Efficient and mild cyclization procedures for the synthesis of novel 2-amino-4H-pyran derivatives with potential antitumor activity

Article abstract of DOI:10.1016/j.cclet.2014.04.026

A series of novel 2-amino-4H-pyran derivatives have been synthesized via a three-component reaction of α, β-unsaturated ketone derivatives, malononitrile, aldehydes in excellent yields, using DBU as a catalyst and ethanol as a cheap, safe, and environmentally benign solvent under mild conditions. Their antitumor activity was evaluated in three human tumor cell lines, including human colon cancer (HCT116), human cervical cancer (Hela), and non-small cell lung cancer (H1975).

Full text of DOI:10.1016/j.cclet.2014.04.026

Accepted Manuscript
Title: Efficient and mild cyclization procedures for the
synthesis of novel 2-amino-4H-pyran derivatives with
potential antitumor activity
Author: Dao-Cai Wang Yong-Mei Xie Chen Fan Shun Yao
Hang Song
PII:
S1001-8417(14)00200-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2014.04.026
CCLET 2970
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
11-3-2014
10-4-2014
16-4-2014
Please cite this article as: D.-C. Wang, Y.-M. Xie, C. Fan, S. Yao, H. Song,
Efficient and mild cyclization procedures for the synthesis of novel 2-amino-4H-
pyran derivatives with potential antitumor activity, Chinese Chemical Letters (2014),
http://dx.doi.org/10.1016/j.cclet.2014.04.026
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Graphical Abstract
Efficient and mild cyclization procedures for the synthesis of novel 2-
amino-4H-pyran derivatives with potential antitumor activity
Dao-Cai Wang a, Yong-Mei Xie b, Chen Fan b, Shun Yao a, Hang Song a,*
a Department of Pharmaceutical and Biological Engineering, College of Chemical Engineering, Sichuan University, Chengdu
610065, China
b State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041,
China
NH2
NC
CN
O
NC
2
O
DBU
+
Ethanol
R
X
CF3
R
CHO
X
CF3
1
3
4a-q
A series of novel 2-amino-4H-pyran derivatives have been synthesized via a three-component reaction of α, β-unsaturated ketone derivatives,
malononitrile, aldehydes in excellent yields, using DBU as a catalyst and ethanol as a cheap, safe, and environmentally benign solvent under
mild conditions. Their antitumor activity was evaluated in three human tumor cell lines, including human colon cancer (HCT116), human
cervical cancer (Hela), and non-small cell lung cancer (H1975).
Page 1 of 6

