Design, synthesis and bioevaluation of 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives as potent neuroprotective agents

Article abstract of DOI:10.1016/j.ejmech.2018.03.052

Diverse of 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives were designed, synthesized and evaluated for their neuroprotective activity against NMDA-induced cytotoxicity in vitro, and 5q exhibited excellent neuroprotective activity. The compound 5q was selected for further investigation. We found that 5q could attenuate Ca²⁺ influx induced by NMDA, meanwhile, 5q could suppress the NR2B up-regulation and increase p-ERK1/2 expression. The molecular docking results showed that 5q might fit well in the binding pocket of 4 and interact with some key residues in the binding pocket of 1 simultaneously. Besides, 5q exhibited acceptable metabolic stability. These results suggested that 5q was a promising lead for further development of new potent and orally bioavailable NR2B-selective NMDAR antagonists.

Full text of DOI:10.1016/j.ejmech.2018.03.052

Accepted Manuscript
Design, synthesis and bioevaluation of 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-
carboxylic acid derivatives as potent neuroprotective agents
Linkui Zhang, Ying Zhao, Jian Wang, Donglin Yang, Chenwen Zhao, Changli Wang,
Chao Ma, Maosheng Cheng
PII:
S0223-5234(18)30294-0
10.1016/j.ejmech.2018.03.052
EJMECH 10317
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 January 2018
Revised Date: 16 March 2018
Accepted Date: 17 March 2018
Please cite this article as: L. Zhang, Y. Zhao, J. Wang, D. Yang, C. Zhao, C. Wang, C. Ma, M. Cheng,
Design, synthesis and bioevaluation of 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid
derivatives as potent neuroprotective agents, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.03.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design,
synthesis
and
bioevaluation
of
1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives as
potent neuroprotective agents
Linkui Zhang a, Ying Zhao a, Jian Wang a, Donglin Yang a, Chenwen Zhao a, Changli
Wang b, Chao Ma a, *, Maosheng Cheng a, *
a
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of
Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
b
Department of Pharmacy, General Hospital of Shenyang Military Area Command,
83 Wenhua Road, Shenhe District, Shenyang 110840, PR China
*
Email address: machao_syphu@163.com (C. Ma); mscheng@syphu.edu.cn (M.
Cheng)
Abstract
Diverse of 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives
were designed, synthesized and evaluated for their neuroprotective activity against
NMDA-induced cytotoxicity in vitro, and 5q exhibited excellent neuroprotective
activity. The compound 5q was selected for further investigation. We found that 5q
could attenuate Ca2+ influx induced by NMDA, meanwhile, 5q could suppress the
NR2B up-regulation and increase p-ERK1/2 expression. The molecular docking
results showed that 5q might fit well in the binding pocket of 4 and interact with some
key residues in the binding pocket of 1 simultaneously. Besides, 5q exhibited
acceptable metabolic stability. These results suggested that 5q was a promising lead
for further development of new potent and orally bioavailable NR2B-selective
NMDAR antagonists.
Key words: Neuroprotective activity, Ca2+ influx, p-ERK1/2, NR2B-selective
NMDAR antagonists
1. Introduction
Glutamate is the principal excitatory neurotransmitter in the central nervous system

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(CNS), which mediates its effects via activation of metabotropic glutamate (mGlu)
and ionotropic glutamate (iGlu) receptors [1, 2], while glutamate accumulation and
excessive stimulation of its receptors induce potent excitotoxicity in the CNS [3]. The
N-methyl-D-aspartate receptors (NMDARs) belong to the family of iGlu receptors [4],
which play a pivotal role in mediating glutamate excitotoxicity [5]. Overactivation of
NMDARs can lead to Ca2+ overload of neurons, which is associated with a number of
acute and chronic neurodegenerative diseases, including stroke, seizures, Parkinson’s
disease, and chronic pain [6-8]. NMDARs are heterotetrameric proteins, consisting of
three types of subunits: NR1 (NR1a-h), NR2 (NR2A-D), and NR3 (NR3A-B) [9]. A
functional NMDAR protein contains at least one NR1 subunit and one NR2 subunit
[10], indeed, excitotoxicity is triggered by the selective activation of NMDARs
containing the NR2B subunits [11]. The ERK signaling pathway is activated by
calcium influx through NMDARs and plays an important role in synaptic plasticity
and cell survival [12, 13]. It has been proposed that NR2B-containing NMDARs are
mainly involved in the inhibition of ERK1/2 activity [14, 15].
The neuroprotective potential of NR2B-selective NMDAR antagonists can be
exploited for the treatment of neurodegenerative diseases like stroke, Alzheimer’s
disease and Parkinson’s disease. Compared to non-subtype selective NMDA receptor
antagonists, NR2B selective antagonists might have less side effects [16]. 1
(ifenprodil) [17] was the first reported NR2B-selective NMDAR antagonist (Figure
1), unfortunately, its selectivity is poor. The interaction of 1 with other receptors (α1,
σ1, σ2, 5-HT2A and 5-HT2C receptor), leads to undesired side effects, e.g. impaired
motor function. Besides, the bioavailability of 1 is rather low due to its fast
biotransformation [18]. Nevertheless, 1 serves as an important lead structure for the
rational design of novel NR2B-selective NMDAR antagonists, such as 2 (traxoprodil,
CP-101,606) [19]. Research efforts have focused on identifying novel structural
classes of NR2B-selective NMDAR antagonists for improving the potency, selectivity
profiles and the metabolic stability, for example, 3 is a potent orally bioavailable,
NR2B-selective NMDAR antagonist with antidepressant effects [20]. In 2016, David
Stroebel et al. reported the crystal structure of the NR1/NR2B heterodimer in complex

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with 4 [21], the binding site of 4 revealed a remarkably different situation with a
shared interdomain cavity but little overlap with the binding site of 1. These data
widen the allosteric and pharmacological landscape of NMDARs and facilitate the
design of NR2B-selective NMDAR antagonists with therapeutic value for brain
disorders.
Figure 1. Chemical structures of several representative NR2B-selective NMDAR
antagonists (1-4), S-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid
derivative (5a) and general structure (6) of designed compounds.
A previous study had found that 5a (Figure 2) could protect against
ischemia-induced cell injury in an oxygen glucose deprivation (OGD) model of
ischemic stroke [22]. We noted that the structure of compound 5a contains the similar
features with many NR2B-selective NMDAR antagonists (e.g. 1-3): one hydrophobic
aromatic part (A) links to another aromatic part (B). Inspired by the neuroprotective
activity and the outstanding structure of 5a, we attempted to search for potent
neuroprotective agents with the hope of identifying structurally distinct
NR2B-selective NMDAR antagonists. In this study,
a
series of
1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives (6) were
designed, synthesized and evaluated for the neuroprotective activity, and 5q was
identified as an effective neuroprotective agent in vitro NMDA-induced cytotoxicity
model. We assume that NR2B-NMDARs were involved in the neuroprotection of 5q.

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To verify the hypothesis, the compound 5q was selected for further investigation. First,
we measured the intracellular calcium concentration change caused by
N-methyl-D-aspartate (NMDA) pretreatment with or without 5q using the laser
scanning confocal microscope. Then the potential effects of 5q on NMDA-induced
apoptosis were verified by western-blot assay. Finally, computational molecular
docking methods were used to predict the potential molecular interaction mechanisms
between 5q and NR2B-NMDARs. Besides, we evaluated the in vitro metabolic
stability of 5q.
2. Results and discussion
2.1. Chemistry
The key intermediates 10a-10d were synthesized through the method described
previously [26] (Scheme 1). The reductive amination of 7a-7b using L- or D-glutamic
acid gave the intermediates 8a-8d. The subsequent intramolecular cyclization and
dehydration in refluxing ethanol produced the intermediates 9a-9d. Finally,
compounds 9a-9d were treated with 80% hydrazine hydrate, FeCl3·6H2O, and
activated carbon in refluxing ethanol for 3 days, yielding the key intermediates 10a–
10d. The target compounds 5a-5s and 12a-12j were prepared via the amidation of
intermediates 10a-10d with 11a-11m. The similar method was used to synthesize the
compounds 13, 14 and 16a-16c (Scheme 2).

