Development of anthraquinone analogues as phosphoglycerate mutase 1 inhibitors

Article abstract of DOI:10.3390/molecules24050845

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based on the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the structure?activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone scaffold was essential for maintaining potency and the modifications of the hydroxyl of alizarin would cause a sharp decrease in potency. In the meantime, we determined the co-crystal structure of PGAM1 and one of the anthraquinone inhibitors 9i with IC₅₀ value of 0.27 μM. The co-crystal structure revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of inhibitors which further validated the SAR. Consistent with the crystal structure, a competitive assay illustrated that compound 9i was a noncompetitive inhibitor. In addition, compound 9i effectively restrained different lung cancer cells proliferation in vitro. Taken together, this work provides reliable guide for future development of PGAM1 inhibitors and compound 9i may act as a new leading compound for further optimization.

Full text of DOI:10.3390/molecules24050845

Communication
Development of Anthraquinone Analogues as
Phosphoglycerate Mutase 1 Inhibitors
Ke Huang, Lulu Jiang, Huiti Li, Deyong Ye * and Lu Zhou *
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, No. 826, Zhangheng Rd.,
Shanghai 201203, China; kehuang13@fudan.edu.cn (K.H.); 18111030012@fudan.edu.cn (L.J.);
1
7211030005@fudan.edu.cn (H.L.)
*
Correspondence: dyye@shmu.edu.cn (D.Y.); zhoulu@fudan.edu.cn (L.Z.); Tel.: +86-21-5198-0117 (D.Y.);
+
86-21-5198-0125 (L.Z.)
Received: 18 December 2018; Accepted: 28 January 2019; Published: 27 February 2019
Abstract: Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote
cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based
on the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the
structure−activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone
scaffold was essential for maintaining potency and the modifications of the hydroxyl of alizarin
would cause a sharp decrease in potency. In the meantime, we determined the co-crystal structure
of PGAM1 and one of the anthraquinone inhibitors 9i with IC50 value of 0.27 μM. The co-crystal
structure revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of
inhibitors which further validated the SAR. Consistent with the crystal structure, a competitive
assay illustrated that compound 9i was a noncompetitive inhibitor. In addition, compound 9i
effectively restrained different lung cancer cells proliferation in vitro. Taken together, this work
provides reliable guide for future development of PGAM1 inhibitors and compound 9i may act as a
new leading compound for further optimization.
Keywords: phosphoglycerate mutase 1; anthraquinone inhibitors; co-crystal structure; cancer
treatment
1
. Introduction
In 1924, Warburg discovered that cancer cells tended to metabolize glucose through aerobic
glycolysis rather than oxidative phosphorylation, even if there was sufficient oxygen [1,2]. This
character of cancer cells was distinct from normal differentiated cells and the phenomenon was
thereafter named the “Warburg effect”. However, the reason why cancer cells adopt such an altered
metabolism remained unclear. In the past decades, reprogramming energy metabolism was
considered as an emerging hallmark of cancer cells [3] and the study of cancer metabolism has
drawn great interest [4–8]. During aerobic glycolysis of cancer cells, adenosine 5´-triphosphate
(
ATP) is produced inefficiently in this metabolic pattern, whereas building blocks such as
nucleotides, and amino acids are generated in large amounts, satisfying the demands of anabolic
biosynthesis which are essential for rapid cell proliferation [9–12]. Nowadays, various enzymes
playing critical roles in cancer metabolism have been reported, including isocitrate dehydrogenase
[
13–15]. Inhibitors targeting these enzymes have been approved by Food and Drug Administration
14,15] which further demonstrated the potential of modulating cancer metabolism for cancer
[
therapy.
Phosphoglycerate mutase 1 (PGAM1) is a key enzyme in the 8th step of glycolysis pathway
converting 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) [16]. Previously we found that
Molecules 2019, 24, 845; doi:10.3390/molecules24050845
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PGAM1 could balance the intracellular concentrations of its substrate 3PG and its product 2PG to
control tumor growth [17]. In detail, 3PG inhibits 6-phosphogluconate dehydrogenase in pentose
phosphate pathway (PPP) and 2PG activates 3-phosphoglycerate dehydrogenase in serine synthesis
[17]. When PGAM1 is inhibited by a small molecule or shRNA, intracellular level of 3PG increased
and 2PG decreased which led to the blockade of PPP, serine synthesis, glycolysis and subsequently
impaired tumor growth. Since PGAM1 regulates both glycolysis and biosynthesis, developing
inhibitors targeting PGAM1 is considered as a way of dual inhibition of catabolism and anabolism
for cancer therapy [18]. Furthermore, PGAM1 is upregulated in many kinds of tumors and the
expression of PGAM1 is correlated with cancer patients’ prognosis [19–21]. Recently, more
functions of PGAM1 in cancer metabolism were described. Posttranslational modulations of
PGAM1 such as Y26 phosphorylation [22] and deacetylation of K100 [23] would promote cancer cell
proliferation and tumor growth. Besides, it is essential for homologous recombination repair by
regulating the deoxyribonucleotide triphosphate nucleoside (dNTP) pool [24] and it facilitates
cancer cell migration independent of its metabolic function [25]. Taken together, PGAM1 is a
promising target for cancer treatment.
However, up to now, only four representative PGAM1 inhibitors were reported (Figure 1). The
first PGAM1 inhibitor 1 (MJE3) was discovered by the Cravatt group in 2005 [26]. It was identified
as a covalent inhibitor labeling the K100 of PGAM1 by spiroepoxides [27]. Another PGAM1
inhibitor described these days was 2((−)-epigallocatechin-3-gallate (EGCG)), a natural product from
green tea [28]. It demonstrated strong inhibition activity towards PGAM1 [28] but it was reported to
display extensive potency on multiple targets [29–31]. We discovered the other two inhibitors, one
of which was anthraquinone derivative 3 (PGMI-004A) [17] and the other was the xanthone
derivative 4 (12r) [32]. Both of them showed moderate inhibition activity on PGAM1 and cancer cell
proliferation.
Figure 1. Reported phosphoglycerate mutase 1 （PGAM1） inhibitors.
Since only a limited amount of anthraquinone inhibitors were reported, the structure−activity
relationship (SAR) of anthraquinone derivatives and PGAM1 was not well studied yet. Besides, up
to these days, the molecular interactions of PGAM1 and anthraquinone inhibitor were not observed
in crystal structure. However, anthraquinones exhibit a variety of pharmacological activities
including anticancer [33–35], antifungal [36], and antibacterial properties [37] and so on. What’s
more, there are many clinically approved anthraquinone drugs such as doxorubicin and
mitoxantrone with strong potency in suppressing cancer cell proliferation [38]. Here we designed
and synthesized a series of new anthraquinone compounds and evaluated their biological activity
on PGAM1 and cancer cell proliferation. In addition, we solved the co-crystal structure of PGAM1
and compound 9i which interpreted the molecular mechanism of compound 9i interacting with
PGAM1 and further confirmed the SAR we investigated. Combined with the crystal structure,
additional competitive assay demonstrated the noncompetitive binding of compound 9i with the
3
2
2
PG substrate.
. Results and Discussion
.1. Chemistry

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Four classes of anthraquinone derivatives were synthesized, with 1,2-substituted,
-substituted, 2,4-substituted, and 2,3-substituted anthraquinone core scaffolds (Figure 2).
3
Figure 2. The optimization strategy and SAR exploration of the anthraquinone derivatives against
PGAM1.
As shown in Scheme 1, starting from commercially available alizarin, compounds 6a–e were
obtained by substitution with ethyl bromoacetate, hydrolysis of the ester, and amidation.
Scheme 1. Synthesis of compounds 6a–e. Reagents and conditions: (a) BrCH
2COOEt, K
2
CO , DMF, 80
3
°
C; (b) LiOH, MeOH, H O, 30 °C; (c)NHMe ·HCl, EDCI, HOBt, DIPEA, DMF.
2
2
In Scheme 2, firstly, alizarin was nitrated by HNO and then reduced by Sn and HCl to afford
3
the key amine intermediate 8, which was reacted with diverse sulfonyl chlorides to give the target
compounds 9a–q.
Scheme 2. Synthesis of compounds 9a–q. Reagents and conditions: (a) HNO
3
, CH COOH, 50 °C; (b)
3
3
Sn, SnCl2·H
2
O, HCl, EtOH; (c) R SO
2
Cl, Py.

