Synthesis of antitumor 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine derivatives via reductive dearomatization-initiated intramolecular cyclization

Article abstract of DOI:10.1016/j.tet.2016.04.036

In light of the importance of N-fused heterocycles in pharmaceuticals, there is continuing interest in research on N-fused heterocycles and their preparation. A new and efficient reductive dearomatization-initiated intramolecular cyclization reaction with

Full text of DOI:10.1016/j.tet.2016.04.036

Accepted Manuscript
Synthesis of antitumor 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine
derivatives via reductive dearomatization-initiated intramolecular cyclization
Wei Li, Xiaotian Zhou, Yang Chen, Shunmin Guo, Feifei Ba, Wei Tian, Chao Yang,
Mingping Wang, Yu Liu, Yunlong Song, Ju Zhu, Youjun Zhou, Feng Zhou, Hao Guo,
Canhui Zheng
PII:
S0040-4020(16)30298-8
10.1016/j.tet.2016.04.036
TET 27679
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 23 December 2015
Revised Date: 11 April 2016
Accepted Date: 13 April 2016
Please cite this article as: Li W, Zhou X, Chen Y, Guo S, Ba F, Tian W, Yang C, Wang M, Liu Y,
Song Y, Zhu J, Zhou Y, Zhou F, Guo H, Zheng C, Synthesis of antitumor 3,4,6,7-tetrahydro-2-
pyrimido[1,6-]quinazolin-2-imine derivatives via reductive dearomatization-initiated intramolecular
cyclization, Tetrahedron (2016), doi: 10.1016/j.tet.2016.04.036.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis of Antitumor 3,4,6,7-Tetrahydro-2H-pyrimido[1,6-
c]quinazolin-2-imine Derivatives via Reductive Dearomatization-
Initiated Intramolecular Cyclization
Wei Li,a,d,† Xiaotian Zhou,c,† Yang Chen,b,† Shunmin Guo,d Feifei Ba,b Wei Tian,a Chao Yang,a
Mingping Wang,a Yu Liu,a Yunlong Song,a Ju Zhu,a Youjun Zhou,a Feng Zhou,a,c,* Hao Guo,b,*
and Canhui Zhenga,*
aSchool of Pharmacy, Second Military Medical University, Shanghai 200433, China
bDepartment of Chemistry, Fudan University, Shanghai 200433, China
cState Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical
Industry, Shanghai 200040, China
dFujian Provincial Key Laboratory of Medical Analysis, Fujian Academy of Medical Sciences,
Fuzhou 350001, China
R1
R1
NaBH4 (2.0 equiv.)
MeOH, rt
N
N
N
HN
N
NH
R2
R2
N
N
H
12 examples, up to 80% yield
8f: R1 = tert-butyl, R2 = 3-OCH2CH2O-4
human breast cancer cell line ZR-75-30, IC50= 0.71 ± 0.3 µM
multi-targeted kinases (FLT3, INSR and VEGFR-2) inhibitor

ACCEPTED MANUSCRIPT
Synthesis of Antitumor 3,4,6,7-Tetrahydro-2H-pyrimido[1,6-
c]quinazolin-2-imine Derivatives via Reductive Dearomatization-
Initiated Intramolecular Cyclization
Wei Li,a,d,† Xiaotian Zhou,c,† Yang Chen,b,† Shunmin Guo,d Feifei Ba,b Wei Tian,a Chao Yang,a
Mingping Wang,a Yu Liu,a Yunlong Song,a Ju Zhu,a Youjun Zhou,a Feng Zhou,a,c,* Hao Guo,b,*
and Canhui Zhenga,*
aSchool of Pharmacy, Second Military Medical University, Shanghai 200433, China
bDepartment of Chemistry, Fudan University, Shanghai 200433, China
cState Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical
Industry, Shanghai 200040, China
dFujian Provincial Key Laboratory of Medical Analysis, Fujian Academy of Medical Sciences,
Fuzhou 350001, China
ABSTRACT: In light of the importance of N-fused heterocycles in pharmaceuticals, there is
continuing interest in research on N-fused heterocycles and their preparation. A new and efficient
reductive dearomatization-initiated intramolecular cyclization reaction with a broad scope has
been developed, affording 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine derivatives.
*Corresponding Author. Tel.: +86-21-81871240; e-mail adress: canhuizheng@smmu.edu.cn (C.
Zheng). E-mail adress: Hao_Guo@fudan.edu.cn (H. Guo). E-mail adress:
zhoujie020202@163.com (F. Zhou).
†W. Li, X. Zhou and Y. Chen contributed equally.
1

ACCEPTED MANUSCRIPT
Notably, this type of compound showed good inhibitory activity against specific kinases and
human cancer cell lines. These results might mean a new molecular scaffold for the development
of new antitumor agents.
Keywords: 3,4,6,7-Tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine; N-fused heterocycles;
cyclization reaction; antitumor agents; kinase inhibitors
1. Introduction
Nitrogen heterocycles are particularly well represented among natural products and biologically
active structures.1 They are also among the most significant structural components of
pharmaceuticals,2-5 present in 59% of all small-molecule drugs5 and 100% of novel small-
molecule kinase inhibitors approved by the U.S. FDA.6,7 Among them, N-fused heterocycles are
a special and important type and have been shown to possess a wide range of pharmacological
activities. Many types of important drugs contain these scaffolds, such as antibiotics of the
penicillin class and cephalosporin class, anxiolytics of the benzodiazepine class, sedative-
hypnotics of pyrazolopyrimidine class, antitumor agents among the vinca alkaloid analogues and
camptothecin analogues,3,5 and novel small-molecule kinase inhibitors of the imidazopyridazin
class (representative drugs shown in Figure 1).6,7 In addition, the cephem of cephalosporins and
the penam of penicillins are the fourth and eighth most commonly used nitrogen heterocycles in
pharmaceuticals, respectively.5,8,9 In light of the importance of these compound classes, there is
continuing interest in research on new N-fused heterocycles and their preparation.10-17 Despite
the numerous achievements that have been reported in this field,10-17 the method for the synthesis
of 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine derivatives remain undeveloped.
2

ACCEPTED MANUSCRIPT
Meanwhile, studies on their biological activities are still in the blank fields. Thus, the
construction of such a molecular skeleton is not only a challenge in synthetic chemistry, but also
interesting in medicinal chemistry. Herein, we wish to report our recent observation on the
synthesis of 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine derivatives as well as their
antitumor activities.
Figure 1. Representative drugs containing N-fused heterocycles.
2. Results and discussion
2.1. Chemistry
At the beginning of our studies, the model reactant N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-
(2-(4-(trifluoromethyl)benzylideneamino)phenyl)pyrimidin-4-amine (1a) was obtained by a
simple three-step synthesis route (Scheme 1). In the presence of concentrated HCl, the 4,6-
dichloropyrimidine (2) reacted with 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (3a) in isopropanol
3

