Syntheses and evaluation of new Quinoline derivatives for inhibition of hnRNP K in regulating oncogene c-myc transcription

Article abstract of DOI:10.1016/j.bioorg.2018.12.020

Aberrant overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key feature in oncogenesis and progression of many human cancers. hnRNP K has been found to be a transcriptional activator to up-regulate c-myc gene transcription, a critical proto-oncogene for regulation of cell growth and differentiation. Therefore, down-regulation of c-myc transcription by inhibiting hnRNP K through disrupting its binding to c-myc gene promoter is a potential approach for cancer therapy. In the present study, we synthesized and screened a series of Quinoline derivatives and evaluated their binding affinity for hnRNP K. Among these derivatives, (E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (compound 25) was determined to be the first-reported hnRNP K binding ligand with its K_D values of 4.6 and 2.6 μM measured with SPR and MST, respectively. Subsequent evaluation showed that the binding of compound 25 to hnRNP K could disrupt its unfolding of c-myc promoter i-motif, resulting in down-regulation of c-myc transcription. Compound 25 showed a selective anti-proliferative effect on human cancer cell lines with IC₅₀ values ranged from 1.36 to 3.59 μM. Compound 25 exhibited good tumor growth inhibition in a Hela xenograft tumor model, which might be related to its binding with hnRNP K. These findings illustrated that inhibition of DNA-binding protein hnRNP K by compound 25 could be a new and selective strategy of regulating oncogene transcription instead of targeting promoter DNA secondary structures such as G-quadruplexes or i-motifs.

Full text of DOI:10.1016/j.bioorg.2018.12.020

Accepted Manuscript
Syntheses and Evaluation of New Quinoline Derivatives for Inhibition of
hnRNP K in Regulating Oncogene c-myc Transcription
Bing Shu, Ping Zeng, Shuangshuang Kang, Peng-Hui Li, Dexuan Hu, Guotao
Kuang, Jiaojiao Cao, Xiaoya Li, Meiling Zhang, Lin-Kun An, Zhi-Shu Huang,
Ding Li
PII:
S0045-2068(18)31200-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.020
YBIOO 2684
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 October 2018
13 December 2018
14 December 2018
Please cite this article as: B. Shu, P. Zeng, S. Kang, P-H. Li, D. Hu, G. Kuang, J. Cao, X. Li, M. Zhang, L-K. An,
Z-S. Huang, D. Li, Syntheses and Evaluation of New Quinoline Derivatives for Inhibition of hnRNP K in Regulating
Oncogene c-myc Transcription, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.020
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Syntheses and Evaluation of New Quinoline Derivatives for
Inhibition of hnRNP K in Regulating Oncogene c-myc Transcription
Bing Shua,b, Ping Zenga, Shuangshuang Kanga, Peng-Hui Lia, Dexuan Hua, Guotao
Kuanga, Jiaojiao Caoa, Xiaoya Lia, Meiling Zhanga, Lin-Kun Ana, Zhi-Shu Huanga, and Ding
Lia*
aSchool of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, 132
Waihuan East Road, Guangzhou 510006, P. R. China.
bSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
* Corresponding author E-mail address: liding@mail.sysu.edu.cn (D. Li).
Abstract
Aberrant overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a
key feature in oncogenesis and progression of many human cancers. hnRNP K has been found
to be a transcriptional activator to up-regulate c-myc gene transcription, a critical
proto-oncogene for regulation of cell growth and differentiation. Therefore, down-regulation
of c-myc transcription by inhibiting hnRNP K through disrupting its binding to c-myc gene
promoter is a potential approach for cancer therapy. In the present study, we synthesized and
screened a series of Quinoline derivatives and evaluated their binding affinity for hnRNP K.
Among
these
derivatives,
(E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (compound
25) was determined to be the first-reported hnRNP K binding ligand with its KD values of 4.6
and 2.6 μM measured with SPR and MST, respectively. Subsequent evaluation showed that
the binding of compound 25 to hnRNP K could disrupt its unfolding of c-myc promoter
i-motif, resulting in down-regulation of c-myc transcription. Compound 25 showed a selective
anti-proliferative effect on human cancer cell lines with IC50 values ranged from 1.36 to 3.59
μM. Compound 25 exhibited good tumor growth inhibition in a Hela xenograft tumor model,
which might be related to its binding with hnRNP K. These findings illustrated that inhibition
of DNA-binding protein hnRNP K by compound 25 could be a new and selective strategy of

regulating oncogene transcription instead of targeting promoter DNA secondary structures
such as G-quadruplexes or i-motifs.
KEY WORDS: hnRNP K; c-myc; Quinoline; i-motif; cancer

1. Introduction
The human c-myc protogene is an important regulator of a wide array of cellular
processes necessary for normal cell growth and differentiation, and its dysregulation is one of
the hallmarks of many cancers [1-3]. The aberrant overexpression of this gene is closely
related to the occurrence and development of various human cancers [4]. Therefore, the
strategy of suppressing c-myc transcription and expression in established cancer cell lines has
been investigated extensively [5-7]. It has been shown that the crystal structure of c-MYC
protein does not have an apparent ligand-binding cavity, which make it difficult to design
protein-binding ligands [8, 9], and therefore it becomes important to modulate c-myc
transcription through c-myc gene promoter. The G-quadruplex and i-motif formed in GC-rich
region of c-myc gene promoter have been shown to suppress c-myc gene transcription, and a
variety of ligands binding to and stabilizing these DNA secondary structures can regulate
gene transcription [10-14]. However, due to overwhelming abundance of these types of DNA
secondary structures in genomic sequence, the selective targeting by small molecule ligands is
a problem. Therefore, development of a new and more selective strategy of regulating
oncogene transcription instead of targeting widely existed DNA secondary structures such as
G-quadruplexes and i-motifs has become more and more important.
Heterogeneous nuclear ribonucleo protein K (hnRNP K) encodes a multifunctional
transcription factor [15-17] that plays a critical role in a broad range of cellular processes,
including the regulation of cell cycle progression, cell growth, differentiation, transformation,
angiogenesis, and apoptosis [15, 18, 19]. Besides, hnRNP K has been suggested to be related
with other transcription factors such as heat shock proteins [20]. It has been shown that the
overexpression of hnRNP K is associated with development of chronic myeloid leukemia,
prostate, colorectal and breast cancer possibly through activation of c-myc gene, indicating
hnRNP K as a potential drug target for cancer treatment [21]. The previous investigations
have implicated that hnRNP K could up-regulate c-myc gene transcription through its binding
interaction with C-rich sequence of c-myc gene promoter [16, 22]. It has been reported that
hnRNP K could recognize and bind to the 3′ lateral and central loops of the i-motif in the
promoter of c-myc gene leading to transcriptional activation [23, 24]. Dash et al have reported
that PBP1 could induce the formation of c-myc promoter i-motif resulting in down-regulation

of c-myc luciferase expression [14]. Recently, we have reported that acridone B19 could
induce the formation of c-myc promoter i-motif selectively without significant effect on its
corresponding G-quadruplex resulting in down-regulation of c-myc transcription [13]. The
existence of i-motifs in cells has recently been demonstrated by using antibodies, which might
play important roles as transcriptional regulators [25-27]. The above findings indicated that
hnRNP K could bind to oncogene promoter i-motifs and act as transcriptional activator.
Hence, disruption of the hnRNP K/i-motif interaction through specific binding of small
molecules to hnRNP K could provide a new and selective method for down-regulating
oncogene transcription instead of targeting widely-existed gene promoter i-motif structures in
cells [6, 7, 28].
It has been reported that Withanone and Withaferin A (Fig. 1) could inhibit cancer cells
growth, migration, angiogenesis and metastasis through targeting hnRNP K [29]. The
unsaturated ketones seem to be the pharmacophores, however the mechanism is uncertain
with only MD simulations. Xu group have reported that Nujiangexathone A (NJXA) could
reduce the expression of hnRNP K by accelerating ubiquitin-proteasome-dependent hnRNP K
degradation [30], however NJXA did not interact with hnRNP K directly. Several other
ligands have been designed based on the KH3 domain of hnRNP K, such as cytosine
derivative, thymine derivatives, and benzimidazole derivatives. These structures also contain
the unsaturated ketones or similar bioisosteres (Fig. 1) [21, 31], however these compounds
have not been shown to interact with hnRNP K. Therefore, discovery and development of
highly selective hnRNP K binding ligands are important to gain new insight for hnRNP K
regulated oncogene transcription.
In this study, we overexpressed and purified hnRNP K, which was able to bind with
C-rich sequence/i-motif of c-myc gene promoter. Then we screened our synthesized
compounds library in our laboratory (110 compounds, including acridone derivatives,
quinoline derivatives, acridine derivatives, imidazole derivatives, and indole derivatives, Fig.
S1) by using SPR. Among these compounds, we identified a Quinoline derivative,
(E)-1-(4-chlorophenyl)-3-(6-methoxy-4-morpholinoquinolin-2-yl)prop-2-en-1-one
(compound Q16, Fig. S1), with the binding affinity IC50 value of 20.1 μM as a hit compound.
In order to obtain stronger hnRNP K binding ligands and study the structure-activity

