Design and synthesis of novel artemisinin derivatives with potent activities against colorectal cancer in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2019.111665

A series of novel derivatives of artemisinin-4-(arylamino)quinazoline have been designed and synthesized, and most of them showing potent in vitro cytotoxic activity against HCT116 and WM-266-4 cell lines. Compound 32 was the most active derivative against HCT116 cell line with an IC₅₀ of 110 nM, and significantly improved the antitumor activity of the parent compounds dihydroartemisinin (DHA) (IC₅₀ = 2.85 μM) and Gefitinib (IC₅₀ = 19.82 μM). In vivo HCT116 xenografts assay showed that compound 32 exhibited potent antitumor activity with obvious tumor growth delay and tumor shrunken after 18 days treatment on xenografted mice, and especially without loss of body weight. Our results indicate that compounds 32 may represent a safe, novel structural lead for developing new chemotherapy of colorectal cancer.

Full text of DOI:10.1016/j.ejmech.2019.111665

European Journal of Medicinal Chemistry 182 (2019) 111665
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of novel artemisinin derivatives with potent
activities against colorectal cancer in vitro and in vivo
,
b
,
c
,
,
b
,
c
,
, b,
Liang-Liang Wang a
1, Lingmei Kong a
1, Hui Liu d, Yunqin Zhang a c, Li Zhang d,
Xingyong Liu d, Feng Yuan a c, Yan Li a
**, Zhili Zuo a
,
b,
,
b
,
c
,
, b, c, d, *
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR China
b Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming, 650201, PR China
c University of Chinese Academy of Sciences, Beijing, 100049, PR China
d School of Chemical Engineering, Sichuan University of Science & Engineering, Zigong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel derivatives of artemisinin-4-(arylamino)quinazoline have been designed and synthe-
sized, and most of them showing potent in vitro cytotoxic activity against HCT116 and WM-266-4 cell
lines. Compound 32 was the most active derivative against HCT116 cell line with an IC50 of 110 nM, and
significantly improved the antitumor activity of the parent compounds dihydroartemisinin (DHA)
Received 20 June 2019
Received in revised form
29 August 2019
Accepted 29 August 2019
Available online 30 August 2019
(IC50 ¼ 2.85
m
M) and Gefitinib (IC50 ¼ 19.82
mM). In vivo HCT116 xenografts assay showed that compound
32 exhibited potent antitumor activity with obvious tumor growth delay and tumor shrunken after 18
days treatment on xenografted mice, and especially without loss of body weight. Our results indicate that
compounds 32 may represent a safe, novel structural lead for developing new chemotherapy of colo-
rectal cancer.
Keywords:
Artemisinin
Quinazoline
Hybridization
Colorectal cancer
Chemotherapy
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
medicinal herb Artemisia annua L. in 1972 by Youyou Tu [7,8]. ART
and its derivatives such as Dihydroartemisinin (DHA), and Artesu-
Colorectal cancer (CRC) has been ranked as the third most
common cancer causing nearly 0.5 million deaths each year and
over 1.3 million new cases annually, while in China, morbidity rate
was continuously increasing in the last decade [1e3]. Though sur-
gical resection combined with chemotherapy for colorectal cancer
has effectively inhibited colorectal cancers, the 5-year survival rate
has been hardly improved due to the drug resistance and unde-
sirable side effects because of lacking in selectivity of currently
available drugs, which makes it highly urgent to develop new and
more effective anti-colorectal cancer medicine [4e6]. One feasible
approach of seeking new chemotherapy drug candidate is to
modify the current clinically available anti-CRC medicines.
nate (AS) etc. are frontline drugs against malarial infections (Fig. 1)
[9e11]. While in the last decades, the broad anticancer activities of
ART derivates including anti Leukemia, breast cancer, lung cancer,
pancreatic cancer, liver cancer etc. have also been extensively
documented [12e20]. Yet, the anticancer mechanism is not fully
understood, it is believed that the 1,2,4-trioxane moiety in the
molecule underlies the potentially similar mechanism for both
antitumor and antimalarial properties via reactive oxygen species
(ROS) and carbon-centered free radicals generated from peroxide
bridge cleavage by intracellular Fe(II) [21e24]. Recently, Krishna's
team reported an oral artesunate neoadjuvant therapy with good
tolerance in a randomized double-blind pilot clinical phase II trial
to treat colorectal cancer (CRC), indicating that ART derivates have
Artemisinin (ART) was isolated from the traditional Chinese
* Corresponding authors: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201,
PR China.
** Corresponding author. State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, PR
China.
E-mail addresses: liyanb@mail.kib.ac.cn (Y. Li), zuozhili@mail.kib.ac.cn (Z. Zuo).
Contribute equally to this work.
1
https://doi.org/10.1016/j.ejmech.2019.111665
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
Fig. 1. The design of Artemisinin-4-(arylamino)quinazoline hybrids.
potential to be developed as an anti-CRC drug [25e28]. To seek
more potent ART derivatives possessing potent pharmacological
properties, we envisioned that one straightforward approach is to
utilize the concept of molecular hybridization [29,30] to hybridize
ART with one pharmacophore existing in approved drug, which
would generate tremendous combinations and produce new ana-
logues with improved bioactivities compared to the parent
compounds.
2. Result and discussion
2.1. Chemistry
2.1.1. Synthesis of artemisinin hybrids with ether linker
We initiated the synthesis of the first series of artemisinin hy-
brids 9a-9h featured with an ether linker from starting materials 2-
amino-5-methylbenzoic acid 1 which was converted to 4-(3-
chloro-4-fluorophenylamino)quinazoline 2 by known procedure
[32], followed by acetylation and radical bromination of bezylic
methyl to afford bromide 3. Subsequent nucleophilic substitution of
3 by water or diol under basic condition delivered 4e7 in good
yields. Subjection of commercially available dihydroartemisinin
(DHA) 8 to alcohol 4e7 in the presence of BF3$Et2O resulted in the
Protein kinases inhibitors for instance Gefitinib, Vandetanib,
Afatinib, etc. have become one type of significant anticancer agents
in various cancer chemotherapy by deregulation of protein kinases
involved in cancer progression in the past fifteen years (Fig. 1) [31].
Many of those kinase inhibitor drugs harbor the same crucial motif
4-(arylamino)quinazoline as the core pharmacophore. In consid-
eration of the great potential of ART derivatives as anticancer agent
as well as their unique antitumor mechanism and the prevalence of
4-(arylamino)quinazoline fragment in the anticancer drug, we have
tried to combine those two entity together in expectation of
discovering novel ART-4-(arylamino)quinazoline hybrids with
more potent anti-CRC effect. Herein, we report the syntheses of a
set of ART-4-(arylamino)quinazoline hybrids with various types of
linkers featured with ether, ester, amide and chiral elements
(Fig. 1). To the best of our knowledge, No ART-4-(arylamino)
quinazoline hybrids have been reported to date. Their antitumor
activities on human colorectal cancer HCT116 cells and human
melanoma cancer WM-266-4 were examined in vitro. The in vivo
study on xenografted mice showed this type of artemisinin hybrids
possessed potent antitumor activities.
hybrid mixtures of 10b-isomer 9a-9d and 10a-isomer 9e-9h, of
which both are readily separated by flash column chromatography
(Scheme 1). The stereochemistry of both diastereomers was
confirmed by analyzing 1He1H NOESY spectrum or by calculating
the vicinal coupling constant JH10-H9. Generally J10 -isomer (7e10 Hz)
with trans configuration is bigger than J10
configuration [33,34].
a
(3e5 Hz) with cis
b
-isomer
2.1.2. Synthesis of artemisinin hybrids with ester and amide linker
10 -Dihydroartemisinin (DHA) 8 coupled with dicarboxylic acid
10e12 to predominately deliver 13e15 respectively. The following
a
esterification reaction with alcohol 4 or 16 could afford the 10
hybrids 17a-17c or 17d-17f (Scheme 2).
a-
10
a-aminoartemisinin
18
could
be
obtained
from

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
3
Scheme 1. Synthesis of hybrids with ether linker.
