Synthesis and Anticancer Activity of 3-(Substituted Aroyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole Derivatives

Article abstract of DOI:10.1002/cbdv.201600219

A series of 3-(substituted aroyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole derivatives were synthesized and determined for their anticancer activity against eleven cancer cell lines and two normal tissue cell lines using MTT assay. Among the synthesized comp

Full text of DOI:10.1002/cbdv.201600219

Received Date: 20-Jun-2016
Revised Date: 26-Sep-2016
Accepted Date: 28-Sep-2016
Article Type: Full Paper
Synthesis and anticancer activity of 3-(substituted aroyl)-4-
(3,4,5-trimethoxy phenyl)-1H-pyrrole derivatives
Xiaoping Zhan a, Lan Lan a, Shuai Wang a, Kai Zhao a, Yuxuan Xin a, Qi Qi a, Yaolin Wang a, and
Zhenmin Mao a, *
a School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China
* Corresponding author: Zhenmin Mao. E-mail: zmmao@sjtu.edu.cn
Abstract:
A series of 3-(substituted aroyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole derivatives were synthesized and
determined for their anticancer activity against eleven cancer cell lines and two normal tissue cell lines using MTT
assay. Among the synthesized compounds, compound 3f was the most potent compound against A375, CT-26, Hela,
MGC80-3, NCI-H460 and SGC-7901 cells (IC50s = 8.2－31.7 μM); 3g, 3n and 3a were the most potent compounds
against CHO (IC50 = 8.2 μM), HCT-15 (IC50 = 21 μM) and MCF-7 cells (IC50 = 18.7 μM), respectively. Importantly, all the
target compounds showed no cytotoxicity towards the normal tissue cell (IC50 > 100 μM). Thus, these compounds with
the potent anticancer activity and low toxicity have potential for the development of new anticancer chemotherapy
agents.
Keywords: Pyrrole derivatives; Anticancer activity; Synthesis; MTT assay; Cytotoxicity
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Introduction.- Cancer is considered a major public health problem worldwide because it is a major cause of death
affecting millions of people worldwide. Chemotherapy has been widely employed for various cancer treatments.
However traditional chemotherapeutic agents also kill normal tissue cells during killing tumor cells, chemotherapeutic
agents in clinical use usually induce multidrug resistance, toxicity and mutation [1]. Hence, design and synthesis of
novel anticancer agents with potent anticancer activity and minimum side effects is still one of the greatest medical
challenges.
The N-containing heterocyclic unit is usually found in some natural anti-cancer products, thus it attracts many
researchers’ interest in the drug discovery. Neda et al. reported a series of 1,2,4-oxadiazole derivatives, these
compounds exhibited excellent potency against many cancer cell lines and even the IC50 values (half maximal inhibitory
concentration) of some compounds are less than 0.1 μM [2-5]. Pyrrole is another important five-membered N-containing
heterocycle, the compounds bearing pyrrole moiety are found in many natural and artificial products with the broad-
spectrum pharmacological effects including antifungal [6-8], antimicrobial [9-11], antivirus [12-15], anticancer [16-18],
cyclooxygenase inhibitors [19-21], antidiabeti [22] and antianxiety [23]. Recently a new range of possible
chemotherapeutic compounds have been studied using pyrrole derivatives, which have been demonstrated to have
good anti-proliferative activity. Temburnikar et al. explored the pyrrolo[3,2-d]pyrimidines which showed significant
antiproliferative activity against cancer cells [24]. Pérez et al. reported the synthesis of a series of ferrocene coupling
with pyrrole complexes and their cytotoxic activity against MCF-7 breast cancer, MCF-10A normal breast and HT-29
colon cancer cell lines [25]. Ghorab et al. published a series of pyrrole and fused pyrrole derivatives including
pyrrolopyrimidines,
pyrazolopyrrolopyrimidine,
triazolopyrrolopyrimidines,
tetrazolopyrrolopyrimidine,
triazinopyrrolopyrimidines and pyrrolopyrimidotriazepines, and their cytotoxic activity against MCF-7 cell [26]. Dyson et
al. synthesized a series of the natural product oroidin analogues, which is a pyrrole alkaloid isolated from the marine
sponge agelas oroides, and evaluated their cytotoxicity against twelve cancer cell lines [27].
