Straightforward synthesis, characterization, and cytotoxicity evaluation of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones

Article abstract of DOI:10.1080/10286020.2018.1540599

Dozens of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones were synthesized in a straightforward method by condensation of substituted 2H-thieno[2,3-d][1, 3]oxazine-2,4(1H)-diones or N-methyl-2H-thieno[2,3-d][1, 3]oxazine-2,4(1H)-dione with 3,4-dihydro-β-carbolines. In vitro cytotoxic assay discovered that compounds 9a, 10e, 11a, 11d, 11f, and 12a could induce antiproliferation against four different types of human cancer cells while compounds 10f and 12e were inactive. Notably, compound 11a displayed potent cell cytotoxicity for human non-small cell lung cancer cells A549, PC-9, human prostate cancer cells PC-3, and human breast cancer cell line MCF-7. Furthermore, compound 11a exhibited strong colony formation inhibition to A549 cells. These results unfold potential anticancer therapeutic applications of hybrids of thieno[2,3-d]pyrimidinones and quinazolinones.

Full text of DOI:10.1080/10286020.2018.1540599

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Straightforward synthesis, characterization,
and cytotoxicity evaluation of hybrids of
natural alkaloid evodiamine/rutaecarpine and
thieno[2,3-d]pyrimidinones
Li-Fei Nie, Si-Si Wang, Jian-Guo Cao, Fei-Ze Liu, Hainimu Xiamuxi, Haji Akber
Aisa & Guo-Zheng Huang
To cite this article: Li-Fei Nie, Si-Si Wang, Jian-Guo Cao, Fei-Ze Liu, Hainimu Xiamuxi, Haji Akber
Aisa & Guo-Zheng Huang (2018): Straightforward synthesis, characterization, and cytotoxicity
evaluation of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2018.1540599
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1540599
Straightforward synthesis, characterization, and
cytotoxicity evaluation of hybrids of natural alkaloid
evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones
c
c
Li-Fei Niea,b , Si-Si Wang , Jian-Guo Cao , Fei-Ze Liua,b, Hainimu Xiamuxia,b
,
ꢀ
ꢀ
Haji Akber Aisaa and Guo-Zheng Huanga,c
aKey Laboratory of Plant Resources and Chemistry of Arid Zone, State Key Laboratory Basis of
Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and
b
Chemistry, Chinese Academy of Sciences, Urumqi 830011, China; University of Chinese Academy of
c
Sciences, Beijing 100049, China; College of Life and Environmental Sciences, Shanghai Normal
University, Shanghai 201418, China
ABSTRACT
ARTICLE HISTORY
Received 15 June 2018
Revised 18 October 2018
Accepted 18 October 2018
Dozens of hybrids of natural alkaloid evodiamine/rutaecarpine
and thieno[2,3-d]pyrimidinones were synthesized in a straightfor-
ward method by condensation of substituted 2H-thieno[2,3-d][1,
3]oxazine-2,4(1H)-diones or N-methyl-2H-thieno[2,3-d][1, 3]oxa-
zine-2,4(1H)-dione with 3,4-dihydro-b-carbolines. In vitro cytotoxic
assay discovered that compounds 9a, 10e, 11a, 11d, 11f, and
12a could induce antiproliferation against four different types of
human cancer cells while compounds 10f and 12e were inactive.
Notably, compound 11a displayed potent cell cytotoxicity for
human non-small cell lung cancer cells A549, PC-9, human pros-
tate cancer cells PC-3, and human breast cancer cell line MCF-7.
Furthermore, compound 11a exhibited strong colony formation
inhibition to A549 cells. These results unfold potential anticancer
therapeutic applications of hybrids of thieno[2,3-d]pyrimidinones
and quinazolinones.
KEYWORDS
Thieno[2,3-d]pyrimidinone;
evodiamine; rutaecarpine;
hybrid; cytotoxicity
CONTACT Haji Akber Aisa
haji@ms.xjb.ac.cn; Guo-Zheng Huang
g.huang@ms.xjb.ac.cn
Key Laboratory of
Plant Resources and Chemistry of Arid Zone, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants
Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi
830011, China.
ꢀ
These authors contribute equally.
Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2018.1540599.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
L.-F. NIE ET AL.
1. Introduction
Quinazolinones are generally regarded as one of the privileged pharmacophores for
drug discovery. Application of quinazolinone skeleton could be found in drug candi-
dates for the evaluation of diverse biological activities including antimicrobial, anti-
inflammatory, antimalarial, antiviral, and last but not least anti-cancer activities [1–4].
Among the two structural isomers, 4-quinazolinones are far more abundantly occur-
ring in synthetic and natural products than 2- quinazolinones [5].
Evodiamine and rutaecarpine (compounds 1 and 2, Figure 1) are two main 4-qui-
nazolinone alkaloids isolated from the fruit of the plant Tetradium ruticarpum, which
is used in traditional Chinese medicine for the treatment of a variety of diseases.
Although their isolation was achieved nearly a century ago, they are still being
actively investigated as leads for drug developments [6], such as for anti-influenza
[7], anti-Alzheimers [8, 9], anti-cancer [10], and vasodilator effect [11]. Wang et al.
[12, 13] recently modified evodiamine to obtain a series of highly potent and multi-
targeting antitumor agents. Rutaecarpine was reported for its effect of antiproliferative
[14], anti-hyperlipidemia and hyperglycemia [15] and protective effect of hepatotox-
icity [16]. Incorporation of evodiamine or rutaecarpine into hybrids was also been
investigated [17, 18].
Molecular hybridization has been widely applied for drug discovery in recent years.
This strategy usually combined two active compounds with or without a linker to
obtain a new drug identity with potential synergistic or even over-additive effect [19].
Alternatively, two compounds can be fused together by replacement the similar moi-
ety in one compound with that of the other compound. Thieno[2,3-d]pyrimidin-
4(3H)-one is a favored building block commonly applied for the drug development
with diversified pharmaceutical activities. It has been widely used for preparation of
new compounds as antibacterial, antitumor agents, etc. Compounds 3 and 4 (Figure
1) represented two good examples of derivative of thieno[2,3-d]pyrimidin-4(3H)-one
as potent anticancer agents [20, 21]. As part of our continuous research in the syn-
thesis of new evodiamine and rutaecarpine derivatives [22, 23], herein we present our
application of molecular hybridization strategy to synthesize dozen of hybrids of
thieno[2,3-d]pyrimidin-4(3H)-one and evodiamine/rutaecarpine and to evaluate their
cytotoxic activities.
