Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

Article abstract of DOI:10.3389/fphar.2020.01294

Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) inhibitors have potential as novel drugs for the (immuno)therapy of cancer. They increase the extracellular concentration of immunostimulatory ATP and reduce the formation of AMP, which can be further hydrolyzed by ecto-5’-nucleotidase (CD73) to immunosuppressive, cancer-promoting adenosine. In the present study, we synthesized analogs and derivatives of the standard CD39 inhibitor ARL67156, a nucleotide analog which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed at the human enzyme with respect to substituents in the N⁶- and C8-position of the adenine core, and modifications of the triphosph(on)ate chain. Capillary electrophoresis coupled to laser-induced fluorescence detection employing a fluorescent-labeled ATP derivative was employed to determine the compounds’ potency. Selected inhibitors were additionally evaluated in an orthogonal, malachite green assay versus the natural substrate ATP. The most potent CD39 inhibitors of the present series were ARL67156 and its derivatives 31 and 33 with K_i values of around 1 μM. Selectivity studies showed that all three nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking studies provided plausible binding modes to both targets. The present study provides a full characterization of the frequently applied CD39 inhibitor ARL67156, presents structure-activity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.

Full text of DOI:10.3389/fphar.2020.01294

Nucleotide Analog ARL67156 as a Lead Structure for
the Development of CD39 and Dual CD39/CD73
Ectonucleotidase Inhibitors
1
1
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1
Laura Schäkel , Constanze C. Schmies , Riham Omer , Xihuan Luo , Sang-Yong Lee ,
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Vittoria Lopez , Salahuddin Mirza , The-Hung Vu , Julie Pelletier , Jean Sevigny ,
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Vigneshwaran Namasivayam , Christa E. Müller
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3
University of Bonn, Germany, Laval University, Canada, PharmaCenter Bonn,
Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn,
Germany
Submitted to Journal:
Frontiers in Pharmacology
Specialty Section:
Experimental Pharmacology and Drug Discovery
ISSN:
1
663-9812
Article type:
Original Research Article
Received on:
0
7 Jun 2020
Accepted on:
4 Aug 2020
0
Provisional PDF published on:
4 Aug 2020
0
Frontiers website link:
www.frontiersin.org
Citation:
Schäkel L, Schmies CC, Omer R, Luo X, Lee S, Lopez V, Mirza S, Vu T, Pelletier J, Sevigny J,
Namasivayam V and Müller CE(2020) Nucleotide Analog ARL67156 as a Lead Structure for the
Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors. Front. Pharmacol. 11:1294.
doi:10.3389/fphar.2020.01294
Copyright statement:
©
2020 Schäkel, Schmies, Omer, Luo, Lee, Lopez, Mirza, Vu, Pelletier, Sevigny, Namasivayam and
Müller. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted,
provided the original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction
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Frontiers in Pharmacology | www.frontiersin.org

Nucleotide Analog ARL67156 as a Lead Structure for the Development
of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors
1
,#
1,#
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1
1
1
2
3
Laura Schäkel , Constanze C. Schmies , Riham M. Idris , Xihuan Luo , Sang-Yong Lee ,
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1
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2,3
Vittoria Lopez , Salahuddin Mirza , The Hung Vu , Julie Pelletier , Jean Sévigny
,
1
1,
Vigneshwaran Namasivayam and Christa E. Müller *
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1
5
6
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB),
Pharmaceutical & Medicinal Chemistry, University of Bonn, Germany
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Centre de Recherche du CHU de Québec – Université Laval, Québec City, QC, Canada
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Départment de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université
Laval, Quebec City, QC, Canada
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These authors contributed equally to this work
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* Correspondence:
Christa E. Müller
Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry,
An der Immenburg 4, D-53121 Bonn, Germany
E-mail: christa.mueller@uni-bonn.de
Tel: +49-228-73-2301
Fax: +49-228-73-2567
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2
1
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Keywords: ARL67156, CD39, CD73, Docking, Dual-target inhibitors, Ecto-5’-nucleotidase,
NTPDase1, Nucleotides
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1

Nucleotide inhibitors of CD39
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5
Abstract
Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) inhibitors have potential as novel
drugs for the (immuno)therapy of cancer. They increase the extracellular concentration of
immunostimulatory ATP and reduce the formation of AMP, which can be further hydrolyzed by ecto-
5’-nucleotidase (CD73) to immunosuppressive, cancer-promoting adenosine. In the present study, we
synthesized analogs and derivatives of the standard CD39 inhibitor ARL67156, a nucleotide analog
which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed
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2
2
2
3
3
3
3
3
3
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3
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4
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0
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5
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1
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with respect to substituents in the N - and C8-position of the adenine core, and modifications of the
triphosph(on)ate chain at the human enzyme. Capillary electrophoresis coupled to laser-induced
fluorescence detection employing a fluorescent-labeled ATP derivative was employed to determine the
compounds’ potency. Selected inhibitors were additionally evaluated in an orthogonal, malachite green
assay versus the natural substrate ATP. The most potent CD39 inhibitors of the present series were
i
ARL67156 and its derivatives 31 and 33 with K values of around 1 µM. Selectivity studies showed
that all three nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking
studies provided plausible binding modes to both targets. The present study provides a full
characterization of the frequently applied CD39 inhibitor ARL67156, presents structure-activity
relationships, and provides a basis for future optimization towards selective CD39 and dual
CD39/CD73 inhibitors.
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2
3
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1
Introduction
Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39, EC 3.6.1.5) catalyzes the hydrolysis
of extracellular nucleoside tri- and diphosphates producing the corresponding monophosphates
(Zimmermann et al., 2012). CD39 is membrane-bound and often co-localized with ecto-5’-
nucleotidase (CD73), another ectonucleotidase that further hydrolyzes the nucleoside monophosphates
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5
6
7
2

Nucleotide Inhibitors of CD39
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4
5
8
9
0
to the corresponding nucleosides (Flögel et al., 2012; Augusto et al., 2013; Bastid et al., 2015). The
main substrate of CD39 is ATP which is cleaved via ADP to AMP, while AMP acts as the main
substrate of CD73 which catalyzes its hydrolysis to adenosine (see Figure 1).
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5
5
5
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6
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2
3
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Many tumor cells overexpress ectonucleotidases (De Marchi et al., 2019; Horenstein et al., 2019),
which metabolize proinflammatory ATP to immunosuppressive, angiogenic, pro-metastatic, and tumor
growth-promoting adenosine (Vitiello et al., 2012). Inhibition of CD39 could reduce the production of
cancer-promoting adenosine, e.g. in the tumor microenvironment, and increase the concentration of
immuno-stimulatory ATP. Due to its pathophysiological role, CD39 represents a promising potential
drug target that requires, however, further validation. For this purpose, potent, selective and
metabolically stable inhibitors need to be identified. Besides selective CD39 inhibitors, dual inhibition
of CD39 and CD73 is of interest and may be synergistic since the substrate of CD73, extracellular
AMP, may additionally be formed by alternative ectonucleotidases, such as nucleotide
pyrophosphatase/phosphodiesterase1 (NPP1) (Lee et al., 2017a).
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6
6
6
1
2
3
4
5
6
7
Up to now, only moderately potent and/or non-selective CD39 inhibitors are available. These can be
6
divided into (i) nucleotide derivatives and analogs, e.g. N -diethyl-β,γ-dibromomethylene-ATP
(ARL67156, I) and 8-butylthio-AMP (8-BuS-AMP, II), and (ii) non-nucleotides, including the
sulfonate dyes, reactive blue 2 (RB-2) and related anthraquinone derivatives (e.g. III),
polyoxometalates (e.g. PSB-POM-142, IV), and tryptamine-derived imines (e.g. V) (Crack et al., 1995;
Müller et al., 2006; Lévesque et al., 2007; Baqi et al., 2009; Lecka et al., 2013; Lee et al., 2015; Kanwal
et al., 2019). A selection of the most potent CD39 inhibitors described so far is depicted in Figure 2.
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7
8
9
0
The nucleotide-based competitive CD39 inhibitor N ,N -diethyl-β,γ-dibromomethylene-ATP
(ARL67156) was developed by Fisons Laboratories (now AstraZeneca, Loughborough, UK) as a probe
to study ecto-nucleotidases and purinoceptors (Crack et al., 1995). The nucleotide analog was proposed
3

Nucleotide inhibitors of CD39
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1
2
3
to be relatively stable towards hydrolysis by ectonucleotidases (CD39; NTPDase2,-3,-8; CD73; NPP1;
NPP3) because the cleavage site is blocked by replacement of the β,γ-oxygen atom of the ATP
triphosphate chain by a dibromomethylene moiety yielding a phosphonate linkage (Lévesque et al.,
2007). ARL67156 (I) was shown to competitively inhibit the mouse and human forms of CD39
i i i
(K (human) 11 μM), NTPDase3 (K (human) 18 μM) and NPP1 (K (human) 12 μM), but was reported
to have a weaker effect on NTPDase2, NTPDase8, NPP3 and CD73 (Lévesque et al., 2007).
Furthermore, in contrast to other NTPDase inhibitors, ARL67156 had no significant effect on P2
receptors due to di-substitution of the exocyclic amino group (Robson et al., 2006). ARL67156 is
currently the only commercially available CD39 inhibitor, claimed to be metabolically stable and
CD39-selective, and it is therefore frequently used for in vitro as well as in vivo studies despite its
moderate potency (Mandapathil et al., 2010; Zhou et al., 2014; Li et al., 2015). Metabolic stability of
ARL67156 has not been sufficiently studied to date, and structure-activity relationships (SARs) are
largely unknown.
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4
5
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In this study, we characterized the CD39 inhibitor ARL67156 (I) and used it as a lead structure for
studying the SARs of ATP analogs and derivatives as inhibitors of CD39 and other ecto-nucleotidases.
6
Derivatization in the N - and 8- position of the adenine ring, as well as replacement of the di-
bromomethylene bridge were performed. Selectivity versus a broad range of ecto-nucleotidases and
metabolic stability were determined for ARL67156 and selected potent inhibitors. Finally, we
performed docking studies to facilitate future drug design efforts.
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9
0
1
2
Materials and Methods
2.1 Syntheses
9
9
2
3
2.1.1 Materials and Instruments
All reagents were commercially obtained from various producers (Acros, Fluorochem, Merck,
9
4
Carbosynth, Santa Cruz, Sigma Aldrich, and TCI) and used without further purification, unless
4

Nucleotide Inhibitors of CD39
9
9
9
9
9
5
6
7
8
9
otherwise stated. Commercial solvents of reagent grade were used without additional purification or
drying. 8-Bromoadenosine was synthesized according to a published procedure (Bhattarai et al., 2015).
Reactions were monitored by thin layer chromatography (TLC) using Merck silica gel 60 F254
aluminum sheets and dichloromethane (DCM)/methanol (9:1 or 3:1) as the mobile phase. The TLC
plates were analyzed by ultraviolet (UV) light at a wavelength (λ) of 254 nm. Column chromatography
was carried out with silica gel 0.040-0.060 mm, pore diameter ca. 6 nm. Anion exchange
chromatography was performed on a fast protein liquid chromatography (FPLC) instrument (ÄKTA
FPLC, from Amersham Biosciences) with a HiPrep Q Fast Flow sepharose column, 16 x 100mm (GE
Healthcare Life Sciences). Elution of the nucleoside triphosphate analogs was achieved with a linear
gradient (5-100%, 0:5 m aqueous ammonium bicarbonate buffer in water, 8 column volumes, flow 1
ml/min). The neutral impurities (e.g. nucleosides) eluted first, followed by charged species (mono-,
and finally triphosphate analogs). Semi-preparative high performance liquid chromatography (HPLC)
was performed on a Knauer Smartline 1050 HPLC system equipped with a Eurospher-100 C18 column,
250 x 20 mm, particle size 10 μm. The UV absorption was detected at 254 nm. Fractions were collected,
and appropriate fractions were pooled, diluted with water, and lyophilized several times, using a
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
4 3
CHRIST ALPHA 1-4 LSC freeze dryer, to remove the NH HCO buffer, yielding the nucleotides as
white powders. Mass spectra were recorded on an API 2000 mass spectrometer (Applied Biosystems,
Darmstadt, Germany) with a turbo ion spray ion source coupled with an Agilent 1100 HPLC system
(Agilent, Böblingen, Germany) using an EC50/2 Nucleodur C18 Gravity 3 μm column (Macherey-
Nagel, Düren, Germany), or on a micrOTOF-Q mass spectrometer (Bruker, Köln, Germany) with an
ESI-source coupled with a HPLC Dionex Ultimate 3000 (Thermo Scientific, Braunschweig, Germany)
using an EC50/2 Nucleodur C18 Gravity 3 μm column (Macherey-Nagel, Düren, Germany). All
compounds containing Br atoms (14-16 and 24-38) showed the expected typical isotope distribution
pattern (see Figure S6 and Figure S7). UV absorption was detected from 220 to 400 nm using a diode
array detector (DAD). Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance
5

