Discovery of a promising agent IQZ23 for the treatment of obesity and related metabolic disorders

Article abstract of DOI:10.1016/j.ejmech.2020.112172

Discovery of novel anti-obesity agents is a challenging and promising research area. Based on our previous works, we synthesized 40 novel β-indoloquinazoline analogues by altering the skeleton and introducing preferential side chains, evaluated their lipid-lowering activity and summarized the structure-activity relationships. In combination with an evaluation of the lipid-lowering efficacies, AMP-dependent activated protein kinase (AMPK) activating ability and liver microsomal stability, compound 23 (named as IQZ23) was selected for further studies. IQZ23 exerted a high efficacy in decreasing the triglyceride level (EC₅₀ = 0.033 μM) in 3T3-L1 adipocytes. Mechanistic studies revealed the lipid-lowering activity of IQZ23 was dependent on the AMPK pathway by modulating ATP synthase activity. This activation was accompanied by mitochondrial biogenesis and oxidation capacity increased, and insulin sensitivity enhanced in pertinent cell models by various interventions. Correspondingly, IQZ23 (20 mg/kg, i.p.) treatment significantly reversed high fat and cholesterol diet (HFC)- induced body weight increases and accompanying clinical symptoms of obesity in mice but without indicative toxicity. These results indicate that IQZ23 could be a useful candidate for the treatment of obesity and related metabolic disorders.

Full text of DOI:10.1016/j.ejmech.2020.112172

Journal Pre-proof
Discovery of a promising agent IQZ23 for the treatment of obesity and related
metabolic disorders
Yong Rao, Zhao Xu, Yu-Tao Hu, Chan Li, Yao-Hao Xu, Qin-Qin Song, Hong Yu,
Bing-Bing Song, Shuo-Bin Chen, Qing-Jiang Li, Shi-Liang Huang, Jia-Heng Tan,
Tian-Miao Ou, Hong-Gen Wang, Guo-Ping Zhong, Ji-Ming Ye, Zhi-Shu Huang
PII:
S0223-5234(20)30139-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112172
EJMECH 112172
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2019
Revised Date: 18 February 2020
Accepted Date: 19 February 2020
Please cite this article as: Y. Rao, Z. Xu, Y.-T. Hu, C. Li, Y.-H. Xu, Q.-Q. Song, H. Yu, B.-B. Song, S.-
B. Chen, Q.-J. Li, S.-L. Huang, J.-H. Tan, T.-M. Ou, H.-G. Wang, G.-P. Zhong, J.-M. Ye, Z.-S. Huang,
Discovery of a promising agent IQZ23 for the treatment of obesity and related metabolic disorders,
European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112172.
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Title page
Discovery of a Promising Agent IQZ23 for the Treatment of Obesity and Related Metabolic
Disorders
a, †,
a, †
a
a
a
a
a
a
Yong Rao *, Zhao Xu , Yu-Tao Hu , Chan Li , Yao-Hao Xu , Qin-Qin Song , Hong Yu , Bing-Bing Song ,
a
a
a
a
a
a
Shuo-Bin Chen , Qing-Jiang Li , Shi-Liang Huang , Jia-Heng Tan , Tian-Miao Ou , Hong-Gen Wang , Guo-Ping
c
b
a,
Zhong , Ji-Ming Ye , Zhi-Shu Huang *
a
Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences,
Sun Yat-Sen University, Guangzhou, Guangzhou 510006, China
b
Molecular Pharmacology for Diabetes Group, School of Health and Biomedical Sciences, RMIT University,
Melbourne, VIC, 3083 Australia
c
Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-Sen
University, Guangzhou, China.
*
To whom correspondence should be addressed. Tel.: 8620-39943056, Fax: 8620-39943056, E-mail addresses:
raoyong0805@126.com (Y. Rao), ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
†
These authors contributed equally
Abstract
Discovery of novel anti-obesity agents is a challenging and promising research area. Based on our previous works,
we synthesized 40 novel β-indoloquinazoline analogues by altering the skeleton and introducing preferential side
chains, evaluated their lipid-lowering activity and summarized the structure-activity relationships. In combination
with an evaluation of the lipid-lowering efficacies, AMP-dependent activated protein kinase (AMPK) activating
ability and liver microsomal stability, compound 23 (named as IQZ23) was selected for further studies. IQZ23
exerted a high efficacy in decreasing the triglyceride level (EC = 0.033 µM) in 3T3-L1 adipocytes. Mechanistic
5
0
studies revealed the lipid-lowering activity of IQZ23 was dependent on the AMPK pathway by modulating ATP
synthase activity. This activation was accompanied by mitochondrial biogenesis and oxidation capacity increased,
and insulin sensitivity enhanced in pertinent cell models by various interventions. Correspondingly, IQZ23 (20
mg/kg, i.p.) treatment significantly reversed high fat and cholesterol diet (HFC)- induced body weight increases
and accompanying clinical symptoms of obesity in mice but without indicative toxicity. These results indicate that
IQZ23 could be a useful candidate for the treatment of obesity and related metabolic disorders.
KEYWORDS
synthesis; 3T3-L1 adipocyte; AMPK activation; obesity; metabolic disorders.
Abbreviations
ACC: acetyl-CoA carboxylase; ACSL: long-chain acyl-CoA synthetase; AMP: adenosine 5′-monophosphate;
AMPK: adenosine 5′-monophosphate activated protein kinase; ATP: adenosine-triphosphate; C/EBPα:
CCAAT-enhancer binding proteins α; CH: chow diet; CPT-1β: carnitine palmitoyl transterase-1β; FAS: fatty acid
synthase; GTT: glucose tolerance test; H&E: hematoxylin-eosin staining; HFC: high fat and cholesterol diet; ITT:

insulin
tolerance
test;
LDH:
lactate
dehydrogenase;
2-NBDG:
2
-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; NRF2: nuclear factor erythroid-2-related factor 2;
OCR: oxygen consumption ratio; ORO: oil-red O; PGC-1α: peroxisome proliferator activated receptor gamma
coactivator-1α; PI3K: phosphatidylinositol 3′-kinase; PPARγ: peroxisome proliferator-activated receptor γ; SCD-1:
stearoyl-CoA desaturase 1; SREBP-1c: sterol responding element binding protein-1c; TFAM: transcription factor A
mitochondria; TG: triglyceride.

