New modification strategy of matrine as Hsp90 inhibitors based on its specific L conformation for cancer treatment

Article abstract of DOI:10.1016/j.bmc.2020.115305

The similarity of spatial structure between radicicol and matrine urged us to perform conformation modification of matrine, followed by L-shaped matrine derivatives, 6, 12, 21a-h and 22a-h were originally designed, synthesized and evaluated for Hsp90^N inhibitors as anticancer agents. TSA (Thermal Shift Assay) results indicated that 21e, 22a-c and 22e-g exhibited strong binding force against Hsp90^N with∣ΔTm∣ > 3, meanwhile, MTT assay also revealed these compounds displayed potent anticancer activity with IC₅₀ values below 25 μM against HepG2, HeLa and MDA-MB-231 cells lines. Then, compound 22g with a high ΔTm = 10.92 was chosen as a representative to perform further mechanism study. It can induce cell apoptosis, arrest the cell cycle at the S phase and decrease the expression level of Hsp90 in Hela cell. These results originally provided targeted modification strategy for matrine derivatives to serve as Hsp90 inhibitors for cancer therapy.

Full text of DOI:10.1016/j.bmc.2020.115305

Journal Pre-proofs
New modification strategy of matrine as Hsp90 inhibitors based on its specific
L conformation for cancer treatment
Yiming Xu, Dewang Jing, Dong Zhao, Yongji Wu, Lu Xing, Haroon ur
Rashid, Haodong Wang, Lisheng Wang, Huiling Cao
PII:
S0968-0896(19)31415-4
https://doi.org/10.1016/j.bmc.2020.115305
BMC 115305
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 August 2019
22 December 2019
2 January 2020
Please cite this article as: Y. Xu, D. Jing, D. Zhao, Y. Wu, L. Xing, H. ur Rashid, H. Wang, L. Wang, H. Cao,
New modification strategy of matrine as Hsp90 inhibitors based on its specific L conformation for cancer
treatment, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115305
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New modification strategy of matrine as Hsp90 inhibitors based on
its specific L conformation for cancer treatment
Yiming Xua,c,†, Dewang Jing , Dong Zhao , Yongji Wu , Lu Xing , Haroon ur
c,†
b
d
b
c,e
c
a,
b,
Rashid , Haodong Wang , Lisheng Wang * and Huiling Cao *
a
Medicinal College, Guangxi University, Nanning 530004, China;
b
Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Institute of Basic Translational Medicine, Xi’an
Medical University, Xi’an 710021, China;
c
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China;
d
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
e
Department of Chemistry, Sarhad University of Science & Information Technology, Peshawar, Khyber
Pakhtunkhwa 25120, Pakistan;
*
Correspondence: lswang@gxu.edu.cn; caohuiling_jzs@xiyi.edu.cn;
†
These authors contributed equally to this work;
Abstract
The similarity of spatial structure between radicicol and matrine urged us to perform
conformation modification of matrine, followed by L-shaped matrine derivatives, 6, 12, 21a-h and
N
2
2a-h were originally designed, synthesized and evaluated for Hsp90 inhibitors as anticancer
agents. TSA (Thermal Shift Assay) results indicated that 21e, 22a-c and 22e-g exhibited strong
N
binding force against Hsp90 with ∣ △Tm ∣ > 3, meanwhile, MTT assay also revealed these
compounds displayed potent anticancer activity with IC values below 25 μM against HepG2, HeLa
5
0
and MDA-MB-231 cells lines. Then, compound 22g with a high △Tm = 10.92 was chosen as a
representative to perform further mechanism study. It can induce cell apoptosis, arrest the cell cycle
at the S phase and decrease the expression level of Hsp90 in Hela cell. These results originally
provided targeted modification strategy for matrine derivatives to serve as Hsp90 inhibitors for
cancer therapy.
Keywords: L-shaped matrine derivatives; Radicicol; Thermal Shift Assay; Anticancer activity;
Mechanism of action;
Introduction
Matrine (Fig. 2d), a quinolizidine natural product extracted from Sophora flavescens (Kushen)
[1-4], was approved by Chinese FDA in 1995 for cancer treatment. Currently, matrine has been
widely used to adjuvant treatment of liver cancer, lung carcinoma and gastric cancer with low side
effects for many years in China [5-6]. Although large-scale application of matrine with reference to
its use for the treatment of cancer was confirmed [7-11], its activity is moderate, which might be
caused by its speculated features such as broad binding sites but low specificity. Thus, enhancing
the sensibility and specificity against anticancer targets of matrine attracted our attention. On the
other hand, matrine possess many advantages such as high solubility, good safety profiles and a
special chemical scaffold, suggesting that it is an ideal lead compound for further modifications and
optimizations to treat cancer.

Recently, our group also conducted a comprehensive literature review (Fig. 1) on modification
strategy of matrine [12]. As illustrated in Fig. 1, lots of matrine derivatives have been synthesized
through various modifications on different positions of matrine. However, up to now, research on
targeted design for matrine derivatives is still not be involved which caused blindness for their
development. Different from traditional cytotoxic chemotherapy, targeted therapy has many
advantages, such as high specificity, good efficacy and low toxicity. Inspired by above suggestions,
targeted design for matrine might be an effective strategy to discover anticancer candidates of
matrine derivatives.
Introduction of double bond, aromatic ring and
appropriate nitrogen-carrying heterocyclic groups
on 14-position of matrine can substantially
improve the anticancer activities of target
compounds
Opening the lactam ring decreases the
Introduction of an appropriate substituent
activity, principally for derivatives with
particularly EWGs at the C-13 position
5
R configuration, but incorporation of an
and/or C-14 position, could increase the
anticancer activity
aliphatic acyl or a chlorobenzyl group at
the N-16 position, or a nitric oxide group
on the carboxyl chain might considerably
increase the anticancer activity
1
8
14
Incorporation of substituents containing
N or S atom at C-13 position of matrine
and its derivatives could improve the
antiproliferative activities of the target
compounds.
O
15
1
3
1
6
6
H
12
The activities of matrine derivatives
with 5S configuration is more
encouraging than those having 5R
configuration
N
H
1
7
11
7
5
4
3
8
H
H
N
1
9
2
10
O
Presnce of oxygen atom is not necessary to
improve anticancer activities of the target
matrine derivatives
Fig. 1 Modification strategy of matrine and SAR study reported in literature review [12]
Interestingly, the specific L-shaped conformation of matrine is similar with radicicol (Fig. 2).
N
Besides, radicicol serves as a Hsp90 inhibitor and its molecular mechanism has been thoroughly
studied, which provided possibility for structural modification of matrine derivatives to target
N
Hsp90 based on radicicol conformation analysis. Moreover, the overexpressed characteristics in
tumor cells and the prospect of stalling a number of oncogenes by inhibiting a single protein have
made Hsp90 an attractive target for cancer therapy [13-15]. Thus, by targeted design and inhibiting
the activity of Hsp90, it might be an effective strategy for matrine modifications to treat cancer.

