Syntheses of certain 3-aryl-2-propenoates and evaluation of their cytotoxicity

Article abstract of DOI:10.1016/S0960-894X(01)00165-2

A series of 3-aryl-2-propenoates including cinnamates, (E)-methyl/ethyl 3-[2-(1,4-dimethoxy-5,8-dione)naphthalenyl]-2-propenoates (8ba, 8bb) and (E)-methyl/ethyl 3-[2-(1,4-dihydroxy-9,10-dione)anthracenyl]-2-propenoates (9aa, 9ab) was synthesized and eval

Full text of DOI:10.1016/S0960-894X(01)00165-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1173–1176
Syntheses of Certain 3-Aryl-2-propenoates and Evaluation of
Their Cytotoxicity
Nguyen-Hai Nam,a Young-Jae You,a Yong Kim,a Dong-Ho Hong,b Hwan-Mook Kimb
and Byung Zun Ahna,*
aCollege of Pharmacy, Chungnam National University, Taejon 305-764, South Korea
bKorea Research Institute of Bioscience and Biotechnology, Taejon 305-600, South Korea
Received 18January 2001; accepted 3 March 2001
Abstract—A series of 3-aryl-2-propenoates including cinnamates, (E)-methyl/ethyl 3-[2-(1,4-dimethoxy-5,8-dione)naphthalenyl]-2-
propenoates (8ba, 8bb) and (E)-methyl/ethyl 3-[2-(1,4-dihydroxy-9,10-dione)anthracenyl]-2-propenoates (9aa, 9ab) was synthesized
and evaluated for antitumor cytotoxicity. It was found that the ortho- or para-dihydroxy funtionality on the aryl ring was essential
for the cytotoxicity of cinnamates. Compounds 8ba, 8bb and 9aa, 9ab showed potent cytotoxicity against various tumor cell lines.
# 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of a,b-unsaturated ketones have demonstrated pre-
ferential activity toward thiol.13 Alkylation with a cel-
Cinnamic acid (1) and its derivatives are widely dis-
tributed in nature and constitute a class of compounds
with a broad spectrum of activities such as antiinflam-
matory,1 anticonvulsant,2 antifungal,3 analgesic1 and
antihypertensive.4,5 They have been also reported to
inhibit human immunodeficiency virus (HIV)6 and a
number of enzymes such as protein tyrosine kinases,7
ras farnesyl transferase.8 Several reports have unveiled
the antiinvasive and antiangiogenic activity of this
compound class.9 However, the evaluation of cinna-
mates and synthesis of related derivatives, 3-aryl-2-pro-
penoates as anticancer agents appear to be unexplored
fields.
lular thiol such as glutathione (GSH) may also occur with
cinnamates like 4a, leading to adducts at the b-position.
Hence, a,b-unsaturated carbonyl-containing com-
pounds may be free from problems of mutagenicity and
carcinogenicity that are associated with many alkylating
agents used in cancer chemotherapy.14 Taking into
consideration this structural feature and the interesting
bioactivity of the compound 4a, the present investiga-
tion aims at preparing a number of 4a analogues and
related 3-aryl-2-propenoates in order to evaluate their
cytotoxicity.
