3,4-Dihydropyrimidin-2(1 H)-ones as Antagonists of the Human A2BAdenosine Receptor: Optimization, Structure-Activity Relationship Studies, and Enantiospecific Recognition

pubs.acs.org/jmc

Article

3,4-Dihydropyrimidin-2(1H)‑ones as Antagonists of the Human A_2B

Adenosine Receptor: Optimization, Structure−Activity Relationship

Studies, and Enantiospeciﬁc Recognition

¶

́

Mar í a Majellaro, Willem Jespers, Abel Crespo, Mar í a J. Nun

̃

ez, Silvia Novio, Jhonny Azuaje,

Ruben Prieto-D í az, Claudia Gioe, Belma Alispahic, Jose Brea,* Mar í a I. Loza, Manuel Freire-Garabal,

Carlota Garcia-Santiago, Carlos Rodr í guez-Garc í a, Xerardo Garc í a-Mera, Olga Caamano,

Christian Fernandez-Masaguer, Javier F. Sardina, Angela Stefanachi, Abdelaziz El Maatougui,

́

̃

́

Ana Mallo-Abreu, Johan Åqvist, Hugo Gutierrez-de-Teran,* and Eddy Sotelo *

Cite This: J. Med. Chem. 2021, 64, 458 −480

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2(1H)-ones as A_2BAR antagonists,

an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A_2BAR, with a number of potent and selective

antagonists further conﬁrmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the

most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure−activity relationship of

this chemotype. The stereospeciﬁc binding modeled for this scaﬀold was conﬁrmed by resolving the two most potent ligands

[( )-47, and ( )-38 K_i= 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the aﬃnity on the corresponding

(S)-eutomers (K_i= 6.30 and 11.10 nM, respectively). The assessment of the eﬀect in representative cytochromes (CYP3A4 and

CYP2D6) demonstrated insigniﬁcant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that

this pair of compounds exhibits a pronounced antimetastatic eﬀect.

myocyte contractility, penile erection, glucose homeostasis,

pulmonary inﬂammation, inﬂammation, and pain).²²Para-

doxically, the reason for the initial low interest in A_2BAR

(namely its low aﬃnity for adenosine) potentially turns this

receptor into an excellent therapeutic target for highly relevant

related pathologies,^12,22since it becomes activated under very

speciﬁc pathological conditions (i.e. micromolar adenosine

concentrations).

INTRODUCTION

■

Since its identiﬁcation and cloning in the early 1990s, A_2B

remains the most enigmatic of the four adenosine receptors

(ARs).^1,2Initially, this receptor was seen as a low-aﬃnity

(adenosine binds with an EC₅₀= 24 μM) version of the

A_2AAR, with which it is often co-expressed in many tissues.

These observations lead to the initial idea that the A_2BAR was

a receptor with scarce physiological relevance,³⁻⁵which was

later ruled out due to the characterization of distinctive

intracellular signaling pathways, body distribution, and

physiological roles between these two subtypes of ARs.⁶⁻¹²

High levels of A_2BAR have been detected in neurons,¹³

astrocytes,¹⁴and various immune cells,¹⁵⁻¹⁸and it has been

conﬁrmed that its expression is heavily aﬀected by environ-

mental cues such as inﬂammation, cell stress, injury, and

hypoxia.¹⁹⁻²¹Accordingly, the A_2BAR is involved in diverse

and relevant biological processes (e.g. vascular tone, cardiac

The relevance of the A_2BAR in cancer progression has been

recognized recently, with increasing evidence demonstrating

Received: August 18, 2020

Published: December 29, 2020

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

458

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Structures of representative A_2BAR antagonists.³⁴⁻⁴³

its protumorigenic role in multiple types of cancer cells.²³⁻²⁵

Growing evidence supports that the A_2BAR promotes tumor

progression in many ways, eliciting key roles during tumor

proliferation, angiogenesis, chemoresistance, metastasis, and

immune suppression.²⁶⁻³¹Consistent with this, high levels of

A_2BAR are generally associated with worse prognosis in several

cancer types.²⁵Comparative analysis has shown that the

A_2BAR is particularly highly expressed in human prostate

cancer tissues as compared to prostatic healthy tissues.³²

Recent studies employing PSB603^32,33(Figure 1, compound

1), a selective sub-nanomolar A_2BAR antagonist, documented

its antitumor eﬀect in PCa cells³²and also the suppression of

tumor growth and metastasis by inhibiting induction of

regulatory T cells.³³Moreover, data emerging from the study

of the adenosine check point in cancer immunotherapy are

progressively demonstrating highly relevant functions for the

A_2BAR.^25,28,29Accordingly, A_2BAR antagonists are emerging as

promising anticancer agents that can take advantage of the

central roles of adenosine in the tumor microenviron-

ment.^25,28,29

The elucidation of the functional roles and therapeutic

potential of the A_2BAR has been partially hampered by the

limited availability of potent and speciﬁc ligands and

molecular probes.⁴⁴⁻⁴⁸While some compounds have reached

clinical trials,^25,46the chemical space explored by the current

A_2BAR agonists is still limited,⁴⁴⁻⁴⁸and the best characterized

A_2BAR antagonists are mainly xanthine congeners (Figure

1).³⁴⁻⁴³Hence, novel prototypic structures providing unex-

plored topologies, physicochemical features, and alternative

binding modes are in demand.^46,47In this scenario, we

recently documented novel series of potent A_2BAR antagonists

assembled by a Biginelli multicomponent approach (Figure 1,

cpds 9−12).⁴¹⁻⁴³The chiral center in these chemotypes

imposes critical topological features, diﬀerentiating themselves

from classical A_2BAR ligands and modifying their biological

proﬁle from quantitative and qualitative points of view. Thus,

in addition to its low nanomolar aﬃnities and selectivity, their

antagonistic eﬀect is mediated by a previously unexplored

stereospeciﬁc recognition at the A_2BAR.^42,43,49

In this work, we report an exhaustive exploration of the

structural determinants governing the A_2BAR antagonistic

eﬀect of the 3,4-dihydropyrimidin-2(1H)-one (DHPM)