Fig. 2 L-shaped 3D conformation of radicicol (a) and matrine (b); Molecular structures of radicicol (c) and matrine
d).
(
Drug design
N
Radicicol, as the Hsp90 inhibitor, its mechanism involves a competition with ATP at N-terminal
ATP-binding pocket of Hsp90, resulting in the inactivation of protein [16-17]. The co-crystal
structures [18] revealed that the binding mode of planar moiety in radicicol with N-terminal ATP-
binding pocket is similar to that of adenine moiety in ATP, through which it can directly form
hydrogen bond with residues or via a water-mediated network of hydrogen bonds (Fig. 3a, c) and
then “occupy” the adenine aisle. Another moiety of the radicicol mainly participated in the specific
L-conformation formation. We speculated that the specific conformation can not only “capture” the
planar moiety to the suitable site at the aisle without penetrating deep into it, but also “close” the
ATP entrance (Fig. 3b, d).
(a)
(b)

(c)
(d)
Fig. 3 (a) Binding mode of ATP with Hsp90 and (b) Modeling of ATP represented as a surface [PDB code: 3T0Z];
(c) Binding mode of radicicol and (d) Modeling of radicicol represented as a surface [PDB code: 4EGK].
The lack of planar structure with hydrophilic and halogen groups is the main difference of
scaffolds between matrine and radicicol (Fig. 2c, d), which act as a targeted warhead to N-terminal
ATP-binding pocket. Additionally, the spatial L conformation of matrine similar with radicicol (Fig.
2
a, b) could block the cavity to prevent ATP entrance. Thus, based on radicicol structure and its
mechanism, planar structure with hydrophilic group and halogen group was introduced into matrine
to enhance the targeting effect towards Hsp90, meanwhile, the hybridization should keep derivatives
in L conformation.
In present study, two modification strategies were adopted. First, we hypothesized that the
variation in size of the conformation might influence the activity. Thus, enlargement and contraction
were performed via D-ring modifications of matrine. Second, planar structure with substituents was
built on matrine scaffold (Scheme 1.).
1
2
. Participate in L conformation
. Possess hydrophilic group
OH
O
and halogen atom
O
O
HO
H
H
O
Cl
O
N
Radicicol
H
H
X=C, N
R=OH or NH2
H
H
N
L-conformation
size variation
N
R
O
H
Hybridization
X
H
H
N
H
H
H
N
H
O
H
N
Matrine
N
H
H
H
N
Scheme 1. The modification strategy for L-shaped matrine derivatives based on radicicol.
Chemistry

The synthetic route of lactam D ring contraction for the synthesis of derivative 6 is depicted in
Scheme 1. Matrine was first treated with n-butylithium to gain carbanion intermediate, followed by
its substitution with 2, and oxidation via IBX to gain 3. Then 3 was subjected to elimination, using
K CO in toluene under reflux to produce sophocarpine 4 [19]. β-amino acid 5 was obtained via the
2
3
cleavage of lactam and alkenyl bond in 4 by its treatment with KMnO in 10% H SO aqueous.
4
2
4
Finally, after screening, Mukaiyama’s reagent [20] was used to activate the carboxyl group for the
formation of β-lactam of 6.
O
S
O
H
O
H
O
H
H
H
H
H
H
H
H
N
N
H
N
H
N
H
OH
a
b
c
S
H
H
S
O
H
H
N
N
N
N
1
2
3
4
5
O
N
d
H
H
H
H
N
6
Scheme 2. Reagents and conditions: (a) (i) diisopropylamine, n-butylithium, matrine, THF, -78 °C to 25 °C, 1h;
diphenyl disulfide, 2h; (ii) IBX, HCl aqueous, 50 °C~70 °C, 3h; (b) K CO , toluene, reflux, 1.5 h; (c) 10% H SO
aqueous, KMnO , reflux, 2 h; (d) 2-chloro-1-methylpyridinium iodide, Et N, CH Cl /1, 4-dioxane, rt, overnight.
IBX = 2-iodoxybenzoic acid.
2
3
2
4
4
3
2
2
The synthetic route of 12 is shown in Scheme 3. Acid 7 was prepared by hydrolysis of matrine
in aqueous KOH under reflux, followed by acidification with HCl. Then, fluorenylmethoxy
carbonyl, as a protecting group, was introduced on nitrogen atom to produce 8. Thionyl chloride
was added to dichloromethane solution of 8 to form corresponding acyl chloride followed by
treatment with TMSCHN , which facilitated the diazotization smoothly in relatively high yield and
2
purity. With silver oxide as a catalyst, α-diazoketone 9 was transformed to acid 10 through Wolff
rearrangement [21]. Deprotection of 10 was completed in 10 % NaOH aqueous to obtain 11. Finally,
HATU was verified as an efficient polypeptide condensation reagent for the synthesis of target 12
working at low concentrations of 11 in order to suppress possible intermolecular side reactions [22].
O
Fmoc
N
Fmoc
H
H
N
N
H
OH
OH
N
a
b
c
N2
H
H
H
H
H
H
O
O
O
H
H
H
H
H
H
H
H
N
N
N
N
1
7
8
9
Fmoc
N
O
H
N
COOH
COOH
f
d
e
N
H
H
H
H
H
H
H
H
H
H
H
N
N
N
1
0
11
12
2 3
Scheme 3. Reagents and conditions: (a) 5 N NaOH aqueous, reflux, overnight; HCl, pH to 5~6; (b) Na CO , Fmoc-Cl,

1
2
, 4-dioxane/H
2
O, rt, 6~8 h; (c) (i) SOCl
2
, CH
2
Cl
2
, reflux, 1~2 h, N
2 2 2 2
protection; (ii) TMSCHN , CH Cl , -15 °C~-
0 °C, 1~2 h; (d) Ag
2
O, Na CO , NaHSO
2
3
3
, 1, 4-dioxane/H
2
O, 50 °C~60 °C, 2~3 h; HCl, pH to 5~6; (e) 10 % NaOH
aqueous, rt, 1~2h; HCl, pH to 5~6; (f) HATU, DIPEA, THF/DCM, rt, overnight.
Aiming at the synthesis of quinolionmatrinic derivatives, a retrosynthetic analysis is first given
in Scheme 4. The target compound 13 could be disconnected directly at 14-position to produce
synthon 14. It is notable that compound 13 can be transformed to 16 by functional group addition
strategy. Then the double bond fragment of 16 could be formed by a base-mediated cyclization of
an amidine 17 (Friedländer type cyclization), bearing an aromatic ester or a nitrile (R) functional
group. Considering the importance of amino and hydroxyl groups as hydrogen bond donor in
medicinal field, compound 17 was chosen as an intermediate synthon. Finally, compound 17 can be
disconnected to produce starting material 1 and 18.
CHO
O
N
N
N
H
H
H
H
CHO
H
H
NH2
N
H
H
N
N
H
1
4
1
15
N
H
H
Y
R
H
O
H
H
N
H
X
H
N
N
N
N
H
H
H
13
R
H
H
H
NH2
H
N
H
N
N
18, R = CN, CO2R'
1
6, Y = NH , OH
17, R = CN, CO2R'
1
2
Scheme 4. Retrosynthetic analysis of ring-constrained quinolinomatrinic derivatives.
As depicted in Scheme 5, matrine was treated with POCl to afford the corresponding α-
3
choroiminiums, which react efficiently with nucleophiles, including amines. However, the
formation, as an intermediate, of a highly strained bridgehead iminium species remains challenging
[
23]. Suitable reaction conditions were gained after screening (POCl 2 eq., CH Cl , reflux for 2.5
3 2 2
h) in order to obtain a useful α-chloroenamine intermediate 19. Considering the role of Schiff base
in pharmaceutical activities, such as antibacterial [24-25], antihypertensive [26], anti-inflammatory
[27] and anticancer [28-30], a series of aromatic amine was first reacted with matrine to yield the
intermediate products 21a-h. Subsequently, products 20a-h were synthesized using anilines bearing
cyan derivatives 18a-h. The crystal data and refinement details of 21g (CCDC 1819315) revealed
the newly synthesized 21a-h have E-configuration (Fig. 4). Then, Lithium diisopropylamide (LDA)
was chosen as a base to mediate the cyclization of 20a-h to produce the corresponding products
2
2a-h.

