Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors

Article

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

pubs.acs.org/jmc

Structure−Activity Relationship of Purine and Pyrimidine

Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors

,

†,‡,§,∇

Christian Renn, Clemens Dobelmann, Vigneshwaran Namasivayam,^‡

‡,∇

§,∇

Anna Junker,*

†

#

¶

†

⊥

†

Shanu Jain, Karolina Losenkova, Heikki Irjala, Sierra Duca, Ramachandran Balasubramanian,

̈

Saibal Chakraborty, Frederik Borgel, Herbert Zimmermann, Gennady G. Yegutkin,

Christa E. Muller, and Kenneth A. Jacobson*

̈

†

∥

#

‡

,†

^†Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney

Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States

‡

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121

Bonn, Germany

§

European Institute for Molecular Imaging (EIMI), University of Mu

̈

nster, Waldeyerstr. 15, D-48149 Mu

Institute for Pharmaceutical and Medicinal Chemistry, University of Munster, Correnstr. 48, D-48149 Mu

Institute of Cell Biology and Neuroscience, Goethe-University, D-60438 Frankfurt am Main, Germany

̈

nster, Germany

∥

̈

nster, Germany

⊥

#

Medicity Research Laboratory, University of Turku, 20520 Turku, Finland

¶

Department of OtorhinolaryngologyHead and Neck Surgery, Turku University Hospital and Turku University, 20520 Turku,

Finland

*

S Supporting Information

ABSTRACT: Cluster of diﬀerentiation 73 (CD73) converts

adenosine 5′-monophosphate to immunosuppressive adenosine,

and its inhibition was proposed as a new strategy for cancer

treatment. We synthesized 5′-O-[(phosphonomethyl)phosphonic

acid] derivatives of purine and pyrimidine nucleosides, which

represent nucleoside diphosphate analogues, and compared their

CD73 inhibitory potencies. In the adenine series, most ribose

modiﬁcations and 1-deaza and 3-deaza were detrimental, but 7-

3

deaza was tolerated. Uracil substitution with N -methyl, but not

larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl

4

modiﬁcations were not tolerated. N -(Aryl)alkyloxy-cytosine de-

rivatives, especially with bulky benzyloxy substituents, showed

increased potency. Among the most potent inhibitors were the 5′-O-

[

(

(phosphonomethyl)phosphonic acid] derivatives of 5-ﬂuorouridine

4l), N -benzoyl-cytidine (7f), N -[O-(4-benzyloxy)]-cytidine (9h), and N -[O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) (K

4

i

values 5−10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes

showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the

most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

INTRODUCTION

structure as catalysis occurs. Two conformational classes have

been determined: an open and a closed form.

Although the substrate, AMP, is not a potent agonist of

■

Ecto-5′-nucleotidase (ecto-5′-NT, eN, CD73, EC 3.1.3.5) is a

glycosylphosphatidylinositol (GPI)-linked cell surface enzyme

that dephosphorylates extracellular nucleoside monophos-

adenosine receptors (ARs), the enzymatic reaction product,

adenosine, activates four AR subtypes (A AR, A AR, A AR,

1

−3

1

2A

2B

phates.

The enzyme can be cleaved from its GPI linker

6

and A AR). One of the important activities of adenosine is the

suppression of inﬂammation. Thus, cluster of diﬀerentiation

3

6

and is also present in a soluble active form in serum. The

vertebrate enzyme selectively hydrolyzes adenosine 5′-mono-

phosphate (AMP) over adenosine 2′- or 3′-monophosphates,

73 (CD73) upregulation and the increased production of

adenosine are beneﬁcial in chronic inﬂammatory diseases. A

1

leading to elevated extracellular concentrations of adenosine.

coexpression of CD73 and A AR found in many tissues

2

A

The X-ray crystallographic structures of the enzyme in complex

4

,5

with either an inhibitor or a substrate have been reported.

There is a marked conformational rearrangement of the

Received: January 27, 2019

©

XXXX American Chemical Society

A

DOI: 10.1021/acs.jmedchem.9b00164

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Journal of Medicinal Chemistry

Article

20

Chart 1. Selected Inhibitors of Ecto-5′-Nucleotidase (eN, CD73)

a

Scheme 1. Synthesis of Adenosine 2a−g and Uridine Derivatives 4a−y

a

Reagents and conditions: (a) DCC (3 equiv), methylene diphosphonic acid (1.5 equiv), DMF, room temp, 3−24 h; for compounds 4w and 4x:

DCC (3 equiv), ethylene diphosphonic acid (1.5 equiv), DMF, room temp, 3 h (b) methylenebis(phosphonic dichloride) (3 equiv), trimethyl

phosphate, 0 °C, 30 min, then TEAC buﬀer pH 8.4−8.6, rt, 30 min.

1

8,19

including the brain and immune cells, especially when

ment of CD73 assays

inhibitors of the enzyme.

has led to reports of diverse

2

0−24

inﬂammation is present, allows the concerted activation of

Among the ﬁrst potent

2

,7,8

this receptor.

elevated CD73 and adenosine counteract the body’s immune

However, in the tumor microenvironment,

inhibitors to be identiﬁed was adenosine-5′-O-

[(phosphonomethyl)phosphonic acid (I, α,β-methylene-ADP,

AOPCP) (Chart 1). Also, various anthraquinone and

sulfonamide derivatives were found to be competitive

inhibitors, whereas polyphenols and polyoxometalates are

noncompetitive CD73 inhibitors. Recently, adenine nucleotide

analogues of I that display low nanomolar potency in

inhibition of CD73 were identiﬁed. Modeling based on the

X-ray structures of CD73 has aided in the design of novel

9

−14

25

21

defense against the tumor.

This is particularly important in

2

cancer immunotherapy, for which coadministration of either

26

27

an A AR antagonist or an inhibitor of CD73 oﬀers synergistic

2

A

antitumor activity. A CD73 inhibitor also potentiates the in

vivo anticancer eﬀect of inhibitors of nicotinamide phosphor-

1

5

12

ibosyltransferase, which is required for the biosynthesis of

+

intracellular NAD . Antibodies against CD73 are currently

16,17

28

undergoing clinical trials for cancer therapy.

The develop-

inhibitors. The present work complements that study

B

DOI: 10.1021/acs.jmedchem.9b00164

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Journal of Medicinal Chemistry

Article

a

Scheme 2. Synthesis of Cytosine-Derived 5′-O-[(Phosphonomethyl)phosphonic Acid] Derivatives 7a−f and 9a−i

a

Reagents and conditions: (a) DCC (3 equiv), methylene diphosphonic acid (1.5 equiv), DMF, room temp, 3−24 h; (b) methylenebis-

3

(phosphonic dichloride) (3 equiv), trimethyl phosphate, 0 °C, 30 min, then TEAC buﬀer pH 8.4−8.6, rt, 30 min: (c) R -O-NH xHCl, pyridine, 80

C, 12 h. (d) alkyl iodide (ol, 1.5 equiv), K CO (1.7 equiv) in DMF/acetone (1:1) at 50 °C for 3 d.

2

°

2

3

through the exploration of the structure−activity relationships

SARs) of both purine and pyrimidine nucleotides as inhibitors

of CD73. An advantage of inhibitors derived from pyrimidines

would be that if hydrolyzed to the parent nucleoside, they

would not activate ARs, unlike adenine nucleotide I and its

congeners.

combination of ion-exchange and reverse-phase C18 chroma-

tography. The SAR of the purine scaﬀold in AOPCP

derivatives was extensively explored in a previous study,

leading to CD73 inhibitors with potency in the low nanomolar

(

12

range. However, the SAR of the ribose moiety of AOPCP

derivatives had not been explored. Therefore, we prepared 2′-

deoxy (2a), 2′-amino-2′-deoxy (2b), and 3′-deoxy (2d)

AOPCP derivatives by reacting the respective nucleoside

with methylene diphosphonic acid in the presence of DCC in

DMF (Scheme 1). In the case of 2b, the reaction proceeded

very slowly, and the 3′-phosphonate side product (2c) was

formed to an equal extent (0.5% yield). Compound 2c was

isolated, and its structure was unequivocally determined using

RESULTS

■

Chemistry. There are several commonly used multistep

methods for the preparation of nucleoside-5′-O-

(phosphonomethyl)phosphonic acid] derivatives, that is,

[

either reacting the protected nucleoside with activated

bisphosphonate or utilizing methylene diphosphonic acid and

1

2

9−31

H− H-COSY NMR spectroscopy. To address ring variations

coupling reagents.

However, despite the use of protecting

of the adenine moiety, the 1-deaza (2e), 3-deaza (2f), and 7-

deaza (2g) AOPCP derivatives were prepared by the reaction

of unprotected nucleosides with methylenebis(phosphonic

dichloride) in trimethyl phosphate to increase yields (Scheme

groups, these synthetic strategies suﬀer overall from very low

yields. Previously, we were able to demonstrate that

phosphonylation reactions of unprotected nucleosides using

methylenebis(phosphonic dichloride) in trimethyl phosphate

provided the nucleoside-5′-O-[(phosphonomethyl)phosphonic

1

).

20

Then, we turned our focus to pyrimidine-derived 5′-O-

acids] as the main products under optimized conditions.

[(phosphonomethyl)phosphonic acids, for example, UOPCP

Furthermore, reacting unprotected nucleosides with 1.5 equiv

methylene diphosphonic acid and 3 equiv dicyclohexylcarbo-

diimide (DCC) in dimethylformamide (DMF) led to the

formation of nucleoside-5′-O-[(phosphonomethyl)phosphonic

acids] as the main products as well. Therefore, we employed

solely unprotected nucleosides as starting materials in our

syntheses. When additional phosphonylation occurred at the

(

4a), prepared by phosphonylation of uridine (3a). Fur-

3

thermore, N -substituted nucleosides 3b −e were prepared via

nucleophilic substitution using 3a (Supporting Information ),

and their subsequent phosphonylation aﬀorded compounds

4b−e. Substitution of the uracil 5-position was explored with

methyl 4f−h and halogen 4l−o derivatives. The reaction of 5-

ethynyl-uridine with methylenebis(phosphonic dichloride) led,

besides the desired 5-ethynyl-uridine derivative 4i, to the

2

′- and/or 3′-position, these side products were easily

separated from the desired 5′-substituted product through a

C

DOI: 10.1021/acs.jmedchem.9b00164

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formation of 5-(1-chlorovinyl)uridine- (4j) and 5-(1-chlor-

ovinyl)-3-methyluridine-5′-O-[(phosphonomethyl)phosphonic

acid] (4k) through the addition of HCl to the alkyne bond. In

order to explore the variations of the 2′-position at the ribose

moiety, 2′-deoxy (4p), 2′-amino-2′-deoxy (4q), 2′-azido-2′-

deoxy (4r), and 2′-ﬂuoro-2′-deoxy (4s) derivatives were

prepared. The role of the stereochemistry at the 2′-position

was addressed through the synthesis of 2′-ara-ﬂuoro-2′-

deoxyuridine- (4t) and 1-(β-D-arabinofuranosyl)-uridine-5′-

O-[(phosphonomethyl)phosphonic acid (4u). Synthesis of 6-

azauridine derivative (4v) allowed the introduction of an H-

bond acceptor at the 6-position. In addition to the methylene

group, the linker between the two phosphonate groups was

extended to an ethylene moiety by reacting ethylene

diphosphonic acid with the respective nucleosides 3a and 3f

to aﬀord uridine- (4w) and 5-methyluridine-5′-O-

Table 1. Inhibitory Potency of Adenine-Based AOPCP

a

Analogues 2a−g at Rat CD73

K ± SEM (nM)

i

(

% inhibition at

indicated

concentration), rat

CD73

compd

substitution

R¹

R²

I, (AOPCP)

OH

H

167 ± 53

2

a

b

c

d

e

f

>1000 (30%)

>1000 (2%)

>1000 (8%)

1970 ± 220

>1000 (36%)

88.6 ± 4.0

[

(phosphonoethyl)phosphonic acid] (4x). 2-Thiouridine

^N^H2

derivative 4y was prepared following a previously published

4

1

5′-OH

OP O CH

5

^N^H2

procedure.

2

5

H

OH

Next, cytidine derivatives varying at the 2′-position (7a and

X = CH

Y = CH

Z = CH

OH

7

(

b), as well as at the 5-position (7c−e), were prepared

4

Scheme 2). The N -benzoyl COPCP derivative 7f displayed

g

gradual decomposition when left at room temperature (rt)

over several weeks in aqueous solution to UOPCP (4a) and

COPCP (7a) (Supporting Information). In order to introduce

bulky aromatic substituents at the 4-position of the cytosine

moiety without the inherent instability issues as seen with the

a

X, Y, Z = N, unless otherwise noted, compound 2c: 5′-OH.

34

by Tyr in rat). Previous studies had shown that the potency

of competitive inhibitors targeting rat CD73 showed

4

12

N -benzoyl COPCP derivative 7f, we turned our focus toward

comparable or higher potency for human CD73. Never-

alkoxyimino derivatives 9a−i. The required nucleosides were

theless, the most potent compounds (4l, 7f, 9d, 9e, 9g, and

prepared by either reacting cytidine (6a), 2′-deoxycytidine

9

h) were analyzed using recombinant soluble human CD73.

(

6b), 5-ﬂuorocytidine (6d), or 5-methylcytidine (6e) with

Furthermore, to study the inhibitors in a less artiﬁcial

environment, they were investigated on membrane-anchored

CD73 using membrane preparations derived from the triple-

negative breast cancer (TNBC) cell line MDA-MB-231. These

cells overexpress CD73, serve as an in vitro model for TNBC,

and had previously been used for the development of

7

respective alkoxyamino derivatives in pyridine, followed by

the phosphonylation reaction to aﬀord compounds 9a−f, or by

reacting cytidine (6a) with benzyloxyamine, followed by

nucleophilic substitution at the 3-position (Supporting

Information) and subsequent 5′-O-phosphonylation of the

nucleosides to aﬀord compounds 9h and 9i (Scheme 2).

Furthermore, 3-deazauridine-5′-α,β-methylene-diphosphate

16,37−40

therapeutic antibodies against CD73.

Structure−Activity Relationships. AOPCP (I) displays

(

10), (S)-methanocarba-5′-α,β-methylene-diphosphate (11),

moderate CD73 inhibitory activity (K , nM) (167, Table 1),

i

and the ethyl ester of 2-thio-uridine diphosphate (UDP)

whereas UOPCP (4a, 1830, Table 2) and COPCP (7a, 898,

Table 3) are 11- to 5-fold less active. Variations at the ribose

moiety 2a−2d, 4p−4u, and 7b are not tolerated, with the

exception for 2′-ara-ﬂuoro-2′-deoxyuridine 5′-α,β-methylene-

diphosphate (4t, 1750 nM) that displays similar inhibitory

activity compared to UOPCP (4a). 3-Deaza (2e) and 1-deaza

(2f) derivatives of AOPCP are inactive, whereas the 7-deaza

analogue (2g, 88.6 nM) displays 2-fold higher CD73 inhibitory

activity than AOPCP.

Introduction of a substituent larger than a methyl group at

the uridine 3-position is not tolerated: 3-methyluridine (4b,

1864 nM) is equipotent to UOPCP (4a), whereas 3-ethyl-

(4c), 3-propyl- (4d), and the 3-benzyl-uridine-5′-α,β-methyl-

ene-diphosphate (4e) are inactive. These results indicate the

limited size of the binding pocket. Substitution at the uridine

5-position showed the following rank order of potency (K_i,

nM): 5-F (4l, 14.8) > 5-Cl (4m, 86.7) = 5-Br (4n, 88.7) > 5-I

(4o, 162) ≥ 5-ethynyl (4i, 276) ≥ 5-methyl (4f, 338) ≥ 5-(1-

chlorovinyl) (4j, 424). Combination of a 5-methyl group with

variations at the ribose 2′-position [2′-deoxy (4g, 639 nM) and

2′-methoxy (4h, inactive)] as well as combining a 5-(1-

chlorovinyl) group with a 3-methyl substituent (4k, 1050 nM)

reduced inhibitory activity. Extending the distance between the

phosphonate groups by introducing an ethylene linker (4w,

3

2

(12) were prepared to extend the exploration of SAR of the

nucleotide analogues as CD73 inhibitors. All phosphonate-

substituted nucleotides were puriﬁed to homogeneity by ion-

exchange chromatography, followed by reverse-phase C18

HPLC.

Pharmacological Evaluation. The potency of the

compounds to inhibit CD73 was determined by a radiometric

3

CD73 assay using [2,8- H]AMP as a substrate and

2

9,33

recombinant soluble rat CD73.

After the enzymatic

reaction, the substrate was separated from its product

3

[

2,8- H]adenosine by precipitation with lanthanum chloride,

34

followed by ﬁltration through glass ﬁber ﬁlters. For

compounds that inhibited CD73 activity by more than 50%

at an initial screening concentration of 1 μM, full

concentration−response curves were determined in at least

three separate experiments performed in duplicates using 10

diﬀerent concentrations of the inhibitor. K values were

i

calculated from the obtained IC values using the Cheng−

50

3

0

Prusoﬀ equation. Results are summarized in Tables 1−3, and

concentration−inhibition curves are shown in Figures 1 and 2.

