Synthesis and anticancer activities of 3-arylflavone-8-acetic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.11.030

This paper describes the synthesis and the antiproliferative activities of compounds 9a-r, 3-aryl analogs of flavone-8-acetic acid that bear diverse substituents on the benzene rings at the 2- and 3-positions of the flavone nucleus. Their direct and indir

Full text of DOI:10.1016/j.ejmech.2014.11.030

Accepted Manuscript
Synthesis and anticancer activities of 3-arylflavone-8-acetic acid derivatives
Guang-Hua Yan , Xiao-Fang Li , Bing-Chen Ge , Xiu-Dong Shi , Yu-Fang Chen , Xue-
Mei Yang , Jiang-Ping Xu , Shu-Wen Liu , Pei-Liang Zhao , Zhong-Zhen Zhou , Chun-
Qiong Zhou , Wen-Hua Chen
PII:
S0223-5234(14)01062-9
10.1016/j.ejmech.2014.11.030
EJMECH 7523
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 August 2014
Revised Date: 25 October 2014
Accepted Date: 15 November 2014
Please cite this article as: G.-H. Yan, X.-F. Li, B.-C. Ge, X.-D. Shi, Y.-F. Chen, X.-M. Yang, J.-P. Xu,
S.-W. Liu, P.-L. Zhao, Z.-Z. Zhou, C.-Q. Zhou, W.-H. Chen, Synthesis and anticancer activities of 3-
arylflavone-8-acetic acid derivatives, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.11.030.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Synthesis and anticancer activities of 3-arylflavone-8-acetic acid derivatives
#
, a
#, b
a
a
a
Guang-Hua Yan , Xiao-Fang Li , Bing-Chen Ge , Xiu-Dong Shi , Yu-Fang Chen ,
a
a
a
a
, a
Xue-Mei Yang , Jiang-Ping Xu , Shu-Wen Liu , Pei-Liang Zhao , Zhong-Zhen Zhou* ,
a
, a
Chun-Qiong Zhou and Wen-Hua Chen*
This paper describes the synthesis and antiproliferative activities of eighteen
-arylflavone-8-acetic acid derivatives 9a-r. Among them, compounds 9p-r bearing methoxy
3
groups showed higher indirect cytotoxicity than DMXAA and could induce TNF-α
production in HPBMCs. In addition, the position of a methoxy group on the
3
-arylflavone-8-acetic acid was found to have a profound impact on the indirect cytotoxicity.

ACCEPTED MANUSCRIPT
Synthesis and anticancer activities of 3-arylflavone-8-acetic acid derivatives
#
, a
#, b
a
a
a
Guang-Hua Yan , Xiao-Fang Li , Bing-Chen Ge , Xiu-Dong Shi , Yu-Fang Chen ,
a
a
a
a
, a
Xue-Mei Yang , Jiang-Ping Xu , Shu-Wen Liu , Pei-Liang Zhao , Zhong-Zhen Zhou* ,
a
, a
Chun-Qiong Zhou and Wen-Hua Chen*
a
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China. Fax:
86 20 61648533; E-mail addresses: zhouzz@smu.edu.cn (ZZZ), whchen@smu.edu.cn (WHC)
+
b
Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China
#
Both authors contributed equally to this work.
Abstract This paper describes the synthesis and the antiproliferative activities of compounds
a-r, 3-aryl analogues of flavone-8-acetic acid that bear diverse subsitiuents on the benzene
9
rings at the 2- and 3-positions of the flavone nucleus. Their direct and indirect cytotoxicities
were evaluated against HT-29 human colon adenocarcinoma cell lines, A549 lung
adenocarcinoma cell lines and Human Peripheral Blood Mononuclear Cells (HPBMCs). The
results indicate that most of the compounds bearing electron-withdrawing substituents (9b-m)
exhibited moderate direct cytotoxicities. And compounds 9e and 9i showed comparable
indirect cytotoxicities with 5, 6-dimethylxanthenone-4-acetic acid (DMXAA), and low direct
cytotoxicities toward HPBMCs. Interestingly, the compounds 9n-r bearing methoxy groups at
the 2- or 3-position of the flavone nucleus exhibited higher indirect cytotoxicities against
A549 cell lines than DMXAA, and lower cytotoxicities against HPBMCs. In addition,
compounds 9p-r were found to be able to induce tumor necrosis factor α (TNF-α) production
in HPBMCs.
Key words: 3-Arylflavone-8-acetic acid; Direct cytotoxicity; Indirect cytotoxicity; tumor
necrosis factor α;
1
. Introduction
Though there has been great advance in the multimodal management of a wide spectrum of
human cancers, they remain to be one of the major global killers. The discovery of new
anti-cancer drugs represents one of the biggest challenges in modern medicinal chemistry. In
1

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these aspects, nature has proved to be a rich source to provide a variety of molecules that can
specifically target cancers [1]. For example, flavonoids, a class of polyphenolic compounds
that are widely distributed in plants [2], have been recognized for their high potentials as
anti-tumor agents. It has been reported that they function at several stages in cancer
progression with distinct structure-activity relationships [3-5]. As a consequence, some
flavonoids have been screened in clinical trials for the treatment of cancers [6-8]. For example,
it has been reported that 5, 6-dimethylxanthenone-4-acetic acid (DMXAA, 1, Figure 1), an
analogue of flavone acetic acid (FAA, 2) is a vascular disrupting agent which specifically
targets immature and unstable vasculature of solid tumors, leading to thrombosis, hemorrhage
and necrosis [9-11]. In addition, it has been shown that DMXAA 1 is safe and well-tolerated
in humans [6, 7, 12].
(Please insert Figure 1 here)
On the other hand, substituents on the flavonoids bearing carboxylic groups have a
profound impact on the antitumor activity. For example, methyl 6,
-dimethylflavone-8-carboxylate 3 [13] and 3-nitroflavone-8-acetic acid 4 [14] exhibited
7
extensive direct cytotoxicity against leukemia cell lines. Isoflavone-8-acetic acid 5 was found
to have high activity against ovarian carcinoma cells [15]. Azidoxanthenone-4-acetic acid 6
showed 8-fold higher in vivo activity than DMXAA [16], but was more toxic to cultured
endothelial cells than DMXAA and 10-fold less active in stimulating tumor necrosis factor α
(TNF-α) production in cultured splenocytes than DMXAA. In addition, compound 7 bearing a
methoxy group at the 3-position of flavone-8-acetic acid skeleton displayed 7-fold higher
indirect cytotoxicity toword solid tumor cell lines than DMXAA [17].
In our recent study we have reported the synthesis and the antiprolificative activities of
7
-methoxy-3-arylflavone-8-acetic acids 8a-m (Figure 1) [18]. We have shown that compound
1
2
8
e (R = 4-MeO, R = H) exhibited comparable indirect cytotoxicity with DMXAA, but much
1
2
lower direct cytotoxicity. In contrast, compound 8i (R = 4-F, R = H) exhibited higher direct
but lower indirect activity than DMXAA. With the aim to systematically study the impact of
substituents on the antitumor activity, herein we describe the synthesis, direct and indirect
2

