Hindawi Publishing Corporation
Research Letters in Organic Chemistry
Volume 2008, Article ID 209830, 4 pages
Synthesisand BiologicalEvaluation of 7-O-Modified
YingYang,Wen-Jun Mao,Huan-QiuLi,Tao-Tao Zhu,LeiShi,
School of Life Sciences, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China
Correspondence should be addressed to Hai-Liang Zhu, email@example.com
Received 15 July 2008; Accepted 27 September 2008
Recommended by Alexander Greer
Three series of novel formononetin derivatives were synthesized, in which formononetin and heterocyclic moieties were separated
screened for antiproliferative activities against Jurkat cell line and HepG-2 cell line. In this paper, compounds prepared were also
screened for their antibacterial activity of six bacterial strains. Compound 3b exihibited promising antibacterial activity against B.
subtilis with minimal inhibitory concentration (MIC) value of 0.78μg/mL, and compound 5e showed significant antiproliferative
(SARs) was also discussed based on the obtained experimental data.
Copyright © 2008 Ying Yang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Isoflavonoids are a broad class of polyphenolic secondary
metabolites that are abundant in plants [1, 2] and in various
common foods such as apples, onions, tea, and red wine [3,
4]. Isoflavonoids also have a potent activity against protein
tyrosine kinase (PTK) . Because of such a broad range
of pharmacological properties, they receive considerable
therapeutic importance. Protein tyrosine kinases (PTKs)
have been intensively investigated because of their role
in the transduction of proliferative signals in mammalian
cells. Many transmembrane growth factor receptors possess
intracellular PTK activity, with initiation of this activity
following external binding of a growth factor, being the
first step in the cellular signal transduction pathway which
controls mitogenesis and cell proliferation [6, 7]. Therefore,
specific inhibitors of PTK activity has recently emerged as a
major new approach for the design of tumor-specific drugs
Formononetin (1, shown in Scheme 1), a kind of
isoflavonoid, is reported to have many biological activities
including antiproliferative, antioxidant, antidiabetic, antie-
strogenic, antibacterial, antiangiogenic effects, and so on
[10–14]. Formononetin is also a potent aryl hydrocarbon
receptor agonist in vitro . The versatile biological activi-
ties of formononetin prompt us to prepare a new series of its
derivatives and evaluate their biological significance. Herein,
we describe the synthesis of formononetin derivatives in
which formononetin and heterocyclic moieties were linked
by spacers,and investigate the effects of the size of the spacers
and substitution patterns of the heterocyclic moieties. All of
the compounds were assayed for their antiproliferative activ-
ities against a panel of two human tumor cell lines (Jurkat
and HepG-2) by applying the MTT colorimetric assay. The
results of this study may be useful to researchers attempting
to gain more understanding of the PTK inhibitory activity of
To our knowledge, this is the first report on the screening
of 7-O-modified formononetin derivatives for their antimi-
crobial and antiproliferative activities.
2.1. Chemical Synthesis
Compounds 2a–c were the key intermediates for the syn-
thesis of the compounds investigated. They were prepared
2 Research Letters in Organic Chemistry
2a, n = 2
2b, n = 3
2c, n = 4
3a-3f, n = 2
4a-4e, n = 3
5a-5f, n = 4
3a R =
3b R =
3c R =
3d R =
3e R =
3f R =
4a R =
4b R =
4c R =
4d R =
4e R =
5a R =
5b R =
5c R =
5d R =
5e R =
5f R =
Scheme 1: Synthesis of 7-O-heterocycle derivatives of formononetin. Reagents and conditions: (i) BrCH2CH2Br, BrCH2CH2CH2Br or
BrCH2CH2CH2CH2Br, K2CO3, DMF; (ii) R, K2CO3, dioxane, DMF, heating.
