Nitrogen-containing apigenin analogs: Preparation and biological activity

Article abstract of DOI:10.3390/molecules171214748

A series of nitrogen-containing apigenin analogs 4a-j was synthesized via Mannich reactions to develop anticancer, antibacterial, and antioxidant agents from plant-derived flavonoids. The chemical structures of these compounds were confirmed using 1H-NMR, 13C-NMR, and ESI-MS. The in vitro biological activities of the analogs were evaluated via assays of their antiproliferative, antibacterial, and antioxidant activities. The prepared apigenin analogs exhibited different antiproliferative activities against four human cancer cell lines, namely human cervical (HeLa), human hepatocellular liver (HepG2), human lung (A549), and human breast (MCF-7) cancer cells. Compound 4i showed the most favorable in vitro antiproliferative activity with IC50 values of 40, 40, 223, and 166 μg/mL against HeLa, HepG2, A549, and MCF-7, respectively. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay also showed that 4i had the most potent antioxidant activity, with the smallest IC50 value (334.8 μg/mL). The antibacterial activities of the analogs were determined using a two-fold serial dilution technique against four pathogenic bacteria, namely Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. All the prepared apigenin analogs exhibited more potent activities than the parent apigenin. Compounds 4h and 4j, in particular, exhibited the best inhibitory activities against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with MIC values of 3.91 and 1.95 μg/mL, respectively.

Full text of DOI:10.3390/molecules171214748

Molecules 2012, 17, 14748-14764; doi:10.3390/molecules171214748
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Nitrogen-Containing Apigenin Analogs:
Preparation and Biological Activity
Rui Liu 1,2, Bin Zhao 1, Dong-En Wang 1, Tianyu Yao 1, Long Pang 1, Qin Tu 1,
Saeed Mahmoud Ahmed 1, Jian-Jun Liu 1,* and Jinyi Wang 1,*
1
Colleges of Science and Forestry, Northwest A&F University, Yangling 712100, Shaanxi, China
2
College of Agronomy and Life Sciences, Shanxi Datong University, Datong 037009, Shanxi, China
* Authors to whom correspondence should be addressed; E-Mails: ljj@nwsuaf.edu.cn (J.-J.L.);
jywang@nwsuaf.edu.cn (J.W.); Tel./Fax: +86-29-8708-2520 (J.W.).
Received: 30 October 2012; in revised form: 30 November 2012 / Accepted: 5 December 2012 /
Published: 11 December 2012
Abstract: A series of nitrogen-containing apigenin analogs 4a–j was synthesized via
Mannich reactions to develop anticancer, antibacterial, and antioxidant agents from
plant-derived flavonoids. The chemical structures of these compounds were confirmed using
1H-NMR, C-NMR, and ESI-MS. The in vitro biological activities of the analogs were
13
evaluated via assays of their antiproliferative, antibacterial, and antioxidant activities. The
prepared apigenin analogs exhibited different antiproliferative activities against four human
cancer cell lines, namely human cervical (HeLa), human hepatocellular liver (HepG2),
human lung (A549), and human breast (MCF-7) cancer cells. Compound 4i showed the most
favorable in vitro antiproliferative activity with IC50 values of 40, 40, 223, and 166 μg/mL
against HeLa, HepG2, A549, and MCF-7, respectively. The 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical scavenging activity assay also showed that 4i had the most potent
antioxidant activity, with the smallest IC50 value (334.8 μg/mL). The antibacterial activities
of the analogs were determined using a two-fold serial dilution technique against four
pathogenic bacteria, namely Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and
Pseudomonas aeruginosa. All the prepared apigenin analogs exhibited more potent
activities than the parent apigenin. Compounds 4h and 4j, in particular, exhibited the best
inhibitory activities against the Gram-positive bacteria Staphylococcus aureus and Bacillus
subtilis with MIC values of 3.91 and 1.95 μg/mL, respectively.

