Synthesis, telomerase inhibitory and anticancer activity of new 2-phenyl-4H-chromone derivatives containing 1,3,4-oxadiazole moiety

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Synthesis, telomerase inhibitory and anticancer

activity of new 2-phenyl-4H-chromone derivatives

containing 1,3,4-oxadiazole moiety

Xu Han, Yun Long Yu, Duo Ma, Zhao Yan Zhang & Xin Hua Liu

To cite this article: Xu Han, Yun Long Yu, Duo Ma, Zhao Yan Zhang & Xin Hua Liu (2021)

Synthesis, telomerase inhibitory and anticancer activity of new 2-phenyl-4H-chromone derivatives

containing 1,3,4-oxadiazole moiety, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1,

344-360, DOI: 10.1080/14756366.2020.1864630

To link to this article: https://doi.org/10.1080/14756366.2020.1864630

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2021, VOL. 36, NO. 1, 344–360

https://doi.org/10.1080/14756366.2020.1864630

BRIEF REPORT

Synthesis, telomerase inhibitory and anticancer activity of new 2-phenyl-4H-

chromone derivatives containing 1,3,4-oxadiazole moiety

Xu Han, Yun Long Yu, Duo Ma, Zhao Yan Zhang and Xin Hua Liu

School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei, P. R. China

ABSTRACT

ARTICLE HISTORY

Received 1 November 2020

Revised 2 December 2020

Accepted 7 December 2020

Based on previous studies, 66 2-phenyl-4H-chromone derivatives containing amide and 1,3,4-oxadiazole

moieties were prepared as potential telomerase inhibitors. The results showed most of the title com-

pounds exhibited significantly inhibitory activity on telomerase. Among them, some compounds demon-

strated the most potent telomerase inhibitory activity (IC₅₀< 1 mM), which was significantly superior to

the staurosporine (IC₅₀¼ 6.41 mM). In addition, clear structure–activity relationships were summarised,

indicating that the substitution of the methoxy group and the position, type and number of the substitu-

ents on the phenyl ring had significant effects on telomerase activity. Among them, compound A33

showed considerable inhibition against telomerase. Flow cytometric analysis showed that compound A33

could arrest MGC-803 cell cycle at G2/M phase and induce apoptosis in a concentration-dependent way.

Meanwhile, Western blotting revealed that this compound could reduce the expression of dyskerin, which

is a fragment of telomerase.

KEYWORDS

2-phenyl-4H-chromone; syn-

thesis; telomerase inhibitor;

anticancer activity; dyskerin

1. Introduction

the linker should be involved in the improvement of inhibitory

activity (Figure 1(B)). In addition, the amount of methoxy groups

on the benzene ring has an essential effect on antitumor activity,

such as natural A4. Therefore, on basis of the above, the optimisa-

tion design of the structure was carried out in this study

(Figure 1(C)).

Telomerase is a ribonucleoprotein that exists in mammalian cells,

playing an important role in maintaining the length of stable telo-

mere and the chromosomal integrity of frequently dividing cells¹.

It is almost undetectable in most somatic cells with the exception

of some adult pluripotent stem cells and male germline cells^2,3

.

As is known to us, 1,3,4-oxadiazole as a privileged scaffold was

used extensively in drugs discovery^25–28. It was often used as bioi-

sosteres for compounds containing carbonyl such as esters and

amides, participating in hydrogen bonding interactions with the

receptors^29–33. Furthermore, different substituted 1,3,4-oxadiazole

derivatives with potent antitumor activity have been confirmed

(Figure 1(D)). Therefore, 2-phenyl-4H-chromone used as a basic

scaffold, following by adjusting the number and substitution posi-

tions of OCH₃and H on the phenyl ring, retaining the amide frag-

ment as a linker, then introducing 1,3,4-oxadiazole heterocycle

and continuing unsaturated substituent. At last, a series of new 2-

phenyl-4H-chromone derivatives were designed and synthesised

in this study (Figure 1(E)). Their telomerase inhibitory activity was

evaluated, and the SAR was widely discussed. In addition, some

compounds were selected to screen for their anticancer activity

and explore the possible mechanism.

However, in 85–90% of primary tumours, telomerase is reacti-

vated, so that the ends of chromosomes are maintained during

cells proliferation, which results in unlimited proliferation and

immortalisation of tumour cells⁴. Therefore, telomerase is regarded

as an effective drug target⁵. Regulating the stability of telomerase

G-quadruplex as anticancer agents have been widely reported^6–13

.

A lot of studies confirmed that dyskerin, fragment protein of

telomerase was essential for telomerase activity, which allowed

the correct assembly and stabilisation of mature human telomer-

ase RNA (hTR)¹⁴. Highly expressed dyskerin was closely related to

the occurrence and development of various tumours^15–17

.

Considering that most cancers rely on the holoenzyme telomerase

to promote tumorigenesis and development, and that dyskerin

was closely related to the maintenance of telomeres. So, this pro-

tein was

a potential target for development of anticancer

therapies¹⁸.

Several studies had shown that some flavonoid derivatives had

strong telomerase inhibitory activity and extensive antitumor

activity^19–24. In our previous work²², myricetin derivatives exhib-

ited moderate telomerase inhibitory activity (Figure 1(A)), and the

preliminary structure–activity relationships (SARs) showed that the

2. Experimental section

2.1. Chemistry

All reagents and solvents were purchased from standard commer-

introduction of amide segment could significantly change the tel- cial suppliers and used without further purification. The reactions

omerase inhibitory activity and cytotoxicity. This indicated that were monitored by thin-layer chromatography (TLC) on pre-coated

CONTACT Xin Hua Liu

xhliuhx@163.com

School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Medical University, Hefei

230032, P. R. China

Supplemental data for this article can be accessed here.

ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

345

Figure 1. Design of the title compounds.

silica GF254 plates and visualised under UV light at 254 and (2 C), 96.5, 92.8, 73.4, 61.1, 56.6, 56.5(2 C), 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₀H₂₈N₃O₁₀: 590.1769; found: 590.1767.

365 nm. Melting points (uncorrected) were determined on a

XT4MP apparatus (Taike Corp., Beijing, China). ¹H and ¹³C NMR

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5–(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A2).

spectral data were recorded on a Bruker 400 MHz or an Agilent

600 MHz spectrometer in CDCl₃or DMSO-d₆using tetramethylsi-

lane (TMS) as the internal standard at room temperature. High-

resolution mass spectrometry (HRMS) was recorded on an Agilent

Technologies LC-TOF instrument (Supporting Material). X-ray crys-

tallographic data were collected on a Bruker SMART APEX-II CCD

diffractometer.

White solid, 47.60% yield, m.p.: 221–223 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.56 (s, 1H), 8.11–8.07 (m, 2H), 7.25 (s, 2H), 7.18 (t,

J ¼ 8.6 Hz, 2H), 6.57 (d, J ¼ 2.1 Hz, 1H), 6.43 (d, J ¼ 2.1 Hz, 1H), 4.41

(s, 2H), 4.00 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H), 3.94 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.7, 166.7, 165.1, 164.6 (d, J ¼ 252.6 Hz),

161.1, 160.7, 159.1, 156.9, 154.4, 153.6 (2 C), 141.1 (2 C), 128.9 (d,

J ¼ 8.8 Hz) (2 C), 124.4, 120.2 (d, J ¼ 3.2 Hz), 116.2 (d, J ¼ 22.4 Hz)

(2 C), 108.5, 105.9 (2 C), 96.5, 92.8, 73.4, 61.06, 56.6, 56.5 (2 C), 55.9.

HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₇FN₃O₁₀: 608.1675;

found: 608.1674.

2.2. General procedure for synthesis of title compounds A1-A33

and B1-B33

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

To a solution of the intermediate 1 (0.5 mmol, in acetone (20 ml),

the intermediate 4 (0.48 mmol), K₂CO₃(0.96 mmol) and KI (cat)

were added. The reaction mixture was stirred at the reflux tem-

perature for 12 h, monitored by TLC. After the reaction was com-

pleted, the reaction mixture was cooled to room temperature,

diluted with water, extracted with CH₂Cl₂(50 ml ꢀ 3), and washed

with saturated sodium chloride. The combined organic layers

were dried over anhydrous sodium sulphate, filtered, and concen-

trated under reduced pressure. The crude residue was purified by

flash chromatography (DCM: MeOH ¼ 25:1, v/v), and then recrys-

tallized by ethanol to give title compounds A1–A33. The title

compounds B1–B33 could be obtained according to the

same procedure.

yloxy)-N-(5–(3-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A3).

White solid, 35.83% yield, m.p.: 217–219 ^ꢁC; ¹H NMR (400 MHz,

CDCl₃) d 12.77 (s, 1H), 7.91–7.87 (m, 1H), 7.79 (ddd, J ¼ 9.2, 2.5,

1.5 Hz, 1H), 7.47 (td, J ¼ 8.1, 5.6 Hz, 1H), 7.25 (s, 2H), 7.21 (tdd,

J ¼ 8.4, 2.6, 0.9 Hz, 1H), 6.57 (d, J ¼ 2.2 Hz, 1H), 6.43 (d, J ¼ 2.2 Hz,

1H), 4.40 (s, 2H), 4.01 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H), 3.94 (s, 3H).

¹³C NMR (151 MHz, CDCl₃)

d 174.6, 166.6, 165.1, 162.8 (d,

J ¼ 247.6 Hz), 161.2, 160.5, 159.1, 157.1, 154.4, 153.6 (2 C), 141.3,

141.1, 130.7 (d, J ¼ 7.2 Hz), 125.8 (d, J ¼ 8.2 Hz), 124.4, 122.4, 118.3

(d, J ¼ 20.9 Hz), 113.7 (d, J ¼ 24.3 Hz), 108.5, 106.2 (2 C), 96.5, 92.8,

73.4, 60.99, 56.5 (3 C), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

C₃₀H₂₇FN₃O₁₀: 608.1675; found: 608.1672.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)acetamide (A1). White solid,

46.23% yield, m.p.: 222–224 ^ꢁC; ¹H NMR (400 MHz, CDCl₃) d 12.62

(s, 1H), 8.12–8.06 (m, 2H), 7.54–7.45 (m, 3H), 7.25 (s, 2H), 6.57 (d,

J ¼ 2.2 Hz, 1H), 6.42 (d, J ¼ 2.2 Hz, 1H), 4.40 (s, 2H), 4.00 (s, 3H),

3.95 (s, 3H), 3.94 (s, 6H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

174.6, 166.6, 165.1, 161.5, 161.1, 159.1, 156.9, 154.4, 153.6 (2 C),

yloxy)-N-(5–(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A4).

White solid, 44.80% yield, m.p.: 213–215 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.73 (s, 1H), 8.04 (t, J ¼ 7.0 Hz, 1H), 7.49 (dd, J ¼ 12.0,

6.8 Hz, 1H), 7.27–7.20 (m, 4H), 6.54 (d, J ¼ 1.4 Hz, 1H), 6.40 (s, 1H),

4.39 (s, 2H), 3.98 (s, 3H), 3.96–3.90 (m, 12H). ¹³C NMR (151 MHz,

CDCl₃) d 174.6, 166.7, 165.1, 161.1, 159.9 (d, J ¼ 258.4 Hz), 159.0,

158.1 (d, J ¼ 4.9 Hz), 157.2, 154.4, 153.5 (2 C), 141.1, 141.0, 133.1 (d,

141.1, 141.1, 131.3, 128.9 (2 C), 126.7(2 C), 124.4, 123.9, 108.5, 105.9 J ¼ 8.2 Hz), 129.6, 124.5 (d, J ¼ 3.3 Hz), 124.4, 116.8 (d,

346

X. HAN ET AL.

J ¼ 20.8 Hz),112.4 (d, J ¼ 11.9 Hz), 108.4, 105.97 (2 C), 96.47, 92.72, 2H), 6.55 (d, J ¼ 1.9 Hz, 1H), 6.41 (d, J ¼ 1.8 Hz, 1H), 4.40 (s, 2H),

73.36, 61.01, 56.50 (3 C), 55.94. HRMS (ESI): m/z [M þ H]^þcalcd for 3.98 (s, 3H), 3.96–3.90 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d

C₃₀H₂₇FN₃O₁₀: 608.1675; found: 608.1671.

174.6, 166.6, 165.1, 161.2, 160.2, 159.0, 157.3, 154.4, 153.6 (2 C),

141.2, 141.1, 134.3, 132.1, 131.6, 127.4, 125.4, 124.4, 121.7, 108.5,

106.1 (2 C), 96.5, 92.8, 73.4, 61.0, 56.6 (2 C), 56.5, 55.9. HRMS (ESI):

N-(5–(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A5).

White solid, 49.06% yield, m.p.: 227–229 ^ꢁC; ¹H NMR (600 MHz, m/z [M þ H]^þcalcd for C₃₀H₂₇BrN₃O₁₀: 668.0874; found: 668.0873.