Scheme 2. Synthesis of hybrids with ester linker.
Dihydroartemisinin (DHA) 8 by azidation and subsequent Stau-
reaction between DHA 8 and
to deliver
and acid 31 afforded the target compound 32. The structure of 10
D
-Phenylglycine derived alkamine 29
dinger reduction [35]. The following amidation reaction between
18 and dicarboxylic acid 10e12 afforded intermediates 19e21
respectively, which then reacted with alcohol 4 or 16 to produce
a-isomer 30. The following coupling reaction between 30
a-
isomer 32 was unambiguously confirmed by full NMR spectrum
hybrids 10
a-hybrids 22a-22c or 22d-22f (Scheme 3).
analysis (Scheme 4) (See supporting information).
2.1.3. Synthesis of artemisinin hybrids with chiral linkers
2.2. Biological evaluation
The preparation of Hybrids (S)-27 began with esterification of
TBS protected (S)-Mandelic acid 23 with alcohol 4, followed by
desilylation to afford alcohol (S)-25. The following acetalation re-
2.2.1. In vitro cytotoxicity activity
To evaluate the cytotoxic effect of the novel hybrids, we inves-
tigated cell viability in human colorectal cancer HCT116 cells and
human melanoma cancer WM-266-4 cells by MTS assay using
Dihydroartemisinin (DHA), Artesunate (AS), Cisplatin (cis-DDP) and
Taxol as control. The results of IC50 were shown in Table 1.
We first evaluated the hybrids 9a-9d with various length of
Et
·
action between 25 and DHA in the presence of BF3 2O delivered
the inseparable and mixture of hybrid (S)-27. Hybrid (R)-28 was
a
b
synthesized by the same way as hybrid (S)-27 from TBS protected
(R)-Mandelic acid 24 (Scheme 4).
The synthesis of hybrids 32 commenced with acetylation

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L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
Scheme 3. Synthesis of hybrids with amide linker.
Scheme 4. Synthesis of hybrids with chiral linkers.
ether linkers against both HCT116 cells and WM-266-4 cells.
Promising antiproliferative activity was observed for 9d with a
anomers 9e-9h showed less potent antiproliferative activity against
both cells in comparison with 9a-9d (Entries 5e8 vs 1e4, Table 1),
indicating that the stereochemistry on the anomeric carbon of
hybrid exerts impact on its bioactivities. Generally, hybrids 9a-9d
selectively inhibited HCT116 cells compared with WM-266-4 cells.
lowest IC50 (0.24
potent than the parent drugs dihydroartemisinin (DHA) with an
IC50 of 2.85 M (Entry 27, Table 1). However, its corresponding
mM) (Entry 4, Table 1), which is 10-fold more
m
b-

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
5
Table 1
respectively. Hence, both the (S)-alkamine linker-contained ART
fragment and 4-(arylamino)quinazoline fragment on hybrid 32 are
crucial to its significantly enhanced potency against HCT116 cell
since the corresponding analogues 30 and 31 of those two frag-
ments showed almost neglectable activities on HCT116 cells with
IC50 > 15 mM, respectively (Entries 25e26, Table 1). With this most
active compound 32 in hand, we are able to further investigate its
anti-CRC activity in vivo.
The IC50 values of artemisinin derivatives to the HCT116 and WM-266-4 cells.
Entry
Compounds No.
IC50(m
M)a
HCT116
WM-266-4
1
2
3
4
5
6
7
8
9a
9b
9c
9d
9e
9f
9g
9h
17a
17b
17c
17d
17e
17f
22a
22b
22c
22d
22e
22f
32
27
28
Gefitinib
30
31
DHA
AS
DDP
Taxol
0.29 0.11
2.11 0.34
0.36 0.02
0.24 0.06
0.42 0.20
6.54 1.20
2.42 0.33
0.35 0.06
0.18 0.03
0.29 0.05
0.31 0.03
0.28 0.11
0.78 0.14
0.66 0.14
2.78 0.22
5.56 0.36
5.68 0.77
12.88 0.76
5.35 0.28
11.78 0.46
0.11 0.03
0.89 0.12
0.68 0.11
19.82 2.54
16.24 1.26
>40
10.75 0.20
5.18 0.57
3.81 0.36
2.91 0.12
4.56 0.91
15.75 1.47
12.60 2.86
5.70 1.11
10.24 1.73
11.36 0.58
3.51 0.68
9.29 0.54
14.99 2.39
18.76 3.36
9.88 0.12
15.29 0.25
25.27 0.32
15.43 1.29
5.08 0.10
27.79 1.27
6.67 0.41
ND
2.2.2. In vivo anti- CRC activity
Compound 32 was used to evaluate the in vivo anti-CRC efficacy
in HCT116 mouse xenograft models. The following treatments were
administered to female nude BALB/c mice inoculated with visible
tumors: (1) control (Solvent), (2) and (3) compound 32, (4) Gefiti-
nib, (5) DHA. Compound 32 showed obvious tumor growth delay in
comparison with both control and drug Gefitinib or DHA treatment
groups, even though the dosage of compound 32 used in the
experiment is less than the control group. 5 mg/kg and 2.5 mg/kg
were used in the test, while 10 mg/kg of Gefitinib and DHA were
used as control. Meanwhile, no difference in body weight was
observed between the Control and 32 treated mice, indicating the
safety of compound 32. These results demonstrated that 32 has
potential anti-tumor activity in vivo (Fig. 2).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
ND
17.42 1.56
15.12 0.25
>40
3. Conclusion
2.85 0.65
0.89 0.02
10.98 1.01
0.11 0.01 pM
>40
A series of novel artemisinin derivatives were designed and
synthesized by junction of ART and 4-(arylamino)quinazoline
pharmacophore via various types of linkers. All those artemisinin
analogues were tested against the human colorectal cancer
HCT116 cells and human melanoma cancer WM-266-4 and showed
potent antiproliferative activities. In particular, the compound 32
was the most active derivative and effectively suppressed the tu-
mor growth in vitro with an IC50 of 110 nM and significantly
improved the antitumor activity of the parent compounds. More-
over, in vivo study on xenografted mice disclosed that compound 32
effectively delayed tumor growth after 18 days treatment, while the
weight of the mice remained unchanged compared with the con-
trol group. This promising results indicated that compound 32
represent a new structural lead for development of new anti-CRC
cancer drug.
8.46 0.24
3.92 1.13
0.32 0.08 nM
a
The IC50 values were calculated from three independent experiments and rep-
resented the mean SD (n ¼ 3). ND: not determined.
The selectivity could be attributed to the Artemisinin pharmaco-
phore. Artemisinin derivatives were reported to selectively atten-
uate colon cancers [28] due to the inhibition of the constitutively
activated STAT3 signaling [36], which is particularly high and
required for the growth of colon cancer cells [37e39].
Then hybrids 17a-17c with diester linker was tested on
HCT116 cell and the sub-micromole level IC50 were observed with
0.18, 0.29 and 0.31 mM, respectively (Entries 9e11, Table 1). In this
case the length of linkers exerted tiny influence on the bioactivities.
The similar tendency was observed that hybrids 17a-17c as well as
N-acetylated 17d-17f showed more potency against HCT116 cells
than WM-266-4 cells, while hybrids 17d-17f were less potent than
the corresponding N-Hydrogen analogues 17a-17c on both types of
cells (Entries 9e11 vs 12e14, Table 1).
