Synthesis and biological evaluation of cinnamyl compounds as potent antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2007.07.033

A series of cinnamyl compounds related to 2′-hydroxycinnamaldehyde were synthesized and their antitumor effects against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon tumor xenograft in

Full text of DOI:10.1016/j.bmcl.2007.07.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5423–5427
Synthesis and biological evaluation of cinnamyl compounds
as potent antitumor agents
Dae-Seop Shin,a,b Jiin Kim,a Dong Cho Han,a Kwang-Hee Son,a Chang Woo Lee,a
Hwan-Mook Kim,a Su Hyung Hongc and Byoung-Mog Kwona,b,
*
aKorea Research Institute of Bioscience and Biotechnology, 52 Uendong Yoosung, Daejeon 305-333, Republic of Korea
bBioMolecular Science, University of Science and Technology, 52 Uendong Yoosung, Daejeon 305-333, Republic of Korea
cDepartment of Dental Microbiology, School of Dentistry, Kyungpook National University, Daegu 700-412, Republic of Korea
Received 29 March 2007; revised 3 June 2007; accepted 11 July 2007
Available online 25 July 2007
Abstract—A series of cinnamyl compounds related to 20-hydroxycinnamaldehyde were synthesized and their antitumor effects
against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon
tumor xenograft in nude mice. Its antitumor effects belong to the induction of apoptosis and arresting cell cycle at G2/M phase,
which is confirmed by detection of apoptosis markers and cell cycle analysis.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Natural products are still major sources of new drug
development, for example, between 1981 and 2002, 5%
of the 1031 new chemical entities approved as drugs
by the US Food and Drug Administration (FDA) were
natural products, and another 23% were natural-prod-
uct-derived molecules.1 Antitumor drugs such as taxol,
topotecan, irinotecan, and vinblastine, isolated from
natural sources, were clinically used.
to the induction of apoptosis and arresting cell cycle at
G2/M phase.11,12
Antitumor agents inhibited the growth of tumor cell and
induced cell death by activating key elements of the
apoptosis program. Proteolytic enzymes (caspases) are
main effectors of apoptosis. Apoptosis was initially de-
scribed by its morphological characteristics, including
cell shrinking, chromatin condensation, and nuclear
fragmentation.13 Defects in apoptotic pathways are con-
sidered to contribute to a number of human diseases,
ranging from neurodegenerative disorders to malig-
nancy. Subsequent studies reveal high frequency of
apoptosis in spontaneously regressing tumors and in tu-
mor treated with anticancer agents.
Cinnamomum cassia Blume (Lauracea) has been tradi-
tionally used to treat dyspepsia, gastritis, blood circula-
tion disturbances, and inflammatory disease in both
Eastern and Western countries.2–4 20-Hydroxycinnamal-
dehyde was isolated from the stem bark of Cinnamomum
cassia and reported to have an inhibitory effect on far-
nesyl protein transferase activity and inhibited the pro-
liferation of several human cancer cell lines including
breast, leukemia, ovarian, lung, and colon tumor cells.5,6
The cinnamaldehyde has also been shown to have
various activities such as anti-angiogenic activity, immu-
nomodulation, inhibition of CDK4/cyclinD1 kinase
activity, and anti-inflammatory activity through the
inhibition of NO production.7–10 Antitumor effects of
20-hydroxylcinnamaldehyde and its derivatives belong
Cinnamaldehydes are very unstable in serum, because
they have an aldehyde group.14 However, 20-hydroxy-
cinnamyl alcohol and acid did not inhibit the growth
of human tumor cells,15 which means that the aldehyde
group is a key functional group for the antitumor
effects. Therefore, we modified the aldehyde group with
hydroxylamines and synthesized the cinnamyl
compounds 4–7 without aldehyde group. The growth
inhibitory activity of the compounds against tumor
cells was evaluated.