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antiproliferative activities of 3-arylflavone-8-acetic acid derivatives 9a-r (Scheme 1) that bear
diverse subsitiuents on the benzene rings at the 2- and 3-positions of flavone nucleus, whereas
no methoxy group at the 7-position. Their structure-activity relationships are also briefly
discussed.
2
. Results and discussion
2
.1 Chemistry
The synthetic route for compounds 9a-r is outlined in Scheme 1. 3-Methyl-2-hydroxy
deoxybenzoins 10a-g were prepared from the microwave-assisted alkali degradation of
-aryl-4-hydroxycoumarins in water [19], starting from methyl 2-hydroxy-3-methylbenzoate
3
and substituted phenylacetic acids. Then, esterification of compounds 10a-g with aromatic
acyl chlorides 11a-d and subsequent heating in freshly distilled anhydrous glycerol at 260 °C
gave the corresponding 8-methyl-3-arylflavones 13a-r, which were then brominated, cyanated
and hydrolyzed to give compounds 9a-r. Their structures were confirmed on the basis of MS,
1
13
NMR ( H and C) and elemental analysis data (see experimental section and supporting
information).
(Please insert Scheme 1 here)
2
.2 Pharmacology
2
.2.1 Direct cytotoxicity
The direct antiproliferative activities of compounds 9a-r were evaluated against HT-29
human colon adenocarcinoma cell lines, A549 human non-small-cell carcinoma cell lines and
Human Peripheral Blood Mononuclear Cells (HPBMCs). The obtained IC50 values that
represent the concentration of each compound resulting in 50% inhibition in cell growth,
together with that of DMXAA as a positive control are listed in Table 1. It can be seen that
most of the compounds bearing electron-withdrawing groups on the benzene rings exhibited
moderate to good direct cytotoxicities toward HT-29 and A549 cells with DMXAA. Among
them, compounds 9c, 9f and 9j exhibited higher direct cytotoxicity toward HT-29 cells than
3

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DMXAA, whereas compounds 9i, 9k and 9l showed higher direct cytotoxicity toward A549
cells than DMXAA. In contrast, compounds 9n-r bearing methoxy groups on the benzene
rings at the 2- or 3-position of flavone nucleus exhibited lower direct cytotoxicities toward
HT-29 and A549 cells than DMXAA. These results suggest that an electron-withdrawing
group on the benzene rings at the 2- or 3-position of flavone nucleus may be favorable. It is
worth noting that compounds 9e, 9i and 9n-r displayed lower direct cytotoxicities toward
HPBMCs than DMXAA.
(Please insert Table 1 here)
2
.2.2 Indirect cytotoxicity
It is reported that the antitumor effects of both DMXAA and FAA are due to their indirect
rather than direct cytotoxicities. In other words, they are deeply involved in the induction of
apoptosis in tumor vascular endothelial cells [20] and the production of a spectrum of
cytokines and chemokines, such as TNF-α [21], interferon-β [22], interferon-inducible protein
[
23] and inducible nitric oxide synthase (iNOS) [24]. Therefore, we evaluated the indirect
cytotoxicities of compounds 9a-r induced in the HPBMCs (in the top well) toward A549 cells
®
(
in the bottom wells) after co-cultured for 24 h in HTS Transwell -96Well Permeable Support
Systems [18]. The indirect cytotoxicities of compounds 9a-r at the concentration of 50 ꢀM are
shown in Figure 2. It clearly indicates that compounds 9a-r could induce indirect
cytotoxicities on the HPBMCs toward A549 cells. Among them, most of compounds bearing
electron-withdrawing groups exhibited lower indirect cytotoxicities than DMXAA, whereas
compounds 9e and 9i showed comparable indirect cytotoxicities with DMXAA. Intrestingly,
compounds 9n-r bearing methoxy groups showed higher indirect cytotoxicities than
DMXAA.
(Please insert Figure 2 here)
Therefore to gain further insight into the pharmacological activities of compounds 9e, 9i
and 9n-r, their IC50 value toward A549 cells were measured (Table 2). Interestingly, the
indirect cytotoxicities (IC = 35.7 ~ 92.6 ꢀM) of compounds 9e, 9i and 9n-r are substantially
50
higher than their corresponding direct cytotoxicities (IC > 239.2 ꢀM, Table 1). This result
5
0
4