Original article
Efficient and mild cyclization procedures for the synthesis of novel 2-amino-4H-pyran
derivatives with potential antitumor activity
Dao-Cai Wang a, Yong-Mei Xie b, Chen Fan b, Shun Yao a, Hang Song a,*1
a
Department of Pharmaceutical and Biological Engineering, College of Chemical Engineering, Sichuan University, Chengdu 610065, China
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
b
ARTICLE INFO
ABSTRACT
Article history:
A series of novel 2-amino-4H-pyran derivatives have been synthesized via a three-
component reaction of α, β-unsaturated ketone derivatives, malononitrile, aldehydes in
excellent yields, using DBU as a catalyst and ethanol as a cheap, safe, and environmentally
benign solvent under mild conditions. Their antitumor activity was evaluated in three
human tumor cell lines, including human colon cancer (HCT116), human cervical cancer
(Hela), and non-small cell lung cancer (H1975).
Received 11 March 2014
Received in revised form 10 April 2014
Accepted 16 April 2014
Available online
Keywords:
Synthesis
2-Amino-4H-pyran derivatives
Multicomponent
Anticancer
1
1. Introduction
2
3
The fused pyran ring skeleton is a well-known heterocycle and important core unit in a number of natural products [1-3]. Natural
products bearing a fused pyran ring system possess distinct properties of general interest, and have a variety of valuable biological
activities and potential medicinal applications [4-8]. It is worthwhile to note that many of these natural products exhibit cancer cell
growth inhibitory activity and have been further investigated as potential anticancer agents [9-10]. As more valuable properties emerge,
these pyran-containing structures begin to inspire chemists to develop efficient synthetic approaches and identify novel biologically
active compounds [11-12].
Recently, multicomponent reactions (MCR) have been used frequently in organic synthesis, and considerable efforts have been
focused on the design and development of environmentally friendly and less expensive procedures for the generation of libraries of
heterocyclic compounds [13-15]. Among them, the construction of pyran ring is particularly interesting and valuable, especially those
methods that provided a feasible strategy for the production of molecules comprised of two heterocyclic nucleuses [11,16]. However,
these methods are limited by the aldehydes tolerated, which greatly restricted the generation of more diverse pyran analogue libraries.
Here, we report a mild method for the synthesis of novel 2-amino-4H-pyran derivatives using ethanol as a cheap, safe, and
environmentally benign solvent. Their in vitro antitumor activity was evaluated using the National Cancer Institute (NCI) in vitro
disease-oriented human cells screening panel assay.
4
5
6
7
8
9
10
11
12
13
14
15
16
2. Experimental
17
18
19
20
21
22
23
24
25
26
27
All reagents were acquired from commercial sources and used without further purification. All reactions were monitored by thin
layer chromatography (TLC silica gel 60 F254 plates) analysis. All products were purificated by column chromatography (200–300
mesh silica gel, Qingdao Marine Chemical Ltd., Qingdao, China). Melting points were measured using an YRT-3 melting point
measuring apparatus (Precision Instrument Plant, Tianjin University) and uncorrected. The 1H NMR spectra and 13C NMR spectra were
recorded on a Bruker AM400 NMR spectrometer and the chemical shifts in ppm were reported relative to tetramethylsilane (TMS) or
residual solvent peaks. Mass spectrometry (ESI-MS) data were mensurated using a Bruker Daltonics amaZon SL mass spectrometer.
Crystal data of 4a were collected on a Xcalibur E diffractometer with monochromated Mo Kα radiation (λ＝0.71073 Å) at 293 K, and
operating in the ω scan mode. The structure was solved with the Superflip structure solution program using Charge Flipping and
refined on F2 by the full matrix least-squares methods using SHELXTL.
General procedures for the synthesis of compounds 4a－4q (Scheme 1): A solution of α, β-unsaturated ketones (1, 0.5 mmol),
malononitrile (2, 0.5 mmol), aldehydes (3, 0.5mmol), DBU (1 mmol) in ethanol (10 mL) was stirred at room temperature for 1 hour or
———
* Corresponding author.
E-mail address: hangsong@scu.edu.cn (H. Song).
Page 2 of 6