Keywords: Cinnamaldehyde; Apoptosis; Cell cycle.
*
In this report, we described the antitumoric activity of
synthesized cinnamyl compounds against human cancer
Corresponding author. Tel.: +82 42 860 4557; fax: +82 42 861
2675; e-mail: kwonbm@kribb.re.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.033

5424
D.-S. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5423–5427
O
(human colon tumor cell), NCI-H23 (human lung
O
tumor cell), SK-OV3 (human ovarian tumor cell),
A549 (human lung tumor cell), and HEK-239 (human
kidney tumor cell) were treated at different concentra-
tions from 0.1 to 100 lM for 24 h (Table 1). Growth
of human tumor cells was strongly inhibited by the
compounds 3–6 with GI50 value of 3–50 lM and
mildly inhibited the cells by compound 7 in compari-
son with the other hydoxylamines. However, imino-
compound 9 and amide 11 did not inhibit growth of
the tumor cells.17
Cl
OH
a
OR1
OR1
2
1
b
O
O
NHOR2
NHOH
c
OR1
4-7
OR1
3
Antitumor agents are considered to mediate cell death
by activating key elements of the apoptosis program.
To characterize the mechanism of synthetic com-
pound-induced apoptosis, activation of casepase-3 and
degradation of its substrates were monitored in a
dose-dependent manner. To determine the effect of
compound 6 on the cell cycle, after treatment of the
compound in SW620 and HCT116 (human colon tumor
cell), the cells were harvested and analyzed with a FAC-
Scalibur. As shown in Table 2 and Figure 1, compound
6 induced a cell cycle arrest at G2/M phase. When the
compound 6 was treated in SW620 and HCT116 cells,
we found apoptosis signals on FACS diagram (see
Fig. 1).18 Even though the cells are human colon tumor
cells, SW620 is less sensitive than HCT116 against com-
pound 6.
Scheme 1. Synthesis of N-hydroxylamine derivatives. Reagents and
conditions: (a) thionyl chloride in chloroform, reflux, 5 h; (b)
NH2OHÆHCl and NaHCO3 in water, 0 ꢁC, 2 h; (c) carbonyl chlorides,
CH3CN, 25 ꢁC, 5 h, K2CO3.
cells. We also investigated biologically relevant aspects
such as apoptotic markers to identify the mechanism
of the antitumor effect and tumor xenografted in nude
for in vivo activity of hydroxylamine derivative 6.
As shown in Scheme 1, the hydroxylamine derivatives
were synthesized from the 20-substituted cinnamic acid
1. The compound 3 was prepared by the reaction of cin-
nammic carbonyl chloride 2 with hydoxylamine in pres-
ence of base and N-hydroxylamine derivatives 4–7 were
obtained from acylation of compound 3 with commer-
cially available carbonyl chlorides.16
In order to study of the antitumor effects of compound 6
in detail, we monitored dose-dependent response of
colon tumor cells after compound 6 treatment. It was
found that compound 6-treated cells were inhibited in
the cell spreading and becoming of round morphology
of the cells. To confirm the effects of 6 on apoptosis,
we investigated the cleavage of apoptosis marker protein
such as PARP (poly ADP-ribose polymerase), which is
involved in DNA repair in response to environmental
stress and one of the apoptosis markers.19 As shown
in Figure 2, PARP degradation was strongly induced
by the compound 6 at 20 lM and, in addition, cleavage
of caspase-3 was also observed by Western analysis
using specific caspase-3 antibody in HCT116.20 As
already mentioned, when 30 lM of compound 6 was
treated, we could observe the apoptosis signals in
SW620 cells. These results strongly support that
compound 6 exerts its antitumor activity against colon
tumor cells through cell cycle arrest at G2/M and apop-
tosis (Table 3).
We also synthesized imino-type compounds 9 from cinn-
amaldehyde 8 for examining antitumor effects against
human tumor cells. Amide 11 was also prepared from
the acylation of N-methylpiperidine with compound 10
in presence of base (see Scheme 2).