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suggests that the antitumor activities of these compounds toward solid tumor may be a result
of the indirect effects. In addition, compounds 9e and 9i exhibited comparable indirect
cytotoxicities with DMXAA, whereas compounds 9n-r showed up to 2.4-fold higher indirect
cytotoxicities than DMXAA.
(Please insert Table 2 here)
Careful analysis on the structure-activity relationship of compounds 9a-r and our previous
-arylflavone-8-acetic acids 8a-m [18] reveals that the position of methoxy groups on the
-arylflavone-8-acetic acid has a profound impact on the indirect cytotoxicity. Firstly,
3
3
methoxy groups on the benzene rings at the 2- or 3-position of the flavone nucleus are
favorable for the indirect cytotoxic activity. For example, compounds 9n-r showed higher
indirect activities than compounds 9a-m. Secondly, the finding that a 3-arylflavone-8-acetic
acid always exhibits higher indirect cytotoxicity than its corresponding analogue with a
7
-methoxy group (Figure 3), suggests that a 7-methoxy group is less favorable for the indirect
cytotoxicity. This may be because the intramolecular hydrogen bond between the 7-methoxy
group and the 8-acetic acid group obstructs the formation of pyrylium-type salts (Figure 4),
which was proposed by Kovacic et al [25].
(Please insert Figures 3 and 4 here)
2
.2.3 The effects of compounds 9p-r on TNF-α production
It is reported that DMXAA induces different cytokines (such as TNF-α, interleukin-6,
macrophage inflammatory protein-1a and interferon-c), chemokines (interferon-inducible
protein 10), and vasoactive factors (nitric oxide). They interact with tumor endothelial cells
resulting in haemorrhagic tumor necrosis [26-28]. As TNF-α plays a critical role in the
antitumor activity of DMXAA [29, 30], we investigated the effects of compounds 9p-r on the
TNF-α production by the HPBMCs (Figure 5). Their responses were compared with that
obtained with lipopolysaccharide (LPS), a known inducer of TNF-α synthesis [31]. As shown
in Figure 5, no cytokine production was stimulated by DMXAA alone at any tested
concentration, which is in agreement with the results by Gobbi [17] and Philpott [32]. In
5

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contrast, the level of TNF-α was significantly increased by compounds 9p-r.
(Please insert Figure 5 here)
3
. Conclusion
In summary, a series of novel flavone-8-acetic acid derivatives 9a-r bearing different
substituents on the phenyl rings at the 2- or 3-position of the flavone nuclei, have been
synthesized and fully characterized. Except compounds 9b and 9e, the derivatives bearing
electron-withdrawing groups exhibited comparable direct cytotoxicities with DMXAA. More
interestingly, compounds 9n-r bearing methoxy group showed higher indirect activities
against A549 cells than DMXAA, and lower direct cytotoxicities. In addition, compounds
9
p-r could induce TNF-α production in the HPBMCs. Analysis on the structure-activity
relationship of compounds 8a-m and 9a-r suggests a methoxy group on the benzene rings at
the 2- or 3-position of the flavone nucleus is favorable for indirect cytotoxicities, whereas less
favorable when it is translocated to the 7-position.
4
. Experimental Section
4
.1 Chemistry
Melting points were determined in open glass capillaries using an X-5 apparatus and are
uncorrected. ESI spectra were measured on a Waters UPLC/Quattro Premier XE mass
1
13
spectrometer. H and C NMR spectra were recorded in CDCl or d -DMSO using a Varian
3
6
Mercury 400 spectrometer and TMS as an internal reference. Element analyses were carried
out on a Vario ELIII CHNSO elemental analyzer.
DMXAA was purchased from Sigma (St. Louis, MO, USA). All the other chemicals were of
analytical grade and used without further purification. Compounds (12-15)a-r were
synthesized using the procedures described in the Supporting Information.
Synthesis of 3-arylflavone-8-acetic acid derivatives 9a-r
General procedures: 3-arylflavone-8-acetonitriles 15a-r (1 mmol) were dissolved in a mixture
of acetic acid (9 mL), H O (9 mL) and concentrated H SO (9 mL). After the mixture was
2
2
4
6

ACCEPTED MANUSCRIPT
refluxed for 2 h, water (375 mL) was added. The formed precipitate was collected by filtration
and re-crystallized from EtOH to give compounds 9a-r.
2
2
7
8
1
1
-(4-Oxo-2,3-diphenyl-4H-chromen-8-yl)acetic acid (9a): Color: White. Yield: 86%. Mp:
1
22.5–223.0 °C. H-NMR (d -DMSO, 400 MHz) δ 3.95 (s, 2H), 7.17–7.20 (m, 2H),
6
.30–7.34 (m, 5H), 7.38–7.41 (m, 3H), 7.48 (t, J = 7.6 Hz, 1H), 7.78 (dd, J = 7.3, 1.6 Hz, 1H),
1
3
.03 (dd, J = 8.0, 1.6 Hz, 1H), 12.60 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 122.4,
6
22.9, 124.4, 125.2, 125.6, 127.7, 128.2, 128. 4, 129.5, 130.5, 131.3, 132.9, 133.1, 135.8,
+
+
54.1, 160.8, 172.0, 176.4. ESI-MS (m/z): 379.3 [M+Na] , 357.4 [M+H] . Anal. Calcd. for
C H O : C, 77.52; H, 4.53. Found: C, 77.30; H, 4.24.
23
16
4
2
-(3-(2-Chlorophenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9b): Color: White.
1
Yield: 78%. Mp: 177.7–178.9 °C. H-NMR (d -DMSO, 400 MHz) δ 3.98 (s, 2H), 7.24–7.32
6
(m, 2H), 7.36–7.45 (m, 6H), 7.48–7.52 (m, 2H), 7.81 (dd, J = 7.2, 1.6 Hz, 1H), 8.03 (dd, J =
1
3
8
1
1
.0, 1.6 Hz, 1H), 12.61 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 120.6, 122.6, 124.3,
6
25.4, 125.7, 127.4, 128.5, 128.7, 129.3, 130.1, 130.9, 132.5, 132.7, 133.1, 134.4, 136.1,
-
54.2, 161.2, 172.0, 175.6. Negative ESI-MS (m/z): 389.1 [M-H] . Anal. Calcd. for
C H ClO : C, 70.68; H, 3.87. Found: C, 70.41; H, 3.65.
23
15
4
2
-(3-(2-Chlorophenyl)-2-(4-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9c): Color:
1
White. Yield: 94%. Mp: 233.3–235.0 °C. H-NMR (d -DMSO, 400 MHz) δ 3.98 (s, 2H),
6
7
.27–7.34 (m, 2H), 7.39–7.41 (m, 3H), 7.46 (d, J = 8.8 Hz, 2H), 7.51 (t, J = 9.0 Hz, 2H), 7.82
1
3
(
d, J = 7.2 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 120.7,
6
1
1
22.6, 124.3, 125.5, 125.8., 127.5, 128.7, 129.4, 130.2, 130.5, 131.4, 132.3, 133.1, 134.2,
-
35.8, 136.1, 154.1, 160.1, 172.0, 175.5. Negative ESI-MS (m/z): 423.7 [M-H] . Anal. Calcd.
for C H Cl O : C, 64.96; H, 3.32. Found: C, 64.75; H, 3.59.
2
3
14
2
4
2
-(2-(3-Chlorophenyl)-3-(4-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9d): Color:
1
White. Yield: 86%. Mp: 97.7–99.3 °C. H-NMR (d -DMSO, 400 MHz) δ 3.99 (s, 2H),
6
7
.29–7.36 (m, 3H), 7.37–7.46 (m, 3H), 7.50–7.55 (m, 3H), 7.83 (dd, J = 7.2, 1.2 Hz, 1H),
1
3
8
.04 (dd, J = 8.0, 1.6 Hz, 1H), 12.61 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 121.0,
6
7

