1-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl) -piperidin-2-one as a new potent apoptosis agent

Article abstract of DOI:10.1016/j.bmc.2009.09.042

Compounds 4a-f, 5a-f and 6-9, showed significant growth inhibition activity against human tumor cell lines. Of these compounds, 1-(2-((Z)-6-(2- (trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperidin-2-one (8) displayed the most potent growth inhibi

Full text of DOI:10.1016/j.bmc.2009.09.042

Bioorganic & Medicinal Chemistry 17 (2009) 7412–7417
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)-
piperidin-2-one as a new potent apoptosis agent
Yu-Sheng Tu a, Tsai-Hui Duh a, Chen-Yi Tseng c, Ying-Ting Lin d, Yu-Hsiang Lo b, Yi-Ling Hu a,
Chen-Hung Chen a, Ching-Ming Chien a, Sheng-Huei Yang a, Shinne-Ren Lin a, Shyh-Chyun Yang b,
Ming-Jung Wu c,
*
a Faculty of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
b Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan
c Department of Chemistry, National Sun Yat-sen University, Kaohsiung, Taiwan
d Faculty of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds 4a–f, 5a–f and 6–9, showed significant growth inhibition activity against human tumor cell
lines. Of these compounds, 1-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperi-
din-2-one (8) displayed the most potent growth inhibition activity. Compound 8 also arrested cancer
cells in G2/M phase and induced apoptosis via activation of caspase-3 and -9. According to western-blot-
ting analysis, compound 8 can up-regulate Bax, down-regulate Bcl-2 and XIAP, as well as promote cyto-
chrome c release.
Received 28 August 2009
Revised 18 September 2009
Accepted 19 September 2009
Available online 25 September 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Acyclic enediynes
Antitumor agents
Antitubulin
Apotosis
1. Introduction
to the a- and b-tubulin in the same manner as colchicine. The trifluo-
romethylphenyl group of 1 sits into the pocket of b-tubulin just like
the trimethoxyphenyl subunit of colchicine and the amino group of
compound 1 would form hydrogen bonding with the amino acids,
Microtubules are cytoskeletal protein polymers formed by highly
dynamic assemblies of
a-tubulin and b-tubulin heterodimers and
play a crucial role in many biological processes, including mitosis,
intracellular transport, exocytosis and cell division.1a Because of
the role of microtubules in mitosis and cell division, they have been
an important target for the development of new anticancer drugs.1b
There are two major groups of these antitumor agents: microtubule
stabilizers such as paclitaxel2 and microtubule destabilizers (colchi-
cine,3 combretastatin A-44and vinca alkaloids5). Most of these com-
pounds arrest cancer cells in G2/M phases of the cell cycle and cause
mitotic catastrophy and finally induce apoptosis.6
179-Thr and 181-Val, of a-tubulin, that is similar to the hydroxyl
group of CA-4 and the carbonyl group of colchicine. (Fig. 1) However,
both of the cell growth and tubulin inhibition activities of compound
1 are around 10-fold lower than that of colchicine and CA-4. As
shown in Figure 1c, the distances between the amino group of 1
0
and the amino acids, 179-Thr and 181-Val, are 4.159 ÅA and
0
3.358 ÅA, respectively. They are longer than that of CA-4 and colchi-
cine. We therefore anticipated that conversion of the amino func-
tionality of compound 1 to the amides would shorten the distances
of these hydrogen bondings and enhance the biological activities.
Thus, a series of amide analogs of 1 and 2, compounds 4a–f, 5a–f
and 6–9, were synthesized. These compounds were evaluated in
the in vitro antitumor protocol of the NCI. They were also evaluated
in the cell cycle analysis, the caspase-3, -8 and -9 colorimetric assay,
microtubule depolymerization assay and Western blot analysis with
apoptotic-associated proteins.
In our earlier reports,7 compounds 1 and 2 were shown to exhibit
good growth inhibition activity against various human tumor cell
lines at the concentration of 10ꢀ6–10ꢀ8 M. Compound 1 was also
found to arrest cancer cells in G2/M via microtubule depolymeriza-
tion and induce apoptosis via activation of caspase family.7a
Comparing the structure of compound 1 with some of the common
microtubule depolymerization agents, we found that the structure
of compound 1 is similar to combretastatin A-4 (CA-4) and colchi-
cine. Accordingto ourligand dockingexperiment, compound1 binds
2. Chemistry
The synthesis of compounds, 4a–f, 5a–f and 6–9, are summarized
in Scheme 1. Treatment of 2-(6-(2-trifluoromethylphenyl)-3(Z)-
* Corresponding author. Tel.: +886 7 5252000x3914; fax: +886 7 5253908.
