Synthesis and biological evaluation of anthraquinone derivatives as allosteric phosphoglycerate mutase 1 inhibitors for cancer treatment

Article abstract of DOI:10.1016/j.ejmech.2019.01.085

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis, pentose phosphate pathway, and serine synthesis to promote tumor growth through the regulation of its substrate 3-phosphoglycerate (3 PG) and product 2-phosphoglycerate (2 PG). Herein, based on our previously reported PGAM1 inhibitor PGMI-004A, we have developed anthraquinone derivatives as novel allosteric PGAM1 inhibitors and the structure?activity relationship (SAR) was investigated. In addition, we determined the co-crystal structure of PGAM1 and the inhibitor 8g, demonstrating that the inhibitor was located at a novel allosteric site. Among the derivatives, compound 8t was selected for further study, with IC₅₀ values of 0.25 and approximately 5 μM in enzymatic and cell-based assays, respectively. Mechanistically, compound 8t reduced the glycolysis and oxygen consumption rate in cancer cells, which led to decreased adenosine 5′-triphosphate (ATP) production and subsequent 5′ adenosine monophosphate-activated protein kinase (AMPK) activation. The inhibitor 8t also exhibited good efficacy in delaying tumor growth in H1299 xenograft model without obvious toxicity. Taken together, this proof-of-principle work further validates PGAM1 as a potential target for cancer therapy and provides useful information on anti-tumor drug discovery targeting PGAM1.

Full text of DOI:10.1016/j.ejmech.2019.01.085

Accepted Manuscript
Synthesis and biological evaluation of anthraquinone derivatives as allosteric
phosphoglycerate mutase 1 inhibitors for cancer treatment
Ke Huang, Lulu Jiang, Ronghui Liang, Huiti Li, Xiaoxue Ruan, Changliang Shan,
Deyong Ye, Lu Zhou
PII:
S0223-5234(19)30110-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.085
EJMECH 11092
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 December 2018
Revised Date: 26 January 2019
Accepted Date: 31 January 2019
Please cite this article as: K. Huang, L. Jiang, R. Liang, H. Li, X. Ruan, C. Shan, D. Ye, L. Zhou,
Synthesis and biological evaluation of anthraquinone derivatives as allosteric phosphoglycerate
mutase 1 inhibitors for cancer treatment, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.085.
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ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of
Anthraquinone Derivatives as Allosteric
Phosphoglycerate Mutase 1 Inhibitors for
Cancer Treatment
‡
‡
‡
Ke Huang a, , Lulu Jiang a, , Ronghui Liang b, , Huiti Li a, Xiaoxue Ruan a, Changliang
Shan b,c*, Deyong Ye a,*, and Lu Zhou a,*
Table of Contents graphic

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Synthesis and Biological Evaluation of
Anthraquinone Derivatives as Allosteric
Phosphoglycerate Mutase 1 Inhibitors for Cancer
Treatment
‡
‡
‡
Ke Huang a, , Lulu Jiang a, , Ronghui Liang b, , Huiti Li a, Xiaoxue Ruan a, Changliang Shan b,c*
,
Deyong Ye a,*, and Lu Zhou a,*
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, No. 826,
Zhangheng Rd., Shanghai 201203, China
bBiomedical Translational Research Institute, Jinan University, Guangzhou, Guangdong 510632,
China
c State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai
University, Tianjin 300350, China.
‡K.H., L.J. and R.L. contributed to the work equally.
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* Corresponding Author
email: changliangshan@nankai.edu.cn (C. Shan); dyye@shmu.edu.cn (D. Ye);
zhoulu@fudan.edu.cn (L. Zhou).
ABSTRACT
Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis, pentose phosphate pathway, and
serine synthesis to promote tumor growth through the regulation of its substrate 3-
phosphoglycerate (3PG) and product 2-phosphoglycerate (2PG). Herein, based on our
previously reported PGAM1 inhibitor PGMI-004A, we have developed anthraquinone
derivatives as novel allosteric PGAM1 inhibitors and the structure−activity relationship (SAR)
was investigated. In addition, we determined the co-crystal structure of PGAM1 and the inhibitor
8g, demonstrating that the inhibitor was located at a novel allosteric site. Among the derivatives,
compound 8t was selected for further study, with IC50 values of 0.25 and approximately 5 µM in
enzymatic and cell-based assays, respectively. Mechanistically, compound 8t reduced the
glycolysis and oxygen consumption rate in cancer cells, which led to decreased adenosine 5´-
triphosphate (ATP) production and subsequent 5' adenosine monophosphate-activated protein
kinase (AMPK) activation. The inhibitor 8t also exhibited good efficacy in delaying tumor
growth in H1299 xenograft model without obvious toxicity. Taken together, this proof-of-
principle work further validates PGAM1 as a potential target for cancer therapy and provides
useful information on anti-tumor drug discovery targeting PGAM1.
Keywords: Phosphoglycerate mutase 1; Anthraquinone derivatives; Allosteric inhibitors;
Cancer treatment
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1. INTRODUCTION
Cancer is one of the most lethal diseases and seriously threatens human life worldwide. One of
the hallmarks of cancer is the deregulation of cellular energetics [1]. Nearly a century ago,
Warburg found that cancer cells prefer anaerobic glycolysis rather than oxidative
phosphorylation, whether in anaerobic or aerobic conditions; this was named the “Warburg
effect” [2,3]. Although adenosine 5´-triphosphate (ATP) is generated inefficiently in this manner,
considerably more glucose is taken in to produce adequate ATP necessary for proliferation [4,5].
More importantly, anaerobic glycolysis provides cancer cells with the building blocks essential
for anabolic biosynthesis, such as amino acids and lipids. In addition, through pentose phosphate
pathway (PPP), the precursors for nucleotides and the reduced form of nicotinamide adenine
dinucleotide phosphate (NADPH), which maintains the cell in an antioxidant status and
contributes to lipid synthesis, are generated [5,6].
Despite this knowledge regarding why cancer cells adopt reprogrammed metabolism, the
detailed mechanisms remain poorly understood. In recent years there has been an emerging
interest in uncovering the secrets of abnormal cancer metabolism and related phenomena [6-10].