ACCEPTED MANUSCRIPT
1
22.5, 124.3, 125.6, 125.8, 127.3, 127.5, 128.5, 129.4, 130.3, 130.5, 130.8, 132.1, 133.1,
+
1
33.3, 134.2, 134.4, 136.2, 154.2, 159.5, 172.0, 175.6. ESI-MS (m/z): 447.2 [M+Na] , 425.2
+
[
M+H] . Anal. Calcd. for C H Cl O : C, 64.96; H, 3.32. Found: C, 64.83; H, 3.58.
23
14
2
4
2
-(3-(4-Fluorophenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9e): Color: White.
1
Yield: 81%. Mp: 219.8–220.9 °C. H-NMR (d -DMSO, 400 MHz) δ 3.94 (s, 2H), 7.13–7.24
6
(m, 4H), 7.34–7.41 (m, 5H), 7.46 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 8.03 (d, J = 8.0
1
3
Hz, 1H), 12.64 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 115.1, 115.3, 121.4, 122.8,
6
1
24.3, 125.2, 125.7, 128.4, 129.3(d, J = 3.3 Hz), 129.5, 130.5, 132.8, 133.3, 133.4, 135.9,
-
1
54.1, 160.5 , 161.0, 162.9, 172.0, 176.4. Negative ESI-MS (m/z): 373.6 [M-H] . Anal. Calcd.
for C H FO : C, 73.79; H, 4.04. Found: C, 73.53; H, 4.19.
2
3
15
4
2
-(2-(4-Chlorophenyl)-3-(4-fluorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9f): Color:
1
White. Yield: 80%. Mp: 205.1–207.3 °C. H-NMR (d -DMSO, 400 MHz) δ 3.95 (s, 2H), 7.17
6
(t, J = 8.8 Hz, 2H), 7.22–7.26 (m, 2H), 7.39–7.50 (m, 5H), 7.79 (dd, J = 7.4, 1.0 Hz, 1H), 8.03
1
3
(
dd, J = 8.0, 1.6 Hz, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.3, 115.2, 115.4, 121.6, 122.8,
6
1
24.3, 125.3, 125.6, 128.6, 129.0 (d, J = 3.2 Hz), 131.4, 131.7, 133.3, 133.4, 135.4, 136.0,
‒
1
54.1, 159.8, 160.5, 163.0 172.0, 176.3. Negative ESI-MS (m/z): 407.4 [M-H] . Anal. Calcd.
for C H ClFO : C, 67.57; H, 3.45. Found: C, 67.62; H, 3.57.
2
4
14
4
2
-(2-(3-Chlorophenyl)-3-(4-fluorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9g): Color:
1
White. Yield: 91%. Mp: 156.2–158.3 °C. H-NMR (d -DMSO, 400 MHz) δ 3.95 (s, 2H), 7.18
6
(t, J = 8.8 Hz, 2H), 7.24–7.27 (m, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H),
7
.46–7.51 (m, , 3H), 7.80 (d, J = 7.2 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 12.60 (s, 1H).
1
3
C-NMR (d -DMSO, 100 MHz) δ 35.4, 115.2, 115.4, 116.3, 121.9, 122.8, 124.4, 125.4,
6
1
25.7, 128.3, 128.9(d, J = 3Hz) 129.3, 130.3, 130.4, 133.1, 133.3, 136.0, 154.1, 159.2, 160.6,
+
1
63.1, 171.9, 176.3. ESI-MS (m/z): 409.5 [M+H] . Anal. Calcd. for C H ClFO : C, 67.57;
2
3
14
4
H, 3.45. Found: C, 67.32; H, 3.56.
2
-(3-(2-Chloro-4-fluorophenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9h): Color:
1
White. Yield: 73%. Mp: 106.5–107.4 °C. H-NMR (d -DMSO, 400 MHz) δ 3.97 (s, 2H),
6
8