CDCl₃) d 12.69 (s, 1H), 8.02 (d, J ¼ 8.5 Hz, 2H), 7.47 (d, J ¼ 8.5 Hz,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

2H), 7.25 (s, 2H), 6.56 (d, J ¼ 1.9 Hz, 1H), 6.42 (d, J ¼ 1.7 Hz, 1H), yloxy)-N-(5-p-tolyl-1,3,4-oxadiazol-2-yl)acetamide (A11). White solid,

4.40 (s, 2H), 4.00 (s, 3H), 3.99–3.90 (m, 12H). ¹³C NMR (151 MHz, 46.21% yield, m.p.: 225–227 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.45

CDCl₃) d 174.7, 166.6, 165.1, 161.1, 160.7, 159.1, 157.0, 154.5, 153.6 (s, 1H), 7.98 (d, J ¼ 8.1 Hz, 2H), 7.29 (d, J ¼ 8.0 Hz, 2H), 7.25 (s, 2H),

(2 C), 141.2, 141.1, 137.6, 129.3 (2 C), 127.9 (2 C), 124.4, 122.3, 6.57 (d, J ¼ 2.0 Hz, 1H), 6.43 (d, J ¼ 1.9 Hz, 1H), 4.41 (s, 2H), 4.00 (s,

108.5, 106.0 (2 C), 96.5, 92.8, 73.4, 61.1, 56.6, 56.5(2 C), 55.9. HRMS 3H), 3.96 (s, 3H), 3.95 (s, 6H), 3.94 (s, 3H), 2.42 (s, 3H). ¹³C NMR

(ESI): m/z [M þ H]^þcalcd for C₃₀H₂₇ClN₃O₁₀: 624.1379; (151 MHz, CDCl₃) d 174.6, 166.6, 165.0, 161.7, 161.1, 159.1, 156.6,

found: 624.1376.

154.4, 153.6 (2 C), 141.8, 141.1 (2 C), 129.6 (2 C), 126.7 (2 C), 124.4,

121.1, 108.5, 105.9 (2 C), 96.5, 92.7, 73.4, 61.1, 56.6, 56.5 (2 C), 55.9,

N-(5–(3-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A6). 21.6. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₁H₃₀N₃O₁₀: 604.1926;

White solid, 52.33% yield, m.p.: 206–208 ^ꢁC; ¹H NMR (400 MHz,

found: 604.1922.

CDCl₃) d 12.81 (s, 1H), 8.07 (t, J ¼ 1.6 Hz, 1H), 7.99 (dt, J ¼ 7.5,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

1.2 Hz, 1H), 7.50–7.46 (m, 1H), 7.43 (t, J ¼ 7.8 Hz, 1H), 7.25 (s, 2H), yloxy)-N-(5-m-tolyl-1,3,4-oxadiazol-2-yl)acetamide (A12). White solid,

6.57 (d, J ¼ 2.0 Hz, 1H), 6.42 (d, J ¼ 2.0 Hz, 1H), 4.40 (s, 2H), 4.01 (s, 42.83% yield, m.p.: 197–199 ^ꢁC; ¹H NMR (400 MHz, CDCl₃) d 12.60

3H), 3.95 (s, 3H), 3.94 (d, J ¼ 2.7 Hz, 6H), 3.93 (s, 3H). ¹³C NMR (s, 1H), 7.92 (s, 1H), 7.88 (d, J ¼ 7.6 Hz, 1H), 7.37 (t, J ¼ 7.6 Hz, 1H),

(151 MHz, CDCl₃) d 174.7, 166.6, 165.1, 161.1, 160.2, 159.0, 157.2, 7.31 (d, J ¼ 7.7 Hz, 1H), 7.25 (s, 2H), 6.57 (d, J ¼ 2.2 Hz, 1H), 6.42 (d,

154.4, 153.6 (2 C), 141.2, 141.1, 135.0, 131.3, 130.2, 126.6, 125.5, J ¼ 2.2 Hz, 1H), 4.40 (s, 2H), 4.00 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H),

124.8, 124.4, 108.4, 106.0 (2 C), 96.5, 92.8, 73.4, 61.0, 56.5 (3 C), 3.93 (s, 3H), 2.42 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6,

55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₇ClN₃O₁₀: 624.1379; 165.1, 161.7, 161.2, 159.1, 156.8, 154.4, 153.6 (2 C), 141.1, 141.1,

found: 624.1375.

138.7, 132.1, 128.8, 127.2, 124.4, 123.9, 123.7, 108.5, 106.0 (2 C),

96.5, 92.8, 73.4, 61.1, 56.5, 56.5 (2 C), 55.9, 21.2. HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₁H₃₀N₃O₁₀: 604.1926; found: 604.1924.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

N-(5–(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A7).

White solid, 43.61% yield, m.p.: 201–203 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.78 (s, 1H), 7.99 (d, J ¼ 7.7 Hz, 1H), 7.53 (d, J ¼ 8.0 Hz, yloxy)-N-(5-o-tolyl-1,3,4-oxadiazol-2-yl)acetamide (A13). White solid,

1H), 7.47–7.36 (m, 2H), 7.25 (s, 2H), 6.56 (d, J ¼ 1.9 Hz, 1H), 6.41 (d, 45.08% yield, m.p.: 205–207 ^ꢁC; ¹H NMR (400 MHz, CDCl₃) d 12.64

J ¼ 1.7 Hz, 1H), 4.40 (s, 2H), 3.99 (s, 3H), 3.97–3.91 (m, 12H). ¹³C (s, 1H), 8.00–7.95 (m, 1H), 7.42–7.36 (m, 1H), 7.34 ꢂ 7.27 (m, 2H),

NMR (151 MHz, CDCl₃) d 174.7, 166.6, 165.1, 161.2, 159.7, 159.0, 7.25 (s, 2H), 6.56 (d, J ¼ 2.2 Hz, 1H), 6.41 (d, J ¼ 2.2 Hz, 1H), 4.40 (s,

157.3, 154.4, 153.6 (2 C), 141.1, 141.1, 133.2, 132.0, 131.2, 131.0, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.94 (s, 6H), 3.93 (s, 3H), 2.72 (s, 3H).

126.9, 124.4, 123.2, 108.5, 106.0 (2 C), 96.5, 92.7, 73.4, 61.1, 56.5 ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6, 165.0, 161.8, 161.2,

(3 C), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₇ClN₃O₁₀: 159.0, 156.7, 154.4, 153.6 (2 C), 141.1, 141.1, 138.3, 131.5, 130.8,

624.1379; found: 624.1377.

128.9, 125.9, 124.4, 122.9, 108.5, 106.0 (2 C), 96.5, 92.7, 73.4, 61.0,

56.5, 56.5 (2 C), 55.9, 21.9. HRMS (ESI): m/z [M þ H]^þcalcd for

C₃₁H₃₀N₃O₁₀: 604.1926; found: 604.1923.

N-(5–(4-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A8).

White solid, 50.89% yield, m.p.: 234–236 ^ꢁC; ¹H NMR (600 MHz,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

CDCl₃) d 12.67 (s, 1H), 7.96 (d, J ¼ 8.4 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz, yloxy)-N-(5–(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A14).

2H), 7.25 (s, 2H), 6.57 (s, 1H), 6.43 (s, 1H), 4.40 (s, 2H), 4.00 (s, 3H), Light yellow solid, 42.89% yield, m.p.: 233–235 ^ꢁC; ¹H NMR

3.96 (s, 3H), 3.95 (s, 6H), 3.94 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d (600 MHz, CDCl₃) d 13.01 (s, 1H), 8.89 (s, 1H), 8.44 (d, J ¼ 7.8 Hz,

174.7, 166.6, 165.1, 161.1, 160.8, 159.1, 157.0, 154.5, 153.6 (2 C), 1H), 8.36 (dd, J ¼ 8.2, 1.1 Hz, 1H), 7.70 (t, J ¼ 8.0 Hz, 1H), 7.25 (s,

141.2, 141.1, 132.2 (2 C), 128.1 (2 C), 125.9, 124.4, 122.8, 108.5, 2H), 6.57 (d, J ¼ 2.0 Hz, 1H), 6.43 (d, J ¼ 1.8 Hz, 1H), 4.41 (s, 2H),

106.0 (2 C), 96.5, 92.8, 73.4, 61.1, 56.6, 56.5 (2 C), 55.9. HRMS (ESI): 4.02 (s, 3H), 3.97–3.92 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d

m/z [M þ H]^þcalcd for C₃₀H₂₇BrN₃O₁₀: 668.0874; found: 668.0873.

N-(5–(3-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A9).

174.8, 166.7, 165.2, 161.2, 159.4, 159.1, 157.6, 154.6, 153.6 (2 C),

148.6, 141.2, 141.1, 132.2, 130.2, 125.7, 125.5, 124.3, 121.5, 108.4,

105.9 (2 C), 96.5, 92.8, 73.5, 61.0, 56.5(2 C), 55.9. HRMS (ESI): m/z

White solid, 45.80% yield, m.p.: 202–204 ^ꢁC; ¹H NMR (400 MHz, [M þ H]^þcalcd for C₃₀H₂₇N₄O₁₂: 635.1620; found: 635.1620.

CDCl₃) d 12.84 (s, 1H), 8.23 (s, 1H), 8.03 (d, J ¼ 7.8 Hz, 1H),

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

7.66–7.61 (m, 1H), 7.36 (t, J ¼ 7.9 Hz, 1H), 7.25 (s, 2H), 6.57 (d, yloxy)-N-(5–(2-nitrophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A15).

J ¼ 2.0 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 1H), 4.40 (s, 2H), 4.01 (s, 3H), Light yellow solid, 45.03% yield, m.p.: 204–206 ^ꢁC; ¹H NMR

3.96 (s, 3H), 3.94 (s, 6H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d (600 MHz, CDCl₃) d 12.99 (s, 1H), 8.08 (dd, J ¼ 8.0, 1.2 Hz, 1H), 7.98

174.7, 166.6, 165.1, 161.2, 160.1, 159.1, 157.2, 154.5, 153.6 (2 C), (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.77 (td, J ¼ 7.6, 1.3 Hz, 1H), 7.73 (td,

141.2, 141.1, 134.3, 130.4, 129.5, 125.7, 125.2, 124.4, 122.9, 108.5, J ¼ 7.8, 1.5 Hz, 1H), 7.24 (s, 2H), 6.56 (d, J ¼ 2.2 Hz, 1H), 6.41 (d,

106.0 (2 C), 96.5, 92.8, 73.4, 61.0, 56.6, 56.5 (2 C), 55.9. HRMS (ESI): J ¼ 2.2 Hz, 1H), 4.38 (s, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.95 (s, 6H),

m/z [M þ H]^þcalcd for C₃₀H₂₇BrN₃O₁₀: 668.0874; found: 668.0871.

N-(5–(2-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A10).

3.93 (s, 3H). HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₇N₄O₁₂:

635.1620; found: 635.1619.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

White solid, 53.94% yield, m.p.: 199–201 ^ꢁC; ¹H NMR (600 MHz, yloxy)-N-(5–(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

CDCl₃) d 12.77 (s, 1H), 7.93 (d, J ¼ 7.7 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz, (A16). White solid, 51.73% yield, m.p.: 231–233 ^ꢁC; ¹H NMR

1H), 7.43 (t, J ¼ 7.6 Hz, 1H), 7.35 (dd, J ¼ 10.9, 4.5 Hz, 1H), 7.24 (s, (600 MHz, CDCl₃) d 12.78 (s, 1H), 8.22 (d, J ¼ 8.2 Hz, 2H), 7.75 (d,

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347

J ¼ 8.3 Hz, 2H), 7.25 (s, 2H), 6.57 (s, 1H), 6.43 (d, J ¼ 1.7 Hz, 1H),

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

4.41 (s, 2H), 4.01 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H), 3.94 (s, 3H). ¹³C yloxy)-N-(5–(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide (A22).

NMR (151 MHz, CDCl₃) d 174.7, 166.6, 165.1, 161.1, 160.3, 159.1, White solid, 38.43% yield, m.p.: 239–241 ^ꢁC; ¹H NMR (600 MHz,

157.4, 154.5, 153.6(2 C), 141.3, 141.1, 132.94 (m),127.1, 126.9 (2 C), CDCl₃) d 12.41 (s, 1H), 8.01 (d, J ¼ 8.5 Hz, 2H), 7.25 (s, 2H), 6.98 (d,

125.9 (d, J ¼ 3.6 Hz) (2 C), 124.4, 123.6, 108.5, 106.1 (2 C), 96.5, 92.8, J ¼ 8.6 Hz, 2H), 6.56 (d, J ¼ 1.5 Hz, 1H), 6.42 (d, J ¼ 1.3 Hz, 1H), 4.40

73.4, 61.0, 56.5 (3 C), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for (s, 2H), 4.00 (s, 3H), 3.98–3.92 (m, 12H), 3.87 (s, 3H). ¹³C NMR

C₃₁H₂₇F₃N₃O₁₀: 658.1643; found: 658.1641.

