1; 78.4; 30.6; 21.9; 19.5; 13.5. EI-MS: 214 (100, M ), 184 (41), 171 (80), 165 (53). HR-EI-
‡
MS: 214.0817 (C14H14S ; calc. 214.0811).
1
2-[(Z)-Dec-3-ene-1,5-diynyl]pyrazine (17). Yield 99%. Oil. H-NMR (200 MHz, CDCl3): 8.66 (d, J ˆ 1.6,
1 H); 8.55 (s, 1 H); 8.45 (d, J ˆ 2.4, 1 H); 5.98( m, 2 H); 2.44 (t, J ˆ 6.8, 2 H); 1.58 1.43 (m, 4 H); 0.88 (t, J ˆ 7.0,
3 H). 13C-NMR (50 MHz, CDCl3): 147.8; 144.4; 142.6; 140.3; 123.5; 116.5; 101.3; 91.7; 90.8; 78.1; 30.5; 21.8; 19.5;
‡
‡
13.5. EI-MS: 210 (39, M ), 181 (100), 168 (14), 127 (17). HR-EI-MS: 210.1158 (C14H14N2 ; calc. 210.1154).
1-[(Z)-Dec-3-ene-1,5-diynyl]-4-(trifluoromethyl)benzene (18). Yield 46%. Oil. 1H-NMR (200 MHz,
CDCl3): 7.57 (s, 4 H); 5.95 (d, J ˆ 1.6, 2 H); 2.46 (t, J ˆ 6.8, 2 H); 1.58 1.49 (m, 4 H); 0.90 (t, J ˆ 7.0, 3 H).
13C-NMR (50 MHz, CDCl3): 146.9; 134.6; 131.8; 127.0; 125.3; 121.6; 117.4; 112.1; 100.2; 89.4; 78.3; 30.6; 21.9;
‡
19.5; 13.5. EI-MS: 276 (100, M ), 233 (59), 207 (31), 192 (24), 165 (47), 49 (43). HR-EI-MS: 276.1174
‡
(C17H15F3 ; calc. 276.1129).

2574
Helvetica Chimica Acta Vol. 85 (2002)
1-[(Z)-Dec-3-ene-1,5-diynyl]-3-(trifluoromethyl)benzene (19). Yield 46%. 1H-NMR (200 MHz, CDCl3):
7.72 (s, 1 H); 7.61 (d, J ˆ 7.6, 1 H); 7.55 (d, J ˆ 8.0, 1 H); 7.45 (t, J ˆ 7.6, 1 H); 5.97 (d, J ˆ 10.8, 1 H); 5.92 (d, J ˆ
10.8, 1 H); 2.46 (t, J ˆ 6.8, 2 H); 1.62 1.45 (m, 4 H); 0.90 (t, J ˆ 7.2, 3 H). 13C-NMR (50 MHz, CDCl3): 146.9;
134.6; 128.8; 128.5; 124.8; 124.8; 124.2; 121.5; 117.4; 100.1; 94.2; 88.6; 78.3; 30.6; 21.8; 19.5; 13.4. EI-MS: 276
‡
‡
(100, M ), 261 (30), 246 (34), 233 (84), 207 (47), 183 (62), 178 (37), 165 (68). HR-EI-MS: 276.1127 (C17H15F3
;
calc. 276.1125).
1-[(Z)-Dec-3-ene-1,5-diynyl]-2-(trifluoromethyl]benzene (20). Yield 33%. Oil. 1H-NMR (200 MHz,
CDCl3): 7.64 (td, J ˆ 7.8, 1.8, 2 H); 7.39 7.37 (m, 2 H); 6.02 5.87 (m, 2 H); 2.45 (t, J ˆ 7.0, 2 H); 1.60 1.51
(m, 4 H); 0.90 (t, J ˆ 7.0, 3 H). 13C-NMR (50 MHz, CDCl3): 146.3; 134.2; 131.2; 128.0; 125.8; 125.7; 121.6; 117.5;
‡
112.1; 100.3; 92.5; 91.5; 78.0; 30.6; 21.9; 19.5; 13.5. EI-MS: 276 (100, M ), 233 (34), 232 (29), 221 (48), 214 (22),
‡
183 (29), 165 (14). HR-EI-MS: 276.1174 (C17H15F3 ; calc. 276.1174).