E-mail address: mijuwu@faculty.nsysu.edu.tw (M.-J. Wu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.042

Y.-S. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7412–7417
7413
Figure 1. Predicted binding model of compound 2 in a-, b-tubulin. (A) Indicate: DAMA–colchicine (gold) and compound 2 (green). (B) Indicate: CA-4 (gold) and compound 2
(green). (C) Comparison of the proposed binding mode of compound 2 with
a- and b-tubulin that are shown in a ribbon representation. Protein comes from the X-ray
structure that complex with DAMA–colchicine (PBD code: 1SA0).
Table 1
R
R
The in vitro testing results of compounds 4c, 5c, 7 and 8
a
GI50
5c
(l
M)
O
Et3N
Panel/cell line
4c
6.36
2.75
10.00
4.27
5.08
4.76
12.00
4.04
8.87
7
8
+
R'
Cl
CH2Cl2
Leukemia-(K-562)
Leukemia-(CCRF-CEM)
Non-small cell lung cancer-(HOP-62)
Colon cancer-(HCT-15)
Colon cancer-(HCC-2998)
CNS cancer-(SNB-75)
2.29
0.79
2.52
3.35
4.19
0.63
1.79
1.69
1.79
4.08
1.93
2.17
0.75
0.44
8.56
1.56
0.89
1.53
0.95
1.61
0.90
1.44
0.87
2.67
1.68
1.16
0.60
0.21
5.02
1.53
1.32
2.34
3.00
1.44
1.03
10.50
10.10
1.19
0.90
O
R'
N
H
H2N
R = CF3
R = SCH3
3a R' = CH3
4a (66%)
4b (90%)
4c (50%)
4d (80%)
4e (81%)
4f (89%)
1 R = CF3
2 R = SCH3
5a (50%)
5b (88%)
5c (88%)
5d (65%)
5e (85%)
5f (95%)
3b R' = CH2Cl
3c R'= CH2CH3
3d R' = Ph
Melanoma-(M14)
Melanoma-(UACC-62)
Ovarian cancer-(OVCAR-3)
Renal cancer-(ACHN)
Prostate cancer-(DU-145)
Breast cancer-(MDA-MB-231/ATCC)
Breast cancer-(MDA-MB-435)
3e R' = CH2CH2CH2Br
3f R' = CH2CH2CH2CH2Br
30.10
40.50
7.92
1.92
R
R
Data obtained from the NCI’s in vitro human tumor cell screen.
a
The concentration produces 50% reduction in cell growth.
K2CO3
DMF
In flow cytometry assay, treatment of K-562 cells with
compounds 4c, 5c, 7 and 8 for 24 h resulted in arrest the cell in
the G2/M phase and induction of apoptosis. (Fig. 2) The percentage
of G2/M phase increased from 21.1% (control) to 51.7% and 78.3%
O
O
Br
N
N
n
H
n
4e n = 1, R = CF3
5e n = 1, R = SCH3
4f n = 2, R = CF3
5f n = 2, R = SCH3
by compounds 7 and 8 at the concentration of 10
lM. At higher
6 n = 1, R = CF3 (85%)
7 n = 1, R = SCH3 (52%)
8 n = 2, R = CF3 (65%)
9 n = 2, R = SCH3 (55%)
concentration of 50 M, compound 8 induced the percentage of
l
sub-G1 area is 31.57% that is 4.5 times higher than the control
(7.5%). It also indicated that the induction of apoptosis by
compound 8 (51.0%) is about three times higher than the lead
compound 1 (19.4%) (Fig. 3).
Scheme 1.