2. Results and discussion
Preparation of evodiamine and its analogs has been well documented and is relatively
straightforward [12, 13], usually via condensation of 3,4-dihydro-b-carboline and
Figure 1. Chemical structure of rutaecarpine (compound 1), evodiamine (compound 2), and deriva-
tives of thieno[2,3-d]pyrimidin-4(3H)-ones (compounds 3 and 4) as potent anticancer reagents.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthesis of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyri-
midinones. Reagents and conditions: i) Lithium hydroxide (LiOH), ethanol/H2O, 70 ꢁC, 4 h; ii) triphos-
gen, 1,4-dioxane, reflux, 20 h, iii) DMF, 95 ꢁC, 24 h; iv) CH2Cl2, 95 ꢁC, 24 h; and v) methyl iodide,
dimethyl acetamide, diisopropylethylamine, 45 ꢁC overnight.
N-methyl isatoic anhydride. On the other hand, methods for synthesis of rutaecarpine
are diversified [6]. We recently developed a one-pot, two-component protocol for fast
preparation of rutaecarpine using isatoic anhydride and 3,4-dihydro-b-carboline,
which is devoid of tedious purification procedures [23]. Considering the structural
similarity between isatoic anhydride and 2H-thieno[2,3-d][1, 3] oxazine-2,4(1H)-
dione, it is envisioned that this method could also applied for thieno -rutarcarpines.
The synthetic process is elucidated in Scheme 1. Substituted ethyl 2-aminothio-
phene-3-carboxylates (compound 5a–5f) were prepared by application of Gewald
reaction to cyanoacetate, sulfur and respectful ketone in the presence of trimethyl-
amine. Hydrolysis of esters (compounds 5a–5f) in hot sodium hydroxide solution
(ethanol/water) afforded 2-aminothiophene-3-carboxylic acids (compounds 6a–6f).
Treatment of acids (compounds 6a–6f) with triphosgene in dry dioxane produced
2H-thieno[2,3-d][1, 3]oxazine-2,4(1H)-diones (compounds 7a–7f). Condensation of
compounds 7a–7f with 3,4-dihydro-b-carboline (compound A) or 6-methoxy-4,9-
dihydro-3H-pyrido[3,4-b]indole (compound B) in DMF at 95 ꢁC yielded smoothly the
desired hybrids of rutaecarpine and thieno[2,3-d]pyrimidinones (compounds 9a–9f
and 10a–10f).
Methylation of intermediates (compounds 7a–7f) with iodomethane yielded
compounds 8a–8f, which was then condensed with 3,4-dihydro-b-carboline (com-
pound A or B) in dichloromethane at room temperature, to produce the desired
hybrids of evodiamine and thieno[2,3-d]pyrimidinones (compounds 11a–11f
and 12a–12f).
In general, preparation of all the intermediates did not require any chromato-
graphic purification. The target hybrids of rutarcarpines can be collected by filtration
of the precipitated solids upon treatment of the reaction mixtures with water. All of

4
L.-F. NIE ET AL.
Figure 2. ORTEP representation of compound 11e (CCDC 1842566).
the targets were purified by column chromatography to ensure the purities for bio-
1
logical assay. The obtained compounds were characterized by melting point, H and
13C NMR to confirm their structures. These data are in total agreement with the
desired structures. Moreover, the HRMS of the synthesized targets display the correct
molecular ion peaks. Compound 11e was further confirmed by the analysis of X-ray
data (Figure 2, CCDC 1842566 contains the supplementary crystallographic data for
compound 11e. The data can be found free of charge at www.ccdc.cam.ac.uk/
getstructures).
Considering that series of quinazolinones natural products had been identified as
cytotoxic agents to tumor cells [4], we investigated the antiproliferative activity of 24
compounds in several types of human cancer cells including human non-small cell
lung cancer cells A549, PC-9, human prostate cancer cells PC-3, and human breast
cancer cell line MCF-7. Topoisomerase I inhibitor camptothecin was used as a posi-
tive control compound [14]. The result revealed that majority of compounds exhib-
ited moderate cytotoxic activity to all of the A549, PC-9, PC-3, and MCF-7 cells,
although compounds 10f and 12e were nearly inactive to the four cell lines (Table 1).
Compounds 9a, 10e, 11a, 11d, 11f, and 12a exhibited promising antiproliferative
activity towards the four cells with individual half maximal inhibitory concentration
(IC50) values of around 10 lM.
The structure–activity relationship study revealed that the nature of the substituted
group on the thieno[2,3-d]pyrimidinone plays an important role in the cytotoxic
activity of the compound. In general, the evodiamine hybrids (compounds 11a–11f
(bearing methoxy group at 9-position of indole ring)) exhibit more potent cytotox-
icity against four cell lines than rutaecparine hybrids (compounds 9a–9f), while evo-
diamine hybrids (compounds 12a–12f (bearing methoxy group at 9-position of
indole ring)) are less potent compared to rutaecparine hybrids (compounds 10a–10f).
Compounds 9d, 10d, 12b, and 12d displayed better activities to A549, MCF-7 cell
lines compared to PC-3 and PC-9 cell lines. Compounds 10f and 12f shared similar
substitution pattern and they were almost inactive to A549, PC-3, and PC-9 cell lines,
although compound 12f was moderately toxic to MCF-7 cell. These results might
indicate those substitution groups were unfavorable for the anti-cancer activity.
Compound 11a showed the greatest activity to inhibit the proliferation of A549,
PC-9, PC-3, and MCF-7 cell lines and there no significant differences among these

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. Cytotoxic activitya (IC50, lM) of compounds 9a–12f.