In our previous work, we found that pyrrole derivatives (Fig. 1) showed potent and broad-spectrum anticancer
activity against human gastric cancer, breast cancer, malignant melanoma, osteosarcoma and large cell lung cancer
[28-30]. When the moieties at the 4th-position of the pyrrole ring is 4-methoxy phenyl, the IC50 values (half maximal
inhibitory concentration) of some pyrrole derivatives are less than 20 μM, these compounds show good anti-proliferative
activity [29]. When the moieties at the 4th-position is 4-methylthio phenyl, the IC50 values of some pyrrole derivatives
achieve micromolar level, these compounds show potent anticancer activity [30].
Figure 1.The structures and bioactivity of the pyrrole derivatives
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Considering methoxy and methylthio belonging to electron-donating groups, we suppose that other electron-
donating groups maybe increase the anticancer activity. Thus, 3, 4, 5-trimethoxy phenyl is condensed at the 4th-position
of the pyrrole ring in this article, and the moieties at the 3rd-position of the pyrrole ring are still substituted aroyl or
hetero aroyl referred to our previous work (Fig. 2). All the synthesized target compounds were confirmed by
1
spectroscopic methods like H NMR, 13C NMR and HRMS spectrometry and screened for their anticancer activity in
vitro.
Figure 2. The structure of the target compounds
Results and Discussion
Chemistry. The target compounds were synthesized according to the methods described in our previous work [28-
30]. All target compounds were obtained through a facile two-steps reaction: (1) preparation of the intermediates,
namely, 3,4,5-trimethoxy benzaldehyde 1 condensing with substituted benzoyl under base-catalyzed conditions to give
the corresponding intermediates 2a-o; (2) synthesis of the target compounds, namely, intermediates reacting with
TosMIC reagent under ice-bath conditions to give the target compounds 3a-o (Scheme 1).
Scheme 1. Synthetic route of the target compounds
Biological evaluation. The cytotoxicity activity of these target compounds was evaluated using the [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay against A375 (human malignant melanoma), CHO
(Chinese hamster ovary), CT-26 (murine colon carcinoma), DU145 (human prostate cancer), HCT-15 (human colorectal
adenocarcinoma), Hela (human cervical carcinoma), MCF-7 (human breast cancer)，MG-63 (human osteosarcoma),
MGC80-3 (human gastric cancer), NCI-H460 (human large cell lung cancer), SGC-7901 (human gastric
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adenocarcinoma), NIH/3T3 (murine embryonic fibroblast) and HUVEC (human umbilical vein endothelial). The
calculated IC50 values of tested compounds are presented in Table 1.
Among the tested target compounds, compound 3f bearing 4-bromophenyl was the most potent against A375 (IC50
= 11.1 μM), CT-26 (IC50 = 14.7 μM), Hela (IC50 = 12.7 μM), MGC80-3 (IC50 = 8.2 μM), NCI-H460 (IC50 = 21.4 μM) and
SGC-7901 (IC50 = 31.7 μM); 3g bearing 4-methyl phenyl was the most potent against CHO (IC50 = 8.2 μM); 3n bearing
2-naphthyl was the most potent against HCT-15 (IC50 = 21 μM); 3a bearing 2-fluro phenyl was the most potent against
MCF-7 (IC50 = 18.7 μM).
All the target compounds showed good anti-proliferative activity against MGC80-3 (IC50s = 8.2－23.4 μM) except the
compounds 3l bearing 3-pyridyl and 3m bearing 2-thienyl (IC50s > 100 μM). However, all the target compounds showed
poor anti-proliferative activity against DU145 (IC50s > 100 μM) except 3f (IC50 = 45.2 μM) and 3g (IC50 = 43.5 μM) with
moderate anticancer activity. And all the target compounds showed poor anticancer activity against MG-63 (IC50s > 100
μM) except 3f (IC50 = 64.7 μM), 3n (IC50 = 49.9 μM), 3o bearing 4-biphenyl (IC50 = 57.1 μM) with moderate anticancer
activity.