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As shown in Scheme 3, the 4-nitro group was reduced to an amino group after the nitrification
of compound 6c, followed by sulfonylation of the amino group and hydrolysis of the ester as
described above.
Scheme 3. Synthesis of compounds 12 and 13. Reagents and conditions: (a) HNO
3, CH3COOH, 50 °C;
(
b) Sn, SnCl ·H O, HCl, EtOH; (c) 4-chlorobenzenesulfonyl chloride, Py; (d) LiOH, MeOH, H
2
2
2O, 30 °C.
Finally, compound 9i was employed as a starting material, and compounds 14a–g were
obtained by substitution with different reagents and further hydrolysis of the ester or amidation of
the carboxyl group (Scheme 4).
Scheme 4. Synthesis of compounds 14a–g. Reagents and conditions: (a) K
substituted reagent; (b) methanesulfonyl chloride, DIPEA, Py; (c) LiOH, MeOH, H
NH NH ·H O, EtOH, reflux.
2
CO
3
, DMF, corresponding
2
O, 30 °C; (d)
2
2
2
2
.2. SAR Exploration of the Anthraquinone Derivatives Against PGAM1
Previously we reported several anthraquinone compounds as PGAM1 inhibitors [17]. Due to
the limited amount of compounds and the lack of chemical diversity, the SAR of anthraquinone
derivatives and PGAM1 remained poorly understood. Here we synthesized four classes of
compounds on the base of bioisosterism to study the SAR of anthraquinone derivatives against
PGAM1 in detail (Figure 2), using an optimized enzymatic assay in which PGMI-004A served as a
control with an IC50 value of 1.2 ± 0.3 μM. Firstly, we found that different modifications of the
hydroxyl of alizarin (6a–e) showed weak PGAM1 inhibition activity at 10 μM (Table 1).
Table 1. Inhibition activity of compounds 6a–e towards PGAM1.
Compound
R 1
R 2
Inhibition of PGAM1 Activity at 10 μM 1
6
a
−0.6 ± 1%

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6
b
14.3 ± 5.9%
0 ± 1.8%
6
c
H
H
6
d
5.5 ± 0.03%
6
e
H
20.2 ± 2.2%
1
The data are presented as mean ±s.d. (n = 2).
In order to explore the SAR of 3-substituents of the anthraquinone scaffold against PGAM1,
compounds 9a–q were synthesized by reversing the sulfonamide group of PGMI-004A. Compounds
9
a–e with different substituents on the phenyl ring displayed comparable inhibition activity towards
PGAM1, with IC50 values of 0.5 μM or so (Table 2). They were much more potent than the
compounds without the sulfonamide group, suggesting the sulfonamide group was essential for
maintaining potency. Then, we studied the impact of substituents of different halogens on the
phenyl ring (9f–q) on the inhibition activity towards PGAM1 (Table 2). Generally, compounds with
Cl, Br and I substituents (9i–q) with IC50 values below 0.5 μM performed better than F substituents
(
9f–h). Among them, we discovered one of the most potent PGAM1 inhibitors 9i with an IC50 value
of 0.27 μM and we solved the co-crystal structure of compound 9i with PGAM1 which would be
discussed below.
Table 2. Inhibition activity of compounds 9a–q towards PGAM1.
O
OH
OH
O
NHS R3
O
O
Compound
R 3
PGAM1 IC50 (μM) 1
9
a
0.54 ± 0.04
9
b
0.47 ± 0.08
0.90 ± 0.07
9
c
9
d
2.2 ± 0.5
9
e
0.61 ± 0.12

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Table 2. Cont.
9
f
0.54 ± 0.07
0.79 ± 0.04
9
g
9
h
0.89 ± 0.13
0.27 ± 0.04
0.28 ± 0.07
9
i
j
9
9
k
0.89 ± 0.33
0.90 ± 0.26
9
l
9
m
0.26 ± 0.02
0.20 ± 0.04
9
n
9
o
p
q
0.35 ± 0.005
0.47 ± 0.07
0.26 ± 0.03
9
9
1
The data are presented as mean ± s.d. (n = 3).
We next moved the sulfonamide to the C4 position of alizarin by substituting the hydroxyl,
followed by nitrification, reduction, and sulfonylation. Probably due to the modification of the
hydroxyl and replacement of the sulfonamide, compounds 12 and 13 failed to maintain potency,
decreasing 10% and 34% of PGAM1 activity at 10 μM, respectively. Finally, considering the good
solubility and potency of inhibitor 9i, we kept the 4-chlorobenzene group and synthesized
compounds 14a–g. However, diverse modifications of the hydroxyl caused a loss of inhibition
activity toward PGAM1 to different extents (Table 3).
In order to exclude the inhibition of the other three enzymes (enolase-pyruvate kinase-LDH) in
the coupled assay, we performed counter screening of selected compounds with IC50 value below 1
μM on PGAM1.
Table 3. Inhibition activity of compounds 14a–14g towards PGAM1.

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Compound
4a
R 5
R 6
PGAM1 IC50 (μM) 1
1
H
>10 μM
1
4b
H
H
>10 μM
1
4c
4d
4e
CH
3
2.92 ± 0.11
7.01 ± 0.92
>10 μM
1
CH
3
2
CH
3
1
-CH
-
-CH -
2
1
4f
H
H
2.0 ± 0.2
>10 μM
1
4g
1
The data are presented as mean ±s.d. (n = 3).
As shown in Table 4, the compounds displayed almost no inhibition on the three downstream
enzymes at 0.5 μM which further confirmed the on-target effect of the compounds.
Table 4. The inhibition in the control linked enzyme assay of selected compounds.
Compound PGAM1 IC50 (μM) Inhibition in the Control Linked Enzyme Assay (0.5 μM) 1
9
a
0.54 ± 0.04
0.47 ± 0.08
0.90 ± 0.07
0.61 ± 0.12
0.54 ± 0.07
0.79 ± 0.04
0.89 ± 0.13
0.27 ± 0.04
0.28 ± 0.07
0.89 ± 0.33
0.90 ± 0.26
0.26 ± 0.02
0.20 ± 0.04
0.35 ± 0.005
0.47 ± 0.07
0.26 ± 0.03
1
8.0 ± 5.0%
−2.9 ± 0.3%
3.7 ± 1.8%
6.7 ± 0.2%
5.7 ± 0.7%
10.2 ± 3.0%
0.06 ± 0.4%
−6.4 ± 5.0%
−2.0 ± 5.1%
0.5 ± 3.2%
0.2 ± 1.7%
−0.1 ± 2.5%
6.1 ± 0.8%
7.7 ± 0.2%
1.9 ± 0.1%
−2.3 ± 2.4%
9
b
9
c
9
e
9
f
9
g
9
h
9
i
9
j
9
k
9
l
9
m
9
n
9
o
p
q
9
9
The data are presented as mean ±s.d. (n = 2).
2
.3. Binding Mode of Compound 9i with PGAM1
To further understand the molecular mechanism of the anthraquinone derivatives interacting
with PGAM1, we determined the X-ray structure of PGAM1 in complex with compound 9i at
resolution of 1.98 Å (Table 5). Compound 9i occupied a novel allosteric site adjacent to substrate
binding site with nice electron density (Figure 3A and Figure 3B). The allosteric pocket was
surrounded by the residues of F22, R90, K100, R116 and R191. In detail, the anthraquinone scaffold
and sulfonamide of compound 9i interacted with the main chain carbonyl of K100 through water
bridges (Figure 3C). In addition, a hydrophobic interaction was observed between F22 and
chlorine-substituted phenyl ring of compound 9i (Figure 3C). Compound 9i also engaged in a