Results and Discussion
Chemistry
In our search for novel antitumor agents from natural
sources, we isolated a phenyl propanoid, methyl 3,4-
dihydroxycinnamate (methyl caffeate 4a, Fig. 1), from
the plant Notopterygium incisum. This compound
showed a significant cytotoxicity against various cancer
cell lines10 and greatly inhibited the invasion of B16
melanoma cells.11 Structurally, 4a possesses an a,b-
unsaturated carbonyl, which can be considered as a
Michael acceptor, an active moiety often employed in
the design of anticancer drugs.12 In addition, a number
Caffeates (4a–4c, Table 1) were obtained by refluxing
caffeic acid in excess alcohol used as solvent in the pres-
ence of a catalytic amount of HCl (Scheme 1). Other
*Corresponding author. Tel.:+82-42-821-5923; fax: +82-42-823-
6566; e-mail: ahnbj@cnu.ac.kr
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00165-2

1174
N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1173–1176
Table 1. Cytotoxicity of synthesized cinnamates and related compounds against some cancer cell linesa
b
Compound
ArCH¼CHCOR/ArCH2CH2COR
Cytotoxicity (IC50) (mg/mL)
E/Z
Ar
R
B16
HCT116
A431
1
E
E
E
E
E
E
E
Z
E
E
E
E
E
E
E
E
E
3,4-(OH)2–C6H3-
3,4-(OH)2–C6H3-
3,4-(OH)2–C6H3-
3,4-(OH)2–C6H3-
3,4-(OH)2–C6H3-
3,4-(OH)2–C6H3-
2,5-(OH)2–C6H3-
2,5-(OH)2–C6H3-
2,5-(OH)2–C6H3-
3,5-(OH)2–C6H3-
2,3-(OH)2–C6H3-
3-OH-4-OCH3–C6H3-
2-OH-5-OCH3–C6H3-
3,4-(OCH3)2–C6H3-
2,5-(OCH3)2–C6H3-
3,4-OCH2O–C6H3-
2,5-(OH)2–C6H3-
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
(Refer to Scheme 2)
DHAQc
H
>30
2.69
>30
2.49
4.26
3.01
1.99
2.12
0.17
>30
0.19
>30
4.15
27.49
>30
>30
>30
>30
4.59
>30
>30
1.31
1.32
>30
3a
3b
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
NHOH
NHC6H5
OCH3
OC2H5
OnC3H7
OCH3
OCH3
OC2H5
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
2.16
4.23
2.96
1.75
2.46
0.12
30.07
0.21
>30
4.02
25.88
5.00
1.79
1.81
1.89
0.15
>30
0.17
>30
4.06
>30
>30
>30
>30
>30
>30
>30
>30
>30
4.43
>30
>30
4m
5
5.00
>30
>30
8aa
8ab
8ba
8bb
9aa
9ab
9ba
9bb
0.50
0.57
0.180.65
0.32
8.69
3.22
2.29
7
0.8
0.71
14.35
9.03
0.91
1.78
0.78
7.16
>30
>30
>30
Adriamycin
0.11
0.17
0.21
aB16, murine melanoma; HCT116, human colon cancer; A431, human epidermoid carcinoma.
bThe sample concentration produces a 50% reduction in cell growth.
cDHAQ, 1,4-dihydroxy-9,10-anthraquinone, was synthesized as described previously.15
cinnamates (4d–4m) were synthesized in good yields by
a Wittig reaction. In all cases, predominant E-isomers
(>90% of the total products) were formed which were
unambiguously characterized based on a coupling con-
stant value of the two olefinic protons (around 16.5 Hz
for E-isomer and 8Hz for Z-isomer) and NOE tech-
niques. Isolation of a Z-isomer attempted for methyl
2,5-dihydroxycinnamate resulted in 7% yield of 4e. The
two caffeamides 3a, 3b were synthesized through a
sequence shown in Scheme 1: protection of the phenol
groups with methoxycarbonyls, coupling of the pro-
tected caffeic acid with hydroxyamine or aniline and
deprotection by sodium methoxide. Reduction of the
double bond of the propenoate 4d was carried out using
hydrogen (1 atm) and Pd/C (10%) to afford the dihy-
drocinnamate 5 in a quantitative yield.
(E)-3-[2-(1,4-Dihydroxy-9,10-dione)anthracenyl]- and
(E)-3-[2-(1,4-dihydroxy-5,8-dione)naphthalenyl]-2-pro-
penoates were synthesized from 2-formyl-1,4,9,10-tetra-
methoxyanthracene15 (6a) and 2-formyl-1,4,9,10-tetra-
methoxynaphthalene16 (6b) as shown in Scheme 2.