Nucleotide inhibitors of CD39
1
1
1
1
1
1
1
1
1
20
21
22
23
24
25
26
27
28
500 and Ascend 600 MHz spectrometers. DMSO-d
6
, CD
3
OD, or D
2
O were used as solvents. 31P-NMR
spectra were recorded at 25°C, and phosphoric acid was used as an external standard. For spectra
recorded in D O, 3-(trimethylsilyl)propionic acid sodium salt-d was used as an external standard.
When DMSO-d was used, spectra were recorded at 30°C. Shifts are given in ppm relative to the
2
4
6
external standard (in 31P-NMR) or relative to the remaining protons of the deuterated solvent used as
internal standard ( H, 13C-NMR). Coupling constants are given in Hertz (Hz). The designation used to
assign the peaks in the spectra is as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), broad (br). Melting points were determined on a Büchi 530 melting point apparatus and are
uncorrected.
1
1
1
29
30
2.1.2 Synthetic procedures
General procedure for the synthesis of compounds 2-7
1
1
1
1
1
31
32
33
34
35
To 6-chloro-9-(β-D-ribofuranosyl)purine (1, 0.5 g, 1.7 mmol, 1.0 eq) in absolute ethanol (15 ml) the
appropriate alkylamine and Et N (0.1 ml, 1.6 mmol, 0.9 eq) were added. The reaction mixture was
3
refluxed for 6-36 h followed by evaporation of the solvent. Yields for intermediate products 3-6 were
estimated to be above 70%; however exact yields were not determined because they were used without
drying and desalting for the subsequent step; only a small amount was purified for analytical purposes.
1
1
36
37
(2R,3R,4S,5R)-2-(6-(Diethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (2)
1
1
1
1
1
1
1
1
1
38
39
40
41
42
43
44
45
46
The compound was synthesized using N,N-diethylamine (0.3 ml, 3.4 mmol, 2.0 eq) and purified by
1
silica gel column chromatography (CH
NMR (500 MHz, DMSO-d
CHN) 5.39 (d, 1H, J = 6.19 Hz, CHOH) 5.33 (dd, 1H, J = 4.59, 7.02 Hz, CH
Hz, CHOH) 4.58 (q 1H, J =6.04 Hz, CHOH) 4.14 (td, 1H, J =3.36, 4.82 Hz, CHOH) 4.03 (br s, 4H,
3
OH/DCM 2:23) yielding a white powder (0.50 g, 100%). H-
6
) δ 8.34 (s, 1H, NCH=N) 8.19 (s, 1H, NCH=N) 5.89 (d, 1H, J = 6.04 Hz,
2
OH) 5.13 (d, 1H, J = 4.61
N(CH
2
CH
3
)
)
2
) 3.95 (q, 1H, J = 3.54 Hz, CHCH
). 13C-NMR (125 MHz, DMSO-d
6
2
) 3.66-3.54 (d m, 2H, CHCH
2
) 1.19 (t, 6H, J = 6.95 Hz,
N(CH
2
CH
3
2
) δ 153.27, 151.95, 150.06, 138.96, 119.47,87.94,
+
85.91, 73.57, 70.70, 61.73, 42.56, 13.48. LC/ESI-MS (m/z): positive mode 324.1 [M+H] . Purity
determined by HPLC-UV (254 nm)-ESI-MS: 99.2%. mp: 180°C.
6

Nucleotide Inhibitors of CD39
1
47
(2R,3R,4S,5R)-2-(6-(Dimethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (3)
1
1
1
1
1
1
1
1
1
48
49
50
51
52
53
54
55
56
The compound was synthesized using N,.N-dimethylamine (0.1 ml, 1.75 mmol, 1.0 eq) and purified
1
by silica gel column chromatography (CH
(500 MHz, DMSO-d
5.39 (d, 1H, J = 6.17 Hz, CHOH) 5.32 (dd, 1H, J = 4.62, 6.95 Hz, CH
CHOH) 4.56 (q, 1H, J = 5.99 Hz, CHOH) 4.14 (m, 1H, CHCH ) 3.95 (q, 1H, J = 3.55 Hz, CHOH)
3.66-3.55 (d m, 2H, CHCH ) 3.45 (br s, 6H, N(CH ) δ 154.46,
). 13C-NMR (125 MHz, DMSO-d
3
OH/DCM 1:49) yielding a white powder (0.52 g). H-NMR
6
) δ 8.35 (s, 1H, N=CHN) 8.20 (s, 1H, N=CHN) 5.90 (d, 1H, J = 5.97 Hz, CHN)
2
OH) 5.13 (d, 1H, J = 4.78 Hz,
2
2
)
3 2
6
151.82, 150.05, 138.69, 119.94, 87.94, 85.88, 73.64, 70.65, 61.68, 11.57. LC/ESI-MS (m/z): positive
+
mode 296.0 [M+H] . Purity determined by HPLC-UV (254 nm)-ESIMS: 98%. mp: 186°C (lit. 184°C)
(Čechová et al., 2011).
1
1
57
58
(2R,3R,4S,5R)-2-(6-(Ethyl(methyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol
(4)
1
1
1
1
1
1
1
1
59
60
61
62
63
64
65
66
The compound was synthesized using N-ethylmethylamine (0.2 ml, 1.75 mmol, 1.0 eq) yielding a white
1
powder (0.93 g). H-NMR (500 MHz, DMSO-d
6
) δ 8.35 (s, 1H, N=CHN) 8.20 (s, 1H, N=CHN) 5.90
OH)
) 4.04 (br s,
) 3.39 (br s, 3H, NCH ) 1.17
) δ 153.82, 151.89, 150.02, 138.82, 119.69,
(d, 1H, J = 6.00 Hz, CHN) 5.39 (d, 1H, J = 6.19 Hz, CHOH) 5.32 (dd, 1H, J = 4.61, 6.96 Hz, CH
5.13 (d, 1H, J = 4.76 Hz, CHOH) 4.57 (q, 1H, J =5.99 Hz, CHOH) 4.14 (m, 1H, CHCH
2H, NCH ) 3.95 (q, 1H, J = 3.51 Hz, CHOH) 3.66-3.54 (d m, 2H, CHCH
(t, 3H, J = 7.00 Hz, CH
). 13C-NMR (125 MHz, DMSO-d
2
2
2
2
3
3
6
87.91, 85.88, 73.59, 70.66, 61.69, 44.78, 35.47, 12.56. LC/ESI-MS (m/z): positive mode 310.0
+
[M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 98.0%. mp: 101°C.
1
1
67
68
(2R,3S,4R,5R)-2-(Hydroxymethyl)-5-(6-(methyl(propyl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-
diol (5)
1
1
1
1
1
1
1
1
1
69
70
71
72
73
74
75
76
77
The compound was synthesized using N-methylpropylamine (0.18 ml, 1.75 mmol, 1.0 eq) and purified
1
by silica gel column chromatography (CH
(500 MHz, DMSO-d
5.41 (d, 1H, J = 6.16 Hz, CHOH) 5.33 (m, 1H, CH
J = 5.76 Hz, CHOH) 4.14 (d, 1H, J =3.62 Hz, CHOH) 3.95 (d, 1H, J = 3.13 Hz, CHCH
m, 2H, CHCH ) 3.16 (br s, 2H, NCH ) [bulb underneath previous peaks: NCH )] 1.64 (q, 2H, J = 7.30
Hz, CH ) 0.87 (t, 3H, J = 7.34 Hz, CH ) δ 154.16, 151.88, 150.10,
). 13C-NMR (125 MHz, DMSO-d
3
OH/DCM 1:9) yielding a white powder (0.66 g). H-NMR
6
) δ 8.35 (s, 1H, N=CHN) 8.19 (s, 1H, N=CHN) 5.89 (d, 1H, J = 5.97 Hz, CHN)
OH) 5.14 (d, 1H, J = 4.64 Hz, CHOH) 4.57 (q, 1H,
) 3.66-3.54 (d
2
2
2
2
3
2
3
6
138.79, 119.71, 87.92, 85.92, 73.62, 70.71, 61.74, 51.32, 48.75, 21.58, 11.06. LC/ESI-MS (m/z):
+
positive mode 324.1 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 97.7%. mp: 178°C.
7

Nucleotide inhibitors of CD39
1
78
(2R,3R,4S,5R)-2-(6-(Dipropylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (6)
1
1
1
1
1
1
1
1
1
79
80
81
82
83
84
85
86
87
The compound was synthesized using N,N-dipropylamine (0.25 ml, 1.75 mmol, 1.0 eq) and purified
1
by silica gel column chromatography (CH
(500 MHz, DMSO-d
5.40 (d, 1H, J = 5.91 Hz, CHOH) 5.33 (dd, 1H, J = 4.63, 6.97 Hz, CH
CHOH) 4.58 (q, 1H, J = 5.66 Hz, CHOH) 4.13 (q, 1H, J = 4.53 Hz, CHOH) 4.06 (m, 4H, N(CH
3.95 (q, 1H, J = 3.50 Hz, CHCH ) 3.65-3.54 (d m, 2H, CHCH ) 1.64 (m, 4H, (CH ) 0.89 (t, 6H, J =
7.37 Hz, (CH ) δ 153.80, 151.88, 150.10, 138.89, 119.50, 87.92,
). 13C-NMR (125 MHz, DMSO-d
3
OH/DCM 1:19) yielding a white powder (0.65 g). H-NMR
6
) δ 8.35 (s, 1H, N=CHN) 8.18 (br s, 1H, N=CHN) 5.89 (d, 1H, J = 6.05 Hz, CHN)
2
OH) 5.14 (d, 1H, J = 4.60 Hz,
2 2
) )
2
2
2 2
)
)
3 2
6
85.92, 73.56, 70.73, 61.92, 56.17, 48.74, 18.70, 11.18. LC/ESI-MS (m/z): positive mode 352.1
+
[M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 98.3%. mp: 145°C.
1
1
88
89
(2R,3R,4S,5R)-2-(6-(Ethyl(propyl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol
(7)
1
1
1
1
1
1
1
1
1
90
91
92
93
94
95
96
97
98
The compound was synthesized using N-ethylpropylamine (0.2 ml, 1.75 mmol, 1.0 eq) and purified by
1
silica gel column chromatography (CH
NMR (500 MHz, CD
4.74 (dd, 1H, J = 5.15, 6.48 Hz, CHOH) 4.30 (dd, 1H, J = 2.45, 5.09 Hz, CHCH
Hz, CHOH) 3.88-3.72 (d m, 2H, CHCH ) overlapping with 4.10-3.72 (br s, 4H, 2x NCH
2H, CH CH CH ) 1.25 (t, 3H, J = 7.04 Hz, CH CH ) 0.95 (t, 3H, J = 7.39 Hz, (CH CH
). 13C-NMR
(151 MHz, CD
3
OH/DCM 1:9) yielding a white powder (0.38 g, 65%). H-
3
OD) δ 8.15 (d, 2H, J = 2.01 Hz, 2x N=CHN) 5.93 (d, 1H, J = 6.55 Hz, CHN)
) 4.16 (q, 1H, J =2.40
) 1.73 (m,
2
2
2
2
2
3
2
3
2
)
2
3
3
OD) δ 155.40, 152.72, 150.70, 140.17, 121.60, 91.21, 88.17, 75.17, 72.77, 63.58,
+
51.25, 44.72, 22.52, 13.90, 11.36. LC/ESI-MS (m/z): positive mode 310.0 [M+H] . Purity determined
by HPLC-UV (254 nm)-ESI-MS: 97.2%. mp: 160°C.
1
99
(2R,3R,4S,5R)-2-(6-(benzylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (8)
2
2
2
2
2
2
2
2
00
01
02
03
04
05
06
07
The compound was synthesized according to a published procedure (Shimazaki et al., 1987) and
3
purified by silica gel column chromatography (CH OH/DCM 1:9) yielding a white powder (3.45 g,
1
96%). H-NMR (500 MHz, DMSO-d
6
) δ: 8.36 (s, 1H, H-8), 8.19 (s, 1H, H-2), 7.33 - 7.17 (m, 5H,
H
arom.), 5.88 (d, J = 6.1 Hz, 1H, H-1’), 5.39 (d, J = 6.2 Hz, 1H, OH-2’), 5.33 (dd, J = 7.1, 4.6 Hz, 1H,
OH-5’), 5.13 (d, J = 4.7 Hz, 1H, OH-3’), 4.71 (s (br), 2H, N-CH ), 4.61 (dd, J = 11.3, 6.0 Hz, 1H, H-
2’), 4.14 (dd, J = 8.2, 4.8 Hz, 1H, H-3’), 3.96 (dd, J = 3.5 Hz, 1H, H-4’), 3.68 – 3.64 (m, 1H, H-5’a),
3.57 – 3.52 (m, 1H, H-5’b), (1H, NH not visible). 13C-NMR (125 MHz, DMSO-d
) δ: 154.7 (C-6,
quat.), 152.5 (C-2, CH), 148.6 (C-4, Cquat.), 140.1 (Carom., Cquat.), 140.0 (C-8, CH), 128.3 (2 x Carom.
2
6
C
,
8