ACCEPTED MANUSCRIPT
affording 4a in a 60% yield.18 The following Suzuki-Miyaura cross-coupling reaction of 4a and
2-aminophenyl boronic acid (5) under the catalysis of Pd(PPh3)2Cl2 in 1,4-dioxane/H2O yielded
6a in a 91% yield. Finally, condensation of 6a with 4-trifluoromethyl benzaldehyde (7a)
produced the desired product 1a in a 73% yield.
Scheme 1. The synthesis of 1a
B(OH)2
H2N
O
O
NH2
O
O
N
N
N
N
5
3a
HCl
Cl
N
H
4a
Cl
Cl
K2CO3, Pd(PPh3)2Cl2
1,4-dioxane/H2O
Concd. HCl
isopropanol
2
91%
60%
CF3
OHC
CF3
O
O
7a
NH2
N
N
O
N
N
N
N
H
1) Concd. HCl/EtOH
2) NaOH
N
O
H
73%
6a
1a
The initial attempt was carried out employing 1a treated by 2 euqiv. of NaBH4 in EtOH at rt. To
our delight, the desired product N-(6-(4-(trifluoromethyl)phenyl)-3,4,6,7-tetrahydro-2H-
pyrimido[1,6-c]quinazolin-2-ylidene)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine
(8a)
was
obtained in a 51% NMR yield (entry 1, Table 1). Then we further optimized the reaction
4

ACCEPTED MANUSCRIPT
conditions (Table 1). Some commonly used solvents were tested. The results indicated that this
cyclization reaction proceeded smoothly in all the tested solvents. The highest yield was
observed in MeOH (entry 5, Table 1). We tried to improve the amount of NaBH4, however, the
yield decreased dramatically (entry 6, Table 1). Then we tried to reduce the amount of NaBH4,
dissappointingly, the yield was even lower (entry 7, Table 1). Next, the reaction was carried out
under reflux, which resulted in a 58% isolated yield (entry 7, Table 1). Given that the reductive
aminations are often conducted by in situ generation of the iminium ion in the presence of a mild
acid, an acidic condition with reducing reagents of good acid-tolerance might improve the N-
fused heterocycle formation. Based on this consideration, some acid-mediated reductive
aminations19-21 were carried out. However, no better results were observed (entries 9-11, Table 1).
Thus, Condition A (1, NaBH4 (2 equiv.), MeOH, and rt) was applied for further studies.
Table 1. Optimization of the Reaction Conditionsa
Entry Additive (equiv.)
Solvent
EtOH
Temperature
Time (h) NMR Yieldb (%)
1
2
3
NaBH4 (2)
NaBH4 (2)
NaBH4 (2)
rt
rt
rt
46
46
46
51
59
41
DCE
DMSO
5

ACCEPTED MANUSCRIPT
4
NaBH4 (2)
acetone
MeOH
MeOH
MeOH
MeOH
MeOH
rt
46
46
46
46
46
7
27
70
37
21
58
18
21
NR
5
NaBH4 (2)
rt
6
NaBH4 (3)
rt
7
NaBH4 (1.2)
NaBH4 (2)
rt
8
reflux
70 oC
9c
10
11
HCOONH4 (5)
HCOONH4 (12)
HCOONH4 (1)
HCOOH 160 oC
4
Toluene reflux
12
a Reaction conditions: 1 (0.1 mmol), additive, and solvent. b Determined by 19F NMR analysis of
c
the crude reaction mixture using trifluoromethylbenzene as an internal standard. 0.5 mol% of
[RhCp*Cl2]2 was applied.
With the optimal conditions in hand, the substrate scope of this cyclization reaction was
examined by applying a series of structurally diverse substrates (Table 2). Firstly, strong
electron-withdrawing groups (entries 1 and 2, Table 2), weak electron-withdrawing group (entry
3, Table 2), hydrogen (entry 4, Table 2), weak electron-donating groups (entries 5 and 6, Table
2), and strong electron-donating group (entry 7, Table 2) were introduced into the 4-position of
the aryl ring in the imide moiety of 1. The corresponding products were formed in moderate to
good yields. No clear electron effect was observed. Secondly, electron effect of the aryl ring in
the amine moiety of 1 was explored similarly (entries 8-12, Table 2). No clear electron effect
was observed, either. These results indicated that this protocol might serve as the general method
for the synthesis of 3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine derivatives.
Based on the above results, two possible reaction pathways for this cyclization were proposed as
shown in Scheme 2. In pathway A, the initial 1,3-H transfer of 1 resulted in its tautomer 9 which
was easier to be partially reduced by NaBH4. Then, the hydrogen anion from NaBH4 attacked at
6

ACCEPTED MANUSCRIPT
the 2-position of the pyrimidine ring to form the nitrogen anion species 10. The subsequent
intramolecular nucleophilic attack resulted in the cyclized nitrogen anion intermediate 11, which
gave the final product 8 after alcoholysis. In pathway B, direct reductive dearomatization of the
pyrimidine ring by NaBH4 started the whole process. The following intramolecular nucleophilic
addition and alcoholysis afforded intermediate 14. The final product 8 was produced by
isomerization via 1,3-H transfer, which led to a more stabled large conjugated π-system.
Table 2. Substrate Scope of the Cyclization Reactiona
R1
R1
NaBH4 (2 equiv.)
MeOH, rt
N
N
N
HN
N
NH
R2
R2
N
H
N
8
1
Isolated
Yield (%)
Entry
R1
R2
Time (h)
1
8
1
2
3
4
5
6
CF3
3-OCH2CH2O-4
3-OCH2CH2O-4
3-OCH2CH2O-4
3-OCH2CH2O-4
3-OCH2CH2O-4
3-OCH2CH2O-4
53
55
73
25
74
77
70
54
72
79
52
52
1a
1b
1c
1d
1e
1f
8a
8b
8c
8d
8e
8f
COOMe
Br
H
Me
tert-butyl
7

ACCEPTED MANUSCRIPT
7
OMe
H
3-OCH2CH2O-4
4-CF3
76
28
31
53
60
60
57
58
71
71
66
80
1g
1h
1i
8g
8h
8i
8
9
H
4-Br
10
11
12
H
4-H
1j
8j
H
4-Me
1k
8k
H
4-OMe
1l
8l
a Reaction conditions: 1 (0.2 mmol), NaBH4 (0.4 mmol), MeOH (6 mL), rt.
Scheme 2. Proposed Mechanism for the Cyclization Reaction
8

ACCEPTED MANUSCRIPT
To gain more information about the mechanism, an analogue of compound 1 with a methyl
group substituted on the amine nitrogen atom (compound 15, Scheme 3) was prepared and
applied in this reaction. If this reaction was initiated via 1,3-H transfer as proposed in pathway
A, no reaction would take place, since this key step should be totally inhibited. On the other
hand, if this reaction underwent via pathway B, the cyclization would proceed. However, the
final isomerization via 1,3-H transfer should be totally inhibited. The result indicated that a
cyclized product 16 was afforded, which clearly supported pathway B as the mechanism.
Scheme 3. The reaction of 15 under Condition A
tBu
tBu
NaBH4 (2 equiv.)
O
O
O
O
HN
N
N
N
N
N
MeOH, rt, 59 h
50%
N
N
16
15
2.2. Biology
In light of the importance of N-fused heterocycles in pharmaceuticals,5 the antitumor activities of
two representative target compounds, 8a and 8f, were screened. Their inhibitory activities
against three common human cancer cell-lines, ZR-75-30 (breast cancer), HCT116 (colon
cancer), and A549 (lung cancer), were evaluated by the MTT assay (Table 3). According to the
results, these compounds showed obvious inhibitory activity against all of the cancer cell lines.
9