Molecules 2012, 17
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Keywords: apigenin analogs; antiproliferative activity; antibacterial activity;
antioxidant activity
1. Introduction
Flavonoids are important components of various traditional Chinese medicines and phytomedicines
that bear C6-C3-C6 skeletons [1,2]. In addition to their physiological roles in plants, these compounds
also exhibit anticancer, antioxidant, anti-aging, and antibacterial activities [3–5]. Flavonoids display
antibacterial activity by inhibiting nucleic acid synthesis [6–8], cytoplasmic membrane function [9–11],
and energy metabolism [12]. They are generally classified as flavones, flavonols, flavanones,
flavanonol, or isoflavones, based on the degree of oxidation of the carbon-3 bond (C-3) and the
connection position of the B-ring [13]. Flavonoids are prevalent in higher plants as well as in the roots,
stems, leaves, and flowers of ferns [14]. To date, over 4,000 flavonoids have been identified, and several
of these compounds showed diverse pharmacological activities, such as antimicrobial [15], antiallergic [16],
antidiabetic [17], anti-inflammatory [18], antiviral [19], antimutagenic [20], antithrombotic [21],
antioxidant [22], anticarcinogenic [23], and hepatoprotective [24] effects.
Apigenin [2-(4-hydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one], which belongs to the flavone
subclass of flavonoids, are found in fruits, vegetables, and traditional medicinal plants, such as
parsley, onion, orange, tea, chamomile, wheat sprouts, and several seasonings [25]. Apigenin exhibits
anti-inflammatory and anti-carcinogenic effects on the skin [26]. In some cells, apigenin displays a
variety of anti-tumor effects, such as stimulation of gap junctional and intracellular communication [27]
as well as inhibition of mutagenesis [28], transformation [29], angiogenesis [30], and tumorigenesis [31].
In addition, apigenin exhibits antibacterial activity against a number of bacterial species [32,33].
Previous studies have shown the potential of apigenin as a bioactive molecule for various clinical
applications. However, compared with existing drugs, the anticancer, antibacterial, antioxidant, and
other activities of apigenin hardly meet the requirements for clinical application. Considerable effort has
thus been exerted to improve the activities of apigenin [34–37]. A simple and effective way is the study
of the structure-activity relationships of apigenin analogs prepared by introducing various functional
groups into different positions in the apigenin skeleton [38–40]. Gunnarsson et al. [40] synthesized
sulfated apigenin analogs, which were all antithrombin activators to accelerate the inhibition of factor Xa.
Mavel et al. [38] synthesized apigenin analogs with halogen on the 4'-position which showed cytotoxic
activity and MDR-reversing capacity. However, all the current studies mainly focused on the 4'- or
7-position of apigenin. The 8-position of apigenin was scarcely examined.
The Mannich reaction is a classic method for the preparation of Mannich bases, namely, β-amino
ketones and β-amino aldehydes. The Mannich reaction is also one of the very important reaction types
and key reaction-steps in the synthesis of numerous drugs and natural products [41,42]. This reaction
has been considered as an effective method in introducing aminomethyl substituents into the desired
molecules to improve their biological activities [43]. Previous studies have demonstrated that Mannich
bases offer a wide range of biological activities, such as anticancer [44], anti-inflammatory [45],
analgesic [46], antibacterial, and antifungal activities [47]. The inhibitory activities of adriamycin

Molecules 2012, 17
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salicylamide Mannich base against MCF-7 and PC-3 cells are four times better than that of adriamycin,
while simultaneously reducing the clinical side effects [48]. In addition, several studies have shown that
8-aminomethylated oroxylin A analogs demonstrated significantly improved α-glucosidase inhibitory
activity [49], nitrogen-containing baicalein/quercetin analogs exhibited potent CDK1/cyclin B
inhibitory activity [50], and 8-aminomethylluteolin derivatives displayed potential anti-inflammatory
activity [51].
Thus, in the present study, a series of new apigenin analogs with aminomethyl groups on the C-8
position was synthesized via Mannich reactions. In addition, the antiproliferative, antibacterial, and
antioxidant activities of these analogs were investigated. The antiproliferative activities of the
compounds were analyzed against four human cancer cell lines, namely the human cervical (HeLa),
human hepatocellular liver (HepG2), human lung (A549), and human breast (MCF-7) cancer cells, using
a standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide (MTT) assay. The antibacterial activities
of the prepared apigenin analogs against four pathogenic bacteria, namely Staphylococcus aureus
(S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa
(P. aeruginosa), were explored using a two-fold serial dilution technique. The antioxidant activities of
the apigenin analogs were determined using the DPPH radical scavenging assay.
2. Results and Discussion
2.1. Chemistry
Ten new nitrogen-containing apigenin analogs, 4a–j, were prepared via Mannich reactions (Scheme 1)
to further explore their antiproliferative, antibacterial, and antioxidant activities. The hydrogen atom
activity of apigenin in the C-8 position increases because of the p-π conjugative effect of the 5- and 7-
hydroxy in the A-ring. Thus, apigenin easily reacts with formaldehyde and primary or secondary
amines to produce different Mannich bases [52]. The reaction conditions, yields, and structures of the
apigenin analogs are summarized in Table 1. Corresponding products were obtained with the
introduction of different primary or secondary amines. The reaction temperatures had a little difference
for different products, varied from 25 °C to 40 °C. Also, different times were required for these
reactions since no or less byproducts and better yields were obtained under these conditions.
Scheme 1. Synthetic route of compounds 4a to 4j.
OH
R
OH
H2C
HO
O
HO
O
O
O
DMF, MeOH
+
+
R
H
H
H
OH
O
OH
2
3
1
4
H3C(H2C)2HN
4b
(H3C)2HCHN
4c
(H3C)3CHN
R =
H3CH2CHN
4a
4d
(H3C)2N
(C2H5)2N
(C3H7)2N
4e
4f
4g
N
N
O
N
H
4h
4i
4j