Nucleotide Inhibitors of CD39
2
2
2
2
08
09
10
11
CH), 127.2 (2 x Carom., CH), 126.7 (Carom., CH), 119.9 (C-5, Cquat.), 88.1 (C-1’, CH), 86.0 (C-4’, CH),
2 2
73.6 (C-2’, CH), 70.8 (C-3’, CH), 61.8 (C-5’, CH ), 43.0 (Cbenzyl, CH ). LC-ESI-MS (m/z): positive
+
mode 358 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 98%. mp: 178 - 180 °C. (Lit.
184 – 186 °C)(Shimazaki et al., 1987)
2
2
12
13
Synthesis of (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-phenethylamino)-9H-purin-9-yl)tetrahydrofuran-
3,4-diol (9)
2
2
2
2
2
2
2
2
2
2
2
2
14
15
16
17
18
19
20
21
22
23
24
25
The compound was synthesized according to a published procedure (Shimazaki et al., 1987) and
3
purified by silica gel column chromatography (CH OH/DCM 1:9) yielding a white powder (3.21 g,
1
86%). H-NMR (500 MHz, DMSO-d
6
) δ: 8.33 (s, 1H, H-8), 8.23 (s, 1H, H-2), 7.87 (s (br), 1H, NH),
7.29 – 7.16 (m, 5H, Harom.), 5.88 (d, J = 6.1 Hz, 1H, H-1’), 5.40 (d, J = 6.2 Hz, 1H, OH-2’), 5.36 (dd,
J = 7.2, 4.5 Hz, 1H, OH-5’), 5.14 (d, J = 4.6 Hz, 1H, OH-3’), 4.61 (dd, J = 6.2, 4.9 Hz, 1H, H-2’), 4.15
(dd, J = 4.8, 3.0 Hz, 1H, H-3’), 3.96 (dd, J = 3.5 Hz, 1H, H-4’), 3.71 (s (br), 2H, N-CH
(m, 1H, H-5’a), 3.57 – 3.53 (m, 1H, H-5’b), 2.92 (t, J = 9.0 Hz, 2H, CH
-Ph). 13C-NMR (125 MHz,
DMSO-d ) δ: 154.7 (C-6, Cquat.), 152.5 (C-2, CH), 148.5 (C-4, Cquat.), 139.9 (C-8, CH), 139.6 (Carom.
quat.), 128.8 (2 x Carom., CH), 128.4 (2 x Carom., CH), 126.2 (Carom., CH), 119.9 (C-5, Cquat.), 88.1 (C-
2
), 3.69 – 3.65
2
6
,
C
2 2
1’, CH), 86.0 (C-4’, CH), 73.6 (C-2’, CH), 70.8 (C-3’, CH), 61.8 (C-5’, CH ), 41.4 (N-CH ), 35.1
+
(CH
2
-Ph). LC-ESI-MS (m/z): positive mode 372 [M+H] . Purity determined by HPLC-UV (254 nm)-
ESI-MS: 96%. mp: 183-185 °C. (Lit. 166 – 168 °C) (Shimazaki et al., 1987)
2
2
26
27
Synthesis of (2R,3R,4S,5R)-2-(6-aAmino-8-(butylthio)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydro-
furan-3,4-diol (12)
2
2
2
2
2
2
2
2
2
2
2
28
29
30
31
32
33
34
35
36
37
38
To a solution of 8-bromoadenosine (10, 0.5 g, 1.44 mmol, 1.0 eq) in absolute ethanol, thiourea (0.2 g,
2.63 mmol, 1.8 eq) was added. After 7 h of refluxing the solution was allowed to cool down and the
resulting precipitate was filtered off. The remaining filtrate was evaporated yielding a yellow oil that
2
was resuspended in a mixture of H O/EtOH 1:1. The solution was adjusted to basic pH with 2 M
NaOH. Butyliodide (0.5 mL, 4.32 mmol, 3.0 eq) was added and the reaction was stirred at rt for 2h.
After extraction with ethylacetate (3 x 100 mL), the organic phase was evaporated. Purification by
1
column chromatography (8% MeOH in DCM) afforded the product as a white solid (0.39 g, 76 %) H-
NMR (500 MHz, DMSO-d
5.59 (dd, 1H, J = 3.47, 8.81 Hz, CHOH) 5.36 (d, 1H, J = 6.14 Hz, CHOH) 5.14 (d, 1H, J = 4.54 Hz,
CH OH) 4.98 (dd, 1H, J = 6.14, 11.88 Hz, CHCH ) 4.16 (m, 1H, CHOH) 3.96 (m, 1H, CHOH) 3.68-
3.50 (d m, 2H, CHCH ) 3.32-3.27 (d m, 2H overlapping with H
6 2
) δ 8.04 (s, 1H, NCH=N) 7.23 (s, 2H, NH ) 5.77 (d, 1H, J = 7.21 Hz, CHN)
2
2
2
2 2 2
O peak, SCH ) 1.68 (m, 2H, CH ) 1.41
9

Nucleotide inhibitors of CD39
). 13C-NMR (126 MHz, DMSO-d
) δ 184.05, 154.67,
2
2
2
2
39
40
41
42
(m, 2H, CH
2
) 0.90 (t, 3H, J = 7.27 Hz, CH
2
CH
3
6
151.39, 150.56, 148.83, 119.74, 89.01, 86.72, 71.40, 71.12, 62.36, 32.22, 31.03, 21.32, 13.56. LC/ESI-
+
MS (m/z): positive mode 356.2 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 99.0%.
mp: 105°C (lit. 171.5°C) (Halbfinger et al., 1999).
2
2
43
44
Synthesis of (2R,3S,4R,5R)-2-(hHydroxymethyl)-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-
3,4-diol (13)
2
2
2
2
2
2
2
2
2
2
45
46
47
48
49
50
51
52
53
54
To 6-chloro-9-(β-D-ribofuranosyl)purine (1, 2.0 g, 7.0 mmol) in absolute ethanol (40 mL), 33 wt %
3
methylamine in absolute ethanol (0.9 mL, 21 mmol, 3 eq) and Et N (2 mL, 14 mmol, 2 eq) were added.
After 4 h of refluxing, the solvent was evaporated. Column chromatography (CH
3
OH/DCM 1:9)
yielded the product as a white powder (2.0 g, 100 %). 1H-NMR (500 MHz, DMSO-d
6
) δ 8.32 (s, 1H,
NCH=N) 8.21 (br s, 1H, NCH=N) 7.77 (br s, 1H, NHCH
1H, CHOH) 5.14 (br s, 1H, CHOH) 4.59 (t, 1H, J =5.33 Hz, CHOH) 4.14 (dd, 1H, J =3.21, 4.75 Hz,
CHOH) 3.95 (q, 1H, J =3.51 Hz, CHCH ) 3.66-3.54 (d m, 2H, CHCH ) 3.05 (m, 3H, NHCH
). 13C-
3
) 5.87 (d, 1H, J =6.17 Hz, CHN) 5.40 (br s,
2
2
3
NMR (125 MHz, DMSO-d
6
) δ 156.52, 152.46, 148.22, 139.74, 119.98, 88.05, 86.02, 73.65, 70.77,
+
61.79, 24.44. LC/ESI-MS (m/z): positive mode 282.3 [M+H] . Purity determined by HPLC-UV (254
nm)-ESI-MS: 99.3%. mp: 132°C (lit. 130-132°C) (Čechová et al., 2011).
2
55
General procedure for the synthesis of 14-16
6
2
2
2
2
2
56
57
58
59
60
To a solution of N -substituted adenosine (2, 3, or 13, 1.0 eq) in 0.1 M sodium acetate buffer pH 4.0
(15 ml) bromine (5.0 eq) was added. The reaction was stirred at rt overnight and monitored by TLC.
3
The solution was decolorized by the addition of a 40% solution of NaHSO , and the pH of the solution
was then adjusted to 7 with concentrated 4-N aq. NaOH. The precipitate was filtered off and washed
with water.
2
2
61
62
(2R,3R,4S,5R)-2-(8-Bromo-6-(methylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-
diol (14)
2
2
2
2
2
2
63
64
65
66
67
68
The compound was synthesized starting from 13 (1.96 g, 7.0 mmol, 1.0 eq) and afforded a white solid
1
(0.60 g, 25%). H-NMR (500 MHz, DMSO-d
6
) δ 8.20 (s, 1H, NCH=N) 8.02 (s, 1H, NH) 5.84 (d, 1H,
J =7.08 Hz, CHN) 5.45 (q, 1H, J = 4.07 Hz, CHOH) 5.41 (d, 1H, J = 6.77 Hz, CHOH) 5.19 (d, 1H, J
= 4.60 Hz, CH OH) 5.07 (dd, 1H, J = 6.55, 11.33 Hz, CHCH ) 4.20 (m, 1H, CHOH) 3.97 (dd, 1H, J =
4.07, 5.66 Hz, CHOH) 3.69-3.49 (d m, 2H, CHCH ) 2.94 (s, 3H, NHCH
). 13C-NMR (125 MHz,
DMSO-d ) δ 154.12, 152.58, 149.04, 126.87, 120.40, 90.57, 86.84, 71.34, 70.99, 62.24, 27.10. LC/ESI-
2
2
2
3
6
1
0

Nucleotide Inhibitors of CD39
+
2
2
69
70
MS (m/z): positive mode 346.1 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 95.6%.
mp: 228°C.
2
2
71
72
(2R,3R,4S,5R)-2-(8-Bromo-6-(dimethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-
3,4-diol (15)
2
2
2
2
2
2
2
73
74
75
76
77
78
79
The compound was synthesized starting from 3 (2.0 g, 7.0 mmol, 1.0 eq) and afforded a white solid
1
(0.60 g, 21%). H-NMR (500 MHz, DMSO-d
6
) δ 8.18 (s, 1H, NCH=N) 5.84 (d, 1H, J = 6.47 Hz, CHN)
OH) 5.08 (dd, 1H, J = 6.48,
) 4.21 (m, 1H, CHOH) 3.97 (m, 1H, CHOH) 3.70-3.49 (d m, 2H, CHCH ) 3.41 (br
). 13C-NMR (125 MHz, DMSO-d
) δ 153.29, 151.72, 150.88, 126.06, 120.37, 90.68,
5.41 (overlapping q and d, 2H, 2x CHOH) 5.19 (d, 1H, J = 4.68 Hz, CH
11.80 Hz, CHCH
s, 6H, N(CH
2
2
2
3
)
2
6
+
86.80, 71.12, 70.96, 62.25, 56.16, 18.68. LC/ESI-MS (m/z): positive mode 374.2 [M+H] . Purity
determined by HPLC-UV (254 nm)-ESI-MS: 96.6%. mp: 152°C.
2
2
80
81
(2R,3R,4S,5R)-2-(8-Bromo-6-(diethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-
diol (16)
2
2
2
2
2
2
2
2
82
83
84
85
86
87
88
89
The compound was synthesized starting from 2 (1.919 g, 5.9 mmol, 1.0 eq) and afforded a white solid
1
(0.52 g, 23%). H-NMR (500 MHz, DMSO-d
6
) δ 8.17 (s, 1H, N=CHN) 5.84 (d, 1H, J = 6.75 Hz, CHN)
5.45 (dd, 1H, J = 3.87, 8.57 Hz, CHOH) 5.42 (d, 1H, J =5.89 Hz, CHOH) 5.20 (d, 1H, J = 4.40 Hz,
CH OH) 5.09 (q, 1H, J =5.92 Hz, CHCH ) 4.19 (td, 1H, J =2.45, 4.76 Hz, CHOH) 3.97 (td, 1H, J
=2.97, 4.04 Hz, CHOH) 4.19-3.7 (br s, 4H, overlapping with previous peaks N(CH CH ) 3.67-3.51
(d m, 2H, CHCH ) 1.18 (t, 6H, J = 6.89 Hz, N(CH CH
). 13C-NMR (125 MHz, DMSO-d6) δ 152.14,
2
2
2
3 2
)
2
2
3 2
)
151.88, 150.94, 126.35, 119.92, 90.70, 86.85, 71.08, 62.29, 56.19, 42.87, 18.70, 13.65. LC/ESI-MS
+
(m/z): positive mode 402.0 [M+H] . Purity determined by HPLCU-V (254 nm)-ESI-MS: 97.6%.
2
90
General procedure for the synthesis of compounds 17-20
6
2
2
2
91
92
93
To the 8-bromo-N -substituted adenosine derivatives 14-16 in absolute ethanol (15 ml) the
3
corresponding alkylamine and Et N (0.1 ml, 1.6 mmol, 0.9 eq) were added. The reaction mixture was
refluxed for 6-36 h followed by evaporation of the solvent.
2
2
94
95
(2R,3R,4S,5R)-2-(8-(Cyclopropylamino)-6-(methylamino)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetrahydrofuran-3,4-diol (17)
2
2
2
96
97
98
The compound was synthesized starting from 14 (0.5 g, 1.4 mmol, 1.0 eq), using cyclopropylamine
3
(0.3 ml, 4.2 mmol, 3.0 eq). Purification by column chromatography (CH OH/DCM 1:49) afforded the
1
desired product as a yellow waxy residue (0.18 g, 37%). H-NMR (500 MHz, DMSO-d
6
) δ 7.98 (s,
1
1