Title page
Discovery of a Promising Agent IQZ23 for the Treatment of Obesity and Related Metabolic
Disorders
a, †,
a, †
a
a
a
a
a
Yong Rao *, Zhao Xu , Yu-Tao Hu , Chan Li , Yao-Hao Xu , Qin-Qin Song , Hong Yu , Bing-Bing Song,
a
a
a
a
a
a
Shuo-Bin Chen , Qing-Jiang Li , Shi-Liang Huang , Jia-Heng Tan , Tian-Miao Ou , Hong-Gen Wang , Guo-Ping
c
b
a,
Zhong , Ji-Ming Ye , Zhi-Shu Huang *
a
Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences,
Sun Yat-Sen University, Guangzhou, Guangzhou 510006, China
b
Molecular Pharmacology for Diabetes Group, School of Health and Biomedical Sciences, RMIT University,
Melbourne, VIC, 3083 Australia
c
Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-Sen
University, Guangzhou, China.
*
To whom correspondence should be addressed. Tel.: 8620-39943056, Fax: 8620-39943056, E-mail addresses:
raoyong0805@126.com (Y. Rao), ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
Abstract
Discovery of novel anti-obesity agents is a challenging and promising research area. Based on our previous works,
we synthesized 40 novel β-indoloquinazoline analogues by altering the skeleton and introducing preferential side
chains, evaluated their lipid-lowering activity and summarized the structure-activity relationships. In combination
with an evaluation of the lipid-lowering efficacies, AMP-dependent activated protein kinase (AMPK) activating
ability and liver microsomal stability, compound 23 (named as IQZ23) was selected for further studies. IQZ23
exerted a high efficacy in decreasing the triglyceride level (EC = 0.033 µM) in 3T3-L1 adipocytes. Mechanistic
5
0
studies revealed the lipid-lowering activity of IQZ23 was dependent on the AMPK pathway by modulating ATP
synthase activity. This activation was accompanied by mitochondrial biogenesis and oxidation capacity increased,
and insulin sensitivity enhanced in pertinent cell models by various interventions. Correspondingly, IQZ23 (20
mg/kg, i.p.) treatment significantly reversed high fat and cholesterol diet (HFC)- induced body weight increases
and accompanying clinical symptoms of obesity in mice but without indicative toxicity. These results indicate that
IQZ23 could be a useful candidate for the treatment of obesity and related metabolic disorders.
KEYWORDS
synthesis; 3T3-L1 adipocyte; AMPK activation; obesity; metabolic disorders.
Abbreviations
ACC: acetyl-CoA carboxylase; ACSL: long-chain acyl-CoA synthetase; AMP: adenosine 5′-monophosphate;
AMPK: adenosine 5′-monophosphate activated protein kinase; ATP: adenosine-triphosphate; C/EBPα:
CCAAT-enhancer binding proteins α; CH: chow diet; CPT-1β: carnitine palmitoyl transterase-1β; FAS: fatty acid
synthase; GTT: glucose tolerance test; H&E: hematoxylin-eosin staining; HFC: high fat and cholesterol diet; ITT:

insulin
tolerance
test;
LDH:
lactate
dehydrogenase;
2-NBDG:
2
-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; NRF2: nuclear factor erythroid-2-related factor 2;
OCR: oxygen consumption ratio; ORO: oil-red O; PGC-1α: peroxisome proliferator activated receptor gamma
coactivator-1α; PI3K: phosphatidylinositol 3′-kinase; PPARγ: peroxisome proliferator-activated receptor γ; SCD-1:
stearoyl-CoA desaturase 1; SREBP-1c: sterol responding element binding protein-1c; TFAM: transcription factor A
mitochondria; TG: triglyceride.

1
. Introduction
Diet and lifestyle contribute to the high incidence of obesity in developed and developing
countries. Currently, approximately 65% of the adult population is overweight, with a body mass
2
index of greater than or equal to 25 kg/m , and over 30% of the adult population is obese (body mass
2
index of greater than or equal to 30 kg/m ) [1]. Over the past 3 decades, the number of overweight
and obese individuals global is increasing exponentially, especially in Europe, America and other
countries and the number have increased over 1 billion [2]. Obesity is associated with an increased
risk for diabetes mellitus, cancer, and heart disease, and it often causes a shortening of human life
and economic burdens [3]. Advances in the treatment of obesity have thus far been rather limited
with few drugs available to control abnormal fat accumulation [4]. Most anti-obesity agents are
based on altering the appetite by acting on receptors in the brain. Some drugs in this class (such as
fenfluramine) have been withdrawn from the market due to their unexpected toxicity or physical side
effects [5]. Recent attempts to develop compounds that inhibit the absorption of fat through the
gastrointestinal tract (such as orlistat) may improve anti-obesity treatment. Nevertheless, even the
most effective drugs can only reduce body weight by up to 5%, and strict dieting is required for
further weight loss [4]. Apart from these treatments, promoting energy expenditure is another
approach to the treatment of obesity and associated diseases [6].
AMP-dependent activated protein kinase (AMPK) is a master metabolic regulator and maintains
energy homeostasis by regulating the balance between catabolism and anabolism involved in all
branches of cellular metabolism. AMPK’s activation is dependent on energy status and upstream
kinase, such as liver kinase B1 (LKB1) and CaMKK (Calcium/calmodulin-dependent protein kinase
kinase). In relation to metabolic diseases, the activation of AMPK has been shown to play a vital role

in increasing glucose uptake, fatty acid oxidation (FAO), mitochondrial biogenesis, and autophagy,
while suppressing the synthesis of fatty acids, cholesterol, and protein [7, 8]. Regarding these
beneficial effects, several direct or indirect AMPK activators have shown efficacy in ameliorating
obesity and related clinical syndromes [9, 10], and recently some of them have been included in
clinical trials, such as Metabolaid and ActivAMP [11, 12].
Indole and quinazoline are common drug pharmacophores, and their derivatives have extensive
bioactivities, such as anticancer, resistant to chronic obstructive pulmonary disease, and anti-hepatitis.
They have been characterized as “dominant structures” in drug design since they have strong
affinities with some receptors. Several compounds containing indole and quinazoline were reported
to have efficacy in treating obesity and diabetes, such as I3C and KZL-1 [13, 14]. Our preliminary
studies have indicated that the natural alkaloid bouchardatine (Figure 1) ameliorated high-fat diet
(HF) induced obesity in mice (~14.6% decrease in body weight) via regulating energy metabolism
[15]. In addition, a series of derivatives were synthesized and some of them, such as compound R17
(Figure 1), were reported to significantly improve its lipid-lowering activity [16, 17], as well as
anti-obesity activity in mice (data not shown), further indicating that these β-indoloquinazoline
analogues should be considered as promising molecules for treating obesity and related metabolic
disorders. Thus, it would be of great significance to develop more detailed information on the
structure-activity relationships (SARs) of these analogues and on candidates likely to respond well to
drug therapy for obesity and related metabolic disorders.
In this study, to explore new molecules with high lipid-lowering activity and good druglike
properties, 4 series of novel β-indoloquinazoline analogues (totally 40 compounds) were designed
and synthesized based on the structures of our previous β-indoloquinazoline derivatives (Figure 1).

First, to analyze the importance of NH (at 13-position) in the indole ring, we replaced the NH with
an oxygen atom, or introduced a substitute (methyl or benzyl group) at the nitrogen atom, to give 7
compounds for Series I (1‒7). Second, we further expanded the types of side chain at the 5-position
by changing the NH into an oxygen or sulfur atom, to generate 10 compounds for Series II (8‒17).
Third, we introduced a methoxy group at 10-position to improve water-solubility and bioavailability
and to give 6 compounds for Series III (18‒23). Finally, we replaced the aldehyde group at the
8-position with a methyl group to further check the necessity of the aldehyde group, since our
previous studies have confirmed that the replacement of the aldehyde with a halogen group would
affect the activity of the compounds [18]; meanwhile, we also replaced the NH at the 13-position
with an oxygen or sulfur atom, to produce 17 compounds for Series IV (24‒40). Their biological
activities and SARs were investigated. After combinational screening assays including the efficacies
in triglyceride (TG) decreasing and AMPK activating, as well as liver microsomal stability, the best
compound was further evaluated. We assessed lipid-lowering activity, adipocyte differentiation, and
lipid synthesis inhibitory activities via AMPK activation, improvement of insulin sensitivity and
mitochondrial biogenesis, pharmacokinetics properties, and anti-obesity and associated metabolic
disorders effects using a mouse model induced by a high fat and cholesterol diet (HFC).
2
. Results and discussion
2.1 Chemistry.
To improve the lipid-lowering activity, physicochemical properties, and further explore the SARs,
we designed and synthesized four series of compounds (totally 40 new compounds) based on our
preliminary studies [17, 18]. The synthetic routes and chemical structures of the target compounds