ACCEPTED MANUSCRIPT
In particular, compound 8f showed good activity against the ZR-75-30 cancer cell line, with an
IC50 value lower than 1 ꢀM. ZR-75-30 is a cell line derived from human estrogen receptor-
negative (ER-) breast cancer, which is a difficult-to-treat type in clinical practice.22,23 Therefore,
compound 8f could serve as a good lead compound for the further development of new drugs to
treat this type of cancer. Furthermore, the possible mechanisms of this compound were explored.
Considering all of the approved kinase inhibitors containing nitrogen heterocycles,6,7 the
inhibitory activity of compound 8f against common kinases was screened. Similar to most of the
approved kinase inhibitors, it was multi-targeted.24,25 As shown in Table 3, it exhibited middling
inhibitory activity against FLT3, INSR and VEGFR-2, with an IC50 value of approximately 1 ꢀM
in all cases. These protein tyrosine kinases are all known to play significant roles in the
development and progression of many cancers,25-27 and INSR and VEGFR-2 are particularly
closely associated with breast cancer.27,28
Table 3. The Biological Activity of 3,4,6,7-Tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine
Derivatives
IC50 (µM) against human cancer
IC50 (µM) against kinases
cell lines
ZR-75-30
2.4
Compounds
HCT-116 A549
FLT3
/
INSR
/
VEGFR-2
2.9
1.1
7.4
2.2
/
8a
8f
0.71
0.94
0.86
1.0
3. Conclusion
10

ACCEPTED MANUSCRIPT
In summary, a novel and efficient reductive dearomatization-initiated intramolecular cyclization
reaction has been developed to construct a valuable N-fused heterocycle, 3,4,6,7-tetrahydro-2H-
pyrimido[1,6-c]quinazolin-2-imine derivatives. The scope was carefully studied. Moreover, this
type of compound showed multi-targeted inhibitory activity against several cancer-associated
kinases and good activity against the human ER- breast cancer cell line. Therefore, these
compounds might provide a new molecular scaffold for the development of new antitumor
agents. Further exploration in this field is underway in our laboratories.
4. Experimental
4.1. Chemistry
Melting points were determined on a WRS-2 apparatus. 1H (300, 400 or 600 MHz),13C (75, 100,
150 MHz), and 19F (376 MHz) of samples were recorded on a Bruker AC-300P, AVANCE II600
or an AVANCE III400 spectrometer, using TMS as an internal standard and CDCl3, CD3OD or
DMSO-d6 as solvents. HRMS (ESI) determinations were carried out on a Bruker Daltonics
micrOTOFⅡspectrometer. Compounds 3a and 7a were commercial available. Compounds 4a18
and 529 were prepared by the known procedures.
Synthesis of 6-(2-aminophenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)pyrimidin-4-amine
(compound 6a). (2-aminophenyl)boronic acid (5) (1.9 g, 14 mmol), compound 4a (3.0 g,
10mmol), K2CO3 (4.14 g, 30 mmol) and Pd(PPh)3Cl2 (0.35 g, 5 mol%) were suspended in a
mixture of 1,4-dioxane (30 mL) and H2O (15mL). After refluxing for 2.5 h under N2, 15 mL
water was added to the whole, and then extracted three times with EtOAc. The combined organic
11

ACCEPTED MANUSCRIPT
layers were washed with brine and dried over Na2SO4. Evaporation of the solvent gave the crude
product, which was purified by Silica gel column chromatography with EtOAc/PE (1:2) as the
1
eluent to afford the product 6a as yellow solid (2.9 g, yield 91%). Mp = 192 - 196 . H NMR
(600 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.61 (d, J = 1.1 Hz, 1H), 7.41 (dd, J = 7.9, 1.5 Hz, 1H),
7.31 (d, J = 2.5 Hz, 1H), 7.12 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.02 (dd, J = 8.7, 2.5 Hz, 1H), 6.95
(d, J = 1.1 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6.75 (dd, J = 8.2, 1.2 Hz, 1H), 6.61 (ddd, J = 8.1,
7.1, 1.2 Hz, 1H), 6.56 (s, 2H), 4.26 – 4.20 (m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 163.8,
160.8, 157.0, 148.3, 143.0, 139.0, 133.3, 130.6, 128.5, 118.4, 116.9, 116.7, 115.8, 113.5, 109.4,
101.9, 64.2, 63.9. HRMS (ESI) calcd for [M+1]+: C18H16N4O2: 321.1346, Found: 321.1348.
Synthesis
of
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(2-((4-
(trifluoromethyl)benzylidene)amino)phenyl)pyrimidin-4-amine (compound 1a). The compound
6a (250 mg, 0.78 mmol) and 4-(trifluoromethyl)benzaldehyde (7a) (0.19 g, 1.09 mmol) were
suspended in the EtOH (5 mL). To the stirring mixture was added dropwise concentrated HCl
(0.5 mL), followed by stirring overnight at room temperature. the precipitate was filtrated and
washed with a small volume of EtOH. After that, the suspension of the resulting precipitate in
MeOH was made alkaline to colourless with NaOH, and then filtered and washed with MeOH to
1
give compound 1a: white powder, Mp = 218 - 221℃. 294.9 mg, 79.3% yield. H NMR (400
MHz, DMSO-d6): δ 9.38 (s, 1H), 8.74 (s, 1H), 8.63 (d, J = 0.8 Hz, 1H), 8.08 (d, J = 8 Hz, 2H),
7.91 (dd, J = 8, 1.6 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.53 (td, J = 7.6, 1.6 Hz, 1H), 7.41 (td, J =
7.6, 1.6 Hz, 1H), 7.24 (dd, J = 8, 0.8 Hz, 1H), 7.11 (s, 1H), 7.05 (d, J = 1.2 Hz, 1H), 6.86 (d, J =
6.4 Hz, 1H), 6.58 (d, J = 8.4, 1H), 4.17 – 4.15 (m, 4H); 13C NMR (101 MHz, DMSO-d6) δ 160.9,
160.2, 157.9, 149.3, 143.1, 139.5, 139.3, 132.9, 131.9, 131.3, 131.0, 131.0, 129.9, 129.5, 126.4,
12

