# Synthesis and Biological Evaluation of 7-O-Modified Formononetin Derivatives

Authors: Ying Yang; Wen-Jun Mao; Huan-Qiu Li; Tao-Tao Zhu; Lei Shi; Peng-Cheng Lv; Hai-Liang Zhu
Journal: Research Letters in Organic Chemistry
 (2008)

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## Abstract

Three series of novel formononetin derivatives were synthesized, in which formononetin and
heterocyclic moieties were separated by 2-carbon, 3-carbon, and 4-carbon spacers. The chemical
structures of these compounds were confirmed. All the derivatives were 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. Compound3b exihibited promising
antibacterial activity against B. subtilis with minimal inhibitory concentration (MIC) value of 0.78 μg/mL, and compound 5e showed significant antiproliferative activities against Jurkat cell growth
with IC50 of 1.35×10−4 μg/mL. The preliminary investigation of structure-activity relationships (SARs)
was also discussed based on the obtained experimental data.


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## Body

## 1. Introduction

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) [5]. 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, selective
interruption of signal transduction in tumor cells by specific inhibitors of
PTK activity has recently emerged as a major new approach for the design of
tumor-specific drugs [8, 9].Formononetin (1,
shown in Scheme 1), a kind of isoflavonoid, is reported
to have many biological activities including antiproliferative, antioxidant, antidiabetic, antiestrogenic, antibacterial,
antiangiogenic effects, and so on [10–14]. Formononetin is also a potent aryl hydrocarbon
receptor agonist in vitro [14]. The versatile biological
activities 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 activities 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 isoflavonoid
derivatives.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.To our
knowledge, this is the first report on the screening of 7-O-modified
formononetin derivatives for their antimicrobial and antiproliferative
activities.


## 2. Results and Discussions

### 2.1. Chemical Synthesis

Compounds2a–c were the
key intermediates for the synthesis of the compounds investigated. They were
prepared from alkylation of 7-OH group by using 1,2-, 1,3-, or
1,4-dihaloalkanes in the presence of K2CO3 in anhydrous DMF
[15]. 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 formononetin 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 [15]. 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
depicted structures.


### 2.2. Biological Evaluation and Discussion

#### 2.2.1. Antiproliferative Activities [16]

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 Table1. 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, 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, and compound 5d showed promising inhibitory activities
on HepG-2 cell growth.Table 1
Antiproliferative activity of the synthesized compounds.
CompoundsIC50 (μg/mL)Jurkat1HepG-22123.5273.032a48.04>100.002b0.214.142c9.507.183a13.935.573b1.823.243c>100.002.673d3.708.143e12.217.153f16.497.544a0.416.144b5.328.584c16.4452.214d18.085.904e3.4957.825a1.946.545b>100.007.525c0.644.945d1.271.495e1.35×10-413.145f7.1113.535-fluorouracil18.412.501Jurkat: Human T cell lymphoblast-like cell line.
2HepG-2: Human hepatocellular liver carcinoma cell line.