When the diester linker was slightly modified to be amide-ester
linker as shown in derivatives 22a-22c as well as 22d-22f, the
antiproliferative potency were dramatically reduced 10e20 fold on
HCT116 cells compared with corresponding counterpart 17a-17c
and 17d-17f (Entries 15e17 vs 9e11, 18e20 vs 12e14), while the
similar tendency of decreased activities on WM-266-4 cells were
also observed in spite of less degree of variation.
Besides the flexible ether, diester and amide linker mentioned
above, we introduced more rigidly chiral linker to adjust the spatial
conformation of hybrids 27e28 and 32 in expectation of restricting
fragment rotation between ART motif and 4-(arylamino)quinazo-
line subunit around the linker (Entries 21e23, Table 1). To our
delight, compound 32 with (S)-alkamine linker showed the best
antiproliferative potency against HCT116 cells with an IC50 of
110 nM. The other two hybrids 27e28 with a chiral liker derived
from (S) or (R)-Mandelic acid also exhibited good antiproliferative
4. Experimental section
All commercially available reagents were used without further
purification unless otherwise stated. All reactions were performed
under an argon atmosphere unless otherwise stated. Flash column
chromatography on silica gel (200e300 mesh) was used for puri-
fication of reaction products. NMR spectra were recorded with
tetramethylsilane as the internal standard. 1H NMR spectra were
recorded at 400 MHz、500 MHz、600 MHz、800 MHz, respec-
tively, and 13C NMR spectra were recorded at 100 MHz、125 MHz、
150 MHz (Bruker Avance), respectively. Chemical shift values of
NMR data are reported as values in ppm relative to (residual
undeuterated) solvent signal as internal standard. Multiplicities for
1H NMR signals are described using the following abbreviations:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet; High
resolution mass spectra were obtained with the Q-TOF-Premier
mass spectrometer.
4.1. Chemical synthesis
4.1.1. General procedure for preparation of hybrids (9a-9h)
To a solution of dihydroartemisinin 8 (1.2 eq.) and correspond-
ing alcohol 4e7 (1 eq.) in dry dichloromethane under argon was
activities against HCT116 cells with an IC50 of 0.89 and 0.68 mM,

6
L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
Fig. 2. In vivo efficacy of compound 32 in human colorectal cancer xenograft models. (A) Two days after subcutaneous implantation of HCT116 cells, female nude mice received the
following treatments: (1) Ctrl (Solvent), (2) (3) compound 32, (4) Gefitinib, (5) DHA via intraperitoneal injection every day, and tumor volumes were estimated every day. Each data
point represents mean tumor volume of 8 animals in each group SE. p value, *<0.05; **<0.01; (B) The individual body weight was measured and expressed as mean SD (n ¼ 8);
(C) (D) Tumors were isolated and tumor weights were measured at the end of treatment. Representative tumor images are shown and tumor weights are presented as mean SD
(n ¼ 8).
added BF3$Et2O (2 eq.) dropwise at 0 ꢀC. The mixture was then
stirred overnight at this temperature and quenched with saturated
NaHCO3 aqueous solution. The reaction mixture was then extracted
with ethyl acetate. After combining with organic phase and dried
over Na2SO4, the solvent was removed under reduced pressure to
132.10, 128.62, 123.88, 121.39, 121.35, 121.07, 118.90, 116.59, 115.03,
102.18, 88.16, 72.12, 67.81, 65.20, 52.48, 44.34, 37.54, 36.33, 34.57,
30.98, 29.79, 25.81, 24.50, 24.49, 20.30, 13.01; HRMS (ESI) Calcd. for
C
33H40ClFN3O6 [MþH]þ: 628.2590; Found: 628.2592.
Hybrid 9c (28%): 1H NMR (400 MHz, CDCl3)
d 8.74 (s,1H), 8.17 (d,
give the crude product. The
in each case by flash chromatography to afford hybrids 9a-9h.
Hybrid 9a (25%): 1H NMR (400 MHz, CDCl3)
8.74 (s, 1H),
a
,
b
-isomers could be readily separated
J ¼ 7.6 Hz, 2H), 8.06 (dd, J ¼ 6.5, 2.5 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H),
7.74e7.66 (m, 2H), 7.16 (t, J ¼ 8.8 Hz, 1H), 5.55 (s, 1H), 4.73e4.72 (m,
2H), 4.56 (d, J ¼ 12.7 Hz, 1H), 3.93 (dd, J ¼ 13.5, 8.1 Hz, 1H), 3.59 (dd,
J ¼ 9.7, 4.7 Hz, 1H), 3.55e3.42 (m, 1H), 3.40e3.27 (m, 1H), 2.70e2.57
(m, 1H), 2.34 (td, J ¼ 14.1, 3.7 Hz, 1H), 2.03e1.90 (m, 2H), 1.90e1.79
(m, 2H), 1.71 (ddd, J ¼ 16.7, 9.4, 5.0 Hz, 4H), 1.65e1.58 (m, 2H),
1.51e1.40 (m, 2H), 1.30 (s, 3H), 1.28e1.20 (m, 2H), 0.94 (d, J ¼ 6.1 Hz,
d
8.01e7.93 (m, 2H), 7.91 (d, J ¼ 8.6 Hz, 1H), 7.77 (d, J ¼ 8.3 Hz, 1H),
7.64e7.54 (m, 1H), 7.17 (t, J ¼ 8.7 Hz, 1H), 5.46 (s, 1H), 4.94 (d,
J ¼ 12.5 Hz, 1H), 4.89 (d, J ¼ 3.3 Hz, 1H), 4.74 (d, J ¼ 12.5 Hz, 1H),
2.73e2.61 (m, 1H), 2.38 (td, J ¼ 14.1, 3.8 Hz, 2H), 2.08e1.99 (m, 1H),
1.94e1.84 (m, 1H), 1.83e1.73 (m, 2H), 1.61 (dd, J ¼ 13.2, 2.6 Hz, 1H),
1.55e1.46 (m, 2H), 1.43 (s, 3H), 1.33e1.22 (m, 3H), 0.94 (s, 3H), 0.92
3H), 0.88 (d, J ¼ 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 156.62,
153.84, 153.63, 148.51, 136.50, 134.19, 131.52, 127.60, 123.24, 120.76,
119.99, 118.39, 115.52, 113.99, 103.15, 101.08, 87.19, 80.13, 71.27,
69.93, 66.89, 51.55, 43.42, 36.50, 35.40, 33.62, 29.97, 26.27, 25.04,
24.95, 23.66, 23.49, 19.36, 12.02; HRMS (ESI) Calcd. for
(s, 3H); 13C NMR (101 MHz, CDCl3)
d 156.56, 153.99, 153.45, 148.01,
135.94, 133.85, 132.19, 127.70, 123.27, 120.83, 120.10, 118.95, 115.6,
113.49, 103.40, 100.23, 87.17, 80.06, 68.52, 51.40, 43.22, 36.45, 35.32,
33.46, 29.82, 25.10, 23.60, 23.50, 19.27, 12.02; HRMS (ESI) Calcd. for
C
34H42ClFN3O6 [MþH ]þ: 642.2746; Found: 642.2743.
C
30H34ClFN3O5 [MþH]þ: 570.2171; Found: 570.2166.