are shown in Scheme 1 and Table 1, respectively. Ethyl 3-methylbenzofuran-2-carboxylate (a),
originated from the reaction with 2-hydroxyacetophenone and ethyl bromoacetate and was treated
with sodium hydroxide solution to obtain the carboxylic acid b. The intermediate b was prepared
into the carbonyl chloride to react with 2-aminobenzamide to give c, which was subjected to
intramolecular cyclization to get the intermediate d. Compound d was reacted with NBS to give the
bromides e which then reacted with DMSO to produce the intermediate f. The compound g, by
chlorinating the carbonyl group of f, reacted with the desired amine to get the Series I compounds
(1‒5). The intermediate h was prepared by 2-aminobenzamide according to the preliminary literature
procedure [15], and it was introduced into the methyl or benzyl group at the nitrogen atom of the
indole ring to get the intermediates i and j, respectively, followed by substitution and formylation to
get Series I compound 6 and 7. 1H-indole-2-carboxylic acid (k and l) was reacted with oxalyl
chloride to give the corresponding acyl chloride and then reacted with 2-aminobenzamide in one-pot
to generate the intermediates m and n, respectively. The compound m could be formylated and
chlorinated in one-pot by the treatment of POCl and DMF to give the compound o. We added the
3
amines, thiols or alcohols with different alkaline terminal groups to obtain the series II compounds
(8‒17). Similarly, starting from compound n, through the intermediate p, the series III compounds
(18‒23) could be obtained. The key intermediate r was prepared according to the literature procedure
[16]. In addition, the intermediate s was prepared by a similar reaction with the preparation of m
from starting material q. The series IV compounds (24‒40) were synthesized by compounds d, r, and
s treated with POCl and DMF (to give compounds t, u, and v, respectively), followed by the amines
3
or alcohols via nucleophilic substitution.
2.2 Evaluation of the Lipid-Lowering Activity of the Derivatives Using the 3T3-L1 Adipocyte Model.

By using the 3T3-L1 adipocyte cell model, the lipid-lowering activities of all the newly
synthesized compounds were evaluated, and the SAR was concluded.
As shown in Figure 2A and Table S1, after a 6-day stimulation using the adipogenic cocktail (a
mixture of dexamethasone, insulin, and IBMX), triglyceride (TG) levels in mature adipocytes were
highly elevated, with a 5-fold increase compared to undifferentiated (UND) cells. As reported,
treatment by compound R17 significantly reversed the stimulation of the adipogenic cocktail as
indicated by an approximate decrease of 79.2% and 89.3% in TG levels at 1 and 5 µM [16, 17],
respectively, compared with differentiation (Control, Ctrl) cells. Meanwhile, metformin, a
well-known AMPK activator, as positive control also led to a decrease of 60.3% in TG level at a
concentration of 1 mM. It was shown that most of the newly synthesized compounds were effective
in inhibiting lipid synthesis and accumulation in a dose-dependent manner. Among them, 11 of the
4
4
0 compounds showed a reduction of over 50% in TG levels at a concentration of 1 µM. Especially,
compounds (11, 20, 21, 23) showed an equal or an improved lipid-lowering efficacy compared
with R17 at the concentration of 1 µM. Furthermore, The TG inhibitory effects of compound 11, 20,
1, and 23 were also confirmed by Oil-red O (ORO) staining (Figure 2B). To exclude the adverse
2
effect of cytotoxicity, we determined the cytotoxicity of these derivatives at the concentration of 5
µM in 3T3-L1 adipocytes after 6 days of incubation by lactate dehydrogenase (LDH) releasing assay.
As noted, the incubation of all the derivatives did not alter the LDH releasing levels in the culture
medium when compared with Ctrl cells (Figure S1), demonstrating that these derivatives were
well-tolerated by 3T3-L1 adipocytes at the concentration of 5 µM, and the lipid-lowering activity of
these derivatives was not due to their toxicity.
Based on the evaluation results, the SAR was summarized as follows. First, we found that the NH

group in the indole ring could be important. As shown in Figure 2A and Table S1, an obvious
reduction of TG level suppression was observed in Series I compounds. For example, with
replacement of the NH at 13-position of compound 10 with an oxygen atom to give compound 5, the
TG level went from 34.9% to 88.7% at 1 µM, and with substitution of hydrogen for the NH of the
positive compound R17 with methyl for compound 6 and benzyl group for compound 7, the TG
levels went from 20.8% to 70.4% and 57.1%, respectively. Second, we investigated the effect of the
NH group at the 5-position. It was shown that the replacement of NH (such as R17, 10, and 3a [17])
at the 5-position with oxygen (14, 17, and 13, respectively) or sulfur atom (8) would be unfavorable
for the lipid-lowering activity of the compounds. Moreover, it was found that the length of the side
chain also affected the activity. Third, the introduction of a methoxy group at the 10-position (Series
III compounds 18‒23) seems to be beneficial for the decrease of the TG level since the TG contents
were kept at a lower level (10.1 ~48.8%) after treatment with the five compounds compared with
those of compounds without a methoxy group. Finally, we explored the importance of the aldehyde
group. For Series IV compounds (24‒40), replacement of the aldehyde group at the 8-position with a
methyl group and/or the NH group at 13-position with an oxygen or sulfur atom, it was shown that
the lipid-lowering activity of derivatives was decreased when the aldehyde group changed into a
methyl group.
2.3 Evaluation of Adipocytes Differentiation Inhibition and AMPK Activation of 6 Potent
Compounds.
On the basis of lipid-lowering activity and the SAR of the compounds above, we selected 4 potent
molecules (11, 20, 21, and 23) for additional studies. In the first experiments, the 4 compounds, plus
R17 for comparison, were tested for their needed concentration for 50% of maximal effect (EC ) on
5
0

decreasing TG levels. As shown in Figure 3A, the treatment of derivatives at 0, 0.01, 0.1, 0.3, 1, 3,
and 10 µM concentrations yielded dose-dependent decreases in TG levels in 3T3-L1 adipocytes, with
EC50 values being at 0.51 µM, 0.92 µM, 0.31 µM and 0.033 µM, respectively. Specifically,
compound 23 has a higher efficacy in decreasing TG levels compared with R17 as judged by a
comparable EC value (0.033 µM vs 0.091 µM).
5
0
Studies have revealed that adipocyte maturation is regulated by a network of genes that strictly
initiate the processes of adipogenesis and lipogenesis [18]. Among them, adipogenic factors, such
as CCAAT/enhancer-binding proteinβ, δ, and α (C/EBP β, δ, and α) and peroxisome
proliferator-activated receptor γ (PPARγ), play an essential role in controlling the early stage of
adipocyte differentiation, namely, adipogenesis, which is followed by an activation of lipogenesis.
Coinciding with the TG-decreasing effect, we next determined the effects of these potent derivatives
on the expression level of adipogenic factors. Firstly, 1 mM of metformin treatment efficiently
decreased the expression level of C/EBP (β, δ, α) and PPARγ (Figure S2A). Cell treated with our
derivatives dose-dependently blocked the stimulation of the adipogenic cocktail as indicated by
decreases in the mRNA level of C/EBP (δ, β, α) and PPARγ after 24 h incubation (Figure 3B-3E). In
comparison with R17, compound 23 showed a stronger inhibition effect at each concentration in
blocking the activation of these adipogenic markers, which were well related to the TG decreasing
ability. Notably, at a lower concentration of 0.01 µM, 23 treatment exerted a higher efficacy in
blocking the activation of these adipogenic markers.
Our previous studies have demonstrated that the
β-indoloquinazoline derivatives which could
inhibit adipocyte differentiation in cells and ameliorate obesity-related metabolic disorders in mice
were association with an activation of the AMPK pathway [8]. We next determined the stimulation