R
N
H
CF3
NH2
CN
O
H
Cl
R
N
H
H
H
H
N
H
N
H
c
a
N
H
d
N
H
H
H
CN
H
NH2
H
H
H
N
N
N
N
1
19
18a-h
20a-h
22a-h
b
2
2
2
2a: R=H
2b: R=Me
2c: R=OMe
R
N
2
2
2
2
2
2
2
2
1a: R=phenyl
1b: R=4-Cl-phenyl
1c: R=4-OEt-phenyl
1d: R=4-iPr-phenyl
1e: R=4-Ph-phenyl
1f: R=4-OPh-phenyl
N
22d: R=F
22e: R=Cl
H
H
2
2
2
2f: R=Br
H
H
2g: R=CF3
N
1g: R=2-CH -3-NO -phenyl
3
2
2h: R=NO2
1h: R=2-CN-phenyl
21a-h
Scheme 5. Reagents and conditions: (a) POCl , DCM, 60 °C, 2~3 h; (b) aniline, DCM, reflux, 12 h; NaOH aqueous,
3
pH to 8~9; (c) DCM, reflux, 12 h; NaOH aqueous, pH to 8~9; (d) LDA, anhydrous THF, 0 °C;
Fig. 4 Crystal structure of compound 21g represented as ORTEP diagrams.
Thermal Shift Assay
N
Table 1. △Tm (°C) values of matrine and its derivatives binding with Hsp90 measured by Thermal
Shift Assay.
Ligand
6
△Tm Mean (°C)
SD (n=8)
0.28
NT
0.44
NT
1
2
2
1a
1b
1c
1d
1e
1f
-0.57
NS
0.32
NS
2
2
0
0.30
0.40
0.39
0.32
2
-1.24
-4.91
-1.30
2
2

2
2
1g
1h
-1.06
-1.44
-3.12
-4.27
-5.48
-2.30
-6.58
-7.58
0.32
0.30
0.40
0.40
0.36
0.42
0.28
0.36
2
2a
2b
2c
2d
2e
2f
2
2
2
2
2
2
2
2g
2h
10.92
-0.24
0.04
0.60
0.28
0.39
0.29
Matrine
Radicicol
21.32
NT means not tested; NS means no fluorescence signal.
TSA (Thermal Shift Assay) is a method for detecting thermal stability of proteins, which
quantitative real-time PCR is used to measure the melting curves of proteins with or without ligands
and calculate Tm and △Tm [31-33]. The larger | △Tm | means stronger binding force between
N
Hsp90 and ligand, whose | △Tm | > 3 was usually considered as a ligand. TSA results indicated
N
N
the Tm of Hsp90 was 47.63 ± 0.35 °C (Fig. 5a). After Hsp90 binding with matrine and its
derivatives, the Tm of all the complexs shifted to different degrees. Thereinto, the thermal shift of
N
matrine, 6, 21c and 22g binding with Hsp90 performed positive deviation with △Tm＞0, indicating
N
improvement in the Hsp90 thermal stability. While other compounds reduced the thermal stability
of protein with △Tm＜0 (Table 1).
N
On the other hand, there existed nine ligands made the Tm shift of Hsp90 more than 3 °C, among
which 22a, 22b and 21e possess | △Tm | 3-5 (orange); 22c, 22e and 22f possess | △Tm | 5-8 (green);
importantly, 22g possess | △Tm | > 10 (red) (Fig. 5c). Meanwhile, the radicicol possess | △Tm | >
2
0 (Fig. 5d). Moreover, matrine possess weak binding force with Hsp90 (Fig. 5b). The results
N
preliminary illustrated the nine ligands exhibited strong binding force and targeted to Hsp90 . It
was originally confirmed that Hsp90 can serve as one directly target for matrine derivatives,
providing theoretical basis for structural optimizations and exploring anticancer pathways of
matrine.

N
N
Fig. 5 Melting curves of the Hsp90 (a), matrine binding with Hsp90 (b), 22g binding with
N
N
Hsp90 (c) and radicicol binding with Hsp90 (d).
Anticancer activity in vitro
Table 2. IC values of compounds 6, 12, 21a-h and 22a-h against three cancer cells.
5
0
IC (μM)
5
0
Entry
R
HeLa
>100
HepG2
>100
MDA-MB-231
>100
6
1
2
>100
>100
>100
2
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
2g
H
>100
>100
>100
2
4-Cl
>100
>100
73.4 ± 8.3
>100
2
4-OEt
4-iPr
4-Ph
4-OPh
>100
97.5 ± 2.6
96.6 ± 4.3
11.0 ± 0.5
51.3 ± 4.5
86.1 ± 8.9
>100
2
93.3 ± 1.5
23.7 ± 1.3
33.7 ± 2.5
>100
77.5 ± 3.7
24.6 ± 2.1
55.2 ± 5.4
>100
2
2
2
2-CH -3-NO
3
2
2
2-CN
H
>100
>100
2
23.3 ± 1.4
13.4 ± 0.5
18.0 ± 1.2
18.8 ± 3.3
8.9 ± 1.2
6.9 ± 0.8
19.6 ± 0.4
14.3 ± 2.1
9.2 ± 1.2
10.9 ± 2.9
13.1 ± 1.9
6.4 ± 0.3
9.7 ± 2.1
16.1 ± 3.2
22.2 ± 1.8
16.9 ± 1.5
21.2 ± 4.2
37.3 ± 2.5
14.3 ± 0.8
9.7 ± 1.5
22.1 ± 1.2
2
Me
OMe
F
2
2
2
Cl
2
Br
2
CF3

2
2h
NO2
69.2 ± 4.6
4332 ± 423
19.2 ± 2.1
35.1 ± 1.9
4509 ± 224
8.4 ± 3.4
24.4 ± 3.4
5372 ± 327
48.9 ± 3.1
Matrine
Cisplatin
a
IC : a concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the
5
0
mean IC from the dose-response curves of at least three independent experiments.
5
0
Keeping in view the importance of Hsp90 as an attractive target for cancer therapy, the anticancer
activity of all the target compounds against HepG2, HeLa and MDA-MB-231 cell lines was
evaluated by MTT assay using cisplatin as positive drug control. Among the arylimidomatrinic
derivatives 21a-h, compounds 21e exhibited potent activity against three cell lines as compared to
matrine. While among quinolinomatrinic derivatives 22a-h, compounds 22a-g exhibited good
activity with their IC below 25 μM against HepG2, HeLa and MDA-MB-231 cell lines. Notably,
5
0
N
these compounds also showed strong binding force with Hsp90 (Table 1). These results also
N
indicated the possibility of matrine modifications to target Hsp90 leading to potent activity for
cancer treatment.
Structure-activity relationship (SAR) analysis of matrine derivatives was also performed. The
TSA and MTT results of compounds 6, 12 indicated the L-conformation change was made less
effective via ring size variation. Among intermediate arylimidomatrinic derivatives, only 21e
N
showed potent anticancer activity and strong binding force with Hsp90 , which indicated that the
introduction of biphenyl could take an effective function. We speculated that the hydrophobic effect
and extensibility of the molecular structure might be factors that affected the binding mode and
biological activity. Structure-activity relationship (SAR) analysis was then focused on the
quinolinomatrinic derivatives 22a-h. Interestingly, all the compounds exhibited potent anticancer
activities. Particularly, the IC values of compounds 22a-g were below 25 μM. The general increase
5
0
N
in activity and binding force with Hsp90 might be associated with the hydrophobic effect and
building of L-conformation with the participation of quinoline ring. Besides, the R substituents
could work on the activity and TSA results. Compounds 22b, 22c (R= electron-donating groups)
and 22e, 22f (R=electron-withdrawing groups) all showed better MTT and TSA results than 22a,
which indicated inoperative electronic effects caused by the R substituents. We deduced that the R
substituents also participated in L-conformation or exhibited hydrophobic effect and hydrogen bond
N
interaction to react with Hsp90 . In order to better understand the binding mode and mechanism of
N
action of the synthesized derivatives, 22g having the strongest binding force with Hsp90 and potent
anticancer activity was chosen for further study.
Docking

(a)
(b)
Fig. 6 (a) Binding mode of compound 22g with Hsp90 and (b) Modeling represented as surface. [PDB code:
UYM]
1
N
The docking results of compound 22g with Hsp90 were depicted in Fig. 6. It’s obvious that the
quinoline ring moiety of 22g could insert into the active cavity and its tert-nitrogen atom could form
a water-mediated network of hydrogen bonds with PHE-138, ASN-51, GLY-137, GLY-135 and
ILE-110. Similarly, the amino group on the quinoline ring, as a hydrogen donor, also interacted with
ASN-51 and ASP-54 via water molecule. In regard to trifluoromethyl substituent, it not only can
exhibit hydrophobic effect, but also form hydrogen bond with TYR-139 (Fig. 6a). More importantly,
the planar quinoline ring and trifluoromethyl substituent participated in the L-conformation
formation with matrine structure, which was analogous to radicicol. The specific conformation
could enable the planar moiety to “occupy” the aisle and “close” the ATP entrance by matrine
moiety (Fig. 6b). The binding mode originally provides theoretical basis for designing novel matrine
derivatives as Hsp90 inhibitors.
Cell cycle analysis
We further analyzed the impact of compound 22g upon cell cycle distribution. After treatment
for 48h, compound 22g induced obvious S phase arrest in HeLa cells (Fig. 7). In control group, the
cells in S phase represented 17.24%, in the presence of 5, 10 and 20 μM of compound 22g, it
increased to 21.64%, 26.67%, and 40.12%, respectively, in a concentration dependent manner.
Accordingly, there was a marked decrease in the percentage of cells in the G0/G1 phase after
compound 22g treatment. These results revealed that compound 22g arrested the HeLa cell cycle at
the S phase.
Fig. 7 Compound 22g induces S phase arrest in HeLa cells. HeLa cells exposure to 5, 10 and 20 µM of compound
2
2g for 48 h, detection and analysis of cell cycle by flow cytometry.