Hybrid 9d (21%): 1H NMR (400 MHz, CDCl3)
d 8.76 (s, 1H),
Hybrid 9b (31%): 1H NMR (400 MHz, CDCl3)
d
8.74 (s, 1H), 8.16 (s,
8.04e7.93 (m, 2H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 8.7 Hz, 1H),
7.65e7.58 (m, 1H), 7.18 (t, J ¼ 8.7 Hz, 1H), 5.44 (s, 1H), 4.73 (d,
J ¼ 3.1 Hz, 1H), 4.68 (d, J ¼ 7.3 Hz, 2H), 3.80 (dd, J ¼ 15.7, 6.6 Hz, 1H),
3.55 (t, J ¼ 6.4 Hz, 2H), 3.45e3.29 (m, 1H), 2.67e2.53 (m, 1H), 2.36
(td, J ¼ 14.1, 3.8 Hz, 1H), 2.05e1.96 (m, 1H), 1.92e1.83 (m, 1H),
1.83e1.77 (m, 1H), 1.77e1.72 (m, 1H), 1.72e1.65 (m, 3H), 1.63e1.57
(m, 3H), 1.46 (dd, J ¼ 11.2, 5.2 Hz, 3H), 1.38 (s, 3H),1.34e1.27 (m, 1H),
1.26e1.18 (m, 2H), 0.93 (d, J ¼ 6.1 Hz, 3H), 0.87 (d, J ¼ 7.3 Hz, 3H);
1H), 8.11e7.97 (m, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz,
2H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.46 (s, 1H), 4.79 (d, J ¼ 3.1 Hz, 1H), 4.67
(s, 2H), 4.09e3.99 (m, 1H), 3.66 (t, J ¼ 6.0 Hz, 2H), 3.57e3.48 (m,
1H), 2.69e2.59 (m, 1H), 2.27 (dd, J ¼ 18.6, 8.8 Hz, 1H), 1.92 (dd,
J ¼ 12.1, 5.9 Hz, 3H), 1.79 (s, 1H), 1.78e1.72 (m, 2H), 1.61e1.54 (m,
1H), 1.49e1.40 (m, 1H), 1.28 (d, J ¼ 5.7 Hz, 1H), 1.24 (s, 3H), 1.21e1.15
(m, 2H), 1.12 (d, J ¼ 7.2 Hz, 1H), 0.89 (d, J ¼ 7.2 Hz, 6H); 13C NMR
(151 MHz, CDCl3)
d
157.43, 154.77, 154.53, 149.34, 137.55, 135.16,
13C NMR (101 MHz, CDCl3)
d 158.99, 156.66, 153.08, 137.01, 131.97,

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
7
127.02, 123.56, 121.12, 120.00, 118.90, 118.28, 115.61, 113.62, 103.16,
101.88, 101.10, 87.04, 80.13, 71.17, 69.80, 67.37, 51.52, 43.41, 36.49,
35.40, 33.61, 29.90, 28.32, 23.24, 25.12, 23.65, 23.45, 21.76, 19.34,
11.99; HRMS (ESI) Calcd. for C35H44ClFN3O6 [MþH]þ: 656.2903;
Found: 656.2901.
ethylcarbodiimide hydrochloride (EDCI, 2 eq.) and DMAP (0.5 eq.)
were added to above mixture at ambient temperature. The reaction
was monitored by TLC. After completion, the reaction was
quenched by saturated NaHCO3 aqueous solution and extracted by
ethyl acetate. The combined organic phase was then removed un-
der reduced pressure and purified by chromatography to give the
products 17a-17c. Products 17d-17f were prepared by the same
procedure as mentioned above with Alcohol 16 and carboxylic acid
intermediates 13e15 respectively.
Hybrid 9e (23%): 1H NMR (400 MHz, CDCl3)
d 8.73 (s,1H), 8.19 (s,
1H), 8.06 (dd, J ¼ 6.5, 2.4 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.71e7.60
(m, 2H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.40 (s, 1H), 5.11 (d, J ¼ 13.0 Hz, 1H),
4.82 (d, J ¼ 13.0 Hz, 1H), 4.64 (d, J ¼ 9.2 Hz, 1H), 2.60e2.52 (m, 1H),
2.44e2.34 (m, 2H), 2.07e1.98 (m, 1H), 1.91 (d, J ¼ 11.1 Hz, 1H), 1.78
(dd, J ¼ 13.4, 3.4 Hz, 1H), 1.73e1.66 (m, 1H), 1.64e1.56 (m, 1H),
1.54e1.43 (m, 1H), 1.37 (s, 3H), 1.29 (dd, J ¼ 9.9, 4.6 Hz, 2H), 1.25 (s,
Hybrid 17a (77%): 1H NMR (400 MHz, CDCl3)
d 8.76 (s, 1H), 8.64
(s, 1H), 8.41 (dd, J ¼ 6.4, 2.2 Hz, 1H), 7.87 (d, J ¼ 9.9 Hz, 2H), 7.67 (dd,
J ¼ 5.2, 3.3 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz, 1H), 7.19 (t, J ¼ 8.7 Hz, 1H),
5.57 (d, J ¼ 14.2 Hz, 1H), 5.46 (d, J ¼ 9.8 Hz, 1H), 5.27 (d, J ¼ 14.2 Hz,
1H), 4.72 (s, 1H), 2.97e2.78 (m, 4H), 2.43 (s, 1H), 2.23 (td, J ¼ 14.1,
3.5 Hz, 1H), 2.12 (s, 1H), 1.90 (d, J ¼ 14.2 Hz, 1H), 1.85e1.73 (m, 1H),
1.50 (d, J ¼ 12.1 Hz, 1H), 1.41 (dd, J ¼ 17.2, 8.1 Hz, 2H), 1.33e1.18 (m,
2H), 1.14 (s, 3H), 0.91 (s, 3H), 0.82 (d, J ¼ 10.0 Hz, 1H), 0.63 (d,
J ¼ 7.0 Hz, 3H), 0.53 (dt, J ¼ 24.5, 8.3 Hz, 1H); 13C NMR (101 MHz,
1H), 0.99e0.93 (m, 6H); 13C NMR (101 MHz, CDCl3)
d 156.56,153.83,
153.24, 148.01, 135.94, 133.85, 132.19, 127.70, 123.27, 120.83, 120.10,
118.95, 115.64, 113.49, 103.40, 100.23, 87.17, 80.06, 68.52, 51.40,
43.22, 36.45, 35.32, 33.46, 29.82, 25.10, 23.65, 23.52, 19.27, 12.02;
HRMS (ESI) Calcd. for C30H34ClFN3O5 [MþH]þ: 570.2171; Found:
570.2169.
Hybrid 9f (27%): 1H NMR (400 MHz, CDCl3)
d
8.72 (s, 1H), 8.14 (s,
CDCl3) d 172.53, 170.24, 156.50, 153.52, 153.42, 148.04, 134.88,
1H), 7.97 (dd, J ¼ 6.4, 2.3 Hz, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.74 (d,
J ¼ 8.5 Hz, 1H), 7.69e7.62 (m, 1H), 7.15 (t, J ¼ 8.8 Hz, 1H), 5.35 (s, 1H),
4.66 (d, J ¼ 4.2 Hz, 2H), 4.44 (d, J ¼ 9.2 Hz, 1H), 4.07 (dt, J ¼ 9.1,
6.8 Hz, 1H), 3.59 (d, J ¼ 6.4 Hz, 2H), 2.41e2.30 (m, 2H), 2.04e1.95
(m, 1H), 1.86 (dd, J ¼ 11.8, 5.7 Hz, 3H), 1.76e1.63 (m, 3H), 1.50 (dd,
J ¼ 11.2, 6.8 Hz, 1H), 1.42 (dd, J ¼ 11.0, 4.6 Hz,1H), 1.34 (s, 3H), 1.26 (s,
1H), 1.25 (s, 2H), 1.19 (d, J ¼ 13.2 Hz, 1H), 0.94 (d, J ¼ 5.4 Hz, 3H), 0.80
133.97, 129.57, 127.47, 122.64, 120.12, 120.06, 116.60, 115.5, 114.19,
103.27, 92.08, 90.04, 78.72, 63.90, 50.17, 43.70, 36.09, 34.96, 32.76,
30.35, 28.56, 28.11, 24.47, 23.44, 20.46, 19.12, 10.82; HRMS (ESI)
Calcd. for C34H38ClFN3O8 [MþH ]þ: 670.2332; Found: 670.2336.