scaﬀold (Figure 1, cmpd 11), discovered by our group

together with the analogous series of 3,4-dihydropyrimidin-

2(1H)-thiones (Figure 1, cmpd 12).⁴⁸A large library,

consisting of 160 derivatives, was conceived to assess the

contribution of the diverse positions in the heterocyclic core,

with particular emphasis in the exploration of bioisosteric

replacements for the ester function at position 5. The study

also included the elucidation of the structure−activity

relationship (SAR) and assessment of the initially proposed

enantiospeciﬁc recognition at the A_2BAR by computational

modeling using intensive free energy perturbation (FEP)

calculations. The impact of the stereochemistry at position 4

in these series was conﬁrmed by separation of the two most

potent ligands (38 and 47) into their enantiomers, followed

by pharmacological evaluation conﬁrming the enantiospeciﬁc

binding to the A_2BAR. Finally, a preliminary exploration of the

antitumoral potential of ligands 38 and 47 demonstrated a

potent antimigratory eﬀect in three cellular models of PCa.

RESULTS AND DISCUSSION

■

Design. Our early report, documenting the original A_2BAR

antagonistic eﬀect of 34 DHPMs, included a preliminary

459

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. General structure of previously explored chemotypes (panel A) and novel series herein documented (panel B, series I−IX).

exploration of some SAR trends and the proposal of a binding

mode for this scaﬀold.⁴³In subsequent studies, we focused on

the evaluation of diﬀerent points of variability around the

DHPM scaﬀold. The deﬁnition of the optimal substituents in

include the replacement of 3,4-dihydropyrimidin-2(1H)-one/

thione (series I/II, respectively, Figure 2 panel B) and the

methylation of N₁(series III). Since the introduction of aryl,

alkyl, or cycloalkyl groups at R₄produced inactive

compounds,⁵⁰we maintained the original set of pentagonal

heteroaryl group (e.g. 2-furyl, 3-furyl and 2-thienyl, and 3-

thienyl) characteristics of the ligands with highest A_2BAR

aﬃnity. For these three early subsets (I−III), seven alkyl

residues (Me, Et, Pr, i-Pr, i-Bu, t-Bu, and Bn) were explored in

the ester function (R₅). Series IV−IX (Figure 2 panel B) were

designed to complete the evaluation of the inﬂuence of R₅on

the A_2BAR aﬃnity and AR selectivity proﬁles. The design of

each subset was conceived to analyze diﬀerent bioisosteric

replacements (subsets IV, V, VI, and VII). We then moved to

evaluate the eﬀect of cycle creation with series VIII. Finally, as

the biological evaluation of series IV−VIII unequivocally

identiﬁed an aliphatic ester as the optimum group in R₅, a new

subset (IX) was designed to expand the diversity of the alkoxy

group in the ester on this position (Figure 2, panel B).

Chemistry. The library, consisting of 160 AR ligands (cpds

16−99 and 105−180), was assembled by following diﬀerent

experimental protocols based on the highly reliable Biginelli

50

R₄was soon followed by scaﬀold hoping, investigating the

eﬀect of fusing other heterocycles at positions 2 and 3 of the

pyrimidin-2-one core (Figure 2, panel A)⁴¹and the impact of

bioisosteric replacements at either position 2 or 3 (Figure 2,

panel A).^41,42The SAR data accumulated in these studies was

rationalized on the basis of the A_2BAR stereoselective binding

mode originally proposed by us⁴³and constituted the basis to

guide the design of 136 new DHPMs, herein synthesized and

characterized, which we have now divided into nine series

(Figure 2, panel B). The integrated synthesis, binding data,

SAR, and computational studies of the complete ligand library

are described in this manuscript. Three initial subsets (Figure

2, panel B, series I−III) were conceived to complement the

SAR exploration of our original report,⁴³by modiﬁcation of

the substituents at positions 1, 2, 4, and 5 (Figure 2, panel B).

The diversity of space explored was envisaged to assess

distinctive structural features (e.g. electronic, steric, or lipo/

hydrophilic) within the heterocyclic core (Figure 2). These

460

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 1. Synthesis of 3,4-Dihydropyrimidin-2-one/thione Series I−III (Cpds 16−99)

Scheme 2. Synthesis of 3,4-Dihydropyrimidin-2-one Series IV−VI (Cpds 105−144)

three-component reaction.^51,52The experimental conditions of

the synthetic pathways are depicted in Schemes 1−3. In a

typical experiment, the urea derivative (13a−c) was combined

with a pentagonal heterocyclic aldehyde (14a−d), a

methylene-activated carbonyl compound [e.g. keto-esters

(15a−n), diketones (100a−d), keto-amides (101a−d), keto-

thioesters (102a−b), keto-phosphonate (103) or keto-1,3-

oxazole (104)], and the appropriate catalyst. The reaction

mixture was heated at 80 °C during variable reaction times

(6−24 h). Due to the reactivity diﬀerences among the

methylene-activated carbonyl compounds (15, 100, 101, 102,

103, and 104) and being aware that the eﬃciency of the

Biginelli reaction is dependent of the catalyst employed,^51,52

the optimal catalyst for each series was identiﬁed during

preliminary experiments (Schemes 1−3). Targeted pyrimidine

derivatives (16−99 and 105−180) were obtained in variable

yields (37−90%) after appropriate puriﬁcation (i.e. chromato-

graphic methods or recrystallization).

The synthesized compounds were organized in nine subsets

(Figure 2, panel B); the structures are depicted in Tables 1−8.

Within the ﬁrst three series (I, II, and III), 8 out of the 28

compounds within each subset correspond to previously

reported compounds (denoted with an asterisk in Tables 1−3

and the corresponding references included in the Supporting

Information). Series IV−IX contained 16, 16, 8, 4, 4, and 28

new ligands, respectively (Tables 4−8). As in previous

studies,^41−43,49all ligands were tested as racemic mixtures

and this data constituted the basis to explore the SAR.