To evaluate the relationship between the G2/M phase arrest
caused by compound 8 and microtubule function, the microtubule
depolymerization assay was carried out. Treatment of Hela cells
hexen-1,5-diynyl)aniline (1)7a and 2-(6-(2-methylthiophenyl)-
3(Z)-hexen-1,5-diynyl)aniline (2)7b with various acyl chlorides 3a–
f in CH2Cl2 in the presence of Et3N gave 4a–f and 5a–f in 50–90%
yields. Compounds 4e, 4f, 5e and 5f were then converted to lactams
6–9 in 52–85% yields using K2CO3 as a base and DMF as the solvent.
with compound 8 at the concentration of 50 lM for two hours
and the results is shown in Figure 4. The results indicate that com-
pound 8 displays significant microtubule depolymerization activity
just as compound 1 does. By comparing with these bioassay
results, compound 8 not only shows the potency of growth inhibi-
tion activity against human tumor cell lines and the activity of
microtubule depolymerization, but also shows apoptotic activity.
Caspases are the family of cysteine proteases and they mediate
apoptotic pathway in mammalian.9 According to the previous re-
ports, apoptosis can be induced in either extrinsic or intrinsic path-
ways. Extrinsic pathway is dependent on death ligands that are
3. Results and discussion
The growthinhibitionactivities of compounds4a–f, 5a–f and 6–9
were submitted to the National Cancer Institute for testing against a
panel of approximately 60 humantumor cell lines. Details of this test
system have been published by others.8 Compounds 4c, 5c, 7 and 8
displayed a broad-spectrum inhibition activity against various can-
cer cell lines and the results are summarized in Table 1. Herein, we
only showed some cancer cell lines for every kind of cancer, includ-
ing leukemia, non-small cell lung, colon, CNS, melanoma, ovarian,
renal, prostate and breast cancer. The average GI50 values of these
compounds are in the range of 10ꢀ7–10ꢀ6 M. Of these compounds,
compound 8 showed the most potent growth inhibitory activity
against all tumor cell lines.
stimulated by death signals, such as Fas, TRAIL or TNFa. After this
process, caspase-8 was activated to trigger apoptosis. In contrast
with extrinsic pathway, intrinsic pathway was caused by intracel-
lular stresses, such as DNA damage, oncogene activation and mito-
tic damage.10 Caspase-3, -8 and -9 in particular are the principal
promoters of apoptosis. Colorimetric assays of caspase-3, -8 and -
9 provided evidence of the relationship between compound 8

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Y.-S. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7412–7417
Figure 2. The analysis of cancer cell cycle. Leukemia (K-562 cells) was cultured with 10
lM and 50 lM of compounds 4c, 5c, 7 and 8 for 24 h and analyzed by flow cytometry.
G0/G1, S, and G2/M individually indicate each phase and sub-G1 area refers to the portion of apoptotic cells.
Figure 3. The analysis of cancer cell cycle. Compounds 1 and 8 (50
(19.4%) and (C) 8 (51.0%).
lM) treated with Leukemia (K-562 cells) for 24 h. The portion of apoptosis area: (A) control (7.17%); (B) 1
Figure 4. The dose effect of compound 8 on microtubules depolymerization. Hela cells were treated with compound 8 at 37 °C in 0
Then cells were fixed and immunostained with anti-ninein (green in merged images) antibody and anti- -tubulin (red in merged images) antibody and counterstained with
DAPI for nucleus staining. Scale bars represent 10 M.
lM (A) and 50 lM (B) for 2 h, respectively.
a
l
and apoptosis pathways: extrinsic (relative to caspase-3 and -8) or
intrinsic pathway (relative to caspase-3 and -9). As shown in Figure
5(A), compounds 1 and 8 induced caspase-3 activity in a concen-
tration-dependent manner. But in Figure 5(B), the activity of
caspase-8 was not relative to the concentrations of compound 1
or 8. In Figure 5(C), compounds 1 and 8 can induce caspase-9 activ-
compound 8 was treated with these proteins by Western blot anal-
ysis and the results are shown in Figure 6. The induced apoptosis
effect of compound 8 was associated with the up-regulation of
Bax and down-regulation of Bcl-2 and XIAP, as well as cytochrome
c release in a concentration-dependent manner. On the other hand,
caspase-3 activation was accompanied by the cleavage of PARP
(Poly (ADP-ribose) polymerase).
ity, particularly at the concentration of 25 lM and 50 lM. These
results demonstrated that the apoptosis pathway of compounds
1 and 8 stemmed from intrinsic pathway and compound 8 showed
higher caspase induction activity than 1.
4. Conclusion
The intrinsic pathway is related to the mitochondrial function.