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π-cation interaction with R116 (Figure 3C), which explains why modifications of the hydroxyl
group led to decreased potency [39]. To validate the binding mode revealed by the co-crystal
structure, we tested the activity of PGAM1 mutants (Supplementary Data, Figure S1) and the
inhibition activity of compound 9i on different mutations of PGAM1. Compound 9i failed to inhibit
mutations of PGAM1 (F22A, R116H and R191H) as effectively as the wild type at concentration of 5
μM which agreed with the results from crystal structure. Furthermore, a substrate competitive assay
demonstrated that compound 9i held a non-competitive property with substrate 3PG which was
also consistent with the binding mode revealed by X-ray structure. The co-crystal structure together
with the molecular biological assays illustrated the binding mode of the anthraquinone inhibitor
with PGAM1 and provided useful information for further optimization.
Figure 3. Binding mode of anthraquinone inhibitor 9i with PGAM1. (a) Chemical structure of
compound 9i and Fo–Fc electron density of compound 9i contoured at 2.0σ; (b) Overlay of
compound 9i (PBD: 6ISN) and 3PG (PBD:2F90) in PGAM1; (c) Interactions of compound 9i and the
critical residues of PGAM1 in the co-crystal structure; (d) Inhibition of compounds 9i on wild-type
and mutations of PGAM1 at concentration of 5 μM; (e) Noncompetitive property of compound 9i
with substrate 3PG. The data are presented as mean ±s.d.
Table 5. Data collection and refinement statistics.
Property
Value
Data Collection
Space group
Cell constants
a, b, c (Å)
P 2
1
2
1
2
1
8
2.54, 82.76, 104.20
α, β, γ (°)
90.00, 90.00, 90.00
50–1.98 (2.05–1.98)
100.0 (100.0)
330639
Resolution *(Å)
Data completeness
No. Observations
%
No. Unique Reflections
Redundancy
50593 (4986)
6.5 (6.6)
Rmerge
0.06 (0.88)
<
I/σ(I) >
2.05 (at 1.98Å)

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CC1/2
0.996 (0.728)
Refinement
Resolution (Å)
Data completeness
No. reflections
38.89–1.98 (2.02–1.98)
%
100.0 (100.0)
50362 (2636)
Rwork/Rfree
0.197/0.225
Rfree test set
2569 reflections (5.08%)
Wilson B-factor (Å2)
39.4
0.96
4046
41.0
98.7
1.3
Fo
, F correlation
c
Total number of atoms
Average B, all atoms (Å2)
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
Rmsd bond lengths (Å)
Rmsd bond angles (°)
0.0
0.007
1.044
6ISN
PDB Accession Code
2
.4. Inhibition Activity of Selected Compounds on Cancer Cell Proliferation
Given PGAM1 plays a crucial part in cancer metabolism, inhibition of PGAM1 by small
molecules is supposed to suppress cancer cell proliferation. We selected the compounds with IC50
values below 10 μM toward PGAM1 to inhibit H1299 cells proliferation. Then the potent inhibitors
with IC50 values below 20 μM on H1299 cells were further tested on A549 and PC9 cells. In general,
proliferation inhibition of the compounds was correlated with PGAM1 inhibition and the inhibitors
performed similarly among different cancer cells, with IC50 values ranging from approximately 6–50
μM (Table 6 and Table 7). Notably, compound 9i effectively suppressed the three different cancer
cells proliferation which was also potent towards PGAM1.
Table 6. Inhibition activity of compounds 9a–q on cancer cell proliferation.
Compound
R3
H1299 IC50 (μM)
A549 IC50 (μM)
PC9 IC50 (μM)1
9a
20.7 ± 0.6
n.d.
n.d.
9
b
25.8 ± 2.2
16.6 ± 1.8
n.d.
n.d.
9
c
8.0 ± 0.8
10.2 ± 0.7
9
d
13.9 ± 3.6
26.3 ± 4.5
17.0 ± 4.9
n.d.
44.2 ± 1.5
n.d.
9
e

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Table 6. Cont.
9
f
24.2 ± 2.6
n.d.
n.d.
n.d.
n.d.
9
g
31.3 ± 1.9
9
h
45.7 ± 2.7
6.9 ± 1.2
23.7 ± 1.1
n.d.
12.7 ± 2.7
n.d.
n.d.
13.8 ± 1.0
n.d.
9
i
9
j
9
k
34.6 ± 2.4
6.2 ± 1.2
n.d.
>50
n.d.
>50
9
l
9
m
28.1 ± 1.9
50 ± 0.6
n.d.
n.d.
n.d.
n.d.
9
n
9
o
p
q
24.5 ± 0.2
17.3 ± 3.9
26.4 ± 3.8
n.d.
10.3 ± 1.4
n.d.
n.d.
13.4 ± 1.8
n.d.
9
9
1
The data are presented as mean ±s.d. (n = 3) n.d. = not determined.
Table 7. Inhibition activity of compounds 14a–g on cancer cell proliferation.
Compound
4c
4d
R5
CH
CH
R6
H
H1299 IC50 (μM)
26.9 ± 2.1
A549 IC50 (μM)
n.d.
PC9 IC50 (μM) 1
n.d.
1
3
3
1
CH
3
10.3 ± 0.8
9.9 ± 0.2
49.1 ± 1.9
1
4f
H
H
>100
n.d.
n.d.
n.d.
n.d.
1
4g
28.5 ± 0.6
1
The data are presented as mean ±s.d. (n = 3) n.d. = not determined.
3
. Materials and Methods