(151 MHz, CDCl₃) d 174.6, 166.6, 165.0, 162.1, 161.6, 161.1, 159.1,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3- 156.4, 154.32, 153.6 (2 C), 141.1, 141.1, 128.5 (2 C), 124.5, 116.4,

yloxy)-N-(5–(3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

114.3 (2 C), 108.5, 105.9 (2 C), 96.5, 92.8, 73.3, 61.1, 56.6, 56.5(2 C),

(A17). White solid, 54.83% yield, m.p.: 220–222 ^ꢁC; ¹H NMR

55.9, 55.4. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₁H₃₀N₃O₁₁:

(600 MHz, CDCl₃) d 12.87 (s, 1H), 8.33 (s, 1H), 8.28 (d, J ¼ 7.7 Hz, 620.1875; found: 620.1871.

1H), 7.76 (d, J ¼ 7.6 Hz, 1H), 7.63 (t, J ¼ 7.7 Hz, 1H), 7.25 (s, 2H),

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

6.56 (d, J ¼ 1.6 Hz, 1H), 6.42 (d, J ¼ 1.6 Hz, 1H), 4.40 (s, 2H), 3.99 (s, yloxy)-N-(5–(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide (A23).

3H), 3.98–3.89 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.8, 166.7, White solid, 43.92% yield, m.p.: 208–210 ^ꢁC; ¹H NMR (400 MHz,

165.1, 161.1, 160.2, 159.1, 157.3, 154.5, 153.6 (2 C), 141.2, 141.1, CDCl₃) d 12.63 (s, 1H), 7.68–7.64 (m, 1H), 7.63–7.61 (m, 1H), 7.39 (t,

131.6, 129.8, 129.6, 127.8, 124.7, 124.3, 123.6, 123.5, 108.4, 106.0 J ¼ 8.0 Hz, 1H), 7.25 (s, 2H), 7.07–7.03 (m, 1H), 6.57 (d, J ¼ 2.2 Hz,

(2 C), 96.5, 92.8, 73.4, 61.0, 56.5 (3 C), 55.9. HRMS (ESI): m/z 1H), 6.42 (d, J ¼ 2.2 Hz, 1H), 4.40 (s, 2H), 4.00 (s, 3H), 3.96 (s, 3H),

[M þ H]^þcalcd for C₃₁H₂₇F₃N₃O₁₀: 658.1643; found: 658.1642.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5–(2-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

(A18). White solid, 49.66% yield, m.p.: 191–193 ^ꢁC; ¹H NMR

3.95 (s, 6H), 3.93 (s, 3H), 3.88 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

174.6, 166.6, 165.0, 161.5, 161.1, 159.9, 159.1, 156.9, 154.4, 153.6

(2 C), 141.1, 141.1, 129.9, 124.9, 124.4, 119.2, 118.1, 111.2, 108.5,

105.9 (2 C), 96.5, 92.8, 73.4, 61.1, 55.5, 56.6, 56.5 (2 C), 55.9. HRMS

(600 MHz, CDCl₃) d 12.90 (s, 1H), 8.04 (d, J ¼ 7.2 Hz, 1H), 7.85 (d, (ESI): m/z [M þ H]^þcalcd for C₃₁H₃₀N₃O₁₁: 620.1875;

J ¼ 7.3 Hz, 1H), 7.72–7.64 (m, 2H), 7.24 (s, 2H), 6.55 (d, J ¼ 1.7 Hz, found: 620.1873.

1H), 6.41 (d, J ¼ 1.4 Hz, 1H), 4.40 (s, 2H), 3.97 (s, 3H), 3.96–3.91 (m,

N-(5–(3-chloro-4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dime-

12H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.5, 165.1, 161.3, 159.4, thoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)aceta-

159.0, 157.9, 154.4, 153.6 (2 C), 141.2, 141.1, 131.9, 131.9, 131.2, mide (A24). White solid, 31.79% yield, m.p.: 228–230 ^ꢁC; ¹H NMR

129.0, 126.8, 124.4, 123.2, 122.5, 108.5, 106.1 (2 C), 96.5, 92.7, 73.4, (600 MHz, CDCl₃) d 12.79 (s, 1H), 8.16 (d, J ¼ 6.7 Hz, 1H), 8.03–7.98

60.9, 56.5 (2 C), 56.4, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for (m, 1H), 7.28 (d, J ¼ 9.2 Hz, 1H), 7.26 (s, 2H), 6.57 (s, 1H), 6.44 (s,

C₃₁H₂₇F₃N₃O₁₀: 658.1643; found: 658.1641.

1H), 4.41 (s, 2H), 4.02 (s, 3H), 3.98–3.93 (m, 12H). ¹³C NMR

(151 MHz, CDCl₃) d 174.7, 166.6, 165.1, 161.2, 160.2, 159.1, 159.1,

157.2, 154.5, 153.6 (2 C), 141.3, 141.1, 129.1, 126.8, 124.34, 122.2,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5–(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

mide (A19). White solid, 51.51% yield, m.p.: 230–232 ^ꢁC; ¹H NMR 121.2, 117.3, 108.5, 106.1 (2 C), 96.5, 92.8, 73.4, 61.0, 56.6 (3 C),

55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₆ClFN₃O₁₀: 642.1285;

(600 MHz, CDCl₃) d 12.62 (s, 1H), 8.13 (d, J ¼ 8.6 Hz, 2H), 7.33 (d,

J ¼ 8.4 Hz, 2H), 7.25 (s, 2H), 6.56 (d, J ¼ 1.4 Hz, 1H), 6.42 (d, found: 642.1281.

J ¼ 1.2 Hz, 1H), 4.41 (s, 2H), 3.99 (s, 3H), 3.96–3.92 (m, 12H). ¹³C

N-(5–(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-4-

NMR (151 MHz, CDCl₃) d 174.6, 166.6, 165.1, 161.2, 160.4, 159.1, oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide (A25).

157.1, 154.4, 153.6 (2 C), 151.3 (d, J ¼ 1.5 Hz), 141.3, 141.1, 128.4 White solid, 36.16% yield, m.p.: 222–224 ^ꢁC; ¹H NMR (600 MHz,

(2 C), 124.4, 122.5, 121.1 (2 C), 120.3, 108.5, 106.1 (2 C), 96.5, 92.8, CDCl₃) d 12.87 (s, 1H), 8.18 (d, J ¼ 1.8 Hz, 1H), 7.94 (dd, J ¼ 8.4,

73.3, 61.0, 56.6 (2 C), 56.5, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for 1.9 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.25 (s, 2H), 6.57 (d, J ¼ 2.0 Hz,

C₃₁H₂₇F₃N₃O₁₁: 674.1592; found: 674.1590.

1H), 6.43 (d, J ¼ 1.9 Hz, 1H), 4.40 (s, 2H), 4.02 (s, 3H), 3.98–3.92 (m,

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3- 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.8, 166.6, 165.1, 161.1, 159.6,

yloxy)-N-(5–(3-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

159.1, 157.3, 154.5, 153.6 (2 C), 141.2, 141.1, 135.8, 133.5, 131.1,

mide (A20). White solid, 46.65% yield, m.p.: 221–223 ^ꢁC; ¹H NMR 128.3, 125.7, 124.3, 123.7, 108.4, 106.0 (2 C), 96.6, 92.8, 73.4, 61.0,

56.6, 56.5 (2 C), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

(600 MHz, CDCl₃) d 12.73 (s, 1H), 8.04 (d, J ¼ 7.7 Hz, 1H), 7.94 (s,

1H), 7.53 (t, J ¼ 8.0 Hz, 1H), 7.37 (d, J ¼ 7.4 Hz, 1H), 7.25 (s, 2H),

6.57 (d, J ¼ 1.9 Hz, 1H), 6.43 (d, J ¼ 1.7 Hz, 1H), 4.41 (s, 2H), 4.00 (s,

C₃₀H₂₆Cl₂N₃O₁₀: 658.0990; found: 658.0986.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

3H), 3.98–3.92 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.6, yloxy)-N-(5–(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide

165.1, 161.2, 160.2, 159.1, 157.2, 154.4, 153.6 (2 C), 149.6, 141.3, (A26). White solid, 47.13% yield, m.p.: 198–200 ^ꢁC; ¹H NMR

141.1, 130.5, 125.8, 124.9, 124.4, 123.6, 120.4, 119.2, 108.5, 106.1 (600 MHz, CDCl₃) d 12.47 (s, 1H), 7.25 (s, 2H), 7.23 (d, J ¼ 2.2 Hz,

(2 C), 96.5, 92.8, 73.4, 61.0, 56.6 (2 C), 56.5, 55.9. HRMS (ESI): m/z 2H), 6.59 (t, J ¼ 2.2 Hz, 1H), 6.56 (d, J ¼ 2.1 Hz, 1H), 6.42 (d,

[M þ H]^þcalcd for C₃₁H₂₇F₃N₃O₁₁: 674.1592; found: 674.1593.

J ¼ 2.1 Hz, 1H), 4.41 (s, 2H), 4.00 (s, 3H), 3.96 (s, 3H), 3.95 (s, 6H),

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3- 3.94 (s, 3H), 3.86 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6,

yloxy)-N-(5–(2-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

165.0, 161.5, 161.1, 161.0 (2 C), 159.1, 156.8, 154.4, 153.6 (2 C),

141.1, 141.0, 125.4, 124.4, 108.5, 105.9 (2 C), 104.5(2 C), 104.3, 96.5,

mide (A21). White solid, 35.65% yield, m.p.: 202–204 ^ꢁC; ¹H NMR

(600 MHz, CDCl₃) d 12.75 (s, 1H), 8.16 (dd, J ¼ 7.7, 1.1 Hz, 1H), 92.8, 73.3, 61.1, 56.6, 56.5 (2 C), 55.9, 55.7 (2 C). HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₂H₃₂N₃O₁₂: 650.1980; found: 650.1976.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

7.59–7.54 (m, 1H), 7.44 (t, J ¼ 7.7 Hz, 2H), 7.25 (s, 2H), 6.55 (d,

J ¼ 2.0 Hz, 1H), 6.41 (d, J ¼ 1.9 Hz, 1H), 4.41 (s, 2H), 3.98 (s, 3H),

3.97–3.90 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.5, yloxy)-N-(5–(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide

165.0, 161.3, 159.0, 158.4, 157.5, 154.3, 153.6 (2 C), 146.4 (d, (A27). White solid, 41.89% yield, m.p.: 220–222 ^ꢁC; ¹H NMR

J ¼ 1.7 Hz),141.2, 141.1, 132.5, 130.5, 127.2, 124.4, 122.1, 120.5, (600 MHz, CDCl₃) d 12.36 (s, 1H), 7.64 (d, J ¼ 8.3 Hz, 1H), 7.60 (s,

118.2, 108.6, 106.1 (2 C), 96.5, 92.7, 73.4, 61.0, 56.5 (2 C), 56.4, 55.9. 1H), 7.25 (s, 2H), 6.94 (d, J ¼ 8.4 Hz, 1H), 6.56 (d, J ¼ 1.5 Hz, 1H),

HRMS (ESI): m/z [M þ H]^þcalcd for C₃₁H₂₇F₃N₃O₁₁: 674.1592; 6.42 (d, J ¼ 1.4 Hz, 1H), 4.41 (s, 2H), 3.99 (s, 3H), 3.98–3.91 (m,

found: 674.1592.

18H). ¹³C NMR (151 MHz, CDCl₃) d 174.5, 166.6, 165.0, 161.7, 161.2,

348

X. HAN ET AL.

159.0, 156.5, 154.3, 153.6 (2 C), 151.8, 149.3, 141.2, 141.0, 124.4,

120.3, 116.6, 111.1, 109.5, 108.6, 106.1 (2 C), 96.5, 92.8, 73.3, 61.0,

56.6 (2 C), 56.5, 56.2, 55.9, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

C₃₂H₃₂N₃O₁₂: 650.1980; found: 650.1978.

(E)-2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chro-

men-3-yloxy)-N-(5-styryl-1,3,4-oxadiazol-2-yl)acetamide (A33). White

solid, 45.31% yield, m.p.: 223–225 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d

12.59 (s, 1H), 7.57 (d, J ¼ 16.7 Hz, 1H), 7.54 (d, J ¼ 7.4 Hz, 2H),

7.43–7.34 (m, 3H), 7.25 (s, 2H), 7.02 (d, J ¼ 16.4 Hz, 1H), 6.57 (d,

J ¼ 2.0 Hz, 1H), 6.43 (d, J ¼ 1.9 Hz, 1H), 4.40 (s, 2H), 4.01 (s, 3H),

3.98 ꢂ 3.92 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.5,

165.1, 161.2, 161.1, 159.1, 156.4, 154.4, 153.6 (2 C), 141.1, 141.2,

138.2, 134.8, 129.7, 128.9 (2 C), 127.4 (2 C), 124.4, 109.9, 108.5,

105.9 (2 C), 96.5, 92.8, 73.4, 61.0, 56.6, 56.6 (2 C), 55.9. HRMS (ESI):

m/z [M þ H]^þcalcd for C₃₂H₃₀N₃O₁₀: 616.1926; found: 616.1924.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)acetamide (B1). White solid,

46.92% yield, m.p.: 237–239 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.57

(s, 1H), 8.09 (d, J ¼ 6.9 Hz, 2H), 7.67 (d, J ¼ 8.5 Hz, 1H), 7.55 (s, 1H),

7.53–7.46 (m, 3H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.56 (s, 1H), 6.41 (s, 1H),

4.40 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). ¹³C

NMR (151 MHz, CDCl₃) d 174.7, 166.8, 164.9, 161.6, 161.2, 159.1,

156.9, 154.6, 151.9, 149.4, 140.8, 131.3, 128.9 (2 C), 126.8 (2 C),

123.9, 122.3, 121.9, 111.4, 111.1, 108.6, 96.5, 92.8, 73.3, 56.6, 56.3,

56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₆N₃O₉:

560.1664; found: 560.1665.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5-(naphthalen-1-yl)-1,3,4-oxadiazol-2-yl)acetamide

(A28).