Oxidative demethylation of 7 with cerium diammonium
nitrate afforded 8 in good yields (85–90%). Here, the
electron-withdrawing effect of the a,b-unsaturated car-
bonyl side chain might be attributed to the occurrence
of demethylation at 5,8-positions but not 1,4-positions
of 7ba and 7bb. This has been fully explained pre-
viously.17 The use of aluminum chloride in nitrobenzene
as a solvent effected a further selective 1,4-demethyla-
tion while the ester moiety remained intact.18
Bioactivity
The synthesized compounds were assayed19 against
three cancer cell lines including murine melanoma
(B16), human colon cancer (HCT116) and human epi-
dermoid carcinoma (A431). From the IC50 values sum-
marized in Table 1, one general observation for the
cinnamates can be drawn: Compounds bearing ortho- or
para-hydroquinone moieties (4a–4d, 4f, 4h, 5) exhibited
significant cytotoxic activity (IC50ꢀ5 mg/mL) while
compounds with otherwise-substituted benzene rings
were all inactive. For example, methylation of one
hydroxy group (compounds 4i, 4j) or both (compounds
4k, 4l, and 4m) abolished cytotoxicity. It has been
reported that hydroquinones are easily autoxidized in
Scheme 1. (a) (i) CH3OCOCl, TEA, THF; (ii) R00NH2, DCC, HOBt,
CH3CN; (iii) CH3ONa, CH3OH; (b) R0OH, HCl, reflux, 3 h; (c)
Ph3PCH2COOR0, benzene, reflux, 12 h; (d) Pd–C (10% Pd), H2, 1 atm,
30 min.

N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1173–1176
1175
vivo to quinones20 which are known to elicit
cytotoxicity per se or through a cascade of a redox
cycle.21 Thus, the possible formation of quinonoid
metabolites from ortho- or para-hydroquinone com-
pounds (4a–4d, 4f, 4h, 5) might be one of the mechan-
isms responsible for their cytotoxicity. Moreover, the
quinonoid metabolites should be more electron-deficient
at the b-position compared to the parent hydro-
quinones, thus more labile to cellular nucleophiles such
as GSH. Compound 4g bearing a 3,5-dihydroxy sub-
stituent, which is impossible for the formation of qui-
nonoid metabolite, was found inactive. The 2,5-
dihydroxy-substituted pattern seemed to be most favor-
able for the cytotoxic activity, followed by the 3,4- and
2,3-dihydroxy-substituted ones (compound 4d was more
potent than 4a which was in turn more cytotoxic than
4h). Compounds 4d and 4f were most potent among the
cinnamates with IC50 values of less than 0.3 mg/mL in all
three tested cell lines. Interestingly, it was found that the
E configuration proved essential for the cytotoxicity;
compound 4e, a Z-isomer of cytotoxic 4f, was inactive
at the highest concentration assayed (30 mg/mL).
Saturation of the double bond of the propene moiety (5)
dramatically reduced the cytotoxicity. Thus, the a,b-
unsaturated carbonyl moiety of cinnamates was
demonstrated to be important for the cytotoxicity and
appears to play an important role in the alkylation of
cellular nucleophiles, preferentially via Michael addi-
tion.14 Furthermore, the ester group was found to be
necessary for the strong activity; replacement of the
ester by amide or hydroxy groups substantially
decreased or abolished cytotoxicity of the related com-
pounds (3a, 3b and 1 vs 4a). Variation of the alkyl size
did not affect the cytotoxicity significantly (4a–4c).
The rationale behind the synthesis of compounds 9aa,
9ab was based on the consideration of the northeastern
part in the structures of doxorubicin (Adriamycin1)
and daunorubicin (Fig. 2), two drugs currently used in
cancer chemotherapy. As shown in Table 1, compounds
9aa, 9ab were almost as cytotoxic as Adriamycin1. The
two compounds 8aa, 8ab, bearing a 2-(1,4-dimethoxy-
9,10-dione)anthracenyl moiety corresponding to the
2-(1,4-dihydroxy-9,10-dione)anthracenyl group in 9aa,
9ab, were found inactive, suggesting the essentiality of
Scheme 2. (a) Ph3PCH2COOR3, benzene, reflux, 12 h; (b) CAN,
CH3CN–H2O; (c) AlCl3, nitrobenzene.
Figure 2.