In order to confirm the relationship of compound 8 with these
apoptotic-associated proteins (Bax, Bcl-2, XIAP, Cyt.C and PARP),
After the structure modification, we found that compound 8,
the lactam analog of compound 1, exhibits good growth inhibition
activity against human tumor cell lines and arrests cancer cell in

Y.-S. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7412–7417
7415
Figure 5. Induction of caspase-3 (A), -8 (B) and -9 (C) activity in different dose of compound 1 and 8.
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 ll of DNA staining solution
(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 ll of PBS,
the DNA contents of 10,000 events 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 apoptosis the containing DNA were
digested by endonuclease then the sub G1 pick appear. The per-
centage in sub G1 were analyzed by gating on cell cycle dot blots
using Windows Multiple Document Interface software (WinMDI).
5.3. Caspases colorimetric assay
After different treatments, K-562 cells (1 ꢁ 106 cells/ml) were
collected and washed three times with PBS. Resuspended in 200 ll
Figure 6. Western blot analyses of expression levels of Bax, Bcl-2, XIAP, Cyt.C and
PARP. Cells were treated with different doses of compound 8 in 24 h.
cell lysis buffer (Biovision) and incubate cells on ice for 10 min. Cell
lysates were clarified by centrifugation at 18,000 g for 3 min. Trans-
fer supernatant (cytosolic extract) to a fresh tube and keep on ice.
The protein concentration in the supernatant was determined with
a BCA protein assay kit (Pierce, Rockford, IL, USA), and clear lysates
the G2/M phase via microtubule depolymerization. The apoptosis
mechanism of compound 8 was demonstrated through the intrin-
sic pathway and compound 8 showed higher caspase induction
activity than 1. According to western-blotting analysis, compound
8 can up-regulate Bax, down-regulate Bcl-2 and XIAP, as well as
promote cytochrome c release. This study provides a new lead
for the development of antitumor agent. We believe that the infor-
mation disclosed in this paper would have a strong impact to the
development of new anticancer drugs.
containing 50 lg of protein were incubated with 100 lM of
enzyme-specific colorigenic substrates (Biovision) and 2X reaction
buffer (Biovision)containing10 mM DTT at 37 °C for 1 h. Theactivity
of caspases were described as the cleavage of colorimetric substrate
by measuring the absorbance at 405 nm.11
5.4. Immunocytochemistry and microscopy
These procedures were modified from previously described pro-
tocols.12 In brief, Hela cells were grown on glass coverslips at a
density of 1 ꢁ 104 cells for 24 h. Cell cultures were rinsed several
times with PBS and fixed in 4% paraformaldehyde for 5 min, per-
meabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed cells
were rinsed in PBS, and non-specific binding was blocked with
5% normal goat serum (NGS)/1% bovine serum albumin (BSA) in
PBS pH 7.4 for at least 30 min at 37 °C. After a brief wash, the cells
were incubated for 45 min at 37 °C with the primary antibodies di-
luted in the same blocking solution. After extensive washes with
PBS, the cultures were then incubated with the appropriate sec-
ondary antibody conjugated to either Alexa 488 or Alexa 568 for
45 min at 37 °C. Finally, the cells were incubated for 5 min with
DAPI (Roche) prior to mounting (Molecular Probes, USA). Confocal
images were obtained using an OLYMPUS IX71 microscope (100x
5. Experimental section
5.1. Cell culture
Human leukemia K-562 cells were obtained from the American
Type Culture Collection (ATCC, Manassas, VA), and human purified
lymphocytes preparation was obtained from blood as described
previously. Cells were maintained in RPMI 1640 medium supple-
mented with 10% fetal calf serum, 2
(100 units/ml penicillin and 100 /ml streptomycin) at 37 °C in a
lM glutamine, and antibiotics
l
humidified atmosphere of 5% CO2.
5.2. Cell cycle analysis
Flow cytometry was used to measure cell cycle profile and
apoptosis. For cell cycle analysis, K-562 cells treated with com-
UPlanFl objective 1.3 NA) at 0.2 lm z-steps, controlled by FLUO-
VIEW software (Universal Imaging). All images were imported into
pounds 4c, 5c, 7 and 8 (10 and 50
lM) for 24 h were harvested
Adobe Photoshop v7.0 for contrast manipulation.
by centrifugation. After being washed with PBS, the cells were

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Y.-S. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7412–7417
5.5. Western blot analysis
J = 7.6 Hz); 13C NMR (CDCl3, 100 MHz) d 172.0, 139.1, 134.5,
134.1, 131.7, 130.7, 129.8, 128.7, 127.8, 127.7, 125.9, 123.2,
119.6, 119.3, 119.1, 111.3, 93.6, 93.2, 92.6, 92.0, 30.6, 9.16; HRMS
calcd for C22H16F3NO, 367.1184; found, 367.1188.