28
29
30
31
32
until 1 was completely consumed (monitored by TLC analysis). After the completion of the reactions, ethanol was removed under
reduced pressure. The crude products were purified by column chromatography on silica gel, eluting with petroleum ether/ethyl
acetate/ triethylamine (35:15:1, v/v/v). Further purification of the products was accomplished by recrystallization. Physical and
chemical data of chosen products are described below.
NH2
NC
CN
O
X
NC
2
O
DBU
+
Ethanol
R
CF3
R
CHO
X
CF3
1
3
4a-q
33
34
Scheme 1. Synthesis of 2-amino-4H-pyran derivatives.
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
(E)-2-Amino-6-methyl-4-phenyl-8-(4-(trifluoromethyl)benzylidene)-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-carbonitrile
(4a):
White solid; mp 235-238 °C; 1H NMR (400 MHz, CDCl3): δ 2.26 (s, 3H), 2.75 (d, 1H, J = 16 Hz), 2.97 (d, 1H, J = 16 Hz), 3.36 (d, 1H, J =
13.8 Hz), 3.53 (d, 1H, J = 13.8 Hz), 4.03 (s, 1H), 4.68 (s, 2H), 6.90 (s, 1H), 7.24-7.39 (m, 7H), 7.61 (d, 2H, J = 8 Hz); 13C NMR (100 MHz,
CDCl3): δ 41.7, 44.9, 54.6, 55.3, 60.5, 114.1, 119.7, 121.4, 122.8, 125.3, 125.4, 125.5, 127.7, 127.9, 128.9, 129.0, 129.3, 129.8, 139.8, 139.9,
142.0, 158.8. MS (ESI): m/z 424.2 [M+H]+.
(E)-2-Amino-4-(3-nitrophenyl)-8-(4-(trifluoromethyl)benzylidene)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (4o): Fulvous solid;
1
mp 211-214 °C; H NMR (400 MHz, CDCl3): δ 1.65-1.66 (m, 2H), 1.88-1.92 (m, 1H), 2.04-2.08 (m, 1H), 2.54-2.58 (m, 1H), 2.69-2.73 (m,
1H), 4.14 (s, 1H), 4.69 (s, 2H), 6.93 (s, 1H), 7.39-7.63 (m, 6H), 8.11-8.17 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 22.1, 27.0, 27.5, 43.6, 59.4,
114.8, 119.3, 122.3, 122.8, 125.2, 128.7, 129.0, 129.4, 129.9, 130.8, 134.2, 140.4, 141.9, 145.0, 148.8, 159.1. MS (ESI): m/z 454.2 [M+H]+.
(E)-tert-Butyl
2-amino-3-cyano-4-(3-nitrophenyl)-8-(4-(trifluoromethyl)benzylidene)-7,8-dihydro-4H-pyrano[3,2-c]pyridine-6(5H)-
carboxylate (4p): Flavescens solid; mp 210-212 °C; 1H NMR (400 MHz, CDCl3): δ 1.30 (s, 9H), 3.63-4.78 (m, 7H), 6.98 (s, 1H), 7.40-7.42 (m,
2H), 7.57-7.66 (m, 4H), 8.13-8.20 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 28.1, 40.0, 41.3, 44.1, 59.5, 80.9, 111.9, 118.8, 122.7, 123.1, 125.5,
129.3, 130.1, 134.1, 148.9, 153.9, 158.9. MS (ESI): m/z 555.3 [M+H]+.
(E)-2-Amino-4-(3-nitrophenyl)-8-(4-(trifluoromethyl)benzylidene)-4,5,7,8-tetrahydropyrano[4,3-b]pyran-3-carbonitrile (4q): Flavescens
solid; mp 238-240 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.77 (d, 1H, J = 16 Hz), 4.23 (d, 1H, J = 16 Hz), 4.44 (s, 1H), 4.54 (d, 1H, J = 13.8
Hz), 4.65 (d, 1H, J = 13.8 Hz), 7.01 (s, 1H), 7.12 (s, 2H), 7.44-7.46 (m, 2H), 7.69-7.77 (m, 4H), 8.09 (s, 1H), 8.17-8.19 (m, 1H); 13C NMR
(100 MHz, DMSO): δ 55.3, 65.3, 65.5, 114.1, 120.4, 120.9, 122.5, 123.0, 123.3, 125.9, 128.8, 130.2, 131.1, 134.9, 138.9, 139.8, 145.7, 148.6,
160.5. MS (ESI): m/z 456.2 [M+H]+.
54
3. Results and discussion
55
56
57
58
59
60
61
62
63
As shown in Table 1, we began our optimization studies from the reaction between (E)-1-methyl-3-(4-
(trifluoromethyl)benzylidene)piperidin-4-one, malononitrile and benzaldehyde, in equimolecular amounts, using DBU as a base and
dichloromethane as a solvent. Under mild conditions at room temperature for 1 h, the domino reaction provided a promising 87%
isolated yield of (E)-2-amino-6-methyl-4-phenyl-8-(4-(trifluoromethyl)benzylidene)-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-
carbonitrile 4a (Entry 1). This model reaction was then performed in acetonitrile and DMF, which led to poorer isolated yields 73%
and 72% of 4a, respectively (Entries 2-3). To our pleasure, the reaction in ethanol gave an isolated yield of 95% of 4a (Entry 6).
Table 1
Optimization of reaction conditions for compound 4a.
Entry
Solvent
CH2Cl2
CH3CN
DMF
Base (equiv.)
DBU (2)
DBU (2)
DBU (2)
Time (h)
Yield of 4a (%)a
1
2
3
4
5
6
7
8
1
1
1
1
1
1
5
4
87
73
72
74
88
95
68
75
EtOH
EtOH
EtOH
EtOH
EtOH
DBU (1)
DBU (1.5)
DBU (2)
Triethylamine (2)
Piperidine (2)
64
65
66
67
68
69
70
71
a Isolated yield after purification by column chromatography.
The nature and the amount of base have significant influence on the yield (Entries 4-8). It was not advisable to reduce the amount
of DBU, which led to a decrease in yields (Entries 4-6). In addition, the efficacy of the bases assayed changed in the order:
triethylamine < piperidine < DBU (Entries 6-8). It was found that the reaction proceeded most efficiently in the presence of 2
equivalents of DBU in ethanol at room temperature for 1 hour, and the desired product was isolated in the best yield.
Under the optimized conditions, various α, β-unsaturated ketones (1) with a diverse set of aldehydes (3) were then applied to the
preparation of a library of 2-amino-4H-pyran analogues (4). In general, the reactions could proceed smoothly with 60%-96% isolated
Page 3 of 6