effect of these derivatives on AMPK. R17 and metformin (Met) were loaded as a positive control. As
expected, both R17 and Met treatment significantly increased the phosphorylated level (Thr-172) of
AMPKα, and activated AMPK pathway (Figure S2B). Cells treated with the 4 derivatives also
significantly increased the activity of AMPK in 3T3-L1 adipocytes as indicated by an increment in
the phosphorylated level of AMPKα, and 23 showed a more enhanced effect on AMPK activity than
R17. And compound 23 supplemented yielded a higher efficacy in activating AMPK pathway at each
concentration compared with R17-treated cells (Figure 3F and Figure S2B). In light of the data, there
seems to be a positive correlation between AMPK activation and TG-lowering activity, compounds
with higher activity on AMPK activation yielding a higher efficacy in decreasing TG levels (Figure
S2C).
2.4 Assay of Aqueous Solubility and Liver Microsomal Stability of 6 Potent Compounds.
To understand the druggability of the compounds, we further determined the aqueous solubility
and liver microsomal stability of the 4 most potent compounds. As shown in Table 2, compared with
R17, compounds 20, 21, and 23, which introduced a methoxy group at 10-position, showed an
increase in aqueous solubility, demonstrating that the introduction of methoxy is prone to improve
the aqueous solubility of derivatives. Especially compared with R17, the water-solubility of
compound 23 with the best lipid-lowering activity was significantly improved from 41.1 to 73.3
µg/mL. For the liver microsomal stability assay, testosterone was selected as a control compound. As
shown in Table 2, compound 11, 20, 23 showed better stability in Sprague Dawley (SD) rat liver
microsomal in comparison with R17. We next determined the stability of the most potent derivative
23 in different species microsomal, including dog, monkey and human liver microsomal. These
results indicated that 23 was stable enough as indicated by the values of t1/2 were 187.3, 630.1,

1155.2 and 630.1 min, respectively (Table 2, Figure S3). Furthermore, pharmacokinetics assay
revealed that 23 had similar orally bioavailability and more favorable maximum plasma drug
concentration compared with R17 (Table 3), and also had less individual difference among mice.
This improved pharmacokinetics feature of 23 may partly due to its increase in aqueous solubility
[
19, 20]. In conclusion, we selected compound 23 (named as IQZ23) as a hit compound from the
library for further study.
2.5 IQZ23 Blocked Adipocytes Differentiation Related to AMPK Pathway Activation.
As reported, 3T3-L1 pre-adipocytes differentiate into mature white adipocytes by undergoing
three pivotal stages, namely, adipogenesis (Day 0-3), lipogenesis (Day 3-6) and terminal
differentiation (Day 6-9). During the stage of adipogenesis, the adipogenic factors, such as C/EBPα,
PPARγ, and SREBP-1c, are activated in a time-dependent manner, which is followed by an initiation
of lipogenesis by increasing the expression of fatty acid synthesis related proteins [21]. Studies have
demonstrated that the interruption of adipogenesis was an effective approach to block the maturation
of 3T3-L1 adipocytes [22]. Correspondingly, cells treated with IQZ23 decreased TG levels in
adipocytes in a time-dependent manner as indicated by an approximate decrease of 74.2%, 61.8%
and 35.3% during the periods of Day 0-3, 3-6 and 6-9, respectively. Furthermore, there was no major
difference in the TG level while IQZ23 was incubated during the periods of Day 0-3, 0-6 and 0-9
(Figure 4A, Figure S4A), demonstrating that IQZ23 inhibited adipocyte differentiation by
interrupting the stage of adipogenesis. Western blot assays revealed that IQZ23 treatment markedly
decreased the protein level of adipogenic factors C/EBPα, PPARγ, and sterol regulatory
element-binding protein 1c (SREBP-1c)) after 24 h treatment as well as the level of fatty acid
synthesis related proteins fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), stearoyl-CoA

desaturase 1 (SCD1) after 6 days of treatment (Figure 4B-C). The inhibition of adipogenic markers
was also observed in metformin-treated cells (Figure S2A). It is worth noting that IQZ23 showed a
stronger inhibitory effect on these proteins compared with R17 at a concentration of 1 µM, which
accorded with the TG-decreasing effect in 3T3-L1 adipocytes.
Moreover, cells treated with IQZ23 and R17 moderately blunted ATP synthase activity,
decreased ATP levels and thus increased the ratio of AMP-to-ATP (Figure 4D-F). These changes in
ATP levels and AMP-to-ATP ratio accorded well with AMPK activation (Figure 3F). To reveal the
relationship between AMPK activation and the TG-decreasing effect mediated by IQZ23, 3T3-L1
adipocytes were transfected with AMPKα siRNA (50 nM) in the presence or absence of IQZ23.
Knockdown of AMPKα blocked AMPK activation and efficiently abolished the stimulation effect of
IQZ23 on the AMPK pathway. This abolishment of AMPK activation led to a reversal in the protein
level of C/EBPα, PPARγ and TG level in 3T3-L1 adipocytes when compared with IQZ23 treatment
alone (Figure 4G-H, Figure S4B), suggesting that IQZ23 blocked 3T3-L1 adipocyte differentiation
via AMPK activation.
2.6 IQZ23 Improved Insulin Sensitivity in C2C12 Myoblasts via the AMPK Pathway.
Studies have linked AMPK activation with the stimulation of glucose uptake and utilization in
muscles and the liver, which are pivotal for maintaining glucose and insulin homeostasis and often
malfunction due to obesity [8, 23]. As shown in Figure 5A-B, similar to insulin, both R17 and
IQZ23 and also metformin alone treatment significantly increased glucose uptake (red foci) in
C2C12 cells by 2-NBDG assay, an additive effect in glucose uptake was observed in the combination
of drug and insulin compared with drug or insulin alone, and IQZ23 showed a slightly enhanced
effect in improving insulin sensitivity compared with R17 in the presence or absence of insulin.

Moreover, the stimulation effect of R17 and IQZ23 was in parallel with AMPK pathway activation
while no major change was observed in the total and phosphorylated (Ser-308) levels of AKT
(Protein kinase B) (Figure 5C), which plays a key role in insulin-mediated phosphatidylinositol
3
′-kinase (PI3K)/AKT pathway activation. Moreover, PI3K pathway inhibitor LY-294002 addition
was able to block glucose uptake and the phosphorylated (Ser308) level of AKT in C2C12 cells,
while its addition did not abolish the stimulation of IQZ23 on glucose uptake as well as the
activation of AMPK (Figure 5D-E). These results strongly demonstrated that IQZ23 stimulated
glucose uptake in C2C12 myoblasts through the AMPK pathway rather than the insulin-signaling
pathway.
2.7 IQZ23 Increased Mitochondria Oxidation Capacity.
Another important beneficial effect mediated by AMPK activation is mitochondria biogenesis and
oxidative capacity increase, which burns and consumes fatty acids [24]. AICAR, a classic AMPK
activator, was viewed as a control compound. As expected, AICAR, R17 and IQZ23 treatment
increased mitochondria copy number (red foci) in 3T3-L1 adipocytes. This stimulation effect of
AICAR, R17, and IQZ23 were partially blocked by addition of AMPK pathway inhibitor Compound
C (CC) (Figure 6A-B). Moreover, cells treated with R17 and IQZ23 activated the expression of
mitochondria biogenesis markers (PGC-1α, TFAM) and a network of genes indicative of
mitochondrial oxidation capacities, such as CPT-1β, ACSL, and NRF2 (Figure 6C). Correspondingly,
mitochondrial oxidation capacity in R17 and IQZ23 treated cells was increased as indicated by an
increment in oxygen consumption in 3T3-L1 adipocytes using an extracellular Flux Analyzer (Figure
6D).

