Development of Anthraquinone Derivatives as Ectonucleoside Triphosphate Diphosphohydrolase (NTPDase) Inhibitors With Selectivity for NTPDase2 and NTPDase3

Article abstract of DOI:10.3389/fphar.2020.01282

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of nucleoside tri- and di-phosphates to mono-phosphates. The products are subsequently hydrolyzed by ecto-5′-nucleotidase (ecto-5′-NT) to nucleosides. NTPDase inhibitors have potential as novel drugs, e.g., for the treatment of inflammation, neurodegenerative diseases, and cancer. In this context, a series of anthraquinone derivatives structurally related to the anthraquinone dye reactive blue-2 (RB-2) was synthesized and evaluated as inhibitors of human NTPDases utilizing a malachite green assay. We identified several potent and selective inhibitors of human NTPDase2 and -3. Among the most potent NTPDase2 inhibitors were 1-amino-4-(9-phenanthrylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (20, PSB-16131, IC₅₀ of 539 nM) and 1-amino-4-(3-chloro-4-phenylsulfanyl)phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (48, PSB-2020, IC₅₀ of 551 nM). The most potent NTPDase3 inhibitors were 1-amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (42, PSB-1011, IC₅₀ of 390 nM) and 1-amino-4-(3-carboxy-4-hydroxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (33, PSB-2046, IC₅₀ of 723 nM). The best NTPDase2 inhibitor 20 showed a non-competitive inhibition type, while the NTPDase3 inhibitor 42 behaved as a mixed-type inhibitor. These potent compounds were found to be selective vs. other NTPDases. They will be useful tools for studying the roles of NTPDase2 and -3 in physiology and under pathological conditions.

Full text of DOI:10.3389/fphar.2020.01282

ORIGINAL RESEARCH
published: 27 August 2020
doi: 10.3389/fphar.2020.01282
Development of Anthraquinone
Derivatives as Ectonucleoside
Triphosphate Diphosphohydrolase
(NTPDase) Inhibitors With Selectivity
for NTPDase2 and NTPDase3
Edited by:
Salvatore Salomone,
University of Catania, Italy
Reviewed by:
Carla Denise Bonan,
†
2
Pontifical Catholic University of Rio
Grande do Sul, Brazil
Younis Baqi1 , Mahmoud Rashed , Laura Schäkel2, Enas M. Malik2, Julie Pelletier3,
*
Jean Sevigny3,4, Amelie Fiene2 and Christa E. Müller2
*
´
Holger Stephan,
Helmholtz-Gemeinschaft Deutscher
Forschungszentren (HZ), Germany
1 Department of Chemistry, Faculty of Science, Sultan Qaboos University, Muscat, Oman, 2 PharmaCenter Bonn,
Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany, 3 Centre de Recherche
4
´
´
´
´
du CHU de Quebec–Universite Laval, Quebec, QC, Canada, Departement de Microbiologie-Infectiologie et d’Immunologie,
*Correspondence:
Younis Baqi
´
´
´
´
Faculte de Medecine, Universite Laval, Quebec, QC, Canada
baqi@squ.edu.om
Christa E. Müller
Ectonucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of
nucleoside tri- and di-phosphates to mono-phosphates. The products are subsequently
hydrolyzed by ecto-5′-nucleotidase (ecto-5′-NT) to nucleosides. NTPDase inhibitors have
potential as novel drugs, e.g., for the treatment of inflammation, neurodegenerative
diseases, and cancer. In this context, a series of anthraquinone derivatives structurally
related to the anthraquinone dye reactive blue-2 (RB-2) was synthesized and evaluated as
inhibitors of human NTPDases utilizing a malachite green assay. We identified several potent
and selective inhibitors of human NTPDase2 and -3. Among the most potent NTPDase2
inhibitors were 1-amino-4-(9-phenanthrylamino)-9,10-dioxo-9,10-dihydroanthracene-2-
sulfonate (20, PSB-16131, IC50 of 539 nM) and 1-amino-4-(3-chloro-4-phenylsulfanyl)
phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (48, PSB-2020, IC50 of 551
nM). The most potent NTPDase3 inhibitors were 1-amino-4-[3-(4,6-dichlorotriazin-2-
ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (42, PSB-
1011, IC50 of 390 nM) and 1-amino-4-(3-carboxy-4-hydroxyphenylamino)-9,10-dioxo-
9,10-dihydroanthracene-2-sulfonate (33, PSB-2046, IC50 of 723 nM). The best
NTPDase2 inhibitor 20 showed a non-competitive inhibition type, while the NTPDase3
inhibitor 42 behaved as a mixed-type inhibitor. These potent compounds were found to be
selective vs. other NTPDases. They will be useful tools for studying the roles of NTPDase2
and -3 in physiology and under pathological conditions.
christa.mueller@uni-bonn.de
†On leave from the Department of
Pharmaceutical and Medicinal
Chemistry, Faculty of Pharmacy,
Al-Azhar University, Cairo, Egypt
Specialty section:
This article was submitted to
Experimental Pharmacology
and Drug Discovery,
a section of the journal
Frontiers in Pharmacology
Received: 05 June 2020
Accepted: 03 August 2020
Published: 27 August 2020
Citation:
Baqi Y, Rashed M, Schäkel L,
´
Malik EM, Pelletier J, Sevigny J,
Fiene A and Müller CE (2020)
Development of Anthraquinone
Derivatives as Ectonucleoside
Triphosphate Diphosphohydrolase
(NTPDase) Inhibitors With Selectivity
for NTPDase2 and NTPDase3.
Front. Pharmacol. 11:1282.
doi: 10.3389/fphar.2020.01282
Keywords: anthraquinone, CD39, inhibitor, metalloenzymes, neuroinflammation, NTPDase2, NTPDase3, synthesis
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
the gray matter of discrete regions, like habenula (Gampe et al.,
2012), satellite astrocytes in the dorsal root ganglion (Braun et al.,
2003), and astrocyte-like progenitor cells of the subventricular zone
(SVZ) of the lateral ventricle (Shukla et al., 2005; Mishra et al., 2006;
Gampe et al., 2015). NTPDase3 is localized in the midline regions:
in the thalamus, hypothalamus, and the medulla oblongata (Belcher
INTRODUCTION
Ectonucleotidases are membrane-bound metalloenzymes that
affect extracellular nucleotide and nucleoside levels by
catalyzing the hydrolysis of nucleotides to the corresponding
nucleosides releasing inorganic phosphate or diphosphate (Dou
et al., 2018; Le et al., 2019; Vuerich et al., 2019). There are four
major subfamilies of ectonucleotidases: the ecto-nucleoside
triphosphate diphosphohydrolases (NTPDases), the ecto-
nucleotide pyrophosphatases/phosphodiesterases (NPPs), the
alkaline phosphatases (APs), and the ecto-5′-nucleotidase
(ecto-5′-NT, CD73) (Bonan, 2012; Al-Rashida and Iqbal, 2015;
Baqi, 2015; Fiene et al., 2016; Le et al., 2019). In inflammatory
processes, there may be a massive increase in extracellular ATP
concentrations causing proinflammatory immune responses via
P2X and P2Y receptors. ATP can be hydrolyzed by NTPDases,
or at very high concentrations also by APs, via ADP to AMP.
Alternatively, ATP can be cleaved directly to AMP and
diphosphate (pyrophosphate) by NPPs (Lee and Müller, 2017).
The resulting AMP can eventually be hydrolyzed by ecto-5’-NT
yielding adenosine, which induces antiinflammatory effects
via activation of P1 (adenosine) receptors (King et al., 2006;
Burnstock, 2018; Antonioli et al., 2019; Müller et al., 2020).
Several studies reported that NTPDase2 is localized in
specialized astrocytes in rodent brain, such as laminar astrocytes
associated with fiber tracts in the brain stem and cerebrum (Braun
et al., 2003; Braun et al., 2004), tanycytes, non-stellate astrocytes in
́
et al., 2006; Grkovic et al., 2016). Both enzymes, NTPDase2, and to a
lesser extent also NTPDase3, preferentially catalyze the
dephosphorylation of ATP to ADP, generating the physiological
ligand for P2Y1, P2Y12, and P2Y13 receptors (Kukulski et al., 2005;
Zimmermann et al., 2012; Burnstock, 2020; Müller et al., 2020).