ACCEPTED MANUSCRIPT
7
7
.18–7.23 (m, 1H), 7.31–7.35 (m, , 1H), 7.37–7.46 (m, 5H), 7.49–7.54 (m, 2H), 7.82 (dd, J =
13
.4, 1.4 Hz, 1H), 8.03 (dd, J = 8.0, 1.6 Hz, 1H), 12.59 (s, 1H). C-NMR (d -DMSO, 100
6
MHz) δ 35.4, 114.7, 114.9, 116.6, 116.9, 119.7, 122.5, 124.3, 125.5, 125.8, 128.6, 128.7,
1
1
6
29.2 (d, J = 4 Hz) , 130.9, 132.4, 134.5, 134.6, 135.2, 135.3, 136.1, 154.2, 160.6, 161.5,
-
63.3, 172.0, 175.6. Negative ESI-MS (m/z): 407.4 [M-H] . Anal. Calcd. for C H ClFO : C,
23
14
4
7.57; H, 3.45. Found: C, 67.40, H, 3.52.
2
-(3-(2-Chloro-4-fluorophenyl)-2-(4-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid
1
(
9i): Color: White. Yield: 83%. Mp: 238.6–241.3 °C. H-NMR (d -DMSO, 400 MHz) δ 3.97
6
(s, 2H), 7.23 (t, J = 7.4 Hz, 1H), 7.34–7.42 (m, 3H), 7.48–7.54 (m, 4H), 7.82 (d, J = 6.8Hz,
1
3
1
1
1
H), 8.03 (d, J = 7.6 Hz, 1H), 12.57 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.3, 114.8,
6
15.0, 116.8, 117.0, 119.9, 122.5, 124.3, 125.6, 125.8, 128.8, 130.5, 131.3, 134.5, 134.6,
35.1, 135.2, 135.8, 136.2, 154.2, 160.4, 160.7, 163.2, 172.0, 175.6. ESI-MS (m/z): 481.2
+
+
+
[
M+K] , 465.2 [M+Na] , 443.3 [M+H] . Anal. Calcd. for C H Cl FO : C, 62.32; H, 2.96.
23 13 2 4
Found: C, 62.15; H, 2.85.
2
-(3-(2-Chloro-4-fluorophenyl)-2-(3-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid
1
(
9j): Color: White. Yield: 89%. Mp: 98.6–99.5 °C. H-NMR (d -DMSO, 400 MHz) δ 3.98 (s,
6
2
H), 7.22–7.27 (m, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.37–7.44 (m, 2H), 7.48–7.57 (m, 4H), 7.83
13
(
d, J = 6.0 Hz, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 12.61 (s, 1H). C-NMR (d -DMSO, 100
6
MHz) δ 35.4, 114.8, 115.0, 116.7, 117.0, 120.1, 122.5, 124.3, 125.6, 125.8, 127.4, 128.4,
1
1
6
28.7(d, J = 4 Hz), 130.6, 130.9, 133.3, 134.4, 134.6, 134.7, 135.1, 135.2, 136.3,154.2, 159.9,
+
60.8, 163.2, 172.0, 175.6. ESI-MS (m/z): 443.3 [M+H] Anal. Calcd. for C H Cl FO : C,
23
13
2
4
2.32; H, 2.96. Found: C, 62.07; H, 2.80.
2
-(3-(4-Chlorophenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9k): Color: White.
1
Yield: 77%. Mp: 242.5–243.2 °C. H-NMR (d -DMSO, 400 MHz) δ 3.94 (s, 2H), 7.21 (d, J =
6
8
7
1
1
.0 Hz, 2H), 7.35–7.44 (m, 7H), 7.48 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 6.8 Hz, 1H), 8.03 (d, J =
1
3
.6 Hz, 1H), 12.61 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 121.2, 122.8, 124.4,
6
25.2, 125.7, 128.3, 128.5, 129.6, 130.6, 132.0, 132.4, 132.7, 133.2, 135.9, 154.1, 161.0,
-
72.0, 176.2. Negative ESI-MS (m/z): 389.5 [M-H] . Anal. Calcd. for C H ClO : C, 70.68;
2
3
15
4
9

ACCEPTED MANUSCRIPT
H, 3.87. Found: C, 70.51, H, 3.72.
2
-(2,3-Bis(4-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9l): Color: White. Yield:
1
8
6%. Mp: 221.2–223.5 °C. H-NMR (d -DMSO, 400 MHz) δ 3.94 (s, 2H), 7.23 (d, J = 8.0 Hz,
6
2
H), 7.40–7.42 (m, 4H), 7.45–7.51 (m, 3H), 7.80 (d, J = 7.2 Hz, 1H), 8.03 (d, J = 7.6 Hz,
13
1
H), 12.57 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.3, 121.5, 122.8.0, 124.4, 125.4,
6
1
25.7, 128.4, 128.6, 131.4, 131.6, 131.7, 132.6, 133.2, 135.5, 136.0, 154.1, 159.9, 172.0,
+
1
76.1. ESI-MS (m/z): 447.6 [M+Na] . Anal. Calcd. for C H Cl O : C, 64.96; H, 3.32. Found:
2
3
14
2
4
C, 65.17; H, 3.42.
2
-(2-(3-Chlorophenyl)-3-(4-chlorophenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9m):
1
Color: White. Yield: 83%. Mp: 194.3–195.5 °C. H-NMR (d -DMSO, 400 MHz) δ 3.95 (s,
6
2
H), 7.24 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 7.6 Hz, 1H), 7.37-7.42 (m, 3H), 7.48–7.51 (m, 3H),
1
3
7
.80 (d, J = 7.2 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 12.58 (s, 1H). C-NMR (d -DMSO, 100
6
MHz) δ 35.4, 121.8, 122.8, 124.4, 125.4, 125.7, 128.4, 129.3, 130.3, 130.5, 131.6, 132.7,
+
1
33.1, 133.2, 134.7, 136.1, 154.1, 159.4, 171.9, 176.2. ESI-MS (m/z): 425.4 [M+H] . Anal.
Calcd. for C H Cl O : C, 64.96; H, 3.32. Found: C, 64.81; H, 3.45.
2
3
14
2
4
2
8
2
1
-(2,3-Bis(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl)acetic acid (9n): Color: White. Yield:
1
4%. Mp: 225.9–227.4 °C. H-NMR (d -DMSO, 400 MHz) δ 3.76(s, 3H), 3.77(s, 3H), 3.95 (s,
6
H), 6.88–6.91 (m, 4H), 7.10 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.45 (t, J = 7.6 Hz,
1
3
H), 7.75 (dd, J = 7.2, 1.2 Hz, 1H), 8.00 (dd, J = 8.0, 1.6 Hz, 1H), 12.56 (s, 1H). C-NMR
(d6-DMSO, 100 MHz) δ 35.4, 55.2, 55.5, 113.9, 121.0, 122.8, 124.3, 124.9, 125.2, 125.3,
1
25.5, 131.2, 132.4, 135.6, 154.0, 158.7, 160.4, 160.8, 172.1, 176.5. Negative ESI-MS (m/z):
-
4
15.5 [M-H] . Anal. Calcd. for C H O : C, 72.11; H, 4.84. Found: C, 71.92; H, 4.61.
2
5
20
6
2
-(2-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl)acetic
acid
1
(
9o): Color: White. Yield: 77%. Mp: 201.2–202.8 °C. H-NMR (d -DMSO, 400 MHz) δ 3.53
6
(s, 3H), 3.77 (s, 6H), 3.97 (s, 2H), 6.90–6.95 (m, 4H), 7.02 (dd, J = 8.6, 2.2 Hz, 1H), 7.12 (d,
J = 8.8 Hz, 2H), 7.45 (t, J = 7.6 Hz, 1H), 7.76 (dd, J = 7.4, 1.4 Hz, 1H), 8.00 (dd, J = 8.0, 1.6
13
Hz, 1H), 12.56 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.5, 55.2, 55.3, 55.7, 111.2, 112.8,
6
1
0