There are only the IC50 values of the compounds 3f inhibiting against CHO and MGC80-3 and 3g inhibiting against
CHO were micromolar level in the Table 1. However, many compounds in the series of 3-(substituted aroyl)-4-
(methylthio phenyl) pyrrole derivatives could achieve this level in the publications [30]. In comparison with the
compounds bearing 4-methylthio phenyl moiety, the anticancer activity of the target compounds bearing 3, 4, 5-
trimethoxy phenyl moiety didn’t significantly increase. But compared with the pyrrole derivatives bearing 4-methoxy
phenyl moiety in the publications [29], the target compounds in this article showed better anticancer activity.
Compared with the in vitro activity of the chemotherapy reagents approved for cancer therapy, namely, taxol and 5-
fluorouracil (5-Fu), no target compounds exhibited stronger anticancer activity against eleven cancer cell lines than
taxol, but compound 3f showed more potent anticancer activity against A375 (1.2-fold difference), CHO (1.2-fold
difference), MGC80-3 (1.8-fold difference) and NCI-H460 (1.3-fold difference) than 5-Fu. Similar to the recent
publications [28-30], compound 3f showed no cytotoxicity towards normal tissue cells. It‘s very important to cancer
therapy that the chemotherapy reagents have the properties of high effectiveness and low toxicity. Thus, compound 3f
has potential for the development of the new chemotherapy agent.
Table 1. The IC50 values of the target compounds
IC50 (μM)
Comp.
A375
CHO
CT-
26
DU1
45
HCT-
15
Hela
MCF-7
MG-63
MGC80
-3
NCI-
H460
SGC-
7901
NIH/3T
3
HUVEC
3a
3b
3c
3d
3e
3f
>100
--
51.8
72.7
20.7
67.3
67.3
9.4
--a)
--
--
>100
>100
>100
>100
>100
12.7
44
18.7b)
--
>100
--
22.5
14.4
23.4
19.5
19.5
8.2
--
--
--
--
--
--
--
>100
--
--
--
--
>100
>100
>100
11.1
50
>100
>100
>100
14.7
>100
>100
--
>100
>100
>100
45
--
--
--
--
--
--
>100
>100
64.7
>100
--
--
--
--
--
--
--
--
--
--
45.2
43.5
49.5
>100
21.4
>100
31.7
76
>100
--
>100
--
3g
8.2
>100
13.9
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3h
--
68.1
11.2
22.1
24.8
62.4
96.5
11.4
27.6
1.4
>100
37
--
>100
>100
>100
>100
>100
>100
21
21.9
14.5
19.1
47.1
89.9
>100
56.6
14
--
>100
>100
>100
>100
--
12.3
19.5
20.7
19.9
>100
>100
22.4
14.3
4.2
>100
--
>100
66
>100
>100
>100
--
--
3i
46.6
>100
>100
59.2
>100
18.4
>100
5.6
>100
--
>100
>100
>100
>100
--
>100
>100
>100
--
3j
45.5
76.6
>100
>100
21.3
16.5
9.7
--
92.5
--
3k
--
>100
--
3l
>100
--
>100
--
--
3m
3n
>100
49.9
57.1
20.5
27.8
--
--
--
99
--
82.8
22.5
6.9
28.7
86.9
--
>100
>100
>100
83.2
80.1
>100
>100
92.3
3o
>100
7.8
8.3
--
21.8
5.6
Taxol
5-Fu
12.5
10.1
1.7
15.1
10.2
13.7
11.4
7.6
9.3
10.2
14.7
a) “-- “ meant IC50 > 300 μM. b) “bold type” meant IC50 < 20 μM
Conclusions
In conclusion, we report the synthesis of a series of 3-(substituted aroyl)-4-(3,4,5-trimethoxy phenyl) pyrrole
derivatives. All the synthesized target compounds were evaluated for their cytotoxicity activity against eleven cancer cell
lines and two normal cell lines. Among the target compounds, compound 3f was the most potent compound against
A375, CT-26, Hela, MGC80-3, NCI-H460 and SGC-7901; 3g, 3n and 3a were the most potent compounds against
CHO, HCT-15 and MCF-7 cells, respectively. Moreover, 3f inhibiting against CHO and MGC80-3 and 3g inhibiting
against CHO achieved micromolar level.