2.8 IQZ23 Ameliorated HFC Induced Obesity and Related Metabolic Disorders.
To assess whether IQZ23 exerts in vivo therapeutic effects on obesity and related metabolic
syndromes devoid of the influence on calorie intake, we used a high fat (60%) containing 1%
cholesterol diet (HFC) induced mouse model to evaluate the anti-obesity, anti-hypoglycemic and
anti-hypolipidemic effects of IQZ23. After 8 weeks of induction, mice were randomly divided into
two subgroups, and one group of mice received vehicle treatment (HFC saline group), while another
group of mice was treated with 20 mg/kg of IQZ23 (HFC-IQZ23 group) by intraperitoneal (i.p.)
injection every other day for 6 weeks.
As shown in Figure 7A-B and Figure S5A, HFC-feeding induced a large increase in body weight
compared to the group fed with chow diet (CH group) and IQZ23 administration significantly
reversed the increase in body weight to the level of the chow group mice as indicated by a 27.4%
decrease compared with vehicle-treated mice. The body weight reduction was accompanied by a
large decrease in fat mass content and lean mass in IQZ23 treated mice (Figure 7C, Figure S5B).
Consistent with the reduction of fat mass, IQZ23-treated mice displayed a marked reduction in the
size of adipocytes as indicated by hematoxylin-eosin (H&E) staining of white adipose tissue (Figure
S5C). Interestingly, the total food intake over 6 weeks in the IQZ23-treated group showed no major
difference compared with the vehicle-treated mice (Figure 7D), indicating that the anti-obesity effect
of IQZ23 is not mediated by limiting calorie intake.
Next, we measured relevant plasma parameters and tissue weights to examine whether there was
any indication of toxicity. Of note, HFC-feeding induced an increase in the size of organs and no
major difference was observed in IQZ23-treated mice regarding the weight of the tissues studied
when compared with vehicle-treated mice, while a significant decrease was observed in the weight of

livers in IQZ23-treated mice (Figure S5D). IQZ23 administration also significantly reversed the
increase in the activity of serum alanine aminotransferase (ALT) and aspartate transaminase (AST)
as well as total bilirubin (TBIL), albumin (ALB), blood urea nitrogen (BUN) and creatine (CRE)
after 14 weeks of HFC stimulation (Table 4). Moreover, acute toxicity experiments revealed the half
lethal dose (LD ) of IQZ23 in mice is over 500 mg/kg in mice, indicating that IQZ23 was
5
0
well-tolerated in vivo.
Dyslipidemia and hyperglycemia are the main clinic syndromes that often accompany obesity [8].
As expected, mice treated with IQZ23 had large decreases in plasma levels of free fatty acids (FFA),
TG, glucose, insulin, LDL-c as well as the ratio of LDL-c/HDL-c, to a similar level to that of chow
mice (Table 4). At the same time, we also determined the acute effect of IQZ23 on improved
hyperglycemia. HFC feeding mice were injected with IQZ23 once, plasma glucose was decreased in
a time-dependent manner, and an obvious anti-hyperglycemic effect occurred 6 h post-injection
(Figure 7E). Consistent with this, GTT and ITT experiments further confirmed the anti-obesity effect
of IQZ23 in mice. After IQZ23 administration, the mean area under curve (AUC) of GTT and ITT
in IQZ23-treated mice was much lower than that of the vehicle-treated mice (Figure 7F-G), strongly
indicating the effectiveness of IQZ23 for treating of obesity and relevant metabolic disorders.
Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disorder accompanying obesity,
and it is characterized by lipid ectopic accumulation (FFA flux and glucose-induced lipogenesis)
leading to hepatocyte injury in the liver [25]. Notably, mice treated with IQZ23 experienced reduced
liver weight and TG levels (Figure 8A-B). H&E and ORO staining assays further confirmed the
efficacy of IQZ23 in protecting the liver from hepatic steatosis (Figure 8C). These results were in
agreement with a reduction in AST and ALT activities (Table 4).

3
. Conclusions
In the present study, we synthesized 4 novel β-indoloquinazoline analogues (a total of 40
compounds) by altering skeleton structures or introducing preferential side chains and substitutes
1
5, 17
based on our previous findings.
The lipid-lowering activity of these compounds was tested, and
the SAR was summarized in line. Based on the evaluation of the efficacies in lipid reduction, liver
microsomal stability, aqueous solubility, and AMPK activation, IQZ23 was selected as the most
potent candidate for further study of the molecular mechanism and evaluation of the anti-obesity
effect. Of note, an introduction of methoxy at the 10-position was effective at improving aqueous
solubility, stability and pharmacokinetic properties of indoloquinazoline analogues, such as IQZ23.
In cells, IQZ23 was effective at decreasing lipid content with an EC value of 0.036 µM. This
50
TG-decreasing effect was associated with AMPK activation by blunting ATP synthase activity and
decreasing ATP levels. Furthermore, compared to controls, IQZ23 treatment improved insulin
sensitivity in myoblasts and increased mitochondrial oxidation capacity in adipocytes, all of which
were beneficial effects accompanying AMPK activation for treating obesity. Accordingly,
administration of IQZ23 significantly reversed HFC induced body weight and fat mass increases.
Analyses of plasma and liver revealed that IQZ23 treatment significantly ameliorated
obesity-induced dyslipidemia, hyperglycemia, and hepatic steatosis without indications of toxicity in
vivo. Pharmacokinetics studies and liver microsomal assays demonstrated that IQZ23 was more
stable and soluble with a preferable bioavailability (~48.6%) in vitro, which was better than control
compound R17. As shown above, IQZ23 should be taken into consideration as a potential
anti-obesity agent for treating obesity and related metabolic disorders.

4
. Experimental section
4.1. Chemistry
All commercial chemicals used as starting materials were analytical grade and utilized without
1
13
further purification. H and C NMR spectra were recorded using TMS as the internal standard in
DMSO-d , MeOD-d or CDCl with a Bruker Avance III spectrometer at 400 MHz; Mass spectra
6
4
3
(MS) were recorded on Agilent 6120 Quadrupole LC/MS instrument with an ESI mass selective
detector, and high-resolution mass spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF. All
synthesized compounds were purified by using flash column chromatography with silica gel
(200-300 mesh). The purities of synthesized compounds were confirmed to be higher than 95% by
using analytical HPLC equipped with a dual pump Shimadzu LC-20AB system with an AnalaRic
C18 column (4.6 × 250 mm, 5 µm), which was eluted with methanol-water (50:50 to 85:15, v:v) at a
-1
flow rate of 0.5 mL·min . Melting points (m.p.) were determined by using capillary tubes with an
MSRS-OptiMelt automated melting point instrument without correction.
Ethyl 3-methylbenzofuran-2-carboxylate (a). To a stirred suspension of 2-hydroxyacetophenon
(
5.4 g，40 mmol) in DMF(16 mL) was added ethyl bromoacetate (20 g，120mmol) and potassium
carbonat e（ 22.1 g，160 mmol）, and the mixture was stirred at 80 ºC for 1 h, and then up to 160 ºC for
h. After the reaction solution was cooled, it was poured into a large amount of water and extracted
2
with dichloromethane three times. The organic phase was washed with saturated sodium chloride
solution for three times, dried with anhydrous sodium sulfate and removed the solvent under vacuum
to give the crude product, which was then purified by using column chromatography with
1
dichloromethane/petroleum ether, resulting in oil compound (3.2 g, 38.4%). H NMR (400 MHz,
CDCl ) δ 7.62 (d, J = 6.6 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.9
3