Rat CD73 is similar to the human isoform (87% sequence

35,36

identity, BLAST algorithm

), and the active sites only diﬀer

in a single amino acid (Phe in the human enzyme is replaced

D

DOI: 10.1021/acs.jmedchem.9b00164

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Journal of Medicinal Chemistry

Article

Table 2. Inhibitory Potency of Uridine-Derived Nucleotides

quality control liquid chromatography−mass spectrometry

(LC−MS) experiments displayed decomposition of compound

a

4

a−y as Rat CD73 Inhibitors

7

f to UOPCP, which was additionally conﬁrmed via NMR

experiments (Supporting Information, Figure S1). In order to

address the hydrophobic subpocket of CD73, we envisaged the

introduction of an alkoxyimino group at the 4-position, leading

to an increase in potency in the following order (K , nM):

i

H CON (9a, 257) < benzyloxy-N (9b, 112) < 4-F C-

3

benzyloxy-N (9d, 30.3) ≤ naphthalen-2-ylmethoxy-N

(

9e, 18.8). Combination of a 4-benzyloxyimino group with a

methyl group at the 5-position (9f, 321 nM) did not improve

the CD73 inhibitory activity, and introduction of a 5-F

substituent had no eﬀect (9g, 85.1 nM). Remarkably,

introduction of a methyl group at the 3-position of the

benzyloxyimino derivative (9h, 3.67 nM) led to a 23-fold

increase in inhibitory activity and provided the most potent

compound of the series. However, the tolerated size of the

substituent remained limited to a methyl group, as the ethyl

derivative (9i, 262 nM) displayed a 100-fold drop in inhibitory

potency. 3-Deazauridine-5′-α,β-methylene-diphosphate (10),

K ± SEM (nM)

i

(

% inhibition at

indicated

concentration), rat

CD73

_R1

_R2

compd

substitution

Y = CH₃

UOPCP 4a

OH

H

OCH₃

OH

H

1830 ± 530

1860 ± 400

>1000 (4%)

>1000 (7%)

>1000 (3%)

338 ± 56

639 ± 65

>1000 (3%)

276 ± 37

4

b

c

d

e

f

g

h

i

H

Y = C H

2

5

7

Y = C H

3

Y = benzyl

X = CH₃

X = ethynyl

X = 1-chlorovinyl

X = 1-chlorovinyl,

Y = CH₃

X = F

X = Cl

X = Br

X = I

(

S)-methanocarba-uridine-5′-α,β-methylene-diphosphate (11),

32

and the ethyl ester of 2-thio-UDP (12) were inactive at

CD73.

Although it is well-known that rat and human CD73 share

identical orthosteric binding sites, and species diﬀerences for

competitive inhibitors are therefore moderate, the most potent

compounds (4l, 7f, 9d, 9e, 9g, and 9h) were additionally

evaluated for the inhibition of human CD73. We employed a

recombinantly expressed soluble enzyme (Figure 3) as well as

membrane preparations of CD73-expressing MDA-MB-231

cells (Figure 4). The comparison of potencies measured with

the diﬀerent sources of CD73 (see Table 4 and Figure 5)

conﬁrmed the observation that AOPCP analogues are usually

j

424 ± 27

1050 ± 290

k

4

l

OH

H

F

OH

H

14.8 ± 1.9

86.7 ± 7.6

88.7 ± 12.5

162 ± 4

m

n

o

p

q

r

>1000 (37%)

>1000 (2%)

>1000 (9%)

>1000 (11%)

1750 ± 380

>1000 (8%)

>1000 (21%)

>1000 (4%)

>1000 (9%)

>1000 (7%)

NH₂

N₃

slightly more potent at human CD73 as compared to the rat

4,20

enzyme.

Compounds 4l, 7f, 9d, and 9e are 2- to 3-fold

s

t

F

H

more potent, whereas 9g is 5-fold more potent. However, the

most potent compound at rat CD73 (9h, 3.67 nM) is slightly

u

v

w

x

less potent at the human enzyme (K , soluble CD73, 10.6 nM;

i

V = N

n = 2

n = 2, X = CH₃

W = S

OH

membrane preparation, 7.96 nM). All tested inhibitors were

similarly potent at soluble CD73 as compared to the

membrane-bound enzyme.

y

a

_R2_,_X_,_Y₌_H_,_W₌_O_,_V₌_C_H_,_Z₌_N_,_n₌₁_,_u_n_l_e_s_s_o_t_h_e_r_w_i_s_e_n_o_t_e_d_.

Selectivity. UDP-Activated Receptors. Various pyrimidine-

derived nucleoside 5′-diphosphonates were previously shown

to activate two subtypes of P2Y receptors, P2Y and P2Y

receptors, both of which are UDP-activated. Therefore, the

activity of representative compounds 4l, 7f, and 9h was tested

6

14

41

4

x) led to a complete loss of CD73 inhibition. Furthermore,

replacement of 2-oxo by 2-thio (4y) or the introduction of a

nitrogen atom at the 6-position was not tolerated.

at human P2Y

summarized in Table 5. A calcium assay of P2Y

activation in astrocytoma cells and a ﬂuorescent binding

and P2Y14 receptors, and the results are

receptor

6

Because the SAR around the uridine scaﬀold appeared to be

rather limited, tolerating only a small methyl group at the 3-

position (4b) and rather small, highly electronegative

substituents at the 5-position, such as ﬂuoro (4l), we decided

to further explore the cytidine scaﬀold. COPCP (7a) was twice

as potent as UOPCP (4a). However, for the COPCP

derivatives, introduction of a substituent at the 5-position

had a less pronounced eﬀect, leading to an increase in potency

in the following rank order (K , nM): 5-CH (7e, 2030) < 5-H

6

42

competition assay in whole mammalian cells (CHO)

43

expressing the P2Y14 receptor were used. 5-Fluorouridine-

5′-α,β-methylene-diphosphate (4l) activated the P2Y receptor

(EC 203 nM) and also showed aﬃnity for the P2Y receptor

6

5

0

14

(IC 362 nM). The 4-benzoylcytidine derivative (7f) was less

5

0

potent at P2Y and P2Y receptors with potencies in the

6

14

micromolar range. However, the benzyloxyimino-3-methylcy-

tidine-5′-α,β-methylene-diphosphate (9h) was completely

inactive at P2Y and P2Y receptors even at high

i

3

(7a, COPCP, 898) ≤ 5-I (7c, 502) ≤ 5-F (7d, 349) but

altogether rather moderate activity. Surprisingly, introduction

of a benzylamide at the 4-position (7f) provided a 5′-α,β-

methylene-diphosphate derivative with a CD73 inhibitory

potency of 13.9 nM. This high potency indicated the presence

of a large hydrophobic subpocket that could be probed by

further structural modiﬁcation of the inhibitor. Unfortunately,

6

14

concentration (3.0 μM) (Table 6).

Cytosolic Nucleotidases. The synthesized compounds are

negatively charged under physiological conditions, and

membrane permeability is therefore expected to be low.

However, cellular uptake via transporters cannot be completely

E

DOI: 10.1021/acs.jmedchem.9b00164

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Journal of Medicinal Chemistry

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Table 3. Inhibitory Potency of Cytosine Derivatives 7a−f and 9a−i at Rat CD73

compd

substitution

R¹

R²

K_i± SEM (nM) (% inhibition at indicated concentration) rat CD73

COPCP 7a

OH

H

benzoyl

CH₃

benzyl

898 ± 63

>1000 (18%)

502 ± 83

349 ± 41

2030 ± 670

13.9 ± 1.6

257 ± 39

112 ± 15

780 ± 15

30.3 ± 4.2

18.8 ± 3.2

321 ± 9

7

9

b

c

d

e

f

a

b

c

d

e

f

X = I

X = F

X = CH₃

OH

H

OH

4-triﬂuoromethylbenzyl

naphth-2-ylmethyl

benzyl

X = CH₃

X = F

Y = CH₃

Y = C H

g

h

i

benzyl

85.1 ± 7.5

3.67 ± 0.26

262 ± 46

2

5

Figure 1. Concentration−inhibition curves of selected compounds at

Figure 2. Concentration−inhibition curves of selected compounds at

soluble rat CD73. Rat enzyme K : 53 μM; AMP concentration: 5

soluble rat CD73. Rat enzyme K : 53 μM; AMP concentration: 5 μM.

m

μM; data points are from three separate experiments performed in

Data points are from three separate experiments performed in

duplicates. For K values, see Tables 1 and 2.

duplicates. For K values, see Table 3.

i

excluded. To test whether the developed CD73 inhibitors can

additionally block cytosolic nucleotidases, we utilized a

cytosolic preparation of CD73 knockout melanoma cells

liberating assays, capillary electrophoresis, chromatography-

based assays, and lead nitrate-based enzyme histochemistry.

The latter technique is of particular importance in deﬁning the

distribution of ecto-nucleotidase activities within a tissue,

taking advantage of the abilities of these enzymes to generate

3

44

and tested inhibition of AMP hydrolysis by selected

compounds 4l, 7f, 9h, and 9e. Results are collected in Table

45

7

. While a mixture of the phosphatase inhibitors NaF and

inorganic phosphorus (P ) when incubated with appropriate

i

4

6

levamisole inhibited adenosine 5′-triphosphate hydrolysis

under the applied conditions by 70%, none of the four

investigated CD73 inhibitors showed any signiﬁcant inhibition

of AMP hydrolysis. Adding one of the CD73 inhibitors to the

mixture of NaF and levamisole did not change the percentage

of AMP hydrolysis. This clearly indicates that the investigated

compounds selectively inhibit AMP hydrolysis by CD73 but

not by cytosolic nucleotidases.

nucleotide substrates in the presence of lead nitrate, Pb-

(NO ) . This technique was originally employed by Wachstein

3

2

and Meisel for the histochemical characterization of hepatic

48

phosphatases. Using this approach, tissue localization of

CD73 and other ecto-nucleotidases has been characterized as

3

4

brown signal in images of the murine brain, thoracic aortas,

as well as other human and rodent tissues.

3

Figure 6 depicts representative staining images of human

tonsillar CD73-mediated AMPase activity, showing selective

and spatially distinct localization of enzymatic activity in the

germinal centers and connective tissues. Compound 9h at 1,

In Situ Ecto-Nucleotidase Activity Assay. Diﬀerent

analytic approaches have been employed for the measurement

of ecto-nucleotidase activities, including colorimetric P_i-

F

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Journal of Medicinal Chemistry

Article

Figure 5. Comparison of pK values of selected compounds 4l, 7f, 9e,

i

9h, 9d, and 9g determined at recombinant soluble rat and human and

at membrane-bound native CD73.

Figure 3. Concentration−inhibition curves of selected compounds at

Table 5. Potencies of Selected Compounds at Human

Soluble and Membrane-Bound CD73

soluble human CD73. Human enzyme K : 17 μM; AMP

m

concentration: 5 μM. Data points are from three separate experiments

performed in duplicates. For K values, see Table 5.

i

human soluble

human

rat soluble CD73

i

CD73 K ± SEM

membrane-bound CD73

i

compd K ± SEM (nM)

(nM)

K ± SEM (nM)

i

4

7

9

l

f

d

e

g

h

14.8 ± 1.9

13.9 ± 1.6

30.3 ± 4.2

18.8 ± 3.2

85.1 ± 7.5

3.67 ± 0.26

5.33 ± 0.73

4.58 ± 0.55

14.0 ± 1.6

6.88 ± 1.05

15.9 ± 1.1

10.6 ± 0.4

4.51 ± 0.13

5.68 ± 0.75

10.1 ± 1.4

6.29 ± 0.45

16.6 ± 0.7

7.96 ± 0.57

Table 6. Potency of α,β-Methylene Analogues of UDP and

CDP as Agonists at the Human P2Y R and Inhibitors of

6

Fluorescent Ligand Binding at the Human P2Y R

14

hP2Y EC ± SEM

6

50

(

nM) (% activation at

indicated

hP2Y₁₄IC ± SEM

CD73

50

(nM) (% inhibition at

K ± SEM

i

compd

concentration)

indicated concentration) (nM) rat eN

Figure 4. Concentration−inhibition curves of selected compounds at

4l

7f

9h

203 ± 30

1390 ± 220

>3000 (3.6%)

362 ± 90

6660 ± 4740

>3000 (inactive)

14.8 ± 1.9

13.9 ± 1.6

3.67 ± 0.26

membrane preparations of the human TNBC cell line MDA-MB-231,

which natively expresses CD73. K : 14.8 μM; AMP concentration: 5

m

μM; data points are from three separate experiments performed in

duplicates. For K values, see Table 5.

i

Molecular Modeling. Recently, a high-resolution (2.0 Å,

PDB ID: 4H2I) CD73−AOPCP complex structure in the

closed form was reported, and this crystal structure provided

insight into the interaction patterns of the purine derivative,

AOPCP, with the amino acid residues and the two zinc ions in

the binding site. In order to gain further insights into the

molecular determinants involved in binding of the pyrimidine

derivatives, we subsequently docked one of the potent and

selective molecules, 9h, into the human CD73 binding site and

1

0, and 100 nM concentrations inhibited the catalytic activity

of CD73 in a concentration-dependent manner, in situ more

potently than AOPCP, but it is a highly reversible CD73

inhibitor. Therefore, it was necessary for 9h to remain in the

assay medium during both the tissue pretreatment with the

inhibitor tested and subsequent incubation with the AMP

substrate.

49

Table 4. Inhibitory Potency at Rat Ecto-5′-Nucleotidase of 3-Deazauridine-Derived (10), (S)-Methanocarba-Based (11)

Nucleotides, and Ethyl Ester 12

compd

K_i(nM) (% inhibition at indicated concentration) rat CD73

10

11

12

>1000 (19%)

>1000 (12%)

>1000 (10%)

G

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Article

Table 7. Inhibition of Cytosolic AMP Hydrolysis by

Selected CD73 Inhibitors Investigated in a CD73-Knockout

Melanoma Cell Line

a

compound (concentration)

inhibition of AMP hydrolysis ± SEM (%)

70 ± 1

levamisole (1 mM) and

NaF (5 mM)

4

7

9

l (100 μM)

f (100 μM)

e (100 μM)

h (100 μM)

4 ± 13

−3 ± 6

−5 ± 5

−2 ± 3

a

Determined in a malachite green assay using a cytosolic extract of

MaMel.65-CD73 cells (for details, see Experimental Section). Data

are from three independent experiments performed in duplicate.

ko

Figure 7. Binding poses of AOPCP and 9h. (A) Binding pose of

AOPCP (yellow) obtained from the X-ray structure (PDB ID: 4H2I

used as a starting point) and (B) docked pose of 9h (cyan) with

important residues (gray) and zinc ions (marine blue) in the binding

site of the human CD73. 2D interaction diagrams of (C) AOPCP and

Figure 6. Histochemical analysis of the distribution of eN/CD73 in

human tonsils. (A) Tissue samples were stained with hematoxylin and

eosin. (B−E) Tonsillar eN/CD73 (AMPase) activity was assayed by

incubating tissue cryosections with 100 μM AMP and 1.5 mM

Pb(PO₄)₂in the absence (control) or presence of the indicated

concentrations of AOPCP and 9h, followed by microscopic detection

(

D) 9h. (E) Binding pose comparison of the AOPCP crystal structure

(yellow) and docked 9h (cyan). The amino acid residues (gray),

AOPCP (yellow), and 9h (cyan) are shown as stick models, and the

zinc ions in the active site are represented as spheres (marine blue).