Cell apoptosis
Given compound 22g could inhibit Hsp90 activity, resulting in the paramorphia of the clients
including protein kinase and transcription factors associated with signals of tumor growth and
metastasis. We further investigated the mechanism of compound 22g to induce HeLa cells death
using annexin-V (AV)/PI binding assay. Compared with the control group, HeLa cells treated with
compounds 22g exhibited an accumulation of early and late apoptotic cells in a time and dose
dependent manner (Fig. 8). For example, the percentage of early apoptotic cells was 3.22% in
untreated cells, while it was 17.51%, 28.57% and 38.07% in the cells treated with 22g at 5, 10, and
2
0 μM for 48 h, respectively. Also, the percentage of late apoptotic cells showed similar change
trend with corresponding percentages of 8.79%, 17.30% and 21.17% in cells treated with 5, 10 and
0 μM of 22g for 48 h, respectively.
2
Fig. 8 Compound 22g induced apoptosis in HeLa cells. AV/PI assay for the detection of apoptotic HeLa cells after
treatment with 5, 10 and 20 μM of 22g for 48 h.
Western bolt analysis
After treatment with compound 22g for 48 h, the levels of apoptosis protein Bax, Bcl-2, caspase
3
and Hsp90 of HeLa cells were detected by western blotting assay. As shown in Fig. 9, the
immunoblot results confirmed that treatment with compound 22g decreased Bcl-2 expression, and
increased Bax in a concentration-dependent manner. Moreover, the proportion of Bax/Bcl-2
increased. Additionally, caspase-3 is an executioner caspase which modifies proteins ultimately
responsible for apoptosis. Consistent with the above PI-FITC/annexin assay, the results showed that
the expression of cleaved caspase-3 was substantially increased, while protected caspase-3
decreased in a dose-dependent manner in HeLa cells treated with 22g, respectively. Besides,
compound 22g performed potent binding force with Hsp90 measured by thermal shift assay and
level of Hsp90 in HeLa cells was detected after their exposure to increasing concentrations of 22g.
The downregulation of Hsp90 was in accordance with the inhibition of 22g.

Fig. 9 HeLa cells were treated with 12.5 μM, 25 μM and 50 μM of compound 22g or vehicle (DMSO) for 48h. As
shown in the immunoblots. Cell lysates were prepared, equal amount of protein was probed with antibodies specific
for Bax, Bcl-2, CLcasp-3, PRcasp-3 and Hsp90. GAPDH was used as a reference.
Conclusion
Using Hsp90 as an effective target to design inhibitors for cancer therapy enhanced antitumor
potency and might help to overcome resistance. In the present study, not only is this the first design
for matrine derivatives via a L-conformation transform, the results also originally verified matrine
derivatives can serve as Hsp90 inhibitors. Most of L-conformation compounds 22a-22h showed
N
strong binding force indicated by∣△Tm∣> 3 with Hsp90 and potent anticancer activity with
IC below 25 μM. Besides, compound 22g was chosen as a representative for further evaluations.
5
0
N
Docking results indicated that the binding mode of 22g with Hsp90 was similar to radicicol,
illustrating the rationality of the design strategy. Moreover, 22g can induce cell apoptosis, arrest the
cell cycle at the S phase in HeLa cells. Western blot assay indicated that 22g increased the
expression of Bax and cleaved-caspase 3, as well as downregulated the levels Bcl-2, pro-caspase 3
and Hsp90. Taken together, all these comprehensive evaluations warranted matrine as a promising
lead compound worth further optimization and development as Hsp90 inhibitors for cancer therapy.
Experimental protocols
Chemistry
Matrine was purchased from Shanxi Undersun Biomedtech Co., Ltd.- All other chemicals and
reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. Thin Layer Chromatography
(TLC) from Yantai Xinnuo chemical Co. Ltd was used for monitoring the operation process and
visualized by UV light (254 nm) or Wagner’s reagent spray. Column chromatography was
performed for purification on silica gel (300-400 mesh). Melting points were recorded on a WRS-
B digital melting-point apparatus without correction. H-NMR, 13C-NMR and 19F-NMR spectra
1
1
were recorded on a Bruker Advance 600 (600 MHz) spectrometer. Chemical shifts are reported in
ppm with TMS as an internal reference and J values are given in Hertz. Signals are abbreviated as
singlet, s; doublet, d; double-doublet, dd; triplet, t; quartet, q; multiplet, m. ESI-MS spectra were
recorded on a WATERS UPLC I-CLASS-XEVOG2-XSQTOF electro-spray ionization mass
spectrometer. Finally the optical density was measured at the 490 nm wavelength on an enzyme-
linked immunosorbent assay microplate reader.
General procedures for the synthesis of 6
The synthetic route of intermediate 4 is described as reported by Chaojie Li et al [34]. To a
solution of 4 (1g, 4 mmol) in 10% H SO (15 mL), KMnO (2 g) was added portion by portion in
2
4
4
an ice bath, the reactants were then heated to reflux for 2h. After reaction completed, the solution
was neutralized to pH 5~6, filtered and removed under vacuum to get crude product 5 without
purification.
Acid 5 (5 mmol) and 2-chloro-1-methylpyridinium iodide (1.4g, 5.5 mmol) were dissolved in
dichloromethane and 1, 4-dioxane (V:V=1:1, 150 mL), then 11mL Et N was added and the mixture
3
was stirred overnight. The solvent was evaporated under vacuo to get 6, which was then purified by