Thereafter, the most appealing A_2BAR antagonists were

separated into its enantiomers, their conﬁguration was

established by circular dichroism (CD), and then, its binding

aﬃnity at all four Ars was evaluated for each stereoisomer. A

full account of the synthesis, structural, and characterization

data for all compounds is provided in the Supporting

Information.

461

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 3. Synthesis of 3,4-Dihydropyrimidin-2-one Series VII−IX (Cpds 145−180)

Biological Evaluation. The aﬃnities for the four human

AR (hAR) subtypes (A₁, A_2A, A_2B, and A₃) of the 160 3,4-

dihydropyrimidin-2-one/thiones obtained (16−99 and 105−

180) were evaluated in vitro (radioligand binding as-

says)^41−43,49and reported in Tables 1−8. hARs expressed in

transfected Chinese hamster ovary (CHO) (A₁AR), HeLa

(A_2AAR and A₃AR), and HEK-293 (A_2BAR) cells were

employed. [³H]DPCPX for A₁AR and A_2BAR, [³H]ZM241385

for A_2AAR, and [³H]NECA for A₃AR were used as

radiotracers during binding assays. The aﬃnity data obtained

for the racemic mixture of each compound is provided in

Tables 1−8. The biological data are expressed as K_i

standard error of the mean (SEM) (nM, n = 3) or as

percentage inhibition of speciﬁc binding at 1 μM (n = 2,

average) for those compounds that did not fully displace

speciﬁc radioligand binding. K_ivalues were obtained by ﬁtting

the data with nonlinear regression using Prism 2.1 software

(GraphPad, San Diego, CA). The binding aﬃnity obtained for

representative AR antagonists (ISAM-140, ZM241385, and

DPCPX), using the binding protocols herein employed, was

included in Tables 1−8 for comparative purposes.

Functional Experiments. Cyclic Adenosine Monophos-

phate Assays. The two most potent compounds of the series

(38 and 47) were evaluated in cyclic adenosine mono-

phosphate (cAMP) assays to determinate their ability to

inhibit NECA-stimulated (100 nM) cAMP production.^41−43,49

The log concentration−response curves of c-AMP accumu-

lation for selected antagonists to hA_2BARs are presented in

Figure 3. These experiments demonstrated that compounds

38 and 47 inhibit NECA-induced c-AMP accumulation,

unequivocally validating their antagonism at hA_2BARs.

Analysis of the K_iand K_Bvalues shows low nanomolar

range data obtained during binding (K_i= 23.6 and 10.2 nM,

respectively) and functional experiments (K_B= 25.2 and 4.7

nM, respectively).

recognition at the A_2BAR binding pocket was also inferred

during our early SAR exploration of the DHPM scaﬀold.⁴³It

was observed that aromatization of the 3,4-dihydropyrimidin-

2(1H)-one/thione nucleus abolished the A_2BAR aﬃnity for

ligands 38 and 47.⁴³In addition, in our recent studies with

other structurally related scaﬀolds,^42,43the most attractive

ligands were separated and the corresponding active stereo-

isomer correlated with these predictions. Following this line of

reasoning, we proceeded to the enantiomeric separation and

assignment of the most attractive compounds within series I−

IX [( )-38 and ( )-47], followed by their biological

evaluation.

Figure 4 shows the results of our joint approach using chiral

high-performance liquid chromatography (HPLC) and CD

spectroscopy. As recently documented by our own results with

structurally related Biginelli-like systems^42,49as well as for

other pharmacologically active 3,4-dihydropyrimidin-2-ones/

thiones,⁶¹⁻⁶³the sign of the distinctive CD activity of the

enamide group (around 300 nm) allows the unequivocal

assignment of the absolute conﬁguration of each enantiomer

(Figure 4). Semipreparative HPLC separation of the selected

racemic ligands [( )-38 and ( )-47] on a chiral stationary

phase (Figure 4 and Experimental Section) aﬀorded each

enantiomer with excellent stereochemical purity (97 and 99%

respectively). At this wavelength, enantiomers showing a

negative Cotton eﬀect (red line) contain the pentagonal

heterocycle (furan or thiophene) backward [which corre-

sponds to (4S)-38 and (4S)-47, respectively] while the

stereoisomers giving a positive Cotton eﬀect (blue line)

contain the heterocyclic core forward [which corresponds to

(4R)-38 and (4R)-47, respectively].

The biological proﬁles of the isolated enantiomers [(R)-38,

(S)-38, (R)-47, and (S)-47] were then evaluated at the four

hARs, and the resulting data (Table 9) are compared to the

corresponding racemic mixtures [( )-38 and ( )-47]. With

this analysis, we could conﬁrm that the A_2BAR aﬃnity

exhibited by the racemic mixture of 38 and 47 is exclusively

due to the (S)-38 and (S)-47 enantiomers (K_i= 11.1 and 6.30

nM). In both cases, the eutomer [the (S) stereoisomer] is

nearly twofold more potent than the racemic mixture [38, (K_i

= 23.6 nM), 47, (K_i= 10.2 nM)], whereas (R)-stereoisomers

are devoid of any aﬃnity at the four ARs (Table 9). To

Enantiospeciﬁc Binding to the A_2BAR. As indicated above,

all compounds here reported were obtained and tested as

racemates (Tables 1−8). However, both our initial molecular

models⁴⁸and results from the FEP calculations (see previous

section) indicate that only one enantiomer should bind the

orthosteric site of the receptor. The importance of the

stereogenic center at the heterocyclic core during the

462

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 16−43 at the hARs (Subset I, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

R₅

hA₁

34%

15%

43%

18%

27%

4%

36%

8%

21%

50%

25%

hA_2A

25%

31%

55%

23%

36%

7%

hA_2B

hA₃

13%

2%

3%

4%

13%

7%

3%

2%

1%

11%

3%

16%

2%

1%

3%

16⁴³

2-furyl

3-furyl

2-thienyl

3-thienyl

Me

Et

2159 232

585 89

206 11

40.8 2.3

199 11

44%

887 30

42%

39.6 1.1

1192 107

1486 62

17⁴³

18⁴³

n-Pr

i-Pr

i-Bu

t-Bu

Bn

19⁴³(SYAF014)