Oxygen atoms are colored in red, nitrogen atoms in blue, and

phosphorus atoms in orange.

of the nucleotide-derived P as a brown precipitate. All images were

i

captured as tile scans of adjacent areas by using the Pannoramic 250

slide scanner. CT, connective tissue; GC, germinal center; IFA,

interfollicular area. Scale bars: 4 mm (left images) and 1 mm (right).

particular, modiﬁcation of the 2′-OH group leads to a large

decrease in potency (2a) and indicates the importance of its

interaction and presumably its role in ligand orientation in the

substrate binding pocket. The docked pose of 9h shows the

same interaction proﬁle and binding orientation in the enzyme

active site. Although the pyrimidine moiety of 9h (like the

adenine ring of AOPCP) is stacked between Phe417 and

Phe500 and stabilized through H-bonding interactions

between the diphosphonate group (PCP) and the enzyme, it

is expected to have weaker π−π interactions with the aromatic

residues than the adenine ring of AOPCP. Because of the

interactions of N3 (H-bond interaction with Asn390) and N1

(interaction with a water molecule) of the adenine ring with

the enzyme, the N1- (2e) and N3-deaza analogues (2f) were

not tolerated. In contrast, an N7-deaza-adenine modiﬁcation

(2g) is tolerated because N7 does not form any interaction

with the amino acid residues or water molecules inside the

binding pocket of CD73. The keto group at position 2 of the

cytidine ring is oriented in the same direction as the N3 of the

adenine ring and likely forms an interaction with the same

residue, Asn390, as seen with AOPCP (Figure 7E). Adjacent to

(

F) Quantiﬁcation of the enzymatic activities using ImageJ are shown

as mean pixel intensities (mean ± SE) of ﬁve germinal centers. **P <

.01 compared with control, determined by one-way analysis of

0

variance with Dunnett’s multiple comparison test.

studied its interactions with the enzyme (Figure 7). Similar to

AOPCP, the results from the docking studies with 9h showed

that it occupies the same binding site (Supporting Information,

Figure S3), and the diphosphonate chain (PCP) is bound

between the two zinc ions, α-phosphonate forming H-bond

interactions with Asn245, Arg354, and Arg395 and the β-

phosphonate group with Asn117, His118, and Arg395 (Figure

7

A−D). The binding orientation of AOPCP obtained from the

crystal structure shows that the ribose hydroxyl groups form H-

bond interactions with Arg354, Arg395, and Asp506. This

interaction could be important for positioning the ligand in the

correct orientation in the binding pocket of CD73. The

importance of the ribose 2′- and 3′-hydroxyl groups was

conﬁrmed with derivatives 2a−d, where modiﬁcation of the

two hydroxyl groups in AOPCP was not tolerated. In

H

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Journal of Medicinal Chemistry

Article

the exocyclic 6-amino group of the adenine ring, a large pocket

is exposed to the surface with bound water molecules. This

pocket was explored with diﬀerent substitutions at the cytidine

ring, and among them, the benzyloxyimino residue at the 4-

position of cytidine (9h) was found to confer high CD73

inhibitory activity (see Table 5). Upon exploring the pocket

further, three loops (Asp121−Val124, Leu184−Asn190, and

Leu415−Thr420) were observed adjacent to the binding

pocket of 9h, which possibly provide hydrophobic as well as

the polar or charged residues as potential interaction partners

modiﬁcation resulted in a loss of potency; only 2′-deoxy

(intermediate) and 2′-deoxy-2′-ﬂuoro (arabino, potent)

analogues retained inhibitory activity. In the uracil series, the

nucleobase N3 could be substituted with methyl but not larger

alkyl groups. 2-Thiouracil, 3-deaza-uracil, 6-aza-uracil, or 1,2-

diphosphono-ethyl modiﬁcations were not tolerated. However,

the uracil-5-position was amenable to various substitutions,

with a rank order of potency of F > Cl, Br > I, Me.

4

The most successful modiﬁcation was found in N -

(aryl)alkyloxy-cytosine derivatives, especially with bulky

benzyloxy substituents, which increased the inhibitory potency

at CD73. The α,β-methylene 5′-diphosphate derivatives of 5-

(Figure S4). On the other hand, the small electronegative

substituent ﬂuorine at the 5-position of the uracil moiety

provided high potency in comparison to the other substituents

4

ﬂuorouridine (4l), 4-benzoylcytidine (7f), N -[O-(naphthalen-

4

including H, I, and CH . A possible explanation for this SAR

2-ylmethoxy)]-cytidine (9e), and N -[O-(4-benzyloxy)]-3-

3

pattern is that the electronegative ﬂuorine atom is located at a

distance of approximately 4 Å from His118 potentially forming

a H-bond interaction directly with, or mediated through, the

water molecules (located at a distance of ∼3 Å) inside the

binding pocket.

methyl-cytidine (9h) are the most potent CD73 inhibitors

prepared in this work. At the human CD73 isoform, most of

the potent inhibitors examined were at least several-fold more

potent than at the rat isoform. Compound 9h displayed K_i

values of (nM) 3.67 (soluble CD73, rat); 10.6 (soluble CD73,

human); and 7.96 (membrane-bound CD73, human). A major

advantage of such pyrimidine-based inhibitors over adenine

nucleotide analogues is that their hydrolysis products, the

parent nucleosides, do not activate ARs, which would

counteract the intended eﬀects in cancer treatment. Also, 7-

deazaadenosine derivatives are inactive or only very weakly

DISCUSSION

■

We have prepared a range of nucleoside 5′-α,β-methylene-

31,41,42,50−53

diphosphates by standard synthetic methods

and

evaluated them in vitro as CD73 inhibitors. The assay method

using the radiolabeled AMP substrate has been validated to

47,71

3

4

have high reproducibility. This indirect approach to

correcting an imbalance of excess adenosine that occurs in

the microenvironment of a wide range of tumors holds promise

as a cotherapy in the immunotherapy of cancer, and potent

CD73 inhibitors (both small molecules and monoclonal

antibodies) are already in preclinical and clinical develop-

active at ARs,

and the 7-deazaadenine scaﬀold is therefore

preferable to adenine for the development of CD73 inhibitors.

In the CD73 crystal structure with bound AOPCP, the adenine

N7 does not participate in any apparent stabilizing protein

interactions, such as H-bonding. Thus, its replacement with

CH in the more potent 7-deaza analogue 2g might conceivably

displace a water molecule, but in the current X-ray structure,

no water is detected in this region.

The observed SAR was rationalized by docking 9h into the

substrate binding site of human CD73. We were also

concerned about oﬀ-target activity of the phosphonate

1

2,17,32,54−58

ment.

Coinhibition of CD73 and the A AR is also

2A

59

being considered for anticancer therapeutic development. In

addition to cancer, CD73 inhibitors might have utility in

preeclampsia, pulmonary edema, infectious disease, and other

3

3,60,61

conditions.

analogues derived from uracil at the UDP-activated P2Y and

The current study presents important new SAR for AOPCP,

UOCPC, and COPCP analogues as inhibitors of CD73

6

^P²^Y14 receptors. The 5-F derivative 4l interacted with these

two P2Y receptors at higher nanomolar concentrations, but the

(Figure 8). Furthermore, the recognition of a potent COPCP

4

-benzoylcytidine derivative (7f) was less potent at P2Y and

6

P2Y₁₄receptors. The potent inhibition by compound 9h of

CD73 in situ in human tonsils was demonstrated, and it was

more potent than the reference CD73 inhibitor AOPCP.

Importantly, benzyloxyimino-3-methyl-α,β-methylene-diphos-

phate (9h) was completely inactive at P2Y₆and P2Y₁₄

receptors and in inhibition of cytosolic CD73, making it a

particularly useful pharmacological tool compound for the in

vitro and in vivo exploration of CD73 inhibition and an

excellent starting point for future development of CD73 drugs

that act parenterally.

Figure 8. Summary of SAR for nucleotide derivatives as CD73

inhibitors in the purine (left) and pyrimidine series (right).

CONCLUSIONS

■

analogue 9h in the enzyme active site was modeled using a

CD73 X-ray crystallographic structure, and this binding mode

can be used to facilitate a structure-based approach in future

SAR studies. While most of the ribose modiﬁcations resulted in

weak inhibition or inactivity, substantial enhancement of CD73

inhibitory activity was observed with various nucleobase, both

purine and pyrimidine, modiﬁcations. In the adenine series,

most ribose modiﬁcations and 1-deaza and 3-deaza sub-

stitutions were detrimental, but 7-deaza was well tolerated. For

example, on the ribose moiety, 2′-methoxy and 2′-amino

modiﬁcations or a [3.1.0]bicyclohexyl (S)-methanocarba

A series of 50 purine- and pyrimidine-based nucleoside 5′-α,β-

methylene-diphosphates were synthesized and obtained in high

purity. The synthesized nucleosides were evaluated as

inhibitors of CD73 in two species: rat and human (selected

analogues). The AOPCP-derived nucleotides were modiﬁed at

the ribose and the adenine moiety to complement the existing

SAR in the adenine series and to explore an alternative series of

pyrimidine bases. These uridine- and cytosine-derived α,β-

methylene diphosphonates represent an entirely new class of

CD73 inhibitors. Analysis of their SAR allowed optimization,

leading to the development of inhibitors with K values in the

i

I

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4

low nanomolar range. N -(Aryl)alkyloxy-cytosine derivatives,

Preparation of Triethylammonium Hydrogen Carbonate

Buﬀer. A 1 M solution of triethylammonium hydrogen carbonate

(

especially with bulky benzyloxy substituents, increased

potency. The most potent inhibitors at rat CD73 were 5-

ﬂuorouridine (4l, 14.8 nM), 4-benzoylcytidine (7f, 13.9 nM),

TEAC) was prepared by adding dry ice slowly to 1 M triethylamine

solution in deionized water for several hours until the pH of

approximately 8.4−8.6 was indicated using a pH meter.

4

N -[O-(naphthalen-2-ylmethoxy)]-cytidine (9e, 18.8 nM), and

Puriﬁcation of Nucleotides. Ion-Exchange Chromatography.

The crude nucleoside-5′-O-[(phosphonomethyl)phosphonic acid]

derivatives were puriﬁed by ion-exchange chromatography on an

HPLC instrument UltiMate 3000 (Dionex Corp.) with a HiScale 26

20 BH, 26 mm × 130 mm length column. The column was packed

with Source 15Q gel, swelled in a 20% EtOH-solution. Before

puriﬁcation, the column was washed and equilibrated with deionized

water. The sample was prepared by dissolving crude product in 0.5−1

mL of aqueous TEAC buﬀer. Separation was achieved by running a

solvent gradient of TEAC buﬀer: deionized water from 0:100 for 5

min, then from 0:100 to 100:0 in 25 min, followed by a gradient from

4

N -[O-(4-benzyloxy)]-3-methyl-cytidine (9h, 3.67 nM) 5′-α,β-

methylene-diphosphates. Compound 9h displayed particularly

high selectivity for CD73 compared to UDP-activated P2Y

P2Y and P2Y ) receptors and cytosolic 5′-nucleotidase.

(

6

14

Thus, we have expanded the SAR of both purine and

pyrimidine nucleotide analogues as inhibitors of CD73 and

achieved low nanomolar aﬃnity. 5-Halo- and alkyl-uracil

derivatives were also potent CD73 inhibitors. The presented

compounds include the most potent CD73 inhibitors reported

to date and are likely to become useful pharmacological tools

to further elucidate the enzyme’s (patho)physiological role and

its potential as a drug target in cancer immunotherapy and in

other conditions.

1

00:0 to 0:100 in 20 min, and holding 0:100 for 10 min with a ﬂow

rate of 5 mL/min. The UV absorption was detected at 254, 210, and

280 nm. Fractions were collected, and appropriate fractions were

pooled, diluted in water, and lyophilized.

General Procedure A for the Synthesis of Nucleotides. To a

solution of DCC (3 equiv) and the unprotected nucleoside in DMF

(2 mL), methylene diphosphonic acid (1.5 equiv) was added at rt,

and the mixture was allowed to stir at rt for 6−24 h. Samples were

withdrawn at 3−12 h interval for LC−MS to check the disappearance

of nucleosides and to monitor the formation of the desired nucleotide.

On the disappearance of a nucleoside, 10 mL of cold TEAC solution

was added. The mixture was stirred at rt for 30 min, followed by

ﬁltration and lyophilization of the aqueous solution. The mixture of

nucleotide and dinucleotide was separated by ion-exchange

chromatography on Source 15Q. Fractions containing the product

were pooled and evaporated to dryness. The compound was then

puriﬁed by reverse-phase (RP)-HPLC using a gradient of 10 mM

EXPERIMENTAL SECTION

■

Chemical Synthesis. General. Reagents and Instrumentation.

All reagents were commercially obtained from various producers (Alfa

Aesar, Carbosynth, and Sigma-Aldrich) and used without further

puriﬁcation. The purity of all compounds including the starting

material was more than 95%, as determined using high-performance

LC (HPLC). Commercial solvents of speciﬁc reagent grades were

used, without additional puriﬁcation or drying. Analytical thin-layer

chromatography was carried out on Sigma-Aldrich TLC plates, and

compounds were visualized with UV light at 254 nm. Silica gel ﬂash

chromatography was performed using 230−400 mesh silica gel.

Unless noted otherwise, reagents and solvents were purchased from

triethylammonium acetate buﬀer−CH CN from 80:20 to 20:80 in 40

3

1

31

13

Sigma-Aldrich (St. Louis, MO). The H, P, and C NMR spectra

were recorded using a Bruker 400 MHz spectrometer and a DD2 400

MHz or DD2 600 MHz NMR spectrometer (Agilent). DMSO-d₆,

MeOD-d , CDCl , or D O were used as solvents. Shifts are given in

min, and suitable fractions were pooled and lyophilized to obtain the

ﬁnal product as a glassy solid.

General Procedure B for the Synthesis of Nucleotides. A

solution of methylenebis(phosphonic dichloride) (3 equiv) in

trimethyl phosphate (2 mL) cooled to 0 °C was added to a

suspension of the corresponding nucleoside in trimethyl phosphate at

0 °C. The reaction mixture was stirred at 0 °C, and samples were

withdrawn at 10 min interval for LC−MS to check the disappearance

of nucleosides. After 30 min, on the disappearance of a nucleoside, 7

mL of cold 1 M aqueous TEAC buﬀer solution (pH 8.4−8.6) was

added. It was stirred at 0 °C for 15 min, followed by stirring at rt for

30 min. Trimethyl phosphate was extracted using (2 × 100 mL) of

tert-butyl methyl ether, and the aqueous layer was lyophilized. The

mixture of mononucleotide and dinucleotide was separated by ion-

exchange chromatography on Source 15Q. Fractions containing the

mononucleotide product were pooled and evaporated to dryness. The

compound was then puriﬁed by RP-HPLC using a gradient of 10 mM

4

3

2

parts per million relative to the remaining protons of the deuterated

1

13

solvents used as internal standard ( H, C NMR). Puriﬁcation of

ﬁnal compounds was performed by semipreparative HPLC (column:

Luna 5 μm C18(2) 100 Å, LC column 250 × 4.6 mm). Eluent: 10

mM triethylammonium acetate buﬀer−CH CN from 80:20 to 20:80

3

in 40 min, with a ﬂow rate of 5 mL/min. Purities of all tested

compounds were ≥95%, as estimated by analytical HPLC: method A:

eluent: 5 mM triethylammonium phosphate monobasic solution−

CH CN from 100:0 to 50:50 in 20 min and then triethylammonium

3

phosphate monobasic solution−CH CN to 100:0 in 5 min with a ﬂow

3

rate of 1 mL/min (column: Zorbax SB-Aq 5 μm analytical column, 50

×

4.6 mm; Agilent Technologies, Inc.). Method B: eluent: 5 mM

triethylammonium phosphate monobasic solution−CH CN from

0:10 to 0:100 in 20 min and then triethylammonium phosphate

3

9

triethylammonium acetate buﬀer−CH CN from 80:20 to 20:80 in 40

3

monobasic solution−CH CN from 0:100 to 90:10 in 5 min with a

min, then 10 mM triethylammonium acetate buﬀer−CH CN from

3

ﬂow rate of 1 mL/min (column: Zorbax SB-Aq 5 μm analytical

column, 150 × 4.6 mm; Agilent Technologies, Inc.). Method C:

eluent: 5 mM triethylammonium phosphate monobasic solution−

100:0 to 90:10 in 40 min, and then 100:0 in 5 min, with a ﬂow rate of

5 mL/min, and suitable fractions were pooled and lyophilized to

obtain the ﬁnal product as a glassy solid.

CH CN from 80:20 to 20:80 in 20 min and then triethylammonium

2′-Deoxyadenosine-5′-O-[(phosphonomethyl)phosphonic acid]

3

phosphate monobasic solution−CH CN from 20:80 to 80:20 in 10

(2a). Method A. The product was obtained as a colorless solid after

3

1

min with a ﬂow rate of 1 mL/min (column: Zorbax SB-Aq 5 μm

analytical column, 150 × 4.6 mm; Agilent Technologies, Inc.). Peaks

were detected by UV absorption (254 nm) using a diode array

detector. All derivatives tested for biological activity showed >95%

purity in the HPLC system. Low-resolution MS was performed with a

JEOL SX102 spectrometer with 6 kV Xe atoms following desorption

from a glycerol matrix or on an Agilent LC/MS 1100 MSD, with a

Waters (Milford, MA) Atlantis C18 column. High-resolution mass

spectroscopic (HRMS) measurements were performed on a

proteomics optimized Q-TOF-2 (Micromass-Waters) using external

calibration with polyalanine. For lyophilization, a freeze dryer

lyophilization (2 equiv Et N-salt, 33.6 mg, 15%). H NMR (400

3

MHz, D O): δ 8.41 (s, 1H), 8.16 (s, 1H), 6.41 (t, J = 5.9 Hz, 1H),

2

4.17 (s, 1H), 4.08−3.90 (m, 3H), 3.10 (q, J = 7.3 Hz, 11H), 2.76 (dt,

J = 13.3, 6.0 Hz, 1H), 2.55−2.43 (m, 1H), 2.04 (t, J = 18.6 Hz, 2H),

3

1

1.18 (t, J = 7.3 Hz, 16H). P NMR (160 MHz, D O): δ 21.8, 11.8.

2

−

MS (ESI, m/z): 408.0 [M − H] ; ESI-HRMS: calcd m/z for

−

C H N O P , 408.0474; found, 408.0479 [M − H] . HPLC purity

1

16

5

8 2

95% (R = 4.8 min, method HPLC-A).

t

2′-Amino-2′-deoxyadenosine-5′-O-[(phosphonomethyl)-

phosphonic acid] (2b). Method A. The product was obtained as a

colorless solid after lyophilization (0.5 equiv Et N-salt, 1.5 mg, 0.6%).