Acknowledgements
This project is supported by National Major Scientific and Technological Special Project of the PR China
(2010ZX09401-404).
Experimental Part
Chemistry. All chemical reagents were purchased from commercial supplies and were of analytical grade. Flash
column chromatography was performed on a column packed with 200-300 mesh silica gel 60 or neutral aluminum oxide.
1
Thin-layer chromatography (TLC) was performed on silica gel 60 F254-plate with a fluorescent indicator. H NMR (400
MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker Advanced III 400 spectrometer with DMSO-d6 as
solvent and TMS as the internal standard. High-resolution mass spectra (HRMS) data were obtained on an Agilent 6230
ToF mass spectrometer using the electrospray ionization (ESI) technique. Melting points were recorded on a
microscopic melting point apparatus (SGW X-4, Shanghai Precision & Scientific Instrument Co., Ltd.) and were
uncorrected.
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General procedure for the synthesis of the target compounds 3a-o. 10.0 mmoles of 3,4,5-trimethoxy benzaldehyde
1 was mixed with 12.0 mmoles of substituted benzoyl under base condition. The mixture was stirred at room
temperature for 2―8 h. The end of the reaction was monitored by TLC. Afterwards, water (20 mL) and ethyl acetate (75
mL, 25 mL × 3) were added into the mixture. The inorganic precipitate was removed, and the organic solution was
washed with brine three times and dried. The crude product was purified by silica gel column chromatography with ethyl
acetate/hexane as the eluent to get the pure intermediate 2a-o.
The above intermediate (10.0 mmoles), toluenesulfonylmethyl isocyanide (TosMIC, 11.0 mmoles) and potassium
tert-butoxide (12.0 mmoles) were added to an anhydrous tetrahydrofuran (20 mL) solution, and the mixture was stirred
in an ice bath for 1 h. Ice water (50 mL) was poured into the mixture, tetrahydrofuran was removed under vacuum, and
the solution was extracted by ethyl acetate (75 mL, 25 mL × 3). The combined organic layer solution was washed with
saturated brine and dried over anhydrous sodium sulfate. The crude product was purified by aluminum oxide column
chromatography with ethyl acetate/hexane as the eluent to get pure target compound.
1
3-(2-fluorobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3a). White solid, yield 87.5 %, mp 170-172 ºC; H NMR
(400 MHz, DMSO-d6)  (ppm): 11.70 (s, 1H, pyrrole-NH), 7.50-7.44 (m, 2H, -CO-Ar-H), 7.22 (dd, J=8.0, 4.0 Hz, 2H, -
CO-Ar-H), 7.17 (s, 1H, pyrrole-H), 7.08 (s, 1H, pyrrole-H), 6.75 (s, 2H, Ar-H), 3.74 (s, 6H, 3’, 5’-OCH3), 3.64 (s, 3H, 4’-
OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 186.61, 158.77 (1C, JCF=247.5 Hz), 152.05 (2C), 136.05, 131.80 (1C,
JCF=8.2 Hz), 130.31, 129.72, 129.66 (1C, JCF=3.9 Hz), 129.53, 125.26, 124.03 (1C, JCF=3.4 Hz), 121.70, 120.14, 115.77
(1C, JCF=21.9 Hz), 106.39 (2C), 59.92, 55.70 (2C); ESI-HRMS m/z: calcd for C20H18FNO4 ([M+H]): 356.1299; found:
356.1305.
1
3-(4-fluorobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3b). Pale yellow solid, yield 81.0 %, mp 209-211 ºC; H
NMR (400 MHz, DMSO-d6)  (ppm): 11.66 (s, 1H, pyrrole-NH), 7.74 (dd, J=8.0, 4.0 Hz, 2H, -CO-Ar-H), 7.28-7.26 (m,
2H, -CO-Ar-H), 7.22 (s, 1H, pyrrole H), 7.12 (s, 1H, pyrrole H), 6.63 (s, 2H, Ar-H), 3.69 (s, 6H, 3’, 5’-OCH3), 3.62 (s, 3H,
4’-OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 189.18, 163.91 (1C, JCF=249.1 Hz), 152.18 (2C), 136.59 (1C,
JCF=2.9 Hz), 135.89, 131.58 (2C, JCF=9.0 Hz), 130.70, 127.67, 125.41, 120.62, 119.42, 114.89 (2C, JCF=21.7 Hz),
106.15 (2C), 59.95, 55.70 (2C); ESI-HRMS m/z: calcd for C20H18FNO4 ([M+H]): 356.1299; found: 356.1305.