relationship, we designed and synthesized a series of Quinoline derivatives (07-37) with
various substitutive groups (Fig. S2). Then the binding affinity of these compounds for
hnRNP
K
was
evaluated,
and
(E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (compound
25) was found to bind tightly to hnRNP K. Our further in vitro and cellular experiments
suggested that compound 25 as the first determined hnRNP K binding ligand could bind to
hnRNP K and disrupt the interaction between hnRNP K and c-myc gene promoter i-motif.
This caused down-regulation of c-myc transcription and translation, resulting in tumor cells
proliferation inhibition and apoptosis. These results could provide a new and selective
strategy of regulating oncogene transcription instead of targeting DNA secondary structures
such as G-quadruplexes or i-motifs.
2. Results and discussion
2.1 Chemistry
The synthetic pathway for Quinoline derivatives 07-37 was shown in Scheme 1.
4-Substituted anilines (1a-d) were used as the starting materials to react with ethyl
acetoacetate to give 2-methyl-6-substituted-quinolin-4-ol (2a-d), which were then reacted
with phosphorus oxychloride immediately to generate Quinoline derivatives 3a-d. Quinoline
derivatives 4a-d were prepared through reaction of 3a-d with Morpholine solution catalyzed
with Ts-OH. The products were used directly for the next step reaction without purification.
Compounds 4a-d reacted with selenium dioxide through Riley oxidation reaction to give key
compounds 5a-d. Finally, the target Quinoline derivatives 07-37 were obtained through
aldol reaction between aldehyde 5a-d and 1-(4-substituted phenyl)ethanone (6a-f) catalyzed
with H2SO4 in AcOH solution or in EtOH solution containing 10% NaOH. Under various
reaction conditions, we obtained Quinoline derivatives with hydroxyl substitutive group
(31-33). Compound 5b reacted with 3-morpholinopropan-1-amine to give a propylamine
derivative (34). Methylation derivative 35 was obtained with compound 07 as the starting
material stirred in methyl iodide solution. Lastly, compound 12 was reduced under hydrogen
atmosphere to give derivatives 36-37. The synthesized Quinoline derivatives were purified,

and their structures were determined by using HPLC, NMR and high resolution mass spectra.
2.2 Cloning, overexpression in E. coli, purification and characterization of hnRNP K
hnRNP K has been cloned, overexpressed in E. coli and purified by using affinity
chromatography methods, which has been identified by using SDS gel electrophoresis as
described previously [32, 33]. In the present study, hnRNP K was purified to apparent
homogeneity based on SDS–PAGE as shown in Fig. S3a, which was confirmed by using
Western blot as shown in Fig. S3b. It has been previously reported that hnRNP K specifically
bound to a single-stranded cis-element comprised of five imperfect CCCT repeats
(CCCTCCCCA) [34]. To confirm the binding activity of our purified hnRNP K protein,
electrophoretic mobility shift assay (EMSA) was used to study the interaction between
hnRNP K and single-stranded CCCT repeat DNA. As shown in Fig. S3c, when increasing the
concentration of hnRNP K in a mixture of protein and single-stranded CCCT repeat DNA, the
concentration of protein-bound DNA increased, which was consistent with that reported
previously [35], and indicated that the purified hnRNP K was good enough for further
biophysical and biochemical studies.
2.3 hnRNP K could unfold the i-motif of c-myc NHE Ⅲ
1
The nuclease hypersensitive element III1 (NHE III1), located at upstream from the P1
promoter of c-myc gene, has controlled 85-90% of c-myc transcription [4, 10]. As mentioned
above, hnRNP K selectively bound to single-stranded CCCT repeat DNA. There is
convincing evidence that this protein has an important role in the control of c-myc gene
expression through binding to the C-rich strand of DNA [36]. In the present study, we used a
33-bp single strand DNA (Table S1) probe in c-myc NHE III1 domain, containing repeats of
known hnRNP K recognition motif (5′-dTCCC) with a single fluorescent dye labeled to the
5′-end of the forward strand [34]. Previously, the hnRNP K KH3 domain has been identified
as the minimal component of the canonical hnRNP-K-DNA binding mode [37]. The
dissociation equilibrium constant for the KH3 form of the protein to its 30 bp DNA
(5′-CTCTCCTTTCTTTTTCTTCTTCCCTCCCTA-3′) calculated from a steady state analysis
has been determined to be KD = 1.5 μM by using SPR experiment [35]. In our present study,

EMSA was used to determine the binding between the full length hnRNP K and the c-myc
DNA probe. Our result showed that the hnRNP K associated with the DNA probe in a
dose-dependent manner with KD of 1.9 μM as shown in Fig. 2a-b, which was consistent with
that reported previously. This result was also supported by the data from our filter binding
assay. As shown in Fig. 2c, when increasing the concentration of hnRNP K, the protein-bound
DNA was also increased. Besides, it has been shown that C-rich strand in c-myc promoter
could form an i-motif structure and affect gene transcription [38, 39]. However, it was not
clear whether hnRNP K could interact with c-myc promoter i-motif or not. In the present
research, we studied the interaction of hnRNP K with c-myc promoter i-motif through circular
dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET). The CD
spectrum of Py33 sequence showed a positive peak near 288 nm and a negative peak near 260
nm in a buffer at pH 6.0, indicating the formation of i-motif structure, as shown in Fig. S4a.
After one equivalent of hnRNP K was added, the above two peaks were both decreased with
the peak at 288 nm shifted toward 276 nm, which were similar to those reported previously by
Qu group and Hurley group, indicating that hnRNP K could unfold the i-motif into an
unordered random coil [40-43]. The unwinding activity of hnRNP K for i-motif was also
confirmed by using FRET assay, as shown in Fig. S4b. The fluorescence intensity at 580 nm
was decreased upon addition of increasing concentration of hnRNP K. All above results
showed that our hnRNP K could unfold the c-myc promoter i-motif into an unordered random
coil in a dose-dependent manner, which could be further used for screening of protein
inhibitors.
2.4 Binding studies of the Quinoline derivatives for hnRNP K
The binding affinities of the synthesized compounds for the hnRNP K were studied
through surface plasmon resonance (SPR) experiments [44, 45], and the dissociation
constants (KD) were determined as shown in Table 1. Quinoline derivatives (07-30) showed
good binding affinity for hnRNP K, especially the derivatives with resonance electron
donating R1 substitutive groups, such as -OCH3, -H, -F, -OH， and –OBn (compounds 7-9,
11-15, 17-21, 23-27, and 29-30), had their KD values ranged from 4.6 to 25.7 μM. In contrast,
the derivatives with resonance electron withdrawing R1 substitutive groups, such as -NO2

(compounds 10, 16, 22, and 28), showed no significant binding with hnRNP K upon addition
of up to 30 μM ligands. In comparison, R substitutive groups seem to have less significant
effect on the binding affinity. The compounds 7, 13, 19, and 25, with R substitutive groups of
-OCH3, -H, -F, and -NO2, had their KD values of 7.2, 9.7, 6.8, and 4.6, respectively. These
results indicated that R1 substituent is important to maintain the binding affinity. We also
modified the unsaturated ketone in the middle of Quinoline derivatives to generate
hydroxylation derivatives (31-33), propylamine derivative (34), and reduced derivatives
(36-37), however, all these derivatives had significantly reduced binding affinity, indicating
that the unsaturated ketone is important for the binding affinity. The methylation derivative 35
also showed significantly reduced binding affinity, possibly because the methylation could
affect the charge distribution of the Quinoline ring. Among our synthetic compounds,
compound 25 exhibited the best binding affinity (4.6 μM, Table 1 and Figure S5) and was
much better than the hit compound Q16 (20.1 μM).
In order to confirm the binding affinity of compound 25 to hnRNP K, microscale
thermophoresis (MST) experiment was performed, since it is a sensitive method for
quantitative analysis of molecular interactions [46]. As shown in Fig. 3a, our results showed
that compound 25 could bind to hnRNP K with KD value of 2.6 μM, which was consistent
with our above SPR result. In order to know whether compound 25 could bind to i-motif
DNA or duplex DNA, we also studied their binding interactions through MST. As shown in
Fig. S6a-b, binding affinities of compound 25 to i-motif DNA and duplex DNA were found to
be much weak, which could be ignored compared with its binding affinity to hnRNP K. These
results indicated that compound 25 could selectively bind to hnRNP K without significant
interaction with c-myc promoter DNA.
2.5 Quinoline derivatives inhibited various carcinoma cells proliferation
We employed MTT assay to evaluate the anti-proliferation activities of the Quinoline
derivatives on various human cancer cell lines, such as human cervical cancer cell line Siha,
human lung adenocarcinoma cell line A549, human squamous cervical cancer line Hela,
human bone osteosarcoma epithelial cell line U2OS, human melanoma cell line A375, human
hepatocellular carcinoma cell line HuH7. Human embryonic kidney cell line HEK293 was

used as a normal cell control (low expression of hnRNP K). The cytotoxicity of the Quinoline
derivatives was determined with their IC50 values as shown in Table 2. Most of the Quinoline
derivatives 07-30 showed diverse cytotoxicity against various tumor cells. The derivatives
with resonance electron donating R1 substitutive groups, such as -OCH3 (7, 13, 19, and 25)
and -OH (11, 17, 23, and 29), showed generally strong cytotoxicity against most types of
cancer cells but weak cytotoxicity against HEK293, indicating that introduction of electron
donating group into the R1 position could increase the selectivity against tumor cells. In
comparison, R substitutive groups seem to have less significant effect on the selectivity. The
compounds 7, 13, 19, and 25, with R substitutive group of -OCH3, -H, -F, and -NO2, showed
similar cytotoxicity against cancer cells. The hydroxylation derivatives (31-33), propylamine
derivative (34), methylation derivative (35), and reduced derivatives (36-37), all showed very
weak cytotoxicity against cancer cells, indicating that unsaturated ketone structure could be
important for proliferation inhibition activity. Generally, the structure-activity relationship for
the Quinoline derivatives against cancer cells was well consistent with that for inhibition of
hnRNP K measured through SPR. Compound 25 is among the best of these compounds
inhibited the proliferation of various carcinoma cell lines, with its IC50 values ranged from
1.36 to 3.59 μM. Our result showed that compound 25 had very low cytotoxicity against
HEK293 normal cells (Table 2). These results indicated that compound 25 had potent and
selective cytotoxicity against tumor cells possibly through inhibition of hnRNP K. As
mentioned before, compound 25 had the best binding affinity (4.6 μM, Table 1) to hnRNP K,
and therefore, was selected for further in-depth investigation.
2.6 The binding of compound 25 to hnRNP K disrupted the interaction between hnRNP K and
c-myc gene promoter i-motif in vitro and in cells
As mentioned above, compound 25 appeared to be the most potent binding ligand to
hnRNP K. In order to understand its effect on the binding interaction between hnRNP K and
c-myc gene promoter i-motif, we performed several other experiments, including
electrophoretic mobility shift assay (EMSA), filter binding assay, FRET assay, and Chromatin
immunoprecipitation experiment (ChIP).
Firstly we used EMSA to study the effect of compound 25 on the binding interaction