3

1

(

Labconco FreeZone 4.5) was used.

H NMR (400 MHz, D O): δ 8.59 (s, 1H), 8.28 (s, 1H), 6.45 (d, J =

2

J

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7

1

.4 Hz, 1H), 4.88−4.84 (m, 1H), 4.64 (t, J = 5.6 Hz, 1H), 4.52 (s,

H), 4.18 (q, J = 11.7, 10.7 Hz, 2H), 3.20 (q, J = 7.3 Hz, 3H), 2.19 (t,

2H), 4.17−4.14 (m, 1H), 4.13−4.02 (m, 2H), 3.10 (q, J = 7.3 Hz,

1

3

12H), 2.04 (t, J = 19.6 Hz, 2H), 1.17 (t, J = 7.3 Hz, 18H). C NMR

1

3

J = 19.0 Hz, 2H), 1.28 (t, J = 7.3 Hz, 5H). C NMR (100 MHz,

D O): δ 155.7, 153.0, 149.3, 140.1, 118.8, 86.0 (d, J = 6.0 Hz), 84.6,

(100 MHz, D O): δ 166.5, 152.0, 141.9, 102.6, 88.7, 83.4 (d, J = 6.9

2

3

1

Hz), 73.9, 69.5, 63.3, 46.8 (6C), 27.8 (t, J = 122.4 Hz), 8.3 (6C). P

2

3

1

7

0.1, 63.7, 56.3, 46.8 (2C), 27.6 (t, J = 124.3 Hz), 8.3 (2C). P NMR

NMR (160 MHz, D O): δ 20.6, 12.7. MS (ESI, m/z): 401.0 [M −

2

−

(

160 MHz, D O): δ 18.6, 14.6. MS (ESI, m/z): 423.1 [M − H] ;

H] ; ESI-HRMS: calcd m/z for C H N O P , 401.0157; found,

2

10 15

2

11 2

−

ESI-HRMS: calcd m/z for C H N O P , 423.0583; found, 423.0590

401.0154 [M − H] . HPLC purity 99% (R = 5.6 min, method

1

17

6

8

2

t

−

[

M − H] . HPLC purity 98% (R = 9.9 min, method HPLC-B).

HPLC-B).

t

2

′-Amino-2′-deoxyadenosine-3′-O-[(phosphonomethyl)-

3-Methyluridine-5′-O-[(phosphonomethyl)phosphonic acid]

phosphonic acid] (2c). Method A. The product was obtained as a

colorless solid after lyophilization (0.66 equiv Et N-salt, 1.1 mg,

0

(4b). Method A. The product was obtained as a colorless solid after

1

lyophilization (2 equiv Et N-salt, 4.8 mg, 19%). H NMR (400 MHz,

3

1

.5%). H NMR (400 MHz, D O): δ 8.37 (s, 1H), 8.28 (s, 1H), 6.50

D O): δ 8.00 (d, J = 8.0 Hz, 1H), 6.02 (d, J = 8.0 Hz, 1H), 5.98 (d, J

2

(

d, J = 7.6 Hz, 1H), 5.22 (t, J = 5.6 Hz, 1H), 4.79 (s, 1H), 4.59 (s,

= 3.0 Hz, 1H), 4.38 (d, J = 3.6 Hz, 2H), 4.27 (s, 1H), 4.20 (s, 2H),

1

H), 3.94 (d, J = 2.7 Hz, 2H), 3.21 (q, J = 7.3 Hz, 4H), 2.29 (t, J =

3.30 (s, 3H), 3.21 (q, J = 7.3 Hz, 12H), 2.12 (t, J = 19.3 Hz, 2H), 1.29

1

3

13

9.4 Hz, 2H), 1.28 (t, J = 7.3 Hz, 6H). C NMR (100 MHz, D O): δ

(t, J = 7.3 Hz, 18H). C NMR (100 MHz, D

2

O): δ 165.7, 152.3,

2

55.9, 152.9, 148.7, 141.0, 119.5, 87.3, 86.4, 73.3, 61.3, 54.8, 46.8

139.7, 101.8, 89.8, 83.1, 74.0, 69.3, 63.1, 46.8 (6C), 27.8, 8.3 (6C).

3

1

31

(

2C), 28.2 (t, J = 124.4 Hz), 8.3 (2C). P NMR (160 MHz, D O): δ

P NMR (160 MHz, D O): δ 20.2, 13.2. MS (ESI, m/z): 415.1 [M −

2

−

1

8.8, 14.2. MS (ESI, m/z): 423.1 [M − H] ; ESI-HRMS: calcd m/z

H] ; ESI-HRMS: calcd m/z for C H N O P , 415.0308; found,

415.0311 [M − H] . HPLC purity 96% (R = 3.7 min, method

HPLC-C).

1

17

2

11 2

−

for C H N O P , 423.0583; found, 423.0582 [M − H] . HPLC

purity 98% (R = 6.9 min, method HPLC-B).

11

17

6

8

2

t

3

′-Deoxyadenosine-5′-O-[(phosphonomethyl)phosphonic acid]

3-Ethyluridine-5′-O-[(phosphonomethyl)phosphonic acid] (4c).

(

2d). Method A. The product was obtained as a colorless solid after

Method A. The product was obtained as a colorless solid after

1

lyophilization (1.5 equiv Et N-salt, 2.5 mg, 6%). H NMR (400 MHz,

lyophilization (2 equiv Et N-salt, 26.1 mg, 11%). H NMR (400

3

D O): δ 8.49 (s, 1H), 8.25 (s, 1H), 6.10 (s, 1H), 4.77−4.65 (m, 1H),

MHz, D O): δ 8.01 (d, J = 7.1 Hz, 1H), 6.00 (d, J = 6.5 Hz, 1H),

2

4

1

(

1

3

1

.25 (d, J = 10.2 Hz, 1H), 4.13−4.01 (m, 1H), 3.19 (q, J = 7.3 Hz,

0H), 2.42 (ddd, J = 14.5, 9.2, 5.7 Hz, 1H), 2.25−2.09 (m, 3H), 1.27

5.97−5.94 (m, 1H), 4.47−4.34 (m, 2H), 4.30−4.16 (m, 3H), 3.93 (q,

J = 6.9 Hz, 2H), 3.19 (q, J = 7.4 Hz), 2.19−1.94 (m, 2H), 1.27 (t, J =

1

3

13

t, J = 7.3 Hz, 15H). C NMR (100 MHz, D O): δ 154.7, 151.4,

7.3 Hz), 1.18 (t, J = 7.1 Hz). C NMR (100 MHz, D O): δ 165.3,

2

48.4, 140.3, 118.8, 90.6, 80.4 (d, J = 7.3 Hz), 75.4, 64.9, 46.8 (4C),

3.0, 27.5 (t, J = 124.5 Hz), 8.3 (4C). P NMR (160 MHz, D O): δ

151.8, 139.8, 102.0, 89.8, 83.1, 74.1, 69.0, 62.9, 46.7 (6C), 36.8, 11.8,

3

1

31

2

8.3 (6C). The signal for PCH P could not be observed. P NMR

2

−

8.4, 14.9. MS (ESI, m/z): 408.0 [M − H] ; ESI-HRMS: calcd m/z

(160 MHz, D O): δ 21.1, 12.0. MS (ESI, m/z): 429.1 [M − H] ;

ESI-HRMS: calcd m/z for C H N O P , 429.0470; found,

429.0470 [M − H] . HPLC purity 97% (R = 9.8 min, method

2

−

for C H N O P , 408.0474; found, 408.0471 [M − H] . HPLC

11

16

5

8

2

12 19

2

11 2

−

purity 99% (R = 17.1 min, method HPLC-B).

t

1

-Deazaadenosine-5′-O-[(phosphonomethyl)phosphonic acid]

HPLC-C).

(

2e). Method B. The product was obtained as a colorless solid after

3

-Propyluridine-5′-O-[(phosphonomethyl)phosphonic acid]

1

lyophilization (1.5 equiv Et N-salt, 1.7 mg, 8%). H NMR (400 MHz,

(4d). Method A. The product was obtained as a colorless solid after

3

1

D O): δ 8.50 (s, 1H), 7.99 (d, J = 5.9 Hz, 1H), 6.67 (d, J = 5.9 Hz,

lyophilization (2 equiv Et N-salt, 11 mg, 5%). H NMR (400 MHz,

2

3

1

2

7

1

2

H), 6.14 (d, J = 5.9 Hz, 1H), 4.53 (s, 1H), 4.37 (s, 1H), 4.15 (s,

D O): δ 8.00 (d, J = 7.8 Hz, 1H), 6.03 (d, J = 7.7 Hz, 1H), 6.00 (d, J

2

H), 3.18 (q, J = 7.3 Hz, 7H), 2.17 (t, J = 19.5 Hz, 2H), 1.26 (t, J =

= 3.5 Hz, 1H), 4.43−4.39 (m, 2H), 4.32−4.27 (m, 1H), 4.26−4.13

(m, 2H), 3.87 (dd, J = 8.7, 6.6 Hz, 2H), 3.22 (q, J = 7.3 Hz, 12H),

.3 Hz, 12H). ¹³C NMR (100 MHz, D O): δ 147.9, 145.3, 144.2,

2

39.7, 104.3, 87.0, 84.0 (d, J = 6.2 Hz), 73.9, 70.4, 63.7, 46.7 (4C),

2.30−2.11 (m, 2H), 1.64 (dq, J = 14.8, 7.5 Hz, 2H), 1.30 (t, J = 7.3

7.4, 8.3 (4C). ³¹P NMR (160 MHz, D O): δ 18.6, 14.6. MS (ESI,

13

2

Hz, 18H), 0.92 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, D O): δ

2

+

m/z): 425.0 [M + H] ; ESI-HRMS: calcd m/z for C H N O P ,

25.0622; found, 425.0626 [M + H] . HPLC purity 98% (R = 8 min,

165.5, 152.1, 139.8, 102.1, 89.5, 83.3, 73.9, 69.5, 63.3, 46.8 (6C), 43.2,

1

2

19

4

9 2

+

4

27.2 (1C, PCH P), 20.3, 10.6, 8.3 (6C). The signal for PCH P could

t

2

1

3

method HPLC-C).

-Deazaadenosine-5′-O-[(phosphonomethyl)phosphonic acid]

not be observed in the one-dimensional (1D) experiment. C NMR

3

shift of PCH P was determined using heteronuclear single quantum

2

1

3

1

(

5

=

2f). Method B. H NMR (400 MHz, D O): δ 8.58 (s, 1H), 7.75

2

correlation (HSQC). P NMR (160 MHz, D O): δ 18.4, 14.5. MS

2

−

s, 1H), 7.32 (d, J = 6.2 Hz, 1H), 6.06 (d, J = 6.2 Hz, 1H), 4.69 (t, J =

.9 Hz, 1H), 4.58−4.52 (m, 1H), 4.41 (s, 1H), 4.20 (s, 2H), 3.21 (q, J

7.3 Hz, 3H), 2.17 (t, J = 19.3 Hz, 3H), 1.28 (t, J = 7.3 Hz, 6H). P

(

ESI, m/z): 443.1 [M − H] ; ESI-HRMS: calcd m/z for

C H N O P , 443.0626; found, 443.0634 [M − H] . HPLC purity

−

1

3

21

2

11 2

31

9

5% (R = 10.3 min, method HPLC-C).

t

NMR (160 MHz, D O): δ 18.8, 14.4. MS (ESI, m/z): 423.1 [M −

3-Benzyluridine-5′-O-[(phosphonomethyl)phosphonic acid]

2

−

H] ; ESI-HRMS: calcd m/z for C H N O P , 423.0476; found,

(4e). Method A. The product was obtained as a colorless solid after

1

2

17

4

9 2

−

1

4

23.0473 [M − H] . HPLC purity 97% (R = 9.9 min, method

HPLC-C).

lyophilization (2 equiv Et N-salt, 3.7 mg, 2%). H NMR (400 MHz,

t

3

D O): δ 8.07 (d, J = 7.9 Hz, 1H), 7.44−7.38 (m, 2H), 7.38−7.32 (m,

2

7

-Deazaadenosine-5′-O-[(phosphonomethyl)phosphonic acid]

3H), 6.09 (d, J = 7.9 Hz, 1H), 5.99 (d, J = 3.7 Hz, 1H), 5.16 (d, J =

15.1 Hz), 5.11 (d, J = 15.2 Hz), 4.39 (d, J = 3.9 Hz, 2H), 4.30−4.26

(m, 1H), 4.20 (q, J = 11.4 Hz, 2H), 3.20 (q, J = 7.3 Hz, 12H), 2.20 (t,

(

2g). Method B. The product was obtained as a colorless solid after

1

lyophilization (1 equiv Et N-salt, 13.5 mg, 14%). H NMR (600

MHz, D O): δ 8.13 (br s, 1H), 7.61 (s, 1H), 6.70 (s, 1H), 6.14 (d, J =

3

1

3

2

J = 18.3 Hz, 2H), 1.28 (t, J = 7.3 Hz, 18H). C NMR (100 MHz,

5

4

3

9

1

=

.4 Hz, 1H), 4.52 (t, J = 5.5 Hz, 1H), 4.47 (t, J = 4.4 Hz, 1H), 4.36−

.31 (m, 1H), 4.29 (d, J = 11.6 Hz, 1H), 4.17 (d, J = 11.5 Hz, 1H),

.18 (q, J = 7.3 Hz, 6H), 2.24 (t, J = 20.5 Hz, 2H), 1.26 (t, J = 7.3 Hz,

D O): δ 165.2, 152.1, 140.1, 136.1, 128.9 (2C), 127.8, 127.1 (2C),

2

102.1, 89.5, 83.3 (1C), 74.0, 69.5, 63.2, 46.7 (6C), 44.6, 27.5 (t, J =

3

1

124.1 Hz, 1C, PCH P), 8.3 (6C). P NMR (160 MHz, D O): δ 18.4,

2

H). ¹³C NMR (100 MHz, D O): δ 150.8, 146.9, 142.9, 124.6, 102.6,

−

2

14.9. MS (ESI, m/z): 491.1 [M − H] ; ESI-HRMS: calcd m/z for

−

02.1, 86.7, 83.7 (d, J = 5.4 Hz), 74.6, 70.3, 63.9, 46.7 (3C), 27.6 (t, J

C H N O P , 491.0626; found, 491.0622 [M − H] . HPLC purity

1

7

21

2

11 2

3

1

124.5 Hz), 8.3 (3C). P NMR (160 MHz, D O): δ 18.5, 15.1. MS

98% (R = 11.2 min, method HPLC-C).

2

t

−

(

ESI, m/z): 423.1 [M − H] ; ESI-HRMS: calcd m/z for

5-Methyluridine-5′-O-[(phosphonomethyl)phosphonic acid]

−

C H N O P , 423.0476; found, 423.0476 [M − H] . HPLC purity

(4f). Method A. The product was obtained as a colorless solid after

12

17

4

9 2

1

>

99% (R = 4.3 min, method HPLC-C).

lyophilization (2 equiv Et N-salt, 54 mg, 24%). H NMR (400 MHz,

t

3

Uridine-5′-O-[(phosphonomethyl)phosphonic acid] (4a). Meth-

D O): δ 7.77 (s, 1H), 5.99 (d, J = 5.0 Hz, 1H), 4.46−4.34 (m, 2H),

2

od A. The product was obtained as a colorless solid after

4.26 (s, 1H), 4.16 (q, J = 4.7, 4.1 Hz, 2H), 3.20 (q, J = 7.3 Hz, 12H),

1

13

lyophilization (2 equiv Et N-salt, 6 mg, 5%). H NMR (400 MHz,

2.20 (t, J = 19.7 Hz, 2H), 1.94 (s, 3H), 1.28 (t, J = 7.3 Hz, 18H).

C

3

D O): δ 7.91 (d, J = 8.1 Hz, 1H), 5.90−5.80 (m, 2H), 4.34−4.24 (m,

2

NMR (100 MHz, D O): δ 166.6, 152.1, 137.3, 111.9, 88.1, 83.6 (d, J

2

K

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8.0 Hz), 73.5, 69.9, 63.6 (d, J = 4.3 Hz), 59.0, 46.7 (6C), 27.6 (t, J =

5.1 Hz), 46.7 (9C), 28.1, 27.5 (t, J = 125.0 Hz), 8.3 (9C). ³¹P NMR

3

1

−

(160 MHz, D O): δ 18.0, 15.4. MS (ESI, m/z): 475.0 [M − H] ;

2

−

ESI-HRMS: calcd m/z for C H ClN O P , 475.0080; found,

1

3

18

2

11 2

−

475.0093 [M − H] . HPLC purity 95% (R = 10.4 min, method

1

17

2

11

2

t

−

HPLC-C).

t

5-Fluorouridine-5′-O-[(phosphonomethyl)phosphonic acid] (4l).