Molecules 2012, 17
Table 1. Physicochemical data of the nitrogen-containing apigenin analogs.
14751
Compounds
R
Time (h) Temperature (°C) Isolated yield (%)
4a
4b
4c
4d
4e
4f
Ethylamino
4
12
4
25
30
35
35
25
25
25
40
40
40
40.5
38.7
61.7
52.3
39.6
60.9
51.2
41.8
42.1
43.4
Propylamino
Isopropylamino
Tert-butylamino
Dimethylamino
Diethylamino
Diisopropylamino
Cyclohexylamino
Pyrrolidinyl
5
4
1.5
12
8
4g
4h
4i
1.5
12
4j
Morpholinyl
2.2. Antibacterial Activity
A preliminary antibacterial screeningof the synthesized compounds was performed using the disk
diffusion method [53]. Table 2 lists the screening results of the tested compounds against the Gram
negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) and Gram positive bacteria
S. aureus (ATCC 25923) and B. subtilis (ATCC 6633). The results showed that most of the compounds
showed antibacterial activities against the microorganisms at a dose of 1000 μg/mL. Compounds
showing inhibition of at least 10 mm were considered active and were further evaluated for their
minimal inhibitory concentration (MIC) [54,55].
Table 2. Inhibition zones (IZ) in mm (Mean a ± S.D b) of the synthesized compounds 4a to 4j.
Compounds
S. aureus
ATCC 25923
11.1 ± 0.3
17.5 ± 0.1
17.9 ± 0.6
14.1 ± 0.5
14.8 ± 0.2
12.1 ± 0.6
18.3 ± 0.1
15.0 ± 0.3
22.1 ± 0.5
18.8 ± 0.4
25.9 ± 0.6
37.3 ± 0.6
26.3 ± 0.5
B. subtilis
ATCC 6633
11.0 ± 1.0
21.3 ± 0.6
17.5 ± 0.4
15.2 ± 0.1
18.0 ± 0.3
15.9 ± 0.6
18.7 ± 0.5
19.0 ± 0.1
22.8 ± 0.2
19.3 ± 0.4
27.2 ± 0.8
36.7 ± 1.5
21.0 ± 1.0
E. coli
P. aeruginosa
ATCC 25922 ATCC 27853
Apigenin
10.1 ± 0.3
10.3 ± 0.1
10.9 ± 0.7
12.5 ± 0.1
12.8 ± 0.4
10.4 ± 0.2
14.8 ± 0.5
11.0 ± 0.3
15.3 ± 0.1
13.0 ± 0.1
15.6 ± 0.3
28.9 ± 0.1
21.7 ± 1.1
10.3 ± 0.2
8.6 ± 0.1
4a
4b
10.6 ± 0.2
10.5 ± 0.4
10.8 ± 0.1
12.8 ± 0.5
12.4 ± 0.3
10.0 ± 0.7
17.0 ± 1.0
10.2 ± 0.8
14.3 ± 1.0
33.7 ± 0.5
24.3 ± 1.1
4c
4d
4e
4f
4g
4h
4i
4j
Ampicillin
Tetracycline
a Mean value of measured diameters of zones of inhibition; b S.D. denotes the standard deviation.
The MIC values of all the compounds are presented in Table 3. All the prepared apigenin analogs
showed relatively higher antibacterial activities than apigenin. Gram-positive strains S. aureus and
B. subtilis showed relatively high sensitivities toward the synthesized compounds, with MIC values
from 1.95 μg/mL to 15.63 μg/mL. Furthermore, 4h and 4j showed prominent activities with MIC values

Molecules 2012, 17
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of 3.91 and 1.95 μg/mL against S. aureus and B. subtilis, comparable with the positive control
tetracycline, although lower than ampicillin. Compounds 4a, 4b, 4f, and 4i showed moderate activities
against S. aureus and B. subtilis with MIC values of 7.81 μg/mL. However, 4c–e, and 4g exhibited weak
antibacterial activities against S. aureus and B. subtilis with MIC values of 15.63 μg/mL to 31.25 μg/mL.
Table 3. In vitro antibacterial activity (MIC, μg/mL) and DPPH radical scavenging activity
(IC50, μg/mL) of 4a to 4j.
MIC (μg/mL) a
Compounds IC50 (μg/mL) a
S. aureus
B. subtilis
E. coli
P. aeruginosa
ATCC 25923 ATCC 6633 ATCC 25922 ATCC 27853
Apigenin
1094.7 ± 2.2
788.0 ± 1.6
782.4 ± 2.3
670.1 ± 1.5
539.5 ± 1.4
542.3 ± 1.3
434.8 ± 1.4
431.1 ± 1.1
887.7 ± 2.5
334.8 ± 1.2
740.1 ± 1.8
--
31.25
7.81
31.25
3.91
7.81
15.63
7.81
15.63
7.81
7.81
3.91
7.81
1.95
0.12
3.91
62.5
62.5
62.5
125
4a
4b
7.81
62.5
62.5
62.5
62.5
31.25
31.25
62.5
15.63
62.5
15.63
0.49
3.91
4c
15.63
15.63
31.25
7.81
31.25
31.25
62.5
4d
4e
4f
15.63
62.5
4g
15.63
3.91
4h
15.63
31.25
15.63
0.98
4i
7.81
4j
1.95
Ampicillin
Tetracycline
Vitamin C
0.06
--
1.95
3.91
44.2 ± 1.1
a Average of three parallel experiments.
To analyze further the structure-activity relationships, the prepared apigenin analogs were divided
into two categories. One category includes the cyclic substituent-containing apigenin analogs, 4h to 4j,
and the other includes the aliphatic chain substituent-containing apigenin analogs, 4a to 4g. From Table 3,
we find that the apigenin analogs showed relatively higher antibacterial activities than apigenin.
Furthermore, in the cyclic substituent-containing apigenin analogs, the order of the MIC values was
4j < 4h < 4i, indicating that the analogs with 6-membered rings had better antibacterial activities than
those with 5-membered rings. Moreover, in the 6-membered ring compounds, compounds containing
two heteroatoms (4j) showed stronger antibacterial activities than compounds containing one
heteroatom (4i). For the aliphatic chain substituent-containing apigenin analogs, straight-chain primary
amine-substituted apigenin analogs displayed stronger activities than the branched-chain substituted
apigenin analogs.
2.3. Antioxidant Activity
The DPPH radical is a stable free radical commonly used as a substrate to evaluate in vitro
antioxidant activities of extracts from fruits, vegetables, and medicinal plants [56]. An antioxidant can
scavenge the radical by hydrogen donation, which results in a decrease in DPPH absorbance at 517 nm [57].
Generally, flavonoids display different DPPH radical scavenging activities with their different