1-[(Z)-Dec-3-ene-1,5-diynyl]-4-nitrobenzene (21). Yield 59%. Oil. 1H-NMR (200 MHz, CDCl3): 8.16
(d, J ˆ 9.0, 2 H); 7.60 (d, J ˆ 9.0, 2 H); 5.97 (s, 2 H); 2.45 (t, J ˆ 6.4, 2 H); 1.61 1.45 (m, 4 H); 0.9 (t, J ˆ 7.0, 3 H).
13C-NMR (50 MHz, CDCl3): 147.0; 132.1; 123.5; 122.6; 118.3; 117.0; 100.9; 93.6; 92.2; 78.2; 30.6; 21.8; 19.5; 13.5.
‡
‡
EI-MS: 253 (100, M ), 238(16), 210 (16), 192 (14), 165 (13), 163 (21). HR-EI-MS: 253.1103 (C 16H15NO2 ; calc.
253.1105).
Methyl 2-[(Z)-Dec-3-ene-1,5-diynyl]benzoate (22). Yield 31%. Oil. 1H-NMR (200 MHz, CDCl3): 7.95
(d, J ˆ 7.8, 1 H); 7.57 (d, J ˆ 7.4, 1 H); 7.46 7.31 (m, 2 H); 6.00 (d, J ˆ 10.8, 1 H); 5.91 (d, J ˆ 10.8, 1 H); 3.91
(s, 2 H); 2.45 (t, J ˆ 6.8, 2 H); 1.62 1.42 (m, 4 H); 0.87 (t, J ˆ 7.0, 3 H). 13C-NMR (50 MHz, CDCl3): 166.5;
134.3; 131.6; 131.5; 130.3; 127.9; 123.7; 120.8; 118.1; 99.6; 94.6; 78.4; 52.0; 30.6; 21.9; 19.5; 13.5. EI-MS: 266 (71,
‡
‡
M ), 237 (75), 224 (51), 223 (70), 209 (82), 191 (21), 181 (49), 176 (24). HR-EI-MS: 266.1307 (C18H18O2 ; calc.
266.1305).
4-[(Z)-Dec-3-ene-1,5-diynyl]acetophenone (23). Yield 96%. Oil. 1H-NMR (200 MHz, CDCl3): 7.92 (d, J ˆ
8.4, 2 H); 7.54 (d, J ˆ 8.4, 2 H); 5.99 5.90 (m, 2 H); 2.60 (s, 3 H); 2.45 (t, J ˆ 7.2, 2 H); 1.63 1.46 (m, 4 H); 0.91
(t, J ˆ 7.2, 3 H). 13C-NMR (50 MHz, CDCl3): 197.2; 136.3; 131.7; 128.2; 128.1; 121.6; 117.6; 100.3; 95.0; 90.4; 78.4;
‡
‡
30.7; 26.6; 21.9; 19.5; 13.6. EI-MS: 250 (100, M ), 235 (37), 165 (29). HR-EI-MS: 250.1358(C 18H18O ; calc.
250.1372).
Evalution of Inhibitory Concentration of Camptothecin for Gel Electrophoresis. Camptothecin, which shows
significant topo I inhibition, is widely used as the standard for the comparison of activities of the resp.
compounds. To affirm the modest conc. of camptothecin that would still appear in the explicit suppression of
topo I, solns. of various conc. were prepared, and the results were obtained from agarose gel electrophresis. It
was suggested that camptothecin showed less topo I inhibitory activity when the conc. was 26.1 mm. On the other
hand, 43.5 mm camptothecin seemed to inhibit topo I. Therefore, the 43.5 mm concentration was used as the
standard conc. of camptothecin, to achieve inhibition of topo I (data not shown).