ACCEPTED MANUSCRIPT
1
14.0, 121.0, 122.8, 122.9, 124.4, 125.0, 125.5, 125.6, 132.3, 135.7, 148.0, 150.5, 154.0,
-
1
58.8, 160.2, 172.0, 176.6. Negative ESI-MS (m/z): 445.5 [M-H] . Anal. Calcd. for C H O :
2
6
22
7
C, 69.95; H, 4.97. Found: C, 70.22; H, 5.19.
2
-(3-(4-Methoxyphenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9p): Color: White.
1
Yield: 88%. Mp: 224.2–225.7 °C. H-NMR (d -DMSO, 400 MHz) δ 3.75 (s, 3H), 3.94 (s, 2H),
6
6
.88 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 6.4 Hz,
H), 7.47 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 12.57 (s, 1H).
3
1
3
C-NMR (d -DMSO, 100 MHz) δ 35.4, 55.2, 113.7, 121.9, 122.8, 124.4, 124.9, 125.1, 125.6,
6
1
3
28.4, 129.5, 130.4, 132.4, 133.2, 135.7, 154.1, 158.7, 160.5, 172.0, 176.6. ESI-MS (m/z):
+
87.8 [M+H] . Anal. Calcd. for C H O : C, 74.60; H, 4.70. Found: C, 74.33; H, 4.86.
2
4
18
5
2
-(3-(3,4-Dimethoxyphenyl)-4-oxo-2-phenyl-4H-chromen-8-yl)acetic acid (9q): Color:
1
White. Yield: 79%. Mp: 151.7–153.0 °C. H-NMR (d -DMSO, 400 MHz) δ 3.58 (s, 3H), 3.74
6
(s, 3H), 3.94 (s, 2H), 6.65–6.71 (m, 1H), 6.78 (d, J = 1.6 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H),
7
.34–7.37 (m, , 2H), 7.39–7.44 (m, 4H),7.77 (d, J = 7.6 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H),
1
3
1
2.52 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 55.5(6), 55.5(7), 111.6, 115.2, 122.1,
6
1
22.9, 123.7, 124.4, 125.0(8), 125.1(2),125.6, 128.4, 129.4, 130.3, 133.2, 135.7, 148.4, 154.1,
+
+
1
60.7, 172.0, 176.5. ESI-MS (m/z): 439.8 [M+Na] , 417.9 [M+H] . Anal. Calcd. for C H O :
25 20 6
C, 72.11; H, 4.84. Found: C, 71.94; H, 5.02.
2
-(3-(3,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)-4-oxo-4H-chromen-8-yl)acetic
acid
1
(
9r): Color: White. Yield: 89%. Mp: 114.3–116.0 °C. H-NMR (d -DMSO, 400 MHz) δ 3.62
6
(s, 3H), 3.76(s, 3H), 3.77(s, 3H), 3.95 (s, 2H), 6.82 (s, 1H), 6.68 (d, J = 8.4 Hz, 1H), 6.90 (d, J
=
8.4 Hz, 3H), 7.38 (d, J = 8.8 Hz, 2H), 7.45 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 8.01
1
3
(
d, J = 7.6 Hz, 1H), 12.54 (s, 1H). C-NMR (d -DMSO, 100 MHz) δ 35.4, 55.5(1), 55.5(6),
6
5
1
5.6(3), 111.7, 113.8, 115.2, 121.2, 122.8, 123.5, 124.4, 124.9, 125.2, 125.4, 125.6, 131.1,
+
35.6, 148.3, 148.5, 153.9, 160.5, 160.8, 172.1, 176.5. ESI-MS (m/z): 469.7 [M+Na] , 447.8
+
[
M+H] . Anal. Calcd. for C H O : C, 69.95; H, 4.97. Found: C, 69.73; H, 4.87.
26
22
7
4
.2 Pharmacology
1
1