White solid, 53.18% yield, m.p.: 238–240 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.59 (s, 1H), 9.27 (d, J ¼ 8.6 Hz, 1H), 8.24 (d, J ¼ 7.1 Hz,

1H), 8.01 (d, J ¼ 8.1 Hz, 1H), 7.91 (d, J ¼ 8.1 Hz, 1H), 7.68 (t,

J ¼ 7.6 Hz, 1H), 7.57 (dd, J ¼ 17.0, 8.2 Hz, 2H), 7.27 (s, 2H), 6.56 (s,

1H), 6.42 (s, 1H), 4.45 (s, 2H), 4.00 (s, 3H), 3.98–3.92 (m, 12H). ¹³C

NMR (151 MHz, CDCl₃) d 174.5, 166.6, 165.0, 161.6, 161.2, 159.1,

156.7, 154.3, 153.6 (2 C), 141.3, 141.1, 133.8, 132.1, 130.0, 128.5,

128.3, 127.9, 126.5, 126.4, 124.8, 124.5, 120.4, 108.6, 106.2 (2 C),

96.5, 92.8, 73.4, 61.0, 56.6 (2 C), 56.5, 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₄H₃₀N₃O₁₀: 640.1926; found: 640.1924.

N-(5–(2,3-dihydrobenzo[b][1,

4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)-

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)acetamide (A29). White solid, 54.62% yield, m.p.: 231–233 ^ꢁC;

¹H NMR (400 MHz, CDCl₃) d 12.51 (s, 1H), 7.60 (d, J ¼ 2.0 Hz, 1H),

7.57 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.24 (s, 2H), 6.93 (d, J ¼ 8.4 Hz, 1H),

6.56 (d, J ¼ 2.2 Hz, 1H), 6.41 (d, J ¼ 2.2 Hz, 1H), 4.39 (s, 2H), 4.30 (q,

J ¼ 5.1 Hz, 4H), 4.00 (s, 3H), 3.95 (s, 3H), 3.94 (s, 6H), 3.93 (s, 3H).

¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6, 165.0, 161.3, 161.1,

159.1, 156.5, 154.3, 153.5 (2 C), 146.4, 143.8, 141.1, 141.0, 124.5,

120.4, 117.8, 117.1, 115.9, 108.5, 105.9 (2 C), 96.5, 92.7, 73.4, 64.6,

64.2, 61.1, 56.6, 56.5 (2 C), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

C₃₂H₃₀N₃O₁₂: 648.1824; found: 648.1821.

N-(5–(3-(benzyloxy)phenyl)-1,3,4-oxadiazol-2-yl)-2–(5,7-dimethoxy-

4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)acetamide

(A30). White solid, 44.01% yield, m.p.: 188–190 ^ꢁC; ¹H NMR

(600 MHz, CDCl₃) d 12.54 (s, 1H), 7.73 (s, 1H), 7.68 (d, J ¼ 7.6 Hz,

1H), 7.46 (d, J ¼ 7.3 Hz, 2H), 7.39 (t, J ¼ 7.7 Hz, 3H), 7.33 (t,

J ¼ 7.2 Hz, 1H), 7.26 (s, 2H), 7.15–7.10 (m, 1H), 6.56 (d, J ¼ 1.7 Hz,

1H), 6.42 (d, J ¼ 1.6 Hz, 1H), 5.14 (s, 2H), 4.41 (s, 2H), 3.99 (s, 3H),

3.98–3.91 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6,

165.1, 161.4, 161.1, 159.1, 156.9, 154.4, 153.6, 141.1, 141.0, 136.5,

130.1, 128.6, 128.1, 127.6, 125.0, 124.4, 119.44, 118.6, 112.4, 108.5,

106.0, 96.5, 92.7, 73.4, 70.3, 61.0, 56.5, 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₇H₃₄N₃O₁₁: 696.2188; found: 696.2183.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)acetamide (A31). White

solid, 49.30% yield, m.p.: 240–242 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d

12.64 (s, 1H), 7.61 (d, J ¼ 0.9 Hz, 1H), 7.24 (s, 2H), 7.16 (d, J ¼ 3.2 Hz,

1H), 6.57 (dd, J ¼ 3.4, 1.7 Hz, 1H), 6.56 (d, J ¼ 2.1 Hz, 1H), 6.42 (d,

J ¼ 1.9 Hz, 1H), 4.39 (s, 2H), 4.00 (s, 3H), 3.97–3.92 (m, 12H). ¹³C

NMR (151 MHz, CDCl₃) d 174.6, 166.6, 165.0, 161.2, 159.1, 156.3,

154.4, 154.4, 153.6 (2 C), 145.3, 141.2, 141.1, 139.3, 124.4, 113.5,

111.9, 108.5, 106.1 (2 C), 96.5, 92.8, 73.3, 61.0, 56.5 (3 C), 55.9.

HRMS (ESI): m/z [M þ H]^þcalcd for C₂₈H₂₆N₃O₁₁: 580.1562;

found: 580.1562.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B2).

White solid, 54.11% yield, m.p.: 236–238 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.61 (s, 1H), 8.12–8.06 (m, 2H), 7.67 (dd, J ¼ 8.5, 2.0 Hz,

1H), 7.54 (d, J ¼ 1.9 Hz, 1H), 7.17 (dd, J ¼ 12.0, 5.3 Hz, 2H), 7.02 (d,

J ¼ 8.6 Hz, 1H), 6.57 (d, J ¼ 2.2 Hz, 1H), 6.41 (d, J ¼ 2.1 Hz, 1H), 4.40

(s, 2H), 4.01–3.96 (m, 9H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

174.6, 166.7, 164.9, 164.6, 161.1, 160.67, 159.0, 156.9, 154.5, 151.9,

149.3, 140.8, 128.9 (2 C), 122.2, 121.8, 120.3, 116.2 (2 C), 111.3,

110.9, 108.5, 96.4, 92.8, 73.2, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₂₉H₂₅FN₃O₉: 578.1569; found: 578.1569.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(3-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B3).

White solid, 34.63% yield, m.p.: 229–231 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.77 (s, 1H), 7.89 (d, J ¼ 6.8 Hz, 1H), 7.79 (d, J ¼ 8.2 Hz,

1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.55 (s, 1H), 7.47 (d, J ¼ 6.2 Hz, 1H),

7.21 (d, J ¼ 7.2 Hz, 1H), 7.02 (d, J ¼ 7.8 Hz, 1H), 6.57 (s, 1H), 6.42 (s,

1H), 4.40 (s, 2H), 4.04–3.90 (m, 12H). HRMS (ESI): m/z [M þ H]^þ

calcd for C₂₉H₂₅FN₃O₉: 578.1569; found: 578.1568.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B4).

White solid, 38.97% yield, m.p.: 235–237 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.70 (s, 1H), 8.07 (t, J ¼ 7.0 Hz, 1H), 7.67 (d, J ¼ 8.2 Hz,

1H), 7.54 (s, 1H), 7.50 (q, J ¼ 11.4, 6.7 Hz, 1H), 7.28 (d, J ¼ 7.6 Hz,

1H), 7.22 (d, J ¼ 9.3 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.56 (s, 1H),

6.41 (d, J ¼ 0.8 Hz, 1H), 4.40 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97

(s, 3H), 3.92 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.7,

164.9, 161.2, 159.9 (d, J ¼ 258.7 Hz), 159.0, 158.2, 157.2, 154.5,

151.9, 149.3, 140.7, 132.9, 129.7, 124.4, 122.2, 121.9, 116.8, 112.6,

111.4, 111.1, 108.6, 96.4, 92.7, 73.2, 56.5, 56.3, 56.1, 55.9. HRMS

(ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅FN₃O₉: 578.1569;

found: 578.1567.

2–(5,7-dimethoxy-4-oxo-2–(3,4,5-trimethoxyphenyl)-4H-chromen-3-

yloxy)-N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)acetamide

(A32).

White solid, 45.68% yield, m.p.: 224–226 ^ꢁC; ¹H NMR (400 MHz,

CDCl₃) d 12.60 (s, 1H), 7.78 (dd, J ¼ 3.7, 1.1 Hz, 1H), 7.52 (dd,

J ¼ 5.0, 1.0 Hz, 1H), 7.25 (s, 2H), 7.15 (dd, J ¼ 5.0, 3.8 Hz, 1H), 6.57

(d, J ¼ 2.2 Hz, 1H), 6.42 (d, J ¼ 2.2 Hz, 1H), 4.39 (s, 2H), 4.00 (s, 3H),

3.96 (s, 3H), 3.94 (s, 6H), 3.94 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

174.6, 166.6, 165.1, 161.1, 159.0, 157.9, 156.3, 154.4, 153.6 (2 C),

141.2, 141.0, 129.6, 129.5, 127.9, 125.2, 124.4, 108.5, 106.1 (2 C),

96.5, 92.8, 73.3, 61.0, 56.5 (3 C), 55.9. HRMS (ESI): m/z [M þ H]^þ

calcd for C₂₈H₂₆N₃O₁₀S: 596.1333; found: 596.1330.

N-(5–(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

(B5).

White solid, 42.10% yield, m.p.: 213–215 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.75 (s, 1H), 8.01 (d, J ¼ 8.1 Hz, 2H), 7.67 (d, J ¼ 8.4 Hz,

1H), 7.54 (s, 1H), 7.46 (d, J ¼ 8.2 Hz, 2H), 7.01 (d, J ¼ 8.5 Hz, 1H),

6.56 (s, 1H), 6.40 (s, 1H), 4.39 (s, 2H), 4.03–3.95 (m, 9H), 3.92 (s,

3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.7, 164.9, 161.1, 160.6,

159.0, 157.1, 154.6, 151.9, 149.3, 140.7, 137.5, 129.2 (2 C), 127.9

(2 C), 122.4, 122.2, 121.8, 111.3, 110.9, 108.4, 96.4, 92.7, 73.2, 56.5,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

349

56.2, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅ClN₃O₉:

594.1274; found: 594.1272.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5-p-tolyl-1,3,4-oxadiazol-2-yl)acetamide (B11). White solid,

41.50% yield, m.p.: 239–241 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.53

(s, 1H), 7.97 (d, J ¼ 8.1 Hz, 2H), 7.67 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.54 (d,

J ¼ 1.6 Hz, 1H), 7.28 (d, J ¼ 7.9 Hz, 2H), 7.01 (d, J ¼ 8.6 Hz, 1H), 6.56

(d, J ¼ 2.0 Hz, 1H), 6.40 (d, J ¼ 1.9 Hz, 1H), 4.39 (s, 2H), 3.99 (s, 3H),

3.98 (s, 3H), 3.96 (s, 3H), 3.92 (s, 3H), 2.41 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.7, 166.7, 164.9, 161.6, 161.1, 159.0, 156.7,

154.5, 151.8, 149.3, 141.8, 140.7, 129.5 (2 C), 126.7 (2 C), 122.2,

121.8, 121.1, 111.3, 110.9, 108.5, 96.4, 92.7, 73.2, 56.5, 56.2, 56.1,

55.9, 21.6. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₈N₃O₉:

574.1820; found: 574.1817.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5-m-tolyl-1,3,4-oxadiazol-2-yl)acetamide (B12). White solid,

35.96% yield, m.p.: 221–223 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.61

(s, 1H), 7.92 (s, 1H), 7.88 (d, J ¼ 7.6 Hz, 1H), 7.68 (d, J ¼ 7.9 Hz, 1H),

7.55 (s, 1H), 7.37 (t, J ¼ 7.6 Hz, 1H), 7.31 (d, J ¼ 7.4 Hz, 1H), 7.02 (d,

J ¼ 8.5 Hz, 1H), 6.57 (s, 1H), 6.41 (s, 1H), 4.40 (s, 2H), 4.02 ꢂ 3.96 (m,

9H), 3.93 (s, 3H), 2.42 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7,

166.7, 164.9, 161.6, 161.1, 159.0, 156.8, 154.5, 151.8, 149.3, 140.7,

138.7, 132.1, 128.7, 127.2, 123.9, 123.7, 122.2, 121.8, 111.3, 110.9,

108.5, 96.4, 92.7, 73.2, 56.6, 56.3, 56.1, 55.9, 21.3. HRMS (ESI): m/z

[M þ H]^þcalcd for C₃₀H₂₈N₃O₉: 574.1820; found: 574.1816.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5-o-tolyl-1,3,4-oxadiazol-2-yl)acetamide (B13). White solid,

49.04% yield, m.p.: 234–236 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.62

(s, 1H), 7.98 (d, J ¼ 7.7 Hz, 1H), 7.68 (dd, J ¼ 8.4, 1.3 Hz, 1H), 7.55 (s,

1H), 7.39 (t, J ¼ 7.4 Hz, 1H), 7.31 (dd, J ¼ 16.6, 8.0 Hz, 2H), 7.02 (d,

J ¼ 8.5 Hz, 1H), 6.56 (d, J ¼ 1.6 Hz, 1H), 6.41 (d, J ¼ 1.4 Hz, 1H), 4.40

(s, 2H), 4.01–3.96 (m, 9H), 3.93 (s, 3H), 2.73 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.6, 166.7, 164.9, 161.7, 161.1, 159.0, 156.7,

154.5, 151.8, 149.3, 140.7, 138.3, 131.5, 130.8, 128.9, 125.9, 123.0,

122.2, 121.9, 111.3, 110.9, 108.5, 96.4, 92.7, 73.2, 56.5, 56.3, 56.1,

55.9, 21.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₀H₂₈N₃O₉:

574.1820; found: 574.1818.