chromatograph.
Characterization of 6
1
Russet oil; yield 37%; H NMR (600 MHz, Chloroform-d) δ 3.63 (dd, J = 10.7, 4.3 Hz, 1H), 3.48
dd, J = 13.2, 6.1 Hz, 1H), 3.18 – 3.02 (m, 2H), 2.90 – 2.75 (m, 2H), 2.48 (dd, J = 14.4, 1.6 Hz, 1H),
.12 (s, 1H), 1.99 – 1.85 (m, 2H), 1.87 – 1.62 (m, 4H), 1.57 – 1.39 (m, 5H), 1.31 – 1.21 (m, 1H);
(
2
13C NMR (151 MHz, CDCl3) δ 166.53, 63.76, 57.66, 57.44, 44.95, 44.28, 40.96, 38.21, 35.18, 27.26,
+
2
2
5.68, 21.10, 20.73; HR-MS (ESI) m/z: calculated for C H N O [M+H] : 221.1654, Found:
1
3
20
2
21.1646.
General procedures for the synthesis of 12
Matrine 1 (1 g, 4 mmol) was hydrolyzed by 5 N NaOH aqueous (20 mL) under reflux for
overnight. After the reaction completed, the solution was cooled to room temperature followed by
its acidification hydrochloric acid to pH 5~6 to gain compound 7. Subsequently, Na CO (4g) and
2
3
Fmoc-Cl (1.24 g, 4.8 mmol) in 1, 4-dioxane (20 mL) were added and stirred at room temperature
for 6~8 h. Then the reaction solution was extracted with CHCl (3 × 20 mL), the combined organic
3
layers were dried over anhydrous MgSO for 12 h and filtered. The solvent was evaporated under
4
reduced pressure to get the crude product 8 in 60~70% yield without purification.
To a solution of 8 (3 mmol) in CH Cl (10 mL), SOCl (3.3 mL, 30 mmol) was added and the
2
2
2
mixture was refluxed for 1~2 h under nitrogen protection. The redundant SOCl and CH Cl were
2
2
2
removed through evaporation. The residue was dissolved in a small amount of CH Cl , followed by
2
2
its dropwise addition to a solution of TMSCHN (9 mmol) in CH Cl (4.5 mL) at -15~-20 °C and
2
2
2
the mixture was stirred for 1~2 h. After reaction completed, the mixture was washed successively
with dilute acetic acid, 5% Na CO and saturated NaCl aqueous, the combined organic layers were
2
3
dried over anhydrous MgSO , filtered and evaporated to give the crude product 9, which purified
4
by column chromatography to gain product in 55% yield, using a mixture of CH Cl and MeOH
2
2
(30:1~40:1) as eluent.
To a solution of 9 (2 mmol) in 1, 4-dioxane (10 mL), Ag O (0.46g, 2 mmol), Na CO (0.5g),
2
2
3
NaHSO (0.3g) and H O (20 mL) were added. The mixture was stirred at 50~60 °C for 2~3h. After
3
2
reaction completed, dilute nitric acid was added to the reactant to adjust its pH to 5~6 and extracted
with CHCl (3 × 20 mL). The organic layer was combined, dried and evaporated to get 10 in 65%
3
yield without purification.
Compound 10 was treated with 10% NaOH aqueous for 1~2 h at room temperature. After
reaction completed, dilute hydrochloric acid was added to adjust the pH to 5~6. The solvent was
evaporated to gain crude 11. To a solution of 11 (1 mmol) in THF/DCM (V/V=1:1), HATU (1.5
mmol) and DIPEA (3 mmol) were added and the mixture was stirred overnight at room temperature.
After reaction completed, the mixture was washed with dilute acidic solution, then the organic layer
was combined, dried, filtered and evaporated, followed by purification with chromatograph eluted
with a mixture of EA and MeOH (10:1) to gain the final target 12 in 21% yield.
Characterization of 12
1
Yellow oil; H NMR (600 MHz, Chloroform-d) δ 4.28 – 4.22 (m, 2H), 3.96 (s, 2H), 3.21 (s, 2H),
2
1
.84 (s, 4H), 2.58 (ddd, J = 14.9, 10.8, 4.1 Hz, 2H), 2.49 (ddd, J = 14.9, 6.9, 3.5 Hz, 2H), 2.02 –
1
3
.30 (m, 11H), 0.93 – 0.83 (m, 1H); C NMR (151 MHz, CDCl ) δ 172.60, 67.77, 64.54, 57.08,
3

5
6.25, 45.12, 41.52, 36.41, 34.48, 29.70, 27.92, 26.74, 26.08, 22.01, 21.19, 21.06. HR-MS (ESI)
+
m/z: calculated for C H N O [M+H] : 263.2123, Found: 223.2121.
1
6
26
2
General procedure for the synthesis of 21a-h.
Matrine 1 (1.24 g, 5 mmol) was first dissolved in DCM (30 mL) followed by the addition of
POCl (2 eq., 10 mmol) and the mixture was stirred at room temperature. When the solution color
3
turned light yellow, the reaction mixture was placed in an oil bath at 60 °C and stirred for 2-3 h until
the solution turned brown. It was then cooled to room temperature and the corresponding aniline (1-
1
.5 eq.) dissolved in DCM was added dropwise, followed by reflux for 12 h at 60 °C. After reaction
completion, NaOH aqueous was added to adjust the pH to 8-9. The mixture was then extracted with
DCM (3 × 20 mL). The organic layers were collected, dried over MgSO , and concentrated under
4
vacuo. The crude product was purified by column chromatography using PE: DCM = 5:1 to DCM:
EA = 1:1 as eluents.
(E)-15-(N-phenyl) matrinic imine (21a).
1
Yellow powder; yield 73%; mp: 116.8-117.6 °C; H NMR (600 MHz, Chloroform-d) δ 7.25 – 7.16
m, 2H, Ar-H), 6.96 – 6.87 (m, 1H, Ar-H), 6.73 – 6.67 (m, 2H, Ar-H), 4.39 (dd, J = 12.9, 4.5 Hz,
(
1
1
1
1
2
3
H, H-17α), 3.70 (m, 1H, H-11), 3.16 (t, J = 12.7 Hz, 1H, H-17β), 2.86 – 2.79 (m, 2H), 2.40 (m,
H), 2.10 (t, J = 3.1 Hz, 1H, H-6), 2.09 – 2.00 (m, 2H), 1.99 – 1.89 (m, 3H), 1.89 – 1.76 (m, 2H),
.73 – 1.61 (m, 3H), 1.52 (m, 2H), 1.50 – 1.25 (m, 5H); 13C NMR (151 MHz, CDCl ) δ 157.78,
3
51.89, 128.60, 122.33, 121.15, 64.14, 57.44, 57.33, 53.78, 43.78, 42.94, 34.68, 27.99, 27.79, 27.38,
+
6.70, 21.37, 20.99, 19.98; HR-MS (ESI) m/z: calculated for C H N [M+H] : 324.2434, Found:
2
1
30
3
24.2432.
E)-15-(N-4-chlorophenyl) matrinic imine (21b).
(
1
White powder; yield 57%; mp: 106.4-107.9 °C; H NMR (600 MHz, Chloroform-d) δ 7.21 – 7.14
m, 2H), 6.72 – 6.61 (m, 2H), 4.38 (dd, J = 12.8, 4.5 Hz, 1H), 3.72 (m, 1H), 3.16 (t, J = 12.7 Hz,
(
1
1
H), 2.84 (m, 2H), 2.38 (m, 1H), 2.12 (t, J = 3.1 Hz, 1H), 2.11 – 2.01 (m, 2H), 2.01 – 1.91 (m, 3H),
1
3
.90 – 1.74 (m, 2H), 1.75 – 1.61 (m, 3H), 1.54 (m, 2H), 1.50 – 1.21 (m, 5H). C NMR (151 MHz,
CDCl3) δ 158.00, 150.57, 128.59, 126.19, 123.66, 64.08, 57.41, 57.30, 53.73, 43.74, 42.99, 34.71,
2
7.95, 27.70, 27.40, 26.66, 21.34, 20.95, 19.79; HR-MS (ESI) m/z: calculated for C H N Cl [M+H]
21 29 3
+:
358.2045, Found: 358.2044.
(E)-15-(N-4-ethoxyphenyl) matrinic imine (21c).
1
Brown solid; yield 55%; mp:107.2-110.2 °C; H NMR (600 MHz, Chloroform-d) δ 6.79 – 6.74 (m,
2
2
1
1
H, Ar-H), 6.67 – 6.56 (m, 2H, Ar-H), 4.38 (dd, J = 12.9, 4.5 Hz, 1H, H-17α), 3.98 (q, J = 7.0 Hz,
H, OCH ), 3.75 – 3.61 (m, 1H, H-11), 3.14 (t, J = 12.7 Hz, 1H, H-17β), 2.83 (m, 2H), 2.41 (m,
2
H), 2.10 (t, J = 3.1 Hz, 1H, H-6), 2.09 – 1.99 (m, 2H), 1.99 – 1.88 (m, 3H), 1.88 – 1.72 (m, 2H),
1
3
.72 – 1.57 (m, 3H), 1.52 (m, 2H), 1.45 – 1.34 (m, 8H, CH, CH , CH ); C NMR (151 MHz, CDCl )
2
3
3
δ 158.55, 153.82, 144.99, 122.99 (2), 114.76 (2), 64.16, 63.64, 57.43, 57.32, 53.81, 43.78, 42.95,
3
4.68, 27.95, 27.80, 27.33, 26.69, 21.36, 20.97, 19.97, 15.00; HR-MS (ESI) m/z: calculated for
+
C H N O [M+H] : 368.2696, Found: 368.2694.
2
3
34
3
(E)-15-(N-4-isopropylphenyl) matrinic imine (21d).