Thymidine-5′-O-[(phosphonomethyl)phosphonic acid] (4g).

Method B. The product was obtained as a colorless solid after

1

lyophilization (2 equiv Et N-salt, 32.2 mg, 6%). H NMR (600 MHz,

3

1

D O): δ 8.19 (d, J = 6.5 Hz, 1H), 5.96 (dt, J = 2.7, 1.6 Hz, 1H),

2

3

4.41−4.37 (m, 2H), 4.31−4.26 (m, 1H), 4.23 (ddd, J = 11.7, 4.8, 2.7

2

Hz, 1H), 4.17 (ddd, J = 11.7, 5.6, 3.0 Hz, 1H), 3.22 (q, J = 7.3 Hz,

12H), 2.24 (t, J = 19.9 Hz, 2H, PCH P), 1.29 (t, J = 7.3 Hz, 18H).

2

13

C NMR (150 MHz, D O): δ 159.5 (d, J = 26.0 Hz), 150.4, 140.9

2

MHz, D O): δ 151.8, 137.5, 111.8, 109.9, 85.5 (d, J = 7.2 Hz), 85.0,

(d, J = 233.4 Hz), 125.7 (d, J = 34.8 Hz), 88.8, 83.5 (d, J = 8.0 Hz),

73.9, 69.5, 63.2 (d, J = 5.3 Hz), 46.7 (6C), 27.4 (t, J = 125.0 Hz,

2

3

1

7

1.0, 63.9, 46.7 (6C), 38.4, 26.9 (t, J = 124.2 Hz), 11.7, 8.3 (6C). P

3

1

NMR (160 MHz, D O): δ 18.1, 14.7. MS (ESI, m/z): 399.0 [M −

PCH P), 8.3 (6C). P NMR (160 MHz, D O): δ 18.0, 15.4. MS

2

−

H] ; ESI-HRMS: calcd m/z for C H N O P , 399.0358; found,

(ESI, m/z): 419.0 [M − H] ; ESI-HRMS: calcd m/z for

1

17

2

10 2

−

3

99.0357 [M − H] . HPLC purity 97% (R = 9.3 min, method

C H FN O P , 419.0062; found, 419.0057 [M − H] . HPLC

t

10 14

2

11 2

HPLC-B).

purity 97% (R = 9.3 min, method HPLC-C).

t

2

′-O-Methyl-5-methyluridine-5′-O-[(phosphonomethyl)-

5-Chlorouridine-5′-O-[(phosphonomethyl)phosphonic acid]

phosphonic acid] (4h). Method A. The product was obtained as a

(4m). Method B. The product was obtained as a colorless solid

1

colorless solid after lyophilization (2 equiv Et N-salt, 21 mg, 9%). H

after lyophilization (2 equiv Et N-salt, 23.1 mg, 10%). H NMR (400

3

NMR (400 MHz, D O): δ 7.79 (d, J = 1.3 Hz, 1H), 6.03 (d, J = 5.1

MHz, D O): δ 8.19 (d, J = 0.7 Hz, 1H), 5.92 (d, J = 4.4 Hz, 1H),

2

Hz, 1H), 4.53 (t, J = 5.0 Hz, 1H), 4.26−4.22 (m, 1H), 4.22−4.14 (m,

4.40−4.34 (m, 2H), 4.28−4.24 (m, 1H), 4.19 (ddd, J = 11.6, 4.6, 2.7

2

7

1

4

H), 4.13 (t, J = 5.2 Hz, 1H), 3.49 (s, 3H), 3.20 (q, J = 7.3 Hz, 12H),

Hz, 1H), 4.13 (ddd, J = 11.8, 5.7, 3.1 Hz, 1H), 3.18 (q, J = 7.3 Hz,

3

1

.20 (td, J = 19.5, 2.0 Hz, 2H), 1.94 (d, J = 1.2 Hz, 3H), 1.28 (t, J =

12H), 2.22 (t, J = 19.9 Hz, 2H), 1.26 (t, J = 7.4 Hz, 18H). P NMR

.3 Hz, 18H). ¹³C NMR (100 MHz, D O): δ 166.6, 151.8, 137.2,

−

2

(160 MHz, D O): δ 18.2, 14.9. MS (ESI, m/z): 435.0 [M − H] ;

2

11.9, 86.7, 83.6 (d, J = 8.1 Hz), 82.4, 68.3, 63.4 (d, J = 5.2 Hz), 58.1,

ESI-HRMS: calcd m/z for C H ClN O P , 434.9767; found,

1

0

14

2

11 2

3

1

−

6.7 (6C), 27.5 (t, J = 124.0 Hz), 11.7, 8.3 (6C). P NMR (160

434.9776 [M − H] . HPLC purity 97% (R = 8.9 min, method

t

−

MHz, D O): δ 18.0, 14.7. MS (ESI, m/z): 429.0 [M − H] ; ESI-

HRMS: calcd m/z for C H N O P , 429.0464; found, 429.0465 [M

−

HPLC-C).

2

1

2

19

2

11 2

5-Bromouridine-5′-O-[(phosphonomethyl)phosphonic acid]

−

H] . HPLC purity 95% (R = 9.5 min, method HPLC-B).

(4n). Method B. The product was obtained as a colorless solid after

t

1

5

-Ethynylyluridine-5′-O-[(phosphonomethyl)phosphonic acid]

lyophilization (2 equiv Et

3

N-salt, 4.1 mg, 2%). H NMR (400 MHz,

(

4i). Method B. The product was obtained as a brown solid after

D O): δ 8.29 (s, 1H), 5.96 (d, J = 4.5 Hz, 1H), 4.46−4.38 (m, 2H),

2

1

lyophilization (2 equiv Et N-salt, 6.7 mg, 3%). H NMR (600 MHz,

4.32−4.28 (m, 1H), 4.25−4.16 (m, 2H), 3.22 (q, J = 7.3 Hz, 12H),

3

1

3

D O): δ 8.26 (s, 1H), 5.92 (d, J = 4.0 Hz, 1H), 4.37 (dt, J = 13.8, 4.9

2

2.25 (t, J = 18.7 Hz, 2H, PCH P), 1.30 (t, J = 7.3 Hz, 18H).

C

2

Hz, 2H), 4.28−4.24 (m, 1H), 4.20 (d, J = 10.0 Hz, 1H), 4.14 (d, J =

NMR (150 MHz, D O): δ 162.0, 151.2, 141.0, 97.0, 89.0, 83.6, 74.0,

2

1

2

1

2

1.6 Hz, 1H), 3.63 (s, 1H), 3.18 (q, J = 7.3 Hz, 12H), 2.25−2.12 (m,

69.6, 63.1, 46.8 (6C), 27.5 (PCH P), 8.3 (6C). The signal for PCH P

2

1

3

13

H), 1.26 (t, J = 7.3 Hz, 18H). C NMR (150 MHz, D O): δ 164.7,

could not be observed in the 1D experiment. C NMR shift of

2

3

1

50.7, 145.4, 98.8, 89.2, 83.7, 83.4, 74.5, 74.1, 69.4, 62.9, 46.7 (6C),

7.3 (t, J = 126.5 Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 17.1

PCH P was determined using HSQC. P NMR (160 MHz, D O): δ

2

3

1

−

2

18.6, 14.8. MS (ESI, m/z): 479.0 [M − H] ; ESI-HRMS: calcd m/z

79 −

−

(

br). MS (ESI, m/z): 429.0 [M − H] ; ESI-HRMS: calcd m/z for

for C H BrN O P , 478.9262; found, 478.9264 [M − H] .

1

0

14

2

11 2

−

C H N O P , 425.0157; found, 425.0165 [M − H] . HPLC purity

HPLC purity 95% (R = 9.3 min, method HPLC-C).

12

15

2

11

2

t

9

9% (R = 8.6 min, method HPLC-C).

5-Iodouridine-5′-O-[(phosphonomethyl)phosphonic acid] (4o).

t

5

-(1-Chlorovinyl)uridine-5′-O-[(phosphonomethyl)phosphonic

Method A. The product was obtained as a colorless solid after

1

acid] (4j). The compound was obtained as a side product during the

lyophilization (2 equiv Et N-salt, 17.5 mg, 9%). H NMR (400 MHz,

3

synthesis of compound 4i via method B. The product was obtained as

D O): δ 8.27 (s, 1H), 5.93 (d, J = 4.7 Hz, 1H), 4.39 (dt, J = 13.0, 5.1

2

1

a brown solid after lyophilization (3 equiv Et N-salt, 2.7 mg, 1%). H

Hz, 2H), 4.31−4.21 (m, 1H), 4.22−4.10 (m, 2H), 3.21 (q, J = 7.3 Hz,

3

1

3

NMR (600 MHz, D O): δ 8.10 (d, J = 0.7 Hz, 1H), 6.01 (dd, J = 1.6,

12H), 2.27 (t, J = 19.7 Hz, 2H), 1.28 (t, J = 7.3 Hz, 18H). C NMR

2

0

4

1

.7 Hz, 1H), 5.95 (d, J = 5.1 Hz, 1H), 5.71 (d, J = 1.7, 0.8 Hz, 1H),

.42 (t, J = 5.2 Hz, 1H), 4.35 (t, J = 4.9 Hz, 1H), 4.27 (q, J = 3.8 Hz,

H), 4.17−4.13 (m, 2H), 3.18 (q, J = 7.3 Hz, 18H), 2.18 (t, J = 19.9

(100 MHz, D O): δ 163.6, 151.9, 145.9, 88.9, 83.6 (d, J = 8.4 Hz),

2

3

1

73.9, 69.7, 68.7, 63.2, 46.7 (6C), 28.0 (t, J = 124.0 Hz), 8.3 (6C).

P

NMR (160 MHz, D O): δ 18.4, 14.7. MS (ESI, m/z): 526.9 [M −

2

1

3

−

Hz, 2H), 1.26 (t, J = 7.3 Hz, 27H). C NMR (150 MHz, D O): δ

1

H] ; ESI-HRMS: calcd m/z for C H N O IP , 526.9118; found,

2

10 14

2

11

2

−

63.1, 151.0, 140.8, 130.3, 119.0, 113.1, 89.2, 83.7 (d, J = 8.1 Hz),

3.8, 69.8, 63.4 (d, J = 5.4 Hz), 46.7 (9C), 27.5 (t, J = 124.5 Hz), 8.3

526.9123 [M − H] . HPLC purity 98% (R = 9.6 min, method

t

7

HPLC-B).

(

[

9C). ³¹P NMR (160 MHz, D O): δ 18.1, 15.2. MS (ESI, m/z): 461.0

2′-Deoxyuridine-5′-O-[(phosphonomethyl)phosphonic acid]

2

−

M − H] ; ESI-HRMS: calcd m/z for C H ClN O P , 460.9923;

(4p). Method A. The product was obtained as a colorless solid after

1

2

16

2

11 2

−

1

found, 460.9923 [M − H] . HPLC purity 95% (R = 9.8 min, method

lyophilization (2 equiv Et N-salt, 16.2 mg, 13%). H NMR (400

t

3

HPLC-C).

MHz, D O): δ 7.98 (d, J = 8.1 Hz, 1H), 6.31 (t, J = 6.8 Hz, 1H), 5.94

2

5

-(1-Chlorovinyl)-3-methyluridine-5′-O-[(phosphonomethyl)-

(d, J = 8.1 Hz, 1H), 4.65−4.51 (m, 1H), 4.21−4.14 (m, 1H), 4.15−

phosphonic acid] (4k). The compound was obtained as a side

product during the synthesis of compound 4i via method B. The

product was obtained as a brown solid after lyophilization (3 equiv

4.07 (m, 2H), 3.20 (q, J = 7.3 Hz, 12H), 2.39 (dd, J = 6.8, 5.0 Hz,

1

3

2H), 2.17 (t, J = 19.9 Hz, 2H), 1.28 (t, J = 7.3 Hz, 18H). C NMR

(100 MHz, D O): δ 166.4, 151.7, 142.2, 102.5, 85.8 (d, J = 7.5 Hz),

2

1

Et N-salt, 5.6 mg, 2%). H NMR (600 MHz, D O): δ 8.10 (d, J = 0.8

85.4, 70.9, 63.8 (d, J = 4.7 Hz), 46.7 (6C), 38.8, 27.5 (t, J = 124.0

3

2

3

1

Hz, 1H), 5.97 (dd, J = 4.6, 0.9 Hz, 1H), 5.96 (dd, J = 1.6, 0.7 Hz,

H), 5.71 (dd, J = 1.6, 1.0 Hz, 1H), 4.41 (t, J = 5.0 Hz, 1H), 4.34 (t, J

Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 18.3, 14.7. MS (ESI, m/

2

−

1

=

3

7

1

z): 385.0 [M − H] ; ESI-HRMS: calcd m/z for C H N O P ,

1

0

15

2

10 2

−

5.2 Hz, 1H), 4.27 (q, J = 3.9 Hz, 1H), 4.20−4.12 (m, 2H), 3.30 (s,

385.0202; found, 385.0201 [M − H] . HPLC purity 99% (R = 16

t

H), 3.18 (q, J = 7.3 Hz, 18H), 2.18 (t, J = 19.6 Hz, 2H), 1.26 (t, J =

min, method HPLC-B).

.3 Hz, 27H). ¹³C NMR (150 MHz, D O): δ 162.6, 151.4, 138.7,

2

2′-Amino-2′-deoxyuridine-5′-O-[(phosphonomethyl)phosphonic

31.0, 119.1, 112.5, 90.2, 83.4 (d, J = 8.0 Hz), 73.9, 69.6, 63.3 (d, J =

acid] (4q). Method A. The product was obtained as a colorless solid

L

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

3

after lyophilization (1 equiv Et N-salt, 4 mg, 8%), containing 8% of

experiment. C NMR shift of PCH P was determined using HSQC.

3

2

1

31

methylenediphosphonic acid. H NMR (400 MHz, D O): δ 8.02 (d, J

P NMR (160 MHz, D O): δ 18.3, 15.0. MS (ESI, m/z): 403.0 [M −

H] ; ESI-HRMS: calcd m/z for C H N O P , 402.0109; found,

402.0098 [M − H] . HPLC purity 96% (R = 9.1 min, method

2

−

=

8.1 Hz, 1H), 6.28 (d, J = 7.6 Hz, 1H), 6.00 (d, J = 8.1 Hz, 1H), 4.70

9

14

3

11 2

−

(

d, J = 4.6 Hz, 1H), 4.44 (s, 1H), 4.23−4.08 (m, 3H), 3.21 (q, J = 7.3

t

3

1

Hz, 6H), 2.18 (td, J = 19.8, 4.0 Hz, 2H), 1.28 (t, J = 7.3 Hz, 9H).

P

HPLC-C).

NMR (160 MHz, D O): δ 18.5, 15.9 (8% methylenediphosphonic

Uridine-5′-O-[(phosphonoethyl)phosphonic acid] (4w). Method

2

−

acid), 14.4. MS (ESI, m/z): 400.0 [M − H] ; ESI-HRMS: calcd m/z

A. The product was obtained as a colorless solid after lyophilization (3

−

1

for C H N O P , 400.0311; found, 400.0304 [M − H] . HPLC

equiv Et N-salt and 1 equiv H CCO H, 60 mg, 8%). H NMR (400

10

16

3

10

2

3

2

purity 96% (R = 1.8 min, method HPLC-A).

MHz, D O): δ 7.93 (d, J = 8.1 Hz, 1H), 6.14−5.71 (m, 2H), 4.32 (p,

t

2

′-Azido-2′-deoxyuridine-5′-O-[(phosphonomethyl)phosphonic

J = 5.1 Hz, 2H), 4.28−4.22 (m, 1H), 4.17−4.01 (m, 2H), 3.18 (q, J =

7.3 Hz, 18H), 1.90 (s, 3H, H CCO H), 1.85−1.64 (m, 4H), 1.26 (t, J

acid] (4r). Method A. The product was obtained as a colorless solid

3

2

1

13

after lyophilization (2 equiv Et N-salt, 11 mg, 8%). H NMR (400

= 7.3 Hz, 27H). C NMR (100 MHz, D O): δ 181.0, 166.2, 151.8,

3

2

MHz, D O): δ 8.02 (d, J = 8.1 Hz, 1H), 6.02 (d, J = 5.2 Hz, 1H), 5.97

141.6, 102.6, 88.6, 83.4 (d, J = 7.7 Hz), 73.9, 69.7, 63.1 (d, J = 5.5

Hz), 46.7 (9C), 23.2, 22.1 (dd, J = 133.5, 5.5 Hz), 20.4 (dd, J = 135.4,

4.2 Hz), 8.3 (9C). P NMR (160 MHz, D O): δ 27.2 (d, J = 73.5

Hz), 24.0 (d, J = 73.5 Hz). MS (ESI, m/z): 415.0 [M − H] ; ESI-

HRMS: calcd m/z for C H N O P , 415.0308; found, 415.0311 [M

2

d, J = 8.1 Hz, 1H), 4.62 (t, J = 5.2 Hz, 1H), 4.40 (t, J = 5.4 Hz, 1H),

3

1

.27−4.09 (m, 3H), 3.20 (q, J = 7.3 Hz, 12H), 2.19 (t, J = 19.7 Hz,

2

1

3

−

H), 1.28 (t, J = 7.3 Hz, 18H). C NMR (100 MHz, D O): δ 166.3,

2

51.7, 141.6, 102.7, 87.1, 83.7 (d, J = 8.0 Hz), 70.1, 65.4, 63.0 (d, J =

1

17

2

11 2

3

1

−

.8 Hz), 46.7 (6C), 27.5 (t, J = 124.6 Hz), 8.3 (6C). P NMR (160

− H] . HPLC purity 99% (R = 8.7 min, method HPLC-B).

t

−

2

5-Methyluridine-5′-O-[(phosphonoethyl)phosphonic acid] (4x).