between hnRNP K and c-myc gene promoter. As shown in Fig. 3b, in the absence of
compound 25 and hnRNP K, only free i-motif DNA appeared on the gel. Upon addition of
hnRNP K, free DNA was depleted and the hnRNP K-DNA complex appeared on the gel.
When increasing the concentration of compound 25, the amount of the hnRNP K-DNA
complex decreased, and free i-motif DNA increased, which indicated that the binding of
compound 25 to hnRNP K could disrupt the binding interaction between hnRNP K and c-myc
gene promoter i-motif. These results were also consistent with our filter binding assay data, as
shown in Fig. 3c. Upon addition of increasing concentration of compound 25, the amount of
the hnRNP K-DNA complex gradually decreased, while the corresponding free i-motif DNA
increased, indicating that the binding of compound 25 to hnRNP K could disrupt the binding
interaction between hnRNP K and c-myc gene promoter i-motif in a dose-dependent manner.
As mentioned above, our hnRNP K could unfold c-myc promoter i-motif as studied by
using CD and FRET assay. In the present investigation, FRET assay was used to study
whether compound 25 could affect hnRNP K’s unwinding function for c-myc promoter
i-motif. As shown in Fig. S7, hnRNP K could unfold c-myc promoter i-motif, as indicated by
decreased fluorescence intensity at 580 nm. After compound 25 was added, the unwinding
activity of hnRNP K for i-motif was suppressed, as indicated by increased fluorescence
intensity at 580 nm. These results suggested that the binding of compound 25 to hnRNP K
could disrupt the interaction between hnRNP K and c-myc promoter i-motif.
Next, we performed ChIP experiment [45] in Hela cervical cancer cells to study whether
compound 25 could inhibit the binding of hnRNP K to c-myc promoter DNA with a Magna
ChIP™ Kit (Millipore) following the manufacturer's protocol. As shown in Fig. 3d, after
incubation with 1.25 and 2.5 μM compound 25 for 24 h, we found that compound 25 could
cause dose-dependent dissociation of hnRNP K from c-myc promoter DNA. Amplification of
these immunoprecipitated samples showed a decrease of hnRNP K-bound DNA with
increasing concentration of compound 25 in a dose dependent manner, indicating that
compound 25 indeed disrupted the binding of hnRNP K with c-myc promoter DNA in Hela
cells. All above results showed that the binding of compound 25 to hnRNP K could disrupt
the interaction between hnRNP K and c-myc promoter DNA in vitro and in cells.

2.7 Molecular docking studies for the effect of compound 25 on the binding of hnRNP K with
c-myc gene promoter NHE Ⅲ
1
It has been known that the full length hnRNP K contains three KH domains (KH1, KH2
and KH3), which are closely related to the biological function of hnRNP K protein in
recognizing RNA and DNA. KH3 domain has shown the strongest binding affinity to C-rich
single-stranded DNA. Only crystal structure of the KH3 domain responsible for binding to
C-rich single-stranded DNA is available for docking study. A molecular docking study was
performed to investigate the binding mode and to help understand the bioactivity results. The
mode was derived by docking and energy minimization of compound 25 in the ssDNA/RNA
binding site of the KH3_hnRNP K (PDB ID: 1ZZI) [47]. The energy-minimized, top-ranked
pose of compound 25 in the protein was displayed in Fig. S8a-b. Compound 25 was found to
bind well with ssDNA-binding site, which interfered with the binding of hnRNP K to ssDNA.
As shown in Fig. S8b, compound 25 was found to interact with residues Asp23, Ala25, Gly26,
Ser27, Ile28, Lys31, Gly33, Gln34, Gln83, and Ser85 of KH3 hnRNP K protein, resulting in
disrupting the binding of the protein to ssDNA. The ketone carbonyl group of compound 25
was found to have two hydrogen bonding interactions with Gln83 and Ser85 with distances of
3.3 Å and 3.1 Å, respectively. The methoxy group of compound 25 was found to bind to a
narrow site of the protein as shown in Fig. S8a-b, which had a hydrogen bonding interaction
with Gln34 with a distance of 3.0 Å. In contrast, the electron-withdrawing nitryl group on the
other side of compound 25 had no strong binding interaction with the protein. These docking
results are well consistent with our experimental results, which explained the importance of
the ketone carbonyl group and methoxy substitutive group. It should be noted that the ketone
carbonyl group is conjugated with the Quinoline ring and surrounded by several rings, which
should make it electronically and sterically unfavorible to become Michael acceptor attacked
by general nucleophiles. These docking results could be useful for further optimization of
compound 25 and comparison with previous docking data of other compounds.
2.8 Compound 25 down-regulated c-myc gene transcription and translation
c-myc proto-oncogene encodes a multifunctional transcription factor that plays a critical

role in a broad range of cellular processes, including the regulation of cell cycle progression,
cell growth, differentiation, transformation, angiogenesis, and apoptosis [2, 48, 49].
Dysregulation of c-myc could arise through a variety of mechanisms, including chromosomal
translocation [50], gene amplification [51], and increased transcription [1] as well as a higher
rate of translation and enhanced protein stability [17]. Since hnRNP K has been shown to be a
positive-acting transcription factor for human c-myc gene, we further studied whether the
effect of compound 25 on hnRNP K could influence c-myc gene transcription. Considering
hnRNP K showed higher level of expression in Hela cells [52], we chose Hela cells to study
the effect of compound 25 on c-myc gene transcription and expression through RT-PCR and
Western blot. After incubation with varying concentration of compound 25 for 3 h, the total
RNA was extracted with a RNA extraction kit and reversely transcripted to cDNA. The cDNA
was then used as a template for quantitative PCR amplification of the c-myc gene, with
β-actin as a control. As shown in Fig. 4a-b, the incubation with increasing concentration of
compound 25 caused a reduction of c-myc mRNA in a dose-dependent manner in Hela cells.
Western blot was also carried out to measure c-MYC protein expression levels, as shown in
Fig. 4c-d. Upon incubation with increasing concentration of compound 25, the expression
levels of c-MYC also decreased in a dose-dependent manner. These results indicated that
compound 25 could down-regulate c-myc gene transcription and expression in Hela cells in a
dose-dependent manner possibly through its inhibition of hnRNP K.
2.9 Effect of compound 25 on Hela cells apoptosis and apoptosis-related protein expressions
It has been previously reported that a decreasing expression of c-MYC may induce
cellular apoptosis of proliferating cancer cells. As our above studies showed that compound
25 could bind to hnRNP K and down-regulate c-myc transcription and expression, we
analyzed Hela cells apoptosis upon incubation with compound 25. As shown in Fig. 5a-b,
upon incubation of Hela cells with compound 25 at increasing concentration of 0, 1.25, 2.5,
and 5.0 μM for 24 h, both early and late cell apoptosis were induced. The percentages of early
apoptosis cells were 1.06% 12.72%, 29.30%, and 48.49%, while the percentages of late
apoptosis cells were 0.33%, 2.74%, 5.59%, and 6.29%, respectively. Besides, we also
investigated the expression of proteins in the cell apoptosis regulation pathway through

Western blot after incubation with increasing concentration of compound 25. As shown in Fig.
5c, dose-dependent increases in apoptosis related proteins, such as cleaved caspase-3 and
cleaved PARP, were observed. Compound 25 down-regulated the expression of caspase-3 and
PARP, which were related to cellular apoptosis process. Our above results of Hela cells
apoptosis induced by compound 25 could be due to its repression of c-myc transcription,
which are well consistent with previously reported results.
2.10 Compound 25 could inhibit tumor cells proliferation, migration and invasion
hnRNP K has been shown to be closely involved in metastasis and related with
gallbladder carcinoma, prostate cancer, and colorectal cancer metastasis [52, 53]. hnRNP K
regulates metastasis in vivo by regulation of extra-cellular matrix components through the
ERK signaling pathway [17]. As mentioned above, compound 25 could have significant
inhibitory effect on the proliferation of Hela cells in short-term proliferation with an IC50
value of 2.21 μM. The inhibitory effect of compound 25 on cell proliferation was further
investigated through colony formation assay, scrape assay, and transwell assay, in order to
study its inhibition of tumor cells long-term proliferation, metastasis, and invasion,
respectively. As shown in Fig. S9a, upon incubation with compound 25, colony formation
obviously decreased, suggesting inhibition of cell proliferation, which was consistent with our
MTT results. In comparison, compound 25 had no significant effect on HEK293 cells (normal
cells), as shown in Fig. S9b. Our results in scrape assay indicated that compound 25 could
inhibit metastasis in a dose-dependent manner as shown in Fig. 6a. Next, the Hela cells were
incubated with 1.25 μM compound 25 for 24 hours, and the cells migration was reduced for
approximately 68% as shown in Fig. 6b. This result was in good agreement with that of the
cell scratch assay, indicating that compound 25 could inhibit Hela cells invasion. The above
results suggested that compound 25 could inhibit proliferation, metastasis, and invasion of
cancer cells, which could be due to its repression of c-myc transcription.
2.11 Compound 25 inhibited tumor growth in a Hela cervical xenograft
To further demonstrate that compound 25 as a hnRNP K inhibitor could be useful for
cancer treatment, we tested compound 25 in a Hela xenograft mouse model of human cervical