In order to investigate whether the synthesized
compounds inhibit cancer cell proliferation, HCT116
OCH3
O
N
H
H
a
O
O
O
8
O
9
Even though SW620 is less sensitive than HCT116
against compound 6, SW620 tumor xenograft model
of nude mouse was well established in our laboratory,
we tested the antitumor effects of the compound 6
in vivo using SW620 human tumor cells. SW620 cells
were implanted subcutaneously into the right flank of
nude mice on day 0 and compound was intraperito-
neously administered at a concentration of 50 mg/kg
per day for 14 days. The mice were sacrificed and the tu-
mors were weighed on day 14. Tumor volume was de-
creased by 33.6% compared to control mice and the
loss of body weight was not observed in the nude mice
at the doses of 50 mg/kg.21
O
O
N
Cl
N
b
O
CH3
O
10
11
O
O
Scheme 2. Reagents and conditions: (a) NH2OCHÆHCl and NaHCO3
in CH3OH, 25 ꢁC, 2 h; (b) N-methylpiperazine for 11 and K2CO3 in
CH3CN, 25 ꢁC, 3 h.

D.-S. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5423–5427
Table 1. Inhibitory activity of cinnamyl compounds against human tumor cells
5425
Compound
R1
R2
HCT-116a
NCI-H23
SK-OV3
A549
HEK-239
3a
3b
3c
4
H
H
16.8
11.2
15.3
21.3
13.6
12.5
40.4
>100
>100
>100
27.8
17.5
26.9
26.3
18.2
12.9
45.2
>100
>100
>100
50.4
35.3
48.8
16.8
12.9
12.7
>100
>100
>100
>100
55.2
44.9
53.5
30.2
8.8
49.8
20.2
16.8
18.6
3.2
Benzoyl
CH3
H
H
Acetyl
Benzoyl
Benzoyl
Benzoyl
Benzoyl
Benzoyl
Benzoyl
Benzoyl
Benzoyl
5
6
4-Morpholine carbonyl
4-Methyl-1-piperazinecarbonyl
CH3
25.2
>100
>100
>100
>100
5.8
7
89.3
>100
>100
>100
9
10
11
CH3
4-Methyl-1-piperazine
a Selective cytotoxicity against human tumor cell line; GI50 (lM).
Table 2. Effects of compound 6 on cell cycle progressiona
Cell line
Concn (lM)
Cell distribution (%)
S
Sub G1
G0/G1
G2/M
HCT116
0
20
30
40
6.35 0.12
32.36 1.36
39.14 1.72
36.83 1.34
42.23 1.54
42.72 2.23
41.40 1.58
31.06 1.76
27.35 1.09
22.05 0.77
20.39 1.05
26.88 1.56
30.42 2.77
35.23 1.98
38.22 2.32
42.06 2.56
SW620
0
20
30
40
0.92 0.03
3.03 0.04
3.50 0.09
12.7 0.07
56.01 2.01
48.88 1.32
46.46 2.31
33.34 1.22
23.13 0.77
27.79 1.43
26.12 1.78
26.52 1.33
20.66 1.64
22.33 1.89
26.41 1.33
40.14 1.81
a All experiments were independently performed three times.
Figure 1. Effects of compound 6 on cell cycle. HCT116 cells were treated with compound 6. Cells were harvested after 24 h and were subjected to
FACScalibur analysis to determine the distribution of cells through the G0/G1, S, and G2/M phase.

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D.-S. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5423–5427
Figure 2. Induction of cleavage of PARP and caspase-3 by compound 6 which was treated in SW620 and HCT 116 cells. Cell lysates were prepared
with RIPA buffer after 24 h and 40 lg of lysates was resolved by SDS–PAGE.