Cell lines
Compound
A549
PC-9
MCF-7
PC-3
9a
9b
9c
9d
9e
9f
12.90 3.40
15.65 2.66
15.99 3.49
12.14 1.49
8.99 1.39
11.60 1.64
>100
12.31 0.24
12.03 2.28
8.35 1.99
12.15 1.25
>100
10.45 1.27
77.85 6.48
11.52 1.00
11.20 0.85
17.21 0.65
11.51 0.94
14.70 1.91
12.71 0.92
13.93 1.90
13.16 0.45
66.90 6.75
74.75 6.21
0.06 0.02
13.30 2.12
12.97 1.65
14.27 4.23
74.09 17.13
41.98 11.15
12.34 1.54
11.44 0.17
19.43 1.40
36.07 6.50
83.39 17.51
13.84 1.82
59.34 8.06
9.46 0.24
12.87 0.11
10.61 0.31
12.20 0.81
11.26 0.34
11.60 1.46
9.94 0.33
12.39 0.81
49.56 1.72
>100
12.91 0.02
14.91 3.89
>100
73.04 23.14
14.31 0.36
12.21 0.82
>100
11.58 1.21
10.47 0.70
50.50 3.45
89.14 0.22
9.10 0.10
11.00 0.59
12.44 1.04
13.18 1.86
95.78 2.07
9.02 1.12
12.68 2.81
14.24 2.89
11.26 1.30
11.66 3.18
10.83 0.65
7.70 1.79
10.72 2.77
12.25 2.60
15.55 3.18
99.29 4.30
14.11 0.66
0.14 0.04
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
>100
10.07 0.19
>100
12.60 2.10
91.86 3.48
7.77 0.77
50.47 8.19
>100
11.45 0.81
41.14 6.07
11.02 0.53
4.21 0.06
48.99 9.96
15.95 4.54
65.89 11.73
47.90 1.30
65.02 15.61
0.52 0.07
70.14 6.34
24.75 2.12
73.41 2.50
77.06 10.34
86.53 3.76
0.15 0.03
Camptothecin
aData are from at least three independent experiments.
Figure 3. Compound 11a could inhibit the colony formation of cancer cell lines. (A) The colony
formation of A549, PC-9, MCF-7, and PC-3 cells could be reduced by compound 11a at a dose
dependent manner. The compound 11a incubated with cancer cells for 10 days. (B) Quantification
of the rate of colony formation described in A. Statistically significant differences are presented as
ꢀꢀ
ꢀꢀꢀ
p < 0.001, and compared with the control group. CA: camptothecin.
p < 0.01,

6
L.-F. NIE ET AL.
four cancer cells. It may demonstrate that less bulky substation group would be better
for the potency, although bulky group was also tolerable in some cases.
Data mentioned above unfolded that compound 11a might serve as a lead com-
pounds for development of potential anticancer agents. For further evaluation of the
active compound, we utilized a colony formation assay to measure inhibition of pro-
liferation on A549, PC-9, PC-3, and MCF-7 cells. Similar to what we observed in a 3-
(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, compound
11a showed strong cell proliferation inhibition activity to the four cell lines
(Figure 3). The number of colonies was much lower after incubating with compound
11a at 10 lM compared to the control groups (p < 0.001) in the four cell lines. Based
on the results, we found that compound 11a could dose-dependently inhibit the
proliferation of cancer cells. The results indicated that derivatives of quinazolinones
possessed potent anti-tumor activities against various types of cancer.
Summarizing, in this research, by taking advantage of our recently reported
method of expeditious preparation of rutaecarpines, we synthesized 12 hybrids of nat-
ural alkaloid rutaecarpine and thieno[2,3-d]pyrimidinones. By condensation of inter-
mediates (compounds 8a–8f) with 3,4-dihydro-b-carbolines, another 12 hybrids of
evodiamine and thieno[2,3-d]pyrimidinones were prepared. The synthetic route is
straightforward and the targets are easy to be purified. The synthetic compounds dis-
played moderate to good anticancer activities. The potency of compound 11a was fur-
ther confirmed by colony formation assay.
3. Experimental
3.1. General experimental procedures
Reagents and solvents were purchased from commercial suppliers and used without
further purification. Thin-layer chromatography was carried out on glass plates coated
with silica gel (Qingdao Haiyang Chemical Co., G60F-254, Qingdao, China) and
visualized by UV light (254 nm). The products were purified by column chromatog-
raphy over silica gel (Qingdao Haiyang Chemical Co., 200–300 mesh, Qingdao,
China). Melting points were determined on a Buchi B-540 apparatus and uncorrected
(Bern, Switzerland). All the NMR spectra were recorded with a Varian 400 or
600 MHz NMR spectrometer, respectively (California, CA, USA) in deuterated chloro-
form (CDCl3) or dimethyl sulfoxide (DMSO)-d6, using tetramethylsilane (TMS) as an
internal standard. High-resolution mass spectra (HRMS) were recorded on AB SCIEX
QSTAR Elite quadrupole time-of-flight mass spectrometry (California, CA, USA).
The detailed synthetic procedure and spectral data of intermediates (compounds 5a-
5f, 6a-6f, 7a-7f, 8a-8f, A, and B) were described in the supplementary material of
this article.
3.2. General procedure of preparation of compounds 9a-9f and 10a-10f
To the solution of thieno[2,3-d][1, 3]oxazine-2,4-dione (1 mmol) in DMF (1 ml) was
added 3,4-dihydro-b-carboline (compound A or B (1.3 mmol)). The reaction mixture
was heated at 95 ꢁC for 24 hours without protection atmosphere. Then 20 ml of water

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
were poured into the solution. The precipitated solid was collected by filtration,
washed with water, and purified to produce the title compounds.
3.2.1. 2-Methyl-7,12-dihydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-
4(6H)-one (compound 9a)
1
Yield 76%, light yellow solid, m.p. 278–280 ꢁC; H NMR (400 MHz, CDCl3) d 9.12 (s,
1H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 8.2 Hz, 1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.18 (t,
J ¼ 7.6 Hz, 1H), 7.14 (s, 1H), 4.56 (t, J ¼ 7.0 Hz, 2H), 3.20 (t, J ¼ 7.0 Hz, 2H), and 2.53
(s, 3H). 13C NMR (100 MHz, CDCl3) d 162.3, 157.8, 144.9, 138.4, 137.3, 127.1, 125.7
(C and CH overlap), 123.4, 120.8, 120.2, 120.2, 118.3, 112.2, 41.1, 19.8, and 16.2.