Cells were harvested and extracted with lysis buffer [50 mM
Tris–HCl (pH 7.5), 137 mM sodium chloride, 1 mM EDTA, 1% Nonidet
P-40, 10% glycerol, 0.1 mM sodium orthovanadate, 10 mM sodium
pyrophosphate, 20 mM b-glycerophosphate, 50 mM sodium fluo-
5.5.6. N-(2-((Z)-6-(2-(Mthylthio)phenyl)hexa-3-en-1,5-diynyl)-
phenyl)propionamide (5c)
ride, 1 mM phenylmethylsulfonyl fluoride, 2
lM leupeptin, and
2
l
g/ml aprotinin]. The lysates were centrifuged at 20,000 g for
1H NMR (CDCl3, 400 MHz) d 8.42 (d, 1H, J = 8.4 Hz), 8.14 (S, 1H),
7.45–7.31 (m, 4H), 7.12–7.03 (m, 3H), 6.23 (d, 1H, J = 10.8 Hz), 6.15
(d, 1H, J = 10.8 Hz), 2.30 (s, 3H), 2.18 (q, 2H, J = 6.4 Hz), 0.97 (t, 3H,
J = 7.6 Hz); 13C NMR (CDCl3, 100 MHz) d 172.2, 142.1, 139.0, 132.5,
131.8, 130.1, 129.4, 124.2, 124.0, 123.7, 123.0, 121.2, 119.8, 119.2,
118.4, 95.0, 93.9, 93.3, 92.2, 30.6, 14.6, 9.57; HRMS calcd for
C22H19NOS, 345.1187; found, 345.1189.
30 min and the protein concentration in the supernatant was deter-
mined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal
amounts of protein were separated by SDS-polyacrylamide gel elec-
trophoresis and then were electrotransferred to the PVDF mem-
brane. The membrane was blocked with a solution containing 5%
nonfat dry milk TBST buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl
and 0.1% Tween 20) for 1 h and washed with TBST buffer. The indi-
cated primary antibodies were incubated, washed, and monitored
by immunoblotting using specific antibodies. These immunoreac-
tive proteins were detected by enhanced chemiluminescence.
5.5.7. N-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5
-diynyl)phenyl)benzamide (4d)
1H NMR (CDCl3, 400 MHz) d 8.85 (s, 1H), 8.66 (d, 1H, J = 8.4 Hz),
7.81 (td, 2H, J = 4.4, 1.6 Hz), 7.70–7.67 (m, 1H), 7.65–7.52 (m, 2H),
7.49–7.30 (m, 3H), 7.24–7.22 (m, 2H), 7.12 (td, 2H, J = 7.6, 0.8 Hz),
6.24 (s, 2H); 13C NMR (CDCl3, 100 MHz) d 171.1, 139.2, 134.6,
134.3, 132.1, 132.0, 131.8, 130.4, 130.3, 129.8, 128.6, 128.5,
127.9, 126.9, 125.8, 125.7, 124.1, 123.5, 119.7, 119.2, 119.1,
111.8, 94.0, 93.3, 92.5, 92.2; HRMS calcd for C26H16F3NO,
415.1184; found, 415.1191.
5.5.1. N-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5
-diynyl)phenyl)acetamide (4a)
1H NMR (CDCl3, 400 MHz) d 8.40 (d, 1H, J = 8.4 Hz), 7.90 (s, 1H),
7.67 (d, 1H, J = 7.6 Hz), 7.58 (d, 1H, J = 7.6 Hz), 7.51–7.42 (m, 3H),
7.35 (td, 1H, J = 7.6, 1.2 Hz), 7.05 (t, 1H, J = 7.6 Hz), 6.37 (d, 1H,
J = 11.2 Hz), 6.23 (d, 1H, J = 11.2 Hz), 1.86 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 168.3, 139.0, 134.1, 131.8, 131.6, 131.4, 130.2, 128.7,
126.1, 126.0, 126.0, 123.3, 120.9, 119.7, 119.3, 119.2, 93.5, 93.1,
92.6, 92.0, 29.6; HRMS calcd for C21H14F3NO, 353.1027; found,
353.1021.