ACCEPTED MANUSCRIPT
125.8, 125.7, 119.4, 116.8, 114.3, 110.2, 106.7, 64.1, 63.9. HRMS (ESI) calcd for [M+1]+
C26H19F3N4O2: 477.1533; Found: 477.1539. 19F NMR (376 MHz, DMSO-d6) -61.3 (s, 3F).
The following compounds were prepared according to the same procedure of compound 1a.
Methyl-4-(((2-(6-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)pyrimidin-4-
yl)phenyl)imino)methyl)benzoate (1b): white powder, Mp = 176 - 180℃. 207 mg, 47.4% yield.
1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.70 (s, 1H), 8.63 (d, J = 0.8 Hz, 1H), 8.06 (d, J
= 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.93 (dd, J = 8, 1.6 Hz, 1H), 7.52 (td, J = 7.6, 1.2 Hz,
1H), 7.40 (td, J = 7.6, 1.2 Hz, 1H), 7.22(d, J = 7.6 Hz, 1H), 7.13 (d, J = 0.8 Hz, 1H), 7.08 (s,
1H), 6.85 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 8.8 Hz, 1H), 4.20 – 4.15 (m, 4H), 3.90(s, 3H); 13C
NMR (101 MHz, DMSO-d6) δ 165.8, 160.8, 160.4, 160.2, 157.8, 149.5, 143.1, 139.8, 139.3,
132.8, 131.8, 131.7, 130.5, 129.8, 129.5, 129.0, 126.2, 119.3, 116.7, 114.2, 110.2, 106.4, 64.1,
63.8, 52.3. HRMS (ESI) calcd for [M+1]+: C27H22N4O4: 467.1714; Found: 467.1713.
6-(2-((4-Bromobenzylidene)amino)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)pyrimidin-
1
4-amine (1c): white powder, Mp = 182 - 182.5℃. 304.9 mg, 80.2% yield. H NMR (400 MHz,
DMSO-d6) δ 9.36 (s, 1H), 8.62 (s, 1H), 8.60 (s, 1H), 7.90 (d, J = 8 Hz, 1H), 7.80 (d, J = 8.4 Hz,
2H), 7.70 (d, J = 8.8 Hz, 2H), 7.53 – 7.49 (m, 1H), 7.38 (t, J = 8 Hz, 1H), 7.19 (d, J = 8 Hz, 1H),
7.09 (s, 1H), 7.07 (s, 1H), 6.85 (d, J = 10 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 4.20 – 4.17 (m, 4H);
13C NMR (101MHz, DMSO-d6) δ 161.0, 160.2, 157.9, 149.6, 143.1, 139.3, 135.1, 132.8, 131.9,
131.7, 130.7, 130.6, 129.8, 126.1, 125.3, 119.4, 116.8, 114.3, 110.2, 106.5, 64.2, 63.9. HRMS
(ESI) calcd for [M+1]+: C25H19BrN4O2: 487.0764; Found: 487.0769.
13

ACCEPTED MANUSCRIPT
6-(2-(Benzylideneamino)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)pyrimidin-4-amine
(1d): white powder, Mp = 174 - 175℃. 206.5 mg, 64.7% yield. 1H NMR (400 MHz, DMSO-d6)
δ 9.37 (s, 1H), 8.62 (d, J = 1.2 Hz, 1H), 8.59 (s, 1H), 7.89 – 7.87 (m, 3H), 7.57 – 7.48 (m, 4H),
7.39 – 7.35 (m, 1H), 7.18 (dd, J = 8, 0.8 Hz, 1H), 7.14 (d, J = 1.2 Hz, 1H), 7.10 (s, 1H), 6.85 (dd,
J =8.8, 2.4 Hz, 1H), 6.54 (d, J = 8.8 Hz, 1H), 4.19 – 4.16 (m, 4H); 13C NMR (101 MHz, DMSO-
d6) δ 161.3, 161.1, 160.2, 157.9, 150.0, 143.1, 139.3, 136.0, 132.9, 131.7, 131.6, 130.6, 129.8,
128.9, 128.8, 125.8, 119.4, 116.8, 114.1, 110.1, 107.0, 64.2, 63.9. HRMS (ESI) calcd for
[M+1]+: C25H20N4O2: 409.1659; Found: 409.1658.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(2-((4-methylbenzylidene)amino)phenyl)pyrimidin-
1
4-amine (1e): white powder, Mp = 190 - 191℃. 220.4 mg, 55.7% yield. H NMR (400 MHz,
DMSO-d6) δ 9.36 (s, 1H), 8.61 (d, J = 1.2 Hz, 1H), 8.53 (s, 1H), 7.88 (dd, J = 7.6, 1.6 Hz, 1H),
7.76 (d, J = 7.7 Hz, 2H), 7.49 (td, J = 7.6, 1.6 Hz, 1H), 7.37 – 7.29 (m, 3H), 7.16 – 7.14 (m, 2H),
7.10 (s, 1H), 6.86 (dd, J = 8.4, 2.4 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H) , 4.19 – 4.16 (m, 4H), 2.37
(s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.1, 161.1, 160.2, 157.9, 150.1, 143.1, 141.7,
139.3, 133.5, 133.0, 131.6, 130.6, 129.8, 129.4, 128.9, 125.6, 119.4, 116.8, 114.1, 110.1, 106.6,
64.2, 63.9, 21.2. HRMS (ESI) calcd for [M+1]+: C26H22N4O2: 423.1816; Found: 423.1819.
6-(2-((4-(Tert-butyl)benzylidene)amino)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-
1
yl)pyrimidin-4-amine (1f): white powder, Mp = 242 - 244℃. 261.8 mg, 72.3% yield. H NMR
(400 MHz, DMSO-d6): δ 9.37 (s, 1H), 8.20 (d, J = 0.8 Hz, 1H), 8.55 (s, 1H), 7.88 (dd, J = 8, 1.6
Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.53 – 7.47 (m, 3H), 7.35 (td, J = 8, 1.6 Hz, 1H), 7.16 – 7.10
(m, 3H), 6.86 (dd, J = 8.8, 2 Hz, 1H), 6.54 (d, J = 8.8Hz, 1H), 4.19 – 4.16 (m, 4H), 1.31 (s, 9H);
13C NMR (101 MHz, DMSO-d6) δ 161.1, 161.0, 160.1, 157.9, 154.7, 150.1, 143.1, 139.3, 133.5,
14