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Scheme 1. Reagents and conditions: (a) L/D-glutamic acid, NaOH, NaBH4, and then
HCl/H2O; (b) CH3CH2OH, reflux, 4 h; (c) 80% NH2NH2·H2O, FeCl3·6H2O, active
carbon, CH3CH2OH, reflux, 2~3 d; (d) BOP-Cl, Et3N, 0 ~r.t., 6 h.
Compound 10a was reacted with thionyl chloride and ethanol to afford the
compound 17 via esterification. Then, treatment of compound 17 with an aqueous
solution of ammonia (25%) at 50
then dehydrated with POCl3 gave the intermediate 19. Then the amines 20a-20c were
prepared via the Pinner synthesis dry HCl (g) was bubbled into an anhydrous ethanol
solution of 19, then reacted with 11a-11c to afford the target compounds 20a-20c.
afforded 18. The compound 18 was dried and
:

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Scheme 2. Reagents and conditions: (a) BOP-Cl, Et3N, 0 ~r.t., 6 h; (b) BOP-Cl,
Et3N, 0 ~r.t., 6 h; (c) BOP-Cl, Et3N, 0 ~r.t., 6 h; (d) SOCl2, CH3CH2OH, -5 ~r.t.,
6 h; (e) Aqueous solution of ammonia, 50 , 3 h; (f) POCl3, DMF, 50 , 4 h; (g) i,
CH3CH2OH, HCl (g); ii, Et3N, CH3CH2OH.
2.2. Biological evaluation
All of the synthesized compounds were evaluated for their neuroprotective effects
on
SH-SY5Y
cells
damaged
by
NMDA
via
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The
results were presented in Table 1 and Table 2.
Based on the in vitro neuroprotective activities shown in Table 1, it was observed
that the S-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid amides
derivatives were considerably more effective than the corresponding esters and
amidines derivatives (5a-5c versus 16a-16b, 20a-20c). The neuroprotection of 5a-5e
were similar to each other, which suggested that R1 could be some small substituent
groups, such as methyl, hydroxyl and halogen. In consideration of increasing the
metabolic stability, we introduced the substituent Br to the benzene ring of part A, this

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modification did not have major influences on the activity (5b-5e versus 5f-5h).
At low to moderate concentration (0.1, 1, 10µM), almost all of the compounds
5m-5q (n=2) exhibited more potent neuroprotection than 5a-5h (n=1), 5i-5l (n=0) and
5r-5s (n=3), so it is obvious that the distance between the acylamino and the benzene
ring of part B played a crucial role in the neuroprotection of the compounds, and the
optimal length of the alkyl spacer was 2 methylene moieties, shorting or lengthening
the length of alkyl spacer would reduce the activity. Among these compounds, the
compound 5q (75.8%, 80.0%, 84.4%, 78.6%) exhibited slightly higher potency than
ifenprodil (71.0%, 72.7%, 79.5%, 65.1%) at the test concentration
.
Given the compounds had chiral center,
a
a
few
R-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives (12a-12j)
were synthesized and evaluated for their neuroprotective activity. Although almost all
of the R-derivatives were less active than their S-configuration counterparts, the
differences in potency were small (Table 2). Therefore, in spite of the S-configuration
was a little better for these derivatives, the chiral center did not affect the
neuroprotective potency significantly. To extend the investigation range, we
introduced a hydroxymethyl substituent into the methylene of 5m or replaced the
benzene ring by indole ring, and the compounds 13 and 14 were synthesized
according to the synthetic routes shown in Scheme 2, both of their neuroprotective
potency were comparable to 5m.
Table 1. The influences of target compounds on NMDA-damaged SH-SY5Y cells
Cell viability a (%)
Compd.
0.1µM
1µM
10µM
100µM
Control
Model
5a
100 b
58.7 c±2.3
71.6 f±0.6
72.0 f±1.6
71.8 f±2.7
72.6 f±0.5
73.4 f±2.7
73.5 f±0.1
68.4 f±2.5
67.6 f±0.6
68.1 f±1.0
67.1 e±3.5
66.2 e±3.2
69.0 f±1.4
70.5 f±2.9
73.8 f±1.3
74.1 f±0.3
78.9 f±3.3
72.5 f±2.9
76.0 f±3.2
64.4e±1.0
70.3 f±1.8
64.4 e±2.2
63.2±1.7
54.5±2.2
60.0±0.4
5b
5c
5d
5e
5f

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68.8 d±3.1
70.4 f±1.6
71.4 f±2.3
68.3 e±2.9
72.7 f±0.4
66.2 f±0.2
77.6 f±4.2
78.1 f±3.0
81.0 f±3.4
75.4 f±4.0
80.0 f±4.5
71.2 f±0.6
76.6 f±0.9
65.5 f±0.9
64.1 f±1.2
68.6 f±4.0
69.2 f±1.5
65.8 f±0.5
72.7 f±0.4
74.3 e±7.0
70.4 f±0.7
60.4±1.7
60.4±0.6
55.3±4.6
60.1±1.3
54.6±1.4
70.9 f±2.9
48.4 d±2.0
68.4 f±0.8
57.5±0.4
67.8 f±2.2
60.0±1.1
33.5 f±1.0
78.6 f±0.8
30.3 f±2.6
42.2 f±2.2
57.4±0.3
63.6 e±1.8
66.4 f±1.8
64.3 e±0.4
56.9±1.8
65.1 e±1.6
5g
5h
5i
64.8 f±0.5
68.7 f±2.6
67.7 e±2.8
64.7±1.9
70.1 f±2.8
66.4 d±5.9
68.0 f±0.8
80.0 f±0.2
81.4 f±1.2
75.1 f±2.7
68.1 e±2.3
84.4 f±4.0
63.4 e±2.1
66.1 f±0.4
66.1 f±1.9
65.3 f±1.1
68.8 f±1.5
70.2 f±3.0
66.1 f±1.0
79.5 f±1.0
5j
5k
5l
62.9 e±2.3
73.7 f±1.3
67.7 f±0.4
73.0 f±2.7
71.4 f±1.6
75.8 f±0.8
67.2 f±1.3
69.1 f±2.1
61.0 d±0.1
61.4 d±0.7
61.6±0.3
5m
5n
5o
5p
5q
5r
5s
16a
16b
20a
20b
20c
1
61.6±1.7
64.8 f±1.7
71.0 f±3.0
a Cell viability was calculated from the results, data are presented as the mean ± SD of
three independent experiments.
b The survival rates of Control group were 100%.
c The survival rates of Model group treated by NMDA (2 mM) alone.
Statistical significance comparison was determined by one-way ANOVA. Compared
with Model group: d p <0.05. e p <0.01. f p <0.001.
Table 2. The influences of target compounds on NMDA-damaged SH-SY5Y cells
Cell viability a (%)
Compd.
0.1µM
1µM
10µM
100µM
Control
Model
12a
100 b
58.7 c±2.3
65.2 e±1.6 68.0 f±3.0
66.6 f±1.0 70.7 f±2.6
71.1 f±3.2 74.7 f±3.5
64.2 e±0.6
76.2 f±2.1
72.9 f±2.1
60.9±0.9
58.4±0.8
63.9 d±1.2
12b
12c

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75.0 f±4.6 78.0 f±2.2
65.6 f±2.1 67.2 f±1.0
71.3 f±2.3
61.7±3.4
65.2 d±0.7 80.7 f±3.6
67.8 f±1.1 74.5 f±3.5
62.2 d±0.5 71.6 f±2.5
67.4 d±3.8
69.6 f±1.6
68.3 f±0.6
70.5 f±4.0
78.2 f±1.2
70.2 f±2.9
65.4 d±4.1
79.6 f±1.9
80.9 f±3.0
56.9±4.1
12d
12e
12f
12g
12h
12i
12j
13
73.3 f±0.6
66.0 e±0.7
68.0 e±0.8
81.5 f±0.7
42.0 f±0.3
44.1 e±0.7
72.4 f±3.9
78.2 f±1.7
72.1 f±3.8
58.1±1.6
67.1 e±2.6 82.6 f±2.5
66.2 d±0.8 72.0 f±4.6
14
a Cell viability was calculated from the results, data are presented as the mean ± SD of
three independent experiments.
b The survival rates of Control group were 100%.
c The survival rates of Model group treated by NMDA (2 mM) alone.
Statistical significance comparison was determined by one-way ANOVA. Compared
with Model group: d p <0.05. e p <0.01. f p <0.001.
2.3. Effect of 5q on NMDA-induced Ca2+ influx in SH-SY5Y cells
Overactivation of NMDARs can lead to Ca2+ overload of neurons. Ca2+ is a key
mediator of excitotoxic damage, which can activate a number of Ca2+-dependent
enzymes and influence a multitude of cellular processes [23]. Overactivation of the
NMDARs induced massive influx of Ca2+ into the neuron, the interaction of
NR2B-selective NMDAR antagonists with the NMDARs could inhibit the influx of
Ca2+. In this study, we evaluated the effects of 5q on calcium influx (Figure 3). The
results showed that compared with the control group, the intracellular Ca2+ increased
markedly after NMDA was added, while the Ca2+ influx could be attenuated
significantly when the SH-SY5Y cells were pretreated with 5q. So, 5q could inhibit
the influx of Ca2+ induced by NMDA.