Some metabolic enzymes are reported to play pivotal roles in cancer metabolism, including
isocitrate dehydrogenase (IDH) [11] and the M2 isozyme of pyruvate kinase (PKM2) [12], to
name a few. Since tumor cell metabolism is regarded as cancer’s Achilles’ heel [6], a trend for
targeting cancer metabolism in efforts to develop cancer therapies is growing [13-15]. Small
molecules targeting IDH [16,17] and PKM2 [18] as anti-cancer agents have been described,
which also illustrates the possibility of regulating cancer cell metabolism to develop cancer
treatments.
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Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that catalyzes the conversion of
3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) [19]. Our previous study showed that
3PG inhibited 6-phosphogluconate dehydrogenase in PPP, while 2PG activated 3-
phosphoglycerate dehydrogenase (PHGDH) in serine synthesis [20]. Inhibition of PGAM1
should give rise to increased intracellular concentration of 3PG and decreased concentration of
2PG, which will result in attenuated glycolysis, biosynthesis, and eventually reduced tumor
growth [20]. Hence, developing small molecules targeting PGAM1 seems to be “killing two
birds with one stone” as a cancer therapeutic strategy [21]. In addition, posttranslational
regulation, such as the phosphorylation of Tyr26 by oncogenic signaling [22] and the
deacetylation of Lys100 by sirtuin 2 under oxidative stress, stimulate PGAM to promote cancer
cell proliferation [23]. Recently, PGAM1 has been reported to facilitate cancer cell migration
through the interaction with α-smooth muscle actin (ACTA2) independent of its metabolic
activity [24] and promote homologous recombination repair by regulating the
deoxyribonucleotide triphosphate nucleoside (dNTP) pool [25]. As well as these diverse roles
PGAM1 engages in cancer metabolism, upregulated expression of PGAM1 is observed in
multiple tumors, including renal clear cell carcinoma [26], urothelial bladder cancer [27], and
pancreatic cancer [28] and it correlates with the prognosis of cancer patients [26-28], which
further demonstrates the possibility of targeting PGAM1 for cancer therapy.
Fig. 1. Representative PGAM1 inhibitors.
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In spite of the urge for potent PGAM1 inhibitors, only a few have been reported to date
(Figure 1). In 2005, Evans, M.J. et al. identified MJE3 as a covalent PGAM1 inhibitor by cell-
based screening and subsequent in situ proteome reactivity profiling [29]. Later, this group found
that MJE3 labeled PGAM1 on Lys100 by spiroepoxides [30]. Another PGAM1 inhibitor
discovered recently is (-)-epigallocatechin-3-gallate (EGCG), the major natural catechin from
green tea extract, which demonstrated strong molecular potency [31]. Previously, we reported
the compounds PGMI-004A [20] and xanthone devivative 12r [32], which inhibited PGAM1
activity with IC50 values of 13.1 and 2.7 µM and cancer cell proliferation with IC50 values of 25.6
and 5 µM, respectively. Since only a limited amount of anthraquinone analogues were
investigated as PGAM1 inhibitors [20], the structure−activity relationship (SAR) of
anthraquinone analogues and PGAM1 remains largely unknown. Here we report a series of
anthraquinone derivatives targeting PGAM1 as anti-cancer agents and studied the SAR. In
addition, we determined the crystal structure of PGAM1 in complex with one of the inhibitors
8g, which revealed the molecular mechanism of anthraquinone derivatives interacting with
PGAM1. The selected compound 8t adopted the same binding mode as compound 8g and
suppressed glycolysis and induced the generation of reactive oxygen species (ROS) in cancer
cells. Finally, we evaluated the pharmacokinetic properties of compound 8t and it significantly
attenuated tumor growth both in vitro and in vivo.
2. RESULTS AND DISCUSSION
2.1 Synthetic Chemistry
As shown in Scheme 1, we started from commercially available alizarin (5). After nitrification
of alizarin, followed by reduction with Sn and HCl, the key intermediate 7 was afforded and
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sulfonylation of the amino group with a variety of sulfonyl chlorides gave the target compounds
8a-v. In the meantime, we synthesized compound 9 and further modification of compound 9 via
Pd-catalyzed Buchwald-Hartwig cross coupling gave compounds 10a-c.
Scheme 1. Synthesis of compounds 8a-v, 10a-ca
aReagents and conditions: (a) HNO3, CH3COOH, 50 °C; (b) Sn, SnCl2·H2O, HCl, EtOH; (c)
R3SO2Cl, Py; (d) Pd2(dba)3, BINAP, NaOBu-t, corresponding amino.
Fig. 2. Design strategy of PGAM1 inhibitors from the lead compound PGMI-004A and SAR
exploration of the anthraquinone derivatives.
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2.2 SAR study of the anthraquinone derivatives
Previously we have discovered that the sulfonamide group of PGMI-004A was essential for its
biological activity but detailed SAR of anthraquinone derivatives and PGAM1 remained unclear.
Here we reversed the sulfonamide group of PGMI-004A to investigate the SAR of 3-
substituents of the anthraquinone scaffold against PGAM1 (Figure 2). Firstly, we synthesized
compounds 8a–i. As shown in Table 1, compounds containing lipophilic groups, such as
cyclohexyl (8c) and phenyl (8f) were much more potent than compounds containing a
hydrophilic group, such as dimethylamino (8d) and morpholine (8e), suggesting that the
substituent at this position lies in a hydrophobic pocket. In addition, introduction of a phenyl
group (8f) demonstrated better activity than methyl (8a) and n-butyl (8b) groups (Table 1), and
methylbenzene (8g) performed better than phenyl group (8f) (Table 1). Therefore, further
modifications of the phenyl ring were explored and compounds 8j–t were synthesized. The
results showed that substitution of bulky moiety, such as cyclohexyl (8s) or benzene (8t) on the
phenyl ring were superior to other substitutions (Table 1). Thus, we synthesized the analogues
8u-v and 10a-c that have other bulky groups substituted on the phenyl ring, most of which
displayed comparable potency with the inhibitors discussed above (Table 1). However, the
hydrophilic substitution of 4-methylpiperazine (10b) resulted in sharply reduced potency, which
was consistent with the theory proposed above. To this end, the SAR of the anthraquinone
derivatives and PGAM1 was investigated, and we have discovered the most potent PGAM1
inhibitor reported to date with an IC50 value of 97 nM.
Table 1. Biological activity of compounds 8a-v, 10a-ca
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Compd.