1
Yellow solid; yield 69%; mp: 134.1-135.6 °C; H NMR (600 MHz, Chloroform-d) δ 7.20 – 6.94
m, 2H , Ar-H), 6.78 – 6.33 (m, 2H, Ar-H), 4.39 (dd, J = 12.9, 4.5 Hz, 1H, H-17α), 3.70 (m, 1H, H-
1), 3.15 (t, J = 12.7 Hz, 1H, H-17β), 2.90 – 2.79 (m, 3H), 2.45 (m, 1H), 2.12 (t, J = 3.1 Hz, 1H,
H-6), 2.11 – 2.00 (m, 2H), 2.00 – 1.91 (m, 3H), 1.91 – 1.75 (m, 2H), 1.76 – 1.58 (m, 3H), 1.54 (m,
(
1
1
3
2
1
2
+:
H), 1.42 (m, 5H), 1.23 (d, J = 6.9 Hz, 6H, 2CH ); C NMR (151 MHz, CDCl ) δ 157.96, 149.30,
3
3
41.55, 126.50 (2), 122.04 (2), 64.21, 57.45, 57.35, 53.82, 43.82, 42.94, 34.67, 33.39, 27.98, 27.83,
7.38, 26.71, 24.23, 24.21, 21.38, 20.99, 20.01; HR-MS (ESI) m/z: calculated for C H N [M+H]
2
4
36
3
366.2904, Found: 366.2903.
(E)-15-(N-4-biphenyl) matrinic imine (21e).
1
Brown oil; yield 49%; H NMR (600 MHz, Chloroform-d) δ 7.64 – 7.59 (m, 2H, Ar-H), 7.52 – 7.47
(m, 2H, Ar-H), 7.42 (t, J = 7.7 Hz, 2H, Ar-H), 7.33 – 7.24 (m, 1H , Ar-H), 6.81 (d, J = 8.1 Hz, 2H,
Ar-H), 4.45 (dd, J = 12.9, 4.5 Hz, 1H, H-17α), 3.76 (m, 1H, H-11), 3.21 (t, J = 12.7 Hz, 1H, H-17β),
2
1
1
4
.95 – 2.81 (m, 2H), 2.50 (m, 1H), 2.19 – 2.08 (m, 3H), 2.06 – 1.80 (m, 5H), 1.80 – 1.63 (m, 3H),
1
3
.61 – 1.52 (m, 2H), 1.52 – 1.25 (m, 5H); C NMR (151 MHz, CDCl ) δ 157.92, 141.31, 134.01,
3
28.60 (2), 127.32 (2), 126.58 (2), 126.36 (2), 122.80, 118.43, 64.16, 57.43, 57.34, 53.84, 43.89,
3.01, 34.75, 27.96, 27.77, 27.54, 26.69, 21.36, 20.98, 19.88; HR-MS (ESI) m/z: calculated for
+
C H N [M+H] : 400.3457, Found: 400.3462.
2
4
36
3
(E)-15-(N-4-phenoxyphenyl) matrinic imine (21f).
1
White powder; yield 58%; mp: 132.0-132.3 °C; H NMR (600 MHz, Chloroform-d) δ 7.34 – 7.24
(m, 2H, Ar-H), 7.09 – 7.01 (m, 1H, Ar-H), 7.00 – 6.95 (m, 2H, Ar-H), 6.95 – 6.86 (m, 2H, Ar-H),
6
1
1
1
5
.74 – 6.66 (m, 2H, Ar-H), 4.41 (dd, J = 12.9, 4.5 Hz, 1H, H-17α), 3.73 (m, 1H, H-11), 3.18 (t, J =
2.7 Hz, 1H, H-17β), 2.86 (m, 2H), 2.47 (m, 1H), 2.11 (m, 3H), 2.05 – 1.92 (m, 3H), 1.85 (m, 2H),
1
3
.76 – 1.68 (m, 3H), 1.54 (m, 2H), 1.51 – 1.22 (m, 5H); C NMR (151 MHz, CDCl ) δ 158.72,
3
58.27, 150.81, 147.95, 129.49 (2), 123.35, 122.12 (2), 120.26 (2), 117.52 (2), 64.15, 57.44, 57.33,
3.81, 43.80, 42.98, 34.70, 27.97, 27.78, 27.40, 26.69, 21.36, 20.98, 19.94; HR-MS (ESI) m/z:
+
calculated for C H N O [M+H] : 416.2696, Found: 416.2694.
2
7
34
3
(E)-15-(N-2-methyl-3-nitrophenyl) matrinic imine (21g).
1
Yellow crystals; yield 84%; m.p: 124.0-124.9 °C; H NMR (600 MHz, Chloroform-d) δ 7.40 (dd, J
=
8.1, 1.2 Hz, 1H, Ar-H), 7.14 (t, J = 8.0 Hz, 1H, Ar-H), 6.84 (dd, J = 7.9, 1.2 Hz, 1H, Ar-H), 4.42
(dd, J = 12.9, 4.5 Hz, 1H, H-17α), 3.78 (m, 1H, H-11), 3.22 (t, J = 12.7 Hz, 1H, H-17β), 2.90 – 2.81
(
m, 2H), 2.23 (s, 3H, CH ), 2.19 (m, 1H), 2.15 (d, J = 3.1 Hz, 1H, H-6), 2.12 – 1.91 (m, 5H), 1.90
3
1
3
–
1
2
1.60 (m, 5H), 1.60 – 1.52 (m, 2H), 1.51 – 1.20 (m, 5H); C NMR (151 MHz, CDCl ) δ 157.67,
3
52.58, 151.21, 126.00, 125.97, 124.35, 116.95, 64.08, 57.38, 57.29, 53.76, 43.95, 42.86, 34.77,
7.94, 27.58, 27.43, 26.63, 21.31, 20.91, 19.51, 13.86; HR-MS (ESI) m/z: calculated for
+
C H N O [M+H] : 383.2442, Found: 383.2439.
2
2
31
4
2
(E)-15-(N-2-cyanophenyl) matrinic imine (21h).
1
Colorless crystals; yield 79%; mp:135.9-136.6 °C; H NMR (600 MHz, Chloroform-d) δ 7.36 (t, J
6.1 Hz, 1H, Ar-H), 7.27 (t, J = 7.6 Hz, 1H, Ar-H), 6.80 (m, 1H, Ar-H), 6.68 (dd, J = 8.7, 3.7 Hz,
=
1
2
H, Ar-H), 4.36 (m, 1H, H-17α), 3.73 (m, 1H, H-11), 3.11 (m, 1H, H-17β), 2.83 – 2.54 (m, 2H),
.17 (m, 1H), 2.03 (m, 2H), 1.98 – 1.91 (m, 1H), 1.91 – 1.78 (m, 3H), 1.78 – 1.53 (m, 4H), 1.53 –