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3
.1. General Procedures
All reagents were purchased commercially. H-NMR and 13C-NMR spectra were recorded on
1
Bruker AC400 and Bruker AC600 NMR spectrometers, respectively (Billerica, MA, USA).
Low-resolution mass spectra were recorded on a 6120 Quadrupole mass spectrometer (Agilent,
Santa Clara, CA, USA) equipped with electrospray ionization (ESI). High-resolution mass spectra
+
were determined on triple TOF 5600 MS/MS system (AB Sciex, Concord, ON, Canada) in negative
ESI mode. The purity of target compounds was determined by high-performance liquid
chromatography (Agilent, Santa Clara, CA, USA, DIKMA Diamonsil Plus C18, 250 × 4.6 mm, 5μm,
2
5 °C, UV 290 nM). All the biologically tested compounds achieved ≥95% purity.
3
.2. Synthesis of diethyl 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetate (6a)
1,2-Dihydroxyanthracene-9,10-dione (1.2 g, 5 mmol), ethyl 2-bromoacetate (2 g, 12 mmol) and
K2
CO
3
(1.66 g, 12 mmol) were added to N,N-dimethylformamide (DMF, 50 mL) and the mixture was
stirred at 80 °C. The mixture was cooled to room temperature and poured into 10% aqueous HCl (350
mL). Then the suspension was extracted with ethyl acetate and the organic phase was washed with
brine, dried over anhydrous Na
2
SO
4
and filtered. The residue was purified by silica chromatography
1
after removal of ethyl acetate to afford 6a as a yellow solid (0.89 g, 43%). H-NMR (400 MHz, DMSO-d
6)
δ 8.16–8.10 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 7.93–7.84 (m, 2H), 7.53 (d, J = 8.8 Hz, 1H), 5.07 (s, 2H), 4.74
13
(
s, 2H), 4.20 (qd, J = 4.0, 7.2 Hz, 4H), 1.23 (td, J = 2.4, 7.2 Hz, 6H). C-NMR (151 MHz, DMSO) δ 181.69,
181.42, 168.27, 167.97, 156.49, 146.23, 134.58, 134.32, 133.94, 132.26, 127.15, 126.94, 126.67, 126.18, 124.64,
−
1
18.20, 68.72, 65.22, 60.99, 60.44, 14.07, 13.98. MS (ESI) (m/z): 413.1 (M − H) . HRMS (ESI) calcd for
−
C22H20O8
[M − H] : 413.1231; found: 413.1239. Purity 95.1% by HPLC.
3
.3. Synthesis of 2,2′-((9,10-dioxo-9,10-dihydroanthracene-1,2-diyl) bis(oxy)) diacetic acid (6b)
Compound 6a (206 mg, 0.5 mmol) and 0.5M aqueous LiOH (8 mL) were added into methanol (8 mL)
and the mixture was stirred at 30 °C. The mixture was added dropwise to 10% aqueous HCl and
1
acidified to pH 1–2. A yellow solid (169 mg, 95%) of 6b was obtained by filtration. H-NMR (400 MHz,
DMSO-d ) δ 12.76 (brs, 2H), 8.14 (d, J = 7.6 Hz, 2H), 8.01 (d, J = 8.4 Hz, 1H), 7.93-7.85 (m, 2H), 7.51 (d, J =
6
13
8
.8 Hz, 1H), 4.98 (s, 2H), 4.67 (s, 2H). C-NMR (151 MHz, DMSO) δ 181.95, 181.45, 169.75, 169.45,
56.76, 146.32, 134.62, 134.31, 133.98, 132.34, 126.97, 126.82, 126.71, 126.20, 124.61, 118.12, 68.62, 65.05.
1
−
−
MS (ESI) (m/z): 355.0 (M − H) . HRMS (ESI) calcd for C18
H
12
O [M − H] : 355.0459; found: 355.0462.
8
Purity 95.1% by HPLC.
3
.4. Synthesis of 2-((1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) oxy)-N, N-dimethylacetamide (6e)
Compound 6d (149 mg, 0.5 mmol), dimethylamine hydrochloride (31 mg, 0.68 mmol),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 120 mg, 0.6 mmol),
1
-hydroxybenzotriazole (HOBt, 80 mg, 0.6 mmol) and diisopropylethylamine (DIPEA, 175 μL, 1
mmol) were added to dry DMF (10 mL) and the mixture was stirred at room temperature. The
mixture was added dropwise to H
2
O (140 mL). An orange solid (114 mg, 70%) of 6e was obtained by
) δ 12.74 (s, 1H), 8.28–8.11 (m, 2H), 7.99–7.87 (m, 2H), 7.68 (d,
1
filtration. H-NMR (400 MHz, DMSO-d
6
1
3
J = 8.4 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 5.09 (s, 2H), 3.02 (s, 3H), 2.87 (s, 3H). C-NMR (151 MHz,
DMSO) δ 188.67, 180.76, 166.18, 152.60, 151.79, 135.20, 134.26, 133.48, 132.95, 126.81, 126.60, 124.89,
−
1
20.20, 118.29, 115.94, 66.12, 35.46, 35.01. MS (ESI) (m/z): 324.0 (M − H) . HRMS (ESI) calcd for
−
C18H
15NO
5
[M − H] : 324.0877; found: 324.0876. Purity 96.4% by HPLC.
3
.5. General Procedure for the Synthesis of Compounds 9a–q
Step 1. Nitric acid (1.5 mL, 33.33 mmol, Sinopharm Chemical Reagent Co., Ltd, Shanghai,
China) was added to the suspension of alizarin (5 g, 20.8 mmol) in acetic acid (350 mL) at 50 °C. A
yellow solid (4.15 g, 70%) of crude product 6 was obtained by filtration [40].
Step 2. Sn (10.5 g, 341 mmol, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China),
SnCl
2
·2H O (12.5 g, 55.4 mmol) and concentrated HCl (50.4 mL, 604.8 mmol) were added to the
2