ACCEPTED MANUSCRIPT
4
.2.1 Compounds
Compounds 9a-r were dissolved in DMSO and stored as stock solutions (100 mM) at
20 °C. For experimental use, all the compounds were prepared from stock solutions, diluted
with growth medium and used immediately.
-
4
.2.2 Cell culture
A549 and HT-29 cells were cultured in RPMI 1640, supplemented with 10% fetal bovine
serum and 100 U/mL penicillin and 100 ꢀg/mL streptomycin (all from Invitrogen) at 37 °C in
a humidified atmosphere of 5% CO and 95% air. HPBMCs were isolated from heparinized
2
whole blood by centrifugation over Ficoll-Paque Plus (TBD Science).
4
.2.3 Direct cytotoxicity toward solid tumor cell lines
Cells were seeded for attachment in 96-well microculture plates overnight and then
incubated with DMXAA and compounds 9a-r of varying concentrations for 24 h. Then, the
medium was discarded and fresh medium with MTT (0.5 mg/mL) was added to each well,
and the cells were further incubated for 3 h. The stained formazan product was determined
spectrophotometrically at 570 nm in GENios Pro microplate reader (TECAN).
4
.2.4 Indirect cytotoxicity toward A549 cell lines
The indirect cytotoxicity was measured using the proceudres previously described by us
6
[
18]. HPBMCs (1×10 cells/well) were placed at the top well of a 3 ꢀm 96-transwell plate
CORNING) for attachment overnight, and then activated by DMXAA and compounds 9a-r
(
of varying concentrations, using triplicate wells per drug dose. After 24 h, the medium was
3
discarded and the A549 cells were seeded at a density of 3×10 cells per well in the bottom
wells for another 24 h. The cell viability of the A549 cells in the bottom wells was assessed
by the MTT assay. The inhibition percentage was calculated according to the following
formula.
1
2

ACCEPTED MANUSCRIPT

OD(A549) of Compounds 
%
inhibition= 1−
×100%


OD (A549) of Blank Control


4
.2.5 Quantification of TNF-α production
HPBMCs were isolated as described above and treated with culture medium, DMXAA 1
and compounds 9p-r at the concentrations of 25, 50, and 100 ꢀM. LPS (from E. coli serotype
0
127; B8, Sigma) was used as a positive control at the final concentration of 1 ꢀg/mL. After
2
4 h incubations, the medium was carefully collected and stored at -70 °C for assay. The
viability of the cells was assessed by Trypan blue dye exclusion and always higher than 95%.
The concentration of TNF-α was determined according to the manufacturer’s instructions of
the commercially available enzymes-linked immunosorbent assay (ELISA) kits (Sigma).
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China
(
No. 21002048) and the Natural Science Foundation of Guangdong Province
9451051501002541).
(
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1
5

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Table Captions
Table 1. Direct cytotoxicities of compounds 9a-r
Table 2. Indirect cytotoxicities of compounds 9e, 9i and 9n-r toward A549 cells
1

ACCEPTED MANUSCRIPT
Tables
Table 1. Direct cytotoxicities of compounds 9a-r
A549
HT-29
HPBMCs
1
2
Compound
R
R
a
a
b
IC (µM)
IC (µM)
50
CC50 (µM)
50
c
d
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
H
H
>500
>500
>500
>500
ND
d
2-Cl
H
ND
d
2-Cl
4-Cl
3-Cl
H
278.4 ± 48.6
311.8 ± 53.0
>500
188.6 ± 30.9
250.7 ± 49.6
415.5 ± 40.6
206.5 ± 53.7
389.0 ± 37.8
229.6 ± 41.3
216.7 ± 39.0
203.2 ± 28.8
331.0 ± 36.8
270.0 ± 8.3
351.5 ± 28.2
>500
ND
d
d
e
f
2-Cl
ND
4-F
> 1000
d
4-F
4-Cl
3-Cl
H
337.0 ± 53.3
328.8 ± 51.0
314.6 ± 24.8
239.2 ± 36.3
290.5 ± 39.2
250.8 ± 28.4
256.4 ± 48.0
265.0 ± 61.0
377.1 ± 14.1
>500
ND
d
g
h
i
4-F
ND
d
2-Cl,4-F
2-Cl,4-F
2-Cl,4-F
4-Cl
ND
4-Cl
3-Cl
H
> 1000
d
j
ND
d
k
l
ND
d
4-Cl
4-Cl
3-Cl
4-MeO
3,4-diMeO
H
ND
d
m
n
o
p
q
r
4-Cl
ND
4-MeO
4-MeO
4-MeO
3,4-diMeO
3,4-diMeO
\
678.2±48.3
790.7±33.8
> 1000
>500
>500
>500
H
>500
>500
881.5±51.5
890.6±56.9
648.2±31.5
4-MeO
\
>500
>500
DMXAA
291.1±14.4
268.4 ± 49.9
a
The IC50 value represents the concentration of each compound resulting in 50% inhibition in cell growth after 24 h
incubation, and was the mean values of three repeated experiments.
b
The CC50 values represent the 50% cytotoxic concentration, defined as the concentration required to reduce
HPBMCs viability by 50% of the control value after 24 h incubation, and was the mean values of three repeated
experiments.
c
Compound 9a has been previously reported [33].
d
ND = not determined.
2

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Tables
Table 2. Indirect cytotoxicities of compounds 9e, 9i and 9n-r toward A549 cells
a
b
Compound
%Inhibition at 50 µM
IC (µM)
PR
5
0
DMXAA
48.4
47.5
49.5
53.7
53.0
62.9
57.0
66.2
84.2±13.5
92.6±7.8
86.0±10.3
61.7±12.2
67.4±12.6
38.5±7.3
44.5±7.7
35.7±7.6
1.0
0.9
1.0
1.4
1.2
2.2
1.9
2.4
9e
9i
9n
9o
9p
9q
9r
a
Measured after co-cultured for 24 h and was the mean values of three repeated experiments.
b
PR denotes the potency ratios (PRs) relative to DMXAA 1.
3

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Figure Captions
Figure 1. Flavonoids bearing carboxylic groups.
Figure 2. Cytotoxicities of compounds 9a-r (50 µM) induced in HPBMCs toward A549 cells
®
after co-cultured for 24 h in HTS Transwell -96 Well Permeable Support Systems.
Figure 3. Pyrylium-type salts formed from compounds 8.
Figure 4. Indirect inhibitory activities of some 3-arylflavone-8-acetic acids 9 and their
corresponding analogues 8 with a 7-methoxy group toward A549 cells at the concentration of
5
0 µM.
Figure 5. TNF-α released by HPBMC treated with compounds 9p-r of varying concentrations
for 24 h. M represents the medium from unstimulated cells.
1