Molecules 2012, 17
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structures. The radical scavenging activities of flavonoids are highly controlled by the number and
configuration of phenolic hydroxyl groups in the molecules and also influenced by the configuration of
other substituents [58]. Table 3 shows the DPPH radical scavenging activities of the prepared apigenin
analogs compared with those of the parent apigenin and vitamin C. Most of the prepared compounds
exhibited potential scavenging activities compared with the parent apigenin and moderate activities
compared with vitamin C. Compound 4i was found to have significant antioxidant activity with the
smallest IC50 value and compound 4h possessed the least activity among these prepared compounds
with the largest IC50 value. IC50 value is negatively related to antioxidant activity such that the lower
the IC50 value is, the higher the antioxidant activity will be [59]. Therefore, the order of DPPH radical
scavenging abilities of the analogs was 4i > 4g > 4f > 4d > 4e > 4c > 4j > 4b > 4a > 4h > apigenin.
Compared with the parent apigenin, 4f, 4g, and 4i exhibited significant antioxidant activities, with IC50
values of 434.8, 431.1, and 334.8 μg/mL, respectively. The concentration-dependent changes in the
antioxidant activities of these compounds were also analyzed. The antioxidant activity was enhanced
with the concentration of every sample was increased (Figure 1). When the concentration of 4f, 4g, and
4i reached 1,250 μg/mL, their scavenging effects were close to that of vitamin C.
Figure 1. DPPH radical scavenging activities of 4f, 4g, 4i, apigenin, and vitamin C.
Apigenin analogs with branched-chain aliphatic substituents showed stronger antioxidant activities
than those with straight-chain primary amine substituents or heteroatom-containing substituents,
except for 4i. Moreover, the alkyl number of the substituent groups determined the antioxidant activity
of the analogs. The more alkyl numbers existed in the substituted apigenin analogs, the higher were the
displayed activities, as shown by 4f and 4g. All these results above suggested that substituted apigenin
analogs exhibited antioxidant activities due to the introduction of different electron donations on the
C-8 of apigenin [58], and the antioxidant activities of flavonoids could be modulated by the number
and configuration of amine groups in the molecules.

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2.4. Antiproliferative Activity
The antiproliferative activities of the prepared apigenin analogs 4a–j were evaluated by performing
in vitro assays of the inhibition ratios of these compounds to the proliferation of human cancer cells
HeLa, HepG2, A549, and MCF-7. Figure 2 shows that the prepared apigenin analogs possessed
different antiproliferative activities depending on the changes in their chemical structures, and high
doses of these compounds showed more potent antiproliferative activities. Compound 4i exhibited the
most significant antiproliferative activity against the four cancer cells. At 1000 μg/mL, the inhibition
rates of 4i for HeLa, HepG2, A549, and MCF-7 cells were 89.97%, 92.60%, 93.33%, and 93.04%,
respectively. All the prepared apigenin analogs possessed stronger inhibition effects on HeLa and
HepG2 cells than on A549 and MCF-7 cells.
Figure 2. Inhibition ratios of apigenin and its analogs to the proliferation of HeLa (A),
HepG2 (B), A549 (C), and MCF-7 (D) cell lines.
Table 4 summarizes the IC50 values of apigenin analogs against HeLa, HepG2, A549, and MCF-7
cells. Compound 4i exhibited the strongest antiproliferative activity, and its IC50 values reached 40, 40,
223, and 166 μg/mL for HeLa, HepG2, A549, and MCF-7 cells, respectively. Moreover, compounds
4f–h, and 4j showed better antiproliferative activities (with lower IC50 values) against HeLa and HepG2