Molecules 2019, 24, 845
12 of 19
suspension of crude product 6 (1.75 g, 6.14 mmol) in ethanol (350 mL, Sinopharm) and the mixture
was stirred at room temperature overnight. Then the mixture was poured into water (1 L) and a red
solid precipitated. A black solid of crude product 7 (1.41 g, 90%) was obtained after filtration and being
dried under vacuum [40].
Step 3. To the solution of crude product 7 (255 mg, 1 mmol) in dry pyridine (5 mL, Sinopharm),
corresponding sulfonyl chloride (1.5 mmol, Energy Chemical, Shanghai, China) was added and the
mixture was stirred at room temperature. Then it was added to 10% aqueous HCl (50 mL,
Sinopharm) and the suspension was extracted with ethyl acetate (Sinopharm). The organic phase
was washed with brine, dried over anhydrous Na
2
SO and filtered. The residue was purified by
4
silica chromatography after removal of ethyl acetate to give compounds 9a–q.
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-(trifluoromethyl) benzenesulfonamide (9a).
1
Yellow solid, 25% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.90 (brs, 1H), 10.67 (brs,
1
H), 8.22–8.12 (m, 2H), 8.09 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.95–7.86 (m, 2H), 7.73 (s, 1H).
13C-NMR (151 MHz, DMSO) δ 187.78, 180.56, 150.37, 144.20, 143.23, 135.02, 134.22, 133.28, 132.79,
1
1
32.68(q, J = 31.7Hz), 130.34, 127.60 (2C), 126.77, 126.61, 126.59, 126.39, 123.71, 123.38(q, J = 273.3 Hz),
−
−
13.49, 113.35. MS (ESI) (m/z): 462.0 (M − H) . HRMS (ESI) calcd for C21
H
12F
3
NO S [M − H] : 462.0265;
6
found: 462.0284. Purity 99.8% by HPLC.
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-nitrobenzenesulfonamide (9b). Orange solid,
1
5
8
1
1
−
0% yield. H-NMR (400 MHz, DMSO-d
6
) δ 12.61 (brs, 1H), 10.98 (brs, 1H), 10.79 (brs, 1H), 8.41 (d, J =
13
.0 Hz, 2H), 8.25–8.06 (m, 4H), 7.97–7.86 (m, 2H), 7.73 (s, 1H). C-NMR (151 MHz, DMSO) δ 187.79,
80.53, 150.41, 149.90, 145.71, 143.61, 135.05, 134.24, 133.27, 132.79, 130.11, 128.22(2C), 126.77, 126.41,
−
24.65(2C), 123.70, 114.04, 113.50. MS (ESI) (m/z): 439.0 (M − H) . HRMS (ESI) calcd for C20
H12
N
2
O S [M
8
−
H] : 439.0242; found: 439.0228. Purity 98.4% by HPLC.
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-(trifluoromethoxy) benzenesulfonamide (9c).
1
Yellow solid, 41% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.61 (brs, 1H), 10.94 (brs, 1H), 10.51 (brs,
1
H), 8.22–8.12 (m, 2H), 8.05–7.99 (m, 2H), 7.95–7.85 (m, 2H), 7.74 (s, 1H), 7.61 (dd, J = 1.2, 9.2 Hz, 2H).
13C-NMR (151 MHz, DMSO) δ 187.78, 180.60, 151.20, 150.35, 142.96, 139.19, 135.02, 134.23, 133.30,
32.82, 130.63, 129.29, 126.78, 126.40, 123.74, 121.47,119.80 (q, J = 259.7 Hz), 113.22, 113.12. MS (ESI)
1
−
−
(
m/z): 478.0 (M − H) . HRMS (ESI) calcd for C21
6.8% by HPLC.
H
12
F
3
NO S [M − H] : 478.0214; found: 478.0207. Purity
7
9
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2-(trifluoromethoxy) benzenesulfonamide (9d).
1
Yellow solid, 40% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.37 (brs,
1
7
1
H), 8.24–8.09 (m, 2H), 8.00 (dd, J = 2.0, 8.0 Hz, 1H), 7.96–7.86 (m, 2H), 7.83–7.75 (m, 1H), 7.71 (s, 1H),
1
3
.63–7.51 (m, 2H). C-NMR (151 MHz, DMSO) δ 187.81, 180.54, 150.37, 145.18, 143.20, 135.55, 135.05,
34.25, 133.28, 132.82, 132.34, 130.65, 130.41, 127.45, 126.80, 126.43, 123.61, 120.97, 119.81(q, J = 259.7
−
NO
S [M − H]−:
Hz), 113.72, 113.26. MS (ESI) (m/z): 477.8 (M − H) . HRMS (ESI) calcd for C21
H12
F
3
7
4
78.0214; found: 478.0225. Purity 95.2% by HPLC.
4
-Cyano-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9e). Red solid,
1
6
0% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.77 (brs, 2H), 8.22–8.12 (m, 2H), 8.09 (d,
1
3
J = 8.0 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.96–7.87 (m, 2H), 7.70 (s, 1H). C-NMR (151 MHz, DMSO) δ
1
1
87.81, 180.57, 150.39, 144.34, 143.46, 135.05, 134.25, 133.49, 133.29, 132.81, 130.22, 127.34, 126.81,
−
26.75, 126.44, 123.71, 117.60, 115.48, 113.78(2C), 113.44. MS (ESI) (m/z): 419.0 (M − H) . HRMS (ESI)
−
calcd for C21
H
12N
2
O6
S [M − H] : 419.0343; found: 419.0348. Purity 95.1% by HPLC.
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-fluorobenzenesulfonamide (9f). Yellow solid,
1
6
2% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.58 (brs, 1H), 10.84 (brs, 1H), 10.43 (brs, 1H), 8.19–8.11
1
3
(
m, 2H), 8.01–7.84 (m, 4H), 7.73 (s, 1H), 7.46 (t, J = 8.8 Hz, 2H). C-NMR (151 MHz, DMSO) δ 187.75,
1
=
−
80.59, 164.47(d, J = 250.7 Hz), 150.32, 142.76, 136.58, 134.99, 134.21, 133.28, 132.81, 130.82, 129.75(d, J
9.1 Hz,2C), 126.76, 126.38, 123.72, 116.57(d, J = 22.7 Hz, 2C), 113.10, 112.89. MS (ESI) (m/z): 412.0 (M
−
−
H) . HRMS (ESI) calcd for C20
H
12FNO S [M − H] : 412.0297; found: 412.0305. Purity 98.7% by HPLC.
6

Molecules 2019, 24, 845
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N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2-fluorobenzenesulfonamide (9g). Yellow solid,
1
4
2% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.61 (brs, 1H), 10.95 (brs, 1H), 10.49 (brs, 1H), 8.21–8.09
13
(
m, 2H), 7.95–7.80 (m, 3H), 7.77–7.67 (m, 2H), 7.46 (t, J = 9.4 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H). C-NMR
151 MHz, DMSO) δ 187.80, 180.56, 158.34(d, J = 256.7Hz), 150.40, 143.42, 136.02, 135.01, 134.21,
(
1
2
4
33.28, 132.81, 130.39, 129.87, 127.94(d, J = 12.1Hz), 126.77, 126.41, 124.90, 123.57, 117.44(d, J =
−
12FNO
S [M − H]−:
1.1Hz), 113.88, 113.32. MS (ESI) (m/z): 412.0 (M − H) . HRMS (ESI) calcd for C20
H
6
12.0297; found: 412.0300. Purity 96.1% by HPLC.
N-(3,4-Dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2,3-difluorobenzenesulfonamide (9h). Red solid,
1
5
2% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.83 (brs, 2H), 8.23–8.11 (m, 2H), 7.96–7.86
13
(
m, 2H), 7.79 (q, J = 8.6 Hz, 1H), 7.69 (s, 1H), 7.62 (t, J = 7.0 Hz, 1H), 7.44–7.34 (m, 1H). C-NMR (151
MHz, DMSO) δ 187.86, 180.52, 150.46,149.96(dd, J = 249.9Hz, J=12.1Hz),146.64(dd, J = 256.7Hz, J =
3.6Hz) 144.45, 135.05, 134.22, 133.29, 132.81, 130.25(d, J = 10.6Hz), 129.75, 126.78(d, J = 10.6Hz), 126.43
d, J = 7.6Hz), 125.26, 124.95, 123.51, 122.86(d, J = 16.6Hz), 115.28(d, J = 6.0Hz), 113.72. MS (ESI) (m/z):
1
−
−
4
30.0 (M − H) . HRMS (ESI) calcd for C20
H
11FNO S [M − H] : 430.0202; found: 430.0206. Purity 95.4% by
6
HPLC.
4-Chloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9i). Yellow solid,
1
5
6% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.61 (brs, 1H), 10.94 (brs, 1H), 10.47 (brs, 1H), 8.22–8.11
13
(
m, 2H), 7.96–7.83 (m, 4H), 7.78–7.63 (m, 3H). C-NMR (151 MHz, DMSO) δ 187.76, 180.58, 150.34,
1
1
4
42.90, 139.07, 138.04, 135.00, 134.22, 133.28, 132.80, 130.63, 129.50(2C), 128.57(2C), 126.77, 126.39,
−
12ClNO
S [M − H]−:
23.72, 113.18, 113.08. MS (ESI) (m/z): 427.8 (M − H) . HRMS (ESI) calcd for C20
H
6
28.0001; found: 428.0005. Purity 99.4% by HPLC.
2,4-Dichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9j). Orange
1
solid, 60% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.56 (brs, 1H),
13
8
.20–8.08 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 7.95–7.84 (m, 3H), 7.68–7.63 (m, 2H). C-NMR (151 MHz,
DMSO) δ 187.76, 180.50, 150.44, 143.33, 138.62, 136.55, 134.99, 134.20, 133.24, 132.79, 132.25, 132.12,
−
1
31.49, 130.24, 127.85, 126.75, 126.38, 123.63, 113.52, 113.36. MS (ESI) (m/z): 461.8 (M − H) . HRMS (ESI)
−
calcd for C20
H
11Cl
2
NO
6
S [M − H] : 461.9611; found: 461.9614. Purity 99.2% by HPLC.
2,6-Dichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9k). Red solid,
1
1
2% yield. H-NMR (400 MHz, DMSO-d ) δ 12.62 (brs, 1H), 11.00 (brs, 1H), 10.56 (brs, 1H), 8.20–8.09
6
13
(
m, 2H), 7.94–7.83 (m, 2H), 7.72–7.52 (m, 4H). C-NMR (151 MHz, DMSO) δ 187.71, 180.43, 150.40,
1
1
4
42.57, 134.95, 134.80, 134.29, 134.17, 134.13 (2C), 133.22, 132.78, 131.85(2C), 130.41, 126.75, 126.36,
−
NO
S [M − H]−:
23.79, 113.20, 112.18. MS (ESI) (m/z): 461.8 (M − H) . HRMS (ESI) calcd for C20
H
11Cl
2
6
61.9611; found: 461.9601. Purity 95.7% by HPLC.
2,4,6-Trichloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9l). Yellow
1
solid, 10% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.62 (brs, 1H), 10.96 (brs, 1H), 10.73 (brs, 1H),
1
3
8
1
1
.21–8.11 (m, 2H), 7.96–7.86 (m, 4H), 7.66 (s, 1H). C-NMR (151 MHz, DMSO) δ 187.77, 180.49, 150.48,
43.28, 137.64, 135.25 (2C), 135.00, 134.43, 134.22, 133.27, 132.83, 131.20 (3C), 126.79, 126.41, 123.78,
−
−
13.43, 113.21. MS (ESI) (m/z): 495.8 (M − H) . HRMS (ESI) calcd for C20
H10Cl
3
NO S [M − H] : 495.9222;
6
found: 495.9220. Purity 99.4% by HPLC.
4-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9m). Yellow solid,
1
6
5% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.88 (brs, 1H), 10.47 (brs, 1H), 8.22–8.11
13
(
m, 2H), 7.95–7.78 (m, 6H), 7.73 (s, 1H). C-NMR (151 MHz, DMSO) δ 187.77, 180.59, 150.33, 142.88,
139.50, 135.01, 134.23, 133.29, 132.81, 132.44 (2C), 130.65, 128.64 (2C), 127.08, 126.78, 126.39, 123.73,
−
−
1
13.18, 113.05. MS (ESI) (m/z): 471.8 (M − H) . HRMS (ESI) calcd for C20
H
12BrNO S [M − H] : 471.9496;
6
found: 471.9495. Purity 96.0% by HPLC.
2-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9n). Yellow solid,
1
4
6% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.63 (brs, 1H), 11.10 (brs, 1H), 10.24 (brs, 1H), 8.20–8.05
13
(
m, 3H), 7.93–7.84 (m, 3H), 7.67–7.53 (m, 3H). C-NMR (151 MHz, DMSO) δ 187.72, 180.55, 150.37,
142.31, 138.96, 135.62, 134.97, 134.74, 134.21, 133.23, 132.80, 130.93, 130.76, 128.28, 126.75, 126.38, 123.75,