HRTOFMS: m/z 308.0840 [M þ H]þ (calcd for C17H13N3OS, 308.0858).
3.2.2. 2,3-Dimethyl-7,12-dihydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-
4(6H)-one (compound 9b)
1
Yield 82%, light yellow solid, m.p. 310–312 ꢁC; H NMR (400 MHz, CDCl3) d 9.02 (s,
1H), 7.61 (d, J ¼ 7.9 Hz, 1H), 7.41 (d, J ¼ 8.1 Hz, 1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 7.18 (t,
J ¼ 7.5 Hz, 1H), 4.54 (t, J ¼ 7.0 Hz, 2H), 3.20 (t, J ¼ 7.0 Hz, 2H), 2.51 (s, 3H), and 2.39
(s, 3H). HRTOFMS: m/z 322.1002 [M þ H]þ (Calcd for C18H15N3OS, 322.1014).
3.2.3.
5,6,9,10,11,14-Hexahydro-8H-cyclopenta[4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido
[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 9c)
1
Yield 81%, light yellow solid, m.p. 305–307 ꢁC; H NMR (400 MHz, CDCl3) d 9.07 (s,
1H), 7.61 (d, J ¼ 7.9 Hz, 1H), 7.42 (d, J ¼ 8.3 Hz, 1H), 7.36–7.30 (m, 1H), 7.21–7.15
(m, 1H), 4.55 (t, J ¼ 7.1 Hz, 2H), 3.20 (t, J ¼ 7.1 Hz, 2H), 3.10 (t, J ¼ 7.2 Hz, 2H), 2.96
(t, J ¼ 7.2 Hz, 2H), and 2.52–2.43(m, 2H). 13C NMR (100 MHz, CDCl3) d 158.2,
144.4, 141.2, 138.4, 138.3, 127.1, 125.8, 125.7, 120.9, 120.2, 118.7, 118.2, 112.2, 110.17,
40.8 (CH2), 29.8 (CH2), 29.2 (CH2), 28.1(CH2), and 19.9 (CH2). HRTOFMS: m/z
334.0987 [M þ H]þ (Calcd for C19H15N3OS, 334.1014).
3.2.4. 5,9,10,11,12,15-Hexahydrobenzo[4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]
pyrido[3,4-b]indol-8(6H)-one (compound 9d)
1
Yield 88%, light yellow solid, m.p. 326–328 ꢁC; H NMR (400 MHz, CDCl3-CD3OD)
d 7.50 (d, J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 8.3 Hz, 1H), 7.21 (t, J ¼ 7.9 Hz, 1H), 7.05 (dd,
J ¼ 7.9, 7.0 Hz, 1H), 4.40 (t, J ¼ 7.0 Hz, 2H), 3.08 (t, J ¼ 7.0 Hz, 2H), 2.93 (t,
J ¼ 5.8 Hz, 2H), 2.66 (t, J ¼ 5.8 Hz, 2H), and 1.84–1.70 (m, 4H). 13C NMR (100 MHz,
CDCl3-CD3OD) d 161.4, 158.4, 144.9, 138.6, 133.0, 132.0, 126.5, 125.5 (CH), 125.2,
120.7, 120.4 (CH), 119.9 (CH), 118.3, 112.2 (CH), 40.6, 25.6, 25.2, 22.8, 22.2, and
19.6. HRTOFMS: m/z 348.1157 [M þ H]þ (calcd for C20H17N3OS, 348.1171).
3.2.5. 5,6,9,10,11,12,13,16-Octahydro-8H-cyclohepta[4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido
[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 9e)
1
Yield 91%, light yellow solid, m.p. 275–277 ꢁC; H NMR (400 MHz, CDCl3) d 9.10 (s,
1H), 7.61 (d, J ¼ 8.0 Hz, 1H), 7.39 (d, J ¼ 8.3 Hz, 1H), 7.34–7.28(m, 1H), 7.20–7.14
(m, 1H), 4.53 (t, J ¼ 7.0 Hz, 2H), 3.4 (t, J ¼ 5.3 Hz, 2H), 3.18 (t, J ¼ 7.0 Hz, 2H), 2.80
(t, J ¼ 5.3 Hz, 2H), 1.96–1.85(m, 2H), and 1.77–1.65(m, 4H). 13C NMR (100 MHz,

8
L.-F. NIE ET AL.
CDCl3) d 160.1, 158.8, 144.3, 138.4, 138.0, 137.3, 127.2, 125.8, 125.6 (CH), 121.7,
120.8 (CH), 120.1 (CH), 118.0, 112.2 (CH), 40.7, 32.8, 30.1, 28.0, 27.9, 27.4, and 19.9.
HRTOFMS: m/z 362.1314 [M þ H]þ (calcd for C21H19N3OS, 362.1327).
3.2.6. 5,6,9,10,12,15-Hexahydro-8H-pyrano[4’’’,3’’’:4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido
[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 9f)
1
Yield 79%, white solid, m.p. 346–348 ꢁC; H NMR (400 MHz, DMSO-d6) d 11.93 (s,
1H), 7.64 (d, J ¼ 8.0 Hz, 1H), 7.43 (d, J ¼ 8.3 Hz, 1H), 7.26 (t, J ¼ 7.5 Hz, 1H), 7.09 (t,
J ¼ 7.5 Hz, 1H), 4.76 (s, 2H), 4.40 (t, J ¼ 7.0 Hz, 2H), 3.92 (t, J ¼ 5.4 Hz, 2H), 3.15 (t,
J ¼ 7.0 Hz, 2H), and 2.98 (t, J ¼ 5.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d 161.7,
157.2, 145.5, 138.7, 129.3, 128.9, 126.8, 124.8 (C and CH overlapped), 120.0 (CH),
119.8 (CH), 119.6, 117.9, 112.5 (CH), 64.3, 63.9, 54.9, 26.1, and 18.9. HRTOFMS: m/z
350.0952 [M þ H]þ (calcd for C19H15N3O2S, 350.0963).