20

21

22

23

26%

3%

Me

Et

24⁴³(SYAF101)

24%

45%

26%

31%

9%

25

n-Pr

i-Pr

i-Bu

t-Bu

Bn

26⁴³

27

3088 88

11%

41%

6%

326

7

28⁵³

4436 182

6650 210

19%

29

24%

1%

30⁵⁴

31⁴³

Me

Et

1%

33%

30%

19%

12%

10%

30%

2%

34%

48%

25%

37%

5%

41%

1%

32

n-Pr

i-Pr

i-Bu

t-Bu

Bn

43%

37%

25%

26%

45%

7%

16%

44%

26%

56%

15%

57%

20%

1519 117

44%

2130 88

38%

1916 110

36%

23.6 1.0

32%

14%

11%

7%

8%

3%

11%

6%

5%

21%

1%

25%

2%

33⁴³

34

35⁵⁵

36⁵⁶

37⁵⁷

Me

Et

38⁴³(SYAF080)

39

n-Pr

i-Pr

i-Bu

t-Bu

Bn

201

3

40⁴³(SYAF020)

41

42⁵⁵

56.6 1.3

1188 110

47%

43

21%

25%

44%

ISAM-140

ZM241385

DPCPX

3.49 0.2

65.7 1.7

73.24 2.0

683

4

1.9 0.1

157 2.9

863

4

2.20 0.2

1722 11

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

provide a further structural rationale, we calculated the

energetic diﬀerence between both enantiomers of compound

47 via direct FEP simulations between the compound pair,

starting from the binding model proposed in each case (see

Figure S4). The simulations agree with the experimental data,

indicating stark binding preference for the most potent

39, 54, 68, 108, 158, 161, 162, 165, and 176), while still

retaining the excellent selectivity proﬁle characteristic of these

series. The most interesting DHPMs contained an exocyclic

carbonyl group at position 2 and no substituent at positions 1

and 3 (series I, IV−IX), while the most potent A_2BAR

antagonist (47, K_i= 10.2 nM) belongs to the bioisosteric 3,4-

dihydropyrimidin-2(1H)-thione series II (Table 2). The ﬁrst

part of the SAR analysis is focused on the ﬁrst three subsets

(Figure 2, series I−III), exploring precisely the eﬀect of the

O/S bioisosteric replacement at position 2 of the DHPM

scaﬀold (series II) as well as methylation on N₁(series III).

All these series maintain a substituted ester group (position

5), and the impact of bioisosteric replacement of this

functionality is further analyzed in subsets IV−VIII. It will

be deduced that collectively, the most appealing A_2BAR

aﬃnities within series I−VIII are indeed within the ﬁrst subset

of DHPMs (Table 1, compounds 16−43). This motivated an

expansion of the diversity of the alkyl residues at the ester

group (subset IX), which will be analyzed in depth through

eutomer (ΔΔG = 6.04

1.06 kcal/mol). These data

constitute a remarkable example of A_2BAR antagonists that

exhibit enantiospeciﬁc recognition proﬁles, which is satisfac-

torily explained with the binding mode hypothesis behind the

design of this series, in agreement with our observations for

structurally related Biginelli-based scaﬀolds.⁵⁰

SAR Analysis and Molecular Modeling. The biological

evaluation of the nine series of DHPMs (Tables 1−8) allowed

us to identify eight potent (K_i< 100 nM) and speciﬁc A_2BAR

ligands, for example, compounds 19, 24, 38, and 40 (Table

1), 47 and 52 (Table 2), and 154 and 155 (Table 8).

Additionally, a dozen of DHPMs exhibited moderate (K_i

100−500 nM) A_2BAR aﬃnity (e.g. compounds 18, 20, 27,

463

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 2. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-thiones 44−71 at the hARs (Subset II, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

R₅

hA₁

hA_2A

hA_2B

hA₃

44⁵⁴

2-furyl

3-furyl

Me

Et

11%

1%

15%

7%

3%

32%

25%

18%

4%

18%

12%

18%

25%

1%

20%

32%

2%

18%

30%

1%

12%

1%

25%

2%

33%

35%

15%

16%

1%

23%

1%

15%

2%

7%

63%

1%

28%

1%

20%

1%

22%

16%

25%

1.9 0.1

157 2.9

984 52

608 31

46%

10.2 0.5

18%

1%

5%

1%

10%

9%

4%

3%

15%

3%

23%

1%

45⁴³

46

n-Pr

i-Pr

i-Bu

t-Bu

Bn

47⁴³(SYAF030)

48

49

50

51

52⁴³

53

54⁴³

55

9%

41%

48%

Me

Et

43.1 1.6

45%

194 12

1%

1893 88

36%

10%

3247 101

3%

1572 93

12%

n-Pr

i-Pr

i-Bu

t-Bu

Bn

56

57

58

19%

20%

1%

3-furyl

2-thienyl

3-thienyl

Me

Et

59⁴³

60⁵⁸

61⁴³

62⁵⁸

63

2%

7%

4%

n-Pr

i-Pr

i-Bu

t-Bu

Bn

10%

20%

4%

22%

24%

3%

2%

1%

3%

2%

23%

1%

22%

34%

3%

23%

5%

16%

8%

26%

45%

38%

64⁵⁸

65

Me

Et

66⁴³

67

1103 94

26%

n-Pr

i-Pr

i-Bu

t-Bu

Bn

68⁴³

69

315

15%

7

70

71

16%

3%

20%

26%

10%

3.49 0.2

65.7 1.7

73.24 2.0

16%

2%

ISAM-140

ZM241385

DPCPX

683

2.20 0.2

4

863

1722 11

4

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

the last section of this SAR study. The eﬀect of all these

structural modiﬁcations on binding aﬃnity will be systemati-

cally assessed by FEP calculations, performed on the basis of

the binding mode previously proposed and here reﬁned for

the A_2BAR.⁴⁹

largest substitution (Me to Bn), with optimal aﬃnities

observed for medium sized substituents (Et, i-Pr).