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Figure 2. Calcium imaging of NMDA-induced Ca2+ influx. SH-SY5Y cells were
loaded with fluo-3, the fluorescence intensity represents cytoplasmic Ca2+
concentration. (A) The green fluorescence under laser scanning microscope at
different times showed the intracellular Ca2+ concentration in SH-SY5Y cells. (B)
Fluorescence intensity of the control group during detection time. The fluorescence
intensity showed that the intracellular Ca2+ concentration was stable in control group
during detection time (n = 9 cells), NMDA (final concentration was 2 mM) was able
to evoke Ca2+ influx markedly (n = 9 cells), while the Ca2+ influx could be attenuated
significantly when the SH-SY5Y cells were pretreated with 5q (n = 9 cells).
2.4. Effects of 5q on the NR2B and the phosphorylated ERK1/2 (p-ERK1/2)
expression
NR2B-containing NMDARs directly link NMDARs to ERK activation [24], and
NMDA-induced ERK signaling is mediated by NR2B subunit [25]. In this study, 5q
was evaluated for the effects on the NR2B and the p-ERK1/2 expression. The results
showed that NMDA could increase the NR2B expression and down-regulate the
p-ERK1/2 expression, but pretreatment with 5q could suppress the NR2B
up-regulation and increase p-ERK1/2 expression. Therefore, the NMDAR-ERK
signaling cascade and NR2B-NMDARs were likely to be involved in the protective
effect of 5q.

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Figure 3. Western-blot analysis of NR2B and p-ERK1/2. (A) The expression of
NR2B. (B) The expression of p-ERK/ERK. Data were presented as means ± S.E.M.
(n = 3). && p < 0.01 and &&& p < 0.001 compared with control group. ** p < 0.01 and
*** p < 0.001 compared with NMDA-treated group.
2.5. Binding affinity study through docking with NR2B subunit of NMDARs
To rationalize the obtained biological results, computational molecular docking
methods were used to predict the potential molecular interaction mechanisms between
5q and NR2B-NMDARs. 5q was docked at the NR1-NR2B subunit interface, the
docking poses of 5q were examined and the pose which had the highest Libdockscore
was shown in Figure 4. The docking results showed that 5q could fit well in the
active pocket of NR2B-selective NMDAR antagonists but the orientation and
interactions network of 5q (yellow) were different from 1 (green), especially the
hydrophobic aromatic part A, but it was very surprising that this part could almost
overlap with 4 (purple). The parts B of 5q and 1 had the similar interactions with
NR1-NR2B subunit interface. These results indicated that 5q might occupy the
binding pocket of 4 and interacted with some residues in the binding pocket of 1
simultaneously. The docking result was consistent with the biological results. When
the length of the alkyl spacer between the acylamino and the benzene ring of the part
B was 2 methylene moieties, the part B of 5q would occupy the pocket of 1 and place
its phenyl ring in parallel to the part B of 1, and the hydroxyl group (OH) was able to

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form the hydrogen bonding interaction with the residues of NR2B (TYR 175, PHE
176 and GLU 236). The docking results suggested that 5q might interact with
NR2B-NMDARs in outstanding ways.
Figure 4. Docking poses of compound 5q at the NMDA receptor (PDB code 5EWJ).
(A) 1 (Ifenprodil, green), 4 (EVT-101, purple), 5q (yellow). (B) The residues that
interact with 5q, the residues are colored in orange (NR1) and cyan (NR2B).
2.6. In Vitro Metabolic Stability Study
Based on the results of the in vitro neuroprotective activity, the compound 5q was
selected for the in vitro metabolic stability study and 1 was selected as the control
medicine. As shown in Table 3, 1 showed weak stability with a clearance rate of
108.6 µL/min/mg in human liver microsomes, while the compound 5q exhibited
higher metabolic stability with a clearance rate of 46.3 µL/min/mg, the ester
derivative 16b exhibited much lower metabolic stability with a clearance rate of
1183.2 µL/min/mg.
Table 3. In vitro metabolic stability study of 5q, 16b and 1
Human Liver Microsomes
Compd.
CL
Remaining
T1/2 (min)
(µL/min/mg) (*NCF=60 min)
29.9
1.2
12.8
46.3
1183.2
108.6
99.3%
3.2%
87.3%
5q
16b
1

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*NCF: the abbreviation of no co-factor. No NADPH regenerating system is added into
NCF sample (replaced by buffer) during the 60 min-incubation.
3. Conclusions
Diverse 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives
were synthesized and their neuroprotective activity against NMDA-induced
cytotoxicity in vitro varied from moderate to excellent. In addition, the Ca2+ influx
induced by NMDA could be attenuated significantly when the SH-SY5Y cells were
pretreated with 5q, and 5q could suppress the NR2B up-regulation and increase
p-ERK1/2 expression. Moreover, the docking results showed that 5q might fit well in
the binding pocket of 4 and has specific key interactions with some residues in the
binding pocket of 1 simultaneously and the results were consistent with the biological
results. According to the experiments results in this study, it is feasible to conclude
that NR2B-NMDARs might be involved in the protective effect of 5q against to
NMDA-induced neuron impairments. Besides, the compound 5q exhibited acceptable
metabolic stability. Although further studies are needed to reveal the mechanistic
details, the extraordinary structure, the distinct interaction mode with
NR2B-NMDARs and the metabolic stability of 5q made it a promising lead for the
development of new potent and orally bioavailable NR2B-selective NMDAR
antagonists.
4. Experimental section
4.1. Chemistry
All commercially available reagents and solvents were used without further
purification. TLC was performed on silica gel plates (Indicator F-254) and visualized
by UV-light. NMR spectra were recorded on Bruker 400 MHz and 600 MHz
instruments, and the chemical shifts were reported in terms of parts per million with
TMS as the internal reference and coupling constants were reported in Hertz.
High-resolution accurate mass determinations (HRMS) for all final target compounds
were obtained on a Bruker Micromass Time of Flight mass spectrometer equipped
with electrospray ionisation (ESI). Column chromatography was performed with
silica gel (200-300 mesh).

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4.1.1 General procedure for the preparation of compounds 10a–10d
An ethanol solution (400 mL) of 2-nitrobenzaldehyde 7a or
5-bromo-2-nitrobenzaldehyde 7b (300 mmol) was dropwise to an aqueous solution of
L-glutamic acid (or D-glutamic acid, 315 mmol) and sodium hydroxide (2 M, 300 mL)
at room temperature. The mixture was stirred at room temperature for 1 h and cooled
to 0~5 . Sodium borohydride (450 mmol) was added in small portions at 0~5 , and
the mixture was then warmed up to room temperature and stirred for 3 h. The
resulting solution was evaporated under vacuum till its volume was reduced to 300
mL, and then extracted with diethyl ether (70 mL × 3). The aqueous layer was
acidified to pH 4~5 with concentrated hydrochloric acid. The resulting suspension of
8a–8b (or 8c-8d) was filtered and then heated in refluxing ethanol (800 mL) for 5 h.
This solution was then filtered and concentrated in vacuum to give an ivory-white
dusty solid 9a–9b (or 9c-9d), which were used in the next reaction without further
purification. To the ethanol solution (600 mL) of the above crude intermediates 9a–9b
(or 9c-9d), activated carbon (6.0 g) and ferric chloride hexahydrate (0.05 g) were
added. The suspension was then heated to 50 and hydrazine hydrate (1200 mmol)
was added dropwise. This mixture was heated at reflux for approximately 72 h. At this
point, the mixture was filtered and the solvent was removed by vacuum evaporation.
The residue was purified by flash chromatography on silica gel using methanol–ethyl
acetate (5:4 v/v) to give compounds 10a–10b (or 10c-10d).
4.1.2. (S)-N-benzyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide (5a)
To a solution of 10a (1.0g, 4.6 mmol) and triethylamine (1.3mL, 9.3 mmol) in
anhydrous DMF (10 mL) was added Bis(2-oxo-3-oxazolidinyl)phosphonic chloride
(BOP-Cl) (1.4g, 5.5 mmol) at 0 , the mixture was stirred for 0.5 h at this
temperature, then phenylmethanamine (0.6mL, 5.5mmol) was added, stirred for 5h at
room temperature. Added 30mL H2O and stirred for 1 h, the solution was filtered and
gave a white solid 0.6g, yield: 42.7%. 1H-NMR (DMSO-d6, 600 MHz) δ: 8.81~8.79 (t,
J=6.0 Hz, 1H), 7.36~7.33 (t, J=7.8Hz, 2H), 7.28~7.24 (m, 3H), 7.10~7.07 (m, 1H),
6.92~6.91 (m, 2H), 6.84~6.83 (d, J=7.8 Hz, 1H), 4.46~4.43 (d, J=13.2 Hz, 1H),
4.41~4.38 (d, J=13.2 Hz, 1H), 4.34~4.33 (m, 2H), 4.05~4.03 (m, 1H), 2.60~2.55 (m,