#### 2.2.2. Antibacterial Activities [17]

The
antibacterial activities of the synthetic compounds were tested againstB. subtilis, S. aureus and S. faecalis (Gram-positive
bacteria), E. coli, P. aeruginosa, and E. cloacae (Gram-negative bacteria) by broth dilution method
recommended 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 Table 2.Table 2
Antimicrobial activity of the synthesized compounds.
CompoundsMinimum inhibitory concentrations（ MICs ） (μg/mL)Gram positiveGram negativeS. faecalisS. aureusB. subtilisE. coliP. aeruginosaE. cloacae13.1225.0050.0050.0025.0050.002a3.126.253.126.251.5650.002b3.1250.0025.006.2525.001.562c3.123.1250.0012.5025.0012.503a3.126.2550.003.123.1225.003b1.5612.500.783.1250.003.123c3.126.256.2525.006.253.123d3.126.256.2512.503.126.253e3.121.5650.0025.0025.006.253f3.1212.5025.006.2550.0025.004a3.123.1250.006.2550.0050.004b3.1225.003.121.566.2512.504c3.1212.5025.006.256.2512.504d3.126.253.1212.503.1225.004e3.123.1250.001.5650.0050.005a3.121.566.2525.006.253.125b3.1212.501.5625.0050.0050.005c3.1250.001.566.256.2550.005d3.1250.0025.006.253.126.255e3.120.7812.506.2525.0025.005f3.1225.0050.0050.003.121.56Penicillin1.561.561.566.256.253.12Kanamycin3.121.560.393.123.121.56Most compounds
in series 1, which contained a 2-carbon spacer, displayed good activities
against the test microorganisms. In this series, compound3b showed pronounced activity against B. subtilis and S. faecalis with MIC
values of 1.56 μg/mL and 0.78 μg/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, compounds4b, 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 (1.56 μg/mL)
superior to the positive control kanamycin.Similarly,
several compounds in series 3, which contain a 4-carbon spacer, showed good
activities against the test microorganisms. Among them, compounds5a
and 5e exhibited strong activities against S.
aureus with their MIC values of 1.56 μg/mL and 0.78 μg/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 (3.12 μg/mL), which were
comparable to the positive control kanamycin.


## 2.1. Chemical Synthesis

Compounds2a–c were the
key intermediates for the synthesis of the compounds investigated. They were
prepared from alkylation of 7-OH group by using 1,2-, 1,3-, or
1,4-dihaloalkanes in the presence of K2CO3 in anhydrous DMF
[15]. 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 formononetin 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 [15]. 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
depicted structures.


## 2.2. Biological Evaluation and Discussion

### 2.2.1. Antiproliferative Activities [16]

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 Table1. 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, 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, and compound 5d showed promising inhibitory activities
on HepG-2 cell growth.Table 1
Antiproliferative activity of the synthesized compounds.
CompoundsIC50 (μg/mL)Jurkat1HepG-22123.5273.032a48.04>100.002b0.214.142c9.507.183a13.935.573b1.823.243c>100.002.673d3.708.143e12.217.153f16.497.544a0.416.144b5.328.584c16.4452.214d18.085.904e3.4957.825a1.946.545b>100.007.525c0.644.945d1.271.495e1.35×10-413.145f7.1113.535-fluorouracil18.412.501Jurkat: Human T cell lymphoblast-like cell line.
2HepG-2: Human hepatocellular liver carcinoma cell line.


### 2.2.2. Antibacterial Activities [17]

The
antibacterial activities of the synthetic compounds were tested againstB. subtilis, S. aureus and S. faecalis (Gram-positive
bacteria), E. coli, P. aeruginosa, and E. cloacae (Gram-negative bacteria) by broth dilution method
recommended 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 Table 2.Table 2
Antimicrobial activity of the synthesized compounds.
CompoundsMinimum inhibitory concentrations（ MICs ） (μg/mL)Gram positiveGram negativeS. faecalisS. aureusB. subtilisE. coliP. aeruginosaE. cloacae13.1225.0050.0050.0025.0050.002a3.126.253.126.251.5650.002b3.1250.0025.006.2525.001.562c3.123.1250.0012.5025.0012.503a3.126.2550.003.123.1225.003b1.5612.500.783.1250.003.123c3.126.256.2525.006.253.123d3.126.256.2512.503.126.253e3.121.5650.0025.0025.006.253f3.1212.5025.006.2550.0025.004a3.123.1250.006.2550.0050.004b3.1225.003.121.566.2512.504c3.1212.5025.006.256.2512.504d3.126.253.1212.503.1225.004e3.123.1250.001.5650.0050.005a3.121.566.2525.006.253.125b3.1212.501.5625.0050.0050.005c3.1250.001.566.256.2550.005d3.1250.0025.006.253.126.255e3.120.7812.506.2525.0025.005f3.1225.0050.0050.003.121.56Penicillin1.561.561.566.256.253.12Kanamycin3.121.560.393.123.121.56Most compounds
in series 1, which contained a 2-carbon spacer, displayed good activities
against the test microorganisms. In this series, compound3b showed pronounced activity against B. subtilis and S. faecalis with MIC
values of 1.56 μg/mL and 0.78 μg/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, compounds4b, 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 (1.56 μg/mL)
superior to the positive control kanamycin.Similarly,
several compounds in series 3, which contain a 4-carbon spacer, showed good
activities against the test microorganisms. Among them, compounds5a
and 5e exhibited strong activities against S.
aureus with their MIC values of 1.56 μg/mL and 0.78 μg/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 (3.12 μg/mL), which were
comparable to the positive control kanamycin.