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Figures
CH COOH
CH COOHMe
CH COOH
CH COOH
2
2
2
COOMe
O
2
HO
O
O
Me
O
H C
O
3
Me
H C
NO2
3
OH
O
O
O
O
O
OMe
1
DMXAA
2 FAA
3
4
5
CH COOHN
2
3
CH COOH
CH COOH
2
2
R2
O
Me
O
MeO
O
CF3
OMe
R1
O
O
O
6
7
8a-m
Figure 1.
2

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Figures
Figure 2.
3

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Figures
Figure 3.
4

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Figures
Figure 4.
5

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Figures
Figure 5.
6

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Scheme
CH3
O
CH3
CH3
R2
R1
R2
O
OH
i
O
O
ii
O
O
Cl
R2
+
R1
R
O
1
10a-g
11a-d
12a-r
13a-r
CH Br
CH CN
CH COOH
R2
2
2
R2
2
R2
O
O
O
iii
iv
v
R1
R1
R1
O
O
O
14a-r
15a-r
9a-r
Scheme 1. Synthetic route for compounds 9a-r. Reagents and conditions: (i) pyridine, room temperature;
(
ii) glycerol, 260 °C, N , 2h; (iii) NBS, AIBN, CCl , reflux; (iv) Et NCN, CH Cl ; (v) H SO , AcOH, H O,
2 4 4 2 2 2 4 2
1
2
1
05 °C. Please refer to Table 1 for specific R and R .
1

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Research Highlights
ꢀ
ꢀ
Eighteen 3-arylflavone-8-acetic acid derivatives were synthesized.
All the compounds bearing methoxy groups showed higher indirect cytotoxicity than
DMXAA.
ꢀ
ꢀ
ꢀ
Compounds 9p-r were able to induce TNF-α production in HPBMCs.
A methoxy group at the 7-position of flavones is less favorable for indirect cytotoxicity.
A methoxy group on the benzene rings is favorable for indirect cytotoxicities.

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Synthesis and anticancer activities of 3-arylflavone-8-acetic acid derivatives
#
, a
#, b
a
a
a
Guang-Hua Yan , Xiao-Fang Li , Bing-Chen Ge , Xiu-Dong Shi , Yu-Fang Chen , Xue-Mei
a
a
a
a
, a
Yang , Jiang-Ping Xu , Shu-Wen Liu , Pei-Liang Zhao , Zhong-Zhen Zhou* , Chun-Qiong
a
, a
Zhou and Wen-Hua Chen*
a
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR Chin
b
Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China
#
Both authors contributed equally to this work.
Supporting Information
1
2
3
4
5
. Synthesis of 2-Methyl-6-(2-arylacetyl) aryl benzoate 12a-r …………….…….….S2~S5
. Synthesis of 3-aryl-8-methylflavones 13a-r ………………….………….…….….S5~S10
. Synthesis of 3-arylflavone-8- acetonitrile derivatives 15a-r……………..…..……S11~S14
. Table S1. Indirect cytotoxicities and selectivity of compounds 9a-r………….…...S15
1
13
. Figures S1~36. H and C NMR spectra of compounds 13a-r and 9a-r………….S16~51
S1

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1
. Synthesis of 3-aryl-8-methylflavones 12a-r
CH
3
CH3
O
R2
O
pyridine
O
O
OH
O
Cl
R2
room temperature
+
R1
R1
12a-r
10a-g
11a-d
General procedures: a solution of compounds 10a-g (1.0 mmol) and benzoyl chlorides 11a-e (2.7
mmol) in anhydrous pyridine (5 mL) was stirred at room temperature for 5 h. The reaction
mixture was treated with cold diluted HCl (1 M) and then extracted with ether (10 mL×2). The
combined organic layer was washed with 5% aqueous HCl (10 mL) and water (10 mL×2), dried
over anhydrous Na SO and concentrated under reduced pressure to yield oily residue.
2
4
Purification was accomplished by chromatography on a silica gel column, eluted with petroleum
ether-acetone (25/l, v/v), to give esters 12a-r.
1
2
-Methyl-6-(2-phenylacetyl) phenyl benzoate (12a): Yield: 78%. H-NMR (400 MHz, CDCl )
3
δ 2.25 (s, 3H), 4.17 (s, 2H), 7.16–7.22 (m, 3H), 7.24 (d, J = 1.6 Hz, 1H), 7.27 (m, 2H), 7.42 (dd, J
7.6, 0.8 Hz, 1H), 7.47–7.54 (m, 2H), 7.61–7.65 (m, 2H), 8.20 (d, J = 7.2 Hz, 2H). ESI-MS
=
+
+
(
m/z): 369.3 [M+K] , 353.4 [M+Na] .
1
2
-(2-(2-Chlorophenyl)acetyl)-6-methylphenyl benzoate (12b): Yield: 66%. H-NMR (400
MHz, CDCl ) δ 2.26 (s, 3H), 4.33 (s, 2H), 7.17 (d, J = 2.8 Hz, 3H), 7.27–7.34 (m, 2H), 7.45–7.52
3
(m, 3H) 7.61–7.65 (m, 1H), 7.72 (dd, J = 7.6, 1.2 Hz, 1H), 8.21 (d, J = 7.2 Hz, 2H). ESI-MS
+
+
(
m/z): 403.5 [M+K] , 387.5 [M+Na] .
1
2
-(2-(2-Chlorophenyl)acetyl)-6-methylphenyl-4-chlorobenzoate (12c): Yield: 37%. H-NMR
(
(
[
400 MHz, CDCl ) δ 2.25 (s, 3H), 4.32 (s, 2H), 7.15–7.21 (m, 3H), 7.28–7.34(m, 2H), 7.44–7.49
3
m, 3H), 7.74 (dd, J = 7.7, 0.9 Hz, 1H), 8.12 (d, J = 8.6 Hz, 2H). ESI-MS (m/z): 421.3
+
M+Na][M+Na] .
1
2
-(2-(2-Chlorophenyl)acetyl)-6-methylphenyl-3-chlorobenzoate (12d): Yield: 58%. H-NMR
S2