5.5.8. N-(2-((Z)-6-(2-(Mehylthio)phenyl)hexa-3-en-1,5-diynyl)-
phenyl)benzamide (5d)
1H NMR (CDCl3, 400 MHz) d 9.04 (s, 1H), 8.66 (d, 1H, J = 8.4 Hz),
7.82–7.80 (m, 2H), 7.53 (dd, 1H, J = 8, 1.6 Hz), 7.42 (td, 2H, J = 8,
1.6 Hz), 7.26–7.09 (m, 5H), 6.76 (td, 1H, J = 7.4, 0.8 Hz), 6.65 (d,
1H, J = 8.4 Hz), 6.24 (d, 1H, J = 8.4 Hz), 6.14 (d, 1H, J = 8.4 Hz),
3.56 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 165.5, 159.7, 139.3,
134.1, 133.5, 131.8, 131.5, 130.4, 130.1, 128.4, 127.0, 123.4,
121.2, 120.9, 120.6, 120.3, 119.1, 117.5, 112.2, 111.5, 110.2, 95.2,
94.5, 91.7, 91.2, 55.4; HRMS calcd for C26H19NOS, 393.1187; found,
393.1180.
5.5.2. N-(2-((Z)-6-(2-(Methylthio)phenyl)hexa-3-en-1,5-diynyl)-
phenyl)acetamide (5a)
1H NMR (CDCl3, 400 MHz) d 8.39 (d, 1H, J = 8.4 Hz), 8.15 (s, 1H),
7.48–7.29 (m, 4H), 7.13–7.03 (m, 3H), 6.24 (d, 1H, J = 10.8 Hz), 6.16
(d, 1H, J = 10.8 Hz), 2.32 (s, 3 h), 1.88 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 168.6, 142.1, 139.0, 131.6, 131.8, 130.0, 129.5, 124.2,
124.0, 123.7, 120.2, 119.8, 119.7, 119.2, 118.5, 94.9, 94.0, 93.3,
92.2, 24.4, 14.2; HRMS calcd for C21H17NOS, 331.1031; found,
331.1029.
5.5.9. 4-Bromo-N-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-
en-1,5-diynyl)phenyl)butanamide (4e)
5.5.3. 2-Chloro-N-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-
en-1,5-diynyl)phenyl)acetamide (4b)
1H NMR (CDCl3, 400 MHz) d 8.38 (d, 1H, J = 8.4 Hz), 8.00 (s, 1H),
7.69 (d, 1H, J = 0.8 Hz), 7.59–7.41 (m, 4H), 7.30–7.37 (m, 2H), 6.22
(s, 2H), 3.32 (t, 2H, J = 6.4 Hz), 2.16–2.00 (m, 4H); 13C NMR (CDCl3,
100 MHz) d 169.8, 138.7, 134.7, 134.2, 133.8, 131.9, 131.5, 131.4,
130.2, 128.7, 127.7, 126.1, 126.0, 125.9, 123.4, 119.3, 93.7, 93.2,
92.5, 92.1, 35.2, 32.6, 27.8; HRMS calcd for C23H17BrF3NO (M++H),
460.0524; found, 460.0526.
1H NMR (CDCl3, 400 MHz) d 9.21 (s, 1H), 8.39 (d, 1H, J = 8.0 Hz),
7.66 (d, 1H, J = 8.0 Hz), 7.60(d, 1H, J = 7.6 Hz),7.53–7.48 (m, 2H),
7.45–7.26 (m, 2H) 7.13 (td, 1H, J = 7.4, 1.2 Hz), 6.20 (s, 2H), 4.13
(s, 2H); 13C NMR (CDCl3, 100 MHz) d 171.2, 137.8, 134.2, 131.7,
131.4, 131.1, 130.2, 130.1, 128.5, 126.0, 125.9, 125.9, 124.3,
119.8, 119.2, 119.1, 94.1, 93.6, 91.9, 91.8, 43.0; HRMS calcd for
C21H13ClF3NO, 387.0638; found, 387.0636.