Hybrid 17b (69%): 1H NMR (400 MHz, CDCl3)
d 8.73 (s, 1H), 8.17
(s, 1H), 8.05e7.96 (m, 1H), 7.89 (d, J ¼ 8.4 Hz, 1H), 7.71 (d, J ¼ 8.5 Hz,
1H), 7.65e7.55 (m, 1H), 7.16 (t, J ¼ 8.7 Hz, 1H), 5.62 (d, J ¼ 9.8 Hz,
1H), 5.29 (d, J ¼ 6.4 Hz, 2H), 5.22 (s, 1H), 2.53e2.38 (m, 5H),
2.36e2.25 (m, 2H), 2.04e1.94 (m, 3H), 1.85 (dd, J ¼ 8.9, 4.6 Hz, 1H),
1.64 (d, J ¼ 11.9 Hz, 2H), 1.54 (dt, J ¼ 13.3, 3.9 Hz, 1H), 1.39 (ddd,
J ¼ 19.7, 12.3, 5.6 Hz, 1H), 1.31 (s, 3H), 1.22 (dd, J ¼ 12.3, 6.0 Hz, 2H),
1.12e0.99 (m, 1H), 0.94 (d, J ¼ 5.3 Hz, 3H), 0.77 (d, J ¼ 7.1 Hz, 3H);
(d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 168.64, 156.94,
154.18, 148.50, 137.00, 133.92, 132.01, 126.78, 123.76, 121.50, 119.95,
118.80, 115.5, 113.68, 103.47, 99.16, 90.25, 79.53, 71.19, 66.19, 64.62,
50.56, 44.24, 36.40, 35.29, 33.13, 31.65, 28.98, 24.96, 23.63, 21.15,
19.22, 11.55; HRMS (ESI) Calcd. for C33H40ClFN3O6 [MþH ]þ:
628.2590; Found: 628.2583.
13C NMR (151 MHz, CDCl3)
d 172.42, 172.26, 157.96, 154.88, 154.77,
Hybrid 9g (33%): 1H NMR (400 MHz, CDCl3)
d
8.74 (s,1H), 8.21 (s,
149.34, 135.07, 134.67, 132.61, 128.46, 124.91, 122.51, 120.71, 120.49,
116.38, 115.02, 104.44, 92.30, 91.35, 79.99, 65.37, 51.29, 44.94, 37.11,
36.02, 33.85, 33.04, 32.73, 31.51, 25.66, 24.41, 21.72, 20.07, 19.57,
11.96; HRMS (ESI) Calcd. for C35H40ClFN3O8 [MþH ]þ: 684.2488;
Found: 684.2484.
1H), 8.07 (s, 1H), 7.98 (dd, J ¼ 6.4, 2.4 Hz, 1H), 7.88 (d, J ¼ 8.5 Hz, 1H),
7.73 (d, J ¼ 8.2 Hz,1H), 7.64e7.58 (m,1H), 7.16 (t, J ¼ 8.8 Hz,1H), 5.33
(s, 1H), 4.64 (s, 2H), 4.41 (d, J ¼ 9.2 Hz, 1H), 4.00e3.92 (m, 1H), 3.53
(t, J ¼ 5.7 Hz, 2H), 3.44 (dd, J ¼ 6.2, 3.0 Hz, 1H), 2.42e2.31 (m, 2H),
2.04e1.95 (m, 2H), 1.92e1.83 (m, 1H), 1.68 (dd, J ¼ 14.0, 6.6 Hz, 5H),
1.55e1.43 (m, 2H), 1.37 (s, 3H), 1.31e1.20 (m, 4H), 0.95 (d, J ¼ 5.7 Hz,
Hybrid 17c (63%): 1H NMR (400 MHz, CDCl3)
d 8.74 (s, 1H), 8.42
(s, 1H), 8.15 (s, 1H), 8.04e7.97 (m, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.75
(d, J ¼ 8.5 Hz, 1H), 7.63e7.53 (m, 1H), 7.15 (t, J ¼ 8.7 Hz, 1H), 5.65 (d,
J ¼ 9.8 Hz, 1H), 5.25 (s, 3H), 2.56e2.45 (m, 1H), 2.38e2.27 (m, 5H),
2.01e1.93 (m, 1H), 1.89e1.79 (m, 1H), 1.64 (d, J ¼ 10.4 Hz, 6H),
1.58e1.50 (m, 1H), 1.45e1.35 (m, 1H), 1.32 (s, 3H), 1.22 (dd, J ¼ 11.1,
5.3 Hz, 2H), 1.11 (dd, J ¼ 27.0, 13.8 Hz, 1H), 0.97 (d, J ¼ 14.3 Hz, 1H),
0.94 (d, J ¼ 5.2 Hz, 3H), 0.75 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz,
3H), 0.83 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 156.70,
153.98, 153.52, 148.36, 136.70, 134.06, 131.69, 127.62, 123.67, 121.26,
119.96,118.20,115.5,113.84,103.35, 99.26, 90.23, 79.44, 71.10, 69.63,
67.83, 50.59, 44.27, 36.38, 35.30, 33.17, 31.64, 25.36, 25.29, 24.96,
23.66, 21.15, 19.24, 11.58; HRMS (ESI) Calcd. for C34H42ClFN3O6
[MþH]þ: 642.2746; Found: 642.2744.
Hybrid 9h (19%): 1H NMR (400 MHz, CDCl3)
d
8.75 (s, 1H), 8.01
CDCl3) d 171.97, 171.63, 156.85, 154.0, 153.81, 148.40, 133.97, 133.81,
(s, 1H), 7.97 (dd, J ¼ 6.5, 2.5 Hz, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.74 (d,
J ¼ 8.4 Hz, 1H), 7.62e7.55 (m, 1H), 7.17 (t, J ¼ 8.7 Hz, 1H), 5.33 (s, 1H),
4.66 (d, J ¼ 3.2 Hz, 2H), 4.41 (d, J ¼ 9.2 Hz, 1H), 3.95 (dd, J ¼ 15.3,
6.4 Hz, 1H), 3.53 (t, J ¼ 5.5 Hz, 2H), 3.41 (dd, J ¼ 15.7, 6.6 Hz, 1H),
2.36 (ddd, J ¼ 22.6, 12.6, 5.4 Hz, 2H), 2.00 (dd, J ¼ 14.1, 3.5 Hz, 1H),
1.93e1.83 (m, 1H), 1.78e1.68 (m, 4H), 1.63 (dd, J ¼ 15.8, 7.4 Hz, 4H),
1.50e1.47 (m, 1H), 1.46 (s, 1H), 1.38 (s, 3H), 1.26 (dd, J ¼ 9.4, 4.8 Hz,
4H), 0.95 (d, J ¼ 5.9 Hz, 3H), 0.84 (d, J ¼ 7.1 Hz, 3H); 13C NMR
132.29, 127.73, 123.77, 121.32, 119.93, 115.5, 113.94, 103.47, 91.15,
90.43, 79.08, 64.61, 50.42, 44.06, 36.18, 35.14, 32.97, 32.65, 30.65,
24.78, 23.50, 23.08, 22.83, 20.81, 19.15, 11.04; HRMS (ESI) Calcd. for
C
36H42ClFN3O8 [MþH ]þ: 698.2645; Found: 698.2642.