ACCEPTED MANUSCRIPT
132.9, 131.6, 130.6, 129.8, 128.8, 125.6, 119.4, 116.8, 114.1, 110.1, 106.5, 64.2, 63.9, 34.7, 30.9.
HRMS (ESI) calcd for [M+1]+: C29H28N4O2: 465.2285; Found: 465.2288.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(2-((4-methoxybenzylidene)amino)phenyl)pyrimidin-
1
4-amine (1g): white powder, Mp = 215 - 216℃. 322.4 mg, 94.2% yield. H NMR (400 MHz,
DMSO-d6): δ 9.36 (s, 1H), 8.63 (d, J = 1.2 Hz, 1H), 8.49 (s, 1H), 7.89 (dd, J = 7.6, 1.6 Hz, 1H),
7.82 (d, J = 8.8 Hz, 2H), 7.48 (td, J = 7.6, 1.6 Hz, 1H), 7.33 (td, J = 7.6, 2.4 Hz, 1H), 7.20 (s,
1H), 7.20 – 7.14 (m, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8 Hz, 1H), 6.59 (d, J = 8.8 Hz,
13
1H), 4.20 – 4.17 (m, 4H), 3.84 (s, 3H); C NMR (101 MHz, DMSO-d6) δ 162.0, 161.1, 160.4,
160.1, 157.8, 150.2, 143.1, 139.2, 132.9, 131.4, 130.7, 130.5, 129.7, 128.9, 125.3, 119.4, 116.7,
114.2, 114.1, 110.1, 106.4, 64.1, 63.9, 55.4. HRMS (ESI) calcd for [M+1]+: C26H22N4O3:
439.1765; Found: 439.1769.
6-(2-(Benzylideneamino)phenyl)-N-(4-(trifluoromethyl)phenyl)pyrimidin-4-amine (1h): white
1
powder, Mp = 218 - 221℃. 375 mg, 97.9% yield. H NMR (400 MHz, DMSO-d6): δ 10.03 (s,
1H), 8.80 (s, 1H), 8.65 (s, 1H), 7.95 – 7.82 (m, 5H), 7.54 – 7.48 (m, 6H), 7.38 (t, J = 7.2 Hz,
1H), 7.33 (s, 1H), 7.21 (d, J = 7.6 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 161.8, 161.5,
159.5, 157.7, 149.9, 143.5, 136.0, 131.6, 131.5, 130.8, 129.9, 129.0, 128.8, 125.9, 125.9, 125.8,
123.1, 122.1, 121.8, 119.4, 119.2, 108.3. HRMS (ESI) calcd for [M+1]+: C24H17F3N4: 419.1478;
Found: 419.1478. 19F NMR (376 MHz, DMSO-d6) -60.1 (s, 3F).
6-(2-(Benzylideneamino)phenyl)-N-(4-bromophenyl)pyrimidin-4-amine (1i): white powder, Mp
= 209 - 211℃. 213.9 mg, 56.7% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.72 (d, J
= 0.8 Hz, 1H), 8.63(s, 1H), 7.93 – 7.91 (m, 3H), 7.56 – 7.48 (m, 6H), 7.40 – 7.31 (m, 3H), 7.24
13
(d, J = 0.8 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 161.4, 159.6,
15

ACCEPTED MANUSCRIPT
157.7, 149.9, 139.1, 136.0, 131.6, 131.5, 131.4, 130.7, 130.0, 129.0, 128.8, 125.8, 121.9, 119.4,
113.9, 107.5. HRMS (ESI) calcd for [M+1]+: C23H17BrN4: 429.0709; Found: 429.0708.
6-(2-(Benzylideneamino)phenyl)-N-phenylpyrimidin-4-amine (1j): white powder, Mp = 170 -
1
172℃. 270 mg, 80.9% yield. H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.69 (s, 1H), 8.62
(s, 1H), 7.92 – 7.89 (m, 3H), 7.55 – 7.49 (m, 6H), 7.37 (t, J = 7.6 Hz, 1H), 7.22 – 7.14 (m, 4H),
13
6.97 (t, J = 7.2 Hz, 1H); C NMR (101 MHz, DMSO-d6) δ 161.4, 161.3, 159.9, 157.8, 149.9,
139.5, 136.0, 131.6, 131.6, 130.6, 129.8, 128.9, 128.8, 128.6, 125.8, 122.6, 120.3, 119.4, 107.0.
HRMS (ESI) calcd for [M+1]+: C23H18N4: 351.1604; Found: 351.1609.
6-(2-(Benzylideneamino)phenyl)-N-(p-tolyl)pyrimidin-4-amine (1k): white powder, Mp = 182 -
184℃. 219 mg, 55% yield. 1H NMR (400 MHz, DMSO-d6): δ 9.47 (s, 1H), 8.66 (s, 1H), 8.59 (s,
1H), 7.92 – 7.88 (m, 3H), 7.55 – 7.48 (m, 4H), 7.38 – 7.34 (m, 3H), 7.18 – 7.15 (m, 2H), 6.93 (d,
J = 8Hz, 2H), 2.20 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.3, 161.1, 160.1, 157.9, 149.9,
136.7, 136.0, 131.8, 131.6, 131.6, 130.5, 129.8, 129.1, 128.9, 128.8, 125.8, 120.7, 119.4, 106.6,
20.4. HRMS (ESI) calcd for [M+1]+: C24H20N4: 365.1761; Found: 365.1768.
6-(2-(Benzylideneamino)phenyl)-N-(4-methoxyphenyl)pyrimidin-4-amine (1l): white powder,
Mp = 174 - 176℃. 315 mg, 96.8% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.63 (s,
1H), 8.59 (s, 1H), 7.91 – 7.88 (m, 3H), 7.56 – 7.49 (m, 4H), 7.36 – 7.30 (m, 3H), 7.15 (d, J = 7.6
13
Hz, 1H), 7.08 (s, 1H), 6.64 (d, J = 8 Hz, 2H), 3.66 (s, 3H); C NMR (101 MHz, DMSO-d6) δ
161.3, 161.0, 160.3, 158.0, 155.3, 149.9, 136.0, 132.0, 131.7, 131.6, 130.5, 129.8, 128.9, 128.8,
125.8, 122.8, 119.4, 113.9, 105.9, 55.1. HRMS (ESI) calcd for [M+1]+: C24H20N4O: 381.1710;
Found: 381.1719.
16

ACCEPTED MANUSCRIPT
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-methyl-6-(2-((4-
(trifluoromethyl)benzylidene)amino)phenyl)pyrimidin-4-amine (15): white powder, Mp = 168 -
1
170℃. 303 mg, 70.5% yield. H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 1.2 Hz, 1H), 8.4 (s,
1H), 7.87 (dd, J = 7.6, 1.2 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.45 (td, J
= 7.2, 1.2 Hz, 1H), 7.34 – 7.30 (m, 1H), 7.05 (d, J = 7.2 Hz, 1H), 6.86 (d, J = 0.8 Hz, 1H), 6.79
(d, J = 2.8 Hz, 1H), 6.61 (dd, J = 8.4, 2.4 Hz, 1H), 6.39 (d, J = 8.4 Hz,1H), 4.16 (s, 4H), 3.34 (s,
13
3H), 1.36 (s, 9H); C NMR (101 MHz, DMSO-d6) δ 161.3, 161.0, 160.3, 157.7, 154.6, 154.2,
150.0, 143.8, 142.0, 136.8, 133.3, 131.4, 130.5, 129.7, 128.6, 125.6, 119.6, 119.4, 117.6, 115.3,
105.4, 64.0, 63.9, 37.9, 34.8, 30.9. HRMS (ESI) calcd for [M+1]+: C30H30N4O2: 479.2442,
Found: 479.2444.
Synthesis of
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-trifluoromethyl)phenyl-3,4,6,7-tetrahydro-2H-
pyrimido[1,6-c]quinazolin-2-imine (8a). To a 25 mL Schlenck tube were added 1a (48 mg, 0.1
mmol) and NaBH4 (8 mg, 0.2 mmol) under air. The mixture was then evacuated and backfilled
with Ar (3 times). anhydrous MeOH (3 mL) was added subsequently. The tube was screw
capped, then stirred at room temperature for 46 hr. After stirring, H2O was added to the solution,
and extracted with dichloromethane, dried over Na2SO4. After evaporation of solvent, the residue
was purified by the alumina column chromatography with CH2Cl2/MeOH (v:v = 100:0 →
100:15) as the eluent to obtain compound 8a: yellow oil. 67 mg, 70% yield. 1H NMR (400 MHz,
CD3OD) δ 7.96 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 8 Hz, 1H),7.31 (t, J =
7.7Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.83 – 6.81 (m, 3H), 6.74 (dd, J =8.5, 2.3 Hz, 1H), 5.93 (s,
1H), 4.66 – 4.64 (m, 2H), 4.25 (s, 4H); 13C NMR (101 MHz, CD3OD) δ 153.8, 143.9, 143.6,
142.7, 142.4, 134.5, 131.1, 130.8, 129.1, 127.0, 125.8, 125.8, 125.0, 122.3, 119.3, 118.2, 117.8,
17