3.2.7. 9-Methoxy-2-methyl-6,7-dihydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido
[3,4-b]indol-4(12H)-one (compound 10a)
Yield 65%, light yellow solid, m.p.273–275 ꢁC;1H NMR (400 MHz, CDCl3) d 9.05 (s,
1H), 7.29 (t, J ¼ 4.8 Hz, 1H), 7.12 (d, J ¼ 1.1 Hz, 1H), 6.98 (dd, J ¼ 4.8, 2.3 Hz, 2H),
4.55 (t, J ¼ 7.0 Hz, 2H), 3.87 (s, 3H), 3.16 (t, J ¼ 7.0 Hz, 2H), and 2.51 (s, 3H).13C
NMR (100 MHz, CDCl3) d 162.3, 157.7, 154.9, 144.9, 137.2, 133.7, 127.6, 126.0, 123.2,
120.2 (CH), 117.8, 116.8 (CH), 113.1 (CH), 100.8 (CH), 55.9 (CH3), 41.1 (CH2), 19.9
(CH2), and 16.2 (CH3). HRTOFMS: m/z 338.0944 [M þ H]þ (calcd for
C18H15N3O2S, 338.0963).
3.2.8. 9-Methoxy-2,3-dimethyl-7,12-dihydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido
[3,4-b]indol-4(6H)-one (compound 10b)
1
Yield 89%, light yellow solid, m.p. 321–323 ꢁC; H NMR (400 MHz, CDCl3) d 8.91 (s,
1H), 7.31 (d, J ¼ 9.7 Hz, 1H), 7.00 (m, 2H), 4.53 (t, J ¼ 7.0 Hz, 2H), 3.87 (s, 3H), 3.16
(t, J ¼ 7.0 Hz, 2H), 2.51 (s, 3H), and 2.39 (s, 3H). HRTOFMS: m/z 352.1105
[M þ H]þ (calcd for C19H17N3OS, 3521.1120).
3.2.9.
3-Methoxy-9,10,11,14-tetrahydro-5H-cyclopenta[4’’,5’’]thieno[2’’,3’’:4’,5’]
pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8(6H)-one (compound 10c)
Yield 83%, light yellow solid, m.p. 308–310 ꢁC;1H NMR (400 MHz, CDCl3) d 8.98 (s,
1H), 7.30 (d, J ¼ 9.7 Hz, 1H), 7.02–6.94(m, 2H), 4.54 (t, J ¼ 7.0 Hz, 2H), 3.87 (s, 3H),
3.15 (t, J ¼ 7.0 Hz, 2H), 3.09 (t, J ¼ 7.2 Hz, 2H), 2.94 (t, J ¼ 7.2 Hz, 2H), and
2.51–2.41(m, 2H).13C NMR (600 MHz, CDCl3) d 167.0, 158.3, 154.9, 144.5, 141.2,
138.1, 133.7, 127.8, 126.1, 118.6, 117.6, 116.7 (CH), 113.0 (CH), 100.8 (CH), 58.6
(CH3), 40.8 (CH2), 29.8 (CH2), 29.2 (CH2), 28.1 (CH2), and 19.9 (CH2). HRTOFMS:
m/z 364.1112[M þ H]þ (calcd for C20H17N3O2S, 364.1120).
3.2.10. 3-Methoxy-5,9,10,11,12,15-hexahydrobenzo[4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido
[1’,2’:1,2]pyrido[3,4-b]indol-8(6H)-one (compound 10d)
1
Yield 87%, light yellow solid, m.p. 274–276 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.78 (s, 1H), 7.31 (d, J ¼ 8.9 Hz, 1H), 7.11 (d, J ¼ 2.4 Hz, 1H), 6.91 (dd, J ¼ 8.9,
2.4 Hz, 1H), 4.39 (t, J ¼ 7.0 Hz, 2H), 3.79 (s, 3H), 3.12 (t, J ¼ 7.0 Hz, 2H), 2.92

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9
(t, J ¼ 6.0 Hz 2H), 2.76 (t, J ¼ 6.0 Hz 2H), and 1.87–1.74 (m, 4H). HRTOFMS: m/z
362.1313 [M þ H]þ (calcd for C21H19N3O2S, 362.1276).
3.2.11. 3-Methoxy-5,6,9,10,11,12,13,16-octahydro-8H-cyclohepta[4’’,5’’]thieno[2’’,3’’:
4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 10e)
1
Yield 95%, white solid, m.p. 270–272 ꢁC; H NMR (400 MHz, CDCl3) d 9.10 (s, 1H),
7.27–7.21 (m, 1H), 6.99–6.91 (m, 2H), 4.51 (t, J ¼ 7.0 Hz, 2H), 3.85 (s, 3H), 3.37 (t,
J ¼ 5.5 Hz, 2H), 3.13 (t, J ¼ 7.0 Hz, 2H), 2.80 (t, J ¼ 5.5 Hz, 2H), 1.93–1.83(m, 2H), and
1.74–1.63 (m, 4H). 13C NMR (100 MHz, CDCl3) d 160.2, 158.8, 154.8, 144.4, 137.9,
137.1, 133.7, 127.7, 126.0, 121.6, 117.5, 116.6 (CH), 113.0 (CH), 100.8 (CH), 55.9
(CH3), 40.7 (CH2), 32.8 (CH2), 30.1 (CH2), 28.0 (CH2), 27.9 (CH2), 27.4 (CH2), and
19.9 (CH2). HRTOFMS: m/z 392.1413 [Mþ H]þ (calcd for C22H21N3O2S, 392.1433).