cancer. When the tumor size reached approximately 100 mm3, the mice were randomly
divided into four groups (seven mice per group) for intraperitoneal injection (ip) daily
including: the vehicle group, compound 25 6.7 mg/kg treated group, compound 25 20.0
mg/kg treated group, and Cisplatin 1.0 mg/kg treated group [29, 30]. The tumors were
collected after 3 weeks of treatment and analyzed. As shown in Fig. 7a-b and Fig. S10a-b,
compared with the vehicle group (mean of 1000.1 mg), the treatment with compound 25 at
6.7 mg/kg and 20.0 mg/kg resulted in a statistically significant reduction in tumor weight with
a tumor growth inhibition rate (IR) of 54.1% and 69.3%, respectively (mean values of 459.7
mg and 307.4 mg, respectively). The wide-spectrum antitumor drug Cisplatin was used as a
positive control (IR = 83.2%, mean, 168.2 mg). Meanwhile, the results of tumor weight from
each group were consistent with results of tumor volume. The treatment with compound 25 at
6.7 mg/kg and 20.0 mg/kg resulted in a significant decrease in the final tumor volume (means
of 474.1 mm3 and 326.3 mm3, respectively) compared to the vehicle group (mean of 1114.9
mm3, Fig. 7a). Our data showed that the inhibition of tumor growth after treatment with
compound 25 was in a time-dependent manner. As shown in Fig. 7c, no significant
differences in body weights were observed among the vehicle group, compound 25 treated
groups, and Cisplatin treated group. Compound 25 neither altered cellular morphology nor
induced pathological changes in the heart, liver, spleen, and kidney (Fig. 8d and Fig. S10c),
indicating that it was tolerated well at these doses. These data showed that compound 25
exhibited a good antitumor activity for the inhibition of Hela cervical cancer growth in
BALB/C-nu/nu mice with Hela xenografts through the suppression of proliferation in vivo.
3. Conclusion
In this study, we synthesized a series of new Quinoline derivatives and evaluated their
binding affinity for hnRNP K. Among these derivatives, we found that compound 25 could
bind to hnRNP K with the KD value determined to be 4.6 and 2.6 μM by using SPR and MST,
respectively. The binding of compound 25 to hnRNP K could disrupt its interaction with
c-myc promoter i-motif and down-regulate c-myc gene expression. Compound 25 could inbit
the proliferation of various human cancer cell lines with IC50 values ranged from 1.36 to 3.59
μM. Compound 25 could induce Hela cells apoptosis, and inhibit tumor cells long-term

proliferation, migration and invasion. Compound 25 exhibited good tumor growth inhibition
in a Hela xenograft tumor model with a statistically significant reduction of tumor weight,
which might be related to its ability of binding to hnRNP K. As the first binding ligand for
protein of hnRNPs family, compound 25 might be further modified for discovery of new
binding ligands for other proteins of hnRNPs family. It should be noted that DNA secondary
structures including G-quadruplexes and i-motifs have been widely characterized and
believed to play key roles as transcriptional repressors, and a variety of ligands binding to and
stabilizing G-quadruplexes and i-motifs have caused the suppression of gene transcription and
expression. However, due to wide abundance of these types of DNA secondary structures in
genomic sequence, the selective targeting by small molecule ligands is a potential problem.
The overexpression of hnRNP K is associated with development of various cancers, and
tumor cells rely on hnRNP K to turn on or up-regulate certain gene transcriptions partly
through its binding with promoter C-rich sequences/i-motifs. hnRNP K could have different
binding affinity and selectivity to various C-rich sequences/i-motifs in transcriptional
regulation, therefore, our present study for inhibition of hnRNP K by small molecule ligands
could offer a potentially new and more selective strategy of regulating oncogene transcription
by taking advantage of the selectivity of hnRNP K for different C-rich sequences/i-motifs in
gene promoter.
4. Materials and Methods
4.1 General Method for Synthesis
All chemicals were purchased from commercial sources, which were analytical grade
without further purification unless otherwise specified. All synthesized compounds were
1
13
1
confirmed by using H, C NMR spectra and HRMS spectrometry. H and 13C NMR spectra
were recorded using TMS as the internal standard in DMSO-d6, CD3OD, or CDCl3 with a
Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. High resolution mass
spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF of MAT95XP mass
spectrometer (Thermo Fisher Scientific, USA). The purity of the synthesized compounds was

confirmed to be higher than 95% by using analytical HPLC performed with a dual pump
Shimadzu LC-20 AB system equipped with an Ultimate XB-C18 column (4.6 mm × 250 mm,
5 μm), eluting with methanol-water (10:90 to 60:40) containing 0.05% TFA at a flow rate of
0.5 mL/min. The synthetic pathway for Quinoline derivatives 07-37 was shown in Scheme 1.
4.1.1 General procedure for the preparation of 2-methyl-6-substituted-quinolin-4-ol (2a-d)
To a solution of 4-substituted aniline (1a-d, 72.4 mmol) in PPA (150 g), was added ethyl
o
acetoacetate (18.8 g, 14.5 mmol) dropwise. The reaction mixture was stirred at 120 C until
o
the 1a-d was consumed monitored with TLC. After cooling down to 70 C, the mixture was
poured into ice water and stirred, with pH adjusted to 9-10 by adding ammonium hydroxide.
Then the mixture was filtered, and the solid was washed with water for three times and dried
under reduced pressure to give the crude 2a-d, which was used directly for the next step
reaction without any purification.
4.1.2 General procedure for the preparation of 4-chloro-2-methyl-6-substituted-quinoline
(3a-d)
To a solution of 2a-d (4.0 g, crude) in dioxane (60 mL), was added POCl3 (10 mL)
dropwise at 0-5 oC, followed with DMF (0.3 mL). The mixture was then heated to 100 oC, and
reacted for 4h. After cooling down to room temperature, the solvent was removed, and the
residue was poured into ice water with stirring. After addition of ethyl acetate (50 mL), pH
was adjusted to 9-10 by adding sodium bicarbonate, the mixture was filtered and the organic
phase was extracted with ethyl acetate (20 mL × 3). The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate, filtered, and then concentrated under
reduced pressure. The residue was purified by using silica gel chromatograph with petroleum
ether / ethyl acetate (10/1 - 3/1) to give the desire compound 3a-d with yield of 45-57 % for
two steps.
4.1.2.1 4-chloro-6-methoxy-2-methylquinoline (3a). The compound was obtained as a yellow
solid with a yield of 57%. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 5.0 Hz, 1H), 7.57 (d, J =
3.1 Hz, 1H), 7.34 (dd, J = 11.0, 2.9 Hz, 1H), 7.13 (s, 1H), 3.81 (s, 3H), 2.68 (s, 3H). ESI-MS

(m/z) 208 [M + H]+.
4.1.2.2 4-chloro-2-methylquinoline (3b). The compound was obtained as a light yellow solid
1
with a yield of 51%. H NMR (400 MHz, CDCl3) δ 8.13 (dd, J = 7.5, 1.6 Hz, 1H), 8.05 (dd, J
= 7.4, 1.5 Hz, 1H), 7.71 – 7.62 (m, 1H), 7.62 – 7.55 (m, 1H), 7.22 (s, 1H), 2.68 (s, 3H).
ESI-MS (m/z) 178 [M + H]+.
4.1.2.3 4-chloro-6-fluoro-2-methylquinoline (3c). The compound was obtained as a light
1
yellow solid with a yield of 54%. H NMR (500 MHz, CDCl3) δ 8.04 (dd, J = 7.5, 5.0 Hz,
1H), 7.74 (dd, J = 7.9, 1.4 Hz, 1H), 7.46 – 7.33 (m, 1H), 7.20 (s, 1H), 2.68 (s, 3H). ESI-MS
(m/z) 196 [M + H]+.
4.1.2.4 4-chloro-2-methyl-6-nitroquinoline (3d). The compound was obtained as a yellow
solid with a yield of 45%. 1H NMR (400 MHz, CDCl3) δ 9.11 (t, J = 3.1 Hz, 1H), 8.49 (dt, J =
9.2, 2.5 Hz, 1H), 8.14 (t, J = 4.6 Hz, 1H), 7.55 (s, 1H), 2.79 (s, 3H). ESI-MS (m/z) 223 [M +
H]+.
4.1.3
General
procedure
for
the
preparation
of
4-(2-methyl-6-substituted-quinolin-4-yl)morpholine (4a-d)
To a solution of 3a-d (2.25 mmol) in morpholine (2 mL), was added
o
4-methylbenzenesulfonic acid (Ts-OH, 50.0 mg). The reaction mixture was stirred at 120 C
for 3h until the TLC showed complete reaction. After cooling down, the mixture was powered
into ice water and stirred. The organic layer was extracted with ethyl acetate (3 × 10 mL). The
combined organic layer was washed with brine for three times, dried over anhydrous sodium
sulfate, filtered, concentrated and the residue was purified by using silica gel chromatograph
with petroleum ether / ethyl acetate (4/1 - 1/1) to give compound 4a-d with yield of 75-82%.
4.1.3.1 4-(6-methoxy-2-methylquinolin-4-yl)morpholine (4a). The compound was obtained as
1
a light yellow solid with a yield of 80%. H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.9 Hz,
1H), 7.30 (d, J = 9.2 Hz, 1H), 7.27 (s, 1H), 6.75 (s, 1H), 4.03 – 3.96 (m, 4H), 3.92 (s, 3H),
3.23 – 3.15 (m, 4H), 2.67 (s, 3H). ESI-MS (m/z) 259 [M + H]+.