1
3-(2-chlorobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3c). White solid, yield 89.9 %, mp 167-169 ºC; H NMR
(400 MHz, DMSO-d6)  (ppm): 11.71 (s, 1H, pyrrole-NH), 7.48 (t, J=8.0 Hz, 1H, -CO-Ar-H), 7.43 (d, J=8.0 Hz, 2H, -CO-
Ar-H), 7.38 (d, J=8.0 Hz, 1H, -CO-Ar-H), 7.10 (s, 1H, pyrrole H), 7.03 (s, 1H, pyrrole H), 6.82 (s, 2H, Ar-H), 3.78 (s, 6H,
3’, 5’-OCH3), 3.67 (s, 3H, 4’-OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 184.50, 152.06 (2C), 140.76, 136.10,
130.43, 130.24, 130.09, 129.55, 129.47, 128.56, 126.70, 125.25, 121.16, 120.45, 106.37 (2C), 59.94, 55.71 (2C); ESI-
HRMS m/z: calcd for C20H18ClNO4 ([M+H]): 372.1003, 374.0974; found: 372.1008, 374.0985.
1
3-(4-chlorobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3d). White solid, yield 77.4 %, mp 200-202 ºC; H NMR
(400 MHz, DMSO-d6)  (ppm): 11.69 (s, 1H, pyrrole-NH), 7.69 (d, J=8.0 Hz, 2H, -CO-Ar-H), 7.47 (d, J=8.0 Hz, 2H, -CO-
Ar-H), 7.29 (s, 1H, pyrrole H), 7.12 (s, 1H, pyrrole H), 6.64 (s, 2H, Ar-H), 3.70 (s, 6H, 3’, 5’-OCH3), 3.63 (s, 3H, 4’-OCH3);
13C NMR (100 MHz, DMSO-d6)  (ppm): 189.31, 152.17 (2C), 138.76, 136.09, 135.95, 130.71 (2C), 130.63, 128.04 (2C),
127.97, 125.45, 120.50, 119.53, 106.21 (2C), 59.98, 55.71 (2C); ESI-HRMS m/z: calcd for C20H18ClNO4 ([M+H]):
372.1003, 374.0974; found: 372.1013, 374.0990.
3-(3-bromobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3e). Yellow solid, yield 71.5 %, mp 202-204 ºC; 1H NMR
(400 MHz, DMSO-d6)  (ppm): 11.71 (s, 1H, pyrrole-NH), 7.69-7.53 (m, 3H, -CO-Ar-H), 7.44-7.35 (m, 1H, -CO-Ar-H),
7.32 (s, 1H, pyrrole H), 7.10 (s, 1H, pyrrole H), 6.60 (s, 2H, Ar-H), 3.68 (s, 6H, 3’, 5’-OCH3), 3.61 (s, 3H, 4’-OCH3); 13
C
NMR (100 MHz, DMSO-d6)  (ppm): 188.93, 152.18 (2C), 142.09, 135.88, 133.85, 131.46, 130.59, 130.19, 127.98,
127.65, 125.53, 121.25, 120.48, 119.48, 106.23 (2C), 59.86, 55.68 (2C); ESI-HRMS m/z: calcd for C20H18BrNO4 ([M+H]):
416.0498, 418.0478; found: 416.0513, 418.0496.
1
3-(4-bromobezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3f). Pale yellow solid, yield 91.3 %, mp 200-202 ºC; H
NMR (400 MHz, DMSO-d6)  (ppm): 11.69 (s, 1H, pyrrole-NH), 7.59 (s, 4H, -CO-Ar-H), 7.28 (s, 1H, pyrrole H), 7.11 (s,
1H, pyrrole H), 6.63 (s, 2H, Ar-H), 3.69 (s, 6H, 3’, 5’-OCH3), 3.62 (s, 3H, 4’-OCH3); 13C NMR (100 MHz, DMSO-d6) 
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(ppm): 189.44, 152.17 (2C), 139.10, 135.94, 130.98 (2C), 130.89 (2C), 130.62, 125.45, 125.06, 120.43, 119.55, 106.21
(2C), 60.00, 55.71 (2C); ESI-HRMS m/z: calcd for C20H18BrNO4 ([M+H]): 416.0498, 418.0478; found: 416.0518,
418.0500.