from alkylation of 7-OH group by using 1,2-, 1,3-, or 1,4-
dihaloalkanes in the presence of K2CO3in anhydrous DMF
. The synthesis of compounds 3a–f, 4a–e, and 5a–f was
accomplished according to the general pathway illustrated
in Scheme 1. To increase the antimicrobial properties of
formononetin, formononetin derivatives in which the for-
mononetin ring system was linked to the alkylamines by
different spacers at C-7 position were investigated, with a
view to modify their lipophilicity. Literature survey revealed
that the compounds containing alkyl amino side chains
showed better activities against the test bacteria than those
containing aromatic ring amino side chains . To further
optimize this activity, seventeen compounds reported in this
paper contain alkyl amino groups. Reaction of 2a–c with
different cyclic and noncyclic alkylamines yielded 3a–f, 4a–
e, and 5a–f, respectively, which were all first reported. All of
the synthesized compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their
2.2. Biological Evaluationand Discussion
The antiproliferative activities of these compounds were
evaluated against a panel of two human tumor cell lines
(Jurkat and HepG-2) by applying the MTT colorimetric
assay. The observed IC50’s are listed in Table 1. From the
results of the in vitro antiproliferative MTT tests of the
prepared compounds, it followed that in series 1, most
of the prepared compounds showed good antiproliferative
activities. Among them compounds 3b and 3d exhibited
strong activities on Jurkat cell growth. Also, compounds
3b and 3c had stronger activities on HepG-2 cell line
than the positive control 5-UF. In series 2, compounds 4a,
Table 1: Antiproliferative activity of the synthesized compounds.
1Jurkat: Human T cell lymphoblast-like cell line.
2HepG-2: Human hepatocellular liver carcinoma cell line.
4b, and 4e displayed remarkable antiproliferative activities
on Jurkat cell, while they just showed moderate activities
against HepG-2 cell. In series 3, compound 5e displayed
significant antiproliferative activities on Jurkat cell growth,
Research Letters in Organic Chemistry3
Table 2: Antimicrobial activity of the synthesized compounds.
Minimum inhibitory concentrations MICs (μg/mL)
S. aureus B. subtilis
and compound 5d showed promising inhibitory activities on
HepG-2 cell growth.
The antibacterial activities of the synthetic compounds were
tested against B. subtilis, S. aureus and S. faecalis (Gram-
positive bacteria), E. coli, P. aeruginosa, and E. cloacae
(Gram-negative bacteria) by broth dilution method rec-
ommended by National Committee for Clinical Laboratory
Standards (NCCLS) [18, 19]. Standard antimicrobial agents
like penicillin and kanamycin were also screened under
identical conditions for comparison. The minimal inhibitory
concentration (MIC) values for the bacteria are listed in
spacer, displayed good activities against the test microor-
ganisms. In this series, compound 3b showed pronounced
activity against B. subtilis and S. faecalis with MIC values
of 1.56μg/mL and 0.78μg/mL, respectively. In addition,
compound 3e showed good activity against S. aureus.
A few compounds in series 2, which contain a 3-
carbon spacer, exhibited great activities against the test
microorganisms. In this series, compounds 4b, 4d, and 4e
showed great activities against S. aureus, B. subtilis, and E.
coli. Among them, 4b and 4e showed strong activities against
E. coli with their MIC value (1.56μg/mL) superior to the
positive control kanamycin.
Similarly, several compounds in series 3, which con-
tain a 4-carbon spacer, showed good activities against the
test microorganisms. Among them, compounds 5a and
5e exhibited strong activities against S. aureus with their
MIC values of 1.56μg/mL and 0.78μg/mL, respectively.
Compounds 5b and 5c displayed great activities against B.
subtilis. Also, compounds 5d and 5f showed strong activities
against P. aeruginosa with the MICs (3.12μg/mL), which
were comparable to the positive control kanamycin.
To investigate the biological activities of formononetin
derivatives, we had synthesized three series of formononetin
derivatives. For all the compounds synthesized, antiprolifer-
ative and antibacterial activities against two cancer cell lines
(Jurkat and HepG-2) and six bacterial strains (three Gram-
positive bacterial strains: Bacillus subtilis, Staphylococcus
aureus, and Streptococcus faecalis and three Gram-negative
bacterial strains: Escherichia coli, Pseudomonas aeruginosa,
and Enterobacter cloacae) were determined. From the bioas-
say results, it may be concluded that those containing long
4Research Letters in Organic Chemistry Download full-text
alkyl amino side chains exhibited better activities against
gram-positive bacteria than those containing short ones,
while it had the opposite rule for gram-negative. Specifically,
the derivatives with dipropylamine moiety were more active
than most of the other analog. In this study, we focused our
attention on the structure-activity relationships. The work
was of interest because this is a preliminary investigation of
SAR, serving as the basis of further more detailed work.
Financial support by the National Natural Science Founda-
tion of China (no. 30772627) is kindly acknowledged.
 V. M. Malikov and M. P. Yuldashev, “Phenolic compounds of
plants of the Scutellaria genus. Distribution, structure, and
properties,” Chemistry of Natural Compounds, vol. 38, no. 5,
pp. 473–519, 2002.