The observed SAR trends generally support the binding

orientation previously proposed for this chemotype, here

illustrated in Figure 5 for the DHPM scaﬀold after further

reﬁnement (see below).⁴³This model resulted from a docking

exploration of compound 38 (subset I) using a homology

model of the hA_2BAR based on the X-ray structure of the

A_2AAR⁶⁵and retained two key interactions conserved in most

AR antagonists: a hydrogen bond with the AR-conserved

residue N254^6.55, achieved via the oxygen atom at position 2

of the ligand, and the π−π stacking of the core ring with

F173^EL2. These interactions are complemented by an

enantiospeciﬁc shape complementarity between the L-shape

formed by the pyrimidinone core and the ring at R₄and the

A_2BAR speciﬁc residue V250^6.51, providing a possible source of

selectivity versus other AR members (containing a bigger

Leu).⁴³A similar binding orientation was found for the

structural analogues bicyclic and tricyclic scaﬀolds,⁴¹including

The ﬁrst general SAR observation is the systematic drop in

aﬃnity to negligible values due to methylation on position N₁

of the pyrimidine scaﬀold observed along subset III (Table 3,

72−99). Notably, this eﬀect is not compensated by any

substituents explored at either R₄or R₅. Within the ﬁrst

subsets, the eﬀect of the pentagonal heterocycle at position 4

also shows some clear tendencies: 2-furyl-, 3-furyl-, and 3-

thienyl-substituted ligands exert higher aﬃnities as compared

to the 2-thienyl substituted ligands. This is clearly exempliﬁed

in Figure 5, showing a comparison of the most active

compound within each series (I−III). In this case, however,

the eﬀect is variable depending on the substituent at R₅. In

general, the exploration of the R₅chain on the ester group

reveals a size dependency, that is, from the smallest to the

464

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 72−99 at the hARs (Subset III, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

R₅

hA₁

45%

17%

3%

29%

13%

44%

hA_2A

1%

2%

37%

3%

20%

37%

5%

24%

1%

2%

4%

16%

37%

49%

1%

24%

1%

12%

3%

13%

23%

3%

hA_2B

31%

hA₃

72

2-furyl

3-furyl

2-thienyl

3-thienyl

Me

Et

9%

73⁴³

74

6544 101

23%

1151 23

4%

25%

8%

n-Pr

i-Pr

i-Bu

t-Bu

Bn

75⁴³

76

18%

23%

54%

11%

7%

77

78

79

26%

41%

7%

1736 89

15%

17%

1%

Me

Et

80⁴³

81

4259 95

2%

1%

n-Pr

i-Pr

i-Bu

t-Bu

Bn

16%

1%

82⁴³

83

23%

4%

21%

51%

30%

1%

9%

22%

9%

18%

50%

24%

32%

2%

35%

8%

18%

46%

20%

37%

1%

16%

49%

17%

11%

1%

28%

2%

24%

50%

26%

53%

1%

51%

1%

16%

12%

25%

15%

2%

84

85

86⁵⁹

87⁴³

88

89⁴³

90

91

92

Me

Et

n-Pr

i-Pr

i-Bu

t-Bu

Bn

4%

17%

5%

22%

18%

22%

3%

11%

14%

4%

14%

2%

93

Me

Et

94⁴³

95

n-Pr

i-Pr

i-Bu

t-Bu

Bn

96⁴³

97

22%

2%

1%

30%

25%

98

99

35%

4521 79

3.49 0.2

65.7 1.7

73.24 2.0

ISAM-140

ZM241385

DPCPX

683

4

1.9 0.1

157 2.9

863

4

2.20 0.2

1722 11

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

a related series consisting of triﬂuorinated pyrimidine-based

A_2BAR antagonists⁴⁹as well as for the monocyclic cyanoimino

derivatives.⁴²In the last two cases, the stereospeciﬁc

recognition hypothesized with molecular modeling was

experimentally conﬁrmed by enantiomeric separation of the

most potent compounds by chiral HPLC followed by

structural characterization by CD and X-ray determination

of the most potent compounds.^42,49It thus seemed reasonable

to retain the equivalent active stereoisomer for modeling

purposes of the current series, something that we would later

evaluate by a similar experimental separation and evaluation of

the stereoisomers of the most potent compounds here

identiﬁed (see below). The binding orientation described for

38⁴³was used to create an analogous complex with the

prototype compound 19, which was here subject to a

molecular dynamics (MD) equilibration. The resulting reﬁned

binding model importantly considers the role of water-

mediated interactions and led to a slight shift of the position

of the ligand, allowing for a second hydrogen bond between

N254^6.55and the NH at position 1 (see Figure 5), in analogy

to the dual hydrogen bond observed between this residue and

most co-crystallized AR ligands. This reﬁned position was

then used for a ﬁrst series of FEP simulations, designed to

evaluate if the proposed binding model could successfully

explain the pronounced detrimental eﬀect on methylation at

N₁(comparison between subsets I and III, Figure 5) as well

as the role of the carbonyl/thiocarbonyl bioisosteric

substitution at position 2, (i.e., comparison between

equivalent ligand pairs of subsets I and II, see Figure 6).

The FEP calculations covered all compound pairs between the

two series to be compared, where at least one of the molecules

involved showed measurable A_2Bbinding aﬃnities (K_i< 1

μM), thus yielding 10 and 12 pair comparisons for subsets I

→ III and I → II.