R
PGAM1
H1299
A549
IC50 (µM)
n.d.
PC9
IC50 (µM)
n.d.
IC50 (µM)
IC50 (µM)
8a
8b
8c
CH3
5.37±0.38
2.05±0.21
1.75±0.37
28.5±6
38.8±2.0
n.d.
n.d.
n.d.
n.d.
21.8±0.4
8d
8e
8f
>10 µM
>10 µM
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.50±0.23
21.3±1.1
8g
8h
0.36±0.15
0.84±0.05
7.7±1.0
25.4±1.1 15.7±1.5
n.d.
n.d.
25.8±2.6
8i
8j
12.2±2.4
16.1±1.7
0.55±0.11
0.48±0.06
17.7±0.2
27.3±1.4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
8k
8l
2.81±0.83
2.86±0.12
26.7±1.5
20±1
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0.63±0.16
8m
8n
n.d.
n.d.
22.6±0.3
13.0±1.2
21.6±0.9
17.5±4.5
0.55±0.06
8o
8p
8.1±0.4
>100
9.5±0.1
4.7±2.0
0.49±0.06
0.19±0.04
10.2±2.2
8.7±0.5
8q
8r
8s
50.5±0.6
n.d.
18.1±0.9
n.d.
1.29±0.47
2.05±0.19
0.097±0.03
7.7±2.1
47.6±2.8
4.6±0.8
11.2±1.7
11.5±0.2
8t
8u
10.3±0.8
12.0±0.8
6.8±0.5
6.3±0.3
n.d.
4.8±0.05
5.4±0.5
6.6±1.2
3.0±0.2
n.d.
0.25±0.07
0.26±0.1
0.14±0.02
0.33±0.05
>10 µM
6.4±1.3
6.2±0.2
6.3±0.5
10.0±2.5
n.d.
8v
10a
10b
10c
9.2±2.2
11.6±0.2
2.6±0.32
10.3±0.7
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cisplatin
n.d.
6.3±0.6
5.6±1.0
2.1±0.3
aThe data are presented as mean ± s.d.(n=3). n.d. = not determined.
2.3 Binding mode of the anthraquinone inhibitors with PGAM1
To illustrate the binding mode of the anthraquinone inhibitors with PGAM1, we determined
the co-crystal structure of PGAM1 with compound 8g (Table 2). Superposition of pPGAM1-8g
complex to bisphosphoglycerate mutase (BPGM, a homolog of PGAM1)-3PG complex
indicated that compound 8g was located in an allosteric pocket adjacent to the substrate site of
3PG (Figure 3A). Binding of compound 8g changed the conformations of the two helixes (99-
106, 109-117) near the substrate pocket, stabilizing PGAM1 at an open state. Meanwhile, C
terminal residues of PGAM1 (237-255) was not observed in the co-crystal structure, suggesting
that C terminus was more flexible owning to the binding with compound 8g (Figure 3B). Our
previous results of molecular dynamics simulation study showed that flexible C-terminal region
influenced cofactor binding during the catalytic cycle and induced PGAM1 to the open state [33],
which suggested compound 8g inhibiting PGAM1’s enzymatic activity in a similar way. This
allosteric pocket is mainly composed of Phe22, Lys100, Arg116, and Arg191 (Figure 3C). In
detail, compound 8g engaged in hydrophobic interactions with the Phe22 of PGAM1 and
interacted with the Arg191 through water bridges (Figure 3C). In addition, a π–cation interaction
was observed between Arg116 of PGAM1 and the anthraquinone scaffold of compound 8g
(Figure 3C). In the meantime, the co-crystal structure of inactive form of PGAM1 (non—
phosphorylated His11) complexed with 8g was also solved, predicting a dual inhibition despite
PGAM1’s status (Figure 3D). To validate the binding mode, we constructed mutations of
PGAM1 and found that the activity of compound 8g, and another selected derivative 8t, toward
PGAM1 was impaired to different extents when Phe22, Arg116, and Arg191 were mutated
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separately (Figure 3E), which demonstrated that compounds 8g and 8t adopted the same binding
mode. In addition, analysis by a substrate competitive assay indicated that compound 8t was a
non-competitive inhibitor with 3PG (Figure 3F), which agreed with the binding mode observed
in the crystal structure. In conclusion, the binding mode of the synthesized anthraquinone
inhibitors was revealed by co-crystal structures and compound 8t, was shown to be an allosteric
inhibitor non-competitive with the PGAM1 substrate 3PG.
Table 2. Data collection and refinement statistics
PGAM1-8g
pPGAM1-8g
Data collection
Space group
Cell constants
a, b, c (Å)
P 21 21 21
P 21 21 21
82.84 85.11 103.05
82.53 82.74 104.36
90.0, 90.0, 90.0
90.0, 90.0, 90.0
α, β, γ (°)
Resolution *(Å)
Data completeness (%)
No. Observations
No. Unique Reflections
Redundancy
50-1.99 (2.06-1.99)
95.3 (100.0)
320116
50-2.15 (2.23-2.15)
99.9 (100.0)
282662
50256
39576
6.7 (7.3)
7.1 (7.3)
Rmerge
0.17 (0.97)
2.19(2.00Å)
0.07 (0.74)
3.01(2.16Å)
< I/σ(I) >1
Refinement
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Resolution (Å)
43.75-1.99 (2.03-1.99)
93.7 (94)
47158
38.37-2.15 (2.20-2.15)
Data completeness (%)
No. reflections
99.7 (97)
39473
R
work/Rfree
0.216/0.244
2423 (5.11%)
25.9
0.203/0.236
1981 (5.02%)
45.5
Rfree test set
Wilson B-factor (Å2)
Fo,Fc correlation
Total number of atoms
Average B,
0.93
0.95
4132
3957
34.0
47.0
all atoms (Å2)
Ligands
MES: 1;
KH1: 1;
CL: 1;
98.5
MES: 1;
KH1: 1;
CL: 1;
99.4
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
R.m.s deviations
1.5
0.6
0
0
Bond lengths (Å)
0.009
1.202
0.009
1.163
Bond angles(º)
5Y35
5Y64
PDB accession code
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Fig. 3. Binding mode of the anthraquinone derivatives with PGAM1. (A) Overlay of compound
8g (PBD: 5Y64) and 3PG (PBD:2F90) in complex with PGAM1; (B) Superposition of
PGAM1(PBD: 5Y64, green) complexed with 8g and BPGM (PBD:2F90, cyan) complexed with
3PG; (C) Interactions between compound 8g and the surrounding residues observed in the co-
crystal structure; (D) Structure overlay of non-phosphorylated PGAM1 (PBD: 5Y35, white) and
phosphorylated PGAM1 (PBD: 5Y64, green). Active site residue His11 and phosphorylated
His11 (p-H11) were shown in sticks. (E) Inhibition of compounds 8g (5 µM) and 8t (5 µM) on
wild-type and mutated PGAM1; (F) Substrate 3-PG competitive assay of compound 8t. The data
are presented as mean ± s.e.m.