ACCEPTED MANUSCRIPT
116.8, 112.8, 68.5, 64.3, 64.2, 57.5. HRMS (ESI) calcd for [M+1]+: C26H21F3N4O2: 479.1689;
Found: 479.1686. 19F NMR (376 MHz, CD3OD) -62.7 (s, 3F). The scaffold of 3,4,6,7-
tetrahydropyrimido[1,6-c]quinazolin-2-imine was completely confirmed by the multi-
dimensional NMR spectrum of compound 8d.
The following compounds were prepared according to the same procedure of compound 8a.
Methyl-4-(2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)imino)-3,4,6,7-tetrahydro-2H-pyrimido[1,6-
c] quinazolin-6-yl)benzoate (8b): yellow oil. 50 mg, 54% yield. 1H NMR (400 MHz, CD3OD) δ
7.96 (d, J = 8 Hz, 2H), 7.56 – 7.54 (m, 3H), 7.25 (t, J = 7.6 Hz, 1H), 6.82 – 6.77 (m, 5H), 6.68
13
(d, J = 8.4 Hz, 1H), 5.71 (s, 1H), 4.49 (s, 2H), 4.20 (s, 4H), 3.88 (s, 3H); C NMR (101 MHz,
CD3OD) δ 166.3, 158.1, 153.7, 143.8, 143.7, 143.1, 142.5, 134.3, 130.6, 130.0, 129.2, 126.7,
125.7, 119.1, 118.1, 117.7, 116.7, 114.0, 112.6, 77.2, 68.7, 64.2, 64.2, 57.3, 52.1. HRMS (ESI)
calcd for [M+1]+: C27H24N4O4: 469.1870; Found: 469.1878.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-bromo)phenyl-3,4,6,7-tetrahydro-2H-
1
pyrimido[1,6-c]quinazolin-2-imine (8c): yellow oil. 70 mg, 72% yield. H NMR (400 MHz,
CD3OD) δ 7.60 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.32 (t, J = 8.4 Hz,
1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 – 6.75 (m, 4H), 5.89 (s, 1H), 4.70 – 4.58 (m, 2H), 4.05 (s,
4H); 13C NMR (101 MHz, CD3OD) δ 158.8, 155.7, 145.8, 145.7, 144.4, 139.4, 135.6, 133.1,
129.8, 129.8, 126.9, 124.3, 120.2, 119.2, 118.7, 117.2, 114.7, 113.6, 70.1, 65.7, 65.6, 57.8..
HRMS (ESI) calcd for [M+1]+: C25H21BrN4O2: 491.0901; Found: 491.0910.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-phenyl-3,4,6,7-tetrahydro-2H-pyrimido[1,6-
1
c]quinazolin-2-imine (8d): yellow oil. 65 mg, 79% yield. H NMR (600 MHz, CDCl3) δ 7.46 –
7.45 (m, 2H), 7.38 – 7.37 (m, 3H), 7.30 – 7.29 (m, 1H), 7.24 (d, J = 7.4 Hz, 1H), 6.85 (d, J = 8.5
18

ACCEPTED MANUSCRIPT
Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 6.76 (t, J = 7.9 Hz, 1H), 6.71 (dd, J =
8.5, 2.4 Hz, 1H), 5.76 (s, 1H), 5.62 (s, 1H), 5.49 (s, 1H), 4.44 (d, J = 9.9 Hz, 1H), 4.35 (d, J = 9.9
Hz, 1H), 4.26 (s, 4H); 13C NMR (150 MHz, CDCl3 ) δ 159.1, 155.0, 144.7, 144.3, 143.2, 136.9,
134.6, 130.6, 129.8, 129.5, 127.8, 126.3, 120.1, 119.1, 118.2, 116.7, 116.6, 115.1, 113.5, 78.6,
70.9, 64.7, 64.7, 56.6. HRMS (ESI) calcd for [M+1]+: C25H22N4O2: 411.1816; Found: 411.1822.
Its multi-dimensional NMR spectrum attached behind.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-methyl)phenyl-3,4,6,7-tetrahydro-2H-
1
pyrimido[1,6-c]quinazolin-2-imine (8e): yellow oil. 44 mg, 52% yield. H NMR (400 MHz,
CDCl3) δ 7.32 – 7.28 (m, 4H), 7.12 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 7.2 Hz, 2H), 6.89 (d, J = 8.0
Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.70 – 6.67 (m, 2H), 5.61 (s, 1H),
5.53 (s, 1H), 4.43 (d, J =9.9 Hz, 1H), 4.37 (d, J = 9.9 Hz, 1H), 4.22 (s, 4H), 2.27 (s, 3H); 13C
NMR (101 MHz, CDCl3 ) δ 158.3, 154.5, 144.4, 143.8, 142.5, 139.9, 134.1, 133.7, 129.8, 129.8,
129.2, 127.2, 127.1, 125.7, 119.2, 118.4, 117.6, 116.2, 114.3, 112.8, 77.2, 70.0, 64.2, 64.2, 56.2,
21.1. HRMS (ESI) calcd for [M+1]+: C26H24N4O2: 425.1972; Found: 425.1983.
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(tert-butyl)phenyl-3,4,6,7-tetrahydro-2H-
1
pyrimido[1,6-c]quinazolin-2-imine (8f): yellow oil. 48 mg, 52% yield. H NMR (400 MHz,
CDCl3) δ 7.38 (s, 4H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 6.85 – 6.81 (m, 2H),
6.78 (d, J = 2.8 Hz, 1H), 6.76 – 6.70 (m, 2H), 6.06 (s, 1H), 5.64 (s, 1H), 5.53 (s, 1H), 4.47 (d, J =
9.9 Hz, 1H), 4.39 (d, J = 9.9 Hz, 1H), 4.25 (s, 4H), 1.28 (s, 9H); 13C NMR (101 MHz, CDCl3 ) δ
158.4, 154.5, 153.1, 144.3, 143.8, 142.6, 134.1, 133.6, 129.2, 126.9, 126.2, 125.8, 119.3, 118.5,
117.7, 116.2, 114.4, 112.8, 77.2, 70.0, 64.2, 64.2, 56.2, 34.6, 31.1. HRMS (ESI) calcd for
[M+1]+: C29H30N4O2: 467.2442; Found: 467.2448.
19