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cells than apigenin, with inhibition ratios reaching more than 50% at 250 μg/mL. However, no obvious
antiproliferative activity was observed for 4a, 4b, and 4d, indicating the ineffectiveness of these
compounds in suppressing the HeLa and HepG2 cell proliferation. Compounds 4b, 4e, and 4i displayed
stronger activities toward A549 cells, whereas 4e, 4f, and 4i had better antiproliferative activities
against MCF-7 cells, compared with apigenin. Moreover, the antiproliferative activities of the apigenin
analogs against HeLa and HepG2 were enhanced with a short and more branched alkyl chain at the
C-8 position of apigenin. The highest potency was obtained when a piperazin-1-yl-ethoxy group was
present at the C-8 position of the apigenin. Combined with the structural features of the prepared
analogs, the presence of nitrogen at the C-8 position of apigenin was critical to the antiproliferative
activities of the apigenin analogs, which was supported by previous reports [60–62].
Table 4. Antiproliferative activities (IC50, μg/mL) of apigenin analogs against HeLa,
HepG2, A549, and MCF-7 cell lines.
IC50 (μg/mL)
Compounds
HeLa
HepG2
460 ± 2.2
470 ± 2,7
410 ± 2.2
270 ± 1.9
620 ± 3.1
270 ± 2.0
150 ± 1.7
190 ± 1.5
180 ± 1.4
40 ± 1.5
A549
MCF-7
>2000 ± 3.6
>2000 ± 4.1
420 ± 2.3
280 ± 1.7
310 ± 2.1
210 ± 1.9
250 ± 2.0
610 ± 3.2
>2000 ± 4.0
166 ± 1.8
1010 ± 2.9
1
450 ± 2.0
450 ± 2.5
430 ± 1.9
270 ± 1.4
570 ± 2.0
230 ± 1.6
160 ± 1.5
210 ± 2.1
170 ± 1.7
40 ± 1.8
1740 ± 3.4
>2000 ± 4.3
1410 ± 3.0
>2000 ± 3.8
>2000 ± 4.0
380 ± 2.8
4a
4b
4c
4d
4e
4f
>2000 ± 3.7
>2000 ± 4.2
>2000 ± 3.5
223 ± 2.1
4g
4h
4i
4j
180 ± 2.0
220 ± 2.0
>2000 ± 4.4
3. Experimental
3.1. General
Apigenin (>98%) was purchased from Xi’an Spring-chem Bio-tech Co., Ltd. (Xi’an, China).
Tetracycline and DPPH were from Sigma-Aldrich (St. Louis, MO, USA). Ampicillin and MTT were
obtained from Amersco Inc. (Solon, OH, USA). Yeast extract and tryptone were from Oxoid Ltd.
(Basingstoke, Hampshire, UK). Dulbecco’s modified eagle medium, RPMI-1640 medium, and fetal
bovine serum (FBS) were from Gibco Invitrogen Corporation (Carlsbad, CA, USA). HeLa and HepG2
cells were provided by the Cell Center of the Fourth Military Medical University (Xi’an, China). A549
and MCF-7 cells were from the Chinese Academy of Sciences (Shanghai, China). Vitamin C and
primary or secondary amines were from Aladdin (Shanghai, China). N,N-Dimethylformamide (DMF)
was purified by vacuum distillation over CaH2 before utilization. All solvents and other chemicals were
supplied by local commercial suppliers and of analytical reagent grade, unless otherwise stated.
Deionized water (Milli-Q, Millipore, Bedford, MA, USA) was used to prepare aqueous solutions.
Column chromatography was performed with silica gel 60 (200 to 300 mesh). Thin-layer
chromatography (TLC) and preparative TLC (PTLC) were used with silica gel 60 GF254 (Qingdao

Molecules 2012, 17
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Haiyang Chem. Co., Ltd., Shandong, China). Product spots were detected under UV light using ZF-6
type III ultraviolet analyzer (Shanghai Jiapeng Technology Co., Ltd., Shanghai, China). Melting points
were determined using a digital melting point apparatus (Zhengzhou Mingze Technology Co., Ltd.,
Zhengzhou, China) and were uncorrected. ESI mass spectra were obtained using a Thermo Scientific
LCQ FLEET mass spectrometer equipped with an electrospray ion source and controlled by Xcalibur
software (Thermo Fisher Scientific, Waltham, MA, USA). Proton nuclear magnetic resonance spectra
(1H-NMR) and carbon-13 nuclear magnetic resonance spectra (13C-NMR) were obtained using a Bruker
Avance DMX 500 MHz/125 MHz spectrometer (Bruker, Billerica, MA, USA), with chemical shifts
reported in parts per million [in dimethyl sulfoxide (DMSO-d6), tetramethylsilane as an internal
standard, J values were given in Hertz]. MIC was detected using a BIO-RAD 680 Microplate reader
(Beijing Yuanye Bio. Co., Ltd., Beijing, China). The absorbance values in DPPH assays were recorded
using a UV-1600PC spectrophotometer (Shanghai Mapada Instruments Co., Ltd., Shanghai, China).
3.2. General Procedure for the Synthesis of Compounds 4a to 4j
The apigenin analogs 4a–j were synthesized via Mannich reactions [63]. A magnetic stirring bar
and apigenin (1, 100 mg, 0.37 mmol) dissolved in anhydrous DMF (1 mL) were first placed into a
two-necked flask. Then, methanol (15 mL) was added and a yellow solution was immediately
obtained. The reaction system was then immersed in an oil bath while being stirred. Then,
formaldehyde solution (0.028 mL, 37%) and ethylamine solution (0.031 mL, 70%) were added
dropwise to the reaction mixture. Stirring was required to keep the reaction mixture homogenized
during these operations. The reaction process was checked using TLC analysis. After 4 h reaction, the
mixture was filtered, and the supernatant was collected. The solvent was removed under reduced
pressure. The residue was then purified using PTLC with ethyl acetate-methanol (1:1, v/v) as
developing agent to obtain 4a. Compounds 4b to 4j were prepared using similar methods, and their
1
13
structures were confirmed via melting point determination, H-NMR, C-NMR, and ESI-MS. The
data are listed as follows.
4',5,7-Trihydroxy-8-(ethylamino)-2-phenyl-4H-chromen-4-one (4a): yellow solid, yield 40.5%, m.p.
>300 °C. 1H-NMR δ: 13.00 (s, 1H, 5-OH), 7.84 (d, 2H, J = 10.0 Hz), 6.92 (d, 2H, J = 10.0 Hz), 6.59
(s, 1H), 6.00 (s, 0.6H), 5.77 (s, 0.4H), 4.14 (s, 0.9H, CH2), 3.96 (s, 1.1H, CH2), 2.81 (m, 2H,
13
-NH-CH2-CH3), 1.29 (t, 3H, -NH-CH2-CH3); C-NMR δ: 182.0, 169.9, 163.5, 162.0, 159.6, 159.2,
128.4, 128.4, 121.7, 116.6, 116.6, 103.1, 102.7, 102.3, 95.7, 41.7, 35.7, 17.6; ESI-MS (m/z): 325.9
[M−H]−.
4',5,7-Trihydroxy-8-(propylamino)-2-phenyl-4H-chromen-4-one (4b): yellow solid, yield 38.7%, m.p.
>300 °C. 1H-NMR δ: 12.99 (s, 1H, 5-OH), 7.85 (d, 2H, J = 10.0 Hz), 6.90 (d, 2H, J = 10.0 Hz), 6.60
(s, 0.3H), 6.56 (s. 0.7H), 6.02 (s, 0.6H), 5.79 (s, 0.4H), 4.14 (s, 0.7H, CH2), 3.95 (s, 1.3H, CH2), 2.71
(t, J = 19.1 Hz, 2H, -NH-CH2-CH2-CH3), 1.54–1.58 (m, 2H, -NH-CH2-CH2-CH3), 0.91 (t, J = 6.7 Hz,
3H, -NH-CH2-CH2-CH3); 13C-NMR δ: 181.8, 169.9, 163.4, 161.5, 159.0, 157.0, 128.8, 128.8, 122.4,
116.7, 116.7, 103.7, 102.8, 102.1, 95.9, 42.3, 36.1, 27.5, 15.9; ESI-MS (m/z): 339.9 [M−H]−.