2.4 Antiproliferative activity of selected inhibitors on cancer cells
We tested the ability of selected compounds, which had IC50 values below 10 µM toward
PGAM1, to inhibit the proliferation of three cancer cell lines H1299, A549, and PC9 (Table 1).
A similar trend in proliferation inhibition was observed as for PGAM1 inhibition, and most of
the selected compounds performed similarly in the three different cancer cells. Most of the
inhibitors with IC50 values above 1 µM toward PGAM1 showed moderate potency toward the
cancer cells with IC50 values of approximately 20 µM or above. The derivatives that were more
potent toward PGAM1 suppressed the proliferation of cancer cells generally more effectively
with IC50 values ranging from approximately 5–20 µM. Notably, four analogues, 8t, 8u, 8v and
10a, effectively restrain cancer cell growth with IC50 values of approximately 6 µM, while
inhibiting PGAM1 with IC50 values less than 0.4 µM. Considering the biological activity of these
four derivatives on PGAM1 and cancer cells, they were selected for further study.
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2.5 Pharmacokinetic properties of compound 8t in vivo
Firstly, we roughly screened the concentrations in the plasma of the four selected compounds,
at three different times, post intraperitoneal administration in Institute of Cancer Research (ICR)
mice at a dose of 100 mg/kg (Figure S1). The concentrations of compounds 8u, 8v and 10a were
much lower than 8t during the recorded time. Then, we investigated the pharmacokinetic
properties of 8t in detail. After a single intraperitoneal administration of 100 mg/kg in ICR mice,
the blood at eight different times was collected and the plasma was analyzed. The results showed
that compound 8t had a half-life of 4.28 h, a high maximum concentration (Cmax) of 131.01
µg/mL, and an area under the curve (AUC0-24h) of 239.02 µg*h/mL, which indicated a good drug
exposure in the blood (Table 3).
Table 3. Pharmacokinetic properties of compound 8t in vivoa
AUC0-24h
(µg*h/mL)
239.02
AUC0-inf
(µg*h/mL)
244.83
CL/F
(mL/h)
11.6
Cmax
Tmax
(h)
V/F
(mL)
71.64
t1/2
(h)
(µg/mL)
131.01
0.25
4.28
an = 3 animals per group at every time point. AUC, area under the curve; CL, clearance; F:
bioavailability; Cmax, maximum concentration; Tmax, time of maximum concentration; V, volume
of distribution; T1/2, half-life
2.6 Compound 8t attenuated tumor growth in vivo safely
To evaluate the pharmacological efficacy of compound 8t in vivo, we developed H1299
xenograft model by implanting H1299 cells at double flanks subcutaneously in mice. When the
average tumor volume reached approximately 200 mm3, the mice were randomized to be treated
with control or compound 8t at a dose of 100 mg/kg. The tumor volume and the weight of mice
were recorded during the test (Figure 4A and Figure 4C). On the day when the mice were
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sacrificed, the tumor volumes and tumor weights of the 8t group were reduced significantly in
comparison with the control group (Figure 4A and Figure 4B), with a tolerated weight loss in the
mice of approximately 2% (Figure 4C). Additionally, decreased Ki67 staining in the 8t group
further confirmed the anticancer effect in vivo (Figure 4D). The kidneys and livers in each group
were harvested and no significant difference was observed between the two groups (Figure 4E).
Taken as a whole, these results imply that compound 8t is capable of delaying tumor growth in
vivo without obvious toxicity.
Fig. 4. Compound 8t attenuated tumor growth in H1299 xenograft model without obvious
toxicity. (A) Tumor volumes of control (n = 9/6) and 8t 100 mg/kg qd, i.p. (n = 11/7) groups
during the study. (B) Tumor weights of control and 8t groups on the last day; (C) The weights of
mice in control and 8t groups; (D) Ki67 staining of the tumor tissues in control and 8t groups;
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(E) Haematoxylin and eosin histology of the kidneys and livers from mice in control and 8t
groups. The data are presented as mean ± s.e.m. and P-values were obtained from the unpaired t-
test (*: 0.01 < P < 0.05, **: 0.001 < P < 0.01, ***: 0.0001 < P < 0.001).
2.7 Compound 8t reprogramed the Warburg effect
Considering that PGAM1 plays a vital role in cancer metabolism, we attempted to determine
the effect of treatment with the PGAM1 inhibitor 8t on the Warburg effect in cancer cells.
Compound 8t decreased glycolysis in H1299 cells (Figure 5A), and we further assessed the
various parameters of glycolysis function by analyzing the extracellular acidification rate
(ECAR) data at several time points. Our results showed that glycolysis, glycolytic capacity, and
glycolytic reserve were markedly decreased in a dose dependent manner in H1299 cells treated
with compound 8t (Figure 5A). We also detected real-time oxidative phosphorylation by
measuring the cellular oxygen consumption rates (OCR). Our results showed that the OCR were
dramatically decreased after H1299 ꢀcells were treated with the indicated concentrations of
compound 8t (Figure 5B), suggesting that treatment with compound 8t led to the inhibition of
mitochondrial respiration. We further assessed the various parameters of mitochondrial function
by analyzing OCR data at several time points. The data showed that compound 8t markedly
decreased the basal respiration, maximal respiration, and spare capacity in H1299 cells (Figure
5B). Treatment with compound 8t for 12 h resulted in increased levels of ROS (Figure 6A), but
decreased lactate production (Figure 6B) and intracellular ATP levels (Figure 6C). Moreover,
treatment with this PGAM1 inhibitor resulted in increased phosphorylation levels of AMPK
(Figure 6D) which coordinates cell growth and metabolism [34]. These results suggested that the
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selected PGAM1 inhibitor 8t decreased glycolysis and oxidative phosphorylation which led to
decreased ATP level and subsequent AMPK activation in H1299 cells.