3-(4-methylbezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3g). White solid, yield 76.0 %, mp 214-216 ºC; 1H NMR
(400 MHz, DMSO-d6)  (ppm): 11.59 (s, 1H, pyrrole-NH), 7.61 (d, J=8.0 Hz, 2H, -CO-Ar-H), 7.23 (s, 1H, pyrrole H), 7.20
(s, 2H, -CO-Ar-H), 7.11 (s, 1H, pyrrole H), 6.65 (s, 2H, Ar-H), 3.68 (s, 6H, 3’, 5’-OCH3), 3.62 (s, 3H, 4’-OCH3), 2.33 (s,
3H, PhCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 190.31, 152.17 (2C), 141.46, 137.38, 135.85, 130.85, 129.12 (2C),
128.54 (2C), 127.38, 125.31, 120.83, 119.27, 106.06 (2C), 59.96, 55.69 (2C), 20.97; ESI-HRMS m/z: calcd for
C21H21NO4 ([M+H]): 352.1550; found: 352.1576.
1
3-(2,4-dimethylbezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3h). White solid, yield 69.1 %, mp 164-166 ºC; H
NMR (400 MHz, DMSO-d6)  (ppm): 11.57 (s, 1H, pyrrole-NH), 7.22 (d, J=8.0 Hz, 1H, -CO-Ar-H), 7.06 (s, 1H, pyrrole H),
7.04 (s, 1H, pyrrole-H), 6.97 (s, 2H, -CO-Ar-H), 6.75 (s, 2H, Ar-H), 3.73 (s, 6H, 3’, 5’-OCH3), 3.64 (s, 3H, 4’-OCH3), 2.27
(s, 3H, PhCH3), 2.22 (s, 3H, PhCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 192.25, 152.06 (2C), 138.62, 138.49,
135.97, 135.17, 131.03, 130.66, 129.09, 128.02, 125.39, 125.22, 122.15, 119.93, 106.27 (2C), 59.96, 55.69 (2C), 20.77,
19.36; ESI-HRMS m/z: calcd for C22H23NO4 ([M+H]): 366.1701; found: 366.1736.
3-(3-methoxybezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3i). White solid, yield 79.1 %, mp 166-168 ºC; 1H NMR
(400 MHz, DMSO-d6)  (ppm): 11.64 (s, 1H, pyrrole-NH), 7.32 (t, J=8.0 Hz, 1H, pyrrole H), 7.28-7.22 ( m, 2H, -CO-Ar-H),
7.15 (s, 1H, pyrrole H), 7.11-7.05 (m, 2H, -CO-Ar-H), 6.63 (s, 2H, Ar-H), 3.72 (s, 3H, -CO-Ar-OCH3), 3.68 (s, 6H, 3’, 5’-
OCH3), 3.61 (s, 3H, 4’-OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 190.22, 158.79, 152.15 (2C), 141.47, 135.86,
130.80, 129.09, 127.85, 125.41, 121.33, 120.73, 119.42, 117.45, 113.63, 106.15 (2C), 59.93, 55.68 (2C), 55.09; ESI-
HRMS m/z: calcd for C21H21NO5 ([M+H]): 368.1499; found: 368.1533.
3-(4-methoxybezoyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3j). White solid, yield 82.8 %, mp 210-212 ºC; 1H NMR
(400 MHz, DMSO-d6)  (ppm): 11.57 (s, 1H, pyrrole-NH), 7.72 (d, J=8.0 Hz, 2H, -CO-Ar-H), 7.21 (s, 1H, pyrrole H), 7.12
(s, 1H, pyrrole H), 6.96 (d, J=8.0 Hz, 2H, -CO-Ar-H), 6.64 (s, 2H, Ar-H), 3.80 (s, 3H, -CO-Ar-OCH3), 3.68 (s, 6H, 3’, 5’-
OCH3), 3.62 (s, 3H, 4’-OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 189.54, 162.00, 152.20 (2C), 135.79, 132.46,
131.24 (2C), 130.90, 126.64, 125.18, 120.93, 119.03, 113.27 (2C), 105.96 (2C), 59.95, 55.68 (2C), 55.34; ESI-HRMS
m/z: calcd for C21H21NO5 ([M+H]): 368.1499; found: 368.1538.