5.5.10. 4-Bromo-N-(2-((Z)-6-(2-(methylthio)phenyl)hexa-3-en-
1,5-diynyl)phenyl)butanamide (5e)
5.5.4. 2-Chloro-N-(2-((Z)-6-(2-(methylthio)phenyl)hexa-3-en-1,5-
diynyl)phenyl)acetamide (5b)
1H NMR (CDCl3, 400 MHz) d 8.43 (s, 1H), 7.46 (dd, 1H, J = 7.6,
1.6 Hz) 7.43–7.38 (m, 1H), 7.34–7.28 (m, 3H), 7.13–7.00 (m, 3H),
6.24 (d, 1H, J = 10.8 Hz), 6.18 (d, 1H, J = 10.8 Hz), 3.32 (t, 2H,
J = 6.4 Hz), 2.16–2.00 (m, 4H); 13C NMR (CDCl3, 100 MHz) d 172.2,
142.2, 139.3, 132.8, 132.7, 132.4, 131.8, 130.6, 129.4, 124.2,
123.8, 122.9, 119.6, 119.4, 118.6, 95.0, 94.1, 93.4, 92.5, 27.7, 16.1,
14.8, 8.17; HRMS calcd for C23H20BrNOS, 437.0449; found,
437.0446.
1H NMR (CDCl3, 400 MHz) d 9.22 (s, 1H), 8.38 (d, 1H, J = 7.6 Hz),
7.53 (dd, 1H, J = 7.6, 1.6 Hz), 7.43–7.28 (m, 3H), 7.14–7.06 (m, 3H),
6.25 (d, 1H, J = 10.8 Hz), 6.15 (d, 1H, J = 10.8 Hz), 4.04 (s, 2H), 2.30
(s, 3H); 13C NMR (CDCl3, 100 MHz) d 164.0, 142.1, 138.0, 132.7,
132.0, 129.9, 129.3, 124.3, 124.1, 124.0, 123.7, 120.5, 119.1,
118.0, 112.9, 95.4, 94.6, 93.3, 91.4, 43.1, 14.7; HRMS calcd for
C21H16ClNOS, 365.0641; found, 365.0635.
5.5.11. 5-Bromo-N-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-
en-1,5-diynyl)phenyl)pentanamide (4f)
5.5.5. N-(2-((Z)-6-(2-(Tifluoromethyl)phenyl)hexa-3-en-1,5-diy-
nyl)phenyl)propionamide (4c)
1H NMR (CDCl3, 400 MHz) d 8.40 (d, 1H, J = 8.4 Hz), 7.98 (s, 1H),
7.68 (d, 1H, J = 7.6 Hz), 7.58 (d, 1H, J = 7.6 Hz), 7.52–7.43 (m, 3H),
7.35 (td, 1H, J = 8, 1.6 Hz), 7.06 (td, 1H, J = 7.6, 0.8 Hz), 6.23 (s,
2H), 3.29 (t, 2H, J = 6.0 Hz), 2.13 (t, 2H, J = 6.8 Hz), 1.88–1.60 (m,
4H); 13C NMR (CDCl3, 100 MHz) d 170.5, 138.8, 134.1, 131.7,
1H NMR (CDCl3, 400 MHz) d 8.43 (d, 1H, J = 8.4 Hz), 7.99 (s, 1H),
7.67 (d, 1H, J = 7.6 Hz), 7.58 (d, 1H, J = 7.6 Hz), 7.51–7.42 (m, 2H),
7.37–7.30 (m, 1H), 7.26–7.21 (m, 1H), 7.05 (td, 1H, J = 8.4,
0.8 Hz), 6.23 (s, 2H), 2.15 (t, 2H, J = 7.2 Hz), 1.98 (d, 3H,

Y.-S. Tu et al. / Bioorg. Med. Chem. 17 (2009) 7412–7417
7417
131.6, 131.5, 131.2, 130.2, 128.8, 126.16, 126.11, 126.06, 126.01,
124.6, 123.3, 121.9, 93.6, 93.1, 92.6, 92.1, 36.3, 32.0, 31.7, 23.6;
HRMS calcd for C24H19BrF3NO, 353.1027; found, 353.1021.
32.5, 23.3, 21.3; HRMS calcd for C24H18F3NO, 393.1340; found,
393.1343.