N-(5–(3-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

(B6).

White solid, 45.24% yield, m.p.: 220–222 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.81 (s, 1H), 8.08 (s, 1H), 7.99 (d, J ¼ 7.7 Hz, 1H), 7.68 (dd,

J ¼ 8.5, 1.8 Hz, 1H), 7.54 (d, J ¼ 1.7 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz, 1H),

7.43 (t, J ¼ 7.9 Hz, 1H), 7.02 (d, J ¼ 8.6 Hz, 1H), 6.57 (d, J ¼ 2.0 Hz,

1H), 6.41 (d, J ¼ 1.9 Hz, 1H), 4.40 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H),

3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.7,

164.9, 161.1, 160.2, 159.0, 157.2, 154.6, 151.9, 149.3, 140.7, 135.0,

131.3, 130.2, 126.6, 125.5, 124.7, 122.2, 121.8, 111.4, 111.1, 108.4,

96.4, 92.8, 73.2, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þ

calcd for C₂₉H₂₅ClN₃O₉: 594.1274; found: 594.1271.

N-(5–(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

(B7).

White solid, 31.57% yield, m.p.: 221–223 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.73 (s, 1H), 8.00 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.67 (dd,

J ¼ 8.5, 1.9 Hz, 1H), 7.56 ꢂ 7.52 (m, 2H), 7.44 (td, J ¼ 7.8, 1.5 Hz, 1H),

7.38 (td, J ¼ 7.6, 0.8 Hz, 1H), 7.02 (d, J ¼ 8.6 Hz, 1H), 6.56 (d,

J ¼ 2.0 Hz, 1H), 6.41 (d, J ¼ 2.0 Hz, 1H), 4.40 (s, 2H), 3.99–3.96 (m,

9H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.7, 164.9,

161.1, 159.6, 159.0, 157.3, 154.5, 151.8, 149.3, 140.8, 133.2, 132.0,

131.2, 131.0, 126.9, 123.3, 122.2, 121.8, 111.3,110.9, 108.5, 96.4,

92.7, 73.3, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

C₂₉H₂₅ClN₃O₉: 594.1274; found: 594.1270.

N-(5–(4-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

(B8).

White solid, 44.05% yield, m.p.: 212–214 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.73 (s, 1H), 7.98–7.94 (m, 2H), 7.68 (dd, J ¼ 8.5, 2.0 Hz,

1H), 7.65–7.61 (m, 2H), 7.54 (d, J ¼ 2.0 Hz, 1H), 7.02 (d, J ¼ 8.6 Hz,

1H), 6.57 (d, J ¼ 2.1 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 1H), 4.39 (s, 2H),

4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.7, 166.7, 164.9, 161.1, 160.7, 159.0, 157.1,

154.6, 151.9, 149.3, 140.8, 132.2 (2 C), 128.1 (2 C), 125.9, 122.8,

122.2, 121.8, 111.3, 110.9, 108.5, 96.4, 92.8, 73.3, 56.5, 56.3, 56.1,

55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅BrN₃O₉: 638.0769;

found: 638.0764.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B14).

Light yellow solid, 36.20% yield, m.p.: 235–237 ^ꢁC; ¹H NMR

(600 MHz, CDCl₃) d 13.10 (s, 1H), 8.91 (s, 1H), 8.46 (d, J ¼ 7.6 Hz,

1H), 8.37 (d, J ¼ 8.1 Hz, 1H), 7.70 (dd, J ¼ 18.7, 8.6 Hz, 2H), 7.55 (s,

1H), 7.03 (d, J ¼ 8.5 Hz, 1H), 6.58 (s, 1H), 6.43 (s, 1H), 4.41 (s, 2H),

4.03 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H), 3.94 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.9, 166.8, 165.0, 161.1, 159.3, 159.1, 157.6,

154.7, 151.9, 149.3, 148.6, 140.8, 132.2, 130.2, 125.7, 125.6, 122.3,

121.7, 121.5, 111.3, 110.9, 108.4, 96.5, 92.7, 73.4, 56.6, 56.2, 56.1,

55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅N₄O₁₁: 605.1514;

found: 605.1511.

N-(5–(3-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

(B9).

White solid, 39.16% yield, m.p.: 204–206 ^ꢁC; ¹H NMR (400 MHz,

CDCl₃) d 12.87 (s, 1H), 8.24 (t, J ¼ 1.7 Hz, 1H), 8.07–8.02 (m, 1H),

7.68 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.65–7.62 (m, 1H), 7.55 (d, J ¼ 2.0 Hz,

1H), 7.37 (t, J ¼ 7.9 Hz, 1H), 7.02 (d, J ¼ 8.6 Hz, 1H), 6.58 (d,

J ¼ 2.2 Hz, 1H), 6.42 (d, J ¼ 2.2 Hz, 1H), 4.40 (s, 2H), 4.01 (s, 3H),

3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

174.7, 166.7, 164.9, 161.1, 160.0, 159.0, 157.2, 154.6, 151.9, 149.3,

140.8, 134.2, 130.4, 129.4, 125.7, 125.2, 122.9, 122.2, 121.8, 111.3,

110.9, 108.4, 96.4, 92.7, 73.3, 56.6, 56.3, 56.1, 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₂₉H₂₅BrN₃O₉: 638.0769; found: 638.0765.

N-(5–(2-bromophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethoxy-

phenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide (B10).

White solid, 48.95% yield, m.p.: 226–228 ^ꢁC; ¹H NMR (600 MHz,

CDCl₃) d 12.80 (s, 1H), 7.94 (d, J ¼ 7.6 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz,

1H), 7.67 (d, J ¼ 8.4 Hz, 1H), 7.54 (s, 1H), 7.43 (t, J ¼ 7.5 Hz, 1H),

7.35 (t, J ¼ 7.7 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.55 (s, 1H), 6.40 (s,

1H), 4.40 (s, 2H), 4.00–3.95 (m, 9H), 3.92 (s, 3H). ¹³C NMR

(151 MHz, CDCl₃) d 174.6, 166.6, 164.9, 161.2, 160.2, 159.0, 157.4,

154.5, 151.9, 149.4, 140.7, 134.3, 132.1, 131.6, 127.3, 125.5, 122.2,

121.9, 121.7, 111.4, 111.1, 108.5, 96.4, 92.8, 73.24, 56.5, 56.3, 56.1,

55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅BrN₃O₉: 638.0769;

found: 638.0766.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(2-nitrophenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B15).

Light yellow solid, 46.53% yield, m.p.: 254–256 ^ꢁC; ¹H NMR

(600 MHz, DMSO-d₆) d 12.31 (s, 1H), 8.18 (d, J ¼ 8.0 Hz, 1H), 8.00 (d,

J ¼ 7.5 Hz, 1H), 7.95 (t, J ¼ 7.5 Hz, 1H), 7.91 (t, J ¼ 7.6 Hz, 1H), 7.75

(d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 8.4 Hz, 1H), 6.87 (s, 1H), 6.52 (s, 1H),

4.80 (s, 2H), 3.91 (s, 3H), 3.87–3.82 (m, 9H). ¹³C NMR (151 MHz,

DMSO-d₆) d 172.8, 167.2, 164.4, 160.8, 158.6, 158.3, 157.5, 152.3,

151.4, 148.9, 148.20, 139.4, 134.2, 133.7, 131.6, 125.3, 122.6, 122.2,

117.5, 112.0, 111.9, 108.5, 96.6, 93.6, 70.7, 56.6, 56.5, 56.1, 56.1.

HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₅N₄O₁₁: 605.1514;

found: 605.1510.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B16). White solid, 41.83% yield, m.p.: 210–212 ^ꢁC; ¹H NMR

(600 MHz, CDCl₃) d 12.94 (s, 1H), 8.22 (d, J ¼ 8.2 Hz, 2H), 7.75 (d,

350

X. HAN ET AL.

J ¼ 8.3 Hz, 2H), 7.68 (dd, J ¼ 8.5, 2.0 Hz, 1H), 7.55 (d, J ¼ 2.0 Hz, 1H), 122.2, 122.1, 121.9, 120.5, 118.2, 111.3, 111.1, 108.5, 96.4, 92.7,

7.02 (d, J ¼ 8.6 Hz, 1H), 6.57 (d, J ¼ 2.2 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 73.3, 56.4, 56.3, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

1H), 4.40 (s, 2H), 4.01 (s, 3H), 3.99 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). C₃₀H₂₅F₃N₃O₁₀: 644.1487; found: 644.1482.

¹³C NMR (151 MHz, CDCl₃) d 174.8, 166.8, 164.9, 161.0, 160.2,

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

159.0, 157.5, 154.7, 151.9, 149.3, 140.8, 132.9, 127.1, 126.9 (2 C), yloxy)-N-(5–(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide (B22).

125.9 (2 C), 123.6, 122.3, 121.7, 111.3, 110.9, 108.4, 96.5, 92.7, 73.3, White solid, 44.52% yield, m.p.: 206–208 ^ꢁC; ¹H NMR (600 MHz,

56.6, 56.2, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

CDCl₃) d 12.51 (s, 1H), 8.02 (d, J ¼ 8.7 Hz, 2H), 7.67 (d, J ¼ 8.5 Hz,

1H), 7.54 (s, 1H), 7.02 (d, J ¼ 8.5 Hz, 1H), 6.98 (d, J ¼ 8.7 Hz, 2H),

6.56 (s, 1H), 6.41 (s, 1H), 4.39 (s, 2H), 4.01–3.96 (m, 9H), 3.93 (s,

3H), 3.87 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.7, 164.9,

C₃₀H₂₅F₃N₃O₉: 628.1537; found: 628.1535.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

(B17). White solid, 50.80% yield, m.p.: 195–197 ^ꢁC; ¹H NMR 162.1, 161.5, 161.1, 159.0, 156.5, 154.5, 151.8, 149.3, 140.7, 128.5

(600 MHz, CDCl₃) d 12.90 (s, 1H), 8.34 (s, 1H), 8.30 (d, J ¼ 7.7 Hz, (2 C), 122.2, 121.9, 116.5, 114.3 (2 C), 111.3, 110.9, 108.5, 96.4, 92.7,

1H), 7.76 (d, J ¼ 7.5 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz, 1H), 7.64 (t, 73.2, 56.5, 56.3, 56.1, 55.9, 55.4. HRMS (ESI): m/z [M þ H]^þcalcd for

J ¼ 7.8 Hz, 1H), 7.54 (s, 1H), 7.02 (d, J ¼ 8.5 Hz, 1H), 6.57 (d, C₃₀H₂₈N₃O₁₀: 590.1769; found: 590.1769.

J ¼ 1.8 Hz, 1H), 6.42 (s, 1H), 4.40 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H),

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.8, 166.8, yloxy)-N-(5–(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)acetamide (B23).