The calculations of binding aﬃnity diﬀerences between

subsets I and III yielded excellent agreement with the

experimental results (Figure 6, compare gray and blue bars)

with a mean absolute error (MAE) of 0.71 kcal/mol over the

465

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 4. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 105−120 at the hARs (Subset IV, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

105⁶⁰

R₄

R₅

hA₁

30%

hA_2A

21%

hA_2B

38%

hA₃

14%

2-furyl

Me

106

107

108

109

110

111

112

113⁶⁰

114

115

116

117⁶⁰

118

2-furyl

3-furyl

2-thienyl

3-thienyl

c-Pr

n-Pr

i-Bu

Me

c-Pr

n-Pr

i-Bu

Me

c-Pr

n-Pr

i-Bu

Me

c-Pr

n-Pr

i-Bu

2%

1%

16%

6%

1%

2%

6%

1%

2%

20%

2%

1%

1062 29

807 12

7%

1%

31%

2%

16%

2%

18%

3%

15%

2%

18%

1%

240

5

15%

10%

22%

1008 25

2%

1%

9%

43%

24%

1%

9%

1002 11

3.49 0.2

65.7 1.7

73.24 2.0

1%

23%

11%

1%

2%

19%

9%

2%

6%

37%

20%

683

23%

1%

4%

119

120

ISAM-140

ZM241385

DPCPX

11%

19%

25%

1.9 0.1

157 2.9

1%

14%

2%

863

1722 11

4

2.20 0.2

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

10 pair comparisons. According to the binding mode

proposed (Figure 5A), the reason for the systematic reduction

in aﬃnity observed upon N₁-methylation can be found in the

to our binding model, the replacement of an oxygen by a

bigger sulfur atom at position 2 produces a slight shift in the

position of the ligand, which can lead to suboptimal

accommodation of the remaining substituents, depending on

the nature of R₄and R₅. Encouragingly, this trend is also

correctly reproduced by our theoretical calculations, and in

most cases, the stabilizing or destabilizing eﬀect of the

bioisosteric replacement is correctly predicted (Figure 7,

ΔΔG_avg,calc= 0.68 0.87 kcal/mol, MAE = 1.17 kcal/mol).

The next cycle of FEP simulations was conceived to explore

the eﬀects of the diﬀerent substitutions at R₅(subsets IV−IX)

on the A_2BAR aﬃnity. This substituent would sit on a narrow

region between TM2 and TM3 in the extracellular side of the

binding cavity (Figure 7). The ester (subsets I−III and IX) or

bioisosteric analogues (series IV−VIII) would displace the

water molecules that eventually ﬁll this region, which were

initially occupying this pocket by analogy with the high-

resolution crystal structure of the A_2AAR.⁶⁶In this way, the

alkyl chain on the functionalized ester (or by analogy on most

of the bioisosteres), would sit in a relatively small and

destabilization of the hydrogen bond with conserved N254^6.55

.

However, given the relative symmetry of the 3,4-dihydropyr-

imidinone scaﬀold, one cannot rule out the possibility of an

alternate binding mode adopted by the methylated DHPM

scaﬀold. This would meanwhile place the substituent at R₅in

the same extracellular binding crevice, as previously ex-

plored,^42,43while satisfying the double hydrogen bond with

N254^6.55as illustrated in Figure 6B. To further analyze this

possibility, we performed the corresponding FEP trans-

formations using the alternative binding mode. The results,

depicted in Figure 6C in orange bars, systematically predicted

incorrect increases in binding aﬃnity for the methylated

compounds in subset III, in stark contrast with the

experimental results (MAE = 3.59 kcal/mol). Thus, the

simulations clearly support the stereospeciﬁc binding mode

previously proposed⁴³and here reﬁned (Figure 6A).

Consequently, we retained this binding mode as the starting

point to explore the diﬀerences in aﬃnity due to the

thiocarbonyl substitution in position 2 (subset II). This single

heteroatom substitution has a more spurious eﬀect along the

two subsets to be compared (I and II), as illustrated by a 4-

fold increase in binding aﬃnity observed between 19 and 47,

while the best compound in series I (38) dramatically loses

aﬃnity (50 fold) when transformed into the corresponding

thienyl derivative 66. On average, for the 12 compound pairs

considered, the experimental eﬀect of this bioisosteric

substitution is milder than in the previous case of N₁

methylation (ΔΔG_avg,exp= 0.94 1.35 kcal/mol). According

hydrophobic cavity deﬁned by residues Ala64^2.61, Ile67^2.64

,

Ala82^3.29, and Val85^3.32. Indeed, on the basis of this binding

mode, we could recently explain the succinct eﬀect of

ﬂuorination in the functionalized ester substituents at R₅.⁴⁹

The limited size of this pocket also correlates with the SAR

observation that larger hydrophilic changes result in loss of

binding (in particular series VII). To assess these qualitative

observations, we performed FEP calculations transforming R₅

between three reference compounds in series I, namely 19 (R₄

= 2-furyl, R₅= CO₂−i-Pr, K_i= 40.8 nM), 24 (R₄= 3-furyl, R₅

= CO₂−Et, K_i= 39.6 nM), and 38 (R₄= 3-thienyl, R₅=

466

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 5. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 121−136 at the hARs (Subset V, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

R₅, R_5′

hA₁

hA_2A

hA_2B

hA₃

121⁴³

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

2-furyl

3-furyl

2-thienyl

3-thienyl

H, H

H, Et

H, i-Pr

Et, Et

H, H

H, Et

H, i-Pr

Et, Et

H, H

H, Et

H, i-Pr

Et, Et

H, H

H, Et

H, i-Pr

Et, Et

2%

3%

11%

1%

15%

2%

1%

2%

13%

2%

3%

8%

11%

20%

1%

9%

3%

2%

1%

6%

1%

22%

15%

18%

17%

1%

2%

1%

2%

31%

6%

2%

3%

12%

3%

1%

40%

22%

1%

3%

11%

2%

3%

4%

19%

2%

1%

3%

37%

1%

4%

1%

31%

1%

35%

2%

9%

25%

1.9 0.1

157 2.9

ISAM-140

ZM241385

DPCPX

3.49 0.2

65.7 1.7

73.24 2.0

683

2.20 0.2

4

863

1722 11

4

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

Table 6. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 137−144 at the hARs (Subset VI, Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