ACCEPTED MANUSCRIPT
N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-methoxy)phenyl-3,4,6,7-tetrahydro-2H-
1
pyrimido[1,6-c]quinazolin-2-imine (8g): yellow oil. 33 mg, 37% yield. H NMR (400 MHz,
CD3OD) δ 7.59 (d, J = 8 Hz, 1H), 7.42 – 7.40 (m, 3H), 7.31 (t, J = 8.4 Hz, 1H), 6.94 – 6.92 (m,
2H), 6.9 (d, J = 8.8 Hz, 1H), 6.80 – 6.78 (m, 3H), 6.73 (dd, J = 8.6, 2.3 Hz, 1H), 5.73 (s, 1H),
4.52 – 4.45 (m, 2H), 4.23 (s, 4H), 3.76 (s, 3H); 13C NMR (101 MHz, CD3OD) δ 162.1, 158.8,
156.3, 146.6, 145.7, 144.3, 135.4, 131.2, 129.7, 126.9, 112.0, 119.2, 118.6, 117.0, 115.4, 114.7,
113.6, 71.0, 65.7, 65.6, 57.2, 55.9. HRMS (ESI) calcd for [M+1]+: C26H24N4O3: 441.1921;
Found: 441.1929.
6-Phenyl-N-(3-(trifluoromethyl)phenyl)-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-
1
imine (8h): yellow oil. 49 mg, 58% yield. H NMR (400 MHz, CDCl3) δ 7.55 – 7.51 (m, 5H),
7.44 (s, 5H), 7.24 – 7.22 (m, 3H), 6.84 (t, J = 7.6 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 5.67 (s, 1H),
5.38 (s, 1H), 4.30 (d, J = 9.2 Hz, 1H), 4.17 (d, J = 9.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ
157.3, 143.7, 132.5, 131.4, 130.3, 129.3, 129.0, 127.9, 126.3, 126.3, 126.3, 125.1, 122.4, 120.3,
120.3, 120.1, 117.5, 117.5, 115.7, 102.0, 71.6, 58.3. HRMS (ESI) calcd for [M+1]+: C24H19F3N4:
421.1635; Found: 421.1639. 19F NMR (376 MHz, CDCl3) -61.9 (s, 3F).
N-(3-bromophenyl)-6-phenyl-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine
(8i):
1
yellow oil. 61mg, 71% yield. H NMR (400 MHz, CD3OD) δ 7.61 – 7.59 (m, 4H), 7.48 – 7.33
(m, 7H), 7.26 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 7.6 Hz, 2H), 5.92 (s, 1H), 5.83 (s, 1H), 4.69 (d, J =
10.8 Hz, 1H), 4.64 (d, J = 10.8 Hz, 1H); 13C NMR (101 MHz, CD3OD) δ 156.5, 152.1, 143.6,
137.1, 132.8, 131.9, 131.6, 129.7, 128.9, 127.2, 125.0, 124.6, 119.3, 117.1, 115.7, 113.7, 70.6,
57.6. HRMS (ESI) calcd for [M+1]+: C23H19BrN4: 431.0866; Found: 431.0877.
20

ACCEPTED MANUSCRIPT
N,6-diphenyl-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine (8j): yellow oil. 51 mg,
71% yield. 1H NMR (400 MHz, CDCl3) δ 7.54 – 7.49 (m, 2H), 7.48 – 7.38 (m, 7H), 7.36 (dd, J =
8.2, 1.4 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.26 (s, 1H), 6.82 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 6.77 (dd,
J = 8.2, 1.0 Hz, 1H), 5.64 (s, 1H), 5.59 (s, 1H), 4.44 (d, J = 9.8 Hz, 1H), 4.33 (d, J = 9.8 Hz, 1H);
13C NMR (101 MHz, CDCl3) δ 158.2, 144.2, 136.3, 134.0, 130.5, 129.5, 129.5, 128.2, 127.7,
126.6, 125.8, 124.7, 120.0, 117.6, 116.1, 113.5, 77.2, 70.9, 56.4. HRMS (ESI) calcd for [M+1]+:
C23H20N4: 353.1761; Found: 353.1769.
6-Phenyl-N-(p-tolyl)-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine (8k): yellow oil.
48 mg, 66% yield. 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.44 (m, 2H), 7.35 – 7.30 (m, 5H), 7.17
(t, J = 7.2 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8 Hz, 1H),
6.72 (t, J = 8 Hz, 1H), 5.59 (s, 1H), 5.49 (s, 1H), 4.34 (d, J = 9.6 Hz, 1H), 4.24 (d, J = 9.6 Hz,
1H), 2.33 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 157.7, 153.2, 144.0, 137.2, 135.7, 135.6,
133.5, 129.9, 129.8, 129.1, 127.4, 125.4, 124.1, 119.4, 116.1, 113.5, 79.7, 70.5, 57.2, 20.9.
HRMS (ESI) calcd for [M+1]+: C24H22N4: 367.1917; Found: 367.1913.
N-(3-methoxyphenyl)-6-phenyl-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-imine (8l):
yellow oil. 61 mg, 80% yield. 1H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.49 – 7.47 (m, 2H),
7.39 – 7.37 (m, 2H), 7.31 (t, J = 7.6Hz, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8 Hz, 2H),
13
6.83 – 6.80 (m, 2H), 5.85 (s, 1H), 4.59 (s, 2H), 3.80 (s, 3H); C NMR (101 MHz, CD3OD) δ
160.3, 158.9, 156.0, 146.3, 139.8, 135.5, 130.5, 130.1, 128.1, 127.2, 126.9, 120.0, 117.1, 116.2,
113.6, 71.0, 57.6, 56.1. HRMS (ESI) calcd for [M+1]+: C24H22N4O: 383.1866; Found: 383.1869.
6-(4-(Tert-butyl)phenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-methyl-6,7-dihydro-4H-
1
pyrimido[1,6-c]quinazolin-2-amine (16): yellow oil. 69 mg, 50% yield. H NMR (400 MHz,
21