1

0

14

5

10 2

Method A. The product was obtained as a colorless solid after

−

1

H] . HPLC purity 99% (R = 17.5 min, method HPLC-B).

lyophilization (2 equiv Et N-salt, 68 mg, 10%). H NMR (400 MHz,

t

3

2

′-Fluoro-2′-deoxyuridine-5′-O-[(phosphonomethyl)phosphonic

D O): δ 7.74 (s, 1H), 5.99 (d, J = 5.3 Hz, 1H), 4.44−4.32 (m, 2H),

2

4

.30−4.23 (m, 1H), 4.16−4.03 (m, 2H), 3.20 (q, J = 7.3 Hz, 12H),

1

13

3

1.94 (s, 3H), 1.90−1.65 (m, 4H), 1.28 (t, J = 7.3 Hz, 17H). C NMR

MHz, D O): δ 7.97 (d, J = 8.1 Hz, 1H), 6.08 (dd, J = 17.8, 1.8 Hz,

2

(100 MHz, D O): δ 166.6, 152.0, 137.1, 112.0, 88.1, 83.6 (d, J = 7.9

2

1

H), 5.93 (d, J = 8.1 Hz, 1H), 5.52 (ddd, J = 52.5, 4.6, 1.8 Hz), 4.52

Hz), 73.6, 70.0, 63.4 (d, J = 5.5 Hz), 46.7 (6C), 22.0 (dd, J = 133.4,

.0 Hz), 20.4 (dd, J = 134.8, 4.8 Hz), 11.8, 8.3 (6C). P NMR (160

MHz, D O): δ 27.3 (d, J = 73.5 Hz), 24.1 (d, J = 73.5 Hz). MS (ESI,

3

1

(

ddd, J = 21.6, 7.8, 4.6 Hz, 1H), 4.38−4.25 (m, 2H), 4.19 (ddd, J =

5

1

2

1

1.7, 5.8, 2.8 Hz, 1H), 3.20 (q, J = 7.3 Hz, 12H), 2.20 (t, J = 19.7 Hz,

2

1

3

−

H), 1.28 (t, J = 7.3 Hz, 18H). C NMR (100 MHz, D O): δ 166.4,

2

m/z): 429.0 [M − H] ; ESI-HRMS: calcd m/z for C H N O P ,

1

2

19

2

11 2

−

51.4, 142.1, 102.3, 93.5 (d, J = 185.9 Hz), 88.4 (d, J = 35.1 Hz), 81.6

4

29.0464; found, 429.0472 [M − H] . HPLC purity 99% (R = 9.1

t

d, J = 7.9 Hz), 67.7 (d, J = 15.9 Hz), 62.1 (d, J = 4.9 Hz), 46.7 (6C),

min, method HPLC-B).

3

1

7

7.5 (t, J = 124.6 Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 18.4,

2

2-Thiouridine-5′-O-[(phosphonomethyl)phosphonic acid] (4y).

−

4.7. MS (ESI, m/z): 403.0 [M − H] ; ESI-HRMS: calcd m/z for

The product was obtained as a colorless solid after lyophilization (1.5

−

1

10

14

2

10

2

equiv Et N-salt). H NMR (400 MHz, D O): δ 8.21 (d, J = 8.2 Hz,

3

2

t

1H), 6.63 (d, J = 2.8 Hz, 1H), 6.24 (d, J = 8.1 Hz, 1H), 4.51−4.40

(m, 1H), 4.35 (t, J = 5.7 Hz, 1H), 4.33−4.26 (m, 2H), 4.25−4.16 (m,

H), 3.20 (q, J = 7.4 Hz, 10H), 2.17 (t, J = 19.6 Hz, 2H), 1.28 (t, J =

2

′-ara-Fluoro-2′-deoxyuridine-5′-O-[(phosphonomethyl)-

phosphonic acid] (4t). Method A. The product was obtained as a

1

colorless solid after lyophilization (1.5 equiv Et N-salt, 11 mg, 8%).

31

3

7.3 Hz, 15H). P NMR (160 MHz, D O): δ 19.2, 14.0.

2

1

H NMR (400 MHz, D O): δ 7.93 (dd, J = 8.1, 1.7 Hz, 1H), 6.32

2

Cytidine-5′-O-[(phosphonomethyl)phosphonic acid] (7a). Meth-

(

3

(

1

dd, J = 15.5, 4.3 Hz, 1H), 5.92 (d, J = 8.1 Hz, 1H), 5.23 (td, J = 51.7,

.6 Hz, 1H), 4.57 (dt, J = 19.8, 3.6 Hz, 1H), 4.25−4.08 (m, 3H), 3.21

q, J = 7.3 Hz, 9H), 2.19 (t, J = 19.7 Hz, 2H), 1.28 (t, J = 7.3 Hz,

4H). ¹³C NMR (100 MHz, D O): δ 166.3, 151.4, 142.9, 101.9, 94.6

od A. The product was obtained as a colorless solid after

1

lyophilization (2 equiv Et N-salt, 10.3 mg, 8%). H NMR (400

MHz, D O): δ 8.02 (d, J = 7.6 Hz, 1H), 6.13 (d, J = 7.5 Hz, 1H), 5.98

(d, J = 4.1 Hz, 1H), 4.35 (dq, J = 9.2, 5.0 Hz, 2H), 4.30−4.10 (m,

3H), 3.20 (q, J = 7.3 Hz, 12H), 2.19 (t, J = 19.7 Hz, 2H), 1.28 (t, J =

7.3 Hz, 18H). C NMR (100 MHz, D O): δ 166.0, 157.4, 141.8,

96.5, 89.4, 82.9 (d, J = 7.3 Hz), 74.3, 69.3, 63.1, 46.8 (6C), 27.5 (t, J =

124.3 Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 18.4, 14.6. MS

3

2

(

6

1

d, J = 191.8 Hz), 83.6 (d, J = 16.9 Hz), 82.1, 73.2 (d, J = 26.0 Hz),

3

1

13

2.7, 46.8 (4C), 27.5, 8.3 (4C). P NMR (160 MHz, D O): δ 20.6,

2

−

2.8. MS (ESI, m/z): 403.0 [M − H] ; ESI-HRMS: calcd m/z for

−

31

C H N O FP , 403.0108; found, 403.0112 [M − H] . HPLC

purity 98% (R = 9.6 min, method HPLC-B).

10

14

2

10

2

−

t

(ESI, m/z): 403.0 [M − H] ; ESI-HRMS: calcd m/z for

−

1

(β-D-Arabinofuranosyl)-uridine-5′-O-[(phosphonomethyl)-

C H N O P , 400.0311; found, 400.0309 [M − H] . HPLC purity

1

0

16

3

10 2

phosphonic acid] (4u). Method A. The product was obtained as a

9

6% (R = 15.6 min, method HPLC-B).

t

colorless solid after lyophilization (2 equiv Et N-salt, 21.3 mg, 9%).

3

2′-Deoxycytidine-5′-O-[(phosphonomethyl)phosphonic acid]

1

H NMR (400 MHz, D O): δ 7.96 (d, J = 8.1 Hz, 1H), 6.23 (d, J =

(7b). Method A. The product was obtained as a colorless solid after

2

1

5

.5 Hz, 1H), 5.94 (d, J = 8.1 Hz, 1H), 4.46 (t, J = 5.5 Hz, 1H), 4.29

lyophilization (2 equiv Et N-salt, 34 mg, 14%). H NMR (400 MHz,

3

(

t, J = 6.0 Hz, 1H), 4.21 (tt, J = 11.6, 6.6 Hz), 4.10 (dt, J = 7.4, 3.9

D O): δ 8.14 (d, J = 7.7 Hz, 1H), 6.28 (t, J = 6.5 Hz, 1H), 6.21 (d, J =

2

Hz, 1H), 3.22 (q, J = 7.3 Hz, 12H), 2.21 (t, J = 19.7 Hz, 2H), 1.30 (t,

7.7 Hz, 1H), 4.60 (dt, J = 7.1, 3.8 Hz, 1H), 4.31−4.18 (m, 1H), 4.16−

4.06 (m, 2H), 3.20 (q, J = 7.3 Hz, 9H), 2.51−2.28 (m, 2H), 2.17 (t, J

J = 7.3 Hz, 18H). ¹³C NMR (100 MHz, D O): δ 166.2, 151.4, 143.0,

2

1

3

1

8

4

01.3, 84.7, 81.2, 75.2, 73.8, 62.4, 46.7 (6C), 27.4 (t, J = 124.8 Hz),

= 19.8 Hz, 2H), 1.28 (t, J = 7.3 Hz, 14H). C NMR (100 MHz,

.3 (6C). ³¹P NMR (160 MHz, D O): δ 18.4, 14.9. MS (ESI, m/z):

D

O): δ 162.2, 152.4, 143.5, 95.7, 86.4, 86.1 (d, J = 7.3 Hz), 70.7,

2

−

01.0 [M − H] ; ESI-HRMS: calcd m/z for C H N O P ,

01.0157; found, 401.0144 [M − H] . HPLC purity 99% (R = 8.6

63.6 (d, J = 4.3 Hz), 46.7 (4C), 39.5, 27.5 (t, J = 124.7 Hz), 8.3 (4C).

1

0

15

2

11 2

−

31

P NMR (160 MHz, D

O): δ 18.3, 14.7. MS (ESI, m/z): 384.0 [M −

t

2

−

min, method HPLC-C).

H] ; ESI-HRMS: calcd m/z for C H N O P , 384.0362; found,

384.0365 [M − H] . HPLC purity 99% (R = 8.8 min, method

HPLC-B).

1

0

16

3

9 2

−

6

-Azauridine-5′-O-[(phosphonomethyl)phosphonic acid] (4v).

t

Method A. The product was obtained as a colorless solid after

lyophilization (2 equiv Et N-salt, 17.7 mg, 7%). H NMR (400 MHz,

1

5-Iodocytidine-5′-O-[(phosphonomethyl)phosphonic acid] (7c).

3

D O): δ 7.66 (s, 1H), 6.15 (d, J = 3.7 Hz, 1H), 4.65 (dd, J = 5.1, 3.7

Method B. The product was obtained as a colorless solid after

2

1

Hz, 1H), 4.48 (t, J = 5.2 Hz, 1H), 4.30−4.21 (m, 1H), 4.16−3.98 (m,

lyophilization (1.75 equiv Et N-salt, 9.4 mg, 5%). H NMR (400

3

2

1

H), 3.22 (q, J = 7.3 Hz, 12H), 2.13 (t, J = 17.8 Hz, 2H, PCH P),

MHz, D O): δ 8.26 (s, 1H), 5.93 (d, 1H, J = 3.2 Hz), 4.41−4.33 (m,

2

1

3

.30 (t, J = 7.3 Hz, 18H). C NMR (100 MHz, D O): δ 158.6, 150.0,

2H), 4.31−4.27 (m, 1H), 4.21 (m, 2H), 3.22 (q, J = 7.3 Hz, 10.5H),

2

1

3

37.2, 89.7, 83.1, 72.6, 70.5, 64.0, 46.7 (6C), 27.1 (1C, PCH P), 8.3

2.29 (t, J = 17.9 Hz, 2H, PCH P), 1.30 (t, J = 7.3 Hz, 15.75H). C

2

(

6C). The signal of PCH P could not be observed in the 1D

NMR (100 MHz, D O): δ 164.6, 156.6, 147.6, 89.8, 83.1, 74.4, 69.2,

2

M

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6

2.9, 46.7 (5.25C), 27.7 (PCH P), 8.3 (5.25C). The signal for PCH P

(150 MHz, D O): δ 151.0, 147.0, 141.8, 132.2, 129.5 (q, J = 31.9 Hz),

128.3 (2C), 125.5 (q, J = 3.8 Hz, 2C), 124.3 (q, J = 271.5 Hz), 98.3,

2

1

3

could not be observed in the 1D experiment. C NMR shift of

PCH P was determined using HSQC. P NMR (160 MHz, D O): δ

1

for C H IN O P , 525.9283; found, 525.9275 [M − H] . HPLC

purity 99% (R = 9.0 min, method HPLC-C).

3

1

87.3, 83.4 (d, J = 6.6 Hz), 74.5, 72.7, 70.2, 63.8, 46.7 (6C), 27.4 (t, J =

2

−

31

8.5, 15.1. MS (ESI, m/z): 525.9 [M − H] ; ESI-HRMS: calcd m/z

124.4 Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 18.2, 15.1. MS

2

−

(ESI, m/z): 574.1 [M + H] ; ESI-HRMS: calcd m/z for

C H F N O P , 574.0609; found, 574.0616 [M − H] . HPLC

purity >99% (R = 12.5 min, method HPLC-C).

1

0

16

3

10 2

−

t

18 21

3

11 2

5

-Fluorocytidine-5′-O-[(phosphonomethyl)phosphonic acid]

t

4

(

7d). Method B. The product was obtained as a colorless solid after

N -[O-(Naphthalen-2-ylmethoxy)]-cytidine-5′-O-

1

lyophilization (1.5 equiv Et N-salt, 15.5 mg, 7%). H NMR (600

[(phosphonomethyl)phosphonic acid] (9e). Method B. The product

3

MHz, D O): δ 8.15 (d, J = 6.3 Hz, 1H), 5.90 (dd, J = 3.9, 1.4 Hz,

was obtained as a colorless solid after lyophilization (2 equiv Et N-

2

3

1

4

2

H), 4.34 (t, J = 5.3 Hz, 1H), 4.30 (dd, J = 5.1, 3.8 Hz, 1H), 4.27−

salt, 4.5 mg, 2%). H NMR (600 MHz, D O): δ 7.97−7.94 (m, 3H),

2

.19 (m, 2H), 4.14 (d, J = 11.8 Hz, 1H), 3.18 (q, J = 7.3 Hz, 9H),

7.93−7.91 (m, 1H), 7.60−7.57 (m, 3H), 7.20 (d, J = 8.2 Hz, 1H),

5.90 (d, J = 5.5 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 5.20 (s, 2H), 4.34−

4.30 (m, 2H), 4.21−4.19 (m, 1H), 4.10−4.07 (m, 2H), 3.17 (q, J =

1

3

.19 (t, J = 19.2 Hz, 2H), 1.26 (t, J = 7.3 Hz, 15H). C NMR (150

MHz, D O): δ 158.1 (d, J = 15.1 Hz), 155.4, 137.6 (d, J = 248.3 Hz),

2

1

3

1

26.0 (d, J = 33.0 Hz), 89.7, 82.9 (d, J = 6.7 Hz), 74.4, 69.1, 62.9 (d, J

7.3 Hz, 12H), 2.14 (t, J = 19.2 Hz, 2H), 1.26 (t, J = 7.3 Hz, 18H). C

3

1

=

3.6 Hz), 46.7 (4.5C), 27.5 (t, J = 119.5 Hz), 8.3 (4.5C). P NMR

NMR (150 MHz, D O): δ 151.1, 147.0, 135.1, 133.0, 132.9, 132.1,

2

−

(

160 MHz, D O): δ 18.9, 15.1. MS (ESI, m/z): 418.0 [M − H] ;

128.4, 128.0, 127.8, 127.2, 126.7, 126.6, 126.2, 98.4, 87.3, 83.4 (d, J =

2

ESI-HRMS: calcd m/z for C H FN O P , 418.0222; found,

7.2 Hz), 75.5, 72.6, 70.2, 63.8, 46.7 (6C), 27.5 (t, J = 127.0 Hz), 8.3

1

0

15

3

10 2

−

31

4

18.0235 [M − H] . HPLC purity 98% (R = 8.8 min, method

(6C). P NMR (160 MHz, D

2

O): δ 18.3, 14.6. MS (ESI, m/z): 556.1

, 556.0892;

= 12.2 min,

t

−

HPLC-C).

[M + H] ; ESI-HRMS: calcd m/z for C21H N O P

found, 556.0901 [M − H] . HPLC purity 99% (R

method HPLC-C).