Nucleotide inhibitors of CD39
2
3
3
3
3
3
3
99
00
01
02
03
04
05
1H, N=CHN) 7.05 (d, 1H, J = 2.63 Hz, NHCH
3
) 6.86 (q, 1H, J = 4.66 Hz, NHCH) 5.87 (d, 1H, J =
7.29 Hz, CHN) 5.82 (dd, 1H, J = 4.35, 6.07 Hz, NHCH) 5.15 (d, 1H, J = 6.68 Hz, CHOH) 5.08 (d, 1H,
J = 4.35 Hz, CHOH) 4.58 (q, 1H, J = 6.98, 12.55 Hz, CH OH) 4.32 (t, 1H, J = 4.96 Hz, CHCH ) 4.09
(m. 1H, CHOH) 3.94 (q, 1H, J =2.52 Hz, CHOH) 3.61 (m, 2H, CHCH ) 2.93 (d, 3H, J = 4.66 Hz,
NHCH ) 0.66 (m, 2H, CH ) 0.45 (m, 2H, CH ) δ 152.26, 151.58,
). 13C-NMR (125 MHz, DMSO-d
2
2
2
3
2
2
6
148.87, 137.05, 117.62, 86.49, 85.75, 71.03, 70.84, 61.75, 25.01, 18.67, 6.83, 6.19. LC-MS (m/z):
+
positive mode 337.1 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 89.4 %. mp: 219°C.
3
3
06
07
(2R,3R,4S,5R)-2-(8-(Butylamino)-6-(methylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetra-
hydrofuran-3,4-diol (18)
3
3
3
3
3
3
3
3
3
3
3
08
09
10
11
12
13
14
15
16
17
18
The compound was synthesized starting from 14 (0.4 g, 1.1 mmol, 1.0 eq) using N-butylamine (0.3 ml,
4.2 mmol, 3.0 eq). Purification by column chromatography (CH
3
OH/DCM 1:9) afforded the desired
) δ 7.95 (s, 1H,
) 5.89 (d, 1H, J = 7.69
OH) 5.19 (br s, 1H, CHOH) 5.11 (br s, 1H, CHOH) 4.62 (br s, 1H,
) 4.11 (br s, 1H, CHOH) 3.95 (br d, 1H, J = 1.98 Hz, CHOH) 3.62 (br s, 2H, CHCH ) 3.36 (m
overlapping with H O, 2H, NHCH ) 2.92 (d, 3H, J = 4.78 Hz, NHCH ) 1.56 (m, 2H, CH ) 1.33 (m,
2H, CH ) 0.89 (t, 3H, J = 7.38 Hz, CH CH ) δ 152.01, 151.35,
). 13C-NMR (125 MHz, DMSO-d
1
product as a slightly yellow solid (0.36 g, 93%). H-NMR (500 MHz, DMSO-d
6
N=CHN) 6.83 (t, 1H, J = 5.51 Hz, NHCH
Hz, CHN) 5.84 (br s, 1H, CH
CHCH
2 3
) 6.77 (q, 1H, J = 4.74 Hz, NHCH
2
2
2
2
2
3
2
2
2
3
6
148.86, 148.59, 117.62, 86.45, 85.78, 71.09, 70.87, 61.79, 42.17, 31.00, 29.44, 27.44, 19.78, 13.19.
+
LC/ESI-MS (m/z): positive mode 353.0 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS:
91.4%. mp: 202°C.
3
3
19
20
(2R,3R,4S,5R)-2-(8-(Butylamino)-6-(dimethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetra-
hydrofuran-3,4-diol (19)
3
3
3
3
3
3
3
3
3
21
22
23
24
25
26
27
28
29
The compound was synthesized starting from 15 (0.5 g, 1.3 mmol, 1.0 eg) using butylamine (0.4 ml,
4.3 mmol, 3.2 eq). Purification by column chromatography (CH
3
OH/DCM 1:24) afforded the desired
OD) δ 8.00 (s, 1H, NCH=N)
) 4.32 (dd, 1H, J = 1.80, 5.60
Hz, CHOH) 4.16 (br d, 1H, J =1.80 Hz, CHOH) 3.88-3.81 (m, 2H, CHCH ) 3.47 (s, 6H, N(CH ) 2.97
(t, 2H, J = 7.47 Hz, NHCH ) 1.71 (m, 2H, CH ) 1.46 (m, 2H, CH ) 1.02 (m, 3H, CH
). 13C-NMR (125
1
product as a slightly yellow solid (0.16 g, 33%). H-NMR (500 MHz, CD
3
6.04 (d, 1H, J =8.08 Hz, CHN) 4.76 (dd, 1H, J = 5.57, 7.43 Hz, CHCH
2
2
3 2
)
2
2
2
3
3
MHz, CD OD) δ 152.09, 150.65, 150.06, 147.41, 118.20, 87.05, 86.16, 71.42, 71.36, 61.69, 42.00,
+
37.40, 31.16, 19.78, 12.79. LC-MS (m/z): positive mode 235.2, 366.9 [M+H] . Purity determined by
HPLC-UV (254 nm)-ESI-MS: 85.9 %. mp: 119°C
1
2

Nucleotide Inhibitors of CD39
(2R,3R,4S,5R)-2-(6-(Diethylamino)-8-(methylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetra-
3
3
30
31
hydrofuran-3,4-diol (20)
3
3
3
3
3
3
3
3
3
3
32
33
34
35
36
37
38
39
40
41
The compound was synthesized starting from 16 (0.52 g, 1.30 mmol, 1.0 eq) using methylamine (8 M,
33% (w/w) in ethanol, (0.06 ml, 1.31 mmol, 1.0 eq). Purification by column chromatography
1
(CH
MHz, DMSO-d
J = 7.23 Hz, CHN) 5.85 (m, 1H, CH
(q, 1H, J =6.71 Hz, CHCH ) 4.11 (br s, 1H, CHOH) 3.95 (d, 1H, J =1.96 Hz, CHOH) 3.87 (q, 4H, J
=6.09 Hz, N(CH ) 3.62 (m, 2H, CHCH ) 3.08 (q, 3H, J = 7.26 Hz, NCH ) 1.16 (m, 6H, N(CH CH ).
3
OH/DCM 2:23) afforded the desired product as a white powder (0.30 g, 67%). H-NMR (500
6
) δ 7.94 (d, 1H, J =0.97 Hz, N=CHN) 6.81 (q, 1H, J = 4.38 Hz, NHCH
3
) 5.87 (d, 1H,
2
OH) 5.17 (d, 1H, J = 6.63 Hz, CHOH) 5.05 (m, 1H, CHOH) 4.65
2
2
)
2
2
3
2
3 2
)
13
C-NMR (125 MHz, DMSO-d6) δ 151.00, 150.62, 150.51, 148.30, 117.27, 86.55, 85.77, 71.08, 70.81,
+
61.78, 45.90, 42.07, 28.98,14.04, 8.74. LC/ESI-MS (m/z): positive mode 352.9 [M+H] . Purity
determined by HPLC-UV (254 nm)-ESI-MS: 98%. mp: 115°C.
3
3
42
43
Synthesis of (2R,3R,4S,5R)-2-(8-(bButylthio)-6-(methylamino)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetra-hydrofuran-3,4-diol (21)
3
3
3
3
3
3
3
3
3
3
3
3
3
44
45
46
47
48
49
50
51
52
53
54
55
56
To a solution of 14 (0.5 g, 1.4 mmol, 1.0 eq) in absolute ethanol, thiourea (0.2 g, 2.49 mmol, 1.8 eq)
was added. After 7 h of refluxing the solution was evaporated yielding a yellow oil that was
2
resuspended in a mixture of H O/EtOH 1:1. The solution was brought to basic pH with 2 M NaOH. 1-
Iodobutane (0.5 ml, 4.32 mmol, 3.0 eq) was added and the reaction was stirred at rt for 5 h. After
extraction with ethyl acetate (3 x 100 mL), the organic phase was evaporated. Purification by column
1
chromatography (CH
DMSO-d ) δ 8.13 (br s, 1H, NCH=N) 7.63 (br s, 1H, NHCH
(dd, 1H, J =3.61, 8.93 Hz, CH OH) 5.37 (d, 1H, J =6.42 Hz, CHOH) 5.16 (d, 1H, J =4.29 Hz, CHOH)
4.98 (q, 1H, J =6.50 Hz, CHCH ) 4.15 (m, 1H, CHOH) 3.96 (q, 1H, J =3.70 Hz, CHOH) 3.68-3.49 (d
m, 2H, CHCH ) 3.26 (m, 2H, SCH ) 2.96 (br s, 3H, NHCH ) 1.67 (m, 2H, CH ) 1.40 (m, 2H, CH
0.89 (t, 3H, J = 7.38 Hz, CH CH ) δ 153.80, 151.47, 148.49, 128.29,
). 13C-NMR (125 MHz, DMSO-d
3
OH/DCM 1:24) afforded a white solid. (0.21 g, 42%). H-NMR (500 MHz,
6
3
) 5.77 (d, 1H, J = 6.89 Hz, CHN) 5.62
2
2
2
2
3
2
2
)
2
3
6
127.32, 89.04, 86.79, 71.54, 71.17, 62.41, 32.27, 31.11, 27.17, 21.37, 13.59. LC/ESI-MS (m/z):
positive mode 370.1 [M+H]+. Purity determined by HPLC-UV (254 nm)-ESI-MS: 90.1%. mp: 144°C.
3
3
57
58
Synthesis of (2R,3R,4S,5R)-2-(8-(bButylthio)-6-(diethylamino)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetra-hydrofuran-3,4-diol (22)
3
3
59
60
Compound 16 (0.74 g, 1.83 mmol, 1.0 eq) was suspended in absolute ethanol (5 ml) and the solution
was basified with 2 M NaOH. Butanethiol (0.4 ml, 3.7 mmol, 2.0 eq) was added and the reaction
1
3