1
3
1
1
2
3
.40 (m, 2H), 1.32 (m, 6H); C NMR (151 MHz, CDCl ) δ 157.63, 155.78, 133.01, 132.52, 123.14,
3
20.86, 118.51, 105.58, 63.97, 57.25, 57.18, 53.40, 44.02, 42.72, 34.50, 27.88, 27.41, 26.93, 26.48,
+
1.23, 20.81, 18.85; HR-MS (ESI) m/z: calculated for C H N [M+H] : 349.2387, Found:
2
2
29
4
49.2388.
General procedure for the synthesis of 20a-h.
Matrine 1 (1.24 g, 5 mmol) dissolved in DCM (30 mL) was treated with POCl (2 eq., 10
3
mmol) at room temperature. When color of the reaction mixture turned yellow, it was then stirred
at 60 °C for 2-3 h. The solution was then cooled to room temperature and the corresponding aniline
bearing cyan derivatives 18a-h (1-1.5 eq.) dissolved in DCM were added dropwise, followed by
reflux for 12 h at 60 °C. After reaction completion, NaOH aqueous was added to adjust the pH to
8
-9. The mixture was then extracted with DCM (3 × 20 mL). The organic layers were collected,
dried over MgSO , and concentrated. The residue was purified by column chromatography to obtain
4
product 20a-h.
General procedure for the synthesis of 22a-h.
Compound 20a-h (2 mmol) was dissolved in dry THF (30 mL), followed by the addition of
LDA (3eq.). The mixture was stirred at 0 °C and reaction progress was monitored by TLC analysis.
After completion of the reaction, the mixture solution was quenched with saturated ammonium
chloride solution and extracted with DCM (3 × 20 mL). The organic layers were combined,
concentrated and the residue was purified by column chromatography on silica gel using EA:
MeOH= 9: 1 as eluent or on alumina using PE: EA= 5: 1 as eluent to afford 22a-h.
4
-amino-quinolinomatrine (22a).
1
Yellow powder; yield 53%; mp: 197.0-197.2 °C; H NMR (600 MHz, Chloroform-d) δ 7.61 (d, J =
.3 Hz, 1H, Ar-H), 7.49 (dd, J = 8.2, 1.4 Hz, 1H, Ar-H), 7.43 (m, 1H, Ar-H), 7.19 – 6.99 (m, 1H,
Ar-H), 4.92 (dd, J = 12.7, 4.5 Hz, 1H, H-17α), 4.33 (s, 2H, NH ), 3.94 (m, 1H, H-11), 3.34 (t, J =
8
2
1
1
1
2
3
2.6 Hz, 1H, H-17β), 2.86 (m, 2H), 2.65 – 2.45 (m, 2H), 2.31 – 2.10 (m, 2H), 2.05 – 1.72 (m, 8H),
1
3
.61 (m, 1H), 1.58 – 1.35 (m, 4H); C NMR (151 MHz, CDCl ) δ 155.78, 147.16, 144.76, 128.32,
3
26.74, 120.44, 119.40, 115.07, 99.18, 64.45, 57.56, 57.46, 51.56, 45.01, 39.68, 34.98, 28.10, 26.59,
+
3.24, 21.54, 21.14, 19.47; HR-MS (ESI) m/z: calculated for C H N [M+H] : 349.2387, Found:
2
2
29
4
49.2385.
4
-amino-6-methylquinolinomatrine (22b).
1
Yellow powder; yield 55%; mp: 182.1-184.5 °C; H NMR (600 MHz, Chloroform-d) δ 7.53 (d, J =
.9 Hz, 1H, Ar-H), 7.35 – 7.06 (m, 2H, Ar-H), 4.89 (dd, J = 12.8, 4.5 Hz, 1H, H-17α), 4.30 (s, 2H,
NH ), 3.92 (m, 1H, H-11), 3.33 (t, J = 12.6 Hz, 1H, H-17β), 2.86 (m, 2H), 2.56 (m, 2H), 2.45 (s,
8
2
1
3
3
H, CH ), 2.26 – 2.12 (m, 2H), 2.02 – 1.69 (m, 8H), 1.64 – 1.35 (m, 5H); C NMR (151 MHz,
3
CDCl ) δ 155.40, 145.37, 144.38, 130.21, 129.77, 126.62, 118.75, 114.83, 99.31, 64.47, 57.58,
3
5
7.47, 51.57, 45.02, 39.70, 34.91, 28.11, 26.62, 23.40, 21.55, 21.48, 21.16, 19.61; HR-MS (ESI)
+
m/z: calculated for C H N [M+H] : 363.2543, Found: 363.2545.
2
3
31
4
4
-amino-6-methoxyquinolinomatrine (22c).
1
Brown solid; yield 35%; mp: 103.2-105.3 °C; H NMR (600 MHz, Chloroform-d) δ 7.58 (d, J = 9.1

Hz, 1H, Ar-H), 7.13 (dd, J = 9.1, 2.7 Hz, 1H, Ar-H), 6.86 (d, J = 2.8 Hz, 1H, Ar-H), 4.83 (dd, J =
1
1
1
1
2
3
2.8, 4.4 Hz, 1H, H-17α), 4.29 (s, 2H, NH ), 3.90 (m, 1H, H-11), 3.88 (s, 3H, OCH ), 3.33 (t, J =
2 3
2.6 Hz, 1H, H-17β), 2.86 (m, 2H), 2.56 (m, 2H), 2.34 – 2.10 (m, 2H), 2.05 – 1.69 (m, 8H), 1.68 –
1
3
.35 (m, 5H); C NMR (151 MHz, CDCl ) δ 154.58, 154.04, 144.28, 142.33, 128.14, 119.05,
3
15.00, 100.12, 99.82, 64.43, 57.56, 57.46, 55.67, 51.59, 45.10, 39.69, 34.85, 28.10, 26.62, 23.45,
+
1.54, 21.15, 19.72; HR-MS (ESI) m/z: calculated for C H N O [M+H] : 379.2492, Found:
2
3
31
4
79.2491.
4
-amino-6-fluoroquinolinomatrine (22d).
1
Brown solid; yield 33%; mp: 199.3-202.8 °C; H NMR (600 MHz, Chloroform-d) δ 7.58 (dd, J =
9
1
2
1
1
3
.1, 5.4 Hz, 1H, Ar-H), 7.20 (t, J = 8.9 Hz, 1H, Ar-H), 7.14 (d, J = 9.8 Hz, 1H, Ar-H), 4.85 (dd, J =
2.7, 4.4 Hz, 1H, H-17α), 4.24 (s, 2H, NH ), 3.94 (m, 1H, H-11), 3.34 (t, J = 12.6 Hz, 1H, H-17β),
2
.87 (dd, J = 32.8, 11.2 Hz, 2H), 2.57 (m, 2H), 2.29 – 2.12 (m, 2H), 2.03 – 1.73 (m, 8H), 1.63 –
1
3
.36 (m, 5H); C NMR (151 MHz, Chloroform-d) δ 157.42 (d, J = 239.3 Hz, F-Ar-C), 155.39,
44.30, 143.97, 128.56, 117.43, 114.94, 103.76, 100.07, 64.40, 57.53, 57.43, 51.53, 44.97, 39.73,
4.93, 28.09, 26.58, 23.24, 21.51, 21.11, 19.58; 19F NMR (565 MHz, CDCl ) δ -121.80; HR-MS
3
+
(
ESI) m/z: calculated for C H N F [M+H] : 367.2293, Found: 367.2296.
2
2
28
4
4
-amino-6-chloroquinolinomatrine (22e).
1
Brown solid, yield 39%; mp: carbonized; H NMR (600 MHz, Chloroform-d) δ 7.53 (d, J = 8.9 Hz,
1
4
H, Ar-H), 7.46 (d, J = 2.3 Hz, 1H, Ar-H), 7.35 (dd, J = 8.9, 2.3 Hz, 1H, Ar-H), 4.87 (dd, J = 12.8,
.5 Hz, 1H, H-17α), 4.27 (s, 2H, NH ), 3.95 (m, 1H, H-11), 3.34 (t, J = 12.6 Hz, 1H, H-17β), 2.87
2
1
3
(
m, 2H), 2.72 – 2.48 (m, 2H), 2.29 – 2.10 (m, 2H), 2.02 – 1.72 (m, 8H), 1.64 – 1.39 (m, 5H); C
NMR (151 MHz, CDCl ) δ 155.86, 145.68, 143.93, 128.77, 128.23, 125.47, 118.89, 115.78, 99.93,
3
6
4.36, 57.52, 57.42, 51.53, 44.94, 39.77, 35.03, 28.07, 26.56, 23.11, 21.50, 21.09, 19.46; HR-MS
+
(
ESI) m/z: calculated for C H N Cl [M+H] : 383.1997, Found: 383.1998.
2
2
28
4
4
-amino-6-bromoquinolinomatrine (22f).
1
White powder; yield 41%; mp: carbonized; H NMR (600 MHz, Chloroform-d) δ 7.61 – 7.50 (m,
1
4
H, Ar-H), 7.27 (dd, J = 8.9, 2.4 Hz, 1H, Ar-H), 7.09 (d, J = 2.3 Hz, 1H, Ar-H), 4.90 (dd, J = 12.8,
.4 Hz, 1H, H-17α), 4.33 (s, 2H, NH ), 3.93 (m, 1H, H-11), 3.34 (t, J = 12.6 Hz, 1H, H-17β), 2.86
2
(m, 2H), 2.57 (m, 2H), 2.30 – 2.09 (m, 2H), 2.08 – 1.93 (m, 3H), 1.94 – 1.67 (m, 5H), 1.66 – 1.38
1
3
(
m, 5H); C NMR (151 MHz, CDCl ) δ 155.02, 154.78, 144.48, 140.42, 128.23, 125.97, 116.19,
3
1
1
14.58, 99.12, 64.47, 57.57, 57.46, 51.67, 48.56, 39.75, 34.97, 28.08, 26.62, 23.39, 21.54, 21.15,
+
9.59; HR-MS (ESI) m/z: calculated for C H N Br [M+H] : 427.1492, Found: 427.1495.
2
2
28
4
4
-amino-6-trifluoromethylquinolinomatrine (22g).
1
Yellow powder; yield 65%; mp: carbonized; H NMR (600 MHz, Chloroform-d) δ 7.77 (d, J = 1.9
Hz, 1H, Ar-H), 7.63 (d, J = 8.8 Hz, 1H, Ar-H), 7.59 (dd, J = 8.8, 1.9 Hz, 1H, Ar-H), 4.93 (dd, J =
1
2
2.8, 4.5 Hz, 1H, H-17α), 4.40 (s, 2H, NH ), 3.99 (m, 1H, H-11), 3.36 (t, J = 12.6 Hz, 1H, H-17β),
2
.86 (m, 2H), 2.69 – 2.46 (m, 2H), 2.30 – 2.11 (m, 2H), 2.03 – 1.69 (m, 8H), 1.66 – 1.37 (m, 5H);
1
3
C NMR (151 MHz, CDCl ) δ 156.81, 148.97, 144.96, 127.16, 124.96, 124.21, 121.62 (q, J = 32.2
3
Hz), 117.58, 114.07, 99.84, 64.30, 57.49, 57.39, 51.57, 44.97, 39.80, 35.20, 28.05, 26.51, 22.85,
1.47, 21.05, 19.26. 19F NMR (565 MHz, CDCl ) δ -61.10; HR-MS (ESI) m/z: calculated for
2
3