## 2.2.1. Antiproliferative Activities [16]

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 Table1. 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, 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, and compound 5d showed promising inhibitory activities
on HepG-2 cell growth.Table 1
Antiproliferative activity of the synthesized compounds.
CompoundsIC50 (μg/mL)Jurkat1HepG-22123.5273.032a48.04>100.002b0.214.142c9.507.183a13.935.573b1.823.243c>100.002.673d3.708.143e12.217.153f16.497.544a0.416.144b5.328.584c16.4452.214d18.085.904e3.4957.825a1.946.545b>100.007.525c0.644.945d1.271.495e1.35×10-413.145f7.1113.535-fluorouracil18.412.501Jurkat: Human T cell lymphoblast-like cell line.
2HepG-2: Human hepatocellular liver carcinoma cell line.


## 2.2.2. Antibacterial Activities [17]

The
antibacterial activities of the synthetic compounds were tested againstB. subtilis, S. aureus and S. faecalis (Gram-positive
bacteria), E. coli, P. aeruginosa, and E. cloacae (Gram-negative bacteria) by broth dilution method
recommended 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 Table 2.Table 2
Antimicrobial activity of the synthesized compounds.
CompoundsMinimum inhibitory concentrations（ MICs ） (μg/mL)Gram positiveGram negativeS. faecalisS. aureusB. subtilisE. coliP. aeruginosaE. cloacae13.1225.0050.0050.0025.0050.002a3.126.253.126.251.5650.002b3.1250.0025.006.2525.001.562c3.123.1250.0012.5025.0012.503a3.126.2550.003.123.1225.003b1.5612.500.783.1250.003.123c3.126.256.2525.006.253.123d3.126.256.2512.503.126.253e3.121.5650.0025.0025.006.253f3.1212.5025.006.2550.0025.004a3.123.1250.006.2550.0050.004b3.1225.003.121.566.2512.504c3.1212.5025.006.256.2512.504d3.126.253.1212.503.1225.004e3.123.1250.001.5650.0050.005a3.121.566.2525.006.253.125b3.1212.501.5625.0050.0050.005c3.1250.001.566.256.2550.005d3.1250.0025.006.253.126.255e3.120.7812.506.2525.0025.005f3.1225.0050.0050.003.121.56Penicillin1.561.561.566.256.253.12Kanamycin3.121.560.393.123.121.56Most compounds
in series 1, which contained a 2-carbon spacer, displayed good activities
against the test microorganisms. In this series, compound3b showed pronounced activity against B. subtilis and S. faecalis with MIC
values of 1.56 μg/mL and 0.78 μg/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, compounds4b, 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 (1.56 μg/mL)
superior to the positive control kanamycin.Similarly,
several compounds in series 3, which contain a 4-carbon spacer, showed good
activities against the test microorganisms. Among them, compounds5a
and 5e exhibited strong activities against S.
aureus with their MIC values of 1.56 μg/mL and 0.78 μg/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 (3.12 μg/mL), which were
comparable to the positive control kanamycin.


## 3. Conclusion

To investigate the biological
activities of formononetin derivatives, we had synthesized three series of
formononetin derivatives. For all the compounds synthesized, antiproliferative 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 bioassay results, it may be concluded that those containing long 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.



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*Source: 209830-2008-12-18.xml*