Molecules 2019, 24, 845
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−
S [M − H]−:
1
19.31, 113.07, 112.14. MS (ESI) (m/z): 471.8 (M − H) . HRMS (ESI) calcd for C20
H
12BrNO
6
417.9496; found: 417.9498. Purity 97.5% by HPLC.
3-Bromo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9o). Yellow solid,
1
5
4% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.99 (brs, 1H), 10.53 (brs, 1H), 8.21–8.11
13
(
m, 2H), 8.10 (s, 1H), 7.94–7.83 (m, 4H), 7.71 (s, 1H), 7.57 (t, J = 8.0 Hz, 1H). C-NMR (151 MHz, DMSO)
δ 187.75, 180.55, 150.33, 142.90, 142.23, 136.02, 134.98, 134.19, 133.27, 132.79, 131.56, 130.49, 129.08,
−
1
26.76, 126.37, 125.61, 123.71, 122.16, 113.22, 112.97. MS (ESI) (m/z): 473.8 (M − H) .HRMS (ESI) calcd for
−
C20
H
12BrNO
6
S [M − H] : 417.9496; found: 417.9500. Purity 96.1% by HPLC.
4-Iodo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9p). Yellow solid, 70%
1
yield. H-NMR (400 MHz, DMSO-d
6) δ 12.60 (brs, 1H), 10.87 (brs, 1H), 10.44 (brs, 1H), 8.23–8.10 (m,
13
2
H), 7.99 (d, J = 8.0 Hz, 2H), 7.96–7.85 (m, 2H), 7.73 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H). C-NMR (151 MHz,
DMSO) δ 187.75, 180.61, 150.31, 142.73, 139.85, 138.23 (2C), 135.00, 134.22, 133.29, 132.82, 130.76, 128.27
−
(
2C), 126.78, 126.39, 123.74, 113.12, 112.85, 101.56. MS (ESI) (m/z): 519.8 (M − H) . HRMS (ESI) calcd for
−
C20
H
12INO
6
S [M − H] : 519.9357; found: 519.9334. Purity 99.3% by HPLC.
2-Iodo-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (9q). Yellow solid, 48%
1
yield. H-NMR (400 MHz, DMSO-d
6) δ 12.64 (brs, 1H), 11.18 (brs, 1H), 10.02 (brs, 1H), 8.19–8.06 (m, 4H),
13
7
.93–7.85 (m, 2H), 7.65 (s, 1H), 7.61 (td, J = 1.2, 8.0 Hz, 1H), 7.33 (td, J = 1.6, 8.0 Hz, 1H). C-NMR (151
MHz, DMSO) δ 187.72, 180.58, 150.30, 142.77, 142.13, 141.83, 134.97, 134.22, 134.21, 133.24, 132.81,
−
1
31.01, 130.16, 128.64, 126.76, 126.38, 123.83, 112.96, 111.59, 93.30. MS (ESI) (m/z): 519.8 (M − H) . HRMS
−
(
ESI) calcd for C20
H
12INO
6
S [M − H] : 519.9357; found: 519.9356. Purity 96.3% by HPLC.
3
2
.6. Synthesis of ethyl 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-
-yl) oxy) acetate (12)
Step 1. Compound 10 was synthesized according to the same route of compound 7 in which
compound 5 was replaced by compound 6c. Yellow solid, 73% yield.
Step 2. Compound 11 was synthesized according to the same route of compound 8 in which
compound 7 was replaced by compound 10. Purple solid, 93% yield.
Step 3. Compound 12 was synthesized according to the same route of 9i in which compound 8
1
was replaced by compound 11. Red solid, 6% yield. H-NMR (400 MHz, DMSO-d
6) δ 13.37 (s, 1H),
1
4
1
1
2.44 (s, 1H), 8.20 (d, J = 7.2 Hz, 2H), 8.00–7.83 (m, 4H), 7.64 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 5.09 (s, 2H),
13
.25 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). C-NMR (151 MHz, DMSO) δ 187.87, 184.15, 167.48,
53.41, 150.30, 138.86, 137.31, 135.41, 135.23, 134.74, 133.52, 132.12, 129.90 (2C), 128.71 (2C), 127.06,
−
26.50, 114.75, 109.81, 107.99, 65.44, 61.24, 14.04. MS (ESI) (m/z): 514.2 (M − H) . HRMS (ESI) calcd for
−
C24
H
18ClNO
8
S [M − H] : 514.0369; found: 514.0364. Purity 96.0% by HPLC.
3
.7. Synthesis of 2-((4-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetic acid (13)
Compound 13 was synthesized according to the same route of 6b in which compound 6a was
1
replaced by compound 12. Red solid, 95% yield. H-NMR (400 MHz, DMSO-d
H), 8.23–8.13 (m, 2H), 7.98–7.84 (m, 4H), 7.64 (d, J = 8.4 Hz, 2H), 7.35 (s, 1H), 4.99 (s, 2H). C-NMR (151
MHz, Pyr) δ 188.75, 185.01, 171.15, 155.76, 152.45, 140.30, 139.12, 137.34, 134.82, 133.21, 130.44 (2C),
6
) δ 13.36 (s, 1H), 12.53 (s,
13
1
1
29.85 (2C), 127.85, 127.27, 121.12, 119.10, 115.52, 110.23, 108.94, 67.24. MS (ESI) (m/z): 485.8 (M − H)−.
−
HRMS (ESI) calcd for C22
H
14ClNO
8
S [M − H] : 486.0056; found: 486.0061. Purity 98.0% by HPLC.
3
2
.8. Synthesis of ethyl 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-
-yl) oxy) acetate (14a)
Compound 14a was synthesized according to the same route of 6c in which compound 5 was
1
replaced by compound 9i. Yellow solid, 24% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.74 (s, 1H), 10.59
(s, 1H), 8.22–8.09 (m, 2H), 8.01–7.87 (m, 4H), 7.83–7.68 (m, 3H), 4.85 (s, 2H), 4.19 (q, J = 7.2 Hz, 2H), 1.22
13
(
t, J = 7.2 Hz, 3H). C-NMR (151 MHz, DMSO) δ 187.53, 180.83, 169.46, 154.03, 139.72, 138.49, 138.21,