Therefore, NTPDase2 and -3 may modulate inflammatory reactions
within the CNS and could represent useful therapeutic targets in
neuroinflammatory and neurodegenerative diseases.
So far only few, moderately potent, NTPDase inhibitors have
been described (Figure 1), which can be divided into nucleotide
derivatives and non-nucleotides. ARL67156 (1, Figure 1) is a
weak, competitive inhibitor of human NTPDase1 (Ki = 11 mM)
and -3 (Ki = 18 mM) but does not inhibit human NTPDase2 and
́
-8 (Levesque et al., 2007). 8-BuS-ATP (2, Figure 1) was shown to
inhibit NTPDase1 (Ki = 0.8 mM) but being a substrate of
NTPDase2, -3, and -8 it is of limited use (Lecka et al., 2013).
The corresponding 8-BuS-ADP and especially 8-BuS-AMP also
inhibited NTPDase1 but appeared to be more stable towards
́
hydrolysis (Levesque et al., 2007). PSB-6426 (3, Figure 1) is a
metabolically stable, uncharged compound derived from
uridine-5′-carboxylate. It was identified as a moderately potent,
FIGURE 1 | Structures of selected NTPDase inhibitors (Iqbal et al., 2005; Müller et al., 2006; Brunschweiger et al., 2008; Baqi et al., 2009b; Zebisch et al., 2014;
Kanwal et al., 2019).
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
selective competitive inhibitor of human NTPDase2 (Ki = 8.2
mM) (Brunschweiger et al., 2008). Non-nucleotide–derived
compounds have also been developed as NTPDase inhibitors
(compounds 4–7, Figure 1). These include suramin (4), reactive
blue-2 (RB-2, 5), and its derivative PSB-071 (6), PPADS (7), and
tryptamine-derived imine 8 (Iqbal et al., 2005; Baqi et al., 2009b;
Zebisch et al., 2014; Kanwal et al., 2019). However, these
compounds are non-selective and showed only moderate
inhibitory activity in the low micromolar range and/or limited
stability (Iqbal et al., 2005; Baqi et al., 2009b; Zebisch et al., 2014;
Kanwal et al., 2019). Another class of NTPDase inhibitors are
the polyoxometalates (POMs) such as [TiW11CoO40]8- (9),
which are inorganic, negatively charged metal complexes.
POM derivative 9 inhibited rat NTPDase1, -2, and -3 in the
submicromolar concentration range, but this highly negatively
charged compound displays limited stability (Müller et al.,
2006). Moreover, specific antibodies have been reported that
inhibit NTPDase2 and -3 activities; however, the inhibition is
not complete (Munkonda et al., 2009; Pelletier et al., 2017).
Previously, we evaluated anthraquinone derivatives at rat
NTPDase1, -2, and -3, and one of the most potent but non-
selective compounds was PSB-071 (6) (Baqi et al., 2009b). In the
present study, we investigated the structure-activity relationships
(SARs) of this class of NTPDase inhibitors with the goal to improve
their inhibitory potency and subtype-selectivity, in particular with
the aim to obtain potent NTPDase2- (and NTPDase3-) selective
inhibitors. Such compounds are required for biological studies
since they are expected to lead to an accumulation of ADP thereby
acting as indirect P2Y1, P2Y12 and P2Y13 receptor agonists.
electrospray ionization mass spectrometry (ESI-MS) and
ultraviolet (UV) detector using the following procedure: the
compounds were dissolved at a concentration of 0.5 mg/mL in
H2O/MeOH = 1:1, containing 2 mM NH4CH3COO. Then, 10 mL
of the sample was injected into an HPLC column (Phenomenex
Luna 3 m C18, 50 mm × 2.00 mm). Elution was performed with a
gradient of water:methanol (containing 2 mM NH4CH3COO)
from 90:10 to 0:100 starting the gradient immediately at a flow
rate of 250 mL/min for 15 min, followed by washing with 100%
methanol for another 15 min. The purity of the compounds
proved to be ≥95%. For microwave reactions, a CEM Focused
Microwave Synthesis Type Discover apparatus was employed.
A freeze-dryer (CHRIST ALPHA 1-4 LSC) was used
for lyophilization.
The synthesis and analysis of compounds 11−22, 24−26, 31
−33, 36, 38−40, 42−44, 46, 49−52, 54−56, and 58 was previously
described (Baqi and Müller, 2007; Weyler et al., 2008; Baqi et al.,
2009b; Baqi et al., 2010; Baqi and Müller, 2010; Baqi et al., 2011;
Baqi and Müller, 2012; Fiene et al., 2016; Malik et al., 2016). All
other compounds (23, 27−30, 34, 35, 37, 41, 45, 47, 48, 53, and
57) were newly prepared in analogy to described methods (Baqi
and Müller, 2010; Baqi and Müller, 2012; Malik et al., 2016;
Pelletier et al., 2017) with modifications as described below.
General Procedure A: Preparation of 4-Substituted
1-Aminoanthraquinone-2-sulfonate Derivatives (11-51)
To a 5 mL microwave reaction vial, equipped with a magnetic
stirring bar, were added 1-amino-4-bromo substituted
anthraquinone compounds [bromaminic acid sodium salt
(10a) or 1-amino-2,4-dibromoanthraquinone (10b)] (0.1−0.3
mmol) and the appropriate aniline or amine derivative
(1.5−9.0 equiv), followed by a buffer solution of Na2HPO4 (pH
9.6) (5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL) and a finely
powdered elemental copper (0.002−0.003 g, 5−10 mol%). The
mixture was capped and irradiated in the microwave oven
(80−100 W) for 5−24 min at 100−120°C. The reaction mixture
was cooled down to room temperature (rt), and the product was
purified using the following procedure. The contents of the vial
were filtered to remove the elemental copper. Then, ca. 200 mL
of water was added to the filtrate, and the aqueous solution was
extracted with dichloromethane (200 mL). The extraction
procedure was repeated until the dichloromethane layer
became colorless (two to three times). The aqueous layer was
reduced by rotary evaporation to a volume of 10−20 mL, which
was subsequently submitted to flash column chromatography
using RP-18 silica gel and water as an eluent. The polarity of the
eluent was then gradually decreased by the addition of acetone in
the following steps: 5, 10, 20, 40, and 60%. Fractions containing
blue product were collected. For some compounds the last step of
purification (RP-18 flash chromatography) had to be repeated
two to three times to obtain pure product (≥95% purity as
determined by HPLC-UV-MS). The pooled product-containing
fractions were evaporated under vacuum to remove the acetone
and reduce the water volume. The remaining water was
subsequently removed by lyophilization to yield up to 80% of
the product as blue powder (Scheme 1 and Table 1).
EXPERIMENTAL SECTION
Chemistry
Material and Methods
All materials were used as purchased (Acros, Alfa Aesar, Merck,
or Sigma-Aldrich, Germany). Thin-layer chromatography was
performed using TLC aluminum sheets silica gel 60 F254 or TLC
aluminum sheets reversed phase (RP) silica gel 18 F254 (Merck,
Darmstadt, Germany). Colored compounds were visible at
daylight; other compounds were visualized under UV light
(254 nm). Flash chromatography was performed on a Büchi
system using silica gel RP-18 (Merck, Darmstadt, Germany). 1H
and 13C NMR data were collected on either a Bruker Avance 500
MHz NMR spectrometer at 500 MHz (1H) or 126 MHz (13C),
respectively or a 600 MHz NMR spectrometer at 600 MHz (1H)
or 151 MHz (13C), respectively. Deuterated dimethyl sulfoxide
(DMSO-d6) or chloroform-d (CDCl3) were used as a solvent.