Hz, 1H), 4.50 – 4.41 (q, J = 7.5 Hz, 2H), 2.59 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). MS (ESI + APCI) m/z:
+
2
05.1 [M+H] .
3
-Methylbenzofuran-2-carboxylic acid (b). The compound a was added to the mixture of a mixture
of 2 M NaOH (20 mL) and methanol (30 mL), stirring at room temperature for 4 h. Removing
methanol under vacuum, regulating the pH to 1 with concentrated hydrochloric acid, precipitating a
large number of white solid, and extracting three times by ethyl ether, and finally removing the
1
organic phase. It resulted in solid (1.77g, 99%). H NMR (400 MHz, CDCl ) δ 10.85 (s, 1H), 7.69 (d,
3
J = 7.8 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 2.67 (s,
+
3
H). MS (ESI + APCI) m/z: 177.1 [M+H] .
N-(2-Carbamoylphenyl)-3-methylbenzofuran-2-carboxamide (c). The intermediate b (0.7 g, 3.98
mmol) dissolving in sulfoxide chloride (6 mL), was stirred at 82 °C for 2 h. After the reaction
solution cooling, removing sulfoxide chloride under vacuum to give the light green solid. It was
dissolved in dichloromethane, following to drop into the solution of aminophenolamide (0.54 g, 4
mmol) in dichloromethane (20 mL) at ice-bath for 10h. The dichloromethane was removed by
filtration. The crude was washed with a small amount of ethanol, and dried to obtain a white solid
+
(
0.89 g, 83%). MS (ESI + APCI) m/z: 295.1 [M+H] .
2
-(3-Methylbenzofuran-2-yl)quinazolin-4(3H)-one (d). The compound c (0.89 g, 3.03 mmol) was
added to the mixture of 2 M KOH and ethanol (v =1:1) at 80℃ for overnight. Removing ethanol
under vacuum, regulating the pH to 3 with diluted hydrochloric acid and filtering to give a large
1
number of white solid (0.7 g, 80%). H NMR (400 MHz, DMSO-d ) δ 8.10 (d, J = 7.7 Hz, 1H), 7.73
6
(d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.2 Hz, 1H), 7.65 – 7.58 (m, 2H), 7.46 – 7.41 (m, 1H), 7.39 (t, J =
+
7
.5 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 2.75 (s, 3H). MS (ESI + APCI) m/z: 277.1 [M+H] .

2
-(3-(Dibromomethyl) benzofuran-2-yl) quinazolin-4(3H)-one (e). The compound d (1 g, 3.62
mmol) dissolving in CCl (60 mL) was added NBS (1.61 g, 9.05 mmol) and catalytic amount of
4
benzoperoxide. Then it was heated at 90 °C for 24 h. The CCl was removed by using a rotary
4
evaporator. The residue was purified by using column chromatography with
1
dichloromethane/petroleum ether to give compound e (0.8 g, 51%). H NMR (400 MHz, DMSO-d )
6
δ 12.88 (s, 1H), 8.41 (s, 1H), 8.20 (t, J = 8.3 Hz, 2H), 7.96 – 7.86 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H),
+
7.60 (dq, J = 15.3, 7.2 Hz, 3H). MS (ESI + APCI) m/z: 432.9 [M+H]
.
2
-(4-Oxo-3,4-dihydroquinazolin-2-yl) benzofuran-3-carbaldehyde (f). The compound e (196 mg，
0.45 mmol) was dissolved in DMSO (4 mL) then was heated at 100 °C for 2 h. Next, the mixture
was poured into ice-water, filtered, and desiccated under vacuum to give the crude product, which
was then purified by using column chromatography with dichloromethane/petroleum ether, resulting
1
in a yellow solid (80 mg, 60%). H NMR (400 MHz, DMSO-d ) δ 13.07 (s, 1H), 10.98 (s, 1H), 8.22
6
(t, J = 7.0 Hz, 2H), 7.90 (t, J = 9.3 Hz, 2H), 7.81 (d, J = 8.1 Hz, 1H), 7.62 (q, J = 7.6 Hz, 2H), 7.55 –
+
7
.49 (m, 1H). MS (ESI + APCI) m/z: 291.1 [M+H] .
2
-(4-Chloroquinazolin-2-yl) benzofuran-3-carbaldehyde (g). The compound f (0.2 g，0.68 mmol)
dissolving in POCl (3 mL) was heated at 60 °C for 2 h. Next, the mixture was poured into ice-water
3
and extracted with dichloromethane. The combined organic layers were dried over anhydrous
Na SO , filtered, and concentrated under reduced pressure. The crude product was purified by using
2
4
column chromatography with dichloromethane/petroleum ether, resulting in a pale yellow solid (30
1
mg, 14%). H NMR (400 MHz, CDCl ) δ 8.71 (s, 1H), 8.32 (d, J = 9.1 Hz, 1H), 8.26 (d, J = 7.8 Hz,
3
1
1
H), 8.22 (d, J = 8.5 Hz, 1H), 8.03 (t, J = 8.4 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.73 (d, J = 8.3 Hz,
+
H), 7.50 (t, J = 7.8 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H). MS (ESI + APCI) m/z: 309.0 [M+H] .

The compound h was prepared by 2-aminobenzamide according to our preliminary literature
procedure [17].
General method A for the preparation of the compounds i-j. To a solution of compound h
dissolving in DMF was added KOH (1.2 eq) and iodomethane (1.2 eq) or benzyl bromide (1.6 eq).
After being stirred for 2 h, the reaction was quenched with water and extracted with dichloromethane.
The combined organic layers were dried over anhydrous Na SO , filtered, and concentrated under
2
4
reduced pressure. The crude product was purified by using column chromatography to give the
desired product.
General method B for the preparation of the compounds m-n, s. To a stirred suspension of
carboxylic acid k, l or q in dry dichloromethane was added acyl chloride (3 eq) and two drops of
DMF, and the mixture was stirred at room temperature for 2 h. Next, the acyl chloride was removed
by using a rotary evaporator. The residue was dissolved in tert-butanol, then anthranilamide (1 eq)
and potassium tert-butoxide (2 eq) were added, and it was heated at 100 °C for 8 h. The solution was
poured into ice-water, and the pH was adjusted to 7.0. The residue was filtered, washed with water,
and desiccated under vacuum to give the crude product, which was then purified by using column
chromatography to give the desired product.
General method C for the preparation of the compounds o-p. Vilsmeier reagent was prepared by
the reaction of phosphorus oxychloride with N, N-dimethylformamide at the volume ratio of 2:1 in
an ice bath for 0.5 h. m or n was slowly added to Vilsmeier reagent and reacted at room temperature
for 1 h. Next, the reaction solution was slowly dripped into the ice water and neutralized with 10%
NaOH to give the product. The crude product was purified by using column chromatography to give
the desired product.