165.0, 161.1, 160.1, 159.1, 157.4, 154.7, 151.9, 149.3, 140.8, 131.6, White solid, 47.70% yield, m.p.: 205–207 ^ꢁC; ¹H NMR (400 MHz,

129.8, 129.5, 127.8, 124.8, 123.6, 123.5, 122.3, 121.7, 111.3, 110.9, CDCl₃) d 12.67 (s, 1H), 7.70–7.64 (m, 2H), 7.63–7.60 (m, 1H), 7.54

108.4, 96.5, 92.7, 73.3, 56.5, 56.2, 56.1, 55.9. HRMS (ESI): m/z (d, J ¼ 2.0 Hz, 1H), 7.38 (t, J ¼ 8.0 Hz, 1H), 7.07–7.03 (m, 1H), 7.01

[M þ H]^þcalcd for C₃₀H₂₅F₃N₃O₉: 628.1537; found: 628.1533.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(2-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)acetamide

(d, J ¼ 8.6 Hz, 1H), 6.56 (d, J ¼ 2.2 Hz, 1H), 6.41 (d, J ¼ 2.2 Hz, 1H),

4.39 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H), 3.88

(s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.8, 164.9, 161.4,

(B18). White solid, 58.25% yield, m.p.: 225–227 ^ꢁC; ¹H NMR 161.1, 159.9, 159.1, 156.9, 154.5, 151.8, 149.2, 140.7, 129.9, 124.9,

(600 MHz, CDCl₃) d 12.99 (s, 1H), 8.05 (d, J ¼ 7.3 Hz, 1H), 7.85 (d, 122.2, 121.8, 119.2, 118.1, 111.2, 111.1, 110.9, 108.4, 96.4, 92.7,

J ¼ 7.4 Hz, 1H), 7.72–7.63 (m, 3H), 7.54 (s, 1H), 7.01 (d, J ¼ 8.6 Hz, 73.2, 56.6, 56.2, 56.1, 55.9, 55.5. HRMS (ESI): m/z [M þ H]^þcalcd for

1H), 6.55 (d, J ¼ 1.7 Hz, 1H), 6.40 (d, J ¼ 1.6 Hz, 1H), 4.39 (s, 2H), C₃₀H₂₈N₃O₁₀: 590.1769; found: 590.1764.

4.00–3.95 (m, 9H), 3.92 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.8,

N-(5–(3-chloro-4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dime-

166.7, 165.0, 161.3, 159.4, 159.1, 158.0, 154.6, 151.9, 149.4, 140.9, thoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

131.9, 131.3, 129.2, 129.0, 126.9, 123.2, 122.6, 122.3, 121.9, 111.4, (B24). White solid, 40.86% yield, m.p.: 232–234 ^ꢁC; ¹H NMR

111.1, 108.6, 96.5, 92.8, 73.4, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z (600 MHz, CDCl₃) d 12.85 (s, 1H), 8.16 (dd, J ¼ 6.9, 2.0 Hz, 1H),

[M þ H]^þcalcd for C₃₀H₂₅F₃N₃O₉: 628.1537; found: 628.1537.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

8.02–7.98 (m, 1H), 7.68 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.54 (d, J ¼ 1.7 Hz,

1H), 7.27 (t, J ¼ 8.6 Hz, 2H), 7.02 (d, J ¼ 8.6 Hz, 1H), 6.57 (d,

J ¼ 2.1 Hz, 1H), 6.42 (d, J ¼ 2.0 Hz, 1H), 4.39 (s, 2H), 4.01 (s, 3H),

mide (B19). White solid, 54.40% yield, m.p.: 217–219 ^ꢁC; ¹H NMR 3.99 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d

(600 MHz, CDCl₃) d 12.78 (s, 1H), 8.13 (d, J ¼ 8.7 Hz, 2H), 7.68 (dd, 174.8, 166.8, 164.9, 161.1, 160.1, 159.1, 159.0, 157.2, 154.7, 151.9,

J ¼ 8.5, 1.8 Hz, 1H), 7.54 (d, J ¼ 1.6 Hz, 1H), 7.33 (d, J ¼ 8.3 Hz, 2H), 149.3, 140.8, 129.1, 126.8, 122.3, 122.3, 121.8, 121.2, 117.3, 111.4,

7.02 (d, J ¼ 8.6 Hz, 1H), 6.57 (d, J ¼ 1.9 Hz, 1H), 6.42 (d, J ¼ 1.9 Hz, 110.9, 108.4, 96.5, 92.8, 73.3, 56.6, 56.3, 56.1, 55.9. HRMS (ESI): m/z

1H), 4.39 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). [M þ H]^þcalcd for C₂₉H₂₄ClFN₃O₉: 612.1180; found: 612.1180.

¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.8, 164.9, 161.0, 160.3,

N-(5–(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dimethox-

159.0, 157.2, 154.6, 151.8, 151.3, 149.2, 140.7, 128.4 (2 C), 122.4, yphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide (B25).

122.2, 121.7, 121.1 (2 C), 120.3, 111.2, 110.9, 108.4, 96.4, 92.7, 73.2, White solid, 35.80% yield, m.p.: 230–232 ^ꢁC; ¹H NMR (600 MHz,

56.5, 56.2, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for DMSO-d₆) d 12.25 (s, 1H), 8.04 (s, 1H), 7.87 (s, 2H), 7.77–7.72 (m,

C₃₀H₂₅F₃N₃O₁₀: 644.1487; found: 644.1484.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(3-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

2H), 7.11 (d, J ¼ 8.4 Hz, 1H), 6.85 (s, 1H), 6.51 (s, 1H), 4.80 (s, 2H),

3.90 (s, 3H), 3.88–3.84 (m, 6H), 3.83 (s, 3H). ¹³C NMR (151 MHz,

DMSO-d₆) d 172.8, 167.3, 164.5, 160.8, 159.3, 158.6, 157.9, 152.4,

mide (B20). White solid, 38.85% yield, m.p.: 207–209 ^ꢁC; ¹H NMR 151.5, 148.9, 139.4, 134.8, 132.7, 132.3, 127.9, 126.5, 124.3, 122.7,

(600 MHz, CDCl₃) d 12.78 (s, 1H), 8.04 (d, J ¼ 7.7 Hz, 1H), 7.94 (s, 122.2, 112.2, 111.9, 108.6, 96.6, 93.6, 70.9, 56.6, 56.5, 56.2, 56.1.

1H), 7.70–7.65 (m, 1H), 7.57–7.51 (m, 2H), 7.36 (d, J ¼ 7.7 Hz, 1H), HRMS (ESI): m/z [M þ H]^þcalcd for C₂₉H₂₄Cl₂N₃O₉: 628.0884;

7.02 (d, J ¼ 8.5 Hz, 1H), 6.57 (d, J ¼ 1.7 Hz, 1H), 6.42 (d, J ¼ 1.6 Hz, found: 628.0884.

1H), 4.40 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H).

N-(5–(3,5-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dime-

¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.7, 164.9, 161.1, 160.1, thoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

159.0, 157.3, 154.6, 151.9, 149.6, 149.4, 140.8, 130.5, 125.8, 124.9, (B26). White solid, 45.40% yield, m.p.: 200–202 ^ꢁC; ¹H NMR

123.5, 122.2, 121.8, 120.4, 119.2, 111.4, 111.1, 108.5, 96.4, 92.8, (600 MHz, CDCl₃) d 12.52 (s, 1H), 7.67 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.55

73.3, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for (d, J ¼ 1.8 Hz, 1H), 7.23 (d, J ¼ 2.2 Hz, 2H), 7.01 (d, J ¼ 8.6 Hz, 1H),

C₃₀H₂₅F₃N₃O₁₀: 644.1487; found: 644.1486.

6.59 (t, J ¼ 2.2 Hz, 1H), 6.56 (d, J ¼ 2.1 Hz, 1H), 6.41 (d, J ¼ 2.0 Hz,

1H), 4.40 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H),

3.85 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.7, 164.9, 161.5,

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

yloxy)-N-(5–(2-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)aceta-

mide (B21). White solid, 43.71% yield, m.p.: 226–228 ^ꢁC; ¹H NMR 161.1, 161.1 (2 C), 159.0, 156.9, 154.5, 151.8, 149.3, 140.7, 125.4,

(600 MHz, CDCl₃) d 12.82 (s, 1H), 8.16 (d, J ¼ 7.5 Hz, 1H), 7.67 (d,

122.2, 121.8, 111.3, 110.9, 108.5, 104.5 (2 C), 104.3, 96.4, 92.7, 73.2,

56.5, 56.3, 56.1, 55.9, 55.6 (2 C). HRMS (ESI): m/z [M þ H]^þcalcd for

J ¼ 8.1 Hz, 1H), 7.59–7.52 (m, 2H), 7.48–7.40 (m, 2H), 7.01 (d,

J ¼ 8.4 Hz, 1H), 6.56 (s, 1H), 6.40 (s, 1H), 4.40 (s, 2H), 4.01–3.90 (m,

12H). ¹³C NMR (151 MHz, CDCl₃) d 174.6, 166.6, 164.9, 161.2, 159.0,

C₃₁H₃₀N₃O₁₁: 620.1875; found: 620.1873.

N-(5–(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dime-

158.3, 157.6, 154.5, 151.9, 149.3, 146.4, 140.8, 132.4, 130.5, 127.2, thoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

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351

(B27). White solid, 42.37% yield, m.p.: 151–153 ^ꢁC; ¹H NMR 56.3, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₃₁H₂₈N₃O₁₁:

(600 MHz, CDCl₃) d 12.62 (s, 1H), 7.66 (d, J ¼ 8.5 Hz, 1H), 7.64 (d, 618.1718; found: 618.1715.

J ¼ 8.3 Hz, 1H), 7.59 (s, 1H), 7.54 (s, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H),

N-(5–(3-(benzyloxy)phenyl)-1,3,4-oxadiazol-2-yl)-2–(2-(3,4-dime-

6.93 (d, J ¼ 8.3 Hz, 1H), 6.55 (s, 1H), 6.40 (s, 1H), 4.38 (s, 2H), thoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-yloxy)acetamide

4.01–3.90 (m, 18H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.8, (B30). White solid, 47.88% yield, m.p.: 156–158 ^ꢁC; ¹H NMR

164.9, 161.6, 161.1, 159.0, 156.6, 154.6, 151.9, 151.8, 149.3(2 C), (600 MHz, CDCl₃) d 12.58 (s, 1H), 7.72 (s, 1H), 7.70–7.65 (m, 2H),

140.7, 122.2, 121.8, 120.3, 116.5, 111.3, 111.1, 111.0, 109.4, 108.5, 7.55 (d, J ¼ 1.5 Hz, 1H), 7.46 (d, J ¼ 7.3 Hz, 2H), 7.39 (t, J ¼ 7.4 Hz,

96.4, 92.8, 73.2, 56.5, 56.3, 56.2, 56.1, 55.9, 55.9. HRMS (ESI): m/z 3H), 7.33 (t, J ¼ 7.2 Hz, 1H), 7.11 (dd, J ¼ 8.1, 1.7 Hz, 1H), 7.01 (d,

[M þ H]^þcalcd for C₃₁H₃₀N₃O₁₁: 620.1875; found: 620.1872.

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

J ¼ 8.6 Hz, 1H), 6.56 (d, J ¼ 1.9 Hz, 1H), 6.40 (d, J ¼ 1.8 Hz, 1H), 5.14

(s, 2H), 4.40 (s, 2H), 4.00–3.90 (m, 12H). ¹³C NMR (151 MHz, CDCl₃)

yloxy)-N-(5-(naphthalen-1-yl)-1,3,4-oxadiazol-2-yl)acetamide

(B28). d 174.7, 166.7, 164.9, 161.4, 161.1, 159.1, 159.0, 156.9, 154.5, 151.9,

White solid, 51.26% yield, m.p.: 230–232 ^ꢁC; ¹H NMR (600 MHz, 149.3, 140.7, 136.5, 130.1, 128.6 (2 C), 128.1, 127.6 (2 C), 125.0,

CDCl₃) d 12.64 (s, 1H), 9.28 (d, J ¼ 8.4 Hz, 1H), 8.24 (d, J ¼ 7.1 Hz, 122.2, 121.8, 119.4, 118.6, 112.3, 111.3, 110.9, 108.5, 96.4, 92.7,

1H), 8.00 (d, J ¼ 7.9 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 7.68 (t, 73.2, 70.3, 56.5, 56.2, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for

J ¼ 7.9 Hz, 2H), 7.61–7.53 (m, 3H), 7.02 (d, J ¼ 8.5 Hz, 1H), 6.56 (s, C₃₆H₃₂N₃O₁₀: 666.2082; found: 666.2079.

1H), 6.41 (s, 1H), 4.44 (s, 2H), 4.03–3.96 (m, 9H), 3.93 (s, 3H). ¹³C

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

NMR (151 MHz, CDCl₃) d 174.7, 166.8, 164.9, 161.6, 161.2, 159.1, yloxy)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)acetamide (B31). White

156.8, 154.5, 151.9, 149.4, 140.8, 133.9, 132.2, 130.1, 128.5, 128.3, solid, 47.78% yield, m.p.: 233–235 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d

128.1, 126.6, 126.5, 124.9, 122.3, 121.9, 120.5, 111.4, 111.2, 108.6, 12.73 (s, 1H), 7.67 (dd, J ¼ 8.5, 1.9 Hz, 1H), 7.61 (d, J ¼ 0.9 Hz, 1H),

96.5, 92.8, 73.3, 56.6, 56.4, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þ7.54 (d, J ¼ 1.7 Hz, 1H), 7.16 (d, J ¼ 2.9 Hz, 1H), 7.01 (d, J ¼ 8.6 Hz,

calcd for C₃₃H₂₈N₃O₉: 610.1820; found: 610.1817.

N-(5–(2,3-dihydrobenzo[b][1,

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-ylox-

1H), 6.58–6.57 (m, 1H), 6.57 (d, J ¼ 2.2 Hz, 1H), 6.41 (d, J ¼ 1.9 Hz,

4]dioxin-6-yl)-1,3,4-oxadiazol-2-yl)- 1H), 4.39 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H).

¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.7, 164.9, 161.1, 159.0,

y)acetamide (B29). White solid, 50.60% yield, m.p.: 228–230 ^ꢁC; ¹H 156.4, 154.6, 154.3, 151.8, 149.3, 145.3, 140.8, 139.3, 122.2, 121.8,

NMR (600 MHz, CDCl₃) d 12.45 (s, 1H), 7.67 (dd, J ¼ 8.5, 1.6 Hz, 1H), 113.5, 111.9, 111.3, 110.9, 108.4, 96.4, 92.7, 73.2, 56.6, 56.2, 56.1,

7.60 (d, J ¼ 1.6 Hz, 1H), 7.57 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.55 (s, 1H), 55.9. HRMS (ESI): m/z [M þ H]^þcalcd for C₂₇H₂₄N₃O₁₀: 550.1456;

7.01 (d, J ¼ 8.6 Hz, 1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 6.56 (d, J ¼ 1.8 Hz, found: 550.1457.

1H), 6.41 (d, J ¼ 1.7 Hz, 1H), 4.39 (s, 2H), 4.30 (dd, J ¼ 12.1, 5.0 Hz,

2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-3-

(B32).

4H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR yloxy)-N-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl)acetamide

(151 MHz, CDCl₃) d 174.6, 166.7, 164.9, 161.3, 161.1, 159.0, 156.5, White solid, 49.73% yield, m.p.: 238–240 ^ꢁC; ¹H NMR (600 MHz,

154.5, 151.9, 149.3, 146.4, 143.8, 140.7, 122.2, 121.9, 120.4, 117.8, CDCl₃) d 12.54 (s, 1H), 7.78 (d, J ¼ 2.7 Hz, 1H), 7.67 (d, J ¼ 8.5 Hz,

117.2, 115.9, 111.3, 111.0, 108.5, 96.4, 92.7, 73.2, 64.6, 64.2, 56.5, 1H), 7.55 (s, 1H), 7.51 (d, J ¼ 4.5 Hz, 1H), 7.15 (t, J ¼ 4.2 Hz, 1H),

Scheme 1. Synthesis of title compounds A1–A33 and B1–B33. Reagent and conditions: (a) K₂CO₃, (CH₃)₂SO₄, acetone, reflux, 48 h; (b) Conc.HCl, EtOH, reflux, 2 h; (c)

AcONa, MeOH/H₂O, rt, K₂CO₃, I₂, 1,4-dioxane, 85 ^ꢁC, 5 h; (d) Chloroacetyl chloride, DMF, rt, 12 h; (e) K₂CO₃, KI, acetone, reflux, overnight.

352

X. HAN ET AL.

7.02 (d, J ¼ 8.5 Hz, 1H), 6.57 (s, 1H), 6.42 (s, 1H), 4.40 (s, 2H), 4.00

(s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR (151 MHz,

CDCl₃) d 174.6, 166.7, 164.9, 161.2, 159.0, 157.9, 156.3, 154.5,

151.9, 149.4, 140.7, 129.5, 129.4, 127.8, 125.3, 122.2, 121.9, 111.4,

111.1, 108.6, 96.4, 92.8, 73.2, 56.5, 56.3, 56.1, 55.9. HRMS (ESI): m/z

[M þ H]^þcalcd for C₂₇H₂₄N₃O₉S: 566.1228; found: 566.1227.

(E)-2–(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-

3-yloxy)-N-(5-styryl-1,3,4-oxadiazol-2-yl)acetamide (B33). White solid,

42.70% yield, m.p.: 226–228 ^ꢁC; ¹H NMR (600 MHz, CDCl₃) d 12.64

(s, 1H), 7.70–7.65 (m, 1H), 7.60–7.52 (m, 4H), 7.43–7.34 (m, 3H),

7.03 (d, J ¼ 2.4 Hz, 1H), 7.01 (d, J ¼ 5.1 Hz, 1H), 6.57 (d, J ¼ 1.9 Hz,

1H), 6.41 (d, J ¼ 1.8 Hz, 1H), 4.39 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H),

3.97 (s, 3H), 3.93 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) d 174.7, 166.6,

164.9, 161.1, 161.1, 159.1, 156.4, 154.6, 151.9, 149.3, 140.8, 138.1,

135.0, 129.6, 128.9, 127.4, 122.2, 121.8, 111.3, 110.9, 109.9, 108.5,

96.4, 92.8, 73.3, 56.6, 56.2, 56.1, 55.9. HRMS (ESI): m/z [M þ H]^þ

calcd for C₃₁H₂₈N₃O₉: 586.1820; found: 586.1816.

2.3. Telomerase activity assay²³

2.4. Cell culture

A375, MDA-MB-231, MGC-803, SMMC-7721, SGC-7901 and L-02

cell lines were cultured in DMEM medium supplemented with

10% (V/V) heat-inactivated fetal bovine serum (FBS) (Biological

Industries, Israel) along with 100 U/mL penicillin and 100 mg/mL

streptomycin (Beyotime, China). Cells were grown in a humidified

5% CO₂atmosphere at 37 ^ꢁC and maintained in a logarithmic

growth phase for all experiments.

2.5. Anticancer assay²³

2.6. Cell cycle assay

For cell cycle analysis, cell cycle kit (Beyotime, China) was per-

formed. MGC-803 cells were treated with compound A33 at differ-

ent concentrations for 48 h. Untreated and treated cells were

harvested, and then MGC-803 cells were washed three times using

cold PBS. And then cells were fixed in 70% ethanol at ꢂ20 ^ꢁC for

Figure 2. ORTEP drawing of compounds A11 and B8.

Table 1. Crystallographical and experimental data of compounds A11 and B8.

Properties

A11

C₃₁H₂₉N₃O₁₀

B8

C₂₉H₂₄BrN₃O₉

Chemical formula

Formula weight

603.57

638.42

Temperature/K

Crystal system

Space group

292.56(16)

Monoclinic

P2₁/n

293(2)

Monoclinic

P2₁/c

a/Å

b/Å

20.5711(10)

7.5520(3)

20.9843(11)

90

116.413(6)

90

2919.7(3)

4

1.369

11.1453(2)

29.8454(7)

8.3870(3)

90

101.156(3)

90

2737.11(12)

4

1.549

c/Å

a/^ꢁ

b/^ꢁ

c/^ꢁ

Volume/Å³

Z

q_calcg/cm³

l/mm¹

0.849

1.564

F(000)

1264.0

1304.0

Crystal size/mm³

2H range for data collection/^ꢁ

Index ranges

0.17 ꢀ 0.04 ꢀ 0.02

0.25 ꢀ 0.22 ꢀ 0.19

8.078 to 133.186

3.72–52

Reflections collected

Data/restraints/parameters

Goodness-of-fit on F²

Final R indexes [I ꢃ 2r (I)]

Final R indexes [all data]

Largest diff. peak/hole/e Å^ꢂ3

11523

5029/0/403

1.036

R₁¼ 0.0529, wR₂¼ 0.1381

R₁¼ 0.0739, wR₂¼ 0.1577

0.25/ꢂ0.23

22575

5385/0/379

1.012

R₁¼ 0.0472, wR₂¼ 0.1039

R₁¼ 0.0705, wR₂¼ 0.1137

0.62/ꢂ0.69

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

353

Table 2. Chemical structures of compounds A1–A33 and B1–B33 and inhibitory activity on telomerase.

Telomerase^a

Compds

A1

R¹

R²

Compds

B1

R¹

H

R²

IC₅₀(μM)

b

OCH₃

21.11 2.28

0.77 0.13

2.57 0.50

12.13 2.01

0.81 0.13

7.35 1.01

12.52 1.88

-

A2

B2

1.38 0.25

4.30 0.77

15.30 2.71

4.22 0.62

15.44 4.20

24.33 2.70

A3

B3

A4

B4

A5

B5

A6

B6

A7

B7

A8

A9

OCH₃

1.22 0.29

2.01 0.39

21.05 4.09

B8

B9

H

4.29 0.83

2.90 0.45

-

A10

B10

A11

A12

A13

A14

A15

OCH₃

2.35 0.48

15.33 2.85

25.08 2.01

4.11 0.60

6.98 0.96

B11

B12

B13

B14

B15

H

5.65 1.77

-

8.09 2.25

8.29 1.33

17.20 2.98

(continued)

354

X. HAN ET AL.

A16

A17

A18

A19

A20

A21

A22

A23

A24

A25

OCH₃

0.62 0.11

4.11 0.62

1.07 0.20

1.21 0.29

0.92 0.17

5.11 1.10

9.11 1.88

1.90 0.42

4.01 0.47

-

B16

B17

B18

B19

B20

B21

B22

B23

B24

B25

H

1.29 0.20

27.55 3.11

2.79 0.55

1.77 0.60

20.01 1.96

9.22 1.01

14.29 1.33

2.44 0.42

4.17 0.70

-

A26

A27

A28

OCH₃

1.06 0.11

0.32 0.07

6.17 0.99

B26

B27

B28

H

3.99 0.85

0.51 0.10

1.19 0.18

A29

A30

A31

A32

A33

OCH₃

-

B29

B30

B31

B32

H

2.98 0.27

-

2.75 0.19

2.22 0.51

2.82 0.25

3.75 0.39

0.44 0.09

6.41 1.38

B33

0.97 0.20

0.29 0.06

Staurosporine^c

BIBR1532^c

^aTelomerase supercoiling activity.

^bNo activity was observed in the concentration range of 0-60 μM.

^cStaurosporine and BIBR1532 were reported as a control.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

355

1 h. After fixation, cells were washed with cold PBS and stained 3.2. Crystal structure analysis

with 0.5 ml of propidium iodide (PI) staining buffer, which contain

200 mg/mL RNase A and 50 mg/mL PI, at 37 ^ꢁC for 30 min in the

dark. Analyses were conducted on FACSVerse Flow Cytometer

(Becton Dickinson). The experiments were repeated three times.

The structure of compounds A11 and B8 was further determined

by X-ray crystallography. The crystal data were presented in Table

1. The molecular structure of compounds A11 and B8 was

showed in Figure 2, respectively. Crystallographic data (excluding

structure factors) for the structure had been deposited with the

Cambridge Crystallographic Data Centre as supplementary publi-

cation No. CCDC 2010105 and 2005763.

2.7. Apoptosis assay

For cell apoptosis analysis, we employed annexin V-FITC/PI apop-

tosis detection kit (BestBio, China). MGC-803 cells in logarithmic

growth phase were treated with compound A33 at different con-

centrations for 48 h. Cells were collected in cold PBS by centrifu-

gation for 5 min at 1000 g. And then cells were re-suspended at a

buffer (1 ꢀ 10⁶cells/mL), stained with FITC-labeled annexin V and

PI for 20 min in the dark and immediately analysed on FACSVerse

Flow Cytometer (Becton Dickinson).

3.3. Telomerase inhibitory activity and SAR

All title compounds were assayed for telomerase activity using

MGC-803 cells extract, Staurosporine and BIBR1532 used as the

references²³. The results were presented as mean SD, summar-

ised in Table 2. Most of the title compounds demonstrated potent

inhibition against telomerase. Among these, compounds A2, A5,

A16, A20, A27, A33, B27 and B33 displayed significant inhibitory

activity (IC₅₀< 1 mM), with IC₅₀values of 0.77, 0.81, 0.62, 0.92,

0.32, 0.44, 0.51 and 0.97 mM, respectively, which were found to be

obviously superior to staurosporine (IC₅₀¼ 6.41 mM), and were

comparable to BIBR1532 (IC₅₀¼ 0.29 mM). Moreover, these com-

pounds have stronger telomerase inhibitory effect than the myri-

cetin²²and 1,3,4-oxadiazole³³derivatives we reported previously.

Based on the data of Table 2, the preliminary SARs analysis

revealed that except compounds A13, A25, A28, A29 and A30,

other compounds of A series (R¹¼ OCH₃) possessed higher tel-

omerase inhibitory activity than B series (R¹¼ H). Therefore, it

2.8. Western blotting

Human MGC-803 cells were lysed with RIPA lysis buffer (Beyotime,

China). Whole extracts were prepared, and protein concentration

was detected using a BCA protein assay kit (Beyotime, China). The

protein samples were separated by SDS-PAGE and blotted onto a

PVDF membrane (Millipore Corp, Billerica, MA). After blockade of

non-specific protein binding, nitrocellulose blots were incubated

at 4 ^ꢁC for 8 h with primary antibodies. After extensive washing in

TBS/Tween-20, the membranes were incubated at room tempera-

ture for 1 h with secondary antibodies. After washed in TBS/

Tween-20, the blots were processed with distilled water for detec-

tion of antigen using the enhanced chemiluminescence system.

Proteins were visualised with ECL-chemiluminescent kit (ECL-plus,

Thermo Scientific).

Table 3. Antiproliferative activity in vitro of compounds with strong telomerase

inhibitory activity (IC₅₀<1 lM) against A375, MDA-MB-231, MGC-803, SMMC-

7721 and SGC-7901 cell lines^a.