Molecules 2012, 17
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4',5,7-Trihydroxy-8-(isopropylamino)-2-phenyl-4H-chromen-4-one (4c): yellow solid, yield 61.7%,
m.p. >300 °C. 1H-NMR δ: 12.99 (s, 1H, 5-OH), 7.86 (d, 2H, J = 10.0 Hz), 6.91 (d, 2H, J = 10.0 Hz),
6.59 (s, 0.6H), 6.56 (s, 0.4H), 6.01 (s, 0.4H), 5.79 (s, 0.6H), 4.14 (s, 1.1H, CH2), 3.96 (s, 0.9H, CH2),
13
3.07–3.14 (m, 1H, -NH-CH(CH3)2), 1.19 (d, 6H, -NH-CH(CH3)2); C-NMR δ: 181.9, 168.2, 163.4,
158.9, 158.0, 155.4, 128.4, 128.4, 121.1, 116.3, 116.3, 102.7, 102.4, 100.0, 95.8, 48.4, 40.4, 20.3;
ESI-MS (m/z): 340.0 [M−H]−.
4',5,7-Trihydroxy-8-(tert-butylamino)-2-phenyl-4H-chromen-4-one (4d): yellow solid, yield 52.3%,
m.p. >300 °C. 1H-NMR δ: 12.98 (s, 1H, 5-OH), 7.84 (d, 2H, J = 10.0 Hz), 6.92 (d, 2H, J = 10.0 Hz),
6.57 (s, 0.4H), 6.54 (s, 0.6H), 6.00 (s, 0.6H), 5.77 (s, 0.4H), 4.14 (s, 1.1H, CH2), 3.96 (s, 0.9H, CH2),
13
1.27 (s, 9H, (CH3)3C-); C-NMR δ: 180.8, 168.7, 162.9, 161.5, 158.0, 155.4, 128.6, 128.6, 125.6,
116.7, 116.7, 102.6, 102.4, 101.5, 95.9, 54.0, 49.1, 26.6; ESI-MS (m/z): 353.9 [M−H]−.
4',5,7-Trihydroxy-8-(dimethylamino)-2-phenyl-4H-chromen-4-one (4e): yellow solid, yield 39.6%,
m.p. >300 °C. 1H-NMR δ: 13.03 (s, 1H, 5-OH), 7.94 (d, 2H, J = 10.0 Hz), 6.96 (d, 2H, J = 10.0 Hz),
6.77 (s, 1H), 6.11 (s, 1H), 3.97 (s, 2H, CH2), 2.43 (s, 6H, -N(CH3)2); 13C-NMR δ: 181.9, 168.2, 163.4,
161.5, 161.1, 155.4, 128.8, 128.8, 121.9, 116.5, 116.5, 103.0, 102.7, 100.0, 99.9, 53.4, 43.9; ESI-MS
(m/z): 325.8 [M−H]−.
4',5,7-Trihydroxy-8-(diethylamino)-2-phenyl-4H-chromen-4-one (4f): yellow solid, yield 60.9%, m.p.
>300 °C. 1H-NMR δ: 13.47 (s, 1H, 5-OH), 7.89 (d, 2H, J = 8.0 Hz), 6.92 (d, 2H, J = 8.0 Hz), 6.71 (s,
1H), 6.29 (s, 1H), 3.92 (s, 2H, CH2), 2.75 (q, 4H, J = 10.0Hz, -N(CH2CH3)2), 1.11 (t, 6H, J = 10.0 Hz,
-N(CH2CH3)2; 13C-NMR δ: 181.8, 169.3, 163.6, 161.6, 159.0, 157.2, 128.7, 128.7, 121.8, 116.4, 116.4,
103.6, 102.9, 102.2, 94.9, 48.5, 46.3, 10.7; ESI-MS (m/z): 354.0 [M−H]−.
4',5,7-Trihydroxy-8-(diisopropylamino)-2-phenyl-4H-chromen-4-one (4g): yellow solid, yield 51.2%,
1
m.p. >300 °C. H-NMR δ: 13.45 (s, 1H, 5-OH), 10.37 (s, 1H, 7-OH), 7.91 (d, 2H, J = 10.0 Hz), 6.93
(d, 2H, J = 10.0 Hz), 6.72 (s, 1H), 6.25 (s, 1H), 3.96 (s, 2H, CH2), 3.18–3.25 (m, 2H,-N(CH(CH3)2)2,
1.15(d, 12H, J = 5.0 Hz, -N(CH(CH3)2)2; 13C-NMR δ: 181.8, 169.9, 163.6, 161.5, 158.3, 157.0, 128.8,
128.8, 121.9, 116.4, 116.4, 104.0, 103.0, 102.1, 94.9, 49.5, 41.9, 19.2; ESI-MS (m/z): 381.8 [M−H]−.
4',5,7-Trihydroxy-8-(cyclohexylamino)-2-phenyl-4H-chromen-4-one (4h): yellow solid, yield 41.8%,
m.p. >300 °C. 1H-NMR δ: 13.01 (1H, s, 5-OH), 7.85 (d, 2H, J = 10.0 Hz), 6.89 (d, 2H, J = 10.0 Hz),
6.58 (s, 0.6H), 6.55 (s, 0.4H), 6.04 (s, 0.4H), 5.80 (s, 0.6H), 4.15 (s, 1.2H, CH2), 3.97 (s, 0.8H, CH2),
13
2.74 (s, 1H, cyclohexylamine-CH), 1.24–1.97 (m, 10H, cyclohexylamine-CH2); C-NMR δ: 181.0,
169.0, 162.8, 162.6, 161.1, 158.7, 128.5, 128.5, 121.2, 116.7, 116.7, 102.4, 101.0, 100.7, 95.5, 55.5,
55.2, 31.2, 25.7, 24.5; ESI-MS (m/z): 379.9 [M−H]−.
4',5,7-Trihydroxy-8-(pyrrolidin-1-ylmethyl)-2-phenyl-4H-chromen-4-one (4i): yellow solid, yield
42.1%, m.p. >300 °C. 1H-NMR δ: 13.53 (1H, s, 5-OH), 7.89 (d, 2H, J = 5.0 Hz), 6.91 (d, 2H, J = 5.0 Hz),
6.69 (s, 1H), 6.28 (s, 1H), 3.95(s, 2H, CH2), 2.80 (s, 4H, pyrrolidine-N(CH2)2), 1.83 (s, 4H,
pyrrolidine-(CH2)2); 13C-NMR δ: 181.5, 170.1, 163.4, 161.7, 159.0, 157.4, 128.7, 128.7, 121.7, 116.5,
116.5, 104.6, 102.7, 101.5, 95.0, 53.2, 49.9, 23.6; ESI-MS (m/z): 351.9 [M−H]−.