General Topoisomerase I Assay. All samples were kept in 23 ml total volume; a) negative control (DNA
alone): contained 10 Â topo I buffer (2ml) and pGEM9zf(À) DNA(1 mg/ml), 0.1% BSA (2 ml) and D2O (18 ml);
b) positive control (DNA ‡ topo I): contained 10  topo I buffer (2 ml) and pGEM9zf(À) DNA (1 mg/ml), 1
units/ml of topoisomerase I, 0.1% BSA (2 ml), and D2O (17 ml); c) camptothecin control (DNA ‡ topo I ‡
camptothecin): contained 10 Â topo I buffer (2 ml) and pGEM9zf(À) DNA (1 mg/ml), 1 units/ml of topo-
isomerase I, 0.1% BSA (2 ml), D2O (15 ml), and camptothecin (2 ml, dissolved in DMSO, final conc. of DMSO
was 8.7% (v/v)); d) experiments for compounds 7 23 (DNA ‡ topo I ‡ compounds): contained 10  topo I
buffer (2 ml) and pGEM9zf(À) DNA (1 mg/ml), 1 units/ml of topoisomerase I, 0.1% BSA (2 ml), D2O (15 ml), and
compounds (2 ml, dissolved in DMSO, final conc. of DMSO was 8.7% (v/v)). a) d) were well-mixed before
incubation. The tubes were incubated in 378 water bath for 30 min.
Gel Electrophoresis. The reaction mixtures were treated with 2% agarose gel in standard TBE buffer (1 Â ,
0.06m Tris, 0.06m boric acid, and 0.5m EDTA), which had been previously added to 2 ml of loading buffer
containing 0.25% bromophenol blue, 0.25% xylene cyanol, 5% SDS, and 0.25% sucrose. The gels were run at
50 V for 1.5 h, and stained with ethidium bromide for 20 min, and then placed into a UV box, and photographic
images of the gels were obtained with Polaroid 665 films.
Cell-Cycle Analysis. Samples of 1 Â 106 Hep G2 cells were placed on a 6-cm tissue-culture dish, and the
cells were allowed to recover for 24 h before any treatment. Then, DMSO or compound 5 (1 mm and 10 mm) was
added to the cells for 1 h in complete medium at 378, washed twice with PBS, and incubated in fresh media. Cells
were harvested with trypsin and washed twice with PBS. Samples were fixed in 70% EtOH and stored at 48 for at
least 24 h and then washed once with McIlvaine×s buffer (0.2m Na2HPO4, 0.1m citric acid, pH 7.5) and once with
PBS. Samples were stained with PI (propidium iodide) staining soln. (PBS containing 100 mg/ml RNase A and

Helvetica Chimica Acta Vol. 85 (2002)
2575
10 mg/ml PI [sigma]), processed on a Coulter Elite flow cytometer, and the data were analyzed with the
Multiplus AV program.
We would like to thank the National Science Council of the Republic of China and National Research
Institute of Chinese medicine for financial support.
REFERENCES
[1] J. C. Wang, Annu. Rev. Biochem. 1996, 65, 635.
[2] M. Gupta, A. Fujimori, Y. Pommier, Biochim. Biophys. Acta 1995, 1262, 1.
[3] a) J. C. Wang, Annu. Rev. Biochem. 1985, 54, 665; b) S. J. Froelich-Ammon, N. Osheroff, J. Biol. Chem.
1995, 270, 21429.
[4] M. M. Heck, W. N. Hittelman, W. C. Earnshaw, Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 1086.
[5] C. F. Lin, P. C. Hsieh, W. D. Lu, H. F. Chiu, M. J. Wu, J. Bioorg. Med. Chem. 2001, 9, 1707.
[6] M. J. Wu, L. J. Chang, L. M. Wei, C. F. Lin, Tetrahedron 1999, 55, 13193.
Received January 18, 2002