Nucleotide inhibitors of CD39
3
3
3
3
3
3
3
3
3
3
3
3
61
62
63
64
65
66
67
68
69
70
71
72
mixture was stirred at rt for 5 days. After evaporation, the crude product was subjected to silica gel
chromatography. However, separation of starting material and product was not possible. Therefore, the
mixture was purified by RP-HPLC (20-100% CH
3
OH in H
2
O in 15 min, 20 ml/min) yielding the
) δ 8.10 (s, 1H,
OH) 5.36 (d, 1H, J =
) 4.15 (s, 1H, CHOH) 3.95
(m, 1H, CHOH) 4.15-3.65 (large bulb, 4H, underneath other peaks, N(CH ) 3.65-3.51 (d m, 2H,
CHCH ) 3.25 (m, 2H, SCH ) 1.72 (m, 2H, CH ) 1.40 (m, 2H, CH ) 1.19 (t, 6H, J = 6.69 Hz,
N(CH CH ) 0.89 (t, 3H, J = 7.39 Hz, S(CH CH ) δ 151.78,
). 13C-NMR (125 MHz, DMSO-d
1
desired product as a white powder (0.09 g, 12%). H-NMR (500 MHz, DMSO-d
6
N=CHN) 5.72 (t, 1H, J =6.89 Hz, CHN) 5.60 (dd, 1H, J = 3.43, 8.71 Hz, CH
2
5.22 Hz, CHOH) 5.16 (m, 1H, CHOH) 4.98 (d, 1H, J = 5.24 Hz, CHCH
2
2 2
)
2
2
2
2
2
3
)
2
)
2 3
3
6
151.54, 150.81, 147.96, 119.80, 88.99, 86.78, 71.31, 71.16, 62.44, 42.61, 31.88, 31.39, 21.56, 13.60
+
(missing: N(CH CH )
2 3 2
). LC/ESI-MS (m/z): positive mode 412.0 [M+H] . Purity determined by
HPLC-UV (254 nm)-ESI-MS: 98.5%. mp: 147°C.
3
73
Preparation of triethylammonium hydrogencarbonate (TEAC) buffer
3
3
74
75
A 1 M solution of TEAC was prepared by adding dry ice slowly to a 1 M triethylamine solution in
water for several hours until a pH of approximately 7.4−7.6 was indicated using a pH meter.
3
76
General procedure for the synthesis of I and 24-38
3
3
3
3
3
3
3
3
3
3
3
3
3
3
77
78
79
80
81
82
83
84
85
86
87
88
89
90
Lyophilized adenosine derivatives and proton sponge (1.5 eq) were dissolved in 5 ml of trimethyl
phosphate under an argon atmosphere at room temperature. The mixture was cooled to 0°C, and
phosphoryl chloride (0.1 ml, 1.3 mmol) was added dropwise. After 5 h of stirring at 0°C, tributylamine
(4 eq) and 0.5 M tri-N-butylammonium dibromomethylenebisphosphonate solution in DMF (2.5 eq)
were added to the mixture simultaneously. After 30 min, a cold 0.5 M aqueous TEAC solution (20 ml,
pH 7.4 - 7.6) was added to the mixture and stirring was continued at room temperature for one hour.
Trimethyl phosphate was extracted with tert.-butylmethylether (3 x 200 ml) and the aqueous solution
was lyophilized. The crude nucleoside triphosphate analogs were purified by fast protein liquid
chromatography (FPLC). After equilibration of the column with deionized water, the crude product
was dissolved in deionized water and injected into the column. The column was first washed with 5%
0.5 M NH
80% 0.5 M NH
NH HCO buffer. Fractions were collected, and appropriate fractions were pooled and lyophilized
several times. The monophosphate and the triphosphate analogs were each purified by preparative
4
HCO
3
buffer to remove unbound components. Elution started with a solvent gradient of 5-
4
HCO
3
buffer over 8 column volumes followed by an isocratic phase at 80% of 0.5 M
4
3
1
4

Nucleotide Inhibitors of CD39
3
3
91
92
HPLC (0%-30% acetonitrile in 50 mM NH
4
HCO
3
buffer within 15 min, 20 ml/min). Fractions were
collected and appropriate fractions pooled and lyophilized.
3
3
93
94
(Dibromo((((((2R,3S,4R,5R)-5-(6-(diethylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid (I)
3
3
3
3
3
4
4
4
4
95
96
97
98
99
00
01
02
03
The compound was synthesized starting from 2 (0.32 g, 1.0 mmol, 1.0 eq) affording a white solid (0.03
1
g, 4%). H-NMR (500 MHz, D
2
O) δ 8.43 (s, 1H, N=CHN) 8.14 (s, 1H, N=CHN) 6.11 (d, 1H, J = 5.83
) 4.33 (m, 2H,
). 13C-NMR (125 MHz, D
O)
δ 156.09, 155.13, 152.30, 140.63, 121.34, 89.38, 86.70, 77.05, 73.12, 68.09, 57.61, 46.64, 15.47. 31P-
Hz, CHN) 4.76 (d, 1H, J = 5.53 Hz, CHOH) 4.63 (m, 1H, CHOH) 4.40 (m, 1H, CHCH
2
CHCH ) 3.85 (br s, 4H, N(CH CH ) 1.24 (t, 6H, J = 7.07 Hz, N(CH
2
2
3
)
2
3
)
2
2
2
NMR (202 MHz, D O) δ 7.61 (d, 1P, J =13.94 Hz, P
γ β
) 0.40 (dd, 1P, J = 13.66, 29.09 Hz, P ) -10.61
+
(d, 1P, J = 29.33 Hz, P
α
). LC/ESI-MS (m/z): positive mode 719.9052 [M+H] (calcd. 719.9054), and
-
negative mode 717.8904 [M-H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 97.5%. mp:
127°C.
4
4
04
05
(Dibromo((((((2R,3S,4R,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydro-furan-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid (24)
4
4
4
4
4
4
4
4
06
07
08
09
10
11
12
13
The compound was synthesized starting from 3 (0.29 g, 1.0 mmol, 1.0 eq) affording a white solid (0.01
1
g, 1%). H-NMR (500 MHz, D
2
O) δ 8.45 (s, 1H, N=CHN) 8.17 (s, 1H, N=CHN) 6.12 (d, 1H, J = 5.92
O, CHCH ) 4.61 (dd, 1H, J = 3.60, 4.99 Hz, CHOH) 4.41
) 3.42 (br s, 6H, N(CH O) δ 156.66,
). 13C-NMR (125 MHz, D
154.25, 152.05, 140.97, 121.92, 89.56, 86.89, 77.12, 73.26, 68.16, 51.04, 48.52, 41.92. 31P-NMR (202
Hz, CHN) 4.78 (m, 1H overlapping with H
2
2
(m, 1H, CHOH) 4.31 (m, 2H, CHCH
2
)
3 2
2
2
MHz, D O) δ 7.48 (d, 1P, J = 14.23 Hz, P
γ β
) -0.73 (dd, 1P, J = 14.24, 27.90 Hz, P ) -10.65 (d, 1P, J =
+
28.38 Hz, P
α
). LC/ESI-MS (m/z): positive mode 691.8745 [M+H] (calcd. 691.8742), and negative
-
mode 689.8587 [M-H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 99.7%. mp: 184°C.
4
4
4
14
15
16
(Dibromo((((((2R,3S,4R,5R)-5-(6-(ethyl(methyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetra-
hydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic
acid
(25)
4
4
4
4
17
18
19
20
The compound was synthesized starting from 4 (0.3 g, 1.0 mmol, 1.0 eq) affording a white solid (0.08
1
g, 12%). H-NMR (500 MHz, D
2
O) δ 8.41 (s, 1H, N=CHN) 8.11 (s, 1H, N=CHN) 6.10 (d, 1H, J =
5.79 Hz, CHN) 4.76 (t, 1H, J = 4.99 Hz, CHOH) 4.61 (t, 1H, J = 3.49 Hz, CHOH) 4.40 (br s, 1H,
CHCH ) 4.31 (m, 2H, CHCH ) 3.88 (br s, 2H, NCH ) 3.30 (br s, 3H, NCH ) 1.20 (t, 3H, J = 7.10 Hz,
2
2
2
3
1
5

Nucleotide inhibitors of CD39
4
4
4
4
4
21
22
23
24
25
NCH
2
CH
3
). 13C-NMR (125 MHz, D
2
O) δ 156.57, 155.00, 152.16, 140.60, 121.57, 89.49, 86.73, 77.08,
73.13, 68.11, 59.78, 48.81, 39.25, 14.75. 31P-NMR (202 MHz, D
2 γ
O) δ 7.58 (d, 1P, J = 14.50 Hz, P )
0.22 (q, 1P, J = 14.29, 29.14 Hz, P
705.8896 [M+H]+ (calcd. 705.8898), and negative mode 703.8737 [M-H] . Purity determined by
β
) -10.62 (d, 1P, J = 29.27 Hz, P
α
). LC/ESI-MS (m/z): positive mode
-
HPLC-UV (254 nm)-ESI-MS: 100%. mp: 199°C.
4
4
4
26
27
28
(Dibromo((((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-(methyl(propyl)amino)-9H-purin-9-yl)tetra-
hydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic
acid
(26)
4
4
4
4
4
4
4
4
4
29
30
31
32
33
34
35
36
37
The compound was synthesized starting from 5 (0.32 g, 1.0 mmol, 1.0 eq) affording a white solid (0.06
1
g, 9%). H-NMR (500 MHz, D
2
O) δ 8.43 (s, 1H, N=CHN) 8.15 (s, 1H, N=CHN) 6.12 (d, 1H, J = 5.96
Hz, CHN) 4.77 (d, 1H, J = 5.58 Hz, CHOH) 4.63 (t, 1H, J =4.23 Hz, CHOH) 4.41 (br s, 1H, CHCH
4.36-4.24 (d m, 2H, CHCH ) 3.90 (br s, 2H, NCH ) 3.55 (br s, 3H, NCH ) 1.69 (m, 2H, NCH CH
0.89 (t, 3H, J = 7.40 Hz, CH CH
). 13C-NMR (125 MHz, D
121.69, 89.60, 86.82, 77.06, 73.22, 68.19, 58.70, 55.17, 39.98, 23.18, 12.99. 31PNMR (202 MHz, D
2
)
)
2
2
3
2
2
2
3
2
O) δ 157.07, 155.07, 152.33, 140.55,
O)
) -10.62 (d, 1P, J = 28.61 Hz, P ).
2
δ 7.56 (d, 1P, J = 13.84 Hz, P
γ
) -0.23 (dd, 1P, J = 14.43, 29.03 Hz, P
β
α
+
LC/ESI-MS (m/z): positive mode 719.905047 [M+H] (calcd. 719.9055), and negative mode 717.8896
-
[M-H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 95.6%. mp: 101°C.
4
4
38
39
(Dibromo((((((2R,3S,4R,5R)-5-(6-(dipropylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydro-furan-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid (27)
4
4
4
4
4
4
4
4
4
40
41
42
43
44
45
46
47
48
The compound was synthesized starting from 6 (0.35 g, 1.0 mmol, 1.0 eq) affording a white solid (0.06
1
g, 8%). H-NMR (500 MHz, D
2
O) δ 8.43 (s, 1H, N=CHN) 8.15 (s, 1H, N=CHN) 6.12 (d, 1H, J = 5.88
) 4.36-4.26 ( d
) 0.91 (t, 6H, J = 7.40
O) δ 156.76, 155.12, 152.44, 140.49, 121.54, 89.36,
86.77, 77.06, 73.10, 68.12, 53.51, 50.89, 23.47, 13.12. 31P-NMR (202 MHz, D
O) δ 7.64 (d, 1P, J =
13.87 Hz, P ) 0.78 (q, 1P, J = 13.82, 29.45 Hz, P ) -10.59 (d, 1P, J = 29.59 Hz, P
Hz, CHN) 4.76 (d, 1H, J = 5.53 Hz, CHOH) 4.64 (m, 1H, CHOH) 4.40 (m, 1H, CHCH
m, 2H, CHCH ) 3.81 (br s, 4H, N(CH CH CH ) 1.68 (m, 4H, N(CH CH CH
Hz, N(CH CH CH
). 13C-NMR (125 MHz, D
2
2
2
2
3
)
2
2
2
3 2
)
2
2
3
)
2
2
2
γ
β
α
). LC/ESI-MS (m/z):
positive mode 747.9349 [M+H]+ (calcd. 747.9368), and negative mode 745.9222 [M-H] . Purity
-
determined by HPLC-UV(254 nm)-ESI-MS: 97%. mp: 189°C.
1
6