Table 2. Cytotoxicity of representative compounds in a panel of cancer cell lines
Organ
Cell line
Cytotoxicity (IC50)a (mg/mL)
4d
4f
8ba
8bb
9aa
9bb
ADRb
Lung
NCI-H23
A549
0.93
7.81
0.17
0.27
0.97
5.43
0.39
1.10
0.680.77
0.280.33
0.56
0.12
0.82
0.37
0.42
2.59
0.73
0.65
7.15
0.19
0.26
2.22
3.21
0.29
2.14
1.01
1.12
1.31
0.97
0.83
0.07.895
0.30
1.11
1.02
0.93
1.32
0.680.66
0.92
4.59
0.11
1.02
0.39
1.13
0.65
1.58 0.53
0.73
5.16
4.78
9.03
0.10
0.20
0.17
Colon
HCT 116
HCT 15
COLO 205
HCC29985.22
SW620
HT 29
DLD-1
LOX-IMVI
UACC62
B16
3.69
0.11
0.17
0.45
0.55
1.47
2.16
0.17
0.21
0.99
0.11
1.15
0.35
9
0.8 3.28 0.32
Melanoma
Breast
0.09
0.680.11
0.57
1.14
0.96
0.64
1.32
0.58
0.91
3.69
0.182.56
0.74
0.46
2.72
0.69
0.42
0.11
0.06
0.11
0.57
0.21
0.53
0.67
0.74
2.26
0.82
0.59
0.50
0.98
1.21
0.49
1.25
0.61
MDA-MB-231
MCF7
PC-3
3.69
4.69
5.67
2.49
3.19
0.24
0.13
0.30
0.29
0.14
Prostate
CNS
Renal
SNB19
ACHN
Mean
1.37
1.480.97
3
0.8 0.69
3.95
0.20
aThe sample concentration produces a 50% reduction in cell growth.
bADR, Adriamycin.

1176
N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1173–1176
the para-dihydroxy functionality for the strong cyto-
toxicity of 9aa and 9ab. Noteworthy however, a 1,4-
dihydroxy-9,10-anthraquinone (DHAQ) was also inactive
up to 30 mg/mL in all cell lines assayed. Thus, the
incorporation of the propenoate moiety onto DHAQ
conferred a strong cytotoxicity to the resulting com-
pounds 9aa and 9ab. To the best of our knowledge, this
practice has not been reported for the anthracyclines
though hundreds of analogues of this class have been
prepared in the past decades. In 9aa and 9ab, the pro-
penoate side chain might play an important role in
enhancing the oxidative formation of an anthracene-
1,4,9,10-tetraone metabolite, which is then available for
rereduction and hence undergoes a futile redox cycling,
a known action mechanism of anthraquinones.21 It is
expected that the introduction of a double bond con-
necting C9–C10 in the structure of doxorubicin could
produce a novel analogue with superior bioactivity
without increasing complication of mutagenic and car-
cinogenic toxicity as explained in the preceding
paragraph. This venture remains to be studied.
comparable cytotoxicity to Adriamycin1 and (E)-methyl/
ethyl 3-[2-(1,4-dimethoxy-5,8-dione)naphthalenyl]-2-pro-
penoates, the first naphthoquinone derivatives showing
strong cytotoxicities against solid tumor cell lines. Eva-
luation against multi-drug resistance cells as well as in
vivo experiments are underway.
References and Notes
1. Komatsu, T.; Awata, H.; Sakai, Y.; Inukai, T.; Kimura,
M.; Inaba, S.; Yamamoto, H. Arzneimittelforschung 1972, 22,
2099.
2. Balsamo, A.; Crotti, P.; Lapucci, A.; Macchia, B.; Mac-
chia, F.; Cuttica, A.; Passerini, N. J. Med. Chem. 1981, 24,
525.
3. Gupta, S. K.; Banerjee, A. B.; Achari, B. Lloydia 1976, 39,
218.
4. Delle Monache, G.; Botta, B.; Delle Monache, F.; Espinal,
R.; De Bonnevaux, S. C.; De Luca, C.; Botta, M.; Corelli, F.;
Carmignani, M. J. Med. Chem. 1993, 36, 2956.
5. Bernier, J. L.; Henichart, J. P.; Warin, V.; Trentesaux, C.;
Jardillier, J. C. J. Med. Chem. 1985, 28, 497.