Chemical shifts are reported in parts per million (ppm) relative
to the deuterated solvent, i.e., DMSO, d 1H 2.49 ppm; 13C
39.7 ppm, coupling constants J are given in Hertz, and spin
multiplicities are given as s (singlet), d (doublet), t (triplet), q
(quartet), sext (sextet), m (multiplet), and br (broad).
The purities of isolated products were determined by high
performance liquid chromatography (HPLC) coupled with
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
SCHEME 1 | General synthesis of 4-substituted anthraquinone derivatives 11−56a. aReagents and conditions: (i) R2-NH2, phosphate buffer (pH 6−7), Cu0,
microwave, 80−120°C, 5−24 min; (ii) m-substituted aniline, CuOAc, KOAc, 110°C, argon, 2–15 h; for R1 and R2, see Table 1.
purified by flash column chromatography on a reversed phase
silica gel (RP-18) using a gradient of acetone in water (5 and
20%) as the eluent. Fractions containing the purple product were
collected and evaporated in vacuum to remove acetone and
decrease the volume of water to ca. 10-20 mL. Complete
drying was achieved with a freeze-dryer, affording purple-
colored products in excellent yields (Scheme 2 and Table 1).
General Procedure B: Preparation of 2-Substituted
1-Amino-4-anilinoanthraquinone Derivatives (52-56)
A round bottom flask (25 mL) equipped with a magnetic stirring
bar was charged with one equivalent of starting material (10b or
1-amino-4-bromo-2-methylanthraquinone (10c)), an excess of
appropriate aniline derivative (15 equiv.) and copper(I) acetate
(10 mol%) in the presence of 2.25 equiv. of potassium acetate
(Scheme 1). The resulting mixture was heated at 110°C under an
argon atmosphere for 2−15 h, and the progress of the reaction
was monitored by TLC using 10% dichloromethane/cyclohexane
as eluent. The reaction mixture was then let to cool down to
room temperature, followed by the addition of ethanol (5 mL),
and the blue-colored precipitate was filtered off and washed
successively with ethanol, 0.1 M HCl, and water (ca. 15 mL each),
and then the solid material was dried at 70°C in the oven for
16 h. The product was then purified by silica gel column
chromatography using dichloromethane/cyclohexane (9:1) as
eluent. The desired products (52−56) were obtained in high
yields (Scheme 1 and Table 1).
Sodium 1-amino-4-(3-iodophenylamino)-9,10-dioxo-9,10-
dihydroanthracene-2-sulfonate (23)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 3-iodoaniline (0.1314 g,
0.6 mmol), a buffer solution of Na2HPO4 (pH 9.6) (5.0 mL) and
NaH2PO4 (pH 4.2) (1.0 mL), and copper metal (0.003–0.005 g,
0.05–0.08 mmol). MW conditions: 100 W, 120°C, 10 min.
Analytical data: blue powder (34% yield), mp >300°C. 1H
NMR (500 MHz, DMSO-d6): d 7.20 (t, J = 7.9 Hz, 1H, 2′-H),
7.29 (m, 1H, 5′-H or 6′-H), 7.51 (m, 1H, 5′-H or 6′-H), 7.65 (t, J =
1.9 Hz, 1H, 4′-H), 7.85 (m, 2H, 6-H, 7-H), 7.98 (s, 1H, 3-H), 8.25
(m, 2H, 5-H, 8-H), 11.79 (s, 1H, 4-NH). 13C NMR (126 MHz,
DMSO-d6): d 95.55, 109.51, 112.66, 121.91, 122.99, 126.12, 126.20,
130.91, 131.53, 132.70, 133.00, 133.51, 133.58, 134.26, 139.56,
141.38, 142.66, 144.66, 182.12, 183.07. LC-MS (m/z): 519.2 [M –
Na]–, 521.4 [M – Na]+. Purity by HPLC-UV (254 nm)-ESI-
MS: 100%.
General Procedure C: Preparation of 4-Substituted
Anthraquinone-2-sulfonate Derivatives (57 and 58)
To a 50 mL round bottom flask equipped with a magnetic
stirring bar, 0.1 mmol of 1-aminoanthraquinone derivative (21
or 33) was added, followed by 5 mL of 1 M hydrochloric acid.
The solution was cooled to 0−5°C in an ice bath, and a previously
cooled solution of NaNO2 (13.8 mg, 0.2 mmol, 2 equiv) in 0.5 mL
of distilled water was added dropwise. After 5 min, the mixture
was allowed to warm up to rt, followed by addition of 30 mg of
zinc powder (1.0 mmol, 10 equiv) and 5 mL of ethanol. The
resulting mixture was then allowed to stir at rt for ca. 30 s. The
mixture was filtered off, and the purple-colored filtrate was then
Sodium 1-amino-4-(2-(hydroxymethyl)phenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (27)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 2-aminobenzyl alcohol
(0.0738 g, 0.6 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
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TABLE 1 | Inhibitory activity of anthraquinone derivatives at human ecto-NTPDases.
IC50 SEM (mM)a (or % inhibition at 2 mM concentration)
NTPDase1
NTPDase2
NTPDase3
NTPDase8
Compd.
R1
R2
R3
5 RB-2
6 PSB-071
For structure see Figure 1
For structure see Figure 1
(17)
(-4)
(42)
(22)
0.942 0.024
(1)
(-8)
(-21)
51.5 (rat)b
12.8 (rat)b
19.1 (rat)b
Structure A
11
–
–
–
–
(-3)
(0)
(6)
(6)
(-4)
(3)
12
(-10)
(11)
13
14
15
–
–
–
–
–
–
(-12)
(-13)
(12)
(15)
(-3)
(-3)
(-15)
(9)
(-10)
(-19)
(31)
(-4)
1.5 (rat)b
>100 (rat)b
>100 (rat)b
16
17
–
–
–
–
(3)
5.62 0.72
(-21)
(3)
(-1)
(15)
1.64 0.26
(-11)
18
19
–
–
–
–
(-8)
(34)
(-9)
(3)
0.328 (rat)b
19.1 (rat)b
2.22 (rat)b
(0)
(13)
4.72 0.40
(-35)
20
–
–
(-7)
0.539 0.290
(-5)
(6)
PSB-16131
Structure B
21
22
23
24
25
26
27
H
H
H
H
H
H
F
Br
I
NO2
H
CO2H
H
H
H
H
H
NH2
H
H
(-8)
(-4)
(0)
(-32)
(-19)
(-17)
(-1)
(15)
(12)
(21)
(19)
(17)
(2)
(0)
8.96 1.08
(2)
15.3 2.5
(-25)
(-1)
(-26)
(4)
(3)
(-12)
(-17)
(4)
3.10 0.45
(11)
(12)
28
29
H
H
H
(-2)
(-4)
(7)
(5)
(-9)
(-3)
(1)
H
(-4)
(Continued)
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TABLE 1 | Continued
IC50 SEM (mM)a (or % inhibition at 2 mM concentration)
NTPDase1
NTPDase2
NTPDase3
NTPDase8
Compd.
30
R1
R2
R3
H
H
(-6)
(7)
1.52 0.28
(-11)
31
32
33
H
H
H
NH2
SO3H
CO2H
SO3H
NH2
OH
(-15)
(10)
(-12)
(1)
(5)
(12)
4.26 0.54
1.73 0.55
0.723 0.032
(-19)
(-20)
(-9)
PSB-2046
34
35
36
37
38
39
Cl
Cl
F
H
H
H
H
H
Cl
OH
Cl
H
(1)
(-21)
(-20)
(-8)
(2)
(36)
(16)
(-3)
(-2)
(-2)
(32)
4.04 0.47
(-12)
(7)
(-10)
(-17)
(-6)
(-1)
(28)
CO2H
CO2H
F
CH3
H
(5)
5.45 0.70
3.59 0.85
(9)
13.1 1.65
40
41
H
H
H
H
(12)
(1)
1.11 0.06
0.984 0.327
(10)
(18)
(10)
(9)
(6)
H
42
SO3H
(-2)
0.390 0.041
(-7)
PSB-1011
43
44
45
H
H
H
H
H
H
(7)
1.32 0.05
0.934 0.136
1.08 0.08
(3)
(7)
(3)
(6)
(15)
(52)
(42)
(21)
46
47
H
H
H
H
(33)
(34)
1.73 0.29
(21)
(16)
(10)
(4)
0.951 0.225
48
H
H
Cl
H
(29)
(46)
0.551 0.195
1.28 0.34
(20)
(36)
(14)
(27)
PSB-2020
49
50
H
H
(38)
1.13 0.27
(34)
(17)
(Continued)
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TABLE 1 | Continued
IC50 SEM (mM)a (or % inhibition at 2 mM concentration)
NTPDase1
NTPDase2
NTPDase3
NTPDase8
Compd.