ACCEPTED MANUSCRIPT
1H), 2.51~2.46 (m, 1H), 2.28~2.23 (m, 1H), 1.89~1.84 (m, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ: 170.09, 162.43, 139.02, 128.27 (3C), 127.81, 127.13 (3C), 126.80,
125.98, 123.39, 119.58, 64.01, 44.80, 42.06, 29.46, 23.89. ESI-HRMS: calcd for
C19H20N3O, [M + H]+, 306.1606, found 306.1598.
4.1.3.
(S)-N-(4-hydroxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5b)
1
Yield: 43.5%; H-NMR (DMSO-d6, 400 MHz) δ: 9.19 (s, 1H), 8.25 (s, 1H), 7.07
(br, 1H), 7.00~6.99 (d, J=8.4 Hz, 2H), 6.93~6.90 (m, 2H), 6.82~6.80 (d, J=8.0 Hz,
1H), 6.69~6.67 (d, J=8.4 Hz, 2H), 4.32~4.29 (d, J=13.2 Hz, 1H), 4.22~4.19 (d, J=13.6
Hz, 1H), 3.90~3.86 (m, 1H), 2.65~2.62 (m, 2H), 2.55~2.39 (m, 2H), 2.20~2.11 (m,
13
1H), 1.78~1.76 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ: 170.37, 162.97, 156.14,
143.74, 130.07 (2C), 129.70, 128.33, 126.55, 123.86, 123.81, 120.25, 115.50 (2C),
64.60, 45.27, 34.59, 30.04, 24.31. ESI-HRMS: calcd for C19H20N3O2, [M + H]+,
322.1556; found 322.1549.
4.1.4.
(S)-N-(4-methoxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5c)
Yield: 45.1%; 1H-NMR (DMSO-d6, 600 MHz) δ: 8.72~8.71 (m, 1H), 7.21~7.19 (d,
J=8.4 Hz, 2H), 7.09~7.07 (m, 1H), 6.92~6.89 (m, 4H), 6.83~6.82 (d, J=7.8 Hz, 1H),
4.44~4.41 (d, J=13.2 Hz, 1H), 4.39~4.37 (d, J=13.2 Hz, 1H), 4.26~4.25 (m, 2H),
4.01~3.99 (m, 1H), 3.73 (s, 3H), 2.59~2.53 (m, 1H), 2.50~2.45 (m, 1H), 2.25~2.21 (m,
13
1H), 1.86~1.82 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ: 169.98, 162.40, 158.17,
143.19, 130.96 (2C), 128.51, 127.78, 125.97, 123.30, 123.28, 119.65, 113.66 (2C),
63.94, 54.95, 44.79, 41.53, 29.48, 23.86. ESI-HRMS: calcd for C20H22N3O2, [M + H]+,
336.1712, found 336.1703.
4.1.5.
(S)-N-(4-fluorobenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5d)
1
Yield: 43.1%; H-NMR (DMSO-d6, 600 MHz) δ: 8.81~8.80 (t, J=6.0 Hz, 1H),

ACCEPTED MANUSCRIPT
7.33~7.30 (m, 2H), 7.18~7.16 (t, J=8.4 Hz, 2H), 7.10~7.07 (m, 1H), 6.92~6.91 (m,
2H), 6.84~6.82 (d, J=7.8 Hz, 1H), 4.44~4.42 (d, J=13.2 Hz, 1H), 4.39~4.37 (d, J=13.2
Hz, 1H), 4.31~4.30 (d, J=6.0 Hz, 2H), 4.02~4.00 (m, 1H), 2.58~2.45 (m, 2H),
2.26~2.21 (m, 1H), 1.87~1.82(m, 1H). 13C-NMR (DMSO-d6, 150 MHz) δ: 170.78,
162.97, 161.69 (d, J=242 Hz), 143.81, 135.87, 129.76, 129.71, 128.37, 126.55, 123.90
(2C), 120.26, 115.66, 115.52, 64.53, 45.40, 41.95, 30.05, 24.47. ESI-HRMS: calcd for
C19H20FN3O, [M + H]+, 324.1512, found 324.1506.
4.1.6.
(S)-N-(4-methylbenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5e)
1
Yield: 45.4%; H-NMR (DMSO-d6, 600 MHz) δ: 8.75~8.73 (t, J=6.0 Hz, 1H),
7.17~7.14 (m, 4H), 7.09~7.07 (m, 1H), 6.92~6.91 (m, 2H), 6.83~6.82 (d, J=7.8 Hz,
1H), 4.44~4.42 (d, J=13.2 Hz, 1H), 4.39~4.37 (d, J=13.2 Hz, 1H), 4.28~4.27 (m, 2H),
4.02~4.00 (m, 1H), 2.58~2.45 (m, 2H), 2.28 (s, 3H), 2.26~2.20 (m, 1H), 1.88~1.82 (m,
1H). 13C-NMR (DMSO-d6, 150 MHz) δ: 170.65, 162.98, 143.81, 136.60, 136.45,
129.40 (3C), 128.37, 127.73 (2C), 126.55, 123.89, 120.25, 64.53, 45.39, 42.40, 30.06,
24.46, 21.15. ESI-HRMS: calcd for C20H22N3O, [M + H]+, 320.1763, found 320.1748.
4.1.7.
(S)-7-bromo-N-(4-hydroxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carb
oxamide (5f)
1
Yield: 48.1%; H-NMR (DMSO-d6, 600 MHz) δ: 9.34 (s, 1H), 8.67 (s, 1H),
7.24~7.23 (m, 1H), 7.16 (s, 1H), 7.08~7.07 (m, 2H), 6.76~6.71 (m, 3H), 4.44~4.42 (d,
J=13.8 Hz, 1H), 4.39~4.36 (d, J=13.8 Hz, 1H), 4.20~4.20 (m, 2H), 4.01 (s, 1H),
2.56~2.46 (m, 2H), 2.24~2.21 (m, 1H), 1.84 (s, 1H). 13C-NMR (DMSO-d6, 150 MHz)
δ: 170.32, 163.72, 156.84, 143.36, 131.10, 129.63, 129.17, 129.14 (2C), 125.71,
122.87, 115.58 (2C), 115.16, 64.46, 44.87, 42.27, 30.14, 24.46. ESI-HRMS: calcd for
C19H19BrN3O2, [M + H]+, 400.0661, 402.0640; found 400.0670, 402.0650.
4.1.8.
(S)-7-bromo-N-(4-methoxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-car
boxamide (5g)

ACCEPTED MANUSCRIPT
1
Yield: 40.1%; H-NMR (DMSO-d6, 600 MHz) δ: 8.73 (s, 1H), 7.24~7.16 (m, 4H),
6.91~6.90 (m, 2H), 6.76~6.75 (m, 1H), 4.44~4.36 (m, 2H), 4.26 (s, 2H), 4.01 (s,
1H),3.73 (s, 3H), 2.55~2.50 (m, 2H), 2.23 (s, 1H), 1.84 (s, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ: 170.39, 163.71, 158.77, 143.29, 131.47, 131.12, 129.18, 129.11 (2C),
125.69, 122.85, 115.19, 114.26 (2C), 64.50, 55.54, 44.88, 42.14, 30.14, 24.47.
ESI-HRMS: calcd for C20H21BrN3O2, [M + H]+, 414.0817, 416.0797; found 414.0823,
416.0806.
4.1.9.
(S)-7-bromo-N-(4-fluorobenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carbo
xamide (5h)
1
Yield: 38.3%; H-NMR (DMSO-d6, 600 MHz) δ: 8.82~8.80 (t, J=6.0 Hz, 1H),
7.32~7.30 (m, 2H), 7.25~7.23 (m, 1H), 7.19~7.16 (m, 3H), 6.77~6.76 (d, J=8.4 Hz,
1H), 4.45~4.43 (d, J=13.8 Hz, 1H), 4.39~4.37 (d, J=14.4 Hz, 1H), 4.31~4.30 (d, J=6.0
Hz, 2H), 4.04~4.02 (m, 1H), 2.59~2.45 (m, 2H), 2.27~2.25 (m, 1H), 1.87~1.82 (m,
1H). 13C-NMR (DMSO-d6, 150 MHz) δ: 170.59, 163.71, 161.70 (d, J=243 Hz),
143.21, 135.79, 131.13, 129.77, 129.72, 129.19 125.68, 122.85, 115.68, 115.54,
115.23, 64.51, 44.91, 41.97, 30.12, 24.49. ESI-HRMS: calcd for C19H18BrFN3O, [M +
H]+, 402.0617, 404.0597; found 402.0620, 404.0402.
4.1.10.
(5i)
(S)-N-phenyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
Yield: 43.4%; 1H-NMR (DMSO-d6, 600MHz) δ: 10.33 (s, 1H), 7.65~7.64 (m, 2H),
7.34~7.32 (t, J=7.8 Hz, 2H), 7.12~7.07 (m, 2H), 6.95~6.91 (m, 2H), 6.87~6.85 (d,
J=7.8 Hz, 1H), 4.53~4.50 (d, J=13.2 Hz, 1H), 4.48~4.46 (d, J=13.2 Hz, 1H),
4.23~4.21 (m, 1H), 2.65~2.60 (m, 1H), 2.57~2.52 (m, 1H), 2.36~2.30 (m, 1H), 2.00
13
~1.95 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ: 169.52, 163.07, 143.25, 139.09,
129.27 (2C), 128.44, 126.64, 124.22, 124.08, 123.65, 120.13, 119.92 (2C), 65.09,
45.38, 30.01, 24.60. ESI-HRMS: calcd for C18H18N3O, [M + H]+, 292.1450; found
292.1456.
4.1.11.
(S)-N-(4-hydroxyphenyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide

ACCEPTED MANUSCRIPT
(5j)
1
Yield: 45.1%; H-NMR (DMSO-d6, 400 MHz) δ: 10.05 (s, 1H), 9.26 (s,
1H),7.43~7.41 (d, J=8.8 Hz, 2H), 7.12~7.07 (m, 1H), 6.95~6.91 (m, 2H), 6.86~6.84
(d, J=7.6 Hz, 1H), 6.73~6.71 (d, J=8.8 Hz, 2H), 4.51~4.43 (m, 2H), 4.15~4.12 (m,
1H), 2.65~2.48 (m, 2H), 2.32~2.27 (m, 1H), 1.99~1.91 (m, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ: 168.80, 163.01, 154.14, 143.75, 130.70, 128.38, 126.59, 123.91, 123.86,
121.66 (2C), 120.23, 115.56 (2C), 64.95, 45.35, 30.08, 24.54. ESI-HRMS: calcd for
C18H18N3O2, [M + H]+, 308.1399; found 308.1390.
4.1.12.
(S)-N-(4-methoxyphenyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5k)
Yield: 51.2%; 1H-NMR (DMSO-d6, 600 MHz) δ: 10.20 (s, 1H), 7.56~7.54 (m, 2H),
7.12~7.09 (m, 1H), 6.96~6.89 (m, 4H), 6.87~6.85 (d, J=7.2 Hz, 1H), 4.53~4.50 (d,
J=13.2 Hz, 2H), 4.48~4.45 (d, J=13.8 Hz, 1H), 4.22~4.18 (m, 1H), 3.72 (s, 3H),
2.66~2.53 (m, 2H), 2.33~2.31 (m, 1H), 2.00~1.94 (m, 1H). 13C-NMR (DMSO-d6, 150
MHz) δ: 168.89, 163.12, 156.00, 132.17, 128.47, 126.67, 124.14, 123.46, 121.47 (2C),
120.06, 114.36 (3C), 65.11, 55.65, 45.37, 30.03, 24.56. ESI-HRMS: calcd for
C18H18N3O, [M + H]+, 322.1556; found 322.1566.
4.1.13.
(S)-7-bromo-N-(4-hydroxyphenyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-car
boxamide (5l)
1
Yield: 35.7%; H-NMR (DMSO-d6, 400 MHz) δ: 10.04 (s, 1H), 9.24 (s, 1H),
7.41~7.49 (d, J=8.4 Hz, 2H), 7.24~7.22 (d, J=8.4 Hz, 1H), 7.16 (s, 1H), 6.77~6.69 (m,
3H), 4.49~4.40k (m, 2H), 4.12 (s, 1H), 2.60~2.49 (m, 2H), 2.30~2.25 (m, 1H), 1.94 (s,
1H). 13C-NMR (DMSO-d6, 150 MHz) δ: 167.98, 163.12, 153.54, 142.64, 130.51,
130.03, 128.59, 125.07, 122.23, 121.06 (2C), 114.95 (2C), 114.59, 64.31, 44.24, 29.53,
23.91. ESI-HRMS: calcd for C18H17BrN3O2, [M + H]+, 386.0504, 388.0484; found
386.0495, 388.0476.
4.1.14. (S)-N-phenethyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5m)

ACCEPTED MANUSCRIPT
1
Yield: 44.1%; H-NMR (DMSO-d6, 600 MHz) δ: 8.32~8.30 (t, J=6.0 Hz, 1H),
7.31~7.29 (t, J=7.8 Hz, 2H), 7.23~7.20 (m, 3H), 7.09~7.06 (t, J=7.2 Hz, 1H),
6.93~6.88 (m, 2H), 6.82~6.81 (d, J=7.8 Hz, 1H), 4.32~4.30 (d, J=13.2 Hz, 1H),
4.22~4.19 (d, J=13.8 Hz, 1H), 3.90~3.87 (m, 1H), 3.39~3.35 (m, 2H), 2.80~2.72 (m,
2H), 2.54~2.48 (m, 1H), 2.46~2.41 (m, 1H), 2.18~2.12 (m, 1H), 1.77~1.71 (m, 1H).
13C-NMR (DMSO-d6, 150 MHz) δ: 169.83, 162.35, 143.07, 139.12, 128.61 (2C),
128.13 (2C), 127.72, 125.97, 125.90, 123.26, 123.19, 119.61, 63.98, 44.64, 34.77,
29.41, 23.71. ESI-HRMS: calcd for C20H22N3O, [M + H]+, 320.1763, found 320.1755.
4.1.15.
(S)-N-(4-hydroxyphenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxami
de (5n)
1
Yield: 38.1%; H-NMR (DMSO-d6, 400 MHz) δ: 9.19 (s, 1H), 8.25 (s, 1H),
7.09~7.05 (m, 1H), 7.01~6.99 (d, J=8.0Hz, 2H), 6.93~6.90 (m, 2H), 6.82~6.80 (d,
J=8.0 Hz, 1H), 6.69~6.67 (d, J=8.0 Hz, 2H), 4.32~4.29 (d, J=13.6 Hz, 1H),
4.22~4.18 (d, J=13.6k Hz, 1H), 3.89~3.86 (m, 1H), 3.33~3.28 (m, 2H), 2.65~2.62 (m,
2H), 2.50~2.39 (m, 2H), 2.20~2.11 (m, 1H), 1.80~1.71 (m, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ: 170.32, 162.89, 156.06, 143.73, 130.00 (2C), 129.63, 128.25, 126.47,
123.78 (2C), 120.19, 115.43 (2C), 64.52, 45.20, 40.67, 34.52, 29.98, 24.24.
ESI-HRMS: calcd for C20H22N3O2, [M + H]+, 336.1712, found 336.1705.
4.1.16.
(S)-N-(4-methoxyphenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxam
ide (5o)
Yield: 46.2%; 1H-NMR (DMSO-d6, 600 MHz) δ: 8.28~8.26 (m, 1H), 7.14~7.12 (d,
J=8.4 Hz, 2H), 7.08~7.06 (t, J=6.6 Hz, 1H), 6.92~6.90 (t, J=7.2 Hz, 1H), 6.88~6.85
(m, 3H), 6.82~6.81 (d, J=7.2 Hz, 1H), 4.31~4.29 (d, J=13.2 Hz, 1H), 4.19~4.17 (d,
J=13.8 Hz, 1H), 3.89~3.87 (m, 1H), 3.71 (s, 3H), 3.37~3.27 (m, 2H), 2.73~2.65 (m,
2H), 2.54~2.49 (m, 1H), 2.46~2.41 (m, 1H), 2.18~2.12 (m, 1H), 1.78~1.73 (m, 1H).
13C-NMR (DMSO-d6, 150 MHz) δ: 169.34, 161.88, 157.07, 142.72, 130.47, 129.10
(2C), 127.25, 125.40, 122.77, 122.74, 119.17, 113.07 (2C), 63.51, 54.35, 44.17, 39.54,
33.44, 28.96, 23.21. ESI-HRMS: calcd for C21H24N3O, [M + H]+, 350.1869; found

ACCEPTED MANUSCRIPT
350.1874.
4.1.17
(S)-N-(4-fluorophenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamid
e (5p)
1
Yield: 40.1%; H-NMR (DMSO-d6, 600 MHz) δ: 8.29~8.28 (t, J=5.4 Hz, 1H),
7.26~7.24 (m, 2H), 7.13~7.10 (t, J=9.0 Hz, 2H), 7.09~7.06 (t, J=7.2 Hz, 1H),
6.92~6.90 (t, J=7.2 Hz, 1H), 6.88~6.87 (d, J=7.2 Hz, 1H), 6.82~6.81 (d, J=7.8 Hz,
1H), 4.31~4.29 (d, J=13.2 Hz, 1H), 4.18~4.16 (d, J=13.2 Hz, 1H), 3.88~3.86 (m, 1H),
3.40~3.34 (m, 2H), 2.79~2.71 (m, 2H), 2.52~2.48 (m, 1H), 2.46~2.39 (m, 1H),
13
2.18~2.12 (m, 1H), 1.76~1.70 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ: 170.49,
162.95 161.33 (d, J=240 Hz), 143.79, 135.89, 131.05, 131.00, 128.34, 126.46, 123.88,
123.86, 120.23, 115.47, 115.33, 64.58, 45.25, 34.49, 30.03, 24.33. ESI-HRMS: calcd
for C20H21FN3O, [M + H]+, 338.1669; found 338.1676.
4.1.18.
(S)-7-bromo-N-(4-hydroxyphenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-c
arboxamide (5q)
1
Yield: 43.8%; H-NMR (DMSO-d6, 600 MHz) δ: 9.19 (s, 1H), 8.28~8.26 (t, J=6.0
Hz, 1H), 7.24~7.22 (m, 1H), 7.13~7.13 (m, 1H), 7.00~6.99 (d, J=8.4 Hz, 2H),
6.75~6.74 (d, J=8.4 Hz, 1H), 6.69~6.67 (d, J=7.8 Hz, 2H), 4.33~4.31 (d, J=14.4 Hz,
1H), 4.23~4.20 (d, J=14.4 Hz, 1H), 3.90~3.88 (m, 1H), 3.31~3.26 (m, 2H), 2.64~2.61
(t, J=7.2 Hz, 2H), 2.47~2.39 (m, 2H), 2.19~2.13 (m, 1H), 1.77~1.72 (m, 1H).
13C-NMR (DMSO-d6, 150 MHz) δ: 170.21, 163.68, 156.15, 143.36, 131.08, 130.07
(2C), 129.69, 129.14, 125.70, 122.87, 115.51 (2C), 115.15, 64.53, 46.16, 44.79, 34.62,
30.12, 24.34. ESI-HRMS: calcd for C20H21BrN3O2, [M + H]+, 414.0817, 416.0797;
found 414.0814, 416.0797.
4.1.19.
(S)-N-(3-phenylpropyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(5r)
1
Yield: 42.9%; H-NMR (DMSO-d6, 600 MHz) δ: 8.32~830 (m, 1H), 7.30~7.27 (d,
J=7.8 Hz 2H), 7.22~7.17 (m, 3H), 7.09~7.07 (m, 1H), 6.93~6.89 (m, 2H), 6.83~6.82