5.5.16. 1-(2-((Z)-6-(2-(Methylthio)phenyl)hexa-3-en-1,5-diynyl)-
phenyl)piperidin-2-one (9)
5.5.12. 5-Bromo-N-(2-((Z)-6-(2-(methylthio)phenyl)hexa-3-en-
1,5-diynyl)phenyl)pentanamide (5f)
1H NMR (CDCl3, 400 MHz) d 7.62 (dd, 1H, J = 7.6, 1.2 Hz), 7.52–
7.36 (m, 2H), 7.32–7.20 (m, 3H), 7.17–7.05 (m, 2H), 6.18 (d, 1H,
J = 11.2 Hz), 6.10 (d, 1H, J = 10.8 Hz), 3.70–3.61 (m, 2H), 2.52–
2.47 (m, 4H), 2.39 (s, 3H), 1.84 (t, 2H, J = 2.8 Hz); 13C NMR (CDCl3,
100 MHz) d 170.2, 133.6, 132.6, 129.9, 129.7, 129.2, 129.1, 128.1,
127.3, 124.1, 124.0, 123.9, 120.8, 119.3, 118.9, 94.7, 93.8, 93.3,
91.0, 51.1, 32.5, 23.4, 21.3, 14.9; HRMS calcd for C24H21NOS,
371.1344; found, 371.1337.
1H NMR (CDCl3, 400 MHz) d 8.38 (d, 1H, J = 8.4 Hz), 8.17 (s, 1H),
7.48–7.28 (m, 4H), 7.16–7.03 (m, 3H), 6.25 (d, 1H, J = 10.8 Hz), 6.19
(d, 1H, J = 10.8 Hz), 3.23 (t, 2H, J = 6.8 Hz), 2.32 (s, 3H), 2.12 (t, 2H,
J = 6.8 Hz), 1.66–1.56 (m, 4H); 13C NMR (CDCl3, 100 MHz) d 170.7,
138.9, 132.8, 132.1, 131.8, 130.0, 129.0, 124.1, 124.0, 123.6,
123.2, 120.1, 119.4, 118.5, 117.5, 94.0, 93.5, 93.0, 36.3, 33.0, 31.9,
23.7, 14.6; HRMS calcd for C24H22BrNOS, 451.0605; found,
451.0612.
Acknowledgments
5.5.13. 1-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5-
diynyl)phenyl)pyrrolidin-2-one (6)
We would like to thank the National Science Council of the
Republic of China for the financial support and National Institutes
of Health (National Cancer Institute) for the cytotoxicity screening.
1H NMR (CDCl3, 400 MHz) d 7.67–7.56 (m, 3H), 7.51 (td, 1H,
J = 4.4, 0.8 Hz), 7.48–7.36 (m, 3H), 7.33–7.24 (m, 1H), 6.14 (s, 2H),
3.89 (dd, 2H, J = 9.6, 7.2 Hz), 2.42 (t, 2H, J = 7.6 Hz), 2.09–2.01 (m,
2H); 13C NMR (CDCl3, 100 MHz) d 174.9, 140.2, 134.2, 133.8,
133.6, 131.5, 129.8, 129.7, 128.4, 128.3, 127.6, 127.3, 127.2,
126.0, 125.8, 120.0, 94.4, 93.1, 92.6, 91.4, 31.9, 31.4, 29.6; HRMS
calcd for C23H16F3NO, 379.1184; found, 379.1192.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.09.042.
5.5.14. 1-(2-((Z)-6-(2-(Methylthio)phenyl)hexa-3-en-1,5-
diynyl)phenyl)pyrrolidin-2-one (7)
References and notes
1H NMR (CDCl3, 400 MHz) d 7.47–7.38 (m, 2H), 7.35–7.25 (m,
2H), 7.17–7.00 (m, 4H), 6.24 (d, 1H, J = 10.8 Hz), 6.17 (d, 1H,
J = 10.8 Hz), 2.42 (t, 2H, J = 8.4 Hz), 2.34 (s, 3H), 2.09–2.01 (m,
4H); 13C NMR (CDCl3, 100 MHz) d 172.1, 142.1, 139.3, 132.6,
132.4, 131.7, 130.1, 129.3, 124.0, 123.7, 123.1, 121.2, 119.8,
119.6, 118.5, 94.9, 93.9, 93.3, 92.4, 29.6, 16.1, 14.7, 8.16.; HRMS
calcd for C23H19NOS, 357.1187; found, 357.1182.
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1H NMR (CDCl3, 600 MHz) d 7.66–7.60 (m, 2H), 7.59–7.58 (m,
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