51
R1
R2
R3
H
H
(51)
0.832 0.053
(44)
(27)
Structure C
52
53
54
55
Br
Br
CH3
CH3
CH3
CO2H
C2H5
F
OCH3
C2H5
–
–
–
–
–
(-2)
(-4)
(-4)
(-6)
(-3)
(-5)
(5)
(7)
(3)
(6)
(1)
(-2)
(-2)
(0)
(3)
(-4)
(-2)
(-2)
(-3)
56
(-1)
Structure D
57
58
SO3H
SO3H
F
H
OH
(14)
(1)
(-6)
(5)
(9)
(32)
(-1)
(5)
CO2H
aIC50 values for potent inhibitors are highlighted in bold. bBaqi et al., 2009b. Data are from at least three separate experiments.
SCHEME 2 | Synthesis of deaminated anilinoanthraquinone derivatives 57 and 58a. aReagents and conditions: (i) NaNO2, HCl (1 M), 0−5°C, 5 min; (ii) Zn (10 equiv.),
ethanol, rt, 30 s; for R1, R2, and R3, see Table 1.
Sodium 1-amino-4-(3-(hydroxymethyl)phenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (28)
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W,
120°C, 10 min. Analytical data: blue powder (44.3% yield), mp
1
>300°C. H NMR (500 MHz, DMSO-d6): d 4.52 (d, J = 4.1 Hz,
Reaction conditions according to general procedure A: Compound
10a (0.1213 g, 0.3 mmol), 3-aminobenzyl alcohol (0.0738 g, 0.6
mmol), a buffer solution of Na2HPO4 (pH 9.6) (5.0 mL) and
NaH2PO4 (pH 4.2) (1.0 mL), and copper metal (0.003–0.005 g,
0.05–0.08 mmol). MW conditions: 100 W, 120°C, 10 min.
Analytical data: blue powder (67.7% yield), mp >300°C. 1H
NMR (500 MHz, DMSO-d6): d 4.52 (d, J = 3.7 Hz, 2H, –
CH2OH), 5.24 (t, J = 5.7 Hz, 1H, –CH2OH), 7.15 (m, 2H, 5′-H,
6′-H), 7.21 (s, 1H, 2′-H), 7.39 (t, J = 7.7 Hz, 1H, 3′-H), 7.85 (m,
2H, -CH2OH), 5.30 (t, J = 5.0 Hz, 1H, -CH2OH), 7.23 (m, 3H, 3′-
H, 5′-H, 6′-H), 7.34 (td, J = 7.7, 1.6 Hz, 1H, 4′-H), 7.50 (dd, J =
7.6, 1.5 Hz, 1H, 3-H), 7.84 (m, 2H, 6-H, 7-H), 8.26 (m, 2H, 5-H,
8-H), 11.94 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d
60.59, 109.31, 111.72, 123.37, 123.96, 124.69, 126.07, 126.15,
128.10, 129.15, 132.85, 133.19, 133.80, 134.31, 135.79, 137.78,
141.26, 142.65, 144.41, 181.95, 182.34. LCMS (m/z): 423.1 [M –
Na]–. Purity by HPLC-UV(220–700 nm)-ESI-MS 100%.
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
2H, 6-H, 7-H), 7.99 (s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 12.06 (s,
1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d 62.73, 109.23,
111.39, 121.37, 121.55, 122.74, 122.92, 126.08, 126.16, 129.46,
132.88, 133.27, 133.73, 134.29, 139.19, 141.21, 142.97, 144.46,
144.71, 181.90, 182.53. LCMS (m/z): 423.1 [M – Na]–. Purity by
HPLC-UV(220–700 nm)-ESI-MS 100%.
139.08, 142.39, 144.85, 182.30, 183.86. LCMS (m/z): 461.1 [M –
Na]–, 463.1 [M – Na]+. Purity by HPLC-UV(220–700 nm)-ESI-
MS 100%.
Sodium 1-amino-4-(2-carboxy-3-fluorophenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (35)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 2-amino-6-fluorobenzoic
acid (0.0930 g, 0.6 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W,
120°C, 10 min. Analytical data: blue powder (40.1% yield),
mp >300°C. 1H NMR (500 MHz, DMSO-d6): d 6.78 (t, J =
8.6 Hz, 1H, 6′-H), 6.94 (d, J = 8.0 Hz, 1H, 4′-H), 7.15 (m, 1H, 5′-
H), 7.81 (m, 2H, 6-H, 7-H), 8.10 (s, 1H, 3-H), 8.26 (m, 2H, 5-H,
8-H), 12.07 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d
109.66, 109.89, 110.08, 113.17, 116.81, 124.44, 126.03, 126.25,
127.21, 132.80, 133.11, 133.84, 134.20, 138.60, 139.02, 141.95,
144.57, 158.56, 160.49, 166.00, 181.84, 182.19. LCMS (m/z):
455.2 [M – Na]–, 457.3 [M – Na]+. Purity by HPLC-UV(220–
700 nm)-ESI-MS 98%.
Sodium 1-amino-4-(4-(hydroxymethyl)phenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (29)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 4-aminobenzyl alcohol
(0.0738 g, 0.6 mmol), a buffer solution of Na2HPO4 (pH 9.6) (5.0
mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal (0.003–
0.005 g, 0.05–0.08 mmol). MW conditions: 100 W, 120°C,
10 min. Analytical data: blue powder (50.9% yield), mp >300°C.
1H NMR (500 MHz, DMSO-d6): d 4.51 (d, J = 5.2 Hz, 2H, –
CH2OH), 5.19 (t, J = 5.8 Hz, 1H, –CH2OH), 7.24 (m, 2H, 3′-H, 5′-
H), 7.39 (m, 2H, 2′-H, 6′-H), 7.84 (m, 2H, 6-H, 7-H), 7.98 (s, 1H, 3-
H), 8.27 (m, 2H, 5-H, 8-H), 12.06 (s, 1H, 4-NH). 13C NMR (126
MHz, DMSO-d6): d 62.73, 109.21, 111.20, 122.78, 123.34, 126.06,
126.16, 128.03, 132.87, 133.23, 133.75, 134.29, 137.77, 139.24,
141.43, 143.00, 144.43, 181.87, 182.40. LCMS (m/z): 423.1 [M –
Na]–. Purity by HPLC-UV(220–900 nm)-ESI-MS 98%.
Sodium 1-amino-4-(2-fluoro-4-hydroxyphenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (37)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 4-amino-3-fluorophenol
(0.0762 g, 0.6 mmol), a buffer solution of Na2HPO4 (pH 9.6) (5.0
mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal (0.003–
0.005 g, 0.05–0.08 mmol). MW conditions: 100 W, 120°C,
10 min. Analytical data: blue powder (38.4% yield), mp >300°C.
1H NMR (500 MHz, DMSO-d6): d 6.72 (m, 2H, 4′-H, 5′-H), 7.23
(s, 1H, 3′-H), 7.58 (d, J = 1.7 Hz, 1H, 3-H), 7.84 (m, 2H, 6-H, 7-H),
8.27 (m, 2H, 5-H, 8-H), 11.68 (s, 1H, 4-NH). 13C NMR (126 MHz,
DMSO-d6):d 103.65, 103.82, 110.51, 112.18, 117.38, 122.18, 126.06,
126.16, 128.64, 132.85, 133.21, 133.71, 134.30, 142.94, 143.25,
144.12, 157.03, 158.34, 181.86, 182.50. LCMS (m/z): 427.3 [M –
Na]–, 429.2 [M – Na]+. Purity by HPLC-UV(220–900 nm)-ESI-
MS 97%.