24 3 11 2

−

5

-Methylcytidine-5′-O-[(phosphonomethyl)phosphonic acid]

t

(

7e). Method B. The product was obtained as a colorless solid after

1

4

lyophilization (1.5 equiv Et N-salt, 47.7 mg, 22%). H NMR (600

N - [ O - ( 4 - B e n z y l o x y ) ] - 5 - m e t h y l - c y t i d i n e - 5 ′ - O -

3

MHz, D O): δ 7.92 (s, 1H), 5.99 (d, 1H, J = 3.9 Hz), 4.42−4.35 (m,

[(phosphonomethyl)phosphonic acid] (9f). Method B. The product

2

H), 4.32−4.25 (m, 1H), 4.25−4.13 (m, 2H, J = 11.6 Hz), 3.21 (q, J

was obtained as a colorless solid after lyophilization (2 equiv Et

3

N-

salt, 17.5 mg, 9%). H NMR (400 MHz, D O): δ 7.48−7.36 (m, 5H),

2

1

=

7.3 Hz, 9H), 2.21 (t, J = 19.0 Hz, 2H, PCH P), 2.07 (s, 3H), 1.29

2

1

3

(

t, J = 7.3 Hz, 13.5H). C NMR (150 MHz, D O): δ 163.2, 153.8,

7.03−7.00 (m, 1H), 5.92−5.88 (m, 1H), 5.07 (s, 2H), 4.37−4.32 (m,

2H), 4.21−4.17 (m, 1H), 4.11−4.06 (m, 2H), 3.18 (q, J = 7.3 Hz,

2

1

39.7, 89.2, 83.3, 74.2, 69.4, 63.3, 46.7 (4.5C), 27.5 (t, J = 122.7 Hz,

3

1

C, PCH P), 12.3, 8.3 (4.5C). P NMR (160 MHz, D O): δ 18.0,

12H), 2.16 (t, J = 19.6 Hz, 2H), 1.80 (s, 3H), 1.26 (t, J = 7.3 Hz,

2

−

31

4.8. MS (ESI, m/z): 414.0 [M − H] ; ESI-HRMS: calcd m/z for

18H). P NMR (160 MHz, D

2

O): δ 18.0, 14.7. MS (ESI, m/z):

−

C H N O P , 414.0473; found, 414.0484 [M − H] . HPLC purity

520.1 [M + H] ; ESI-HRMS: calcd m/z for C18H N O P ,

520.0892; found, 520.0911 [M − H] . HPLC purity >99% (R =

t

10.9 min, method HPLC-C).

24 3 11 2

11

19

3

10 2

−

9

9% (R = 7.0 min, method HPLC-C).

t

4

-Benzoylcytidine-5′-O-[(phosphonomethyl)phosphonic acid]

4

(

7f). Method B. The product was obtained as a colorless solid after

N - [ O - ( 4 - B e n z y l o x y ) ] - 5 - ﬂ u o r o - c y t i d i n e - 5 ′ - O -

1

lyophilization (5 equiv Et N-salt, 39.2 mg, 13%). H NMR (600

[(phosphonomethyl)phosphonic acid] (9g). Method B. The product

3

MHz, D O): δ 8.47 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.7 Hz, 2H), 7.67

was obtained as a colorless solid after lyophilization (2 equiv Et

3

N-

and 1 H CO -salt, 40.0 mg, 11%). H NMR (600 MHz, D O): δ

2 3 2

2

1

(

t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.7 Hz, 2H), 7.52−7.44 (m, 1H),

.96−5.94 (m, 1H), 4.39−4.33 (m, 2H), 4.33−4.28 (m, 2H), 4.20 (d,

J = 11.6 Hz, 1H), 3.18 (q, J = 7.3 Hz, 30H), 2.29−2.10 (m, 2H), 1.26

5

7.45−7.36 (m, 6H), 5.89 (d, J = 5.3 Hz, 1H), 5.09 (s, 2H), 4.35−4.31

(m, 1H), 4.29 (t, J = 5.5 Hz, 1H), 4.24−4.17 (m, 1H), 4.14−4.07 (m,

2H), 3.57 (q, J = 7.2 Hz, 6H), 3.17 (q, J = 7.3 Hz, 6H), 2.24−2.05

1

3

(

t, J = 7.3 Hz, 45H). C NMR (150 MHz, D O): δ 169.5, 163.2,

2

1

3

1

56.7, 145.8, 133.6, 132.6, 129.0 (2C), 128.1 (2C), 98.8, 90.9 (d, J =

.4 Hz), 82.8, 74.8, 68.7, 62.7, 46.7 (15C), 27.4 (t, J = 120.2 Hz), 8.3

(m, 2H), 1.33 (t, J = 7.2 Hz, 9H), 1.26 (t, J = 7.3 Hz, 9H). C NMR

3

(

(150 MHz, D O): δ 149.7, 140.6 (d, J = 21.7 Hz), 137.9 (d, J = 235.3

2

15C). ³¹P NMR (160 MHz, D O): δ 18.5, 15.1. MS (ESI, m/z):

Hz), 137.0, 128.8 (2C), 128.5, 128.3 (2C), 116.4 (d, J = 35.1 Hz),

2

−

5

1

04.0 [M − H] ; ESI-HRMS: calcd m/z for C H N O P ,

87.6, 83.4, 75.9, 72.8, 70.1, 63.7, 58.5 (3C), 46.7 (3C), 27.2 (t, J =

1

7

20

3

11 2

−

31

04.0579; found, 504.0588 [M − H] . HPLC purity 90% (R =

124.4 Hz), 8.3 (3C), 7.19 (3C). P NMR (160 MHz, D

2

O): δ 16.5.

t

−

0.5 min, method HPLC-C). However, the compound displayed

MS (ESI, m/z): 524.1 [M + H] ; ESI-HRMS: calcd m/z for

−

decomposition in aqueous solution (Supporting Information) and was

C

H

17

21FN

3

O

11

P

2

, 524.0641; found, 524.0645 [M − H] . HPLC

= 11.4 min, method HPLC-C).

t

tested for its CD73 inhibition at a purity of 75%.

purity 99% (R

4

N -[O-(Benzyloxy)]-2′-deoxycytidine-5′-O-[(phosphonomethyl)-

N - [ O - ( 4 - B e n z y l o x y ) ] - 3 - m e t h y l - c y t i d i n e - 5 ′ - O -

phosphonic acid] (9c). Method B. The product was obtained as a

[(phosphonomethyl)phosphonic acid] (9h). Method B. The product

colorless solid after lyophilization (1.5 equiv Et N-salt, 4.3 mg, 4.4%).

was obtained as a colorless solid after lyophilization (2 equiv Et N-

3

1

salt, 8.5 mg, 4%). H NMR (600 MHz, D O): δ 7.48−7.44 (m, 2H),

H NMR (400 MHz, D O): δ 7.50−7.37 (m, 5H), 7.20 (d, J = 8.3

Hz, 1H), 6.28 (t, J = 7.1 Hz, 1H), 5.71 (d, J = 8.3 Hz, 1H), 5.03 (s,

2

7

6

4

.44−7.41 (m, 2H), 7.40−7.37 (m, 1H), 7.32 (d, J = 8.4 Hz, 1H),

.44 (d, J = 8.3 Hz, 1H), 5.94 (d, J = 5.2 Hz, 1H), 5.01 (s, 2H), 4.34−

.30 (m, 2H), 4.20 (q, J = 3.1 Hz, 1H), 4.12−4.08 (m, 2H), 3.21−

2

7

3

H), 4.57 (dt, J = 5.9, 2.8 Hz, 1H), 4.16−3.97 (m, 3H), 3.19 (q, J =

.3 Hz, 9H), 2.33 (dt, J = 14.1, 7.0 Hz, 1H), 2.23 (ddd, J = 14.1, 6.4,

1

3

3.14 (m, 15H), 2.16 (t, J = 19.1 Hz, 2H), 1.26 (t, J = 7.4 Hz, 18H).

.3 Hz, 1H), 2.14 (t, J = 19.8 Hz, 2H), 1.27 (t, J = 7.3 Hz, 14H).

C

1

3

C NMR (150 MHz, D O): δ 153.9, 151.5, 136.9, 132.7, 128.9 (2C),

NMR (100 MHz, D O): δ 147.2, 137.3, 132.3, 130.2, 128.8 (2C),

2

1

6

28.7 (2C), 128.5, 94.1, 88.4, 83.2 (d, J = 7.3 Hz), 75.6, 73.0, 70.0,

3.7 (d, J = 4.4 Hz), 46.7 (6C), 29.3, 27.4 (t, J = 124.2 Hz), 8.3 (6C).

1

4

1

28.5, 128.4 (2C), 98.2, 85.1 (d, J = 7.8 Hz), 84.3, 75.6, 71.2, 63.9,

3

1

6.7 (4C), 37.7, 13.6, 8.3 (4C). P NMR (160 MHz, D O): δ 21.3,

2

−

31

2.0. MS (ESI, m/z): 492.1 [M + H] ; ESI-HRMS: calcd m/z for

P NMR (160 MHz, D

2

O): δ 18.4, 15.0. MS (ESI, m/z): 520.1 [M +

H] ; ESI-HRMS: calcd m/z for C18 , 520.0892; found,

= 11.6 min, method

−

C H N O P , 492.0937; found, 492.0928 [M + H] . HPLC purity

H N O P

24 3 11 2

17

24

3

10 2

−

9

9% (R = 10.9 min, method HPLC-B).

N -[O-(4-Triﬂuoromethylbenzyloxy)]-cytidine-5′-O-

520.0889 [M − H] . HPLC purity >99% (R

t

4

HPLC-C). The synthesis was further optimized: a solution of

methylenebis(phosphonic dichloride) (719 mg, 2.88 mmol, 1.5

equiv) in trimethyl phosphate (10.5 mL) cooled to 0 °C was added

to a suspension of 8h (697 mg, 1.92 mmol, 1.0 equiv) in trimethyl

phosphate at 0 °C. The reaction mixture was stirred at 0 °C, and

samples were withdrawn at 10 min intervals for LC−MS. After 45

+ +

[(phosphonomethyl)phosphonic acid] (9d). Method B. The product

was obtained as a colorless solid after lyophilization (2 equiv Et N-

3

1

salt, 32.1 mg, 17%). H NMR (600 MHz, D O): δ 7.69 (d, J = 8.0 Hz,

2

4

2

1

H), 7.54 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.1 Hz, 1H), 5.91 (d, J =

.8 Hz, 1H), 5.71 (d, J = 8.1 Hz, 1H), 5.10 (s, 2H), 4.35−4.31 (m,

H), 4.22−4.19 (m, 1H), 4.12−4.07 (m, 2H), 3.18 (q, J = 7.3 Hz,

−

min, on the disappearance of 8h formate [M + CHO ] ([M − H ]

2

1

3

2H), 2.16 (t, J = 18.8 Hz, 2H), 1.26 (t, J = 7.3 Hz, 18H). C NMR

disappeared after ∼30 min), 7 mL of cold 1 M aqueous TEAC buﬀer

N

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

solution (pH 8.4−8.6) was added. It was stirred at 0 °C for 15 min,

followed by stirring at rt for 30 min. Because of the high lipophilicity

of the product, no extraction was performed, and instead, the aqueous

layer was lyophilized directly. Separation of the mixture of nucleotide

and dinucleotide was done following method B to obtain the ﬁnal

ﬂasks and centrifuged for 5 min at 2000g. The supernatant (1.5 mL,

viral stock) was added to 75 cm cell culture ﬂasks containing Sf9 cells

2

(60−70% conﬂuence), and the cells were incubated for 4 days at 27

°C. Then, 1.5 mL of the supernatant was taken and added to

2

uninfected Sf9 cells in a 75 cm ﬂask. This was repeated ﬁve more

product as a glassy solid (2 equiv Et N-salt, 423 mg, 0.584 mmol,

times, using more cells and larger ﬂasks after the third round of

3

2

3

0%).

infections (175 cm to which 3.0 mL of the supernatant was added).

4

N -[O-(4-Benzyloxy)]-3-ethyl-cytidine-5′-O-[(phosphonomethyl)-

The ﬁnal stock solution was used for infection of the cells. For

phosphonic acid] (9i). Method B. The product was obtained as a

colorless solid after lyophilization (2 equiv Et N-salt, 9.0 mg, 5%). H

protein expression, 3 mL of the virus solution was used to infect 150

1

6

3

mL of cell media containing 2 × 10 cells/mL in a 500 mL

NMR (600 MHz, D O): δ 7.46 (dt, J = 7.2, 1.3 Hz, 2H), 7.42 (tq, J =

Erlenmeyer ﬂask, and the cells were incubated for 4 days at 27 °C

with shaking (150 rpm). Then, cell suspensions were transferred to 50

mL Falcon tubes and centrifuged at 15 min at 5000g at 4 °C. The

supernatants were subjected to ultraﬁltration using Amicon Ultra-15,

10 kDa cutoﬀ (Merck Millipore, MA, USA) at 5000g for 15−30 min

2

6

2

7

1

7

.4, 1.1 Hz, 2H), 7.40−7.36 (m, 1H), 7.27 (dd, J = 8.4, 1.1 Hz, 1H),

.41 (dd, J = 8.5, 1.2 Hz, 1H), 5.92 (dd, J = 5.4, 1.2 Hz, 1H), 5.00 (s,

H), 4.34−4.29 (m, 2H), 4.19 (q, J = 3.2 Hz, 1H), 4.12−4.08 (m,

H), 3.18 (q, J = 7.3 Hz, 12H), 2.16 (t, J = 18.9 Hz, 2H), 1.26 (t, J =

.3 Hz, 18H). ¹³C NMR (150 MHz, D O): δ 153.0, 151.1, 137.0,

2+

2

at 4 °C. The concentrated protein was puriﬁed with HisPur Ni −

32.7, 129.1 (2C), 128.7 (2C), 128.5, 94.3, 88.3, 83.2 (d, J = 7.4 Hz),

5.6, 72.9, 70.0, 63.7 (d, J = 4.6 Hz), 46.7 (6C), 27.4 (t, J = 124.4

NTA spin columns (#: 88226, Thermo Fisher Scientiﬁc, MA, USA).

The elution of the columns was performed as recommended in the

instruction manual with adjusting the incubation time for protein

binding to 1 h at 4 °C with an end-over-end mixer and an additional

incubation step of 5 min with the elution buﬀer before eluting. Eluates

were pooled and dialyzed (Membra-Cel, 14 kDa cutoﬀ, 250 mm × 44

mm × 0.02 mm; Carl Roth, Germany) at 4 °C in 25 mM Tris buﬀer,

pH 7.4, with a volume adjusted to 40 times the volume of the elution

fraction. The buﬀer was exchanged after 8 h. The enzyme was

aliquoted and stored at −80 °C until use.

31

Hz), 8.3 (6C). P NMR (160 MHz, D O): δ 18.3, 15.0. MS (ESI, m/

z): 534.1 [M + H] ; ESI-HRMS: calcd m/z for C H N O P ,

2

−

1

9

26

3

11 2

−

5

34.1048; found, 534.1074 [M − H] . HPLC purity >99% (R = 11.9

t

min, method HPLC-B).

-Deazauridine-5′-O-[(phosphonomethyl)phosphonic acid]

10). Method A. The product was obtained as a colorless solid after

3

(

1

lyophilization (2 equiv Et N-salt, 19 mg, 15%). H NMR (400 MHz,

3

D O): δ 7.92 (d, J = 7.8 Hz, 1H), 6.31 (d, J = 7.6 Hz, 1H), 6.19 (d, J

2

=

4.3 Hz, 1H), 5.87 (d, J = 2.6 Hz, 1H), 4.37 (dt, J = 18.0, 5.1 Hz,

Cell Culture. TNBC cells (MDA-MB-231), which natively express

CD73, were grown in Dulbecco’s modiﬁed Eagle medium (#: 41966,

Thermo Fisher Scientiﬁc, MA, USA), and melanoma cancer cells with

2

H), 4.33−4.27 (m, 1H), 4.27−4.09 (m, 2H), 3.21 (q, J = 7.3 Hz,

2H), 2.20 (t, J = 19.7 Hz, 2H), 1.28 (t, J = 7.3 Hz, 18H). C NMR

1

3

1

7

0

ko

(

100 MHz, D O): δ 169.7, 165.7, 134.7, 104.2, 88.4, 82.9 (d, J = 7.8

2

CRISPR-Cas9 knockout of CD73 (MaMel.65-CD73 ) were

cultivated in Roswell Park Memorial Institute medium 1640 (Thermo

Fisher Scientiﬁc, Langerwehe, Germany) plus 2 mM L-glutamine

(PAN Biotech, Germany). Both media were supplemented with 100

U/mL penicillin/100 μg/mL streptomycin (PAN Biotech, Germany)

and 10% of fetal bovine serum (PAN Biotech, Germany) and

3

1

Hz), 74.7, 69.4, 63.2, 46.7 (6C), 27.5 (t, J = 124.3 Hz), 8.3 (6C).

P

NMR (160 MHz, D O): δ 18.3, 14.7. MS (ESI, m/z): 400.0 [M −

2

−

H] ; ESI-HRMS: calcd m/z for C H NO IP , 400.0199; found,

1

16

11

2

−

4

00.0203 [M − H] . HPLC purity 96% (R = 9.4 min, method

t

HPLC-C).