Molecules 2019, 24, 845
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136.81, 135.08, 134.58, 132.87, 132.72, 129.71(2C), 128.70(2C), 128.46, 126.88, 126.45, 113.29, 109.22, 68.53,
−
−
6
0.98, 13.98. MS (ESI) (m/z): 514.0 (M − H) . HRMS (ESI) calcd for C24
H
18ClNO S [M − H] : 514.0369;
8
found: 514.0363. Purity 95.6% by HPLC.
3
.9. Synthesis of 2-((3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
oxy) acetic acid (14b)
Compound 14b was synthesized according to the same route of 6b in which compound 6a was
1
replaced by compound 14a. Yellow solid, 95% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.72 (s, 1H),
13
8
.19–8.10 (m, 2H), 7.97–7.87 (m, 4H), 7.75 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 4.80 (s, 2H). C-NMR (151
MHz, DMSO) δ 187.49, 180.86, 171.92, 154.23, 139.93, 138.53, 138.14, 137.00, 135.06, 134.59, 132.86,
32.74, 129.76 (2C), 128.67 (2C), 128.58, 126.89, 126.45, 113.28, 108.86, 68.92. MS (ESI) (m/z): 486.0 (M −
1
−
−
H) . HRMS (ESI) calcd for C22
H
14ClNO S [M − H] : 486.0056; found: 486.0049. Purity 99.0% by HPLC.
8
3
.10. Synthesis of 4-chloro-N-(4-hydroxy-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzene-
sulfonamide (14c)
Compound 9i (215 mg, 0.5 mmol), K
2
CO (70 mg, 0.5 mmol) and iodomethane (70 mg, 12 mmol)
3
were added to DMF (2.5 mL) and mixture was stirred at room temperature. The mixture was added
to 10% aqueous HCl (20 mL). The suspension was extracted with ethyl acetate and the organic phase
was washed with brine, dried over anhydrous Na
silica chromatography after removal of ethyl acetate to afford 14c as a yellow solid (115 mg, 52%).
1H-NMR (400 MHz, DMSO-d
) δ 12.68 (brs, 1H), 11.02 (brs, 1H), 8.28–8.14 (m, 2H), 7.99–7.89 (m, 2H),
2SO
4
and filtered. The residue was purified by
6
1
3
7
1
1
.70 (q, J = 8.5 Hz, 4H), 7.45 (s, 1H), 3.20 (s, 3H). C-NMR (151 MHz, DMSO) δ 188.19, 180.25, 151.50,
50.13, 138.19, 137.11, 135.30, 134.32, 133.37, 132.82, 132.10, 129.46 (2C), 129.16 (2C), 126.88, 126.62,
−
22.74, 121.75, 115.43, 37.30. MS (ESI) (m/z): 442.0 (M − H) . HRMS (ESI) calcd for C21
H
14ClNO S [M −
6
−
H] : 442.0158; found: 442.0162. Purity 100% by HPLC. Purity 97.0% by HPLC.
3
.11. Synthesis of 4-chloro-N-(4-hydroxy-3-methoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-N-methyl-
benzenesulfonamide (14d)
Compound 14d was synthesized according to the same route of 14c in which room temperature
1
was raised to 40 °C. Orange solid, 50% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.79 (s, 1H), 8.28–8.13
13
(
m, 2H), 8.00–7.92 (m, 2H), 7.76 (q, J = 8.4 Hz,4H), 7.40 (s, 1H), 3.80 (s, 3H), 3.23 (s, 3H). C-NMR (151
MHz, DMSO) δ 188.22, 180.56, 155.48, 150.66, 138.57, 138.37, 136.79, 135.40, 134.67, 132.96, 132.83,
29.62 (2C), 129.23 (2C), 127.10, 126.96, 126.72, 119.91, 116.60, 60.65, 38.12. MS (ESI) (m/z): 456.0 (M −
1
−
−
H) . HRMS (ESI) calcd for C22
H
16ClNO S [M − H] : 456.0314; found: 456.0305. Purity 100% by HPLC.
6
3
6
.12. Synthesis of 4-((4-chlorophenyl) sulfonyl)-12-hydroxy-3,4-dihydro-2H-anthra[2 ,3-b][1,4]-oxazine-
,11-dione (14e)
Compound 14e was synthesized according to the same route of 14c in which iodomethane was
1
replaced by 1,2-dibromoethane. Orange solid, 32% yield. H-NMR (400 MHz, DMSO-d
6) δ 12.63 (s,
1
2
1
1
4
H), 8.18 (t, J = 8.2 Hz, 2H), 8.11 (s, 1H), 7.98–7.88 (m, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz,
13
H), 4.06 (dd, J = 4.4, 16.8 Hz, 4H). C-NMR (151 MHz, DMSO) δ 187.52, 180.70, 151.78, 140.17, 139.33,
36.21, 135.07, 134.43, 133.27, 132.79, 130.21(2C), 129.30, 129.04(2C), 126.92, 126.55, 124.47, 113.41,
+
14ClNO
S [M + H]+:
12.82, 64.03, 43.77. MS (ESI) (m/z): 456.0 (M + H) . HRMS (ESI) calcd for C22
H
6
56.0303; found: 456.0307. Purity 100% by HPLC.
3
2
.13. Synthesis of 4-chloro-N-(3-(2-hydrazinyl-2-oxoethoxy)-4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-
-yl) benzenesulfonamide (14f)
Compound 14a (20 mg, 0.04 mmol) and hydrazine hydrate (85%, 5 μL, 0.16 mmol) were added to
ethanol (1.5 mL) and mixture was stirred at 76 °C. A yellow solid (14 mg, 72%) of 14f was obtained by
1
13
filtration. H-NMR (400 MHz, DMSO-d
6
) δ 12.95 (brs, 1H), 8.43–7.36 (m, 9H), 4.53 (s, 2H). C-NMR (151
MHz, DMSO) δ 187.43, 180.96, 168.15, 154.79, 140.49, 138.43, 135.10, 134.98, 134.67, 132.89, 132.81,