Fig. 5. Compound 8t decreased the ECAR and OCR. (A) The ECAR (a proxy for the rate of
glycolysis) was used to assess the glycolysis using a Seahorse 96XF extracellular flux analyzer
following sequential addition of glucose (10 mM), oligomycin (inhibitor of ATP synthase, 1
µM), and 2-deoxyglucose (2-DG, glucose analog, 50 mM), as indicated by the arrows, in H1299
cells in the presence of increasing concentrations of compound 8t. Data are presented as the
mean ± s.d. (ns, not significant, *P < 0.05, **P < 0.01). The representative images represent the
mean ECAR ± s.d. of triplicate replicates. (B) H1299 cells were cultured with increasing
concentrations of compound 8t, and the OCR was measured in real time using the Seahorse
XF96 Extracellular Flux Analyzer after the basal OCR was measured at three time points,
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followed by sequential injection of oligomycin (1 µM), carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP, uncoupler of oxidative phosphorylation, 1 µM), and
rotenone/ antimycin A (inhibitor of complex I, III; 0.5 µM). The overall OCR curves were
plotted as the mean OCR ± s.d. of triplicate replicates. Basal respiration, maximal respiration,
and spare capacity were assessed. Data are presented as the mean ± s.d. (*P < 0.05, **P < 0.01,
***P < 0.001).
Fig. 6. Compound 8t increased ROS and decreased lactate and ATP levels, as well as
activating AMPK. (A) H1299 cells treated with increasing concentrations of compound 8t were
assayed for general ROS levels. (B) H1299 cells treated with increasing concentrations of
compound 8t were assayed for lactate production. (C) H1299 cells treated with increasing
concentrations of compound 8t were assayed for the levels of ATP, calculated from the OCR.
(D) H1299 cells were treated with increasing concentrations of compound 8t, followed by
western blotting to detect the phosphorylation level of AMPK (pT172).
3. CONCLUSIONS
PGAM1 coordinates glycolysis, PPP pathway, and serine synthesis to promote tumor growth,
and is thought to be a potential target for cancer therapy. In this study, we focused on the 3-
substituents of the anthraquinone scaffold and investigated the SAR of the synthesized
19

ACCEPTED MANUSCRIPT
anthraquinone derivatives with regard to PGAM1. Among them, we discovered the most potent
PGAM1 inhibitor with IC50 value of 97 nM. In addition, we solved the crystal structure of
PGAM1 in complex with the inhibitor 8g, which revealed a novel allosteric site and showed that
Phe22, Arg116 and Arg191 are critical residues for binding. These investigations provide a
reliable guide for further optimization of anthraquinone derivatives. Furthermore, a competitive
assay demonstrated noncompetitive binding of the anthraquinone derivatives with the PGAM1
substrate 3PG. Compound 8t demonstrated potent inhibition of PGAM1 and cancer cell
proliferation with good pharmacokinetic properties, as well as an ability to alter the Warburg
effect and good efficacy in reducing tumor growth in vivo. These results illustrate the possibility
of targeting PGAM1 for cancer therapy, and compound 8t is a promising lead compound for
further optimization.
4. EXPERIMENTAL SECTION
4.1 Chemistry
All reagents were purchased commercially. Column chromatography was conducted on silica
1
gel (300−400 mesh). H NMR and 13C NMR spectra were recorded on Bruker AC400 and
Bruker AC600 NMR spectrometer respectively and tetramethylsilane was used as an internal
reference. Low-resolution mass spectra were performed on Agilent 6120 Quadrupole mass
spectrometer with electrospray ionization (ESI). High-resolution mass spectra were determined
on triple TOF 5600+ MS/MS system (AB Sciex, Concord, Ontario, Canada) in negative ESI
mode. The purity of target compounds was determined by high-performance liquid
chromatography (HPLC, Agilent ChemStation 1260, DIKMA Diamonsil Plus C18, 250 × 4.6
mm, 5µm, 25 °C, UV 290 nM). Mobile phase A was water and mobile phase B was methanol
20

ACCEPTED MANUSCRIPT
containing 0.1% trifluoroacetic acid. Flow rate was 1mL/min using linear gradients as follow: 0-
2 min was 60% B, 2-10 min was from 60% B to 95%B, 10-13 min was 95%B, 13-14 min was
from 95%B to 60% B,14-18 min was 60% B. All the biologically tested compounds achieved
≥95% purity.
4.1.1 General procedure for the synthesis of compounds 8a-v
Step 1. Nitric acid (1.5mL, 33.33mmol) was added dropwise to the suspension of 1,2-
dihydroxyanthracene-9,10-dione (5g, 20.8 mmol) in acetic acid (350 mL) at 50 .Then the
mixture was cooled to room temperature and a yellow solid (4.15 g, 70 %) of crude product 6
was obtained by filtration [35].
Step 2. Sn (10.5 g, 341 mmol), SnCl2·2H2O (12.5 g, 55.4 mmol) and concentrated HCl (50.4
mL, 604.8 mmol) were added to the suspension of crude product 6 (1.75 g, 6.14 mmol) in
ethanol (350 mL) and the mixture was stirred at room temperature overnight. After getting rid of
part of ethanol under reduced pressure, the residue 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 [35].
Step 3. Corresponding sulfonyl chloride (1.5 mmol) was added to the solution of crude product
7 (255 mg, 1 mmol) in dry pyridine (5 mL) and the mixture was stirred at room temperature for
4h. Then it was added to 10% aqueous HCl (50 mL) and the suspension was extracted with ethyl
acetate. The organic phase was washed with brine, dried over anhydrous Na2SO4 and filtered.
The residue was purified by silica chromatography after removal of ethyl acetate to afford
compounds 8a-v.
4.1.1.1 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) methanesulfonamide (8a)
21

ACCEPTED MANUSCRIPT
Yellow solid, yield 45%.1H NMR (400 MHz, DMSO-d6) δ 12.68 (brs, 1H), 11.00 (brs, 1H),
9.59 (brs, 1H), 8.28-8.09 (m, 2H), 7.99 – 7.85 (m, 2H), 7.81 (s, 1H), 3.17 (s, 3H). 13C NMR (151
MHz, DMSO) δ 187.75, 180.68, 150.49, 142.37, 135.00, 134.22, 133.32, 132.81, 131.84, 126.79,
126.39, 123.95, 112.97, 112.91, 40.80. MS (ESI) (m/z): 332.0 (M-H)-. HRMS (ESI) calcd for
C15H11NO6S [M-H]-: 332.0234; found: 332.0237. Purity 96.9% by HPLC.