IC₅₀(lM)^b

Compounds

A375

MDA-MB-231 MGC-803 SMMC-7721 SGC-7901

2.9. Statistical analysis

A2

A5

11.03 1.54 25.06 2.10 17.26 2.21 8.07 1.30 56.91 1.24

20.09 0.62

10.09 0.52

c

–

15.22 0.41

–

25.36 0.59

All data are expressed as means SD. Student’s t-test was used to

determine statistical significance at p < 0.05. SPSS 17.0 and

Graphpad Prism 5 software were used for the statistical analyses.

A16

A20

A27

A33

B27

B33

–

8.92 0.69 19.50 1.00

6.29 0.36

10.22 0.65

–

11.21 0.69 9.89 0.44

8.76 0.25 9.67 0.82 10.01 0.51

–

3. Results and discussion

BIBR1532^d57.58 0.21

ADM^d

0.58 0.20 0.51 0.12

0.42 0.08 0.79 0.13 0.82 0.43

3.1. Chemistry

Negative control 0.1% DMSO, no activity.

Myricitrin and Rutin are used as raw materials. The hydroxyl

groups on the benzene ring were protected by methylation with

dimethyl sulphate, and the glycosides were removed under strong

acidic and reflux conditions to obtain 3-hydroxy-5,7-dimethoxy-

2–(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (1)²²and 2–(3,4-

dimethoxyphenyl)-3-hydroxy-5,7-dimethoxy-4H-chromen-4-one (2)³⁴,

respectively. Secondly, a series of 5-substituted-1,3,4-oxadiazol-2-

amines (3) were synthesised by the condensation of semicarbazide

hydrochloride and the corresponding aldehydes and following by

I₂4)³⁵were prepared from reacting of the intermediate 3 with

chloroethyl acid chloride in the presence of anhydrous DMF.

Finally, title compounds, 2-phenyl-4H-chromone derivatives con-

taining 1,3,4-oxadiazole and amide moieties, were synthesised by

refluxing the key intermediate 1 with 4 in the presence of K₂CO₃

and KI in acetone. The synthetic route of title compounds A1-A33

and B1-B33 was showed in Scheme 1. All title compounds were

characterised by means of ¹H NMR, ¹³C NMR and HR-MS spec-

tral analysis.

^aThe data represented the mean of three experiments in triplicate and were

expressed as means SD.

^bThe IC₅₀value was defined as the concentration at which 50% survival of cells

was observed. The results are listed in the table.

^cNot observed in the tested concentration range (>100 lM).

^dUsed as a positive control.

Table 4. Toxicity of compounds with strong telomerase inhibitory activity (IC₅₀

< 1 lM) against human normal liver cells L-02^a.

Compounds

L-02 (IC₅₀, mM)

A2

A5

1.37 0.43

1.99 0.13

0.90 0.11

0.62 0.24

1.38 0.21

2.21 0.17

1.67 0.25

1.01 0.14

A16

A20

A27

A33

B27

B33

^aMTT assays were used for evaluation, and values were expressed as mean IC₅₀

of the triplicate experiment.

356

X. HAN ET AL.

could be seen that the methoxy group (OCH₃) as a substituent at

Interestingly, as compared to compounds A22 and A23, com-

the R¹position played a vital role in telomerase inhibitory activity. pounds A26 and A27 bearing two the methoxy groups on the

The position, type and number of the substituents on the phe- phenyl ring at R²exhibited stronger activity. A similar trend was

nyl ring at R²and electronic effect had significant effects on inhib- also observed at compounds B22, B23, B26 and B27. However,

ition of telomerase. Firstly, by comparing compounds A2–A10, compounds A30 and B30 with a benzyl group at the meta pos-

when the phenyl ring at R²was substituted with halogen (F, Cl, ition of the phenyl ring at R²completely lost inhibitory activity,

Br), the inhibitory activity was para > meta > ortho. In addition, which might be affected by steric hindrance. Finally, we found

the substitution of halogen in the para and ortho position at the that replacement of the phenyl group at R²with aromatic fused

phenyl ring increased the activity with the increase of electro- rings and different aromatic heterocycles was also greatly import-

negativity (F > Cl > Br). A similar trend was also observed by com- ant for activity.

paring compounds B2-B7, B8, B10. Furthermore, compounds

As compared to compound A1, compounds A28, A31, A32

A24, A25, B24 and B25 disubstituted in the para and meta pos- substituted by naphthalene ring, furan ring and thiophene ring at

ition at the phenyl ring demonstrated significant reduction or R², respectively, displayed more potent inhibitory activity. A simi-

even complete loss of inhibitory activity as compared to the para- lar trend was also observed by comparing compounds B1, B28,

substituted compounds A2, A5, B2 and B5. Secondly, by compar- B31 and B32. In particular, replacement of the phenyl group at

ing compounds A2-A21, it was found that compounds with elec- R²with styryl yielded compound A33 and B33, which significantly

tron-withdrawing groups (F, Cl, Br, NO₂, CF₃, OCF₃) on the phenyl increased inhibitory activity as compared to compounds A1 and

ring at R²displayed higher inhibitory activity than those with elec- B1. It can be seen that styryl is crucial for activity and should be

tron-donating groups (CH₃).

further optimised in the future study.

Figure 3. Cell cycle distribution induced by compound A33 was measured in MGC-803 cells. Cells were treated with compound A33 of 3, 6 and 9 lM for 48 h.

Samples were analysed by flow cytometry and received results were analysed by modifit software.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

357

3.4. In vitro anticancer activity

lines as compared to other compounds. The results suggest that

compound A33 may have different mechanisms from BIBR1532 in

inhibiting telomerase activity, which supports that this compound

deserves further study.

The most active compounds A2, A5, A16, A20, A27, A33, B27

and B33 (IC₅₀< 1 mM) were selected to screen their in vitro anti-

cancer activity against A375 (human melanoma cell), MDA-MB-231

(human breast cancer cell), MGC-803 (human gastric cancer cell),

SMMC-7721 (human hepatoma cell) and SGC-7901 (human gastric

cancer cell) cell lines using MTT assay. Adriamycin (ADM) and

BIBR1532 were used as the references²². The IC₅₀values were

summarised in Table 3. In general, similar to the telomerase inhibi-

tor BIBR1532, most of compounds possessed excellent telomerase

inhibitory activity but no obvious antiproliferative activity against

solid cancer cells (A5, A16, A27, B27, B33). However, many title

compounds exhibited moderate antiproliferative activity on

3.5. Assay of human normal cell

In order to determine the selective cytotoxicity of selected com-

pounds, we subsequently conducted a proliferative inhibition

assay with human normal liver cell (L-02). The results were sum-

marised in Table 4. It was observed that the selected eight title

compounds all showed lower cytotoxicity. In particular, compound

human melanoma A375 cells (A2, A5, A16, A20, A33), which may A33 manifested an obvious non-toxic effect on L-02, with IC₅₀of

be due to the high expression of telomerase in human melanoma 2.21 mM. The data indicated that compound A33 displayed excel-

A375 cells³⁶. Besides, what should be of most concern was that lent selectivity against tumour cells over the normal somatic cells.

compound A33 with styryl, which demonstrated moderately Moreover, this compound exhibited lower cytotoxicity than 1,3,4-

effective antiproliferative activity against all tested five cancer cell oxadiazole derivatives reported previously³³. Therefore, in

Figure 4. Percentage of apoptotic cells was determined in MGC-803 cells by Annexin-V FITC/PI staining. MGC-803 cells were treated with increasing concentrations of

compound A33 for 48 h and stained with Annexin-V FITC/PI. Apoptotic ratio increased, accompanied with the increase of concentration.

358

X. HAN ET AL.

combination with the above points, it is quite meaningful to fur- induced by mechanical forces, environmental stimulus and so on;

ther explore the mechanisms of this compound.

the second quadrant generally denotes later period apoptotic cells

and necrotic cells; the third quadrant often represents early apop-

totic cells; and the fourth quadrant customarily denotes normal

cells. The percentage of AnnexinV-FITC binding MGC-803 cells sig-

nificantly increased from 4.27% to 11.39, 57.31 and 86.96%,

respectively, after 48 h of treatment with increasing concentrations

of compound A33. The results show that compound A33 can

induce apoptosis of MGC-803 cells in a concentration-dependent

manner. This is consistent with the fact that telomerase inhibitors

can induce apoptosis and thus inhibit the unlimited proliferation

of tumour cells³⁷.

3.6. Cell cycle analysis

The results of anticancer activity showed that compound A33

could inhibit proliferation of MGC-803 cells. To verify whether cell

cycle arrest leads to decrease cells proliferation, we used flow

cytometric analysis to measure the effect of this compound on

induction of cell cycle. As shown in Figure 3, treatment of MGC-

803 cells with increasing concentrations (3, 6, 9 mM) of compound

A33 for 48 h, increased the G2/M phase distribution by 45.37%

(from 9.76 to 55.13%), whereas the G0/G1 and S phase distribu-

tion decreased from 52.71 to 32.62% and from 37.61 to 12.25% in

MGC-803 cells, respectively. In a word, this compound can induce

cell cycle arrest at G2/M phase in a concentration-dependent

manner, delaying cell cycle progression, thereby resulting in cell

proliferation inhibition.

3.8. Down-regulated expression of Dyskerin-NOP10-NHP2

Dyskerin-NOP10-NHP2, trimer proteins are the core components

of telomerase, playing a key role in the stabilisation, activation

and assembly of telomerase, and the loss of dyskerin function can

influence telomerase activity. Dyskerin over-expression associated

with a variety of tumour types has been reported³⁸. To test

whether compound A33 can modulate the expression of the tri-

mer proteins, we used Western blotting. As shown in Figure 5,

treatment with different concentrations (3, 6, 9 mM) of compound

A33 for 48 h (MGC-803 cells were selected), expression level of

3.7. Cell apoptosis analysis

To determine whether compound A33 meditated inhibition of

proliferation was related with apoptosis, MGC-803 cells was

selected for examination. The Annexin V-FITC/PI apoptosis detec-

tion kit was used in cell apoptosis analysis. As shown in Figure 4, dyskerin protein was reduced in a concentration-dependent man-

the first quadrant usually represents damaged cells which was ner. Meanwhile, NHP2³⁹and NOP10⁴⁰, as the important

Figure 5. Compound A33 inhibited Dyskerin expression in MGC-803 cells. MGC-803 cells were treated with compound A33 of 3, 6 and 9 lM for 48 h. The proteins

expression of Dyskerin, NOP10 and NHP2 were analysed by Western blotting. The results are expressed as relative expression against control expression. n ¼ 3. Results

ꢄ

ꢄꢄ

are shown as mean SD from three independent experiments. p < 0.05, p < 0.01.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

359

components of dyskerin-NHP2-NOP10 trimer, had also been

assessed together. The results indicated that expressions of

NOP10 and NHP2 were also lower level than control group.

Therefore, compound A33 may be an efficient dyskerin regulator.

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on telomere targeting in cancer. Mol Carcinogen 2019;58:

1581–8.

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4. Conclusions

With the aim to discover highly efficient telomerase inhibitors,

upon extensive optimisation, a total of 66 2-phenyl-4H-chromone

derivatives containing amide and 1,3,4-oxadiazole moieties were

designed and synthesised. Most of the title compounds demon-

strated potent telomerase inhibitory activity. SARs studies showed

that the substitution of the methoxy group at R¹was very advan-

tageous for telomerase activity, and the substitution of halogen

for the para position of the phenyl ring at R²significantly

improved the telomerase inhibitory activity. However, replacing

phenyl ring at R²with aromatic fused rings, aromatic heterocycles

and other substituents had also the significant effect on telomer-

ase activity. In particular, compound A33 substituted by styryl at

R²not only possessed strong activity against telomerase, but also

exhibited moderately effective antiproliferative activity against all

tested five human cancer cell lines, which was superior to tel-

omerase inhibitor BIBR1532. Furthermore, it had no obvious tox-

icity towards human normal L-02 cell with IC₅₀of 2.21 mM. Flow

cytometric analysis indicated that MGC-803 cell cycle was arrested

in the G2/M phase by this compound, inducing MGC-803 cells

apoptosis. Western blotting revealed that compound A33 could

significantly decrease the expression of dyskerin. In conclusion, it

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Disclosure statement

No potential conflict of interest was reported by the author(s).

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Funding

This work was supported by the National Natural Science Funding

of China [21977001].

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Supporting information

The following files are available free. ¹H NMR and ¹³C NMR spec-

tra and HRMS of all compounds. Fitting plot of compounds A2,

A5, A16, A20, A27, A33, B27 and B33 for telomerase activity.

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Article Doi

Synthesis, telomerase inhibitory and anticancer activity of new 2-phenyl-4H-chromone derivatives containing 1,3,4-oxadiazole moiety

DOI: 10.1080/14756366.2020.1864630

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Article abstract of DOI:10.1080/14756366.2020.1864630

Full text of DOI:10.1080/14756366.2020.1864630

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