Table 3. Antitumoric effects of compound 6 in nude mice
Treatment
Dose (mg/kg)
Number of
mice
Tumor volume (mm3)
Start
End
Day 5
Day 7
Day 9
Day 12
Day 14
Control (0.5% Tween 80)
Compound 6
Doxifluridine
0
50
50
8
8
8
8
8
8
8.0 3.9
1.4 1.3
3.8 3.0
18.0 9.8
4.3 3.5
15.0 6.2
48.0 14.2
17.3 7.1
41.4 19.6
85.4 18.7
45.0 22.3
77.9 33.0
134.1 34.3
89.0 32.6
120.7 32.3
9. Jeong, J. H.; Kim, M. R.; So, K. H.; Han, M. Y.; Ha, J.
H.; Garnier, M.; Meijer, L.; Kwon, B. M. Bioorg. Med.
Chem. Lett. 2000, 10, 1819.
10. Lee, S. H.; Lee, S. Y.; Son, D. J.; Lee, H.; Yoo, H. S.;
Song, S.; Oh, K. W.; Han, D. C.; Kwon, B. M.; Hong, J.
T. Biochem. Pharm. 2005, 69, 791.
11. Jeong, H. W.; Han, D. C.; Son, K. H.; Han, M. Y.; Lim, J.
S.; Ha, J. H.; Lee, C. W.; Kim, H. M.; Kim, H. C.; Kwon,
B. M. Biochem. Pharmacol. 2003, 65, 1343.
It can be concluded that the hydroxylamine derivatives
of 20-hydroxycinnamaldehyde will be a new class of anti-
tumor agents inducing apoptosis and arresting cell cycle.
Even though the compound 6 showed relatively weak
activity in tumor cells and nude mice experiment in com-
parison with 20-hydroxycinnamaldehyde,5,6 these results
suggest that hydroxylamine derivatives could be a good
lead molecule for the design of new antitumor agents.
12. Han, D. C.; Lee, M. Y.; Shin, K. D.; Jeon, S. B.; Kim, J.
M.; Son, K. H.; Kim, H. C.; Kim, H. M.; Kwon, B. M.
J. Biol. Chem. 2004, 279, 6911.
Acknowledgments
13. Shi, Y. Mol. Cell 2002, 9, 459.
14. The half life of 20-hydroxycinnamaldehyde is about
10 min, however, that of compound 6 is more than 2 h
in rat serum at 37 ꢁC.
This research was financially supported by grants from
Plant Diversity Research Center of 21st Century Fron-
tier Research Program, KRIBB Research Initiative Pro-
gram, and the National Chemical Genomics Research
Program.
15. Kwon, B. M.; Lee, S. H.; Choi, S. U.; Park, S. H.; Lee, C. O.; Cho,
Y. K.; Sung, N. D.; Bok, S. H. Arch. Pharm. Res. 1998, 2, 147.
16. Typical procedure for the synthesis of compound 6. To a
solution of 1 (10 mmol of 20-hydroxylcinnamic acid or
20-benzoylcinnamic acid) in chloroform (100 mL), thionyl
chloride (15 mmol) was added and the solution was
refluxed for 2 h. The reaction mixture was evaporated
and then dried in vacuo to give 2. To a solution of
NH2OHÆHCl (10 mmol) in saturated NaHCO3 water
(100 mL), a solution of 2 in chloroform (50 mL) was
added at 0 ꢁC. After stirring for 30 min, the mixture was
extracted two times with EtOAc. The combined organic
layer was washed with brine, dried over anhydrous
Na2SO4, filtered, and evaporated to give crude product
3. The crude compound 3 was used for synthesis of
compounds 4–7 without further purification, because the
compound 3 was not very stable at room temperature.