ACCEPTED MANUSCRIPT
CDCl3) δ 7.40 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 8
Hz, 1H), 6.93 – 6.87 (m, 2H), 6.77 (s, 1H), 6.72 – 6.64 (m, 2H), 6.46 (s, 1H), 5.60 (s, 1H), 5.13
(s, 1H), 4.64 – 4.62 (m, 2H), 4.30 (s, 4H), 3.54 (s, 3H), 1.24 (s, 9H); 13C NMR (101 MHz,
CDCl3) δ 158.4, 152.5, 152.3, 144.1, 144.1, 143.4, 136.5, 134.7, 133.5, 126.6, 126.0, 125.2,
119.8, 118.7, 118.0, 116.3, 115.6, 79.6, 79.4, 77.3, 69.6, 64.2, 57.9, 41.3, 34.5, 31.1. HRMS
(ESI) calcd for [M+1]+: C30H32N4O2: 481.2598, Found: 481.2613.
4.2. Biology.
Tumor cell growth inhibitory activity test. Tumor cell growth inhibitory activities were tested
against human cancer cell-lines, ZR-75-30 (breast cancer), HCT116 (colon cancer), and A549
(lung cancer), by the known procedure.30 Briefly, For each well of a 96-well microplate, 100 ꢀL
of cell dilution was seeded, allowed to attach overnight, and then exposed to varying
concentrations of compounds for 72 h (37 °C, 5% CO2 atmosphere). The number of living cells
was estimated by the MTT assay. The IC50 values were determined by a nonlinear regression
analysis. Average values were reported.
Kinases inhibitory activity test. Kinases inhibitory activities were tested against several common
kinases, such as ABL, FGFR1, FLT3, MET, INSR, EGFR, VEGFR-2, PDK1, CHK1, etc., by the
known procedure.31 The activities of the kinases were detected by mobility shift assay on EZ
Caliper Reade. For the determination of IC50, the compounds were tested at 10 concentrations.
Average values were reported.
22

ACCEPTED MANUSCRIPT
Acknowledgements
We thank the Shanghai Rising-Star Program (16QA1404800), State Scholarship Fund of China,
International Science & Technology Cooperation Program of China (2014DFE40130), the
National Natural Science Foundation of China (Grant No. 21172260 and 21572266), “Chen
Guang” project supported by the Shanghai Municipal Education Commission, Shanghai
Education Development Foundation (12CG42), and National Basic Research Program of China
(973 Program, 2012CB720300) for financial support.
Supplementary data
Supplementary data related to this article can be found at.
References and notes
1.
2.
3.
4.
Zhang, B.; Studer, A. Chemical Society Reviews 2015, 44, 3505-3521.
Patel, D. R.; Patel, K. C. Journal of Saudi Chemical Society 2015, 19, 347-359.
Taylor, R. D.; MacCoss, M.; Lawson, A. D. J Med Chem 2014, 57, 5845-59.
Reddy, M. K.; Rao, Y. J.; Krupadanam, G. L. Journal of Saudi Chemical Society 2015,
19, 372-378.
5.
6.
7.
8.
Vitaku, E.; Smith, D. T.; Njardarson, J. T. J Med Chem 2014, 57, 10257-74.
Wu, P.; Nielsen, T. E.; Clausen, M. H. Drug Discov Today 2015.
Wu, P.; Nielsen, T. E.; Clausen, M. H. Trends Pharmacol Sci 2015, 36, 422-39.
Tomi, I. H. R.; Tomma, J. H.; Al-Daraji, A. H. R.; Al-Dujaili, A. H. Journal of Saudi
Chemical Society 2015, 19, 392-398.
9.
Miniyar, P. B.; Barmade, M. A.; Mahajan, A. A. Journal of Saudi Chemical Society
2015, 19, 655-660.
10.
11.
Bagdi, A. K.; Santra, S.; Monir, K.; Hajra, A. Chemical Communications 2015, 51, 1555-
1575.
Jia, F. C.; Xu, C.; Zhou, Z. W.; Cai, Q.; Li, D. K.; Wu, A. X. Org lett 2015, 17, 2820-3.
23

ACCEPTED MANUSCRIPT
12.
13.
Xu, T.; Zhang, L.; Xu, S.; Yang, C. Y.; Luo, J.; Ding, F.; Lu, X.; Liu, Y.; Tu, Z.; Li, S.;
Pei, D.; Cai, Q.; Li, H.; Ren, X.; Wang, S.; Ding, K. Angew Chem Int Ed Engl 2013, 52,
8387-90.
Arnold, D. M.; Laporte, M. G.; Anderson, S. M.; Wipf, P. Tetrahedron 2013, 69, 7719-
7731.
14.
15.
16.
17.
Zhu, J.; Xie, H.; Chen, Z.; Li, S.; Wu, Y. Chem Commun (Camb) 2011, 47, 1512-4.
Seregin, I. V.; Schammel, A. W.; Gevorgyan, V. Tetrahedron 2008, 64, 6876-6883.
Seregin, I. V.; Schammel, A. W.; Gevorgyan, V. Org lett 2007, 9, 3433-6.
Ziarani, G. M.; Nasab, N. H. Rahimifard, M.; Soorki, A. A. Journal of Saudi Chemical
Society 2015, 19, 676-681.
18.
19.
Hartung, C. G.; Backes, A. C.; Felber, B.; Missio, A.; Philipp, A. Tetrahedron 2006, 62,
10055-10064.
Kitamura, M. Lee, D.; Hayashi, S.; Tanaka, S.; Yoshimura, M. J. Org. Chem. 2002, 67,
8685-8687.
20.
21.
22.
23.
Akimova, T. I.; Kravchenko, N. S.; Denisenko, V. A. Tetrahedron 2008, 64, 4204-4208.
O’Connor, D.; Lauria, A.; Bondi, S. P.; Saba , S. Tetrahedron Letters 2011, 52, 129-132.
Sestak, I.; Cuzick, J. Curr Opin Obstet Gynecol 2015, 27, 92-7.
Mayer, I. A.; Abramson, V. G.; Lehmann, B. D.; Pietenpol, J. A. Clin Cancer Res 2014,
20, 782-90.
24.
Gross, S.; Rahal, R.; Stransky, N.; Lengauer, C.; Hoeflich, K. P. J Clin Invest 2015, 125,
1780-9.
25.
26.
27.
28.
Hojjat-Farsangi, M. Int J Mol Sci 2014, 15, 13768-801.
Belfiore, A.; Malaguarnera, R. Endocr Relat Cancer 2011, 18, R125-47.
Shibuya, M. J Biochem 2013, 153, 13-9.
Guo, S.; Colbert, L. S.; Fuller, M.; Zhang, Y.; Gonzalez-Perez, R. R. Biochim Biophys
Acta 2010, 1806, 108-21.
29.
30.
31.
Groziak, M. P.; Ganguly, A. D.; Robinson, P. D. Journal of the American Chemical
Society 1994, 116, 7597-7605.
Jiang, J.; Zheng, C.; Zhu, K.; Liu, J.; Sun, N.; Wang, C.; Jiang, H.; Zhu, J.; Luo, C.;
Zhou, Y. J Med Chem 2015, 58, 2538-2546.
Wang, Y.; Cai, Y.; Ji, J.; Liu, Z.; Zhao, C.; Zhao, Y.; Wei, T.; Shen, X.; Zhang, X.; Li,
X.; Liang, G. Cancer Lett 2014, 344, 82-89.
24