Nucleotide Inhibitors of CD39
(Dibromo((((((2R,3S,4R,5R)-5-(6-(ethyl(propyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetra-
4
4
4
49
50
51
hydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid
(28)
4
4
4
4
4
4
4
4
4
52
53
54
55
56
57
58
59
60
The compound was synthesized starting from 7 (0.33 g, 1.0 mmol, 1.0 eq) affording a white solid (0.05
1
g, 6%). H-NMR (500 MHz, D
2
O) δ 8.42 (s, 1H, N=CHN) 8.14 (s, 1H, N=CHN) 6.10 (d, 1H, J = 5.70
) 4.33 (m, 2H,
) 1.20 (t, 3H, J = 7.05 Hz,
O) δ 156.41, 155.11, 152.37, 140.54,
121.41, 89.36, 86.32, 77.05, 73.16, 68.13, 61.65, 53.08, 47.05, 23.53, 15.39, 13.13. 31P-NMR (202
Hz, CHN) 4.75 (t, 1H, J = 5.41 Hz, CHOH) 4.63 (m, 1H, CHOH) 4.39 (s, 1H, CHCH
CHCH ) 3.78 (br d, 4H, J = 56.7 Hz, N(CH ) 1.68 (m, 2H, NCH CH CH
CH ) 0.91 (t, 3 H, J =7.39 Hz, CH
). 13C-NMR (125 MHz, D
2
2
)
2 2
2
2
3
3
3
2
MHz, D2O) δ 7.68 (d, 1P, J = 7.68 Hz, P
γ β
) 1.10 (dd, 1P, J = 13.61, 29.77 Hz, P ) -10.59 (d, 1P, J =
+
29.75 Hz, P
α
). LC/ESI-MS (m/z): positive mode 734.1371 [M+H] (calcd. 734.1373), and negative
-
mode 731.9086 [M-H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 97.1%. mp: 128°C.
4
4
61
62
(((((((2R,3S,4R,5R)-5-(6-(benzylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)dibromomethyl)phosphonic acid (29)
4
4
4
4
4
4
4
4
4
4
4
63
64
65
66
67
68
69
70
71
72
73
The compound was synthesized starting from (8) (0.36 g, 1.0 mmol, 1.0 eq) affording a white solid
1
(0.001 g, recovered from NMR). H-NMR (600 MHz, D
2
O) δ: 8.52 (s, 1H, H-8), 8.24 (s, 1H, H-2),
), 4.81 (t, J = 5.4 Hz,
1H, H-2’), 4.63 (dd, J = 5.4, 3.6 Hz, 1H, H-3’), 4.42 (m, 1H, H-4’), 4.36 – 4.32 (m, 1H, H-5’a), 4.28
– 4.24 (m, 1H, H-5’b), (OHs and NH are not visible). 13C-NMR (125 MHz, D
O) δ: 157.5 (C-6, Cquat.),
7.44 - 7.33 (m, 5H, Harom.), 6.15 (d, J = 6.6 Hz, 1H, H-1’), 4.84 (s (br), 2H, N-CH
2
2
155.7 (C-2, CH), 142.3 (C-8, CH), 141.3 (C-4, Cquat.), 131.6 (2 x Carom., CH), 130.2 (Carom., CH), 129.7
(2 x Carom., CH), 124.7 (Carom., Cquat.), 121.8 (C-5, Cquat.), 117.8 (Br-C-Br), 89.5 (C-1’, CH), 87.0 (C-4’,
CH), 77.2 (C-2’, CH), 73.3 (C-3’, CH), 68.2 (C-5’, CH
2
), 46.8 (Cbenzyl, CH
2
). 31P-NMR (243 MHz,
β
), -0.45 (dd, J = 14.34, 28.43 Hz, 1P, P ), -10.52 (d, J = 28.43 Hz,
D
2
O) δ: 7.67 (d, J = 14.34 Hz, 1P, P
1P, P
ESI-MS: 99.9%.
γ
+
α
). LC-ESI-MS (m/z): positive mode 753.7 [M+H] . Purity determined by HPLC-UV (254 nm)-
4
4
74
75
(Dibromo((((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-(phenethylamino)-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid (30)
4
4
4
4
76
77
78
79
The compound was synthesized starting from (9) (0.37 g, 1.0 mmol, 1.0 eq) affording a white solid
1
(0.018 g, 4.8 %). H NMR (600 MHz, D
2
O) δ 8.51 (s, 1H, C8-H), 8.22 (s, 1H, C2-H), 7.28 (s, 5H,
aryl), 7.21 (s, 1H, NH), 6.09 (d, J = 5.7 Hz, 1H, C1ʹ-H), 4.59 (t, J = 4.1 Hz, 1H, C3ʹ-H), 4.41 (t, 1H,
C4ʹ-H), 4.35 – 4.28 (m, 2H, C5ʹ-H), 3.87 (s, 2H, CH
2
), 3.01 (s, 2H, CH
2
), 13C NMR (151 MHz, D
2
O)
1
7

Nucleotide inhibitors of CD39
4
4
4
4
80
81
82
83
δ 143.24 (1C, Cq-aryl), 131.95 (2C, CH-aryl), 131.41 (1C, CH-aryl), 129.48 (1C, CH-aryl), 90.06 (1C,
C1ʹ), 86.95 (1C, C2ʹ), 77.35 (1C, C3ʹ), 73.18 (1C, C4ʹ), 68.08 (1C, C5ʹ). 31P NMR (243 MHz, D
2
O) δ
7.59 (d, J = 14.7 Hz, P
), -0.60 (dd, J = 28.7, 14.8 Hz, P ), -10.50 (d, J = 28.2 Hz, P ). LC-ESI-MS
γ β α
+
(m/z): positive mode 766.9 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 99.9 %.
4
4
84
85
(((((((2R,3S,4R,5R)-5-(6-Amino-8-(butylthio)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)dibromomethyl)phosphonic acid (31)
4
4
4
4
4
4
4
4
4
86
87
88
89
90
91
92
93
94
The compound was synthesized starting from 12 (0.27 g, 0.76 mmol, 1.0 eq) affording a white solid
1
(0.014 g, 2.5%). H-NMR (500 MHz, D
2
O) δ 8.17 (s, 1H, N=CHN) 6.10 (d, 1H, J =6.23 Hz, CHN)
) 4.33
) 0.90 (t, 3H, J = 7.39 Hz,
O) δ 155.14, 154.91, 153.42, 152.48, 121.74, 90.88, 86.35, 79.70, 72.54,
68.28, 57.53, 35.40, 33.48, 24.09, 15.69. 31P-NMR (202 MHz, D
O) δ 7.46 (d, 1P, J =14.53 Hz, P ) -
0.69 (dd, 1P, J =14.69, 29.01 Hz, P ) -10.62 (d, 1P, J =28.16 Hz, P
5.19 (t, 1H, J = 6.19 Hz, CHOH) 4.61 (m, 1H, CHOH) 4.39 (dd, 1H, J =6.34, 10.22 Hz, CHCH
(m, 2H, CHCH ) 3.29 (m, 2H, SCH ) 1.73 (m, 2H, CH ) 1.44 (m, 2H, CH
CH
). 13C-NMR (125 MHz, D
2
2
2
2
2
3
2
2
γ
β
α
). LC/ESI-MS (m/z): positive mode
751.8752 [M+H]+ (calcd. 751.8775), and negative mode 749.8619 [M-H] . Purity determined by
-
HPLC-UV (254 nm)-ESI-MS: 100%. mp: 167°C.
4
95
4
4
4
96
97
98
(Dibromo((((((2R,3S,4R,5R)-5-(8-(cyclopropylamino)-6-(methylamino)-9H-purin-9-yl)-3,4-di-
hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-
phosphonic acid (32)
4
5
5
5
5
5
5
5
5
99
00
01
02
03
04
05
06
07
The compound was synthesized starting from 17 (0.14 g, 0.41 mmol, 1.0 eq) affording a white solid
1
(7.0 mg, 2%). H-NMR (500 MHz, D
2
O) δ 8.16 (s, 1H, N=CHN) 5.96 (d, 1H, J = 7.36 Hz, CHN) 4.63
) 4.41 (m, 1H, CHOH) 4.35 (br s, 1H, CHOH) 4.24 (d, 2H, J = 11.92
) 3.10 (s, 3H, NHCH ) 2.76 (m, 1H, NHCH) 0.88 (m, 2H, CHCH ) 0.8-0.72 (d m, 2H,
). 13C-NMR (125 MHz, D
O) δ 155.34, 152.79, 150.67, 150.23, 117.66, 89.45, 87.11, 73.81,
72.65, 63.36, 50.90, 30.49, 27.18, 9.67. 31P-NMR (202 MHz, D2O) δ 7.51 (d, 1P, J = 14.60 Hz, P
) -
). LC/ESI-MS (m/z): positive
(dd, 1H, J =2.7, 5.7 Hz, CHCH
Hz, CHCH
CHCH
2
2
3
2
2
2
γ
β α
0.84 (dd, 1P, J = 14.74, 27.48 Hz, P ) -11.16 (d, 1P, J = 27.67 Hz, P
+
-
mode 732.8970 [M+H] (calcd. 732.9007), and negative mode 730.8852 [M-H] . Purity determined by
HPLC-UV (254 nm)-ESI-MS: 100%. mp: 232°C.
1
8

Nucleotide Inhibitors of CD39
5
5
5
08
09
10
(Dibromo((((((2R,3S,4R,5R)-5-(8-(butylamino)-6-(methylamino)-9H-purin-9-yl)-3,4-dihydroxy-
tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-phosphonic
acid (33)
5
5
5
5
5
5
5
5
5
5
11
12
13
14
15
16
17
18
19
20
The compound was synthesized starting from 18 (0.32 g, 1.0 mmol, 1.0 eq) affording a white solid
1
(0.017 g, 2.3%). H-NMR (500 MHz, D
2
O) δ 8.13 (s, 1H, N=CHN) 6.04 (d, 1H, J =7.76 Hz, CHN)
4.78 (t, 1H, J = 7.82 Hz, CHOH) 4.66 (dd, 1H, J = 2.16, 5.70 Hz, CHOH) 4.45 (m, 1H, 1x CHCH
4.38 (br s, 1H, CHCH ) 4.24 (m, 1H, 1x CHCH ) 3.50 (m, 2H, NHCH ) 3.04 (s, 3H, NHCH ) 1.67 (m,
2H, CH ) 1.39 (q, 2H, J = 7.48 Hz, CH ) 0.93 (t, 3H, J = 7.40 Hz, CH O) δ
). 13C-NMR (125 MHz, D
154.90, 152.87, 150.47, 150.25, 118.58, 89.15, 87.28, 73.33, 72.84, 68.44, 57.70, 45.31, 33.43, 30.46,
2
)
2
2
2
3
2
2
3
2
22.31, 16.07. 31P-NMR (202 MHz, D
2
O) δ 7.48 (d, 1P, J = 16.02 Hz, P
γ
) -0.87 (dd, 1P, J = 14.47, 26.89
+
Hz, P ) -11.26 (d, 1P, J = 27.48 Hz, P
β
α
). LC/ESI-MS (m/z): positive mode 748.9324 [M+H] (calcd.
-
748.9320), and negative mode 746.9163 [M-H] . Purity determined by HPLC-UV (254 nm)-ESI-MS:
99.0%. mp: 178°C.
5
5
5
21
22
23
(Dibromo((((((2R,3S,4R,5R)-5-(8-(butylamino)-6-(dimethylamino)-9H-purin-9-yl)-3,4-di-
hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-
phosphonic acid (34)
5
5
5
5
5
5
5
5
5
24
25
26
27
28
29
30
31
32
The compound was synthesized starting from 19 (0.1 g, 0.27 mmol, 1.0 eq) affording a white solid (6.0
1
mg, 1.8%). H-NMR (500 MHz, D
2
O) δ 8.07 (s, 1H, N=CHN) 6.06 (d, 1H, J = 7.83 Hz, CHN) 4.71
) 4.45 (m, 1H, CHOH) 4.38 (br s, 1H, CHOH) 4.24 (d, 1H, J = 11.78 Hz, CHCH ) 3.54
(d m, 2H, CHCH ) 3.42 (s, 6H, N(CH ) 1.68 (m, 2H, CH ) 1.38 (m, 2H, CH ) 0.93 (t, 3H, J = 7.40
Hz, CH O) δ 163.50, 154.52, 152.23, 149.19, 119.98, 89.00, 87.24, 73.35,
). 13C-NMR (125 MHz, D
72.84, 68.47, 56.93, 45.12, 41.61, 33.75, 22.88, 16.03. 31P-NMR (202 MHz, D
O) δ 6.15 (d, 1P, J =
(m, 2H, NCH
2
2
2
)
3 2
2
2
3
2
2
14.67 Hz, P
γ
) -2.22 (dd, 1P, J = 14.72, 27.57 Hz, P
β
) -12.61 (d, 1P, J = 27.71 Hz, P
α
). LC/ESI-MS
+
-
(m/z): positive mode 762.9478 [M+H] (calcd. 762.9477), and negative mode 760.9331 [M+H] . Purity
determined by HPLC-UV (254 nm)-ESI-MS: 98%. mp: 193°C.
5
5
5
33
34
35
(Dibromo((((((2R,3S,4R,5R)-5-(6-(diethylamino)-8-(methylamino)-9H-purin-9-yl)-3,4-di-
hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-
phosphonic acid (35)
5
5
5
36
37
38
The compound was synthesized starting from 20 (0.08 g, 0.23 mmol, 1.0 eq) affording a white solid
1
(9.0 mg, 4%). H-NMR (500 MHz, D
2
O) δ 8.04 (s, 1H, N=CHN) 6.06 (d, 1H, J = 7.82 Hz, CHN) 4.72
2
(m, 1H, CHOH) 4.60 (dd, 1H, J =1.99, 5.68 Hz, CHOH) 4.45 (dd, 1H, J = 6.43, 10.55 Hz, CHCH )
1
9