Molecules 2012, 17
14758
4',5,7-Trihydroxy-8-(morpholinomethyl)-2-phenyl-4H-chromen-4-one (4j): yellow solid, yield 43.4%,
m.p. >300 °C. 1H-NMR δ: 12.98 (s, 1H, 5-OH), 8.03 (d, 2H, J = 5.0 Hz), 6.91 (d, 2H, J = 5.0 Hz), 6.83
(s, 1H), 6.58 (s, 1H), 4.11 (s, 2H, CH2), 3.81 (s, 2H, morpholine-O-CH2), 3.75 (s, 2H,
morpholine-O-CH2), 2.52 (s, 4H, morpholine-N(CH2)2); 13C-NMR δ: 182.5, 165.0, 164.0, 161.9,
160.7, 154.9, 128.9, 128.9, 121.7, 116.6, 116.6, 104.3, 103.3, 102.9, 101.5, 66.6, 53.3, 50.9; ESI-MS
(m/z): 368.0 [M−H]−.
3.3. Antibacterial Assay
The bacterial strains were kept in liquid nitrogen (−196 °C) in a Luria-Broth (LB) medium (5 g/L
yeast extract, 10 g/L bactopeptone, and 10 g/L sodium chloride) containing 15% glycerol. Prior to the
experiment, the bacterial strains were grown on LB agar plates at 37 °C. A single colony of bacteria
from an overnight culture was inoculated into 150 mL of fresh LB medium. Each bacterial colony was
then continuously incubated for 12 h at 37 °C in a rotary shaker set to 200 rpm. The cell density was
determined via the normal plate count method [64]. The inocula were then diluted with 0.9% sterile
normal saline to obtain a cell density of approximately 1.5 × 108 CFU/mL to match the 0.5 McFarland
standard [65].
Antibacterial activity was done by the disk diffusion method according to the National Committee
for Clinical Laboratory Standards (NCCLS) [66]. Ten μL of bacterial suspension was mixed with
sterile LB agar medium (10 mL) at 40 °C and poured onto an agar plate. Sterile paper discs (6.0 mm in
diameter) impregnated with compound dissolved in dimethylsulfoxide (DMSO) at concentrations of
1000 μg/mL were prepared. Then, the impregnated paper discs were placed on the surface of the media
inoculated with the microorganism. Ampicillin and tetracycline were used as positive controls. DMSO
poured disk was used as negative control. The diameter of zone of growth inhibition around the disc was
measured after 18 h of incubation at 37 °C. An average of three independent determinations was recorded.
The MICs for the synthesized compounds were also determined using the National Committee for
Clinical Laboratory Standards in a LB medium in 96-well tissue culture plates [67]. All the tested
compounds were dissolved in DMSO and diluted in the growth media, resulting in the final
concentration ranging from 0.06 μg/mL to 500 μg/mL. Afterward, 100 μL of this solution was pipetted
into the first well of each line in the 96-well tissue culture plate, which contained 100 μL of the LB
medium. The solution was then serially diluted to obtain two-fold serial dilutions of the test
compounds and positive controls in the subsequent wells, which contained 100 μL LB medium. The
0.5 McFarland-matched bacterial suspension (1 mL) was then diluted with 100 mL of the medium. The
diluted bacterial suspension (100 μL) was added to each well and then kept for incubation. One well
containing bacterial cells and DMSO without any test compounds (growth control), and one well
containing only growth medium (sterility control), were used as controls. Ampicillin and tetracycline
were used as positive controls. The maximum concentration of the test compounds was 125 μg/mL.
The sealed microtiter plates were incubated at 37 °C in a moist, dark chamber. MIC values were
recorded spectrophotometrically at 630 nm after 24 h incubation. All compounds were tested in
triplicate. The experiments were repeated at least thrice.