3.2.12. 3-Methoxy-5,6,9,10,12,15-hexahydro-8H-pyrano[4’’’,3’’’:4’’,5’’]thieno[2’’,3’’:
4’,5’]pyrimido [1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 10f)
1
Yield 82%, white solid, m.p. 306–308 ꢁC; H NMR (400 MHz, DMSO-d6) d 11.80 (s,
1H), 7.32 (d, J ¼ 8.9 Hz, 1H), 7.11 (d, J ¼ 2.4 Hz, 1H), 6.91 (dd, J ¼ 8.9, 2.4 Hz, 1H),
4.76 (s, 2H), 4.40 (t, J ¼ 7.0 Hz, 2H), 3.92 (t, J ¼ 5.5 Hz, 2H), 3.79 (s, 3H), 3.13 (t,
J ¼ 7.0 Hz, 2H), 2.98 (t, J ¼ 5.5 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d 161.8,
157.2, 153.8, 145.5, 134.0, 129.1, 128.9, 127.1, 125.0, 119.5, 117.4, 116.1 (CH), 113.3
(CH), 100.5 (CH), 64.3, 63.9, 55.3, 39.1, 26.1, 19.0. HRTOFMS: m/z 380.1057
[M þ H]þ (calcd for C20H17N3O3S, 380.1069).
3.3. General procedure of preparation of compounds 11a–11f and 12a–12f
To the solution of 1 mmol of thieno[2,3-d][1,3]oxazine-2,4-dione in 3 ml of
dichloromethane (CH2Cl2), 1.3 mmol of 3,4-dihydro-b-carboline (compound A or B)
was added. The reaction mixture was stirred at room temperature for 24 hours with-
out protection atmosphere. The solvent was evaporated off and the residue was puri-
fied to produce the title compounds.
3.3.1.
2,13-Dimethyl-7,12,12b,13-tetrahydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]
pyrido[3,4-b]indol-4(6H)-one (compound 11a)
1
Yield 67%, light yellow solid, m.p. 251–253 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.31 (s, 1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 7.5 Hz,
1H), 7.03 (t, J ¼ 7.5 Hz, 1H), 6.78 (d, J ¼ 1.1 Hz, 1H), 6.10 (s, 1H), 4.56 – 4.48 (m,
1H), 3.12 – 3.02 (m, 1H), 2.88 – 2.75 (m, 2H), 2.59 (s, 3H), 2.36 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 161.4, 159.5, 136.7, 130.0, 128.5, 125.7, 122.0, 121.1, 120.4,
118.9, 118.4, 112.1, 111.6, 70.6, 39.2, 37.8, 19.9, 15.0. HRTOFMS: m/z 322.1018 [M-
H]þ (calcd for C18H17N3OS, 322.1014).
3.3.2. 2,3,13-Trimethyl-7,12,12b,13-tetrahydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]
pyrido[3,4-b]indol-4(6H)-one (compound 11b)
1
Yield 82%, light yellow solid, m.p. 229–331 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.31 (s, 1H), 7.52 (d, J ¼ 7.8 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 7.5 Hz,

10
L.-F. NIE ET AL.
1H), 7.03 (t, J ¼ 7.5 Hz, 1H), 6.04 (s, 1H), 4.57–4.49(m, 1H), 3.12–3.03(m, 1H),
2.89–2.72(m, 2H), 2.57 (s, 3H), 2.26 (s, 3H), and 2.23 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) d 162.1, 158.4, 136.7, 130.2, 128.5, 125.7, 122.6, 122.0, 119.0, 118.9, 118.4,
112.1, 111.6, 70.1, 70.6, 39.2, 37.8, 19.9, 15.0, 13.0, and 12.2. HRTOFMS: m/z
336.1177 [M-H]þ (calcd for C19H19N3OS, 336.1093).
3.3.3 13-Methyl-5,6,9,10,11,13,13a,14-octahydro-8H-cyclopenta[4’’,5’’]thieno[2’’,3’’:
4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 11c)
1
Yield 78%, white solid, m.p. 283–285 ꢁC; H NMR (600 MHz, DMSO-d6) d 11.32 (s,
1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.36 (d, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 7.4 Hz, 1H), 7.03 (t,
J ¼ 7.4 Hz, 1H), 6.13 (s, 1H), 4.56–4.48(m, 1H), 3.12– 3.05 (m, 1H), 2.90–2.82 (m,
2H), 2.82–2.70(m, 4H), 2.60 (s, 3H), 2.35–2.28 (m, 2H). 13C NMR (125 MHz, DMSO-
d6) d 164.1, 161.8, 141.5, 136.7, 130.3, 128.5, 125.7, 122.0 (CH), 118.9 (CH), 118.4
(CH), 115.8, 112.1, 111.6 (CH), 70.8, 39.7, 37.8, 28.9, 28.5, 27.3, and 19.9.
HRTOFMS: m/z 348.1164 [M þ H]þ (calcd for C20H19N3OS, 348.1171).
3.3.4.
14-Methyl-5,6,9,10,11,12,14,14a-octahydrobenzo[4’’,5’’]thieno[2’’,3’’:4’,5’]
pyrimido[1’,2’:1,2] pyrido[3,4-b]indol-8(15H)-one (compound 11d)
1
Yield 93%, light yellow solid, m.p. 321–323 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.31 (s, 1H), 7.51 (d, J ¼ 7.9 Hz, 1H), 7.36 (d, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 7.6 Hz,
1H), 7.02 (t, J ¼ 7.4 Hz, 1H), 6.06 (s, 1H), 4.51 (ddd, J ¼ 12.9, 5.1, 2.1 Hz, 1H), 3.07
(ddd, J ¼ 12.6, 10.8, 4.6 Hz, 1H), 2.97–2.72 (m, 3H), 2.67–2.57 (m, 5H), and 1.80–1.62
(m, 4H). 13C NMR (100 MHz, DMSO-d6) d 162.1, 159.1, 136.7, 132.4, 128.6, 125.7,
125.6, 122.0, 118.9, 118.4, 117.8, 112.1, 111.6, 70.3, 39.0, 37.7, 25.4, 24.2, 22.8, and
22.0 19.9. HRTOFMS: m/z 362.1317 [M-H]þ (calcd for C21H21N3OS, 362.1327).
3.3.5.