ACCEPTED MANUSCRIPT
(d, J=7.2 Hz, 1H), 4.43~4.41 (d, J=13.2 Hz, 1H), 4.38~4.36 (d, J=13.8 Hz, 1H),
3.96~3.94 (m, 1H), 3.14~3.12 (m, 2H), 2.60~2.45 (m, 4H), 2.24~2.19 (m, 1H),
13
1.85~1.80 (m, 1H), 1.76~1.71 (m, 2H). C-NMR (DMSO-d6, 150 MHz) δ: 170.54,
163.01, 143.85, 142.09, 128.78 (4C), 128.35, 126.54, 126.25, 123.88, 123.86, 120.25,
64.58, 45.34, 38.70, 32.97, 31.31, 30.08, 24.46. ESI-HRMS: calcd for C21H24N3O, [M
+ H]+, 334.1919; found 334.1930.
4.1.20.
(S)-7-bromo-N-(3-phenylpropyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carbo
xamide (5s)
1
Yield: 55.3%; H-NMR (DMSO-d6, 600 MHz) δ: 8.32 (s, 1H), 7.30~7.27 (m, 2H),
7.24~7.16 (m, 5H), 6.76~6.75 (d, J=8.4 Hz, 1H), 4.43~4.41 (d, J=13.8 Hz, 1H),
4.38~4.35 (d, J=13.8 Hz, 1H), 3.97~3.95 (m, 1H), 3.14~3.10 (m, 2H), 2.60~2.44 (m,
4H), 2.25~2.19 (m, 1H), 1.84~1.80 (m, 1H), 1.75~1.72 (m, 2H). 13C-NMR (DMSO-d6,
150 MHz) δ: 170.36, 163.73, 143.39, 142.08, 131.09, 129.17, 128.79 (2C), 128.77
(2C), 126.25, 125.72, 122.88, 115.14, 64.55, 44.85, 38.71, 32.97, 31.28, 30.16, 24.47.
ESI-HRMS: calcd for C21H23BrN3O, [M + H]+, 412.1024, 414.1004; found 412.1013,
414.1013.
4.1.21.
(R)-N-(4-hydroxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(12a)
1
Yield: 39.9%; H-NMR (DMSO-d6, 400 MHz) δ: 9.31 (s, 1H), 8.65 (s, 1H),
7.08~7.06 (m, 3H), 6.92~6.91 (m, 2H), 6.83~6.81 (d, J=7.6 Hz, 1H), 6.73~6.71 (d,
J=8.0 Hz, 2H), 4.44~4.35 (m, 2H), 4.21~4.20 (m, 2H), 4.01~3.98 (m, 1H), 2.61~2.43
13
(m, 2H), 2.26~2.19 (m, 1H), 1.88~1.80 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ:
169.89, 162.40, 156.23, 143.19, 129.12, 128.54 (2C), 127.77, 125.95, 123.28, 123.25,
119.64, 114.96 (2C), 63.91, 44.76, 41.65, 29.47, 23.83. ESI-HRMS: calcd for
C19H20N3O2, [M + H]+, 322.1556; found 322.1549.
4.1.22.
(R)-N-(4-methoxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(12b)

ACCEPTED MANUSCRIPT
1
Yield: 46.1%; H-NMR (DMSO-d6, 600 MHz) δ: 8.72~8.71 (t, J=6.0 Hz, 1H),
7.21~7.19 (d, J=9.0 Hz, 2H), 7.09~7.07 (m, 1H), 6.92~6.89 (m, 4H), 6.83~6.82 (d,
J=7.8 Hz, 1H), 4.44~4.41 (d, J=13.2 Hz, 1H), 4.39~4.37 (d, J=13.2 Hz, 1H),
4.26~4.25 (m, 2H), 4.01~3.99 (m, 1H), 3.73 (s, 3H), 2.57~2.53 (m, 1H), 2.50~2.47 (m,
1H), 2.26~2.21 (m, 1H), 1.87~1.81 (m, 1H). 13C-NMR (DMSO-d6, 150 MHz) δ:
170.54, 163.01, 158.76, 143.62, 131.54, 129.10 (2C), 128.39, 126.57, 123.93, 123.78,
120.20, 114.25 (2C), 64.55, 55.53, 45.37, 42.12, 30.06, 24.45. ESI-HRMS: calcd for
C20H21N3O2, [M + H]+, 336.1712; found 336.1712.
4.1.23.
(R)-N-(4-fluorobenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(12c)
1
Yield: 45.9%; H-NMR (DMSO-d6, 600 MHz) δ: 8.81~8.79 (t, J=6.0 Hz, 1H),
7.33~7.30 (m, 2H), 7.19~7.15 (m, 2H), 7.10~7.07(m, 1H), 6.92~6.90 (m, 2H),
6.84~6.82 (d, J=7.8 Hz, 1H), 4.45~4.42 (d, J=13.2 Hz, 1H), 4.39~4.37 (d, J=13.8 Hz,
1H), 4.31~4.30 (m, 2H), 4.03~4.00 (m, 1H), 2.61~2.54 (m, 1H), 2.51~2.45 (m, 1H),
13
2.27~2.21 (m, 1H), 1.88~1.82 (m, 1H). C-NMR (DMSO-d6, 150 MHz) δ: 170.76,
163.00, 161.70 (d, J=242 Hz), 143.67, 135.87, 129.77, 129.71, 128.38, 126.56, 123.94,
123.83, 120.23, 115.66, 115.52, 64.56, 45.40, 41.96, 30.04, 24.47. ESI-HRMS: calcd
for C19H19FN3O, [M + H]+, 324.1512; found 324.1514.
4.1.24.
(R)-7-bromo-N-(4-hydroxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-car
boxamide (12d)
1
Yield: 47.1%; H-NMR (DMSO-d6, 600 MHz) δ: 9.34 (s, 1H), 8.68 (s, 1H),
7.25~7.23 (m, 1H), 7.17 (s, 1H), 7.08~7.07 (m, 2H), 6.77~6.71 (m, 3H), 4.45~4.37 (m,
2H), 4.21~4.20 (m, 2H), 4.02 (s, 1H), 2.57~2.50 (m, 2H), 2.23 (s, 1H), 1.85 (s, 1H).
13C-NMR (DMSO-d6, 150 MHz) δ: 170.24, 163.76, 156.85, 142.94, 131.15, 129.61,
129.22, 129.14 (2C), 125.50, 122.77, 115.58 (2C), 115.28, 64.54, 44.87, 42.28, 30.13,
24.47. ESI-HRMS: calcd for C19H19BrN3O2, [M + H]+, 400.0661, 402.0640; found
400.0666, 402.0649.
4.1.25.