4.1.3.2 4-(2-methylquinolin-4-yl)morpholine (4b). The compound was obtained as a yellow
solid with a yield of 80%. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 8.3 Hz, 1H), 7.98 (d, J =
8.3, 1H), 7.68 – 7.36 (m, 2H), 7.08 (s, 1H), 3.71 (t, J = 9.2 Hz, 4H), 3.17 (t, J = 9.3 Hz, 4H),
2.52 (s, 3H). ESI-MS (m/z) 229 [M + H]+.
4.1.3.3 4-(6-fluoro-2-methylquinolin-4-yl)morpholine (4c). The compound was obtained as a
1
light yellow solid with a yield of 82%. H NMR (400 MHz, CDCl3) δ 7.99 (dd, J = 11.9, 5.0
Hz, 1H), 7.61 (dd, J = 11.0, 5.3 Hz, 1H), 7.52 – 7.26 (m, 1H), 7.10 (s, 1H), 3.72 (t, J = 8.4 Hz,
4H), 3.18 (t, J = 8.4 Hz, 4H), 2.53 (s, 3H). ESI-MS (m/z) 247 [M + H]+.
4.1.3.4 4-(2-methyl-6-nitroquinolin-4-yl)morpholine (4d). The compound was obtained as a
1
yellow solid with a yield of 75%. H NMR (400 MHz, CDCl3) δ 8.91 (d, J = 2.6 Hz, 1H),
8.38 (dt, J = 5.7, 2.8 Hz, 1H), 8.05 (dd, J = 9.2, 5.0 Hz, 1H), 6.86 (s, 1H), 4.04 (t, J = 9.2 Hz,
4H), 3.27 (t, J = 8.8 Hz, 4H), 2.73 (s, 3H). ESI-MS (m/z) 274 [M + H]+.
4.1.4
General
procedure
for
the
preparation
of
4-morpholino-6-substituted-quinoline-2-carbaldehyde (5a-d)
To a solution of 4a-d (1.47 mmol) in dioxane (10 mL), was added selenium dioxide (244
o
mg, 2.20 mmol). The reaction mixture was stirred at 100 C for 3h. After cooling down to
room temperature, the mixture was filtered, concentrated and the residue was purified by
using silica gel chromatograph with petroleum ether / ethyl acetate (6/1 - 2/1) to give
compound 5a-d with yield of 61-78%.
4.1.4.1 6-methoxy-4-morpholinoquinoline-2-carbaldehyde (5a). The compound was obtained
1
as a light yellow solid with a yield of 78%. H NMR (400 MHz, CDCl3) δ 10.12 (s, 1H), 8.12
(d, J = 9.2 Hz, 1H), 7.51 (s, 1H), 7.43 (dd, J = 9.2, 2.8 Hz, 1H), 7.33 (d, J = 2.8 Hz, 1H), 4.00
(d, J = 4.6 Hz, 4H), 3.98 (s, 3H), 3.26 (d, J = 4.6 Hz, 4H). ESI-MS (m/z) 273 [M + H]+.
4.1.4.2 4-morpholinoquinoline-2-carbaldehyde (5b). The compound was obtained as a light
1
yellow solid with a yield of 70%. H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 8.21 (d, J =
8.4 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.80 – 7.73 (m, 1H), 7.66 – 7.60 (m, 1H), 7.49 (s, 1H),

4.01 (d, J = 4.6 Hz, 4H), 3.30 (d, J = 4.6 Hz, 4H). ESI-MS (m/z) 243 [M + H]+.
4.1.4.3 6-fluoro-4-morpholinoquinoline-2-carbaldehyde (5c). The compound was obtained as
1
a yellow solid with a yield of 69%. H NMR (400 MHz, CDCl3) δ 10.09 (s, 1H), 8.25 (d, J =
8.4 Hz, 1H), 7.89 (d, J = 9.2 Hz, 1H), 7.80 – 7.73 (m, 1H), 7.62 – 7.44 (m, 1H), 7.30 (s, 1H),
4.01 (t, J = 5.8 Hz, 4H), 3.30 (t, J = 5.8 Hz, 4H). ESI-MS (m/z) 261 [M + H]+.
4.1.4.4 4-morpholino-6-nitroquinoline-2-carbaldehyde (5d). The compound was obtained as a
1
yellow solid with a yield of 61%. H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H), 9.00 (d, J =
2.5 Hz, 1H), 8.53 (dd, J = 9.2, 2.5 Hz, 1H), 8.43 – 8.27 (m, 1H), 7.57 (s, 1H), 4.07 (t, J = 5.6
Hz, 4H), 3.41 (t, J = 5.4 Hz, 4H). ESI-MS (m/z) 288 [M + H]+.
4.1.5
General
procedure
for
the
preparation
of
(E)-1-(4-substituted-phenyl)-3-(4-morpholino-6-substituted-quinolin-2-yl)prop-2-en-1-ones
(7-11, 13-17, 19-23, and 25-29)
To a solution of 1-(4-substituted-phenyl)ethanone (6a-e, 1.84 mmol, 1.2 equiv) in 10 mL
AcOH, was added aldehyde (5a-d, 1.54 mmol) with stirring slowly. After cooling down to 5
oC, concentrated H2SO4 (0.3 mL) was added slowly and carefully. The mixture was heated to
90 oC and reacted overnight. After cooling down to room temperature, the mixture was poured
into ice water with stirring, and pH was adjusted to 9-10 by adding sodium carbonate. The
solution was filtered, and the solid was washed with saturated sodium bicarbonate, dried and
purified by using silica gel chromatograph with dichloromethane/MeOH (100/1 - 30/1) to
give desire Quinoline derivative (7-11, 13-17, 19-23 and 25-29).
4.1.6
General
procedure
for
the
preparation
of
(E)-1-(4-(benzyloxy)phenyl)-3-(4-morpholino-6-substituted-quinolin-2-yl)prop-2-en-1-ones
(12, 18, 24 and 30)
To a solution of 1-(4-(benzyloxy)phenyl)ethanone (6f, 416 mg, 1.84 mmol, 1.2 equiv) in
o
10 mL EtOH, was added aldehyde (5a-d, 1.54 mmol) at 5 C. The reaction mixture was

stirred until TLC indicated complete reaction. The solution was filtered under reduced
pressure, and the solid was washed with cold EtOH (3×2 mL), dried and recrystallized in
EtOH to give desired compound (12, 18, 24, and 30).
4.1.6.1 (E)-3-(6-methoxy-4-morpholinoquinolin-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one
(7). Compound 5a was reacted with 1-(4-methoxyphenyl)ethanone (6a) according to general
1
procedure to afford compound 7 as a yellow solid with a yield of 75%. H NMR (400 MHz,
DMSO) δ 8.23 (d, J = 15.6 Hz, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 9.2 Hz, 1H), 7.76 (d,
J = 15.6 Hz, 1H), 7.61 (s, 1H), 7.41 (dd, J = 9.2, 2.7 Hz, 1H), 7.28 (d, J = 2.7 Hz, 1H), 7.13 (d,
13
J = 8.8 Hz, 2H), 3.97 – 3.92 (m, 7H), 3.89 (s, 3H), 3.24 (t, J = 4.0 Hz, 4H). C NMR (101
MHz, DMSO) δ 188.08, 163.88, 157.85, 156.20, 152.20, 145.29, 144.15, 132.18, 131.52,
130.72, 125.91, 124.28, 122.17, 114.61, 109.69, 102.55, 66.68, 56.07, 55.87, 52.46. Purity
was determined to be 99.7% by using HPLC. HRMS (ESI; m/z) calcd for C24H24N2O4, [M
+ H]+, 405.1764; found 405.1831.
4.1.6.2
(E)-3-(6-methoxy-4-morpholinoquinolin-2-yl)-1-phenylprop-2-en-1-one
(8).
Compound 5a was reacted with acetophenone (6b) according to general procedure to afford
compound 8 as a pale yellow solid with a yield of 61%. 1H NMR (400 MHz, CDCl3) δ 8.11 (d,
J = 7.4 Hz, 2H), 8.04 (d, J = 9.8 Hz, 1H), 7.87 (d, J = 15.5 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H),
7.54 (d, J = 7.7 Hz, 2H), 7.51 (s, 1H), 7.38 (dd, J = 9.2, 2.8 Hz, 1H), 7.29 (d, J = 2.8 Hz, 1H),
13
7.13 (s, 1H), 4.02 (d, J = 4.6 Hz, 4H), 3.96 (s, 1H), 3.26 (d, J = 4.6 Hz, 4H). C NMR (101
MHz, CDCl3) δ 190.87, 157.98, 151.53, 144.43, 137.96, 132.98, 132.17, 128.82, 128.67,
126.07, 126.00, 124.39, 121.94, 110.07, 101.96, 99.87, 66.95, 55.53, 52.34. Purity was
determined to be 99.4% by using HPLC. HRMS (ESI; m/z) calcd for C23H22N2O3, [M +
H]+, 375.1658; found 375.1715.
4.1.6.3 (E)-1-(4-fluorophenyl)-3-(6-methoxy-4-morpholinoquinolin-2-yl)prop-2-en-1-one (9).
Compound 5a was reacted with 1-(4-fluorophenyl)ethanone (6c) according to general
1
procedure to afford compound 9 as a yellow solid with a yield of 51%. H NMR (400 MHz,
DMSO) δ 8.32 – 8.16 (m, 3H), 7.96 (d, J = 9.2 Hz, 1H), 7.78 (d, J = 15.6 Hz, 1H), 7.63 (s,
1H), 7.50 – 7.36 (m, 3H), 7.29 (d, J = 2.7 Hz, 1H), 3.99 – 3.87 (m, 7H), 3.24 (t, J = 4.0 Hz,