1
3-(2-pyridylformyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3k). Pale yellow solid, yield 68.3 %, mp 155-157 ºC; H
NMR (400 MHz, DMSO-d6)  (ppm): 11.65 (s, 1H, pyrrole-NH), 8.62 (d, J=8.0 Hz, 1H, pyridine H), 7.95 (td, J1=8.0 Hz,
J2=2.0 Hz, 1H, pyridine H), 7.80 (td, J=8.0 Hz, J2=0.8 Hz, 1H, pyridine H), 7.70 (dd, J1=4.0 Hz, J2=2.0 Hz, 1H, pyrrole H),
7.54 (m, 1H, pyridine H), 7.06 (t, J=4.0 Hz, 1H, pyrrole H), 6.72 (s, 2H, Ar-H), 3.75 (s, 6H, 3’, 5’-OCH3), 3.67 (s, 3H, 4’-
OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 187.75, 156.93, 152.04 (2C), 148.21, 137.00, 135.92, 131.08, 130.38,
126.07, 125.60, 123.04, 119.38, 119.25, 106.36 (2C), 59.95, 55.73 (2C); ESI-HRMS m/z: calcd for C19H18N2O4 ([M+H]):
339.1346; found: 339.1387.
1
3-(3-pyridylformyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3l). Pale yellow solid, yield 50.0 %, mp 163-165 ºC; H
NMR (400 MHz, DMSO-d6)  (ppm): 11.77 (s, 1H, pyrrole-NH), 8.80 (s, 1H, pyridine H), 8.68 (dd, J1=4.0 Hz, J2=2.0 Hz,
1H, pyridine H), 8.01 (dt, J1=8.0 Hz, J2=2.0 Hz, 1H, pyridine H), 7.48-7.41 (m, 1H, pyridine H), 7.36 (t, J=4.0 Hz, 1H,
pyrrole H), 7.15 (t, J=4.0 Hz, 1H, pyrrole H), 6.68 (s, 2H, Ar-H), 3.71 (s, 6H, 3’, 5’-OCH3), 3.64 (s, 3H, 4’-OCH3); 13C
NMR (100 MHz, DMSO-d6)  (ppm): 188.78, 152.18 (2C), 151.65, 149.36, 136.16, 135.96, 135.58, 130.46, 128.61,
125.46, 123.21, 120.59, 119.84, 106.30 (2C), 59.94, 55.73 (2C); ESI-HRMS m/z: calcd for C19H18N2O4 ([M+H]):
339.1346; found: 339.1390.
3-(2-thienylformyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3m). White solid, yield 79.0 %, mp 161-163 ºC; 1H NMR
(400 MHz, DMSO-d6)  (ppm): 11.66 (s, 1H, pyrrole-NH), 7.92 (dd, J1=4.0 Hz, J2=1.2 Hz, 1H, thiophene H), 7.65 (dd,
J1=4.0 Hz, J2=1.2 Hz, 1H, thiophene H), 7.50 (t, J=4.0 Hz, 1H, pyrrole H), 7.18 (dd, J1=4.0 Hz, J2=1.2 Hz, 1H, thiophene
H), 7.14 (t, J=4.0 Hz, 1H, pyrrole H), 6.70 (s, 2H, Ar-H), 3.73 (s, 6H, 3’, 5’-OCH3), 3.65 (s, 3H, 4’-OCH3); 13C NMR (100
MHz, DMSO-d6)  (ppm): 182.09, 152.25 (2C), 145.65, 135.92, 133.12, 132.98, 130.70, 128.11, 126.50, 125.03, 120.42,
119.47, 105.98 (2C), 59.97, 55.73 (2C); ESI-HRMS m/z: calcd for C18H17NO4S ([M+H]): 344.0957; found: 344.1008.