72
73
74
75
76
77
78
79
80
yields, which were remarkable considering the large number of individual steps involved (Table 2). Firstly, different aromatic
aldehydes were applied to the cyclization reaction (Entries 1-11). By varying substituent(s) at the benzoyl moiety, we found that the
yields varied greatly within an acceptable range. Next, cyclohexanecarboxaldehyde and two heteroaromatic aldehydes were used for
the three-component reaction to enrich the diversity of aldehydes (Entries 12-14). Finally, (E)-1-methyl-3-(4-
(trifluoromethyl)benzylidene)piperidin-4-one was replaced by other α, β-unsaturated ketones to verify the scope of the three-
component cyclization reaction. As expected, these reactions afforded good yields of desired products (Entries 15-17).
Table 2
Synthesis of 2-amino-4H-pyran derivatives
Entry
X
R
Compound
Yield (%)a
95
1
2
3
4
5
6
7
8
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
N-CH3
CH2
C6H5
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
3-CH3C6H4
3,4-(CH3)2C6H3
2-FC6H4
2-F-4-BrC6H3
3-ClC6H4
65
83
89
94
79
60
62
84
71
78
77
68
96
93
67
69
2,4-Cl2C6H3
3-BrC6H4
9
2-Br-4,5-(CH3O)2C6H2
3-NO2C6H4
2-Naphthyl
Cyclohexyl
2-Thienyl
10
11
12
13
14
15
16
17
3-Pyridyl
3-NO2C6H4
3-NO2C6H4
3-NO2C6H4
N-Boc
O
81
82
83
84
85
86
87
a Isolated yield after purification by column chromatography.
1
The structures of the targeted compounds 4a-4q were fully characterized by melting points, MS (ESI), H NMR and 13C NMR.
The mass spectrum of compound 4a (Table 2, entry 1) displayed the molecular ion peak at m/z 424.2, which is consistent with the
1
proposed structure. The H NMR spectrum of 4a contained singlets for NH2 and N–CH3 at 4.68 and 2.26 ppm, respectively. The
geometrical configuration of 4a was confirmed by a single crystal X-ray crystallographic study (Fig. 1). [17] A proposed mechanism
for the reaction is shown in Scheme 2.
88
89
Fig. 1. Single crystal structure of compound 4a.
90
O
N
CF3
CN
CHO
NC
CN
NC
N
+
CN
A
NH2
NC
O
O-
N
N
CF3
CF3
B
91
92
Scheme 2. The proposed mechanism for the reaction.
93
94
95
The compounds were screened for their in vitro antitumor activity in the full NCI 96 cell panel, including human colon cancer
(HCT116), human cervical cancer (Hela), and non-small cell lung cancer (H1975). In the protocol, all compounds were tested initially
Page 4 of 6

96
97
98
at 20 μmol/L, and their percentage growth inhibition (GI%) were presented in Table 3. Compounds 4k and 4o exhibited noticeable
growth inhibitory activity against the subtotal tested subpanel tumor cell lines, and other compounds also showed some antitumor
activity. However, when the concentrations of the test compounds decreased to 10 μmol/L or lower, none of the above compounds
showed satisfying activity.
99
100
101
102
Table 3
In vitro percentage growth inhibition (GI%) caused by the test compounds at dose of 20 μmol/L.
Subpanel tumor cell lines (% growth inhibitory activity)
Compound
HCT116
10.8
22.4
43.5
8.1
7.2
11.9
9.3
Hela
4.4
5.9
15.1
4.0
37.5
17.3
4.9
H1975
16.9
39.0
69.5
17.2
41.9
42.4
7.4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
13.7
3.5
5.4
58.8
7.1
29.1
3.9
2.0
6.9
17.0
31.4
39.9
78.6
52.0
46.9
3.6
56.4
45.6
0.6
16.1
2.6
43.7
25.7
20.7
78.6
9.8
67.1
77.5
47.7
27.2
103
104
4. Conclusion
105
106
107
108
109
110
In summary, an efficient and mild cyclization procedure for the synthesis of novel 2-amino-4H-pyran derivatives using ethanol as
a cheap, safe, and environmentally benign solvent and DBU as a base was identified. The present methodology provides a direct and
convenient route to construct more diverse pyran analogue libraries. The antitumor activity of the synthesized compounds was
evaluated in three human tumor cell lines, including human colon cancer (HCT116), human cervical cancer (Hela), and non-small cell
lung cancer (H1975).
111
References
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