Molecules 2012, 17
14759
3.4. Antioxidant Assay
DPPH free radical scavenging activity was measured to evaluate the antioxidant activity via the
previously reported method [68]. The chemical compounds were dissolved in methanol to obtain a
final concentration ranging from 78 μg/mL to 1,250 μg/mL to determine IC50 (a high concentration
results in 50% inhibition of DPPH color). A methanol solution of DPPH (0.2 mM) was incubated with
a methanol solution of each of the test samples (78 μg/mL to 1250 μg/mL) for 30 min at room
temperature (25 °C) in the dark. The DPPH radical scavenging activity was determined by measuring
the absorbance at 517 nm using a spectrophotometer. The DPPH radical scavenging activity of
Vitamin C was also assayed for control, whereas that of methanol was used as a blank. The percentage
of DPPH radical scavenging effect was calculated as follows:
[(Acontrol − Atest)/Acontrol] × 100%
where Acontrol is the absorbance of the control (DPPH solution without any test sample) and Atest is the
absorbance of the test sample (DPPH solution plus test sample). All tests were performed in triplicate.
3.5. Antiproliferative Assay
The inhibition effects of 4a–j on the HeLa, HepG2, A549, and MCF-7 cell proliferation were
evaluated in vitro via MTT staining according to the procedures reported previously [69,70], with
slight modification. The four selected cancer cell lines, HeLa, HepG2, A549, and MCF-7 were routinely
cultured using suitable medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL
streptomycin at 37 °C and 5% CO2 with 95% humidity. The cells were normally passaged at a ratio of
1:3 every 3 days to maintain them in the exponential growth phase. Before use, the cells were harvested
through trypsinization with 0.25% trypsin in Ca2+- and Mg2+-free Hanks’ balanced salt solution at 37 °C.
Trypsinization was stopped through the addition of fresh supplemented medium. The cell suspension
was centrifuged at 1000 rpm for 5 min. The cells were then resuspended in supplemented medium
(1.0 × 106 cells/well in 6-well plates) for further use. Then, the cells were seeded into 96-well microtiter
plates (4.0 × 103 cells per well) with fresh medium (150 μL). After 24 h incubation, the tested compounds
(150 μL, final concentrations of 62.5, 125, 250, 500, 1000, or 2000 μg/mL in the culture medium) were
added to each well and continuously cultured for another 24 h. Afterward, MTT (20 μL, 5 mg/mL) was
added to each well, which was then cultured for another 4 h under similar conditions. Finally, DMSO
(150 μL) was added to terminate the reaction. The survival rate of the cancer cells was evaluated by
measuring the optical density (A) on a microplate reader (model 680, BIO-RAD, Hercules, CA, USA) at
490 nm. All in vitro results were expressed as the cancer cell proliferation inhibition ratio according to
the formula below:
[(Acontrol − Atest)/Acontrol] × 100%
where Acontrol and Atest are the optical densities of the control and the test groups, respectively. All
assays were done in triplicate.

Molecules 2012, 17
14760
4. Conclusions
In summary, we have synthesized a series of 8-aminomethylated apigenin analogs. Bioactivity assays
showed that all the synthesized compounds exhibited greater potential antiproliferative, antibacterial,
and antioxidant activities compared with the parent apigenin. Among these apigenin analogues,
compound 4j was found to be the most active, with MIC values of 1.95, 1.95, 15.63, and 15.63 μg/mL
against S. aureus, B. subtilis, E. coli, and P. aeruginosa, respectively. Compound 4i exhibited the
strongest antiproliferative activities against HeLa, HepG2, A549, and MCF-7 cells. Compound 4i may
also act as an antioxidant agent because of its potent DPPH scavenging activity.
Previous reports [49–51,60,61] have shown that the bioactivity increased when aminomethyl groups
were incorporated at the C-8 position. Our results are consistent with this. The above screening results
also showed that the increased biological activities were due to the introduction of aminomethyl groups
into the C-8 position of the parent apigenin. However, further pharmaceutical studies on the
mechanisms of the antiproliferative, antibacterial, and antioxidant activities of the apigenin analogs are
still necessary. In addition, the synthesis yields of these apigenin analogs should be improved. All these
studies are currently under way in our laboratory.
Acknowledgments
This work was financially supported by the State Forestry Administration of the People’s Republic of
China (Grant No. 200904004), the National Natural Science Foundation of China (Grant Nos.
20975082, 21175107, and 31100726), the Ministry of Education of the People’s Republic of China
(Grant No. NCET-08-0464), and the Northwest A&F University.
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