6. Artico, M.; Di Santo, R.; Costi, R.; Novellino, E.; Greco,
G.; Massa, S.; Tramontano, E.; Marongiu, M. E.; De Montis,
A.; La Colla, P. J. Med. Chem. 1998, 41, 3948.
7. Buzzetti, F.; Brasca, M. G.; Crugnola, A.; Fustinoni, S.;
Longo, A.; Penco, S.; Dalla Zonca, P.; Comoglio, P. M.
Farmaco 1993, 48, 615.
8. Poradosu, E.; Gazit, A.; Reuveni, H.; Levitzki, A. Bioorg.
Med. Chem. 1999, 7, 1727.
9. Ken-ichi, S.; Tohru, O.; Naomi, A.; Hirotsuku, M.; Masao,
H.; Ikuo, S. Bio. Pharm. Bull. 1997, 20, 345.
10. Nam, N. H.; Huong, H. T. T.; Kim, H. M.; Ahn, B. Z.
Kor. J. Pharmacogn. 2000, 31, 77.
Encouraged by the interesting results obtained from 9aa
and 9ab, we went on to synthesize two naphthoquinone
derivatives 8ba and 8bb. Previously, Song et al.16 repor-
ted several series of highly cytotoxic naphthoquinones
against leukemic cell lines but none of them was active
against solid tumor cells. Our result showed that naph-
thoquinones 8ba and 8bb were significantly cytotoxic
against all three solid tumor cell lines with IC50 values
as low as 0.5 mg/mL. No naphthoquinone derivatives
exerting notable cytotoxicity against solid tumor cells
have been documented at the time of writing. 1,4-
Demethylation of 8ba and 8bb slightly enhanced their
activity toward A431 but substantially decreased cyto-
toxicity against B16 and HCT116. This shift is still not
fully understood though it has been noted for other
naphthoquinones on leukemia cells previously.16
11. Personal unpublished results.
12. Ahn, B. Z.; Sok, D. E. Curr. Pharm. Design 1996, 2, 247.
13. Lee, K. H.; Huang, E. S.; Piantadosi, C.; Pagano, J.;
Geissman, T. A. Cancer Res. 1971, 31, 1649.
14. Lee, K. H.; Hall, I. H.; Starness, C. D.; Elgebaly, S. A.;
Waddell, T. G.; Hadgraft, R. T.; Ruffler, C. G.; Weidner, I.
Science 1977, 196, 533.
15. Guang, Z. J.; Song, G. Y.; Zheng, S. G.; Kim, Y.; Sok,
D. E.; Ahn, B. Z. Arch. Pharm. Res. 1998, 21, 208.
16. Song, G. Y.; Zheng, X. G.; Kim, Y.; You, Y. J.; Sok,
D. E.; Ahn, B. Z. Bioorg. Med. Chem. Lett. 1999, 9, 2407.
17. Yasuhiro, T.; Akira, T. Bull. Chem. Soc. Jpn. 1988, 61,
2039.
Further evaluation of the most active compounds was
carried out in a panel of cancer cell lines and the results
are shown in Table 2. The naphthoquinones 8ba and
8bb and anthraquinone 9aa showed IC50 values of less
than 1 mg/mL in most of the cell lines. It is noteworthy
to mention that two cell lines A549 (lung carcinoma)
and HCT116 (colon cancer) were rather resistant
toward the two cinnamates 4d and 4f.
18. All newly synthesized compounds gave satisfactory ana-
lytical and spectroscopic data.
19. The cytotoxicity assay was performed as described in ref
10.
20. Ollinger, K.; Llopis, J.; Cadenas, E. Archiv. Biochem.
Biophys. 1989, 275, 514.
21. Monks, T. J.; Hanzlik, R. P.; Cohen, G. M.; Ross, D.;
Graham, D. G. Toxicol. Appl. Pharmacol. 1992, 112, 2.
In conclusion, we have found potent cytotoxicities dis-
played by methyl and ethyl 2,5-dihydroxycinnamates
against a variety of cancer cell lines. We also discovered
simple analogues (E)-methyl/ethyl 3-[2-(1,4-dihydroxy-
9,10-dione)anthracenyl]-2-propenoates with an almost