4H). 13C NMR (101 MHz, DMSO) δ 188.57, 166.89, 164.38, 157.95, 156.22, 151.98, 145.22
(d, J = 18.0 Hz), 134.51, 132.16 (t, J = 7.2 Hz), 125.72, 124.36, 122.23, 116.51, 116.29,
109.75, 102.57, 66.67, 55.89, 52.47. Purity was determined to be 99.6% by using HPLC.
HRMS (ESI; m/z) calcd for C23H21FN2O3, [M + H]+, 393.1609; found 393.1622.
4.1.6.4 (E)-3-(6-methoxy-4-morpholinoquinolin-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (10).
Compound 5a was reacted with 1-(4-nitrophenyl)ethanone (6d) according to general
procedure to afford compound 10 as a yellow solid with a yield of 46%. 1H NMR (400 MHz,
CDCl3) δ 8.45 – 8.35 (m, 2H), 8.30 – 8.21 (m, 2H), 8.15 – 8.02 (m, 2H), 7.92 (d, J = 15.4 Hz,
1H), 7.41 (dd, J = 9.2, 2.8 Hz, 1H), 7.32 (d, J = 2.7 Hz, 1H), 7.14 (s, 1H), 4.04 (d, J = 4.6 Hz,
4H), 3.99 (s, 3H), 3.28 (d, J = 4.6 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 189.34, 158.22,
156.20, 150.83, 150.18, 146.08, 145.71, 142.75, 132.23, 129.69, 124.97, 124.71, 123.90,
122.20, 110.49, 101.92, 66.93, 55.55, 52.33. Purity was determined to be 98.2% by using
HPLC. HRMS (ESI; m/z) calcd for C23H21N3O5, [M + H]+, 420.1553; found 420.1566.
4.1.6.5
(E)-1-(4-hydroxyphenyl)-3-(6-methoxy-4-morpholinoquinolin-2-yl)prop-2-en-1-one
(11). Compound 5a was reacted with 1-(4-hydroxyphenyl)ethanone (6e) according to
1
general procedure to afford compound 11 as a yellow solid with a yield of 56%. H NMR
(400 MHz, DMSO) δ 10.49 (s, 1H), 8.21 (d, J = 15.6 Hz, 1H), 8.10 (d, J = 8.6 Hz, 2H), 7.95
(d, J = 9.2 Hz, 1H), 7.73 (d, J = 15.6 Hz, 1H), 7.61 (s, 1H), 7.41 (dd, J = 9.2, 2.4 Hz, 1H),
7.29 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 3.99 – 3.89 (m, 7H), 3.24 (t, J = 3.6 Hz,
13
4H). C NMR (101 MHz, CDCl3) δ 189.30, 162.33, 157.87, 156.34, 151.71, 144.98, 142.60,
131.37, 130.92, 129.37, 126.22, 124.30, 122.05, 115.40, 109.58, 101.94, 66.74, 55.29, 52.07.
Purity was determined to be 95.3% by using HPLC. HRMS (ESI; m/z) calcd for
C23H22N2O4, [M + H]+, 391.1652; found 391.1662.
4.1.6.6
(E)-1-(4-(benzyloxy)phenyl)-3-(6-methoxy-4-morpholinoquinolin-2-yl)prop-2-en-1-one (12).
Compound 5a was reacted with 1-(4-(benzyloxy)phenyl)ethanone (6f) according to general
1
procedure to afford compound 12 as a pale yellow solid with a yield of 64%. H NMR (400
MHz, CDCl3) δ 8.14 (d, J = 8.8 Hz, 2H), 8.12 – 8.01 (m, 2H), 7.86 (d, J = 15.4 Hz, 1H), 7.56

– 7.34 (m, 6H), 7.30 (d, J = 2.8 Hz, 1H), 7.16 – 7.05 (m, 3H), 5.17 (s, 2H), 4.02 (d, J = 4.6 Hz,
13
4H), 3.96 (s, 3H), 3.26 (d, J = 4.6 Hz, 4H). C NMR (101 MHz, CDCl3) δ 188.91, 162.74,
157.88, 156.03, 151.81, 145.69, 143.47, 140.79, 136.23, 132.15, 131.16, 128.71, 128.25,
127.51, 125.80, 124.44, 121.82, 114.74, 110.17, 101.95, 70.20, 66.96, 55.50, 52.34. Purity
was determined to be 99.0% by using HPLC. HRMS (ESI; m/z) calcd for C30H28N2O4, [M
+ H]+, 481.2121; found 481.2135.
4.1.6.7
(E)-1-(4-methoxyphenyl)-3-(4-morpholinoquinolin-2-yl)prop-2-en-1-one
(13).
Compound 5b was reacted with 6a according to general procedure to afford compound 13 as
a yellow solid with a yield of 43%. 1H NMR (400 MHz, DMSO) δ 8.29 (d, J = 15.6 Hz, 1H),
8.19 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.81 – 7.72 (m,
2H), 7.61 (s, 1H), 7.58 (d, J = 7.3 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 3.92 (t, J = 7.3 Hz, 4H),
3.89 (s, 3H), 3.27 (t, J = 7.3 Hz, 4H). 13C NMR (101 MHz, DMSO) δ 188.13, 163.95, 157.43,
154.55, 149.52, 143.95, 131.59, 130.63, 130.48, 130.15, 127.04, 126.70, 124.25, 123.05,
114.64, 109.09, 66.63, 56.10, 52.78. Purity was determined to be 99.5% by using HPLC.
HRMS (ESI; m/z) calcd for C23H22N2O3, [M + H]+, 375.1703; found 375.1713.
4.1.6.8 (E)-3-(4-morpholinoquinolin-2-yl)-1-phenylprop-2-en-1-one (14). Compound 5b was
reacted with compound 6b according to general procedure to afford compound 14 as a yellow
solid with a yield of 30%. 1H NMR (400 MHz, CDCl3) δ 8.21 – 8.09 (m, 4H), 8.01 (d, J = 8.4
Hz, 1H), 7.88 (d, J = 15.5 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.64 – 7.59 (m, 1H), 7.56 – 7.49 (m,
3H), 7.11 (s, 1H), 4.01 (d, J = 4.4 Hz, 3H), 3.35 – 3.24 (m, 1H). 13C NMR (101 MHz, CDCl3)
δ 190.82, 157.40, 153.88, 149.75, 144.17, 137.83, 133.09, 130.57, 129.81, 128.85, 128.70,
126.94, 126.23, 123.46, 123.27, 109.38, 66.91, 52.62. Purity was determined to be 98.8% by
using HPLC. HRMS (ESI; m/z) calcd for C22H20N2O2, [M + H]+, 345.1597; found
345.1609.
4.1.6.9
(E)-1-(4-fluorophenyl)-3-(4-morpholinoquinolin-2-yl)prop-2-en-1-one
(15).
Compound 5b was reacted with compound 6c according to general procedure to afford
1
compound 15 as a yellow solid with a yield of 55%. H NMR (400 MHz, CDCl3) δ 8.23 –
8.10 (m, 4H), 8.03 (dd, J = 8.4, 0.9 Hz, 1H), 7.89 (d, J = 15.4 Hz, 1H), 7.72 (ddd, J = 8.4, 6.9,

1.4 Hz, 1H), 7.53 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.22 (ddd, J = 8.7, 5.9, 2.4 Hz, 4H), 7.12 (s,
1H), 4.03 (d, J = 4.6 Hz, 1H), 3.30 (d, J = 4.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 189.05,
167.07, 164.53, 157.44, 153.72, 149.79, 144.24, 134.23 (d, J = 2.8 Hz), 131.46 (d, J = 9.3 Hz),
130.59, 129.81, 126.33 (d, J = 19.0 Hz), 123.46, 123.31, 115.82 (d, J = 21.8 Hz), 109.55,
66.90, 52.62. Purity was determined to be 100% by using HPLC. HRMS (ESI; m/z) calcd for
C22H19FN2O2, [M + H]+, 363.1503; found 363.1510.
4.1.6.10
(E)-3-(4-morpholinoquinolin-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one
(16).
Compound 5b was reacted with compound 6d according to general procedure to afford
1
compound 16 as a yellow solid with a yield of 62%. H NMR (400 MHz, CDCl3) δ 8.35 (d, J
= 8.7 Hz, 2H), 8.23 (d, J = 8.7 Hz, 2H), 8.13 (d, J = 10.8 Hz, 1H), 8.11 (d, J = 3.6 Hz, 1H),
8.02 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 15.4 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.6 Hz,
13
1H), 7.10 (s, 1H), 4.02 (d, J = 4.4 Hz, 4H), 3.29 (d, J = 4.4 Hz, 4H). C NMR (101 MHz,
CDCl3) δ 189.25, 157.59, 153.11, 150.21, 149.73, 145.78, 142.56, 130.59, 129.99, 129.71,
126.51, 125.97, 123.89, 123.52, 123.38, 109.73, 66.87, 52.62. Purity was determined to be
99.6% by using HPLC. HRMS (ESI; m/z) calcd for C22H19N3O4, [M + H]+, 390.1448;
found 390.1458.
4.1.6.11
(E)-1-(4-hydroxyphenyl)-3-(4-morpholinoquinolin-2-yl)prop-2-en-1-one
(17).
Compound 5b was reacted with compound 6e according to general procedure to afford
1
compound 17 as a yellow solid with a yield of 41%. H NMR (400 MHz, DMSO) δ 10.50 (s,
1H), 8.26 (d, J = 15.6 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 8.4 Hz, 1H), 8.02 (d, J =
8.4 Hz, 1H), 7.78 – 7.71 (m, 2H), 7.60 (s, 1H), 7.57 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 8.6 Hz,
2H), 3.91 (d, J = 4.0 Hz, 4H), 3.27 (d, J = 4.0 Hz, 5H). 13C NMR (101 MHz, DMSO) δ
187.80, 163.00, 157.44, 154.66, 149.52, 143.55, 131.87, 130.66 – 130.50 (m), 130.15, 129.30,
127.16, 126.67, 124.25, 123.03, 116.03, 109.02, 66.64, 52.79. Purity was determined to be
98.6% by using HPLC. HRMS (ESI; m/z) calcd for C22H20N2O3, [M + H]+, 361.1547;
found 361.1558.
4.1.6.12 (E)-1-(4-(benzyloxy)phenyl)-3-(4-morpholinoquinolin-2-yl)prop-2-en-1-one (18).
Compound 5b was reacted with compound 6f according to general procedure to afford