15-Methyl-9,10,11,12,13,15,15a,16-octahydro-5H-cyclohepta[4’’,5’’]thieno
[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8(6H)-one (compound 11e)
1
Yield 95%, light yellow solid, m.p. 321–323 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.28 (s, 1H), 7.50 (d, J ¼ 7.9 Hz, 1H), 7.34 (d, J ¼ 8.1 Hz, 1H), 7.15 – 7.06 (t,
J ¼ 7.4 Hz, 1H), 7.00 (t, J ¼ 7.4 Hz, 1H), 6.00 (s, 1H), 4.55 – 4.46 (m, 1H), 3.21–3.12
(m, 1H), 3.10–2.96(m, 2H), 2.84 (d, J ¼ 15.1 Hz, 1H), 2.79–2.71(m, 1H), 2.70–2.62(m,
2H), 2.50 (s, 3H), 1.84–1.73(m, 2H), and 1.61–1.47(m, 4H). 13C NMR (100 MHz,
DMSO-d6) d d 162.1, 157.5, 137.9, 136.7, 129.8, 128.4, 125.6, 122.0, 119.5, 118.9,
118.5, 112.14, 111.6, 69.9, 38.8, 37.6, 32.0, 28.7, 27.6, 27.3, 26.9, and 19.9. HRTOFMS:
m/z 378.1269 [M þ H]þ (calcd for C22H23N3OS, 378.1640).
3.3.6. 14-Methyl-5,6,9,10,12,14,14a,15-octahydro-8H-pyrano[4’’’,3’’’:4’’,5’’]thieno
[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 11f)
1
Yield 86%, white solid, m.p. 300–302 ꢁC; H NMR (600 MHz, DMSO-d6) d 11.31 (s,
1H), 7.51 (d, J ¼ 7.8 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.16–7.11 (m, 1H), 7.06–7.01
(m, 1H), 6.11 (s, 1H), 4.66–4.57(m, 2H), 4.55–4.49(m, 1H), 3.87–3.80 (m, 2H),
3.11–3.05 (m, 1H), 3.01–2.93 (m, 1H), 2.87–2.76 (m, 2H), 2.77–2.68 (m, 1H), and
2.65 (s, 3H). 13C NMR (125 MHz, DMSO-d6) d 162.0, 160.0, 136.7, 130.1, 128.6,
125.7, 123.0, 122.1 (CH), 118.9 (CH), 118.45 (CH), 117.0, 112.2, 111.6 (CH), 70.5,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
64.1, 64.0, 39.1, 37.9, 26.1, and 19.8. HRTOFMS: m/z 366.1257 [M þ H]þ (calcd for
C20H19N3O2S, 366.1276).
3.3.7.
2,13-Dimethyl-6,7,12b,13-tetrahydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]
pyrido[3,4-b]indol-4(12H)-one (compound 12a)
1
Yield 72%, light yellow solid, m.p. 321–323 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.13 (s, 1H), 7.26 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 2.4 Hz, 1H), 6.80–6.75 (m, 2H),
6.07 (s, 1H), 4.56–4.47(m, 1H), 3.76 (s, 3H), 3.06 (td, J ¼ 11.9, 4.5 Hz, 1H), 2.86–2.72
(m, 2H), 2.59 (s, 3H), and 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 161.4,
159.4, 153.4, 131.7, 129.9, 129.0, 126.0, 121.2, 121.1, 120.3, 112.3, 112.2, 112.2, 111.9,
100.2, 70.7, 55.4, 39.3, 37.7, 20.0, and 15.0. HRTOFMS: m/z 352.1124 [M þ H]þ (calcd
for C19H19N3OS, 352.1120).
3.3.8. 3-Methoxy-14-methyl-5,6,9,10,12,14,14a,15-octahydro-9-methoxy-2,3,13-tri-
methyl-7,12,12b,13-tetrahydrothieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]
indol-4(6H)-one (compound 12b)
1
Yield 79%, light yellow solid, m.p. 213–215 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.13 (s, 1H), 7.25 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 2.2 Hz, 1H), 6.77 (dd, J ¼ 8.8,
2.2 Hz, 1H), 6.00 (s, 1H), 4.57– 4.47 (m, 1H), 3.76 (s, 3H), 3.11–3.01 (m, 1H),
2.86–2.71 (m, 2H), 2.56 (s, 3H), 2.25 (s, 3H), and 2.23 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 162.1, 158.4, 153.4, 131.7, 130.2, 129.1, 125.9, 122.4, 118.8, 112.3, 112.2,
111.9, 100.21, 70.2, 55.3, 38.9, 37.5, 120.0, 13.0, and 12.1. HRTOFMS: m/z 366.1272
[M-H]þ (calcd for C20H21N3O2S, 366.1276).
3.3.9. 3-Methoxy-13-methyl-5,6,9,10,11,13,13a,14-octahydro-8H-cyclopenta[4’’,5’’]
thieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2] pyrido[3,4-b]indol-8-one (compound 12c)
1
Yield 85%, light yellow solid, m.p. 205–207 ꢁC; H NMR (600 MHz, DMSO-d6) d
11.15 (s, 1H), 7.25 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 1.6 Hz, 1H), 6.90 – 6.74 (m, 1H),
6.10 (s, 1H), 4.54–4.47 (m, 1H), 3.76 (s, 3H), 3.10–3.01(m, 1H), 2.99–2.69 (m, 6H),
2.60 (s, 3Hand 2.35–2.27 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d 164.1, 161.8,
153.4, 141.5, 131.7, 130.2, 129.1, 126.0, 115.7, 112.3 (CH), 112.2 (CH), 111.9, 100.2
(CH), 70.8, 55.4, 39.1, 37.8, 28.9, 28.5, 27.34, and 19.94. HRTOFMS: m/z 380.1421
[M þ H]þ (calcd for C21H21N3O2S, 380.1433).
3.3.10.