(

S)-Methanocarbauridine-5′-O-[(phosphonomethyl)phosphonic

cultivated at 37 °C with 5% CO . MDA-MB-231 and MaMel.65-

2

acid] (11). Method A. The product was obtained as a colorless solid

ko

CD73 cells were split 1:20 and 1:5, respectively, every 72 h (at 80−

90% cell conﬂuence). To detach the adherent cells, growth medium

1

after lyophilization (2 equiv Et N-salt, 1.4 mg, 5%). H NMR (400

3

MHz, D O): δ 7.76 (d, J = 7.8 Hz, 1H), 5.83 (d, J = 7.8 Hz, 1H), 4.67

2

was removed, and cells were washed with phosphate-buﬀered saline

(

d, J = 6.3 Hz, 1H), 4.12 (d, J = 6.5 Hz, 1H), 4.10−3.98 (m, 2H),

.21 (q, J = 7.3 Hz, 12H), 2.35 (s, 1H), 2.22−2.02 (m, 2H), 1.86 (dd,

J = 9.5, 4.8 Hz, 1H), 1.68 (t, J = 5.5 Hz, 1H), 1.28 (t, J = 7.3 Hz,

2

(

PBS, 25 cm ﬂask: 2.5 mL, for larger ﬂasks correspondingly larger

3

amounts) and incubated with trypsin/ethylenediaminetetraacetic acid

2

(EDTA) (0.05%/0.6 mM, PAN Biotech, Germany; 1 mL for a 25 cm

1

7

8H). ¹³C NMR (100 MHz, D O): δ 166.9, 152.7, 148.2, 101.8, 75.0,

2

ﬂask) for 5 min in an incubator at 37 °C. Detached cells were diluted

3

1

1.9, 65.4, 51.6, 48.3, 46.8 (6C), 24.8, 15.7, 8.3 (6C). P NMR (160

2

with growth media (2 mL for a 25 cm ﬂask) and transferred to new

−

MHz, D O): δ 19.3, 14.1. MS (ESI, m/z): 411.0 [M − H] ; ESI-

HRMS: calcd m/z for C H N O P , 411.0358; found, 411.0363 [M

−

2

culture ﬂasks containing growth media (5 mL for a 25 cm ﬂask).

1

2

17

2

10

2

Membrane Preparation of CD73 from MDA-MB-231 and

−

ko

H] . HPLC purity 96% (R = 2.9 min, method HPLC-A).

Preparation of the Cytosolic Extract from MaMel.65-CD73

t

2

Soluble CD73 Enzyme Preparations. Soluble rat CD73 was

Cells. For both preparations, cells were expanded in 175 cm culture

expressed in Spodoptera frugiperda 9 (Sf9) insect cells and puriﬁed as

ﬂasks to 80−90% cell conﬂuence. After detachment by trypsin/EDTA

29

6

previously described. The cDNA for the soluble human CD73

genbank accession no. NM_002526) was obtained from Prof. Dr.

(0.05%/0.6 mM), 10 cells per dish were transferred to cell culture

2

(

dishes (150 cm ) and incubated for 4 days at 37 °C with 5% CO .

2

49

Norbert Strat

̈

er (University of Leipzig, Germany). In order to

The culture medium was removed, and cells were washed with 10 mL

of PBS and frozen at −20 °C. For membrane preparation, cells were

treated with 1 mL of ice-cold buﬀer (50 mM Tris, 2 mM EDTA, pH

7.4), scraped oﬀ, collected in a conical tube, and centrifuged for 10

min at 1000g (4 °C). The pellet was resuspended in membrane buﬀer

[0.5 mL/dish; 25 mM Tris, 1 mM EDTA, 320 mM sucrose, 1:1000

protease inhibitor cocktail (Sigma-Aldrich, MO, USA), pH 7.4] and

homogenized three times for 30 s each (20 500 rpm, ULTRA-

TURRAX, IKA-Labortechnik, Germany). After centrifugation for 10

min, at 1000g (4 °C), the supernatants were collected and centrifuged

for 30 min at 48 000g (4 °C). The resulting supernatants containing

the cytosolic proteins were discarded, and the pellet was resuspended

in washing buﬀer (0.5 mL/dish; 50 mM Tris, pH 7.4) and centrifuged

again (same conditions). This step was repeated three times. Finally,

the pellet was resuspended in washing buﬀer (0.1 mL/dish),

aliquoted, and stored at −80 °C until use. For producing the

generate a soluble enzyme, the signaling sequence for anchoring the

protein to the membrane via a GPI anchor had been omitted (N-

terminal residues: 1−27, C-terminal residues: 550−574 including

49

GPI-anchor attachment site). In addition, a 6× His-tag was fused to

the C-terminus and the construct was cloned into the vector

pACGP67B, which provides an N-terminal signal peptide for the

protein secretion. Sf9 insect cells were grown in Insect-XPRESS

media (#: BE12-730Q, Lonza, Switzerland) with 10 mg/L gentamicin

and split at a ratio of 1:3 every fourth day. For transfection, cells were

2

seeded into cell culture ﬂasks (25 cm ) at 60−70% conﬂuence. Cell

medium (100 μL) and 1 μL of vector DNA (1000 ng/μL) were

mixed with 2.5 μL of baculovirus genomic ProEasy vector DNA (AB

vector, CA, USA) and combined with premixed 100 μL of cell

medium and 8 μL of Cellfectin II Reagent (Thermo Fisher Scientiﬁc,

MA, USA). The transfection mixture was left for 30 min at rt and then

added dropwise to the cells into the cell culture ﬂasks. The cells were

incubated for 30 min at rt and for further 4 days at 27 °C. Cells from

the transfection procedure were detached from the bottom of the

ko

cytosolic extract from MaMel.65-CD73 cells, a published procedure

62

was adopted. The protein of the supernatant that was obtained after

ultracentrifugation was precipitated with 40% aq ammonium sulfate

O

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

and centrifuged for 10 min at 12 000g (4 °C). The resulting pellet was

solubilized in 4 mL of N-(2-hydroxyethyl)piperazine-N′-ethanesul-

fonic acid (HEPES) buﬀer (40 mM HEPES, 1 mM EDTA, 5 mM

9h were analyzed at 100 μM, and a mixture of the phosphatase

inhibitors levamisole (1 mM) and sodium ﬂuoride (5 mM) was used

as a positive control. As negative controls, reactions in the absence of

any inhibitor or without AMP were performed. In addition, to the

mixture of levamisole and NaF, one of the CD73 inhibitors (4l, 7f, 9g,

or 9h, respectively, 100 μM) was added; however, the CD73

inhibitors did not result in increased inhibition of AMP hydrolysis as

compared to levamisole and NaF alone, even after an extended

incubation time of up to 120 min.

MgCl , 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl ﬂuoride,

2

1

5% glycerol, pH 7.0), transferred to Amicon Ultra-0.5 tubes (10 kDa

cutoﬀ, Merck Millipore, MA, USA), and centrifuged for 20 min at 16

00g (Mikro 200R, Hettich, Germany) for the removal of ammonium

0

sulfate, phosphate, and nucleotide contaminants. The protein samples

were washed three times with 4 mL of the HEPES buﬀer by using the

Amicon ﬁltration tubes under the described centrifugation conditions,

aliquoted, and stored at −80 °C.

Human P2Y Receptor Assay. Calcium mobilization induced by

6

the nucleotide derivatives was measured in a human astrocytoma cell

CD73 Enzyme Inhibition Assays. The assay was performed

line (1321N1) expressing the human P2Y receptor, as previously

6

34

42

essentially as previously described. Stock solutions (10 mM) of the

compounds were prepared in demineralized water, and further

dilutions were performed in assay reaction buﬀer (25 mM Tris, 140

mM sodium chloride, 25 mM sodium dihydrogen phosphate, pH 7.4).

The inhibitor solution (10 μL) was added to 70 μL of assay reaction

buﬀer. After the addition of 10 μL of CD73-containing solution or

suspension (rat CD73: 1.63 ng; human CD73: 0.365 ng; membrane

preparation of MDA-MB-231 cells expressing CD73: 7.4 ng of protein

per vial), the reaction was initiated by the addition of 10 μL of

described.

Human P2Y₁₄Receptor Assay. Inhibition of binding of a high-

aﬃnity ﬂuorescent antagonist was measured using ﬂow cytometry in a

CHO cell line expressing the human P2Y receptor, as previously

1

4

37

described.

In Situ Ecto-5′-Nucleotidase Activity Assay. For localization of

AMPase activities in human tonsils, a modiﬁcation of the lead nitrate

63,64

method was employed.

In brief, palatine tonsils were obtained

from adult patients with chronic tonsillitis undergoing routine

tonsillectomy at Turku University Hospital (permission # TO6/

033/18 from the Hospital Research Council). The tonsils were

washed with physiological salt solution, embedded in the cryo-mold

with Tissue-Tek O.C.T. compound (Sakura Finetek Europe B.V. The

Netherlands), cut using a cryostat and stored at −80 °C. Tonsil

cryosections were preincubated for 30 min in Trizma-maleate sucrose

buﬀer (TMSB; 40 mM Trizma maleate; 0.25 M sucrose, pH 7.4)

supplemented with the alkaline phosphatase inhibitor levamisole (2

mM) and diﬀerent concentrations of CD73 inhibitor AOPCP or 9h.

The enzymatic reaction was then performed for 45 min at 37 °C in a

ﬁnal volume of 20 mL of TMSB-buﬀered substrate solution

3

8

[

2,8- H]AMP [speciﬁc activity 7.4 × 10 Bq/mmol (20 mCi/mmol)],

American Radio-labeled Chemicals, MO, USA, distributed by

Hartman Analytic, Germany), resulting in a ﬁnal substrate

concentration of 5 μM. The enzymatic reaction was performed for

2

5 min at 37 °C in a shaking water bath. Then, 500 μL of cold

precipitation buﬀer (100 mM lanthanum chloride, 100 mM sodium

acetate, pH 4.0) was added to stop the reaction and to facilitate the

3

precipitation of free phosphate and unconverted [2,8- H]AMP. After

the precipitation was completed (after at least 30 min on ice), the

mixture was separated by ﬁltration through GF/B glass ﬁber ﬁlters

using a cell harvester (M-48, Brandel, Gaithersburg, MD, USA). After

washing each reaction vial three times with 400 μL of cold (4 °C)

demineralized water, 5 mL of the scintillation cocktail (ULTIMA

Gold XR, PerkinElmer, MA, USA) was added, and radioactivity was

measured by scintillation counting (Tri-Carb 2900 TR, Packard/

PerkinElmer; counting eﬃcacy: 49−52%). Two controls were

included and measured as duplicates. One reaction was performed

without the inhibitor, resulting in 100% enzyme activity (positive

control), and the other was incubated without the inhibitor and the

enzyme and served as background control. The resulting data were

subtracted from the background and were normalized to the positive

control. The results were plotted, and concentration−inhibition

curves were ﬁtted with GraphPad Prism 5 (GraphPad Software, La

Jolla, USA). The mean IC₅₀± standard error of the mean (SEM)

containing 1.5 mM Pb(NO ) , 1 mM CaCl , 100 μM AMP, and

3 2 2

tested CD73 inhibitor at the same concentration. The lead

orthophosphate precipitated in the course of nucleotidase activity

was visualized as a brown deposit by incubating sections in 0.5%

(NH ) S for 30 s, followed by three washes in Trizma-maleate buﬀer

4 2

for 5 min each. Slides were mounted with Aquatex medium (Merck,

Germany). Multiple images of adjacent tissue areas were captured

using a Pannoramic 250 slide scanner (3DHistech Ltd., Budapest,

Hungary) and further stitched to a larger overview using the

accompanying Pannoramic Viewer 1.15.4 software. The images of

control and treated tissue were captured at identical exposure times

and other settings and further acquired in parallel using Adobe

Photoshop CS6 software. Nucleotidase activities were quantiﬁed as

mean pixel intensities after grayscale conversion using ImageJ 1.52h

software.

CD73 is highly expressed in the germinal centers, connective

tissues, and, to lesser extent, in the interfollicular area. Such

heterogeneity precludes proper quantiﬁcation of “global” AMPase

staining in the whole tissue. Therefore, we selected the same matched

areas of germinal centers in the control and treated tonsils and further

compared their mean pixel intensities. Also, the inhibitor concen-

trations are given as nanomolar for matched volumes, as we have

noticed that the actual amount of inhibitor per tissue slide is an

important factor.

from three independent experiments was used to calculate the K value

i

3

0

with the Cheng−Prusoﬀ equation (K , rat CD73: 53.0 ± 4.1 μM;

K , human CD73: 17.0 ± 2.1 μM; K , (MDA-MB-231): 14.8 ± 2.1

m

μM; Supporting Information, Figure S2).

To show that the triethylammonium salt form of the inhibitors did

not aﬀect is inhibitory activity, triethylammonium chloride was tested

at 500 μM concentration and no reduction of enzymatic activity was

detected (data not shown).

Analysis of AMP Hydrolysis by the Cytosolic Extract

ko

Derived from MaMel.65-CD73 Cells. Reactions were performed

in 50 mM Tris, pH 7.0, buﬀer containing 100 mM KCl, 5 mM MgCl ,

2

ko

and 15.25 μg of the cytosolic extract derived from MaMel.65-CD73

Molecular Docking Studies. The recent cocrystal structure of

70

49

cells. After preincubation of 5 min at 37 °C, the reactions were

initiated by the addition of 5 mM AMP as a substrate. After 60 min,

the reactions were terminated by heat inactivation (5 min at 95 °C),

and samples were placed on ice. Reaction tubes were centrifuged for 5

min at 23 000g (4 °C), and the supernatant was transferred into the

wells of a 96-well half-area microplate (clear, Greiner Bio-One,

Austria). To enable phosphate detection, 20 μL of detection reagent I

the human CD73 (PDB ID: 4H2I) with the antagonist AOPCP was

obtained from the Research Collaboratory for Structural Bioinfor-

65

matics Protein Data Bank. The downloaded crystal structures were

prepared using the protein preparation tool, and the hydrogen atoms

were assigned according to Protonate 3D implemented in Molecular

66

Operating Environment (MOE 2018.01). The crystal structure of

the human CD73 was applied for ﬂexible ligand docking using

67−69

(ﬁnal concentration 120 μM malachite green oxalate, 0.06% polyvinyl

AutoDock 4.2.

During the docking simulations, the ligands were

alcohol) and 30 μL of detection reagent II (ﬁnal concentration: 6 mM

fully ﬂexible, while the residues of the enzyme were treated as rigid.

The selected potent compound 9f was docked into the active site of

the enzyme to predict the binding mode of the compounds. The

atomic partial charges were added and three-dimensional energy

scoring grids for a box of 60 × 60 × 60 points with a spacing of 0.375

ammonium heptamolybdate, 0.45 M H SO ) were added. The plate

2

4

was incubated at rt and 500 rpm for 20 min. The absorption of the

formed colorimetric complex was analyzed at 600 nm (PHERAstar

FS, BMG Labtech, Ortenberg, Germany). Compounds 4l, 7f, 9g, and

P

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6

7,68

Å were computed using AutoDockTools.

The grids were centered

DMEM, Dulbecco’s modiﬁed Eagle medium; DMAP, 4-

dimethylaminopyridine; DMF, N,N-dimethylformamide;

DMSO, dimethyl sulfoxide; EC, enzyme commission; EDTA,

ethylenediaminetetraacetic acid; ESI, electrospray ionization;

FBS, fetal bovine serum; GPI, glycophosphatidylinositol;

HPLC, high-performance liquid chromatography; LC−MS,

based on the cocrystallized ligand, AOPCP. Fifty independent

docking calculations using the varCPSO-ls algorithm from PSO@

Autodock implemented in AutoDock4.2 were performed and

terminated after 500 000 evaluation steps. Parameters of var-

CPSO-ls algorithm, the cognitive and social coeﬃcients c and c ,

were set at 6.05 with 60 individual particles as swarm size. All other

parameters of the algorithm were set at their default values. Possible

binding modes of the compounds were explored by visual inspection

of the resulting docking poses.

69

1

2

liquid chromatography−mass spectrometry; MeOD-d , deu-

4

terated methanol; MOE, Molecular Operating Environment;

eN, ecto-5′-nucleotidase; eNPPs, ecto-nucleoside pyrophos-

phatases/phosphodiesterases; eNTPDases, ecto-nucleoside

triphosphate diphosphohydrolases; PBS, phosphate-buﬀered

saline; pdb, Protein Data Bank; POMs, polyoxometalates; PSB,

Pharmaceutical Sciences Bonn; SAR, structure−activity

relationship; Sf9, Spodoptera frugiperda 9; TEA, triethylamine;

TEAC, triethylammonium hydrogen carbonate; THF, tetrahy-

drofuran; TLC, thin-layer chromatography; TNBC, triple-

negative breast cancer; Tris, tris(hydroxymethyl)-

aminomethane

ASSOCIATED CONTENT

Supporting Information

■

*

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.9b00164.

1

13

31

H, C, and P NMR spectra of all compounds, plots of

pharmacological data, molecular modeling ﬁgures, and

atomic coordinates of the model of 9h bound to CD73

(

PDF)

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S

DOI: 10.1021/acs.jmedchem.9b00164

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors

DOI: 10.1021/acs.jmedchem.9b00164

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.9b00164

Full text of DOI:10.1021/acs.jmedchem.9b00164

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