Nucleotide inhibitors of CD39
5
5
5
5
5
5
39
40
41
42
43
44
4.33 (d m, 2H, CHCH
N(CH CH
). 13C-NMR (125 MHz, D
73.36, 72.91, 68.66, 57.89, 46.34, 31.89, 15.61. 31P-NMR (202 MHz, D
2
) 3.88 (m, 4H, N(CH
2 3 2 3
CH ) ) 3.09 (s, 3H, NHCH ) 1.24 (t, 6H, J = 7.06 Hz,
2
3
)
2
2
O) δ 155.08, 152.57, 151.93, 149.89, 119.72, 89.05, 87.04,
2
O) δ 7.14 (s, 1P, P
γ
) 0.27 (br
+
s, 1P, P
β
) -10.77 (d, 1P, J = 26.2 Hz, P
α
). LC/ESI-MS (m/z): positive mode 748.9295 [M+H] (calcd.
-
748.9320), and negative mode 746.9181 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS:
93.7%. mp: 249°C.
5
5
5
45
46
47
(Dibromo((((((2R,3S,4R,5R)-5-(8-(butylthio)-6-(methylamino)-9H-purin-9-yl)-3,4-dihydroxy-
tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-phosphonic
acid (36)
5
5
5
5
5
5
5
5
5
48
49
50
51
52
53
54
55
56
The compound was synthesized starting from 21 (0.2 g, 0.54 mmol, 1.0 eq) affording a white solid
1
(13.0 mg, 3%). H-NMR (500 MHz, D
2
O) δ 8.19 (s, 1H, N=CHN) 6.11 (d, 1H, J = 6.70 Hz, CHN)
5.20 (q, 1H, J = 6.30 Hz, CHOH) 4.62 (dd, 1H, J = 4.10, 6.09 Hz, CHOH) 4.37 (m, 1 H, CHCH
(d m, 2H, CHCH ) 3.26 (m, 2H, SCH ) 3.08 (s, 3H, NCH ) 1.71 (m, 2H, CH ) 1.44 (m, 2H, CH
(t, 3H, J = 7.40 Hz, CH
86.17, 73.53, 72.52, 68.30, 50.37, 35.76, 33.60, 30.30, 24.06, 15.69. 31P-NMR (202 MHz, D
2
) 4.32
2
2
3
2
2
) 0.91
). 13CNMR (125 MHz, D2O) δ 156.01, 154.39, 153.99, 152.30, 122.21, 90.78,
O) δ 7.49
3
2
(d, 1P, J = 14.51 Hz, P
γ
) 0.70 (dd, 1P, J =14.28, 27.73 Hz, P
β
) -10.64 (d, 1P, J = 28.37 Hz, P
α
). LC/ESI-
+
-
MS (m/z): positive mode 765.8919 [M+H] (calcd. 765.8931), and negative mode 763.8787 [M-H] .
Purity determined by HPLC-UV (254 nm)-ESIMS: 95.4%. mp: 172°C.
5
5
5
57
58
59
(Dibromo((((((2R,3S,4R,5R)-5-(8-(butylthio)-6-(diethylamino)-9H-purin-9-yl)-3,4-dihydroxy-
tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)methyl)-phosphonic
acid (37)
5
5
5
5
5
5
5
5
5
5
60
61
62
63
64
65
66
67
68
69
The compound was synthesized starting from 22 (0.1 g, 0.24 mmol, 1.0 eq) affording a white solid (7.0
1
mg, 4%). H-NMR (500 MHz, D
2
O) δ 8.18 (s, 1H, N=CHN) 6.13 (d, 1H, J = 6.41 Hz, CHN) 5.16 (t,
1H, J = 6.26 Hz, CHCH
CHCH ) 3.92 (br s, 4H, N(CH
1.26 (t, 6H, J = 7.03 Hz, N(CH
2
) 4.63 (m, 1H, CHOH) 4.38 (dd, 1H, J = 4.92, 10.90 Hz, CHOH) 4.32 (m, 2H,
2
)
2 2
2 2 2
) 3.30-3.22 (d m, 2H, SCH ) 1.72 (m, 2H, CH ) 1.42 (m, 2H, CH )
). 13C-NMR (125 MHz, D2O) δ
2
CH ) 0.89 (t, 3H, J =7.39 Hz, CH
3
)
2
3
153.66, 153.37, 152.57, 151.90, 122.31, 90.78, 86.33, 73.72, 72.61, 68.29, 50.92, 47.16, 36.19, 34.10,
24.22, 15.75, 15.38. 31P-NMR (202 MHz, D2O) δ 7.48 (d, 1P, J = 13.83 Hz, P ) -0.74 (dd, 1P, J =
γ
12.88, 25.51 Hz, P
β
) -10.64 (d, 1P, J = 28.45 Hz, P
α
). LC/ESI-MS (m/z): positive mode 807.9381
+
-
[M+H] (calcd. 807.9401), and negative mode 805.9304 [M+H] . Purity determined by HPLC-UV (254
nm)-ESI-MS: 92%. mp: 190°C.
2
0

Nucleotide Inhibitors of CD39
(((((((2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)-
5
5
70
71
(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)dibromomethyl)phosphonic acid (38)
5
5
5
5
5
5
5
5
72
73
74
75
76
77
78
79
The compound was synthesized starting from 23 (0.2 g, 0.75 mmol, 1.0 eq) affording a white powder
1
(0.12 g, 24%). H-NMR (500 MHz, D
2
O) δ 8.53 (s, 1H, N=CHN) 8.25 (s, 1H, N=CHN) 6.14 (d, 1H,
) 4.30 (d m, 2H,
O) δ 158.49, 155.69, 152.04, 142.81, 121.51, 89.63, 86.95, 77.21,
73.33, 68.20, 57.26. 31P-NMR (202 MHz, D
O) δ 7.56 (d, 1P, J = 14.45 Hz, P ) -0.50 (dd, 1P, J =
) -10.58 (d, 1P, J = 28.56 Hz, P ). LC/ESI-MS (m/z): positive mode 663.8407
J = 6.0 Hz, CHN) 4.79 (s, 1 H, CHOH) 4.62 (m, 1H, CHOH) 4.41 (m, 1H, CHCH
2
CHCH
2
). 13C-NMR (125 MHz, D
2
2
γ
14.40, 28.55 Hz, P
β
α
+
-
[M+H] (calcd. 663.8406), and negative mode 661.8256 [M+H] . Purity determined by HPLC-UV (254
nm)-ESI-MS: 100%. mp: degradation >250°C.
5
5
80
81
Synthesis
of
(((((((2R,3S,4R,5R)-5-(6-aAmino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
yl)methoxy)-(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)dichloromethyl)phosphonic acid (39)
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
Adenosine (23, 0.2 g, 0.75 mmol, 1.0 eq) and proton sponge (0.24 g, 1.13 mmol, 1.5 eq) were dissolved
in 5.0 ml of trimethyl phosphate under an argon atmosphere at room temperature. The mixture was
cooled to 0°C and phosphoryl chloride (0.1 ml, 1.3 mmol, 1.7 eq) was added dropwise. After 5 h of
stirring
at
0°C,
tributylamine
(4.0
eq)
and
0.5
M
tri-N-butylammonium
dichloromethylenebisphosphonate solution in DMF (2.5 eq) were added to the mixture simultaneously.
After 30 min, a cold 0.5 M aqueous TEAC solution (20 ml, pH 7.4 - 7.6) was added to the mixture and
stirring was continued at room temperature for one hour. Trimethyl phosphate was extracted with tert.-
butylmethylether (3 x 200 ml), and the aqueous solution was lyophilized. The crude nucleoside
triphosphate analogs were purified by FPLC. After equilibration of the column with deionized water,
the crude product was dissolved in deionized water and injected into the column. The column was first
washed with 5% 0.5 M NH
solvent gradient of 5-80% 0.5 M NH
phase at 80% 0.5 M NH HCO buffer. Fractions were collected, appropriate fractions were pooled and
lyophilized several times. The nucleotide analog was further purified by preparative HPLC (0%-30%
4
HCO
3
buffer to remove unbound components. Elution started with a
4
HCO buffer over 8 column volumes followed by an isocratic
3
4
3
acetonitrile in 50 mM NH
4
HCO
3
buffer in 15 min, 20 ml/min). Fractions were collected and
1
appropriate fractions were pooled and lyophilized yielding a white solid (0.05 g, 8%). H-NMR (500
MHz, D
CHOH) 4.61 (m, 1H, CHOH) 4.41 (br s, 1H, CHCH
O) δ 158.54, 155.74, 152.05, 142.79, 121.52, 89.62, 86.99, 77.21, 73.26, 68.16, 37.53. 31P-NMR
2
O) δ 8.53 (s, 1H, N=CHN) 8.25 (s, 1H, N=CHN) 6.14 (d, 1H, J = 5.95 Hz, CHN) 4.78 (s, 1H,
) 4.28 (d m, 2H, CHCH
2
). 13C-NMR (125 MHz,
2
D
2
2
1

Nucleotide inhibitors of CD39
6
6
6
01
02
03
(202 MHz, D
2
O) δ 7.83 (d, 1P, J = 18.36 Hz, P
γ
β
) 0.16 (dd, 1P, J = 18.58, 29.06 Hz, P ) -10.55 (d, 1P,
+
J = 29.64 Hz, P
α
). LC/ESI-MS (m/z): positive mode 573.9446 [M+H] (calcd. 573.9445), and negative
-
mode 571.9304 [M+H] . Purity determined by HPLC-UV (254 nm)-ESI-MS: 98.1%. mp: 205°C.
Synthesis of (((((((2R,3S,4R,5R)-5-(6-aAmino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-
6
6
04
05
yl)methoxy)-(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)difluoromethyl)phosphonic acid (40)
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Adenosine (23, 0.2 g, 0.75 mmol, 1.0 eq) and proton sponge (0.24 g, 1.13 mmol, 1.5 eq) were dissolved
in 5.0 ml of trimethyl phosphate under an argon atmosphere at room temperature. The mixture was
cooled to 0°C and phosphoryl chloride (0.1 ml, 1.3 mmol, 1.7 eq) was added dropwise. After 5 h of
stirring
at
0°C,
tributylamine
(4.0
eq)
and
0.5
M
tri-N-butylammonium
difluoromethylenebisphosphonate solution in DMF (2.5 eq) were added to the mixture simultaneously.
After 30 min, a cold 0.5 M aqueous TEAC solution (20 ml, pH 7.4 - 7.6) was added to the mixture and
stirring was continued at room temperature for one hour. Trimethyl phosphate was extracted with tert.-
butylmethylether (3 x 200 ml) and the aqueous solution was lyophilized. The crude nucleoside
triphosphate analog was purified by FPLC. After equilibration of the column with deionized water, the
crude product was dissolved in deionized water and injected into the column. The column was washed
with 5% 0.5 M NH
gradient of 5-80% 0.5 M NH
80% 0.5M NH HCO buffer. Fractions were collected, appropriate fractions were pooled and
lyophilized several times. The product was further purified by preparative HPLC (0%-30% acetonitrile
4
HCO
3
buffer to remove unbound components. Elution started with a solvent
4
HCO buffer over 8 column volumes followed by an isocratic phase at
3
4
3
in 50 mM NH
4
HCO
3
buffer within 15 min, 20 ml/min). Fractions were collected and appropriate
1
fractions pooled and lyophilized yielding a white solid (0.025 g, 6%). H-NMR (500 MHz, D
2
O) δ
8.52 (s, 1H, N=CHN) 8.25 (s, 1H, N=CHN) 6.14 (d, 1H, J = 6.02 Hz, CHN) 4.78 (d, 1H, J = 5.60 Hz,
CHCH ) 4.57 (m, 1H, CHOH) 4.41 (br s, 1H, CHOH) 4.25 (d m, 2H, CHCH
). 13C-NMR (125 MHz,
O) δ 158.39, 155.55, 152.01, 142.77, 121.48, 89.58, 86.87, 71.17, 73.24, 68.07. 31P-NMR (202 MHz,
O) δ 3.40 (td, 1P, J = 58.87, 79.05 Hz, P ) -4.56 (tdd, 1P, J = 28.07, 56.21, 84.20 Hz, P ) -10.68 (d,
). 19F-NMR (202 MHz, D
O) δ -19.76 (t, 2F, J = 82.12 Hz). LC/ESI-MS (m/z):
2
2
D
2
2
D
γ
β
1P, J = 30.49 Hz, P
α
2
+
-
positive mode 542.0017 [M+H] (calcd. 542.0049), and negative mode 539.9888 [M+H] . Purity
determined by HPLC-UV (254 nm)-ESI-MS: 100%. mp: >231°C (decomposition).
6
6
29
30
2.2 Biological assays
2
2