3-Methoxy-14-methyl-5,6,9,10,11,12,14,14a-octahydrobenzo[4’’,5’’]thieno
[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8(15H)-one (compound 12d)
1H NMR (400 MHz, DMSO-d6) d 11.13 (s, 1H), 7.25 (d, J ¼ 8.8 Hz, 1H), 7.00
(d, J ¼ 2.4 Hz, 1H), 6.77 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.03 (s, 1H), 4.54–4.46(m, 1H), 3.76
(s, 3H), 3.05 (ddd, J ¼ 12.6, 10.9, 4.6 Hz, 1H), 2.96–2.85(m, 1H), 2.86–2.68 (m, 2H),
2.67–2.56 (m, 2H), 2.59 (s, 3H), and 1.80–1.63 (m, 4H). 13C NMR (101 MHz, dmso)
d 162.1, 159.1, 153.3, 132.4 131.7, 129.2, 126.0, 125.5, 117.7, 112.3, 112.18, 111.9,
100.2, 70.4, 55.4, 39.0, 37.7, 25.4, 24.2, 22.8, 22.0, and 20.0. HRTOFMS: m/z 392.1421
[M-H]þ (calcd for C22H23N3O2S, 392.1433).

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L.-F. NIE ET AL.
3.3.11. 3-Methoxy-15-methyl-9,10,11,12,13,15,15a,16-octahydro-5H-cyclohepta[4’’,5’’]
thieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8(6H)-one (compound 12e)
1
Yield 88%, light yellow solid, m.p. 321–323 ꢁC; H NMR (400 MHz, DMSO-d6) d
11.13 (s, 1H), 7.25 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 2.4 Hz, 1H), 6.77 (dd, J ¼ 8.8,
2.4 Hz, 1H), 6.00 (d, J ¼ 1.5 Hz, 1H), 4.56–4.47 (m, 1H), 3.76 (s, 2H), 3.22–3.12 (m,
1H), 3.11–2.97 (m, 2H), 2.87–2.80 (m, 1H), 2.79–2.65(m, 3H), 2.53 (s, 3H),
1.84–1.77(m, 2H), and 1.64–1.50 (m, 4H). 13C NMR (100 MHz, DMSO-d6) d 162.1,
157.5, 153.4, 137.9, 131.8, 129.6, 129.0, 125.9, 119.3, 112.3, 112.2, 111.9, 100.2, 69.9,
55.4, 39.5, 38.9, 37.5, 32.0, 28.7, 27.6, 27.3, 26.9, and 20.0. HRTOFMS: m/z 406.1764
[M-H]þ (calcd for C23H25N3O2S, 406.1589).
3.3.12.
3-Methoxy-14-methyl-5,6,9,10,12,14,14a,15-octahydro-8H-pyrano[4’’’,3’’’:
4’’,5’’]thieno[2’’,3’’:4’,5’]pyrimido[1’,2’:1,2]pyrido[3,4-b]indol-8-one (compound 12f)
1
Yield 88%, white solid, m.p. 262–264 ꢁC; H NMR (600 MHz, DMSO-d6) d 11.13 (s,
1H), 7.26 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 2.3 Hz, 1H), 6.78 (dd, J ¼ 8.8, 2.3 Hz, 1H),
6.08 (s, 1H), 4.65–4.57(m, 2H), 4.54–4.49 (m, 1H), 3.87–3.80 (m, 2H), 3.76 (s, 3H),
3.09–3.0.3(m, 1H), 3.01–2.94 (m, 1H), 2.83–2.75(m, 2H), 2.73– 2.67(m, 1H), and 2.65
(s, 3H). 13C NMR (125 MHz, DMSO-d6) d 162.0, 160.0, 153.4, 131.7, 130.1, 129.1,
126.0, 122.9, 116.9, 112.3, 112.2, 112.0, 100.2, 70.6, 64.1, 64.0, 55.4, 39.9, 37.9, 26.2,
and 19.9. HRTOFMS: m/z 396.1361 [M þ H]þ (calcd for C21H21N3O3S, 396.1382).
3.4. Biological activity
3.4.1. Cell culture
All human cell lines were mycoplasma-tested and obtained from the American Type
Culture Collection. The human non-small cell lung cancer (NSCLC) cell lines A549
and PC-9 were cultured in Roswell Park Memorial Institute medium (RPMI) 1640
medium while the human breast cancer cell line MCF-7 were maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inacti-
vated fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). The
human prostate cancer cell line PC-3 cells were incubated with Ham’s F-12 medium
supplemented with 10% FBS, 1% penicillin/streptomycin and 1% glutamine. All cells
were cultured at 37 ꢁC in a humidified 5% carbon dioxide atmosphere.
3.4.2. MTT assay
Cells were seeded into 96-well plates (4ꢂ 103 cells/well) and cultured in medium for
24h respectively and then exposed to 24 chemotherapeutic agents with various concen-
trations (100, 10, 1, 0.1, and 0.01lM). Besides, camptothecin and DMSO served as a
positive control and a negative control, respectively. After 72h of incubation, MTT
solution (5 mg/ml in sterilized phosphate-buffer saline; PBS) of 20ll/well was added
into each well and incubated for another four hours. Sequentially, 100ll of lysis solu-
tion (10% sodium dodecyl sulfate- 5% isobutyl alcohol-0.01M HCl) was added into per
well in order to dissolve the formazan crystals. After dissolving overnight, the optical
density (OD) was measured using a microplate reader at 570 nm. The inhibitory rates
of these compounds were calculated by the following formula: (ODcontrol ꢃ ODtreated)/
ODcontrol ꢂ 100%. The activity of compounds were evaluated by IC50 values.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
3.4.3. Colony formation assays
Briefly, after being washed, trypsinized and counted, cells (4000 cells/well) were
seeded in six-well plates and cultured in medium overnight. After being treated with
drugs (1 and 10 lM) and camptothecin (0.1 lM) for 10 days, the supernatant of per
well was discarded and fresh PBS (1 ml/well) was added to wash the cells twice.
Subsequently, 1 ml of staining solution (10% acetic acid- 10% methanol- 80% purified
water) was added to fix the cells at room temperature for 30 min. Sequentially, the
staining solution was poured out and washed with running water. Then, 1% crystal
violet staining solution (dissolved in methanol) was added to per well and co-incu-
bated with cells at room temperature for 30 min. The number of colonies purple was
counted for cell viability.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by 12th Chinese Recruitment Program of Global Experts (Thousand
Talents Program) and Research Program of Shanghai Normal Univeristy (DYL201702).
ORCID
Haji Akber Aisa
Guo-Zheng Huang
http://orcid.org/0000-0003-4652-6879
http://orcid.org/0000-0002-5730-9549
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