1
compound 18 as a yellow solid with a yield of 74%. H NMR (400 MHz, CDCl3) δ 8.20 –
8.08 (m, 4H), 8.00 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 15.4 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H),
7.54 – 7.33 (m, 6H), 7.09 (s, 2H), 7.06 (s, 1H), 5.16 (s, 2H), 4.01 (d, J = 4.4 Hz, 4H), 3.27 (d,
J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 188.87, 162.82, 157.36, 154.08, 149.79,
143.29, 136.20, 131.23, 131.03, 130.56, 129.74, 128.73, 128.28, 127.54, 126.79, 126.11,
123.44, 123.25, 114.78, 109.51, 70.21, 66.92, 52.63. Purity was determined to be 99.3% by
using HPLC. HRMS (ESI; m/z) calcd for C29H26N2O3, [M + H]+, 451.2016; found
451.2026.
4.1.6.13
(E)-3-(6-fluoro-4-morpholinoquinolin-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one
(19). Compound 5c was reacted with compound 6a according to general procedure to afford
1
compound 19 as a yellow solid with a yield of 52%. H NMR (400 MHz, CDCl3) δ 8.24 –
8.10 (m, 4H), 7.86 (d, J = 15.4 Hz, 1H), 7.62 (dd, J = 9.9, 2.8 Hz, 1H), 7.49 (ddd, J = 9.2, 8.0,
2.9 Hz, 1H), 7.14 (s, 1H), 7.02 (d, J = 8.9 Hz, 2H), 4.03 (d, J = 4.4 Hz, 4H), 3.92 (s, 3H), 3.26
(d, J = 4.4 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 188.72, 163.71, 161.66, 159.19, 156.99,
153.54, 146.77, 142.81, 133.06 (d, J = 9.3 Hz), 131.19, 126.81, 124.23 (d, J = 9.0 Hz), 120.02,
119.76, 113.93, 110.27, 107.25 (d, J = 23.4 Hz), 66.87, 55.53, 52.46. Purity was determined to
be 99.8% by using HPLC. HRMS (ESI; m/z) calcd for C23H21FN2O3, [M + H]+, 393.1609;
found 393.1619.
4.1.6.14
(E)-3-(6-fluoro-4-morpholinoquinolin-2-yl)-1-phenylprop-2-en-1-one
(20).
Compound 5c was reacted with compound 6b according to general procedure to afford
1
compound 20 as a yellow solid with a yield of 62%. H NMR (400 MHz, CDCl3) δ 8.16 –
8.07 (m, 4H), 7.86 (d, J = 15.5 Hz, 1H), 7.67 – 7.58 (m, 2H), 7.57 – 7.43 (m, 3H), 7.13 (s,
1H), 4.01 (d, J = 4.4 Hz, 1H), 3.25 (d, J = 4.4 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 190.67,
161.73, 159.26, 157.00, 153.33, 146.81, 143.72, 137.80, 133.12, 128.82, 128.72, 126.94,
124.28 (d, J = 9.0 Hz), 119.98 (d, J = 25.6 Hz), 110.16, 107.28 (d, J = 23.4 Hz), 66.87, 52.46.
Purity was determined to be 99.6% by using HPLC. HRMS (ESI; m/z) calcd for
C22H19FN2O2, [M + H]+, 363.1503; found 363.1513.
4.1.6.15 (E)-3-(6-fluoro-4-morpholinoquinolin-2-yl)-1-(4-fluorophenyl)prop-2-en-1-one (21).

Compound 5c was reacted with compound 6c according to general procedure to afford
1
compound 21 as a yellow solid with a yield of 30%. H NMR (400 MHz, CDCl3) δ 8.22 –
8.09 (m, 4H), 7.86 (d, J = 15.4 Hz, 1H), 7.61 (dd, J = 9.8, 2.8 Hz, 1H), 7.54 – 7.43 (m, 1H),
13
7.20 (t, J = 8.6 Hz, 2H), 7.12 (s, 1H), 4.01 (d, J = 4.2 Hz, 4H), 3.26 (d, J = 4.2 Hz, 4H). C
NMR (101 MHz, CDCl3) δ 188.90, 167.10, 164.57, 161.74, 159.27, 157.11, 153.11, 143.79,
134.15 (d, J = 2.9 Hz), 133.08 (d, J = 9.2 Hz), 131.47 (d, J = 9.3 Hz), 126.43, 124.33, 120.05
(d, J = 25.5 Hz), 115.86 (d, J = 21.9 Hz), 110.34, 107.30 (d, J = 23.4 Hz), 66.85, 52.46. Purity
was determined to be 99.4% by using HPLC. HRMS (ESI; m/z) calcd for C22H18F2N2O2,
[M + H]+, 381.1409; found 381.1425.
4.1.6.16 (E)-3-(6-fluoro-4-morpholinoquinolin-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (22).
Compound 5c was reacted with compound 6d according to general procedure to afford
1
compound 22 as a yellow solid with a yield of 37%. H NMR (400 MHz, CDCl3) δ 8.37 (d, J
= 8.6 Hz, 2H), 8.24 (d, J = 8.5 Hz, 2H), 8.17 – 8.06 (m, 2H), 7.89 (d, J = 15.4 Hz, 1H), 7.62
(dd, J = 9.7, 2.3 Hz, 1H), 7.54 – 7.45 (m, 1H), 7.13 (s, 1H), 4.02 (d, J = 3.8 Hz, 4H), 3.26 (s,
13
4H). C NMR (101 MHz, CDCl3) δ 189.16, 161.89, 159.41, 157.22, 152.60, 150.27, 146.81,
145.45, 142.52, 133.21 (d, J = 9.1 Hz), 129.71, 125.91, 123.93, 120.22 (d, J = 25.7 Hz),
110.50, 107.36 (d, J = 23.4 Hz), 66.83, 52.46. Purity was determined to be 96.7% by using
HPLC. HRMS (ESI; m/z) calcd for C22H18FN3O4, [M + H]+, 408.1354; found 408.1364.
4.1.6.17
(E)-3-(6-fluoro-4-morpholinoquinolin-2-yl)-1-(4-hydroxyphenyl)prop-2-en-1-one
(23). Compound 5c was reacted with compound 6e according to general procedure to afford
1
compound 23 as a yellow solid with a yield of 32%. H NMR (400 MHz, DMSO) δ 10.51 (s,
1H), 8.26 (d, J = 15.6 Hz, 1H), 8.10 (d, J = 8.0 Hz, 3H), 7.84 – 7.62 (m, 4H), 6.95 (d, J = 7.7
13
Hz, 2H), 3.91 (d, J = 6.0 Hz, 4H), 3.21 (d, J = 6.0 Hz, 4H). C NMR (101 MHz, DMSO) δ
187.25, 162.50, 156.64, 153.83, 146.15, 142.72, 132.94, 131.36, 128.78, 126.76, 123.63,
119.79, 115.52, 109.37, 107.55, 107.32, 66.09, 52.11. Purity was determined to be 95.4% by
using HPLC. HRMS (ESI; m/z) calcd for C22H19FN2O3, [M + H]+, 379.1452; found
379.1464.
4.1.6.18 (E)-1-(4-(benzyloxy)phenyl)-3-(6-fluoro-4-morpholinoquinolin-2-yl)prop-2-en-1-one

(24). Compound 5c was reacted with compound 6f according to general procedure to afford
1
compound 24 as a yellow solid with a yield of 79%. H NMR (400 MHz, CDCl3) δ 8.18 –
8.06 (m, 4H), 7.85 (d, J = 15.4 Hz, 1H), 7.61 (dd, J = 9.9, 2.8 Hz, 1H), 7.53 – 7.33 (m, 6H),
7.12 (s, 1H), 7.08 (d, J = 8.8 Hz, 2H), 5.17 (s, 2H), 4.01 (d, J = 4.4 Hz, 4H), 3.25 (d, J = 4.4
Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 188.76, 162.85, 153.56, 146.81, 142.90, 136.18,
133.13, 133.04, 131.19, 130.99, 128.72, 128.28, 127.51, 126.78, 124.21, 120.01, 119.75,
114.80, 110.27, 107.24 (d, J = 23.6 Hz), 70.22, 66.87, 52.46. Purity was determined to be
96.0% by using HPLC. HRMS (ESI; m/z) calcd for C29H25FN2O3, [M + H]+, 469.1921;
found 469.1927.
4.1.6.19 (E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (25).
Compound 5d was reacted with compound 6a according to general procedure to afford
compound 25 as a yellow solid with a yield of 31%. 1H NMR (400 MHz, DMSO) δ 8.80 (d, J
= 2.4 Hz, 1H), 8.41 (dd, J = 9.2, 2.4 Hz, 1H), 8.34 (d, J = 15.6 Hz, 1H), 8.17 (dd, J = 9.0, 5.2
Hz, 3H), 7.77 (s, 1H), 7.73 (s, 1H), 7.14 (d, J = 8.8 Hz, 2H), 3.94 (t, J = 8.4 Hz, 4H), 3.89 (s,
3H), 3.38 (t, J = 8.5 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 188.35, 163.92, 159.14, 157.36,
152.12, 144.64, 141.77, 132.05, 131.25, 130.57, 128.64, 123.21, 122.05, 120.79, 114.03,
111.07, 66.66, 55.57, 52.85. Purity was determined to be 98.5% by using HPLC. HRMS (ESI;
m/z) calcd for C23H21N3O5, [M + H]+, 420.1554; found 420.1567.
4.1.6.20 (E)-3-(4-morpholino-6-nitroquinolin-2-yl)-1-phenylprop-2-en-1-one (26). Compound
5d was reacted with compound 6b according to general procedure to afford compound 26 as a
1
yellow solid with a yield of 33%. H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H), 8.50 – 8.39 (m,
1H), 8.26 – 8.07 (m, 4H), 7.82 (d, J = 15.4 Hz, 1H), 7.67 – 7.49 (m, 13H), 7.17 (s, 1H), 4.04
13
(s, 4H), 3.35 (s, 4H). C NMR (101 MHz, CDCl3) δ 193.51, 159.72, 155.46, 155.44, 151.81,
145.93, 145.89, 133.42, 132.89, 128.87, 128.82, 123.93, 123.48, 120.94, 120.82, 105.75,
66.56, 52.83. Purity was determined to be 99.9% by using HPLC. HRMS (ESI; m/z) calcd for
C22H19N3O4, [M + H]+, 390.1448; found390.1458.
4.1.6.21 (E)-1-(4-fluorophenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (27).
Compound 5d was reacted with compound 6c according to general procedure to afford