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3-(2-naphthylformyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3n). Brown solid, yield 89.1 %, mp 157-159 ºC; 1H
NMR (400 MHz, DMSO-d6)  (ppm): 11.51 (s, 1H, pyrrole-NH), 7.97 (dd, J1=8.0 Hz, J2=2.8 Hz, 1H, naphthalene H),
7.91 (d, J=8.0 Hz, 2H, naphthalene H), 7.51-7.46 (m, 3H, naphthalene H), 7.40 (t, J=8.0 Hz, 1H, naphthalene H), 7.14 (t,
J=4.0 Hz, 1H, pyrrole H), 7.03 (t, J=4.0 Hz, 1H, pyrrole H), 6.57 (s, 2H, ArH), 3.68 (s, 6H, 3’, 5’-OCH3), 3.63 (s, 3H, 4’-
OCH3); 13C NMR (100 MHz, DMSO-d6)  (ppm): 192.13, 152.47 (2C), 139.19, 136.38, 133.60, 131.11, 130.64, 130.26,
129.81, 128.70, 127.14, 126.82, 126.52, 126.09, 125.80, 125.05, 123.14, 120.46, 106.83 (2C), 60.36, 56.05 (2C); ESI-
HRMS m/z: calcd for C24H21NO4 ([M+H]): 388.1550; found: 388.1609.
3-(4-biphenylformyl)-4-(3,4,5-trimethoxy phenyl)-1H-pyrrole (3o). Pale yellow solid, yield 61.7 %, mp 211-213 ºC;
1H NMR (400 MHz, DMSO-d6)  (ppm): 11.66 (s, 1H, pyrrole-NH), 7.79 (d, J=6.0 Hz, 2H, Ar-H), 7.72 (d, J=6.0 Hz, 2H,
Ar-H), 7.70 (d, J=6.0 Hz, 2H, Ar-H), 7.48 (t, J=6.0 Hz, 2H, Ar-H), 7.40 (t, J=6.0 Hz, 1H, Ar-H), 7.30 (t, J=6.0 Hz, 1H,
pyrrole H), 7.13 (t, J=6.0 Hz, 1H, pyrrole H), 6.67 (s, 2H, Ar-H), 3.69 (s, 6H, 3’, 5’-OCH3), 3.59 (s, 3H, 4’-OCH3); 13C
NMR (100 MHz, DMSO-d6)  (ppm): 190.06, 152.19 (2C), 142.94, 139.20, 138.89, 135.91, 130.79, 129.70 (2C), 129.01
(2C), 128.04, 127.80, 126.81 (2C), 126.25 (2C), 125.43, 120.76, 119.47, 106.16 (2C), 59.94, 55.72 (2C); ESI-HRMS
m/z: calcd for C26H23NO4 ([M+H]): 414.1706; found: 414.1769.
In vitro cytotoxicity studies. Cell culture. All cell lines: A375, CHO, CT-26, DU145, HCT-15, Hela, MCF-7, MG-63,
MGC80-3, NCI-H460, SGC-7901, NIH/3T3 and HUVEC were purchased from Type Culture Collection of Chinese
Academy of Science (Shanghai, China). The cells were cultured in RPMI-1640 (Hyclon) or high glucose DMEM (Hyclon)
medium with 10% heat-inactivated fetal bovine serum (Hyclon) and 1% penicillin-streptomycin (Hyclon), and incubated
at a standard culture condition (37 °C, 5% CO2 in air) (Thermo Fisher Scientific, USA). The culture medium was
refreshed every 2 days.
MTT assay. The logarithmic growth phase cells were collected and seeded into 96-well plates at a seeding density
5000 cells/well and incubated at 37 °C for 24 h. The cells were treated with different concentration of drugs, and four
parallel wells were arranged for each concentration. The blank control groups were prepared by the same procedure
without sample treatment. After 24 h treatment, remove the treatment medium, and then add 150 μL of culture medium
and 20 μL of MTT solution (5 mg/mL in PBS) to each well. After 4 h incubation, discard the medium and add 100 μL of
DMSO to each well, and then shake plate for 10 min for dissolving the formazan crystals. The percentage of cell viability
was determined by measuring the absorption at 570 nm using a Multiskan MK3 microplate reader (Thermo, USA).
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