+
C H N F [M+H] : 417.2261, Found: 417.2262.
2
3
28
4 3
4
-amino-6-nitroquinolinomatrine (6h).
1
Brown powder; yield 39%; mp: carbonized; H NMR (600 MHz, Chloroform-d) δ 8.54 (d, J = 2.5
Hz, 1H, Ar-H), 8.19 (dd, J = 9.2, 2.5 Hz, 1H, Ar-H), 7.51 (d, J = 9.3 Hz, 1H, Ar-H), 4.96 (dd, J =
1
1
5
9
2.8, 4.5 Hz, 1H, H-17α), 4.57 (s, 2H,NH ), 4.10 – 3.96 (m, 1H, H-11), 3.39 (t, J = 12.6 Hz, 1H, H-
2
7β), 2.87 (m, 2H), 2.66 – 2.44 (m, 2H), 2.22 – 2.13 (m, 2H), 2.08 – 1.70 (m, 8H), 1.67 – 1.39 (m,
1
3
H); C NMR (151 MHz, CDCl ) δ 157.44, 151.32, 145.64, 140.09, 126.84, 122.77, 117.71, 113.63,
3
9.68, 64.17, 57.42, 57.34, 51.78, 45.07, 40.01, 35.58, 27.99, 26.45, 22.45, 21.41, 20.97, 18.97;
+
HR-MS (ESI) m/z: calculated for C H N O [M+H] : 394.2238, Found: 394.2235.
2
2
28
5
2
Thermal shift assay
N
Mix Hsp90 protein, buffer, compounds dissolved in DMSO (if applicable) and PTS dye in a 20
μL reaction system. The molar ratio of protein to ligand is 1:5. Then run a melt-curve experiment
on a real-time PCR instrument (7500, ABI Company, USA). Plates were run from 25 °C to 95 °C
with a ramp rate of 1 °C/min. The protein unfolds as it is heated and the environmentally-sensitive
PTS dye binds exposed hydrophobic regions and fluoresces. The melting temperature (Tm) was
calculated from the melt curve and △Tm was correlated to changes in protein stability or ligand
binding.
Anticancer activity assay in vitro
HepG2 and HeLa cell lines were obtained from the American Type Culture Collection (ATTC).
The cells were prepared in DMEM supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin and were then cultured in 96-well plates. After a 24 h incubation period in
5
% CO at 37 °C, compounds with different concentrations (1, 5, 10, 20, 50 and 100 μM) made by
2
serial dilution in culture medium of stock solutions of test compounds prepared in DMSO, were
added and incubated for 48 h. Then, 20 μL MTT (5 mg/mL) was added to each well and the wells
were incubated for 4 h. Supernatant from each well was carefully removed and 150 μL DMSO was
added to each well for the colorimetric reaction. Finally, the absorbance was detected at 490 nm
wavelength on an enzyme-linked immunosorbent assay microplate reader and each concentration
was tested in threefold.
Docking
The chemical structures were constructed on the sketch module of Sybyl 21.0 software (Tripos
Company, USA). The molecular modeling studies require the minimization of structure, calculation
of the force field and charges. The energy minimization and charge calculation done by Tripos force
N
field and Pullman charge (or other) respectively. Human Hsp90 structure (PDB ID: 1UYM)
downloaded from Protein Data Bank was utilized in docking, ligands and water molecules were
removed, polar hydrogen atoms, charges were added and energy minimization executed. The
Surflex-Dock module used to dock ligands into a ligand-protein’s extracted binding site. The
protomol bloat value was set as 1 and the protomol threshold value as 0.5 to obtained meaningful
binding pocket. Other parameters are established by default in software.
Cell apoptosis

For Annexin V-propidium iodide assay, HeLa cells were seeded in a six-well plates in a density
5
of 2 × 10 cells per well, incubated overnight and then treated with graded concentrations of
compound 22g for 48 h. Cellular apoptosis was determined by annexin V-PI apoptosis detection kit
(Beyotime, CN) following its instructions Cells in early or late apoptotic phases were represented
as cumulative percentage compared with control.
Cell cycle
HeLa cells were treated with certain concentrations of compound 22g for 48 h. The cells were
then collected, washed, and fixed with 75% ethanol for 24 h at 4 °C. The cells were then stained
with 4 mg/mL PI and 0.1 mg/mL RNaseA for 30 min in the dark. After staining, cell cycle
distribution of 10,000 cells from each group was analyzed using an Aria FACS flow cytometry
system (Bection Dickinson, USA).
Western boltting
HeLa cells were treated with different concentrations of compound 22g, the cells were collected,
lysed in RIPA buffer containing a protease inhibitor cocktail for 30 min, followed by centrifugation
at 12,000 rpm for 15 min at 4 ℃. Proteins were subjected to 6-15% SDS-PAGE, electrophoresed
and transferred on to a nitrocellulose membrane. After blocking with 5% non-fat milk in Tris-
buffered saline, the membrane was washed and incubated with the indicated primary and secondary
antibodies and detected using the syngene (G:BOXChemiXR5, UK).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by Innovation Project of Guangxi Graduate Education (YCBZ2019026),
the National Natural Science Foundation of China (21403225, U1932130), the Talents Program of
Xi'an Medical University (2015RCYJ01), the Key Program of Shaanxi Provincial Science and
Technology Department (2017ZDXM-SF-029); the Key Program of Shaanxi Provincial Education
Department (18JS102).
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2
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9

Conflict of interest
The authors declare no conflict of interest.