R₅

Et

i-Pr

Et

i-Pr

Et

i-Pr

Et

hA₁

hA_2A

2%

hA_2B

29%

22%

1%

hA₃

14%

137

138

139

140

141

142

143

144

2-furyl

3-furyl

2-thienyl

3-thienyl

6%

5%

3%

9%

6%

1%

11%

24%

20%

683

2%

1%

2%

8%

1%

2%

25%

1%

3%

1%

2%

1%

2%

863

637 28

1%

17%

14%

i-Pr

ISAM-140

ZM241385

DPCPX

3.49 0.2

65.7 1.7

73.24 2.0

4

1.9 0.1

157 2.9

4

2.20 0.2

1722 11

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

CO₂−Et, K_i= 23.6 nM) and their counterparts in series IV−

IX. In other words, each parent compound in series I was

compared to an array of 19 compounds which diﬀer on R₅,

while maintaining the corresponding R₄substituent in each

case (see Tables S1−S3 in the Supporting Information). The

replacement of the ester function to diﬀerent bioisosteres,

namely ketone (subset IV), amide (subset V), thioamide

(subset VI), phosphate (series VII), or oxazolyl (subset VIII)

functions, results in all cases in a moderate to complete loss of

experimental aﬃnity (Tables 4−7), which is correctly

modeled by the corresponding FEP transformations in all

cases (Tables S1 −S3). The mildest reduction in aﬃnity is

observed in subset IV, where a ketone in R₅in combination

with 2-furyl in R₄(Table 4, compounds 105−108) yields

467

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 7. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 145−152 at the hARs (Series VII and VIII,

Figure 2)

a

K_i(nM) or % at 1 μM

b

c

d

e

compound

R₄

hA₁

hA_2A

4%

8%

5%

3%

23%

14%

7%

20%

25%

hA_2B

hA₃

145

146

147

148

149

150

151

152

2-furyl

3-furyl

2-thienyl

3-thienyl

2-furyl

3-furyl

2-thienyl

3-thienyl

3%

1%

2%

4%

27%

21%

34%

20%

683

2%

3%

7%

1%

4%

2%

5%

1%

4%

1%

2%

905 39

801 13

45%

1126 45

3.49 0.2

65.7 1.7

73.24 2.0

ISAM-140

ZM241385

DPCPX

4

1.9 0.1

157 2.9

863

4

2.20 0.2

1722 11

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

compounds that can be almost as active as their counterparts

in subset I (R₅= CO₂R, e.g. compare compound 20 with

108), something that is well captured by the FEP calculations

(see Supporting Information S1). The systematic drop in

aﬃnity observed when an amide function replaces the ester in

R₅(subset V) is also correctly reproduced in our models, with

calculated loss in aﬃnity close to or higher than the

experimental threshold of the binding experiment (e.g.

ΔΔG_exp≥ 3.27 kcal/mol, see Tables S1 −S3), indicating

that this function does not ﬁt on the designated binding site.

The same applies for the phosphate and oxazolyl containing

compounds (series VII and VIII), and in the former case, the

ligand cannot even be accommodated in the binding site to

run any FEP calculation.

Finally, the extensive exploration of the alkoxy substituent

on the ester function resulted in a broad range of eﬀects in

aﬃnity, as illustrated Figure 7, showing a bar plot

representation of the experimental aﬃnities within subsets I

and IX. This eﬀect is, in most cases, correctly reproduced and

rationalized by FEP simulations, particularly in the 3-furyl

(Supporting Information S2) and 3-thienyl (Supporting

Information S3) containing compounds. The structural reason

of the observed eﬀects can be traced back to the presence of

two buried water molecules adjacent to the hydrophobic

pocket (see Figure 8B,C), which were previously characterized

as highly stable.⁶⁷Indeed, along the MD simulations

associated to the FEP calculations, the compounds with

bulkier alkyl chains at R₅= CO₂R trend to selectively displace

one or the two water molecules (depicted as water 2 and 3 in

Figure 8) as compared to compounds with optimized R

chains, which only displace the unstable water 1 (see Figure

8). The overall agreement of our FEP calculations and

experiment is very reasonable, with a MAE of 1.15 kcal/mol

for all perturbations with measurable experimental binding

aﬃnities, which fall well within values previously reported by

others on homology models with much smaller datasets and

provide a better signal for the described SAR observations

than more intuitive relationships, such as correlation of aﬃnity

shifts with MW or SASA of the substituent in R₅(data not

shown). Altogether, the experimental data and computational

modelling suggest a combined eﬀect due to the optimal shape

complementarity and speciﬁc water displacement elicited by

the R₅substituent, which is also dependent on the positioning

of the heteroatom within the ring at R₄.

Preliminary ADME Exploration. With the aim to early

identify metabolic liabilities of herein explored series, the

inhibitory activity of ( )-38 and ( )-47 at two prototypical

cytochrome P450 isoforms (CYP3A4 and CYP2D6) was

assessed.⁶⁸The selected subfamilies (CYP3A4 and CYP2D6)

are abundant in the liver, shows broad substrate speciﬁcity,

and are responsible for the metabolization of numerous

drugs.⁶⁹⁻⁷¹This data would enable to anticipate potential side

eﬀects and to examine the tendency for drug−drug

interactions. The assays (done in duplicates) use ﬂuores-

cence-based detection and employed 7-benzyloxy-4-(triﬂuor-

omethyl)-coumarin (IC₅₀= 0.008 μM), ketoconazole (IC₅₀

=

0.027 μM), and quinidine (IC₅₀= 0.0073 μM) as references

for CYP3A4 and CYP2D6. The obtained data is presented in

Table 10. As observed, at the evaluated concentrations (1 and

10 μM), ligands ( )-38 and ( )-47 do not show substantial

interaction with the studied cytochrome isoforms (CYP3A4

and CYP2D6). The ADME proﬁle (in vitro) of the novel

potent A_2Bantagonists herein identiﬁed (38 and 47) was

completed by evaluating its stability in human microsomes.

The obtained data revealed that both ligands exhibited

satisfactory microsomal stability, with 65 and 77% of ( )-38

and ( )-47 remaining unaltered after 60 min of exposure to

human microsomes. The emerging ADME data highlight the

attractiveness of the identiﬁed lead compounds.