ACCEPTED MANUSCRIPT
(R)-N-(4-hydroxyphenyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(12e)
1
Yield: 50.2%; H-NMR (DMSO-d6, 400 MHz) δ: 10.05 (s, 1H), 9.26 (s, 1H),
7.43~7.41 (d, J=8.8 Hz, 2H), 7.12~7.07 (m, 1H), 6.95~6.90 (m, 2H), 6.86~6.84 (d,
J=7.6 Hz, 1H), 6.73~6.71 (d, J=8.8 Hz, 2H), 4.50~4.43 (m, 2H), 4.15~4.12 (m, 1H),
2.65~2.48 (m, 2H), 2.32~2.27 (m, 1H), 1.98~1.91 (m, 1H). 13C-NMR (DMSO-d6, 150
MHz) δ: 168.21, 162.42, 153.54, 143.15, 130.10, 127.78, 126.00, 123.31, 123.26,
121.07 (2C), 119.64, 114.97 (2C), 64.35, 44.76, 29.48, 23.94. ESI-HRMS: calcd for
C18H18N3O2, [M + H]+, 308.1399; found 308.1389.
4.1.26.
(R)-7-bromo-N-(4-hydroxyphenyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-car
boxamide (12f)
1
Yield: 35.6%; H-NMR (DMSO-d6, 400 MHz) δ: 10.05 (s, 1H), 9.25 (s, 1H),
7.42~7.40 (d, J=7.6 Hz, 2H), 7.25~7.17 (m, 2H), 6.78~6.70 (m, 3H), 4.51~4.41 (m,
2H), 4.14 (s, 1H), 2.67~2.50 (m, 2H), 2.32~2.27 (m, 1H), 1.95 (s, 1H). 13C-NMR
(DMSO-d6, 150 MHz) δ: 167.99, 163.15, 153.57, 142.63, 130.54, 130.06, 128.62,
125.08, 122.24, 121.08 (2C), 114.97 (2C), 114.63, 64.34, 44.27, 29.55, 23.93.
ESI-HRMS: calcd for C18H17BrN3O2, [M + H]+, 386.0504, 388.0484; found 386.0496,
388.0478.
4.1.27.
(R)-N-(4-hydroxyphenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxam
ide (12g)
1
Yield: 40.1%; H-NMR (DMSO-d6, 400 MHz) δ: 9.19 (s, 1H), 8.25~8.23 (m, 1H),
7.09~7.05 (m, 1H), 7.01~6.99 (d, J=8.4 Hz, 2H), 6.93~6.88 (m, 2H), 6.82~6.80 (d,
J=8.0 Hz, 1H), 6.69~6.67 (d, J=8.4 Hz, 2H), 4.32~4.29 (d, J=13.6 Hz, 1H), 4.22~4.19
(d, J=13.6 Hz, 1H), 3.90~3.87 (m, 1H), 3.32~3.23 (m, 2H), 2.65~2.62 (t, J=7.2 Hz,
2H), 2.56~2.40 (m, 2H), 2.20~2.11 (m, 1H), 1.80~1.72 (m, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ:170.37, 162.97, 156.13, 143.73, 130.07 (2C), 129.70, 128.33, 126.55,
123.87, 123.81, 120.25, 115.50 (2C), 64.60, 45.27, 40.74, 34.59, 30.05, 24.31.
ESI-HRMS: calcd for C20H22N3O2, [M + H]+, 336.1712; found 336.1707.

ACCEPTED MANUSCRIPT
4.1.28.
(R)-7-bromo-N-(4-hydroxyphenethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-
carboxamide (12h)
1
Yield: 40.5%; H-NMR (DMSO-d6, 400 MHz) δ: 9.18 (s, 1H), 8.27~8.24 (t, J=5.2
Hz, 1H), 7.24~7.21 (m, 1H), 7.13~7.12 (m, 1H), 7.00~6.98 (d, J=8.0 Hz, 2H),
6.76~6.73 (d, J=8.4 Hz, 1H), 6.69~6.67 (d, J=8.4 Hz, 2H), 4.34~4.30 (d, J=14.0 Hz,
1H), 4.23~4.20 (d, J=14.0 Hz, 1H), 3.91~3.88 (m, 1H), 3.32~3.24 (m, 2H), 2.65~2.61
(t, J=7.2 Hz, 2H), 2.55~2.39 (m, 2H), 2.21~2.11 (m, 1H), 1.79~1.71 (m, 1H).
13C-NMR (DMSO-d6, 150 MHz) δ: 170.21, 163.68, 156.15, 143.34, 131.08, 130.07
(2C). 129.65, 129.12, 125.70, 122.87, 115.51 (2C), 115.16, 64.53, 46.16, 44.79, 34.63,
30.11, 23.34. ESI-HRMS: calcd for C20H21BrN3O2, [M + H]+, 414.0817, 416.0797;
found 414.0817, 416.0795.
4.1.29.
(R)-N-(3-phenylpropyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide
(12i)
1
Yield: 47.0%; H-NMR (DMSO-d6, 600 MHz) δ: 8.32~830 (m, 1H), 7.30~7.27 (d,
J=7.2 Hz 2H), 7.22~7.17 (m, 3H), 7.09~7.07 (m, 1H), 6.92~6.89 (m, 2H), 6.83~6.82
(d, J=7.8 Hz, 1H), 4.43~4.41 (d, J=13.2 Hz, 1H), 4.38~4.36 (d, J=13.8 Hz, 1H),
3.96~3.94 (m, 1H), 3.14~3.11 (m, 2H), 2.60~2.44 (m, 4H), 2.24~2.18 (m, 1H),
1.85~1.80 (m, 1H), 1.76~1.71 (m, 2H). 13C-NMR (DMSO-d6, 150 MHz) δ:170.54,
163.01, 143.83, 142.09, 128.78 (3C), 128.36, 126.55, 126.25, 123.87 (2C), 120.24,
64.58, 45.34, 38.70, 32.97, 31.31, 30.09, 24.47. ESI-HRMS: calcd for C21H24N3O2,
[M + H]+, 334.1919; found 334.1932.
4.1.30.
(R)-7-bromo-N-(3-phenylpropyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carb
oxamide (12j)
1
Yield: 46.9%; H-NMR (DMSO-d6, 600 MHz) δ: 8.32~830 (m, 1H), 7.30~7.27 (t,
J=7.2 Hz 2H), 7.24~7.16 (m, 5H), 6.76~6.75 (d, J=8.4 Hz, 1H), 4.43~4.41 (d, J=13.8
Hz, 1H), 4.38~4.35 (d, J=14.4 Hz, 1H), 3.97~3.95 (m, 1H), 3.14~3.10 (m, 2H),
2.60~2.46 (m, 4H), 2.25~2.19 (m, 1H), 1.85~1.79 (m, 1H), 1.76~1.71 (m, 2H).

ACCEPTED MANUSCRIPT
13C-NMR (DMSO-d6, 150 MHz) δ: 170.36, 163.73, 143.38, 142.09, 131.09, 129.17,
128.80 (2C), 128.78 (2C), 126.26, 125.72, 122.88, 115.14, 64.55, 44.85, 38.71, 32.97,
31.28, 30.16, 24.47. ESI-HRMS: calcd for C21H23BrN3O, [M + H]+, 412.1024; found
412.1034.
4.1.31.
(S)-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoli
ne-1-carboxamide (13)
Yield: 32.8%; 1H-NMR (DMSO-d6, 400 MHz) δ: 8.15~8.13 (m, 1H), 7.32~7.21 (m,
5H), 7.08~7.04 (t, J=7.2 Hz, 1H), 6.93~6.89 (t, J=7.2 Hz, 1H), 6.80~6.78 (m, 2H),
4.89 (s, 1H), 4.11~4.04 (m, 2H), 3.86~3.84 (m, 1H), 3.74~3.70 (d, J=13.6 Hz, 1H),
3.44~3.34 (m, 2H), 2.94~2.90 (m, 1H), 2.64~2.58 (m, 1H), 2.51~2.37 (m, 2H),
2.15~2.07 (m, 1H), 1.79~1.74 (m, 1H). 13C-NMR (DMSO-d6, 150 MHz) δ:169.40,
162.22, 143.09, 138.99, 129.02 (3C), 127.89 (3C), 127.64, 125.78, 125.72, 123.17,
123.15, 119.45, 63.84, 63.00, 52.23, 44.23, 36.60, 29.45, 23.28. ESI-HRMS: calcd for
C21H24N3O2, [M + H]+, 350.1869; found 350.1863.
4.1.32.
(S)-N-(2-(1H-indol-3-yl)ethyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carbox
amide (14)
1
Yield: 38.0%; H-NMR (DMSO-d6, 400 MHz) δ: 10.83 (s, 1H), 8.33 (s, 1H),
7.56~7.54 (d, J=7.6 Hz 1H), 7.35~7.33 (d, J=8.0 Hz, 1H), 7.16 (s, 1H), 7.08~7.04 (m,
2H), 7.00~6.96 (t, J=7.6 Hz, 1H), 6.92~6.85 (m, 2H), 6.82~6.80 (d, J=7.6 Hz, 1H),
4.31~4.20 (m, 2H), 3.92~3.89 (m, 1H), 3.45~3.40 (m, 2H), 2.89~2.85 (t, J=6.4 Hz,
2H), 2.55~2.44 (m, 2H), 2.21~2.14 (m, 1H), 1.82~1.75 (m, 1H). 13C-NMR (DMSO-d6,
150 MHz) δ:170.43, 162.99, 143.75, 136.70, 128.32, 127.75, 126.55, 123.84, 123.80,
123.26, 121.37, 120.25, 118.76, 118.69, 112.07, 111.84, 64.62, 45.23, 30.05, 25.51,
24.34. ESI-HRMS: calcd for C20H23N4O, [M + H]+, 359.1872; found 359.1871.
4.1.33. benzyl (S)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylate (16a)
To a solution of 10a (1.0g, 4.6 mmol) and triethylamine (1.3mL, 9.3 mmol) in
anhydrous DMF (10 mL), BOP-Cl (1.4g, 5.5 mmol) was added at 0 , the mixture
was stirred for 0.5 h at this temperature, then added benzyl alcohol (0.6mL, 5.8mmol),