Hybrid 17d (68%): 1H NMR (400 MHz, CDCl3)
d 9.24 (s, 1H), 8.11
(d, J ¼ 8.7 Hz, 1H), 7.95 (s, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.53 (dd,
J ¼ 6.2, 2.2 Hz, 1H), 7.38e7.30 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 5.76 (d,
J ¼ 9.8 Hz, 1H), 5.41 (s, 1H), 5.30 (d, J ¼ 6.2 Hz, 2H), 2.81e2.64 (m,
4H), 2.60e2.48 (m, 1H), 2.36 (td, J ¼ 14.1, 3.7 Hz, 1H), 2.17 (s, 3H),
2.02 (d, J ¼ 14.5 Hz, 1H), 1.92e1.82 (m, 1H), 1.70 (d, J ¼ 9.9 Hz, 3H),
1.63e1.54 (m, 1H), 1.52e1.42 (m, 1H),1.40 (s, 3H),1.36e1.23 (m, 3H),
0.95 (d, J ¼ 5.8 Hz, 3H), 0.79 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz,
(151 MHz, CDCl3)
d 157.55, 155.87, 154.38, 137.92, 134.85, 132.69,
130.88, 128.79, 124.59, 122.17, 121.01, 118.72, 116.62, 114.68 (s),
104.30, 100.23, 91.22, 80.41, 72.08, 70.88, 68.91, 51.61, 45.28, 37.37,
36.30, 34.19, 32.62, 29.30, 29.22, 25.99, 24.66, 22.73, 22.17, 20.26,
12.59; HRMS (ESI) Calcd. for C35H44ClFN3O6 [MþH]þ: 656.2903;
Found: 656.2902.
CDCl3)
d 170.72, 170.09, 169.89, 160.68, 156.42, 153.99, 151.59,
136.19, 136.03, 133.28, 129.17, 128.62, 126.94, 122.22, 120.84, 120.41,
116.26, 103.45, 91.32, 90.47, 79.10, 64.50, 50.52, 44.17, 36.22, 35.19,
33.05, 30.75, 28.10, 27.88, 24.91, 23.54, 22.64, 20.90, 19.18, 10.99;
HRMS (ESI) Calcd. for C36H40ClFN3O9 [MþH]þ: 712.2437; Found:
712.2440.
4.1.2. General procedure for preparation of hybrids 17a-17f
Alcohol
4 (1 eq.) and corresponding carboxylic acid in-
termediates 13e15 (1.2 eq.) were dissolved in dry dichloromethane
under argon respectively. Then N-(3-dimethylaminopropyl)-N0-
Hybrid 17e (74%): 1H NMR (400 MHz, CDCl3)
d 9.23 (s, 1H), 8.11

8
L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
(d, J ¼ 8.6 Hz, 1H), 7.96e7.86 (m, 2H), 7.50 (dd, J ¼ 6.4, 2.5 Hz, 1H),
7.35e7.27 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 5.77 (d, J ¼ 9.8 Hz, 1H),
5.42 (s, 1H), 5.28 (d, J ¼ 6.1 Hz, 2H), 2.59e2.50 (m, 1H), 2.47 (t,
J ¼ 7.3 Hz, 4H), 2.36 (td, J ¼ 14.1, 3.9 Hz, 1H), 2.17 (s, 3H), 2.06e1.94
(m, 3H), 1.92e1.83 (m, 1H), 1.78e1.68 (m, 3H), 1.61 (dt, J ¼ 13.8,
4.2 Hz, 1H), 1.53e1.43 (m, 1H), 1.40 (s, 3H), 1.37e1.22 (m, 3H), 0.95
(d, J ¼ 5.9 Hz, 3H), 0.82 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz,
11.93; HRMS (ESI) Calcd. for C35H41ClFN4O7 [MþH]þ: 683.2648;
Found: 683.2643.
Hybrid 22c (58%): 1H NMR (400 MHz, CDCl3)
d 9.28 (s, 1H), 8.71
(s, 1H), 8.41 (s, 1H), 8.02 (d, J ¼ 2.3 Hz, 1H), 7.88 (d, J ¼ 8.1 Hz, 1H),
7.71 (d, J ¼ 8.4 Hz,1H), 7.63e7.52 (m,1H), 7.15 (t, J ¼ 8.7 Hz,1H), 6.87
(d, J ¼ 9.3 Hz, 1H), 5.35 (d, J ¼ 12.9 Hz, 1H), 5.18e5.15 (m, 2H), 5.08
(s, 1H), 2.42e2.23 (m, 4H), 2.19e2.01 (m, 2H), 1.95 (d, J ¼ 14.5 Hz,
1H), 1.82 (d, J ¼ 13.5 Hz, 1H), 1.60 (d, J ¼ 10.9 Hz, 4H), 1.49 (dd,
J ¼ 9.6, 4.1 Hz, 2H), 1.43e1.21 (m, 6H), 1.21e1.11 (m, 2H), 1.09e0.97
(m, 1H), 0.92 (d, J ¼ 4.5 Hz, 3H), 0.69 (d, J ¼ 7.0 Hz, 3H); 13C NMR
CDCl3) d 171.42, 170.61, 169.88, 160.60, 156.41, 153.99, 151.59, 136.17,
136.10, 133.37, 129.07, 128.68, 126.83, 122.36, 120.88, 116.28, 103.45,
90.96, 90.48, 79.10, 64.32, 50.54, 44.20, 36.24, 35.19, 33.05, 32.05,
30.73, 24.91, 23.55, 22.68, 20.96, 19.19, 18.73, 11.10; HRMS (ESI)
Calcd. for C37H42ClFN3O9 [MþH]þ: 726.2594; Found: 726.2599.
(151 MHz, CDCl3)
d 173.26, 173.25, 158.04, 155.06, 154.85, 149.86,
135.48, 134.45, 132.75, 128.79, 124.92, 122.51, 121.38, 120.68, 116.42,
115.25, 104.34, 91.42, 80.29, 76.09, 65.99, 51.48, 45.28, 37.14, 36.15,
36.09, 33.85, 33.72, 32.20, 25.81, 24.46, 24.35, 24.17, 21.30, 20.16,
12.97; HRMS (ESI) Calcd. for C36H43ClFN4O7 [MþH]þ: 697.2804;
Found: 697.2801.
Hybrid 17f (71%): 1H NMR (400 MHz, CDCl3)
d 9.24 (s, 1H), 8.12
(d, J ¼ 8.5 Hz, 1H), 7.91 (d, J ¼ 8.5 Hz, 2H), 7.50 (dd, J ¼ 6.4, 2.4 Hz,
1H), 7.34e7.27 (m,1H), 7.18 (t, J ¼ 8.6 Hz,1H), 5.77 (d, J ¼ 9.8 Hz,1H),
5.42 (s, 1H), 5.28 (d, J ¼ 9.0 Hz, 2H), 2.59e2.48 (m, 1H), 2.45e2.31
(m, 5H), 2.17 (s, 3H), 2.02 (dd, J ¼ 13.9, 3.6 Hz, 1H), 1.93e1.84 (m,
1H), 1.74e1.66 (m, 7H), 1.63e1.56 (m, 1H), 1.53e1.44 (m, 1H), 1.41 (s,
3H), 1.37e1.21 (m, 3H), 0.96 (d, J ¼ 5.9 Hz, 3H), 0.82 (d, J ¼ 7.1 Hz,
Hybrid 22d (53%): 1H NMR (400 MHz, CDCl3)
d 9.23 (s, 1H), 8.10
(d, J ¼ 8.6 Hz, 1H), 7.94 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.53 (d,
J ¼ 4.1 Hz, 1H), 7.39e7.30 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 6.36 (d,
J ¼ 9.3 Hz, 1H), 5.38 (s, 1H), 5.31e5.27 (m, 3H), 2.86e2.74 (m, 1H),
2.72e2.61 (m, 1H), 2.57 (d, J ¼ 6.6 Hz, 2H), 2.37 (s, 2H), 2.17 (s, 3H),
2.01 (d, J ¼ 17.5 Hz, 2H), 1.87 (dd, J ¼ 7.2, 3.4 Hz, 2H), 1.69 (d,
J ¼ 12.2 Hz, 2H), 1.56 (d, J ¼ 13.3 Hz, 1H), 1.39 (s, 3H), 1.34e1.18 (m,
3H), 0.94 (d, J ¼ 5.8 Hz, 3H), 0.77 (d, J ¼ 7.0 Hz, 3H); 13C NMR
3H); 13C NMR (101 MHz, CDCl3)
d 171.79, 170.96, 169.85, 160.59,
156.39, 154.00, 151.60, 136.16, 133.42, 129.06, 128.69, 126.71, 122.37,
120.89, 120.32, 116.26, 103.44, 90.84, 90.48, 79.12, 64.27, 50.55,
44.22, 36.25, 35.20, 33.07, 32.75, 30.76, 24.93, 23.56, 23.20, 22.99,
22.67, 20.97, 19.19, 11.10; HRMS (ESI) Calcd. for C38H44ClFN3O9
[MþH]þ: 740.2750; Found: 740.2745.