Nucleotide Inhibitors of CD39
6
6
31
32
2.2.1 Chemicals and materials
ATP, calcium chloride, magnesium chloride, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
(HEPES), ammonium heptamolybdate, dimethyl sulfoxide (DMSO), malachite green, α,β-methylene-
ATP (41), α,β-methylene-ADP (42), β,γ-methyleneadenosine-ATP (43) and polyvinyl alcohol were
obtained from Sigma (Steinheim, Germany). Disodium hydrogenphosphate and sulfuric acid were
6
6
6
6
6
6
6
33
34
35
36
37
38
39
6
purchased from Carl Roth (Karlsruhe, Germany). N -[6-(Fluoresceinyl-5′-carboxamido)hexyl]-ATP
(PSB-170621A) was obtained from Jena Bioscience (Jena, Germany). The polyacrylamide-coated
capillary [30 cm (10 cm effective length) × 50 µm (id), × 360 µm (od)] was purchased from
Chromatographie Service GmbH (Langerwehe, Germany).
6
6
6
6
6
6
6
6
6
6
6
6
6
40
41
42
43
44
45
46
47
48
49
50
51
52
Expression of the enzymes
The cDNAs of human enzymes NPP1, 3, 5, CD38 and CD73 (Genbank accession no. NM_006258,
NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene
(Rockville, USA). Soluble enzymes were produced as previously reported with some modifications
(Lee et al. 2015; Junker et al. 2019). Briefly, the catalytic domains of the enzymes were amplified and
sub-cloned into the expression vector pACGP67 A/B modified with the addition of 9 x histidine tag
(His-tag) at the C-terminus (except for NPP1). The plasmids were transfected in Sf9 insect cells using
Cellfectin™ II Reagent ((Thermo Fisher Scientific, MA, USA) and ProEasy™ baculovirus linearized
DNA (Cat. #A10S , AB Vector, LLC). Protein expression was conducted for 48 h at 27°C. The signal
peptide sequence of the expression vector shuttled the proteins into the supernatant. The supernatant
medium was collected, and the enzymes were purified using HisPurTM Ni -NTA spin columns
according to the manufacturer’s protocol. The protein concentration was determined by the Lowry
method previously described by Randall and Lewis (Randall et al. 1951).
2+
6
53
2
3

Nucleotide inhibitors of CD39
6
6
54
55
2.2.2 Human CD39 preparation
Human umbilical cords were obtained under approved institutional review board protocol (Comité
d’Éthique de la Recherche du CHU de Québec – Université Laval) following written consent as
previously described (Sévigny et al., 1997). They were minced and homogenized with a polytron in 95
mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 45 mM Tris solution, pH 7.6. The
homogenates were then filtered through a cheese cloth, centrifuged for 15 min at 600g, and the
supernatants were subsequently centrifuged for 1 h at 100,000g. The pellets were resuspended in 5 mM
Tris buffer solution, pH 8.0 and 10% glycerol. All purification steps were performed at 4 °C. The
preparations were kept at −80 °C.
6
6
6
6
6
6
6
56
57
58
59
60
61
62
6
6
63
64
2.2.3 Fluorescence capillary electrophoresis assay for CD39
The enzyme activity assay was performed as previously described (Lee et al., 2018). For inhibition
6
6
6
6
6
6
6
6
6
6
6
6
65
66
67
68
69
70
71
72
73
74
75
76
screening, three independent experiments were performed. The concentration of the fluorescent
substrate PSB-017621A was 0.5 µM (K
m
= 19.6 µM); the assay is highly sensitive and therefore allows
value which facilitates the identification and
the use of low substrate concentrations below the K
m
characterization of moderately potent competitive inhibitors. Ttest compounds were initially
investigated at a concentration of 10 µM, and 40 ng protein from human umbilical cord membrane
preparations containing CD39 were added to initiate the reaction. The reaction buffer contained 10 mM
2 2
HEPES, 2 mM CaCl , 1 mM MgCl , pH 7.4. The samples were incubated at 37°C for 4 min, and the
enzymatic reaction was terminated by heating at 90°C for 5 min. The solution was then diluted 1:20
with reaction buffer to perform separation of nucleotides by capillary electrophoresis (CE) followed
by laser-induced fluorescence (LIF) detection. For compounds showing ≥70% inhibition of enzymatic
activity, compared to the positive control without inhibitor, concentration-inhibition curves were
generated at concentrations ranging from 0.01 to 300 µM. Three independent experiments were
2
4

Nucleotide Inhibitors of CD39
6
6
77
78
performed, and curves were calculated by GraphPad Prism 8 software (GraphPad software, San Diego,
CA, USA).
6
6
6
6
6
6
6
6
6
79
80
81
82
83
84
85
86
87
Analysis was carried out using a P/ACE MDQ capillary electrophoresis system (Beckman Instruments,
Fullerton, CA, USA). The separation was performed in a polyacrylamide-coated capillary [30 cm (10
cm effective length) × 50 µm (id), × 360 µm (od)]. Before each run, the capillary was rinsed with the
background electrolyte (50 mM phosphate buffer (pH 6.5)) for 1 min at 30 psi. Samples were
electrokinetically injected by applying a voltage of -6 kV for 30 s at the capillary outlet, and the
fluorescent nucleotide derivatives were separated by voltage application of -15 kV. Detection was
performed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Data
collection and peak area analysis were performed by the P/ACE MDQ software 32 KARAT obtained
from Beckman Coulter (Fullerton, CA, USA).
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6
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89
2.2.4 Malachite green assay for CD39 and human NTPDases2, -3 and -8
The enzyme activity assay was determined essentially as previously described (Cogan et al., 1999)
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6
6
6
6
6
6
6
6
6
7
90
91
92
93
94
95
96
97
98
99
00
2 2
with a few adaptations. The reaction buffer contained 10 mM HEPES, 2 mM CaCl , 1 mM MgCl , pH
7.4 in a final volume of 50 μL in transparent 96-well half area plates. For CD39 (NTPDase1), we made
use of human umbilical cord membranes preparations which express high levels of the enzyme. For
the other human NTPDase isoenzymes, we had to resort to recombinant expression. Human umbilical
cord membrane preparations (250 ng) natively expressing high amounts of CD39, or the respective
recombinant COS-7 -cell membrane preparations expressing the appropriate NTPDase isoenzyme (ca.
100 ng of protein depending on enzyme activity) (Sévigny et al., 1997; Lecka et al., 2013) with or
without inhibitor were preincubated at 37°C and gentle shaking (Eppendorf Thermomixer comfort at
500 rpm) for 5 min. The amount of enzyme preparation was adjusted to ensure 10 – 20 % of substrate
conversion. The reaction was initiated by the addition of 50 µM ATP (K
m m
(CD39) = 17 µM; K
(NTPDase2) = 70 µM; K (NTPDase3) = 75 µM; K (NTPDase8) = 46 µM) (Kukulski et al., 2005).
m
m
2
5

Nucleotide inhibitors of CD39
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7
7
7
7
7
7
7
01
02
03
04
05
06
07
08
After 15 min of incubation at 37°C with gentle shaking, the reaction was stopped by adding the
detection reagents (20 µL malachite green solution, 0.6 mM, and 30 µL ammonium molybdate
solution, 20 mM, in sulfuric acid, 1.5 M). 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 denatured enzyme (90 °C, 15 min), and the inhibition was
calculated as follows:
(
ꢆ − ꢇ)
7
09
% ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ =
∗ 100%
ꢆ
7
7
10
11
where B is the average corrected absorption of the positive control without inhibitor and T the corrected
absorption in the presence of test compound.
7
7
7
7
12
13
14
15
Full concentration-inhibition curves were determined with inhibitor concentrations ranging from 0.1
to 300 µM in the presence of 2% DMSO. Three independent experiments were performed (n=3) and
i
curves were calculated by GraphPad Prism 8 software. The K value was calculated using the Cheng-
Prusoff equation for competitive inhibitors:
ꢀ
ꢊ50
7
16
ꢈꢉ =
[ꢋ]
1
+
ꢈꢌ
7
17
7
7
18
19
2.2.5 CD73 assay
The assay was performed as previously described (Freundlieb et al., 2014). Briefly, the assay was
7
7
20
21
performed with 0.09 µg/ml of soluble human CD73 recombinantly expressed in Sf9 insect cells as
3
described (Junker et al., 2019), the respective test compound, and 5.0 µM [2,8- H]AMP (specific
2
6

Nucleotide Inhibitors of CD39
7
7
7
7
7
7
7
7
7
22
23
24
25
26
27
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30
activity 7.4 x 108 Bq/mmol, 20 mCi/mmol) as radioactive substrate in assay buffer consisting of 25
2 4
mM Tris buffer, 140 mM NaCl, 25 mM NaH PO pH 7.4. The enzymatic reaction was performed for
3
25 min at 37°C in a shaking water bath. Then, 500 µl of cold precipitation buffer (100 mM LaCl , 100
3
mM sodium acetate, pH 4.0) were added to precipitate free phosphate and unconverted [2,8- H]AMP.
After 30 min on ice, filtration through GF/B glass fiber filters using a cell harvester was used to separate
AMP from adenosine. After washing each reaction vial three times with 400 µl of cold (4°C)
demineralized water, aliquots of the filtrate were taken, and 5 ml of the scintillation cocktail (ULTIMA
Gold XR9) was added. The amount of formed adenosine was quantified by liquid scintillation counting
(TRICARB 2900 TR, Packard/PerkinElmer).
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7
31
32
2.2.6 NPP1 assay
Inhibition of NPP1 was determined as previously described (Lee et al., 2017b). p-Nitrophenyl-5’-
7
7
7
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7
7
7
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33
34
35
36
37
38
39
40
41
thymidine monophosphate (p-Nph-5’-TMP) was used as an artificial substrate which results in the
formation of the p-nitrophenolate anion with an absorption maximum of 400 nm. Purified soluble
NPP1 (0.36 µg, expressed in Sf9 insect cells as previously described (Lee et al., 2015)) was mixed with
test compound (20 µM final concentration for initial screening, 0.1 – 200 µM for determining
concentration-dependent inhibition curves), 2% DMSO and 400 µM of p-Nph-5’-TMP as a substrate
in a final volume of 100 µL. The mixture was incubated for 30 min at 37°C with gentle shaking, and
the enzyme reaction was terminated by the addition of 20 µL of 1 M NaOH. The absorption was
measured at 405 nm using a BMG PheraStar FS plate reader (BMG Labtech GmbH, Ortenberg,
Germany).
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7
42
43
2.2.7 NPP4 assay
Soluble NPP4 was expressed in Sf9 insect cells as recently described in detail (Lopez et al., 2020).
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7
44
45
Diadenosine tetraphosphate (AP
4
A) was employed as a substrate which is cleaved by NPP4 to ATP
and AMP. The reaction product ATP was quantified by luciferin-luciferase reaction (Lopez and Müller,
2
7

Nucleotide inhibitors of CD39
7
7
7
7
7
7
7
7
46
47
48
49
50
51
52
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2020). A mixture of 1.4 µg of NPP4 (soluble form expressed in insect cells and purified) (Lopez and
Müller, 2020), 10 µM of test compound, 2 % DMSO and 20 µM of AP
for 60 min at 37°C with gentle shaking. The reaction was terminated by heating at 90°C for 5 min, and
after cooling down on ice, 50 µl of D-luciferin dissolved in buffer (300 mM Tris-HCl, 15 mM MgCl
100 ng D-luciferin, pH 7.8) and 50 µl luciferase (50 ng dissolved in H O) were added. The firefly
4
A as a substrate were incubated
2
,
2
luciferase reacts with D-luciferin in the presence of ATP produced by NPP4. The resulting
luminescence was measured between 10-14 min at 560 nm using a BMG PheraStar FS plate reader
(BMG Labtech GmbH, Ortenberg, Germany).
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7
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55
2.2.8 NPP3 and NPP5 assays
The assays were performed in analogy to published procedures (Blacher et al., 2015). The enzymatic
7
7
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7
7
7
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57
58
59
60
61
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64
activity of human NPP3 and NPP5 (soluble forms expressed in insect cells and purified as previously
6
described (Lopez et al., 2020; Lee et al., 2015) was measured using 1,N -etheno-nicotinamide adenine
+
6
dinucleotide (ε-NAD ) as a substrate, which is hydrolyzed to fluorescent 1,N -etheno-AMP (ε-AMP).
The enzymatic reactions were performed in reaction buffer (10 mM N-cyclohexyl-2-
2 2 2
aminoethanesulfonic acid (CHES), 2 mM CaCl , and 1 mM MgCl , pH 9.0 in H O). Purified NPP3
+
(90 ng) or NPP5 (400 ng), 20 µM of ε-NAD and 10 µM of the test compound were incubated for 30
min at 37°C. The relative fluorescence at 270 nm excitation and 420 nm emission was detected by a
fluorescence microplate reader (Flexstation, Medical Devices LLC. USA, Softmax Pro software to
collect the data).
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7
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66
2.2.9 CD38 assay
The assay operation was analogous to the NPP3 and NPP5 assays. The enzymatic reactions were
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7
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68
performed in 10 mM HEPES reaction buffer (pH 7.2) using 8 ng of human CD38 (expressed in Sf9
insect cells) in analogy to a published procedure (Blacher et al., 2015).
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2
8