ACCEPTED MANUSCRIPT
and stirred for 5h at room temperature. 30mL H2O was added to the resulting solution
and stirred for 0.5 h, the solution was then extracted with ethyl acetate (20 mL × 3),
the organic layers were combined and purified by flash chromatography on silica gel
1
using ethyl acetate-petroleum ether (1:3 v/v) to give an oil, yield 38.2%. H-NMR
(DMSO-d6, 600 MHz) δ: 7.42~7.38 (m, 5H), 7.17~7.15 (m, 1H), 7.03~7.00 (m, 2H),
6.90~6.89 (d, J=7.8 Hz, 1H), 5.22 (s, 2H), 4.62~4.60 (d, J=13.8 Hz, 1H), 4.58~4.56 (d,
J=13.8 Hz, 1H), 4.42~4.40 (m, 1H), 2.71~2.60 (m, 2H), 2.43~2.36 (m, 1H), 2.07~2.02
13
(m, 1H). C-NMR (DMSO-d6, 150 MHz) δ:171.02, 163.06, 136.05, 129.04, 129.02
(2C), 128.79, 128.60 (2C), 126.81, 124.99, 119.57, 67.08, 63.81, 45.56, 29.55, 23.82.
ESI-HRMS: calcd for C19H19N2O2, [M + H]+, 307.1447; found 307.1449.
4.1.34.
4-methoxybenzyl
(S)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylate (16b)
Yield: 35.0%; 1H-NMR (DMSO-d6, 600 MHz) δ: 7.37~7.35 (m, 3H), 7.27~7.24 (m,
2H), 7.13~7.12 (d, J=7.8 Hz, 1H), 6.96~6.94 (m, 2H), 5.21~5.19 (d, J=12.0 Hz, 1H),
5.17~5.15 (d, J=12.0 Hz, 1H), 4.87~4.78 (m, 3H), 3.76 (s, 3H), 3.14~3.08 (m, 1H),
3.05~3.00 (m, 1H), 2.62~2.54 (m, 1H), 2.25~2.21 (m, 1H). 13C-NMR (DMSO-d6, 150
MHz) δ:169.35, 164.91, 159.91, 130.74 (3C), 129.74, 127.72, 127.55, 127.45, 117.43,
114.39 (3C), 67.59, 65.67, 55.63, 45.68, 29.25, 23.70. ESI-HRMS: calcd for
C20H21N2O2, [M + H]+, 337.1552; found 337.1562.
4.1.35. (S)-N-benzyl-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboximidamide
(20a)
Thionyl chloride (30 mL) was added dropwise to a stirred solution of 10a (8.0g,
37.0 mmol) in anhydrous ethanol (300 mL) at 0~5 . Then the mixture was stirred at
room temperature for 6 h and then concentrated under vacuum. The residue was
alkalified to pH 7~8 with a 5% aqueous solution of potassium carbonate and then
extracted with ethyl acetate (50 mL × 3). After being washed with water and brine, the
organic layers were dried over Na2SO4 and concentrated under reduced pressure to
yield 17. 100mL aqueous solution of ammonia (25%) was added, reacted at 50 for
3h, the mixture was filtered and gave a while solid 18, dried and added 30mL POCl3,
stirred at 50 for 4h, cooled to room temperature, then the reaction solution was

ACCEPTED MANUSCRIPT
poured into ice slowly, the mixture was alkalified to pH 7~8 with a 10% aqueous
solution of potassium carbonate and then extracted with ethyl acetate (40 mL × 3).
The organic layers were combined and purified by flash chromatography on silica gel
using ethyl acetate-petroleum ether (1:2 v/v) to give a white solid 19. Dry HCl(g) was
bubbled into an anhydrous ethyl alcohol solution of 19 (0.6g, 3.0mmol) for 0.5h, then
the mixture was evaporated to dry under reduced pressure. Added 15 mL anhydrous
ethyl alcohol and 5 mL Et3N to the residue, stirred and phenylmethanamine (0.6mL,
5.5mmol) was added, stirred for 5 h at room temperature. Added 5 mL water and then
the reaction solution extracted with ethyl acetate (40 mL × 3). The organic layers were
combined and purified by flash chromatography on silica gel using ethyl acetate to
1
give an oil, yield 35.1%. H-NMR (DMSO-d6, 400 MHz) δ: 9.56 (br, 2H), 7.47~7.33
(m, 5H), 7.14~7.01 (m, 1H),7.01~6.98 (m, 2H), 6.92~6.90 (d, J=8.0 Hz, 1H),
4.61~4.52 (m, 2H), 4.45~4.42 (m, 2H), 4.36~4.32 (d, J=13.2 Hz, 1H), 2.66~2.58 (m,
2H), 2.53~2.44 (m, 1H), 1.96~1.87 (m, 1H). 13C-NMR (DMSO-d6, 100 MHz) δ:
165.78, 163.98, 135.47, 129.48, 129.18 (3C), 128.47 (4C), 127.47, 127.06, 117.90,
64.60, 49.06, 46.08, 45.41, 29.63, 25.30. ESI-HRMS: calcd for C19H21N4, [M + H]+,
305.1761; found 305.1758.
4.1.36.
(S)-N-(4-hydroxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboximida
mide (20b)
Yield: 37.3%; 1H-NMR (DMSO-d6, 400 MHz) δ: 9.67 (br, 3H), 7.21~7.19 (d, J=8.8
Hz, 2H), 7.15~7.11 (m, 1H), 7.00~6.95 (m, 2H), 6.91~6.89 (d, J=7.6 Hz, 1H),
6.80~6.78 (d, J=8.4 Hz, 2H), 4.50~4.39 (m, 4H), 4.32~4.28 (d, J=13.2 Hz, 1H),
2.63~2.59 (m, 2H), 2.51~2.41 (m, 1H), 1.92~1.83 (m, 1H). 13C-NMR (DMSO-d6, 100
MHz) δ: 165.86, 163.02, 157.72, 129.76 (3C), 128.57, 126.59, 125.85, 124.65, 120.36,
115.83 (3C), 99.99, 63.01, 49.04, 45.45, 29.66, 25.41. ESI-HRMS: calcd for
C19H21N4O, [M + H]+, 321.1710; found 321.1708.
4.1.37.
(S)-N-(4-methoxybenzyl)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboximida
mide (20c)

ACCEPTED MANUSCRIPT
Yield: 33.9%; 1H-NMR (DMSO-d6, 400 MHz) δ: 9.46 (br, 2H), 7.34~7.32 (d, J=8.4
Hz, 2H), 7.16~7.12 (m, 1H), 6.99~6.95 (m, 4H), 6.92~6.90 (d, J=8.0 Hz, 1H),
4.53~4.41 (m, 4H), 4.34~4.30 (d, J=13.2 Hz, 1H), 3.75 (s, 3H), 2.68~2.61 (m, 2H),
2.51~2.42 (m, 1H), 1.93~1.84 (m, 1H).13C-NMR (DMSO-d6, 100 MHz) δ: 165.86,
163.13, 159.45, 129.82 (3C), 128.63, 127.66, 126.63, 124.77, 120.28, 114.51 (3C),
63.13, 55.64, 49.05, 45.29, 29.65, 25.46. ESI-HRMS: calcd for C20H23N4O, [M + H]+,
335.1866; found 335.1875.
4.2. Cell culture
SH-SY5Y cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine
serum (Hyclone). The cells were incubated in a humidified atmosphere of 5% CO2 at
37 . All of the compounds were dissolved in dimethyl sulfoxide (DMSO) to 100
mM concentration and stored at -20
Amresco (Solon, OH, USA).
4.3. MTT assay
until the assay. DMSO was purchased from
SH-SY5Y (1 × 5000) cells were seeded into 96-well culture plates in a final volume
of 100 µL. The cells were grown for 24 h, then pretreated with different
concentrations of target compounds (final concentrations: 0, 0.1, 1, 10, 100 µM) for 6
h. After these treatments, the medium was replaced with HEPES buffer (1×HMF,
Mg2+-free, CC0074, Leagene, China) with or without containing NMDA (2 mM) for
30 min. And then, HEPES buffer was replaced with fresh DMEM for another 12 h. At
the
end
of
indicated
treatments,
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 0.5 mg/ml,
M-2128, Sigma) was added to the medium and incubated for another 4 h at 37 , then
the medium was replaced by 150 µL DMSO. The absorbance was measured at 490
nm with a microplate reader (ELX 800, Bio-TEK instruments, Inc.). Cell viability was
expressed as a percentage with the control group as 100%. Ifenprodil (I2892, Sigma)
was the control medicine.
4.4. Measurement of Intracellular Calcium Concentration
The concentration of intracellular calcium was measured with Fluo-3 AM
(MA0195, meilunbio, China). SH-SY5Y cells were cultured in 3.5-cm plates at a