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129.78 (2C), 129.08, 128.64 (2C), 128.51, 126.95, 126.49, 113.41, 109.56, 70.47. MS (ESI) (m/z): 500.0 (M −
−
−
H) . HRMS (ESI) calcd for C22
H
16ClN
3
O S [M − H] : 500.0325; found: 500.0340. Purity 95.1% by HPLC.
7
3
.14. Synthesis of 3-((4-chlorophenyl) sulfonamido)-1-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl
methanesulfonate (14g)
Compound 9i (107 mg, 0.25 mmol) and DIPEA (50 μL, 0.3mmol) were added to pyridine (1.5
mL), and then methanesulfonyl chloride (23 μL,0.3 mmol) was added to the mixture. After being
stirred at room temperature for 4 h, the mixture was added to 10% aqueous HCl (20 mL). The
suspension was extracted with ethyl acetate and the organic phase was washed with brine, dried
over anhydrous Na
2
SO
4
and filtered. The residue was purified by silica chromatography after
1
removal of ethyl acetate to afford 14 g as a yellow solid (83 mg, 65%). H-NMR (400 MHz, DMSO-d
6)
δ 12.81 (s, 1H), 8.23–8.11 (m, 2H), 8.00–7.90 (m, 4H), 7.72 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 3.61 (s, 3H).
13C-NMR (151 MHz, DMSO) δ 186.86, 180.93, 155.49, 138.86, 138.32, 135.07, 134.76, 132.83 (2C),
1
31.81, 131.31, 129.70 (3C), 128.79, (3C) 126.98, 126.50, 109.55, 40.45. MS (ESI) (m/z): 505.8 (M − H)−.
−
HRMS (ESI) calcd for C21
H
14ClNO8S
2
[M − H] : 505.9777; found: 505.9791. Purity 99.5% by HPLC.
3
.15. PGAM1 Enzymatic Assay
Firstly, 1 μL inhibitor in dimethyl sulfoxide (DMSO) incubated with 49 μL 4.6 nM recombinant
PGAM1, then 50 μL enzyme mix containing 3 units/mL enolase (Sigma-Aldrich, Saint Louis, MO,
USA), 3 units/mL recombinant pyruvate kinase M2 (Sigma-Aldrich), 0.6 units/mL recombinant
lactatdehydrogenase (LDH, Sigma-Aldrich), 100 mM Tris-HCl, 100 mM KCl, 5 mM MgCl
adenosine diphosphate (ADP), 0.2 mM the reduced form of nicotinamide adenine dinucleotide
NADH) and 4 mM 3PG was added. PGAM1 activity was measured as the decrease in OD at 340 nm.
2
, 1 mM
(
In addition, we performed a counter screening in which 4mM 2PG was added to 50 μL reaction mix
containing the indicated inhibitor, 3 units/mL enolase (Sigma-Aldrich), 3 units/mL recombinant
pyruvate kinase M2 (Sigma-Aldrich), 0.6 units/mL recombinant LDH (Sigma-Aldrich), 100 mM
Tris-HCl, 100 mM KCl, 5 mM MgCl
2, 1 mM ADP and 0.2 mM NADH. The other three enzyme
activity was measured as the decrease in OD at 340 nm [17].
3
.16. Constructions of Mutations of PGAM1
The wild-type plasmid was a gift from Department of Chemistry and Institute for Biophysical
Dynamics, University of Chicago (Chicago, IL, USA). To obtain PGAM1 F22A/R116H/R191H mutant
construct, the quick change kit (#KOD-401, TOYOBO, Osaka, Japan) was used to make site-specific
mutagenesis, which confirmed by standard DNA sequencing methods. The primers used for the
mutation were listed below:
PGAM1 F22A Forward: GAACCTGGAGAACCGCGCCAGCGGCTGGTACGAC
PGAM1 F22A Reverse: GTCGTACCAGCCGCTGGCGCGGTTCTCCAGGTTC
PGAM1 K116H Forward: CAGGTGAAGATCTGGCACCGCTCCTATGATGTCC
PGAM1 K116H Reverse: GGACATCATAGGAGCGGTGCCAGATCTTCATCTG
PGAM1 K119H Forward: CATGGCAACAGCCTCCACGGCATTGTCAAGCAT
PGAM1 K119H Reverse: ATGCTTGACAATGCCGTGGAGGCTGTTGCCATG
3
.17. Protein Purification, Crystallization, Data Collection and Structure Determination
The C-terminal His -tagged PGAM1 was expressed and affinity purified following the reported
6
protocols [22]. The crystals of PGAM1 were obtained using the hanging drop vapor-diffusion
method at 16 °C in a crystallization buffer containing of 8% (w/v) PEG3350 and 100 mM MES 6.0. To
obtain the co-crystal of PGAM1 with compound 9i, the crystals of PGAM1 were soaked in stock
solution containing 500 μM 9i for 2 h. Crystals were then cryo-protected by brief soaking in a

Molecules 2019, 24, 845
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mixture solution of crystallization buffer:glycerol (76:24) and quickly cooled in liquid nitrogen.
Diffraction data were collected at beamline BL17U1, BL18U1 and BL19U1 in the Shanghai
Synchrotron Radiation Facility (SSRF, Shanghai, China) using an X-ray beam of wavelength 0.97851
Å. The data was handled with HKL3000 [41] and the structure was determined by using molecular
replacement in CCP4 [42] via an initial model of PGAM1 derived from PDB entry 4GPZ [22]. The
model was then refined to 1.98 Å resolution using Phenix. The ligand restraints were generated
using eLBOW in Phenix and manual rebuilding of the model was completed using the molecular
graphics program COOT [43] according to the electronic density. All the graphs were plotted by
Pymol (DeLano Scientific LLC, San Carlos, CA, USA).
3
.18. Cell Viability Assays
The H1299 cell line was obtained from Guangzhou Jenniobio Biotechnology Co., Ltd.
(Guangzhou, China), PC9 and A549 cells were gifted from Deng Jiong’s lab in the School of
Medicine, Shanghai Jiao Tong University, Shanghai, China). H1299, PC9 cells were cultured in
RPMI-1640 medium and A549 cells were cultured in F-12K medium containing 10% fetal bovine
serum (FBS), 100 units/mL of Penicillin and 100 μg/mL Streptomycin. In cell viability assays, 2000
H1299 cells, A549 cells or 1000 PC9 cells per well were seeded in 96-well plate. After attachment for
2
4 h, the cells were treated with indicated inhibitor for 72 h. After incubation with 0.5 mg/mL
methylthiazolyldiphenyl-tetrazolium bromide (MTT) for 4 h, cell viability was measured as the OD
at 570 nm.
4
. Conclusions
In summary, we designed and synthesized 31 anthraquinone derivatives as PGAM1 inhibitors.
The SAR study focused on the effect of 1,2-substituents, 3-substituents, 2,4-substituents, and
,3-substituents on the anthraquinone core scaffold activity against PGAM1. Among them, 3-
2
sulfonamide substituents but not 4-substituents on the anthraquinone scaffold were essential for
maintaining potency, which was further revealed by the X-ray structure of PGAM1 complexed with
compound 9i with IC50 value of 0.27 μM. In addition, modifications of the hydroxyl of alizarin
disrupted the π-cation interaction of PGAM1 and the anthraquinone derivatives which led to the
loss of potency. Taken as a whole, the SAR study of anthraquinone derivatives against PGAM1
provided useful information for further discovery of PGAM1 inhibitors.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: The activity
of PGAM1 mutant.
Author Contributions: conceptualization, K.H., D.Y. and L.Z.; chemistry, K.H. and H.L.; biological assay, K.H.
and L.J.; structure determination, L.J. and L.Z.; writing—original draft preparation, K.H.; writing—review and
editing, K.H., D.Y. and L.Z.
Funding: This research was funded by the Chinese National Natural Science Foundation (grant no. 21472026,
2
1877014), the Shanghai Municipal Committee of Science and Technology(14XD1400300), the program for
Shanghai Rising Star (15QA1400300), and the open grant of the State Key Laboratory of Bio-organic and Natural
Products Chemistry, CAS.
Acknowledgments: We thank Deng Jiong’s lab in the School of Medicine, Shanghai Jiao Tong University for
providing the A549 and PC9 cells.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).