Sodium 1-amino-4-(3-(carboxymethyl)phenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (30)
Reaction conditions according to general procedure A: Compound
10a (0.1213 g, 0.3 mmol), 3-aminophenylacetic acid (0.0906 g, 0.6
mmol), a buffer solution of Na2HPO4 (pH 9.6) (5.0 mL) and
NaH2PO4 (pH 4.2) (1.0 mL), copper metal (0.003–0.005 g, and
0.05–0.08 mmol). MW conditions: 100 W, 120°C, 10 min.
1
Analytical data: blue powder (73% yield), mp >300°C. H NMR
(500 MHz, DMSO-d6): d 3.40 (s, 2H, -CH2CO2H), 7.07 (m, 2H, 4′-
H, 6′-H), 7.18 (s, 1H, 2′-H), 7.31 (t, J = 7.8 Hz, 1H, 5′-H), 7.83 (m,
2H, 6-H, 7-H), 8.02 (s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.11 (s,
1H, -CH2CO2H), 12.08 (s, 1H, 4-NH). 13C NMR (126 MHz,
DMSO-d6): d 43.74, 109.23, 111.29, 120.49, 122.96, 124.25, 125.76,
126.09, 126.16, 129.11, 132.87, 133.24, 133.76, 134.28, 138.84,
141.24, 142.93, 144.44, 150.43, 173.43, 181.89, 182.46. LCMS (m/
z): 451.4 [M – Na]–, 453.3 [M – Na]+. Purity by HPLC-UV(220–
700 nm)-ESI-MS 100%.
Sodium 1-amino-4-(3-(phenylsulfanyl)phenylamino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (41)
Reaction conditions according to general procedure A:
Compound 10a (0.1213 g, 0.3 mmol), 3-(phenylsulfanyl)aniline
(0.0664 g, 0.33 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W,
120°C, 7 min. Analytical data: blue powder (14.7% yield), mp
Sodium 1-amino-4-(2,3-dichlorophenylamino)-9,10-dioxo-
9,10-dihydroanthracene-2-sulfonate (34)
Reaction conditions according to general procedure A:
Compound 10a (0.3639 g, 0.9 mmol), 2,3-dichloroaniline
(0.4374 g, 2.7 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W,
120°C, 10 min. Analytical data: blue powder (20% yield), mp
>300°C. 1H NMR (500 MHz, DMSO-d6): d 7.40 (m, 3H, 4′-H, 5′-
H, 6′-H), 7.86 (m, 2H, 6-H, 7-H), 7.93 (s, 1H, 3-H), 8.27 (m, 2H,
5-H, 8-H), 11.90 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-
d6): d 109.75, 113.86, 121.13, 123.01, 124.14, 125.11, 126.22,
126.25, 128.64, 132.84, 133.10, 133.40, 133.79, 134.26, 138.08,
1
>300°C. H NMR (500 MHz, DMSO-d6): d 7.07 (m, 1H, 6′-H),
7.12 (t, J = 1.9 Hz, 1H, 2′-H), 7.18 (dd, J = 7.8, 1.9 Hz, 1H, 4′-H),
7.34 (m, 1H, 5′-H), 7.45 (m, 5H, 2″-H, 3″-H, 4″-H, 5″-H, 6″-H),
7.85 (m, 2H, 6-H, 7- H), 8.01 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-
H), 11.84 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d
109.24, 112.04, 121.11, 122.70, 123.10, 125.00, 125.92, 126.00,
127.94, 129.79, 130.59, 131.82, 132.78, 133.13, 133.25, 133.43,
134.08, 137.12, 139.86, 140.44, 142.58, 144.41, 181.88, 182.72.
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
LCMS (m/z): 501.0 [M – Na]–, 503.2 [M – Na]+. Purity by
HPLC-UV(220–700 nm)-ESI-MS 99.4%.
536.1 [M – Na]+. Purity by HPLC-UV(220–700 nm)-ESI-
MS 97.7%.
1-Amino-2-bromo-4-(3-ethylphenylamino)anthracene-9,10-
dione (53)
Sodium 1-amino-4-[4-(4-chlorophenylthio)phenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (45)
Reaction conditions according to general procedure B:
Compound 10b (0.1143 mg, 0.3 mmol, 1 equiv.), 3-ethylaniline
(0.5453 mg, 4.5 mmol, 15 equiv.), copper(I) acetate (0.0037 mg,
10 mol%) and potassium acetate (0.066 mg, 0.68 mmol) at 110°C
for 2 h. Analytical data: dark blue powder (57.6% yield), mp =
229–230°C. 1H NMR (500 MHz, Chloroform-d): d 1.29 (t, J = 7.6
Hz, 3H, –CH2-CH3), 2.68 (q, J = 7.6 Hz, 2H, –CH2-CH3), 7.05 (d,
1H, 4′-H), 7.10 (m, 2H, 2′-H, 6′-H), 7.33 (m, 1H, 5′-H), 7.76 (m,
2H, 6-H 7-H), 7.88 (s, 1H, 3-H), 8.35 (m, 2H, 5-H, 8-H), 11.90 (s,
1H, 4-NH). 13C NMR (126 MHz, Chloroform-d): d 15.65, 28.94,
111.50, 111.73, 121.23, 122.93, 123.61, 124.79, 126.51, 126.76,
127.74, 129.68, 133.09, 133.15, 133.90, 134.45, 139.47, 142.81,
143.07, 146.32, 183.77, 183.88. LC-MS (m/z): 419.2 [M – H]–,
421.2 [M + H]+. Purity by HPLC-UV(254 nm)-ESI-MS 95%.
Reaction conditions according to general procedure A: Compound
10a (0.1213 g, 0.3 mmol), 4-(4-chlorophenylsulfanyl)aniline
(0.0778 g, 0.33 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W, 120°C,
1
8 min. Analytical data: blue powder (6% yield), mp >300°C. H
NMR (500 MHz, DMSO-d6): d 7.30 (m, 2H, 2′-H, 6′-H), 7.32 (m,
2H, 3″-H, 5″-H), 7.42 (m, 2H, 3′-H, 5′-H), 7.46 (m, 2H, 2″-H, 6″-
H), 7.86 (m, 2H, 6-H, 7-H), 8.07 (s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-
H), 11.91 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d 109.40,
112.62, 123.00, 125.96, 126.03, 127.22, 129.36, 130.82, 131.44,
132.84, 133.34, 133.42, 133.83, 134.08, 135.46, 139.19, 139.95,
142.45, 144.55, 181.96, 182.92. LCMS (m/z): 535.0 [M – Na]–,
536.1 [M – Na]+. Purity by HPLC-UV(220–400 nm)-ESI-MS 99%.
4-(3-Fluorophenylamino)-9,10-dioxo-9,10-
dihydroanthracene-2-sulfonic acid (57)
Sodium 1-amino-4-[4-(4-methoxyphenylthio)phenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (47)
Reaction conditions according to general procedure A: Compound
10a (0.1213 g, 0.3 mmol), 4-(4-methoxyphenylsulfanyl)aniline
(0.0763 g, 0.33 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W, 120°C,
10 min. Analytical data: blue powder (14.2% yield), mp >300°C. 1H
NMR (500 MHz, DMSO-d6): d 3.79 (s, 3H, –OCH3), 7.01 (m, 2H,
2′-H, 6′-H), 7.23 (s, 4H, 2″-H, 3″-H, 5″-H, 6″-H), 7.44 (m, 2H, 3′-H,
5′-H), 7.85 (m, 2H, 6-H, 7-H), 7.99 (s, 1H, 3-H), 8.26 (m, 2H, 5-H,
8-H), 11.95 (s, 1H, 4-NH). 13C NMR (126 MHz, DMSO-d6): d
55.26, 109.21, 111.78, 115.36, 122.71, 123.54, 123.77, 125.92, 126.01,
129.91, 132.43, 132.77, 133.20, 133.48, 134.08, 134.70, 137.83,
140.16, 142.62, 144.39, 159.55, 181.82, 182.55. LCMS (m/z): 531.1
[M – Na]–, 532.2 [M – Na]+. Purity by HPLC-UV(220–400 nm)-
ESI-MS 98.9%.