4.1.1.2 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) butane-1-sulfonamide (8b)
1
Orange solid, 39% yield. H NMR (400 MHz, DMSO-d6) δ 12.68 (brs, 1H), 11.01 (brs, 1H),
9.62 (brs, 1H), 8.24 – 8.13 (m, 2H), 7.97 – 7.86 (m, 2H), 7.82 (s, 1H), 3.23 (t, J = 7.6Hz, 2H),
1.72 (p, J = 7.6 Hz, 2H), 1.39 (h, J = 7.6 Hz, 2H), 0.87 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz,
DMSO) δ 187.73, 180.69, 150.47, 142.36, 134.99, 134.21, 133.33, 132.82, 131.88, 126.79,
126.38, 123.89, 113.22, 112.88, 52.12, 25.06, 20.71, 13.44. MS (ESI) (m/z): 374.0 (M-H)-.
HRMS (ESI) calcd for C18H17NO6S [M-H]-: 374.0704; found: 374.0707. Purity 98.1% by HPLC.
4.1.1.3 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) cyclohexanesulfonamide
(8c)
Orange solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.71 (brs, 1H), 11.05 (brs, 1H),
9.52 (brs, 1H), 8.25 – 8.12 (m, 2H), 7.96 – 7.89 (m, 2H), 7.86 (s, 1H), 3.10 (tt, J = 3.6, 12.0 Hz,
1H), 2.10 (d, J = 12.4 Hz, 2H), 1.77 (d, J = 12.4 Hz, 2H), 1.60 (d, J = 12.0 Hz, 1H), 1.44 (qd, J =
3.2, 12.6 Hz, 2H), 1.30 – 1.06 (m, 3H). 13C NMR (151 MHz, DMSO) δ 187.72, 180.74, 150.43,
142.21, 134.98, 134.21, 133.35, 132.84, 132.15, 126.79, 126.38, 123.86, 113.53, 112.85, 60.89,
25.94(2C), 24.72, 24.40(2C). MS (ESI) (m/z): 400.0 (M-H)-. HRMS (ESI) calcd for C20H19NO6S
[M-H]-: 400.0860; found: 400.0864. Purity 97.3% by HPLC.
4.1.1.4
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-N’,
N’-dimethyl-4-
sulfonamide(8d)
22

ACCEPTED MANUSCRIPT
Orange solid, 45% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.67 (brs, 1H), 10.94 (brs, 1H),
9.67 (brs, 1H), 8.24 – 8.11 (m, 2H), 7.96 – 7.87 (m, 2H), 7.84 (s, 1H), 2.77 (s, 6H). 13C NMR
(151 MHz, DMSO) δ 187.69, 180.76, 150.33, 141.73, 134.95, 134.20, 133.35, 132.87, 132.37,
126.78, 126.37, 123.84, 112.99, 112.58, 37.74(2C). MS (ESI) (m/z): 361.0 (M-H)-. HRMS (ESI)
calcd for C16H14N2O6S [M-H]-: 361.0500; found: 361.0503. Purity 99.6% by HPLC.
4.1.1.5 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) morpholine-4-sulfonamide
(8e)
Red solid, 49% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.68 (brs, 1H), 10.99 (brs, 1H), 9.87
(brs, 1H), 8.25 – 8.12 (m, 2H), 7.97 – 7.87 (m, 2H), 7.85 (s, 1H), 3.58 (t, J = 4.6 Hz, 4H), 3.12 (t,
J = 4.6 Hz, 4H). 13C NMR (151 MHz, DMSO) δ 187.58, 180.56, 150.20, 142.11, 134.81, 134.05,
133.18, 132.72, 131.75, 126.62, 126.22, 123.59, 113.60, 112.68, 65.25(2C), 45.84(2C). MS
(ESI) (m/z): 403.0 (M-H)-. HRMS (ESI) calcd for C18H16N2O7S [M-H]-: 403.0605; found:
403.0603. Purity 95.0% by HPLC.
4.1.1.6 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) benzenesulfonamide (8f)
Red solid, 55% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.58 (brs, 1H), 10.89 (brs, 1H), 10.36
(brs, 1H), 8.21 – 8.09 (m, 2H), 7.97-7.81 (m, 4H), 7.75 (s, 1H), 7.69-7.52 (m, 3H).13C NMR (151
MHz, DMSO) δ 187.70, 180.61, 150.26, 142.34, 140.19, 134.96, 134.20, 133.29, 133.19, 132.82,
131.15, 129.36(2C), 126.76, 126.59(2C), 126.36, 123.72, 112.90, 112.34. MS (ESI) (m/z): 394.0
(M-H)-. HRMS (ESI) calcd for C20H13NO6S2 [M-H]-: 394.0391; found: 394.0388. Purity 98.8%
by HPLC.
4.1.1.7 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-methylbenzene sulfonamide
(8g)
23

ACCEPTED MANUSCRIPT
Orange solid, 50% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.59 (brs, 1H), 10.90 (brs, 1H),
10.25 (brs, 1H), 8.21 – 8.09 (m, 2H), 7.93 – 7.85 (m, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.76 (s, 1H),
7.40 (d, J = 8.0 Hz, 2H), 2.35 (s, 3H).13C NMR (151 MHz, DMSO) δ 187.67, 180.62, 150.23,
143.63, 142.03, 137.29, 134.94, 134.18, 133.27, 132.81, 131.30, 129.78(2C), 126.75,
126.68(2C), 126.34, 123.75, 112.78, 112.02, 20.99. MS (ESI) (m/z): 408.0 (M-H)-. HRMS (ESI)
calcd for C21H15NO6S [M-H]-: 408.0547; found: 408.0545.