 M. J. del Ba˜ no, J. Lorente, J. Castillo, et al., “Flavonoid distri-
bution during the development of leaves, flowers, stems, and
roots of Rosmarinus officinalis. Postulation of a biosynthetic
pathway,” Journal of Agricultural and Food Chemistry, vol. 52,
no. 16, pp. 4987–4992, 2004.
 P. L. Whitten, S. Kudo, and K. K. Okubo, “Isoflavonoids,” in
Handbook of Plant and Fungal Toxicants, pp. 117–137, CRC
Press, Boca Raton, Fla, USA, 1997.
 G. M. Boland and D. M. X. Donnelly, “Isoflavonoids and
related compounds,” Natural Product Reports, vol. 15, no. 3,
pp. 241–260, 1998.
 K. T. Papazisis, D. Zambouli, O. T. Kimoundri, et al., “Protein
tyrosine kinase inhibitor, genistein, enhances apoptosis and
cell cycle arrest in K562 cells treated with γ-irradiation,”
Cancer Letters, vol. 160, no. 1, pp. 107–113, 2000.
 A. Ullrich and J. Schlessinger, “Signal transduction by recep-
tors with tyrosine kinase activity,” Cell, vol. 61, no. 2, pp. 203–
 S. R. Hubbard and J. H. Till, “Protein tyrosine kinase structure
 F. M. Uckun and C. Mao, “Tyrosine kinases as new molecular
targets in treatment of inflammatory disorders and leukemia,”
Current Pharmaceutical Design, vol. 10, no. 10, pp. 1083–1091,
 P. Traxler, J. Green, H. Mett, U. S´ equin, and P. Furet,
“Use of a pharmacophore model for the design of EGFR
tyrosine kinase inhibitors: isoflavones and 3-phenyl-4(1H)-
quinolones,” Journal of Medicinal Chemistry, vol. 42, no. 6, pp.
 X. Yu, W. Wang, and M. Yang, “Antioxidant activities of
compounds isolated from Dalbergia odorifera T. Chen and
their inhibition effects on the decrease of glutathione level of
rat lens induced by UV irradiation,” Food Chemistry, vol. 104,
no. 2, pp. 715–720, 2007.
ity of genistein and daidzein and its effect on their antioxidant
activity,” Journal of Agricultural and Food Chemistry, vol. 51,
no. 15, pp. 4394–4399, 2003.
 S. Sato, J. Takeo, C. Aoyama, and H. Kawahara, “Na+-glucose
cotransporter (SGLT) inhibitory flavonoids from the roots of
Sophora flavescens,” Bioorganic & Medicinal Chemistry, vol. 15,
no. 10, pp. 3445–3449, 2007.
ity of formononetin by two bioassay systems,” Gynecological
Endocrinology, vol. 22, no. 10, pp. 578–584, 2006.
 S. Medjakovic and A. Jungbauer, “Red clover isoflavones
biochanin A and formononetin are potent ligands of the
human aryl hydrocarbon receptor,” The Journal of Steroid
Biochemistry and Molecular Biology, vol. 108, no. 1-2, pp. 171–
 L.-N. Zhang, Z.-P. Xiao, H. Ding, et al., “Synthesis and cyto-
toxic evaluation of novel 7-O-modified genistein derivatives,”
Chemistry & Biodiversity, vol. 4, no. 2, pp. 248–255, 2007.
P. E. Verweij, “Comparison of NCCLS and 3-(4,5-dimethyl-2-
thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) meth-
ods of in vitro susceptibility testing of filamentous fungi and
development of a new simplified method,” Journal of Clinical
Microbiology, vol. 38, no. 8, pp. 2949–2954, 2000.
 M. Hajd´ uch, V. Mih´ al, J. Minaˇ r´ ık, et al., “Decreased in vitro
chemosensitivity of tumour cells in patients suffering from
malignant diseases with a poor prognosis,” Cytotechnology,
vol. 19, no. 3, pp. 243–245, 1996.
 National committee for clinical laboratory standards, “Ref-
erence methed for broth dilution antifungal susceptibility
testing of yeasts: approved standard,” M27-A, NCCLS, Wayne,
Pa, USA, 1997.
 National committee for clinical laboratory standards, “Devel-
opment of in vitro susceptibility testing criteria and quality
control parameters: tentative guideline,” M23-T3, NCCLS,
Villanova, Pa, USA, 1998.