Reaction conditions according to general procedure C:
Compound 21 (0.0434, 0.1 mmol) was dissolved in 5 mL of 1
M HCl then cooled down to 0–5°C in an ice bath. Subsequently
NaNO2 (14 mg, 0.2 mmol) dissolved in water (0.5 mL) was
added portion-wise, and the mixture was stirred for 5 min. It was
then warmed up to rt followed by the addition of ethanol (5 mL)
and zinc (65 mg, 1 mmol, 10 equiv.) and left stirring at rt for 30 s.
Analytical data: dark violet powder (75% yield), mp >300°C. 1H
NMR (600 MHz, DMSO-d6): d 7.06 (td, J = 8.6, 2.5 Hz, 1H, 5′-
H), 7.22 (m, 2H, 4′-H, 6′-H), 7.50 (m, 1H, 2′-H), 7.79, 7.85 (2 d,
J = 1.5 Hz, each 1H, 1-H, 3-H), 7.89, 7.93 (2 td, J = 7.5, 1.5 Hz,
each 1H, 6-H, 7-H), 8.19, 8.24 (2 dd, J = 7.7, 1.3 Hz, 1H, each 1H,
5-H, 8-H), 11.20 (s, 1H, 4-NH). 13C NMR (151 MHz, DMSO-
d6): d 110.34, 110.50, 111.64, 111.78, 114.12, 115.71, 116.23,
119.36, 119.38, 126.69, 126.84, 131.44, 131.51, 132.67, 134.16,
134.35, 134.47, 134.86, 141.12, 141.19, 147.90, 154.52, 162.16,
163.78, 182.48, 184.68. LCMS (m/z): 396.0 [M – H]–, 398.1 [M +
H]+. Purity by HPLC-UV(220–800 nm)-ESI-MS 99%.
Sodium 1-amino-4-(3-chloro-4-phenylsulfanyl)-
phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-
sulfonate (48)
Malachite Green Assay to Investigate
NTPDase Inhibitors
Reaction conditions according to general procedure A: Compound
10a (0.1213 g, 0.3 mmol), 3-chloro-4-(phenylsulfanyl)aniline
(0.0778 g, 0.33 mmol), a buffer solution of Na2HPO4 (pH 9.6)
(5.0 mL) and NaH2PO4 (pH 4.2) (1.0 mL), and copper metal
(0.003–0.005 g, 0.05–0.08 mmol). MW conditions: 100 W, 120°C,
Membrane preparations expressing human NTPDase1, -2, -3, or
́
-8, respectively, were obtained as previously described (Sevigny
et al., 1997; Cogan et al., 1999; Kukulski et al., 2005; Lecka et al.,
2013; Lee et al., 2018). Enzyme inhibition assays were performed
using the malachite green assay in analogy to published
procedures with some modifications (Dou et al., 2018). The
reaction buffer contained 10 mM HEPES, 2 mM CaCl2, and 1
mM MgCl2 (pH 7.4) in a final volume of 50 mL in transparent 96-
well half-area plates. The compounds were initially tested at a
final concentration of 2 µM using a COS-7-cell membrane
preparation expressing the appropriate NTPDase isoenzyme
(protein amount: 143 ng for NTPDase1, 175 ng for NTPDase2,
1
8 min. Analytical data: blue powder (6.5% yield), mp >300°C. H
NMR (500 MHz, DMSO-d6): d 7.21 (dd, J = 8.5, 2.3 Hz, 1H, 6′-H),
7.25 (d, J = 8.5 Hz, 1H, 5′-H), 7.34 (m, 3H, 3″-H, 4″-H, 5″-H), 7.42
(m, 2H, 2″-H, 6″-H), 7.51 (d, J = 2.2 Hz, 1H, 2′-H), 7.86 (m, 2H, 6-
H, 7-H), 8.03 (s, 1H, 3-H), 8.25 (m, 2H, 5-H, 8-H), 11.68 (s, 1H, 4-
NH). 13C NMR (126 MHz, DMSO-d6): d 109.60, 113.55, 121.02,
122.52, 123.29, 125.99, 126.05, 126.99, 127.60, 129.71, 130.51, 132.92,
133.33, 133.49, 133.64, 133.69, 134.06, 135.28, 138.17, 140.95,
142.21, 144.71, 182.12, 183.21. LCMS (m/z): 535.0 [M – Na]–,
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Baqi et al.
Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
152 ng for NTPDase3, and 175 ng for NTPDase8). Preincubated
of the enzyme preparations was perfomred at 37°C in the presence
or absence of test compounds with gentle shaking (Eppendorf
Thermomixer comfort at 500 rpm) for 5 min. The reaction was
initiated by the addition of 50 µM ATP [Km (CD39) = 17 µM] for
NTPDase1 or 100 µM ATP for NTPDase2, -3, and -8 [Km
(NTPDase2) = 70 µM; Km (NTPDase3) = 75 µM; Km
(NTPDase8) = 46 µM] (Kukulski et al., 2005). After 15 min of
incubation at 37°C with gentle shaking, the reaction was stopped by
the addition of the detection reagents (20 µL malachite green
solution, 0.6 mM, and 30 µL of ammonium molybdate solution,
20 mM, in 1.5 M sulfuric acid). The released inorganic phosphate
was quantified after 20 min of gentle shaking at 25°C by measuring
the absorption of the malachite green-phosphomolybdate complex
at 600 nm using a BMG PheraStar FS plate reader (BMG Labtech
GmbH, Ortenberg, Germany). The corrected absorption was
calculated by subtracting the absorption of the negative control
samples, which were incubated with previously denatured enzyme
(90°C, 15 min). Full concentration-inhibition curves were
determined with inhibitor concentrations ranging from 0.03 to 30
µM in the presence of 2% DMSO. Inhibition-type experiments
were performed with 25, 50, 100, 150 and 200 µM ATP as substrate
for NTPDase2 in the presence of inhibitor 20 (0, 0.25, 0.5, and
1µM)and25, 50,100and150µMATPsubstrateforNTPDase3and
compound 42 (0.25, 0.5, and 1 µM). For all of the presented data, at
least three independent experiments were performed, and IC50
values were calculated by GraphPad Prism 8 software.
to excellent isolated yields. Anthraquinones 11–51 bearing a
sulfonate substitution at the 2-position were synthesized starting
from compound 10a in sodium phosphate buffer (pH 6−7) in the
presence of a catalytic amount of elemental copper (Cu0) under
microwave reaction conditions at 80−120°C for 5–24 min (Baqi
and Müller, 2007; Baqi and Müller, 2010).
Compounds 52–56, bearing a bromo or methyl residue at
the 2-position, were synthesized starting from 10b or 10c,
respectively, with excess of the appropriate aniline derivatives
(15 eq.) under argon in the presence of potassium acetate and
copper(I) acetate (CuOAc) as a catalyst, upon heating at 110°C
for 2–15 h (Scheme 1).
In order to investigate the role of the amino group at the 1-
position of the anthraquinone moiety, two anilinoanthraquinone
derivatives (21 and 33) were treated with sodium nitrite in
hydrochloric acid solution (1 M) at 0–5°C for 5 min, then
allowed to warm up to room temperature, followed by the
addition of ethanol and an excess of zinc powder (10 equiv.) to
achieve deamination within 30 seconds (Baqi and Müller, 2012),
affording the desired products 57 and 58 in excellent yields
(Scheme 2).