1
compound 27 as a yellow solid with a yield of 35%. H NMR (400 MHz, CDCl3) δ 8.94 (d, J
= 2.0 Hz, 1H), 8.45 (dd, J = 9.2, 2.1 Hz, 1H), 8.31 – 8.12 (m, 4H), 7.85 (d, J = 15.3 Hz, 1H),
7.23 (dd, J = 16.9, 8.4 Hz, 2H), 7.15 (s, 1H), 4.06 (d, J = 3.8 Hz, 4H), 3.36 (s, 4H). 13C NMR
(101 MHz, CDCl3) δ 188.56, 167.26, 164.72, 159.25, 156.96, 152.10, 144.73, 142.76, 133.94,
132.11, 131.53 (d, J = 9.4 Hz), 128.17, 123.31, 122.11, 120.81, 115.99 (d, J = 21.9 Hz),
111.14, 66.64, 52.86. Purity was determined to be 98.9% by using HPLC. HRMS (ESI; m/z)
calcd for C22H18FN3O4, [M + H]+, 408.1354; found 408.1366.
4.1.6.22 (E)-3-(4-morpholino-6-nitroquinolin-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (28).
Compound 5d was reacted with compound 6d according to general procedure to afford
1
compound 28 as a yellow solid with a yield of 30%. H NMR (400 MHz, CDCl3) δ 8.95 (d, J
= 2.5 Hz, 1H), 8.47 (dd, J = 9.3, 2.5 Hz, 1H), 8.41 (dd, J = 6.9, 1.9 Hz, 2H), 8.27 (ddd, J =
26.3, 8.9, 5.7 Hz, 4H), 7.90 (d, J = 15.4 Hz, 1H), 7.24 (s, 1H), 4.08 (d, J = 4.4 Hz, 4H), 3.41
13
(d, J = 4.4 Hz, 4H). C NMR (101 MHz, CDCl3) δ 189.03, 159.40, 156.43, 151.82, 150.36,
144.75, 144.09, 142.05, 131.72, 129.75, 127.75, 123.92, 123.44, 122.07, 120.77, 111.09,
66.52, 52.69. Purity was determined to be 95.3% by using HPLC. HRMS (ESI; m/z) calcd for
C22H18N4O6, [M + H]+, 435.1299; found 435.1311.
4.1.6.23 (E)-1-(4-hydroxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (29).
Compound 5d was reacted with compound 6e according to general procedure to afford
1
compound 29 as a yellow solid with a yield of 34%. H NMR (400 MHz, DMSO) δ 10.56 (s,
1H), 8.80 (d, J = 2.5 Hz, 1H), 8.41 (dd, J = 9.2, 2.5 Hz, 1H), 8.34 (d, J = 15.6 Hz, 1H), 8.16
(d, J = 9.2 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 10.8 Hz, 1H), 6.95 (d, J = 8.7 Hz,
13
2H), 3.93 (t, J = 4.0 Hz, 4H), 3.55 – 3.23 (m, 4H). C NMR (101 MHz, DMSO) δ 187.14,
162.69, 158.62, 157.52, 151.43, 144.12, 142.02, 131.61, 131.50, 128.65, 128.54, 123.05,
121.32, 120.89, 115.61, 110.20, 66.01, 52.45. Purity was determined to be 96.2% by using
HPLC. HRMS (ESI; m/z) calcd for C22H19N3O5, [M + H]+, 406.1397; found 406.1409.
4.1.6.24 (E)-1-(4-(benzyloxy)phenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one
(30). Compound 5d was reacted with compound 6f according to general procedure to afford
compound 30 as a yellow solid with a yield of 69%. 1H NMR (400 MHz, DMSO) δ 8.81 (d, J

= 2.3 Hz, 1H), 8.42 (dd, J = 9.2, 2.3 Hz, 1H), 8.36 (d, J = 15.6 Hz, 1H), 8.18 (dd, J = 9.0, 3.7
Hz, 3H), 7.76 (d, J = 14.9 Hz, 2H), 7.50 (d, J = 7.2 Hz, 2H), 7.43 (t, J = 7.3 Hz, 2H), 7.37 (t, J
= 7.2 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 5.27 (s, 2H), 3.92 (d, J = 5.0 Hz, 4H), 3.40 (t, 4H).
13C NMR (101 MHz, CDCl3) δ 188.38, 163.06, 159.15, 157.34, 152.10, 144.66, 141.77,
136.10, 132.03, 131.26, 130.90, 130.76, 128.74, 128.31, 127.50, 123.23, 122.05, 120.78,
114.89, 111.05, 70.26, 66.65, 52.85. Purity was determined to be 95.4% by using HPLC.
HRMS (ESI; m/z) calcd for C29H25N3O5, [M + H]+, 496.1876; found 496.1867.
4.1.7 General procedure for the preparation of 3-hydroxy-3-(6-substituted
-4-morpholinoquinolin-2-yl)-1-(4-substituted-phenyl)propan-1-ones (31-33)
To a solution of 1-(4-substituted-phenyl)ethanone (6d, 6f, 1.84 mmol, 1.2 equiv) in 10
mL EtOH, was added aldehyde (5a-b, 1.54 mmol), followed with 1% NaOH (0.5 mL). The
reaction was stirred at room temperature, and monitored by using TLC. The reaction mixture
was quenched with NH4Cl solution. The organic layer was extracted with ethyl acetate (3×10
mL). The combined organic layer was washed with brine for three times, dried over
anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by using silica
gel chromatograph with dichloromethane/MeOH (100/1 - 20/1) to give compound 31-33.
4.1.7.1 3-hydroxy-3-(6-methoxy-4-morpholinoquinolin-2-yl)-1-(4-nitrophenyl)propan-1-one
(31). Compound 5a was reacted with compound 6d according to general procedure to afford
1
compound 31 as a yellow solid with a yield of 70%. H NMR (400 MHz, CDCl3) δ 8.29 (d, J
= 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 9.1 Hz, 1H), 7.33 (dd, J = 9.1, 2.8 Hz,
1H), 7.27 (d, J = 1.6 Hz, 1H), 7.05 (s, 1H), 5.47 (dd, J = 7.4, 4.3 Hz, 1H), 4.75 (s, 1H), 3.99 (t,
13
J = 4.6 Hz, 4H), 3.93 (s, 3H), 3.59 (qd, J = 16.6, 5.9 Hz, 2H), 3.23 (d, J = 4.6 Hz, 4H). C
NMR (101 MHz, CDCl3) δ 198.29, 159.02, 157.27, 156.71, 150.39, 143.85, 141.53, 130.83,
129.42, 123.81, 123.59, 121.37, 106.31, 102.29, 70.35, 66.91, 55.50, 52.38, 46.91. Purity was
determined to be 99.2% by using HPLC. HRMS (ESI; m/z) calcd for C23H23N3O6 , [M +
H]+, 438.1659; found 438.1664.
4.1.7.2
1-(4-(benzyloxy)phenyl)-3-hydroxy-3-(6-methoxy-4-morpholinoquinolin-2-yl)propan-1-one

(32). Compound 5a was reacted with compound 6f according to general procedure to afford
1
compound 32 as a yellow solid with a yield of 84%. H NMR (400 MHz, CDCl3) δ 7.89 (d, J
= 8.9 Hz, 2H), 7.84 (d, J = 9.1 Hz, 1H), 7.32 (q, J = 7.9 Hz, 4H), 7.26 (ddd, J = 11.8, 6.7, 2.3
Hz, 2H), 7.22 – 7.18 (m, 1H), 7.02 (s, 1H), 6.91 (d, J = 8.9 Hz, 2H), 5.38 (dd, J = 7.6, 4.2 Hz,
1H), 5.04 (s, 2H), 3.90 (t, J = 4.5 Hz, 4H), 3.85 (s, 3H), 3.43 (qd, J = 16.8, 6.0 Hz, 2H), 3.24
– 3.03 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 198.50, 162.94, 160.06, 157.10, 156.46,
144.10, 136.10, 130.97, 130.71, 130.26, 128.72, 128.29, 127.50, 123.53, 121.15, 114.62,
106.48, 102.22, 70.67, 70.17, 66.96, 55.49, 52.37, 45.95. Purity was determined to be 99.4%
by using HPLC. HRMS (ESI; m/z) calcd for C30H30N2O5, [M + H]+, 499.2227; found
499.2235.
4.1.7.3 1-(4-(benzyloxy)phenyl)-3-hydroxy-3-(4-morpholinoquinolin-2-yl)propan-1-one (33).
Compound 5b was reacted with compound 6f according to general procedure to afford
1
compound 33 as a yellow solid with a yield of 79%. H NMR (400 MHz, CDCl3) δ 8.05 –
7.96 (m, 4H), 7.68 (ddd, J = 8.5, 6.9, 1.3 Hz, 1H), 7.56 – 7.33 (m, 6H), 7.12 (s, 1H), 7.06 –
6.99 (m, 2H), 5.50 (dd, J = 7.6, 4.2 Hz, 1H), 5.15 (s, 2H), 4.01 (t, J = 4.6 Hz, 4H), 3.54 (qd, J
= 16.8, 6.0 Hz, 2H), 3.37 – 3.18 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 198.40, 162.95,
162.52, 157.52, 148.40, 136.12, 130.70, 130.28, 129.56, 129.33, 128.71, 128.27, 127.47,
125.25, 123.51, 122.62, 114.64, 105.84, 70.75, 70.18, 66.92, 52.65, 45.94. Purity was
determined to be 99.4% by using HPLC. HRMS (ESI; m/z) calcd for C29H28N2O4, [M +
H]+, 469.2122; found 469.2127.
4.1.8
General
procedure
for
the
preparation
of
3-morpholino-N-((4-morpholinoquinolin-2-yl)methyl)propan-1-amine (34)
To a solution of 3-morpholinopropan-1-amine (59.5 mg, 0.41 mmol) in 5 mL
trichloromethane, was added 4-morpholinoquinoline-2-carbaldehyde (5b, 50.0 mg, 0.21
mmol) and NaBH3CN (52.0 mg, 0.83 mmol), followed with HAc (61.9 mg, 1.03 mmol). The
mixture was stirred at room temperature, and monitored by using TLC. The reaction mixture
was quenched with saturated sodium bicarbonate solution. The organic layer was extracted
with dichloromethane (3×5 mL). The combined organic layers were washed with brine for