4.1.1.8 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) thiophene-2-sulfonamide (8h)
Yellow solid, 50% yield.1H NMR (400 MHz, DMSO-d6) δ 12.58 (brs, 1H), 10.71 (brs, 2H),
8.23 – 8.08 (m, 2H), 7.96 (d, J = 4.8 Hz, 1H), 7.94 – 7.85 (m, 2H), 7.83 (s, 1H), 7.66 (d, J = 3.6
13
Hz, 1H), 7.17 (t, J = 4.4 Hz, 1H). C NMR (151 MHz, DMSO) δ 187.74, 180.59, 150.32,
142.90, 140.53, 134.98, 134.19, 133.77, 133.28, 132.81, 132.57, 130.77, 127.74, 126.76, 126.37,
123.65, 113.13, 112.98. MS (ESI) (m/z): 400.0 (M-H)-. HRMS (ESI) calcd for C18H11NO6S2 [M-
H]-: 399.9955; found: 399.9953. Purity 95.2% by HPLC.
4.1.1.9
5-Chloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
thiophene-2-
sulfonamide (8i)
Yellow solid, 52% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.63 (brs, 1H), 11.00 (brs, 1H),
10.72 (brs, 1H), 8.25 – 8.14 (m, 2H), 7.97 – 7.89 (m, 2H), 7.78 (s, 1H), 7.50 (d, J = 4.0 Hz, 1H),
7.25 (d, J = 4.0 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 187.87, 180.65, 150.40, 143.66, 139.00,
135.46, 135.07, 134.27, 133.35, 132.88, 132.37, 130.24, 128.02, 126.83, 126.46, 123.71, 113.84,
113.52. MS (ESI) (m/z): 433.8(M-H)-. HRMS (ESI) calcd for C18H10ClNO6S2 [M-H]-: 433.9565;
found: 433.9562. Purity 96.5% by HPLC.
4.1.1.10
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-3-
methoxybenzenesulfonamide (8j)
24

ACCEPTED MANUSCRIPT
1
Orange solid, 51% yield. H NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.96 (brs, 1H),
10.36 (brs, 1H), 8.20 – 8.08 (m, 2H), 7.93 – 7.83 (m, 2H), 7.76 (s, 1H), 7.56 – 7.44 (m, 3H), 7.22
(dt, J = 2.0, 8.0 Hz, 1H), 3.81 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.69, 180.62, 159.37,
150.24, 142.22, 141.38, 134.93, 134.17, 133.27, 132.81, 131.12, 130.53, 126.75, 126.34, 123.73,
119.19, 118.69, 112.88, 112.15, 111.55, 55.59. MS (ESI) (m/z): 424.0 (M-H)-. HRMS (ESI)
calcd for C21H15NO7S [M-H]-: 424.0496; found: 424.0501. Purity 97.4% by HPLC.
4.1.1.11
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-3,4-
dimethoxybenzenesulfonamide (8k)
Red solid, 45% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 10.95 (s, 1H), 10.12 (s,
1H), 8.18 – 8.08 (m, 2H), 7.93 – 7.82 (m, 2H), 7.78 (s, 1H), 7.55 – 7.46 (m, 2H), 7.12 (d, J = 8.4
Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H). 13C NMR (151 MHz, DMSO) δ 187.63, 180.65, 152.41,
150.20, 148.58, 141.83, 134.89, 134.15, 133.25, 132.79, 131.48, 131.42, 126.73, 126.31, 123.76,
120.55, 112.68, 111.76, 111.13, 109.61, 55.83, 55.72. MS (ESI) (m/z): 454.0 (M-H)-. HRMS
(ESI) calcd for C22H17NO8S [M-H]-: 454.0602; found: 454.0606. Purity 95.0% by HPLC.
4.1.1.12
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-2,4-
dimethoxybenzenesulfonamide (8l)
Yellow solid, 66% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (brs, 1H), 11.03 (brs, 1H),
9.11 (brs, 1H), 8.20 – 8.08 (m, 2H), 7.93 – 7.83 (m, 2H), 7.79 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H),
6.69 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 2.4, 8.8 Hz, 1H), 3.85 (s, 3H), 3.79 (s, 3H). 13C NMR (151
MHz, DMSO) δ 187.62, 180.64, 164.91, 158.12, 150.15, 141.11, 134.88, 134.16, 133.25, 132.82,
131.82, 131.53, 126.73, 126.32, 123.85, 118.65, 112.62, 111.12, 105.15, 99.39, 56.35, 55.82. MS
(ESI) (m/z): 454.0 (M-H)-. HRMS (ESI) calcd for C22H17NO8S [M-H]-: 454.0602; found:
454.0605. Purity 95.6% by HPLC.
25

ACCEPTED MANUSCRIPT
4.1.1.13 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-4-fluoro-3-(trifluoromethyl)
benzenesulfonamide (8m)
Yellow solid, 50% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.99 (brs, 1H),
10.60 (brs, 1H), 8.29 (dd, J = 2.4, 6.8 Hz, 1H), 8.22 – 8.12 (m, 3H), 7.97 – 7.86 (m, 2H), 7.77 (t,
J = 9.6 Hz, 1H), 7.70 (s, 1H). 13C NMR (151 MHz, DMSO) δ 187.82, 180.54, 161.12(d, J =
261.2 Hz), 150.43, 143.67, 137.31, 135.06, 134.25, 134.07(d, J = 10.6 Hz), 133.28, 132.80,
130.03, 126.80, 126.42, 123.71, 121.75(q, J = 273.3 Hz), 118.83(d, J = 21.1 Hz), 117.66(d, J =
13.6 Hz), 117.44(d, J = 13.6 Hz), 114.08, 113.55. MS (ESI) (m/z): 480.0 (M-H)-. HRMS (ESI)
calcd for C21H11F4NO6S [M-H]-: 480.0170; found: 480.0167. Purity 98.5% by HPLC.
4.1.1.14 4-Chloro-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-3-(trifluoromethyl)
benzenesulfonamide (8n)
1
Yellow solid, 71% yield. H NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H), 11.02 (brs, 1H),
10.66 (brs, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.22 – 8.12 (m, 2H), 8.09 (dd, J = 2.0, 8.4 Hz, 1H),
7.98 (d, J = 8.8 Hz, 1H), 7.97 – 7.86 (m, 2H), 7.70 (s, 1H). 13C NMR (151 MHz, DMSO) δ
187.83, 180.53, 150.44, 143.75, 139.87, 135.67, 135.07, 134.25, 133.28, 133.02, 132.80, 132.07,
129.91, 127.51(q, J = 31.7 Hz), 126.80, 126.43, 126.19-126.16 (m), 123.71, 122.06 (q, J = 273.3
Hz), 114.19, 113.60. MS (ESI) (m/z): 495.8 (M-H)-. HRMS (ESI) calcd for C21H11ClF3NO6S [M-
H]-: 495.9875; found: 495.9871. Purity 98.1% by HPLC.