(151 MHz, CDCl3)
d 172.31, 170.95, 170.93, 161.61, 157.40, 154.95,
152.54, 137.15, 134.25, 130.16, 129.55, 127.89, 123.10, 121.82, 121.36,
117.24, 104.31, 91.59, 80.38, 76.04, 65.37, 51.64, 45.51, 37.26, 36.22,
33.99, 32.64, 30.80, 29.10, 26.00, 24.56, 23.65, 21.56, 20.21, 12.92;
HRMS (ESI) Calcd. for C36H41ClFN4O8 [MþH]þ: 711.2597; Found:
711.2591.
4.1.3. General procedure for preparation of hybrid 22a-22f
Alcohol
4 (1 eq.) and corresponding carboxylic acid in-
termediates 19e21 (1.2 eq.) were dissolved in dry dichloromethane
under argon respectively. Then N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDCI, 2 eq.) and DMAP (0.5 eq.)
were added to above mixture at ambient temperature. The reaction
was monitored by TLC. After completion, the reaction was
quenched by saturated NaHCO3 aqueous solution and extracted by
ethyl acetate. The combined organic phase was then removed un-
der reduced pressure and purified by chromatography to give the
products 22a-22c. Products 22d-22f were prepared by the same
procedure as mentioned above with Alcohol 16 and carboxylic acid
intermediates 19e21 respectively.
Hybrid 22e (63%): 1H NMR (400 MHz, CDCl3)
d 9.23 (s, 1H), 8.11
(d, J ¼ 8.7 Hz, 1H), 7.95 (s, 1H), 7.90 (dd, J ¼ 8.7, 1.5 Hz, 1H), 7.51 (dd,
J ¼ 6.3, 2.5 Hz, 1H), 7.37e7.29 (m, 1H), 7.19 (t, J ¼ 8.6 Hz, 1H), 6.33 (d,
J ¼ 9.5 Hz, 1H), 5.38 (s, 1H), 5.35e5.23 (m, 3H), 2.46 (td, J ¼ 7.1,
1.9 Hz, 2H), 2.38e2.29 (m, 2H), 2.26 (td, J ¼ 7.3, 3.9 Hz, 2H),
2.21e2.15 (m, 3H), 2.02 (s, 1H), 1.97 (dd, J ¼ 14.4, 7.1 Hz, 2H), 1.87
(ddd, J ¼ 13.2, 6.3, 3.1 Hz, 1H), 1.80 (s, 1H), 1.76e1.66 (m, 2H),
1.60e1.53 (m, 1H), 1.50e1.39 (m, 2H), 1.37 (s, 3H), 1.29e1.18 (m, 2H),
0.95 (d, J ¼ 6.1 Hz, 3H), 0.78 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz,
Hybrid 22a (64%): 1H NMR (400 MHz, CDCl3)
d
9.43 (s, 1H), 8.77
CDCl3) d 171.69, 170.75, 170.15, 160.64, 156.49, 154.02, 151.51, 136.10,
(s, 1H), 8.60 (d, J ¼ 4.5 Hz, 1H), 7.85 (d, J ¼ 8.2 Hz, 1H), 7.76 (s, 1H),
7.55 (d, J ¼ 8.1 Hz, 1H), 7.17 (t, J ¼ 8.4 Hz, 1H), 6.66 (s, 1H), 5.59 (d,
J ¼ 13.4 Hz, 1H), 5.28 (d, J ¼ 13.9 Hz, 1H), 4.94 (t, J ¼ 10.0 Hz, 1H),
4.56 (s, 1H), 3.03 (d, J ¼ 14.6 Hz, 1H), 2.85e2.61 (m, 3H), 2.31e2.12
(m, 2H), 1.97 (t, J ¼ 24.0 Hz, 3H), 1.79 (d, J ¼ 7.8 Hz, 1H), 1.48 (d,
J ¼ 12.3 Hz, 1H), 1.33 (s, 1H), 1.28 (s, 3H), 1.18 (d, J ¼ 12.1 Hz, 1H), 1.07
(d, J ¼ 5.2 Hz, 1H), 0.91 (s, 4H), 0.80 (s, 1H), 0.47 (d, J ¼ 6.5 Hz, 3H),
133.52, 129.22, 128.64, 127.00, 122.61, 120.91, 120.33, 116.31, 103.28,
90.61, 79.39, 74.90, 64.28, 50.68, 44.56, 36.28, 35.24, 34.15, 33.02,
32.24, 31.62, 24.99, 23.59, 22.63, 20.61, 19.33, 19.22, 12.03; HRMS
(ESI) Calcd. for C37H42ClFN4O8Na [MþNa]þ: 747.2573; Found:
747.2570.
Hybrid 22f (58%): 1H NMR (400 MHz, CDCl3)
d 9.23 (s, 1H), 8.11
(d, J ¼ 8.6 Hz, 1H), 7.95e7.84 (m, 2H), 7.51 (dd, J ¼ 6.4, 2.5 Hz, 1H),
7.36e7.27 (m, 1H), 7.18 (t, J ¼ 8.6 Hz, 1H), 6.20 (d, J ¼ 9.2 Hz, 1H),
5.39 (s, 1H), 5.32 (t, J ¼ 10.1 Hz, 1H), 5.27 (s, 2H), 2.40 (s, 2H),
2.36e2.28 (m, 2H), 2.25e2.19 (m, 2H), 2.17 (s, 3H), 2.05e1.97 (m,
1H), 1.92e1.83 (m, 2H), 1.74e1.65 (m, 6H), 1.60e1.53 (m, 1H), 1.46
(dd, J ¼ 13.0, 3.1 Hz, 1H), 1.38 (s, 3H), 1.29e1.20 (m, 2H), 0.95 (d,
J ¼ 6.1 Hz, 3H), 0.78 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
0.38 (d, J ¼ 12.7 Hz, 1H); 13C NMR (101 MHz, CDCl3)
d 171.72, 171.04,
156.55, 153.46, 153.3, 148.22, 135.44, 134.16, 128.91, 127.41, 122.47,
120.05, 119.77, 116.25, 115.30, 114.36, 103.25, 90.21, 79.08, 75.29,
63.43, 50.32, 43.92, 36.07, 35.09, 32.69, 31.03, 29.80, 27.99, 24.86,
23.48, 19.93, 19.17, 11.68; HRMS (ESI) Calcd. for C34H39ClFN4O7
[MþH]þ: 669.2491; Found: 669.2484.