The compound r was prepared by 2-aminobenzamide according to preliminary literature
procedure [18].
General method D for the preparation of the compounds t-v. The compound d, r-s dissolving in
excess POCl was heated at 50 ~ 80 °C for 6 ~ 10 h. The solution was poured into ice-water, and pH
3
was adjusted to 7.0 by saturated solution of NaHCO . The residue was filtered, washed with water,
3
and desiccated under vacuum to give the crude product, which was purified by using column
chromatography to give the desired product.
4
-Chloro-2-(1-methyl-1H-indol-2-yl)quinazoline (i). Following general method A, the compound i
1
was obtained from h (0.50 g, 1.79 mmol) as a yellow solid (136 mg, 27%). H NMR (400 MHz,
CDCl ) δ 8.27 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.95 (t, J = 7.0 Hz, 1H), 7.83 – 7.71 (m,
3
2H), 7.71 – 7.64 (m, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.39 – 7.33 (m, 1H), 7.18 (t, J = 7.4 Hz, 1H), 4.40
+
(
s, 3H). MS (ESI + APCI) m/z: 294.1[M+H] .
2
-(1-Benzyl-1H-indol-2-yl)-4-chloroquinazoline (j). Following general method A, the compound j
1
was obtained from h (200 mg , 0.72 mmol) as a pale red solid (200 mg, 76%). H NMR (400 MHz,
CDCl ) δ 8.21 (d, J = 8.3 Hz, 1H), 7.89 (dd, J = 18.2, 8.9 Hz, 3H), 7.78 (d, J = 7.8 Hz, 1H), 7.62 (t, J
3
=
6.8 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.36 – 7.27 (m, 2H), 7.24 (d, J = 6.9 Hz, 1H), 7.21 (s, 1H),
+
7
.19 (s, 1H), 7.18 – 7.11 (m, 2H), 6.30 (s, 2H). MS (ESI + APCI) m/z: 370.1[M+H] .
2
-(1H-Indol-2-yl)quinazolin-4(3H)-one (m). Following general method B, the compound m was
1
obtained from k (500 mg，3.11 mmol) as a white solid (0.63 g, 78%). H NMR (400 MHz, DMSO-d )
6
δ 12.61 (s, 1H), 11.80 (s, 1H), 8.16 (dd, J = 7.9, 1.0 Hz, 1H), 7.88 – 7.83 (m, 1H), 7.75 (d, J = 7.9 Hz,
1H), 7.67 (d, J = 1.5 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.56 – 7.48 (m, 2H), 7.26 – 7.21 (m, 1H),
+
7
.07 (t, J = 7.2 Hz, 1H)；MS (ESI + APCI) m/z: 262.1[M+H] .

2
-(5-Methoxy-1H-indol-2-yl)quinazolin-4(3H)-one (n). Following general method B, the
1
compound n was obtained from l (0.96 g, 5 mmol) as a yellow solid (1.02 g, 70%). H NMR (400
MHz, DMSO-d ) δ 12.56 (s, 1H), 11.64 (s, 1H), 8.16 (d, J = 7.4 Hz, 1H), 7.84 (t, J = 7.1 Hz, 1H),
6
7.73 (d, J = 7.9 Hz, 1H), 7.59 (s, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.11 (s, 1H),
+
6
.89 (d, J = 7.9 Hz, 1H), 3.79 (s, 3H). MS (ESI + APCI) m/z: 292.1[M+H] .
2
-(4-Chloroquinazolin-2-yl)-1H-indole-3-carbaldehyde (o). Following general method C, the
1
compound o was obtained from m (100 mg, 0.38 mmol) as a pale yellow solid (113 mg, 97%). H
NMR (400 MHz, DMSO-d ) δ 12.79 (s, 1H), 11.22 (s, 1H), 8.35 (d, J = 8.1 Hz, 1H), 8.30 (d, J = 7.8
6
Hz, 1H), 8.20 (q, J = 8.0 Hz, 2H), 7.93 (t, J = 6.8 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.5
1
3
Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ 189.6, 162.3, 153.7, 151.2,
6
140.4, 136.9, 136.7, 130.6, 129.0, 126.5, 126.3, 125.6, 123.6, 122.6, 122.6, 117.7, 113.5. MS (ESI +
+
APCI) m/z: 308.2[M+H] .
2
-(4-Chloroquinazolin-2-yl)-5-methoxy-1H-indole-3-carbaldehyde (p). Following general method
1
C, the compound p was obtained from n (1 g，3.13 mmol) as a yellow solid (0.87 g, 75%). H NMR
(
400 MHz, DMSO-d ) δ 12.70 (s, 1H), 11.20 (s, 1H), 8.34 (d, J = 8.2 Hz, 1H), 8.24 – 8.14 (m, 2H),
6
7
3
3
.91 (t, J = 6.1 Hz, 1H), 7.78 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 2.5 Hz, 1H),
+
.83 (s, 3H). Purity: 99.1% by HPLC. HRMS (ESI + APCI) m/z: calcd for C H N O Cl, [M+H]
1
8
12
3
2
38.0691, found 338.0694.
2
-(3-Methylbenzo[b]thiophen-2-yl)quinazolin-4(3H)-one (s). Following general method B, the
1
compound s was obtained from q (8 g, 41.7 mmol) as a white solid (5.3 g, 43%). H NMR (400 MHz,
DMSO-d ) δ 12.49 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.08 – 8.02 (m, 1H), 7.97 – 7.91 (m, 1H), 7.86
6
(t, J = 7.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.52 (d, J = 3.1 Hz, 1H), 7.51

+
(
d, J = 3.3 Hz, 1H), 2.65 (s, 3H). MS (ESI + APCI) m/z: 293.1[M+H] .
4
-Chloro-2-(3-methylbenzofuran-2-yl) quinazoline (t). Following general method D, the
compound t was obtained from d (1 g，3.13 mmol) at 50 °C for 10 h , resulting in a yellow solid
1
(
0.68 g, 64%). H NMR (400 MHz, DMSO-d ) δ 7.73 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.2 Hz, 1H),
6
7.65 – 7.58 (m, 2H), 7.46 – 7.41 (m, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 2.75 (s,
+
3
H). MS (ESI + APCI) m/z: 295.1 [M+H] .
4
-Chloro-2-(3-methyl-1H-indol-2-yl) quinazoline (u). Following general method D, the compound
u was obtained from r (60 mg, 0.22 mmol) with two drops DMF at 80 °C for 6 h, resulting in a pale
1
yellow solid (50 mg, 78%). H NMR (400 MHz, CDCl ) δ 9.39 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H),
3
8.02 (d, J = 8.4 Hz, 1H), 7.97 – 7.88 (m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.62 (t, 1H), 7.43 (d, J = 8.2
+
Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 2.94 (s, 3H). MS (ESI) m/z 294.1 [M+H] .
4
-Chloro-2-(3-methylbenzo[b]thiophen-2-yl) quinazoline (v). Following general method D, the
compound v was obtained from s (1 g, 3.42 mmol) with two drops DMF at 80 °C for 6 h, resulting in
1
a white solid (0.91 g, 86%). H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.4
3
Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.81 – 7.74 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.35 – 7.29 (m, 2H),
+
2
.96 (s, 3H). MS (ESI) m/z: 311.0[M+H] .
General method E for the preparation of the target compounds 1-5 (series I) A mixture of
intermediate g (30 mg, 0.1 mmol), triethylamine (30 µL), and the respective amine (1 mmol) in
toluene (10 mL) was heated at 80 °C for 1 h. Next, most of the toluene was removed by using a
rotary evaporator. The crude product was purified via flash chromatography to give the desired
product.
General method F for the preparation of the target compounds 6-7 (series I) A mixture of