Biological Studies
Inhibition of human NTPDases was performed using the
malachite green assay, which was established on a robotic
system (Z’ factors > 0.70) (Baykov et al., 1988; Fiene et al.,
2015). The malachite green assay enables the detection of the
phosphate produced by the enzymatic hydrolysis of nucleotides.
A fixed substrate concentration of 50 µM ATP for NTPDase1
and 100 µM for NTPDase2, -3, and -8 was employed. Test
compounds were initially screened at a concentration of 2 mM.
For compounds that showed about 50% inhibition or more,
concentration-dependent inhibition curves were determined,
and IC50 values were calculated. A total of 48 synthesized
anthraquinone derivatives including 14 new compounds not
previously described in the literature were evaluated for their
inhibitory activity at human NTPDase1, -2, -3, and -8 (for results
see Table 1).
RESULTS AND DISCUSSION
A library of 48 anthraquinone derivatives was synthesized and
tested at human NTPDase1, -2, -3, and -8, which are ecto-
enzymes hydrolyzing extracellular nucleotides, using the
malachite green assay. Subsequently, inhibition curves for
compounds showing above 50% inhibition at 2 µM test
concentration were determined.
Chemistry
Structure-Activity Relationships (SARs)
The anthraquinone derivative reactive blue-2 (RB-2 (5), Figure 1
and Table 1) showed the highest potency at NTPDase3 (IC50 of
0.942 µM) followed by NTPDase2 and was inactive at NTPDase8
(Table 1). RB-2 is a relatively large molecule (molecular weight
of >800 g/mol) with high polarity bearing three negatively
charged sulfonate (SO3Na) groups. Therefore, smaller and less
polar anthraquinone derivatives were designed, synthesized, and
evaluated as NTPDases inhibitors (see Table S1 in Supplementary
Materials for clogD values of all anthraquinone derivatives
discussed in the present study).
The target compounds (11–58) were synthesized as depicted in
Schemes 1 and 2. The syntheses of compounds 11–22, 24–26,
31–33, 36, 38–40, 42–44, 46, 49–52, 54–56, and 58 had been
previously described (Baqi and Müller, 2007; Weyler et al., 2008;
Baqi et al., 2009b; Baqi et al., 2010; Baqi and Müller, 2010; Baqi
et al., 2011; Baqi and Müller, 2012; Fiene et al., 2016; Malik et al.,
2016). In addition to previously reported AQ derivatives, a series
of 14 new compounds (23, 27–30, 34, 35, 37, 41, 45, 47, 48, 53,
and 57) was prepared. Condensation of sodium 1-amino-4-
bromo-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (R1
=
SO3Na, 10a, Scheme 1), 1-amino-2,4-dibromo-9,10-dioxo-
9,10-dihydroanthracene (R1 = Br, 10b, Scheme 1), or 1-amino-
In our previous study, we had investigated a smaller series of
anthraquinone derivatives at ecto-NTPDases of rat, which had
led to the identification of PSB-071 (6) bearing a m-methyl
substituent on the 4-anilino group. This inhibitor was slightly
4-bromo-2-methyl-9,10-dioxo-9,10-dihydroanthracene (R1
=
CH3, 10c, Scheme 1) with the appropriate (ar)alkylamine or
aniline derivatives yielded the target compounds in satisfactory
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
selective for rat NTPDase2 (12.8 mM) (Baqi et al., 2009b; Zebisch
et al., 2014) vs. rat NTPDase1 and -3, while in the present study,
it showed no significant inhibitory activity on all tested human
NTPDases (compound 6, Table 1), except for NTPDase2, at
which it displayed very moderate potency.
Introducing of an (ar)alkyl group, such as propyl (11), benzyl
(12) and phenethyl (13) at the 4-amino group of 1-amino-2-
sulfoanthraquinone abolished the inhibitory activity on all tested
NTPDases (entry 3–5, Table 1). Moreover, unsubstituted
aromatic rings, such as phenyl, 1-naphthyl and 2-naphthyl, 14,
15, and 18 (Table 1), all showed no inhibitory activity as well.
The naphthylamino-substituted anthraquinone derivatives 15
and 18 had shown good potency in our previous study at rat
NTPDase3 (both) and at rat NTPDase1 (compound 18)
indicating considerable species differences between rat and
human NTPDases.
para-position of the phenyl ring restored the inhibitory potency
towards NTPDase2, especially compound 33 showing inhibitory
potency at submicromolar concentration. Any polar substituent in
the ortho-position and in combination with a substituent in the
meta- or para-position led to inactive derivatives. The
introduction of lipophilic substituents in the ortho- and para-
position shifted the inhibitory potency towards NTPDase2, see
compound 38 (Table 1).
Next, we introduced an additional aromatic residue, ring E.
Lipophilic substitution in the meta- and para-position resulted in
moderate to good potency at NTPDase2 (39–41 and 43–51,
Table 1), with potencies reaching the submicromolar range (IC50
of 0.551 mM, 48), while a m-dichlorotriazinyl moiety in
combination with a p-SO3H group furnished the most potent
compound of the present anthraquinone series at NTPDase3 (42,
IC50 of 0.390 mM, Table 1).
Interestingly, a combination between structures of 1-naphthyl
and 2-naphthyl resulting in phenanthryl derivative 20, yielded a
potent inhibitor of NTPDase2 which displayed no activity vs.
NTPDase1, -3, and -8 at the tested concentration. This is
probably due to the presence of a large lipophilic pocket present
in human NTPDase2. This presence of a lipophilic pocket in
NTPDase2 was confirmed with compound 16 (IC50 of 5.62 µM,
Table 1), which is bearing an extra lipophilic methyl group in the 2-
position of the 1-naphthyl moiety; again, this compound was found
to be selective vs. the other investigated human NTPDases (-1, -3,
and -8). Introduction of polar and negatively charged groups,
SO3H (17) or CO2H (19) on the naphthyl moiety shifted the
inhibitory activity towards human NTPDase3.
In the next step, we introduced different substituents on phenyl
ring D (compounds 21–38, Table 1). Mono-substitution of the
aromatic ring D with Br (22), NO2 (24), CO2H (26), or CH2CO2H
(30) in the meta-position led to selective inhibition of NTPDase3,
while other mono-substitutions including m-F, p-NH2, o-CH2OH,
m-CH2OH, and p-CH2OH resulted in no inhibition at all tested
NTPDases. On the other hand, di-substitution with polar
functions, e.g., NH2, SO3H, and OH, on the meta- and
Any modification on the anthraquinone moiety, such as
removal of the amino group in position 1 or replacement of
the sulfonate function in position 2 of the anthraquinone core by
bromo or methyl abolished the inhibitory activity (see
compounds 52–58, Table 1).
Concentration−response curves for selected potent
anthraquinone derivatives 20, 44, 48, and 51 on NTPDase2 and
for 17, 30, 33, and 42 on NTPDase3 are depicted in Figure 2.
The most potent inhibitors were found to be selective vs.
other tested human NTPDases. For examples, the NTPDase2
inhibitors 20 and 44 were found to be selective vs. NTPDase1, -3,
and -8, while the other two most potent NTPDase2 inhibitors 48
and 51 showed lower selectivity (Figure 3). The identified
NTPDase3 inhibitors 17, 30, 33 and 42 were selective vs.
NTPDase1, -2, and -8 (Figure 3).
The SARs for human NTPDase2 and -3 are summarized in
Figure 4. Large and lipophilic substituents have led to selectivity
for NTPDase2 (Figure 4A), while smaller and polar substituent
have provided selectivity for NTPDase3 (Figure 4B).
We previously published articles highlighting the fact that the
anthraquinone scaffold represents a privileged scaffold in
A
B
100
80
60
100
80
60
5
5
20
17
44
30
48
33
51
42
40
20
0
40
20
0
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
log [Compound], M
log [Compound], M
FIGURE 2 | Concentration-inhibition curves of selected anthraquinone derivatives (A) determined using the malachite green assay on recombinant human
NTPDase2 expressed in COS7 cell membrane preparations. ATP at a concentration of 100 µM (Km = 70 µM) was used as substrate and (B) determined using the
malachite green assay on recombinant human NTPDase3 expressed in COS7 cell membrane preparations. ATP at a concentration of 100 µM (Km = 75 µM) was
used as substrate. Data points shown are mean values of at least three independent experiments. IC50 values are collected in Table 1.