Hybrid 22b (67%): 1H NMR (400 MHz, CDCl3)
d
9.33 (s, 1H), 8.71
d 171.90, 171.20, 169.95, 160.58, 156.42, 153.99, 151.53, 136.22,
(s, 1H), 8.32 (s, 1H), 8.07e8.00 (m, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H),
7.68e7.56 (m, 2H), 7.15 (t, J ¼ 8.8 Hz, 1H), 6.66 (d, J ¼ 9.5 Hz, 1H),
5.31 (q, J ¼ 13.7 Hz, 2H), 5.08 (t, J ¼ 10.0 Hz, 1H), 4.99 (s, 1H),
2.58e2.47 (m, 1H), 2.42 (dd, J ¼ 14.8, 7.9 Hz, 1H), 2.37e2.20 (m, 4H),
2.00 (dt, J ¼ 20.6, 13.3 Hz, 4H), 1.82 (dd, J ¼ 7.6, 3.6 Hz, 1H), 1.56 (d,
J ¼ 10.5 Hz, 1H), 1.49e1.41 (m, 2H), 1.31 (s, 3H), 1.19e1.04 (m, 2H),
0.92 (d, J ¼ 5.5 Hz, 3H), 0.89e0.74 (m, 2H), 0.68 (d, J ¼ 7.0 Hz, 3H);
136.14, 133.44, 129.12, 128.64, 126.85, 122.34, 120.88, 120.31, 116.28,
103.29, 90.64, 79.41, 74.89, 64.23, 50.67, 44.56, 36.28, 35.24, 35.15,
33.01, 32.80, 31.67, 25.01, 23.61, 23.58, 23.38, 22.68, 20.62, 19.23,
12.05; HRMS (ESI) Calcd. for C38H45ClFN4O8 [MþH]þ: 739.2910;
Found: 739.2903.
4.1.4. Preparation of hybrid 32
13C NMR (101 MHz, CDCl3)
d
172.01, 171.60, 157.19, 153.94, 153.80,
To a solution of dihydroartemisinin 8 (310.9 mg, 1.09 mmol) and
(S)-alkamine 29 (100 mg, 0.729 mmol) in dry dichloromethane
under argon was added BF3$Et2O (155.2 mmol, 1.09 mmol) drop-
wise at 0 ꢀC. The mixture was then stirred overnight at this
148.35, 134.51, 133.86, 130.69, 127.43, 124.27, 121.89, 119.69, 118.76,
115.35, 114.27, 103.37, 90.45, 79.29, 75.20, 64.33, 50.47, 44.26, 36.16,
35.16, 34.60, 32.82, 32.18, 31.13, 24.88, 23.49, 20.25, 19.31, 19.14,

L.-L. Wang et al. / European Journal of Medicinal Chemistry 182 (2019) 111665
9
temperature and quenched with saturated NaHCO3 aqueous solu-
tion. The reaction mixture was then extracted with ethyl acetate.
After combining with organic phase and dried over Na2SO4, the
solvent was removed under reduced pressure to give the crude
Animal Ethics Committee of Kunming Institute of Botany (Kunm-
ing, China). Three-week-old female BALB/C nude mice, purchased
from Vital River Laboratory Animal Technology (Beijing, China) and
kept in a pathogen-free environment, were used to establish the
HCT116 xenograft model. Briefly, 3 ꢁ 106 HCT116 cells were sub-
cutaneously injected into the right flank of nude mice for tumor
formation. Each group consisted of 8 mice. Animals were daily
treated by intraperitoneal injection for 17 days. Tumor size was
measured in two dimensions with a digital caliper and calculated
using the formula (length ꢁ width ꢁ width ꢁ 0.5). At the end of the
experiment, all mice were sacrificed. Tumors were resected,
weighed and photographed.
product. The pure b-isomers could be predominately isolated by
flash chromatography to afford the amine 30 (118 mg, 40%). To a
solution of amine 30 (118 mg, 0.292 mmol) and carboxylic acid 31
(112 mg, 0.35 mmol) in dry DMF under argon were added DMAP
(36 mg, 0.292 mmol), EDCI (84 mg, 0.438 mmol) and DIPEA (95 mg,
0.735 mmol) at room temperature. The reaction mixture was stir-
red at 40 ꢀC overnight. Then the reaction was quenched by satu-
rated NaHCO3 aqueous solution and extracted by ethyl acetate. The
combined organic phase was then removed under reduced pres-
sure and purified by chromatography to give the hybrids 32
(86.9 mg, 42%).
Conflict of interest
Hybrid 32: 1H NMR (600 MHz, Acetone-d6)
d
9.50 (s, 1H, NH),
The authors declare that we have no conflict of interest.
Acknowledgements
8.85 (s, 1H, H50), 8.70 (s, 1H, H80), 8.53 (d, J ¼ 7.08 Hz, 1H, CONH),
8.36 (dd, J ¼ 1.62, 8.64 Hz, 1H, H110), 8.31 (dd, J ¼ 2.55, 6.75 Hz, 1H,
H130), 7.94e7.92 (m,1H, H170), 7.86 (d, J ¼ 8.64 Hz,1H, H100), 7.50 (d,
J ¼ 7.50 Hz, 2H, H220, H180), 7.36e7.32 (m, 3H, H160, H190, H210), 7.25
(t, J ¼ 7.35 Hz, 1H, H200), 5.61 (s, 1H, H12), 5.32 (dd, J ¼ 5.70,
12.54 Hz, 1H, H20), 4.73 (d, J ¼ 9.36 Hz, 1H, H10), 4.10 (d, J ¼ 5.52 Hz,
2H, H10), 2.36e2.33 (m, 1H, H9), 2.24 (dt, J ¼ 3.72, 13.97 Hz, 1H,
H4a), 1.95e1.93 (m, 1H, H4b), 1.82e1.79 (m, 1H, H5b), 1.72e1.70 (m,
1H, H8a), 1.66e1.63 (m, 1H, H7b), 1.51 (td, J ¼ 4.20, 13.63 Hz, 1H,
H8a0), 1.44e1.40 (m, 1H, H8b), 1.34e1.26 (m, 2H, H6, H5a), 1.19 (t,
J ¼ 7.11 Hz, 1H, H5a0), 1.12 (s, 3H, H14), 1.01e0.95 (m, 1H, H7a), 0.88
(d, J ¼ 6.36 Hz, 3H, H15), 0.81 (d, J ¼ 7.08 Hz, 3H, H16); 13C NMR
We thanks for the financial support from the National Natural
Science Foundation of China [Grant No. 21772207]; CAS‘Light of
West China’Program and Youth Innovation Promotion Association
CAS; “The Thousand Talents Program”of Yunnan Province (L.-L.
Wang); and the Strategic Priority Research Program of the Chinese
Academy of Sciences [Grant No. XDA12030206].
Appendix A. Supplementary data
(151 MHz, Acetone-d6)
d
166.09 (C30), 159.19 (C70), 156.42 (C80),
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111665.
155.08 (d, J CeF ¼ 244.0 Hz, C150), 152.61 (C90), 141.53 (C230), 137.39
(d, J CeF ¼ 3.2 Hz, 1C120), 133.58 (C40), 131.94 (C11’), 129.28 (100),
128.99 (2C, C190, C210), 128.18 (2C, C180, C220), 127.91 (C200), 124.66
(C130), 123.39 (C50), 123.08 (d, J CeF ¼ 6.9 Hz, C170), 120.59 (d, J
CeF ¼ 18.5 Hz, C140), 117.29 (d, J CeF ¼ 23.4 Hz, C160), 115.67 (C60),
104.78 (C3), 101.23 (C10), 91.97 (C12), 80.95 (C12a0), 71.79 (C10),
55.35 (C20), 52.30 (C5a0), 46.15 (C8a’), 37.88(C6), 36.84 (C4), 34.93
(C7), 32.89(C9), 25.92 (C14), 25.33(C5), 22.48 (C8), 20.41 (C15),
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