intermediate i or j (100 mg), triethylamine (30 µL), and the respective amine (1 mmol) in toluene (1
mL) was stirred for 1 h. Next, most of the toluene was removed by using a rotary evaporator, then
dissolved it in the prepared Vilsmeier reagents. Next, the mixture solution was poured into ice-water
and be well stirred, then the pH was adjusted to 7~8, filtered, and desiccated under vacuum to give
the crude product, which was purified via flash chromatography to give the desired product.
General method G for the preparation of the target compounds 8-9 (series
Ⅱ) To a solution of
the respective thiol chain (3.3 mmol) in DMF (5 mL) were added sodium hydride (72 mg, 3 mmol).
Until the solution changed from colorless clarification to white turbidity, the compound o (100 mg,
0.33 mmol) was added and the reaction was heated at 90 °C for 8 h. Next, the mixture solution was
poured into ice-water and be well stirred, then the pH was adjusted to 7, filtered, and desiccated
under vacuum to give the crude product, which was purified by column chromatography to give the
desired product.
General method H for the preparation of the target compounds 10-12 (series
Ⅱ) To a solution of
o (100 mg, 0.33 mmol) in DMF (5 mL) were added K CO (91 mg, 0.66 mmol) and the respective
2
3
amine (3.3 mmol). After being stirred for 1~4 h, the reaction was quenched with water, then the pH
value was adjusted to 8, filtered, and desiccated under vacuum to give the crude product, which was
purified by column chromatography to give the desired product.
General method I for the preparation of the target compounds 13-17 (series
) A solution of the respective alcohol chain (1 mmol) in tetrahydrofuran (4 mL) was added sodium
hydride (24 mg, 1 mmol). Until no bubbles were bubbled up, the compound o (59 mg, 0.2 mmol) or t
59 mg, 0.2 mmol) was added and the reaction was stirred at room temperature for 1 h. Next, most of
the tetrahydrofuran was removed by using a rotary evaporator. The residual solution was poured into
Ⅱ) and 31-35 (series
Ⅳ
(

ice-water and be well stirred, filtered, and desiccated under vacuum to give the crude product, which
was purified by column chromatography to give the desired product.
General method J for the preparation of the target compounds 18-23 (series III) A mixture of
intermediate p (150 mg，0.44 mmol), triethylamine (30 µL), and the respective amine (4.4 mmol) in
toluene (5 mL) was heated at 60 °C for 6 h. Next, the mixture solution was poured into ice-water and
be well stirred, then the pH was adjusted to 7~8, filtered, and desiccated under vacuum to give the
crude product, which was purified via flash chromatography to give the desired product.
General method K for the preparation of the target compounds 24-30 (series IV) A mixture of
intermediate u (100 mg，0.34 mmol), triethylamine (30 µL), and the respective amine (2.5 mmol) in
toluene (5 mL) was heated at 80 °C for 6 h. Next, the mixture solution was poured into ice-water and
well stirred, then the pH was adjusted to 9, filtered, and desiccated under vacuum to give the crude
product, which was then purified via flash chromatography on a silica gel to give the desired product.
General method L for the preparation of the target compounds 36-40 (series IV) A mixture of
intermediate v (100 mg, 0.32 mmol), triethylamine (30 µL), and the respective amine (3.2 mmol) in
toluene (5 mL) was heated under 25 ~ 60 °C for 2 h. Next, the mixture solution was poured into
ice-water and be well stirred, then the pH was adjusted to 7, filtered, and desiccated under vacuum to
give the crude product, which was purified via flash chromatography to give the desired product.
2
-(4-((2-(Dimethylamino)ethyl)amino)quinazolin-2-yl)benzofuran-3-carbaldehyde (1). Following
general method E, the compound 1 was obtained from g and N,N-dimethylethane-1,2-diamine as a
o
1
pale yellow solid (21 mg, 59%): m.p. 124.8-126.0 C; H NMR (400 MHz, CDCl ) δ 11.40 (s, 1H),
3
8
.42 (d, J = 8.5 Hz, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H),
.71 (d, J = 7.7 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.49 – 7.43 (m 1H), 7.43 – 7.37 (m, 1H), 4.13 (br, 2H),
7

1
3
3
1
.35 (br, 2H), 2.86 (s, 6H). C NMR (101 MHz, DMSO-d ) δ 190.6, 160.3, 160.0, 153.8, 153.0,
6
49.4, 134.1, 128.7, 127.7 (2C), 125.7, 125.2, 123.9, 123.1, 120.8, 115.0, 112.4, 56.1, 43.5 (2C), 37.3.
+
Purity: 98.5% by HPLC. HRMS (ESI) m/z: calcd for C H N O , [M+H] 361.1659, found 361.1649.
2
1
20
4
2
2
-(4-((3-(Dimethylamino)propyl)amino)quinazolin-2-yl)benzofuran-3-carbaldehyde
(2).
Following general method E, the compound was obtained from
2
g
and
o
1
N,N-dimethylpropane-1,3-diamine as a pale yellow solid (26 mg, 70%): m.p. 99.4-102.1 C; H
NMR (400 MHz, CDCl ) δ 11.48 (s, 1H), 8.95 (s, 1H), 8.36 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 8.3 Hz,
3
1
H), 8.00 (d, J = 7.9 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.6 Hz,
H), 7.48 – 7.43 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 3.88 (q, J = 5.1 Hz, 2H), 2.95 – 2.91 (m, 2H), 2.65
1
1
3
(
s, 6H), 2.18 – 2.11 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 190.8, 160.1, 160.1, 153.8, 153.2,
6
1
2
3
49.4, 133.9, 128.7, 127.6 (2C), 125.7, 125.3, 123.5, 123.1, 120.8, 114.9, 112.4, 56.3, 44.2 (2C), 40.2,
+
5.2. Purity: 99.8% by HPLC. HRMS (ESI) m/z: calcd for C H N O , [M+H] 375.1816, found
2
2
22
4
2
75.1807.
-(4-((4-(Dimethylamino)butyl)amino)quinazolin-2-yl)benzofuran-3-carbaldehyde (3). Following
general method E, the compound 3 was obtained from g and N,N-dimethylbutane-1,4-diamine as a
2
o
1
yellow solid (22 mg, 58%): m.p. 121.6-123.9 C; H NMR (400 MHz, CDCl ) δ 11.50 (s, 1H), 8.37
3
(d, J = 7.1 Hz, 2H), 8.03 (t, J = 9.4 Hz, 2H), 7.79 (t, J = 7.5 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.54 (t,
J = 7.5 Hz, 1H), 7.48 – 7.37 (m, 2H), 3.72 (q, J = 5.2 Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 2.50 (s, 6H),
1
3
2
1
.01 – 1.91 (m, 2H), 1.90 – 1.82 (m, 2H). C NMR (101 MHz, CDCl ) δ 191.3, 159.9, 159.8, 154.2,
3
53.6, 149.5, 133.0, 129.0, 126.9, 126.8, 125.5, 124.9, 123.4, 121.9, 121.5, 114.8, 112.0, 58.8, 44.9
(2C), 41.6, 26.3, 24.8. Purity: 99.8% by HPLC. HRMS (ESI) m/z: calcd for C H N O ,
23 24 4 2
+
[
M+H] 389.1972, found 389.1962.