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
6.5
6.0
5.5
5.0
4.5
NTPDase1
NTPDase2
NTPDase3
NTPDase8
20
44
48
51
17
30
33
42
Compound
FIGURE 3 | Selectivity of selected anthraquinone derivatives on different human NTPDase ecto-enzymes determined using the malachite green assay. Shown are
the pIC50 values of compounds 20, 44, 48 and 51, active on NTPDase 2 and compounds 17, 30, 33 and 42 active on NTPDase3.
A
B
FIGURE 4 | Summary of SARs of anthraquinone derivatives (A) at human NTPDase2 and (B) at human NTPDase3.
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Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
medicinal chemistry targeting different nucleotide-binding
proteins including ecto-5’-nucleotidase, P2Y12 and P2X2
receptors (Baqi, 2016; Malik and Müller, 2016). However, this
does not mean that potent compounds are non-selective. In fact,
selectivity for specific targets has been achievable (Baqi et al.,
2009a; Baqi, 2016; Malik and Müller, 2016; Rafehi et al., 2017a;
Rafehi et al., 2017b). Compounds that are highly potent at a
specific target typically also have shown selectivity. In a study
published in 2010, we reported the first SARs of anthraquinone
derivatives as inhibitors of rat ecto-5’-nucleotidase (CD73) (Baqi
et al., 2010). The observed SARs were clearly different from the
SARs of anthraquinone derivatives as NTPDase inhibitors. For
example, compound 15 displayed an IC50 of 0.53 µM at rat
CD73 but was virtually inactive at NTPDases, while compound
20, found to be a potent inhibitor of human NTPDase2 in the
present study, was shown to be only weakly active against CD73
(58% inhibition at 1 mM concentration) (Baqi et al., 2010). The
compounds have not yet been tested at alkaline phosphatase, but
this enzyme has a very high Km value for adenine nucleotides,
and its significance in the context of extracellular nucleotide
metabolism and signaling in inflammation is therefore
questionable. Nevertheless, ancillary activities of NTPDase
inhibitors as blockers of CD73 or alkaline phosphatase would
not be detrimental, but might even enhance their over-all effects
leading to an accumulation of immunostimulatory, pro-
inflammatory nucleotides while inhibiting the final production
of immunosuppressive adenosine. Future studies might therefore
be directed at multi-target drugs inhibiting more than one
single ectonucleotidase.
20, and at NTPDase3, compound 42, were investigated with
regard to their inhibition mechanism (see Figure 5).
NTPDase2 inhibitor 20 displayed non-competitive inhibition,
while the larger NTPDase3 inhibitor 42 showed a mixed
inhibition type. Together with previous results (Baqi et al.,
2009b; Zebisch et al., 2014), these data show that anthraquinone
derivatives may inhibit NTPDase isoenzymes with different
inhibition mechanisms depending on the compound’s
substitution pattern and perhaps also the NTPDase subtype and
the species.
CONCLUSIONS
Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase)
plays a major role in controlling extracellular nucleotide levels.
NTPDase inhibitors have potential as novel drugs, for example,
for the treatment of inflammation, neurodegenerative diseases
and cancer. In the present study, we synthesized and investigated
a series of 48 anthraquinone derivatives as potential inhibitors of
NTPDases, 14 of which are novel compounds. The synthesized
compounds showed no inhibitory activity on NTPDase1 (CD39)
or NTPDase8, while potent inhibitors for NTPDase2 or -3 were
identified. The most potent inhibitors exhibited selectivity
for either NTPDase2 or -3. It was noticed that human
NTPDase2 features a lipophilic pocket that accommodates
polynuclear-aromatic rings such as phenanthryl or naphthyl
bearing lipophilic substituents such as chloro or methyl. In
contrast, NTPDase3 was found to accommodate smaller
hydrophilic functions such as hydroxyl, carboxyl or sulfonate.
These NTPDase3-inhibitors were selective (>10-fold) vs. other
NTPDases. Although inhibitors bearing polar sulfonate
functions cannot be expected to be brain-penetrant, they will
be useful tools for studying peripheral effects, or maybe even
used to study central effects after direct application to the brain.
Mechanism of Enzyme Inhibition
In previous studies at rat NTPDase2 and -3, selected small 1-
amino-4-anilino-2-sulfoanthraquinone derivatives were found
to display a competitive inhibition mechanism (Baqi et al.,
2009b; Zebisch et al., 2014). In the present study at human
NTPDases, the most potent inhibitors at NTPDase2, compound
A
B
1.5×10-6
1.2×10-6
Compd. 20
Compd. 42
0
µM
0
µM
0.25 µM
0.50 µM
1.00 µM
0.25 µM
0.50 µM
1.00 µM
1×10-6
5×10-7
0
8×10-7
4×10-7
0
-5×10-7
-4×10-7
-1×10-4
0
1×10-4
2×10-4
-1×10-4 -5×10-5
0
5×10-5 1×10-4 1.5×10-4
[ATP], M
[ATP], M
FIGURE 5 | (A) Hanes-Woolf plot for NTPDase2 inhibition by 20, determined using the malachite green assay and recombinant human NTPDase2 expressed in
COS7 cell membranes. ATP at concentrations of 25, 50, 100, 150 and 200 µM (Km = 70 µM) was used as a substrate. Data points shown are the mean values
SEM of at least three independent experiments, each performed in triplicates (n = 3). (B) Hanes-Woolf plot for NTPDase3 inhibition by 42, determined using the
malachite green assay and recombinant human NTPDase3 expressed in COS7 cell membranes. ATP at concentrations of 25, 50, 100, and 150 µM (Km = 75 µM)
was used as a substrate. Data points shown are the mean values SEM of at least three independent experiments, each performed in triplicates (n = 3).
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Baqi et al.
Anthraquinones as Selective Inhibitors for NTPDase2 and NTPDase3
Academy of Sciences and Humanities (AGYA) (01DL16002 &
01DL20003). EM is grateful to the Deutscher Akademischer
Austauchdienst (DAAD) for a PhD scholarship. JS received
support from the Natural Sciences and Engineering Research
Council of Canada (NSERC; RGPIN-2016-05867) and was the
recipient of a “Chercheur National” Scholarship from the Fonds
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors.
́
́
de Recherche du Quebec–Sante (FRQS).
AUTHOR CONTRIBUTIONS
CM and YB designed the study. YB, MR, and EM synthesized the
compounds. LS and AF performed the biological assays. JS and JP
expressed the NTPDases and produced the enzyme-containing
membrane preparations. YB, CM, and MR wrote the manuscript
with contributions from all coauthors. All authors contributed to
the article and approved the submitted version.
ACKNOWLEDGMENTS
We thank Marion Schneider for LCMS analyses and Sabine
Terhart-Krabbe and Annette Reiner for NMR spectra.
SUPPLEMENTARY MATERIAL
FUNDING
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2020.
01282/full#supplementary-material
This study was supported by the DFG (SFB 1328, ID 335447717),
the BMBF (BIGS DrugS), and the Arab-German Young
Baykov, A. A., Evtushenko, O. A., and Avaeva, S. M. (1988). A malachite green
procedure for orthophosphate determination and its use in alkaline
phosphatase-based enzyme immunoassay. Anal. Biochem. 171, 266–270.
doi: 10.1016/0003-2697(88)90484-8
Belcher, S. M., Zsarnovszky, A., Crawford, P. A., Hemani, H., Spurling, L., and
Kirley, T. L. (2006). Immunolocalization of ecto-nucleoside triphosphate
diphosphohydrolase 3 in rat brain: implications for modulation of multiple
homeostatic systems including feeding and sleep-wake behaviors. Neuroscience
137, 1331–1346. doi: 10.1016/j.neuroscience.2005.08.086
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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August 2020 | Volume 11 | Article 1282