4-Morpholinecarbonyl chloride (10 mmol) was added to a
solution of 3 in CH3CN with K2CO3. After stirring 5 h at
room temperature, the mixture was filtered and the filtrate
was concentrated in vacuo. The residue was chromato-
graphed over silica gel eluting with 30% EtOAc in hexane
to give 6 as a pale yellow solid (1.2 g, 30.3%): mp 154–
156 ꢁC; C21H20N2O6, MS (EI): 396.1325 (Calcd 396.1342),
mp: 154–156 ꢁC, 1H NMR (CDCl3/TMS) (ppm); d 3.52
References and notes
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K.; Kwon, B. M.; Kim, H. M. Planta Med. 1999, 65, 263.
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D.-S. Shin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5423–5427
5427
(broad, 4H), 3.70 (t, 4H, J = 4.8 Hz), 6.47 (d. 1H,
J = 15.9 Hz), 7.22–7.68 (m, 7H), 7.88 (d, 1H,
J = 15.9 Hz), 8.20 (d, 2H, J = 7.5 Hz), 9.16 (broad, 1H).
17. Growth inhibitory assay. Cells were seeded at a density of
5000 cells/well in a 96-well microtiter plate. Cells were
counted with a hemocytometer. After 24 h, cells were
replenished with fresh complete medium containing com-
pounds or 0.1% DMSO. After incubation for 48 h, cell
proliferation reagent WST-1 (Roche) was added to each
well. The amount of WST-1-formazan produced was
measured at 450 nm by ELISA Reader (Bio-Rad).
18. Cell cycle analysis. To analyze the DNA content by flow
cytometry, cells were trypsinized from the culture flask.
After centrifugation at 300g for 5 min at room tempera-
ture, the supernatant was removed. The cells were then
washed twice with PBS solution and fixed with 3 mL of
ice-cold 70% EtOH overnight. Fixed cells were harvested
by centrifugation at 300g for 3 min at room temperature
and washed twice with PBS containing 1% FBS. Collected
cells were resuspended in PBS (100 ll/1 · 105 cells) and
treated with 100 lg/mL of RNase A at 37 ꢁC for 30 min.
Propidium iodide was then added to a final concentration
of 50 lg/mL for DNA staining and 20,000 fixed cells were
analyzed on a FACScalibur (Becton–Dickinson, San Jose,
CA). Cell cycle distribution was analyzed using the
Modifit’s program (Becton–Dickinson).
was resolved by 15% SDS–PAGE and transferred to the
PVDF membrane (Roche, Germany). The membrane was
blocked with 5% nonfat dried milk in TBS-T (50 mM
Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20).
The primary antibodies used were from Cell Signaling.
The secondary antibodies used were horseradish peroxi-
dase-conjugated goat anti-rabbit IgG (1:3000) from Jack-
son immunology. The antibodies were used at dilution
recommended by the manufacturers. Membrane was
incubated with primary antibody for 2 h at room temper-
ature, washed five times with TBS-T. And the proteins
were developed using a chemiluminescence peroxidase
reagent (Roche Applied Science) and exposed to X-ray
films.
21. For the evaluation of in vivo antitumor activity, SW620
human colon adenocarcinoma cells (3 · 107 cells/mL)
were implanted subcutaneously into the right flank of
nude mice on day 0. Compounds were dissolved in 0.5%
Tween 80 and were intraperitoneously administered at a
concentration of 50 mg/kg per day for 20 days. Doxiflu-
ridine was used as a reference compound and its dosage
was 50 mg/kg. Test substances were administrated in a
volume of 0.2 mL per 20 g body weight of animals. On
day 14, the mice were sacrificed and tumor volumes were
estimated [length (mm) · width (mm) · height (mm)/2].
Animal experiments were performed under the permis-
sion according to ‘Institutional Guideline of Animal
Experiments’ of Korea Research Institute of Bioscience
& Biotechnology.
19. Soldani, C.; Scovassi, A. I. Apoptosis 2002, 7, 321.
20. SW620 and HCT 116 cells were treated with 10–40 lM of
6 for 24 h and a 20 lg protein isolated from cell lysates