4.1.1.15
4-(Tert-butyl)-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
benzenesulfonamide (8o)
1
Orange solid, 65% yield. H NMR (400 MHz, DMSO-d6) δ 12.60 (brs, 1H), 10.93 (brs, 1H),
10.30 (brs, 1H), 8.18 – 8.09 (m, 2H), 7.92 – 7.79 (m, 5H), 7.63 (d, J = 8.4 Hz, 2H), 1.26 (s, 9H).
13C NMR (151 MHz, DMSO) δ 187.64, 180.63, 156.22, 150.23, 141.80, 137.44, 134.91, 134.16,
26

ACCEPTED MANUSCRIPT
133.26, 132.79, 131.44, 126.73, 126.51(2C), 126.32, 126.23(2C), 123.81, 112.70, 111.64, 34.92,
30.71(3C). MS (ESI) (m/z): 450.0 (M-H)-. HRMS (ESI) calcd for C24H21NO6S [M-H]-:
450.1017; found: 450.1026. Purity 100% by HPLC.
4.1.1.16
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
naphthalene-2-
sulfonamide(8p)
Red solid, 65% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.54 (brs, 1H), 10.67 (brs, 2H), 8.57
(s, 1H), 8.24 – 7.75 (m, 9H), 7.69 (p, J = 7.0 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 187.68,
180.58, 150.25, 142.52, 137.29, 134.92, 134.37, 134.14, 133.26, 132.77, 131.52, 131.09, 129.53,
129.31, 129.08, 127.85, 127.76, 127.71, 126.73, 126.31, 123.71, 122.09, 112.95, 112.67. MS
(ESI) (m/z): 444.0 (M-H)-. HRMS (ESI) calcd for C24H15NO6S [M-H]-: 444.0547; found:
444.0560. Purity 99.8% by HPLC.
4.1.1.17
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
naphthalene-1-
sulfonamide(8q)
1
Yellow solid, 65% yield. H NMR (400 MHz, DMSO-d6) δ 12.55 (brs, 1H), 10.88 (brs, 1H),
10.77 (brs, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.32 – 8.22 (m, 2H), 8.17 – 8.05 (m, 3H), 7.90 – 7.82
(m, 2H), 7.78 – 7.64 (m, 4H). 13C NMR (151 MHz, DMSO) δ 187.55, 180.60, 150.10, 141.90,
135.23, 134.89, 134.59, 134.17, 133.84, 133.24, 132.82, 132.53, 129.18, 128.99, 128.16, 127.45,
127.08, 126.72, 126.32, 124.66, 124.47, 123.68, 112.53, 111.66. MS (ESI) (m/z): 444.0 (M-H)-.
HRMS (ESI) calcd for C24H15NO6S [M-H]-: 444.0547; found: 444.0557. Purity 98.1% by HPLC.
4.1.1.18 N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl) quinoline-8-sulfonamide (8r)
Red solid, 40% yield. 1H NMR (400 MHz, DMSO-d6) δ 12.48 (brs, 1H), 11.16 (brs, 1H), 9.48
(brs, 1H), 9.11 (s, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 8.0 Hz,
1H), 8.12 (t, J = 6.4 Hz, 2H), 7.96 – 7.73 (m, 5H). 13C NMR (151 MHz, DMSO) δ 187.64,
27

ACCEPTED MANUSCRIPT
180.60, 151.71, 142.16, 137.39, 134.88, 134.83, 134.64, 134.14, 133.85, 133.23, 132.77, 131.55,
131.32, 128.57, 126.71, 126.30, 125.78, 124.00, 122.96, 112.96, 112.85, 110.33. MS (ESI) (m/z):
445.0 (M-H)-. HRMS (ESI) calcd for C23H14N2O6S [M-H]-: 445.0500; found: 445.0504. Purity
95.9% by HPLC.
4.1.1.19
4-Cyclohexyl-N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)
benzenesulfonamide (8s)
1
Orange solid, 52% yield. H NMR (400 MHz, DMSO-d6) δ 12.58 (brs, 1H), 10.95 (brs, 1H),
10.37 (brs, 1H), 8.20 – 8.09 (m, 2H), 7.94 – 7.76 (m, 5H), 7.45 (d, J = 8.0 Hz, 2H), 2.61 – 2.54
(m, 1H), 1.80 – 1.62 (m, 5H), 1.45 – 1.13 (m, 5H). 13C NMR (151 MHz, DMSO) δ 187.65,
180.63, 153.08, 150.22, 141.86, 137.71, 134.92, 134.17, 133.27, 132.80, 131.44, 127.62(2C),
126.75(3C), 126.33, 123.78, 112.71, 111.72, 43.54, 33.40(2C), 26.11(2C), 25.39. MS (ESI)
(m/z): 476.0 (M-H)-. HRMS (ESI) calcd for C26H23NO6S [M-H]-: 476.1173; found: 476.1183.
Purity 99.5% by HPLC.
4.1.1.20
N-(3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-[1,1'-biphenyl]-4-
sulfonamide (8t)
1
Orange solid, 60% yield. H NMR (400 MHz, DMSO-d6) δ 12.59 (brs, 1H), 10.76 (brs, 1H),
10.61 (brs, 1H), 8.21 – 8.09 (m, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.94 – 7.83 (m, 4H), 7.82 (s, 1H),
7.72 (d, J = 7.6 Hz, 2H), 7.52 – 7.38 (m, 3H). 13C NMR (151 MHz, DMSO) δ 187.68, 180.62,
150.27, 144.52, 142.22, 138.95, 138.19, 134.94, 134.17, 133.27, 132.80, 131.21, 129.09(2C),
128.62, 127.46(2C), 127.32(2C), 127.07(2C), 126.74, 126.34, 123.78, 112.87, 112.17. MS (ESI)
(m/z): 470.0 (M-H)-. HRMS (ESI) calcd for C26H17NO6S [M-H]-: 470.0704; found: 470.0704.
Purity 100% by HPLC.
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