468

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 8. Structure and Binding Data for the 3,4-Dihydropyridin-2(1H)-ones 153−180 at the hARs (Subset IX, Figure 2)

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

Evaluation of the Antimetastatic Eﬀect. It is well

accepted that A_2BAR activation has to play a crucial role in

promoting cell migration and motility^32,72and also in early

events of the multistep process of cell invasion and

metastasis.^25,30,73In addition, adenosine binding to A_2BAR

on tumor cells enhance their metastatic capabilities, while

A_2BAR activation enhances tumor chemotaxis and metastasis

in animal models of melanoma and breast cancer.^9,74,75These

precedents aimed us to brieﬂy investigate a potential

antimetastatic eﬀect of the most appealing ligands identiﬁed

in this work on three representative PCa cell lines (LNCaP,

PC-3, and DU145). The selected ligands were tested as

racemates [( )-38 and ( )-47]. Elected cell lines are derived

from PCa metastasis (isolated from the lymph nodes, bone,

and brain, respectively) and represent states with diﬀerent

metastatic potential and androgen responses. To rule out the

potential cytotoxic eﬀect of ( )-38 or ( )-47 in LNCaP, PC-

3, and DU145, the cell growth inhibitory activity of these

ligands was preliminarily evaluated. Both compounds were

tested (10 and 100 μM) following described protocols (MTT

assays) using cisplatin as the control.^76,77These experiments

unequivocally conﬁrmed that ( )-38 or ( )-47 do not exhibit

a cytotoxic eﬀect at the tested concentrations. The transwell

migration assay⁷⁸was used as a marker of A_2BAR

antimetastatic activity. The cytopathic changes induced by

selected compounds in the PC-3 cell line were analyzed using

confocal microscopy.⁷⁹For the sake of comparison, the

prototypic A_2BAR antagonist PSB603³⁴was also included in

469

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

■

▲

Figure 3. Radioligand binding and functional assays. (Left) Concentration−response curves of 47 ( ) and 38 ( ) at hA_2BARs labeled with 22

3

■

▲

nM [ H]DPCPX. (Right) Concentration−response curves of 47 ( ) and 38 ( ) over 100 nM NECA-elicited cAMP formation. Points represent

the mean SD (vertical bars) of three independent experiments.

Figure 4. Chiral HPLC traces, CD spectra, and absolute conﬁguration for ( )-38 and ( )-47 and its enantiomers.

the study. Figure 9 shows the results of the transwell

migration assay after 72 h of exposure to ( )-38, ( )-47,

and PSB603 (at their respective K_ivalues) compared to

untreated cells (controls). As observed, the optimized 3,4-

dihydropyridine derivatives ( )-38 and ( )-47 signiﬁcantly

(p < 0.05) decreased PC-3, DU145, and LNCaP cells

migration in comparison to controls (Figure 9A). Moreover,

the antimigratory eﬀect of this pair of ligands is indeed

470

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 9. Structure and Binding Data for the Enantiomers of the 3,4-Dihydropyridin-2(1H)-ones/thiones 38 and 47 at the

hARs

K_i(nM) or % at 1 μM

a

b

c

d

compound

R₄

R₅

Et

hA₁(%)

hA_2A(%)

hA_2B

hA₃(%)

( )-38

(R)-38

(S)-38

( )-47

(R)-47

(S)-47

3-thienyl

2-furyl

16

5

10

15

10

8

34

21

9

18

6

23.6 1.0

19%

11.1 1.4

10.2 0.5

12%

11

3

6

1

2

Et

i-Pr

13

6.30 1.1

1

a

b

n = 3 for K_ivalues, or n = 2 for percentage displacement of speciﬁc binding. Displacement of speciﬁc [³H]DPCPX binding in adenosine A₁

c

receptors expressed in human CHO cells. Displacement of speciﬁc [3]ZM2421385 binding in adenosine A_2Areceptors expressed in human HeLa

d

e

cells. Displacement of speciﬁc [³H]DPCPX binding in human HEK-293 cells. Displacement of speciﬁc [³H]NECA binding in adenosine A₃

receptors expressed in human HeLa cells.

Figure 5. Proposed binding mode of the 3,4-dihydropyrimidin-

2(1H)-one/thione scaﬀold (left) and graphical analysis of the SAR

from tables I−III (right). The bars represent the pK_ivalue on the

A_2BAR for the highest aﬃnity compound within each of the R₄(top

graph) or R₅(bottom graph) substituents, as extracted from each of

the three subsets (blue: subset I, compounds 16−43 in Table 1;

orange, subset II, compounds 44−71 in Table 2; gray, subset III,

compounds 72−99 in Table 3).

superior to that observed for PSB603 (Figure 9A).

Interestingly, the antimetastatic eﬀect was in all cases lightly

superior on PC-3 cells compared to DU145 and LNCaP.

Here, the antimigratory activity of ligand ( )-47 was clearly

superior to ligand ( )-38 (50 vs 35% in PC-3 migration

assay), which correlates with the observed structural and

aﬃnity diﬀerences between the two ligands.

Figure 6. Eﬀect of methylation on R₁. The binding modes

considered are depicted in panels (A,B) for the compound pair 19

(R₁= NH) and 75 (R₁= N−CH₃). Panel (C) depicts the result of

the FEP simulations, performed on each binding pose for 10 pairs of

Cytopathic changes are considered an indicator of tumor

progression,^80,81and these changes are usually employed to

compounds. Color code corresponds to the binding pose, blue for

evaluate the anti-invasive eﬀect of antitumoral com-

pounds.⁷⁹⁻⁸²It is already known that migration is

comparatively faster in PC-3 cells that have an elongated

shape compared to rounded ones.⁸⁰Changes in the

morphology may be driven by modiﬁcation in the actin

cytoskeleton organization and this, in turn, can target cell

motility.^81,82As a complement of the previous data, it was

decided to study the cytopathic changes induced by the herein

developed pyrimidin-2-ones [( )-38 and ( )-47] and the

reference ligand PSB603 in PC-3 cells using confocal laser

scanning microscopy.⁷⁹⁻⁸²

pose A, and orange for pose B. * = no detectable binding.

As depicted in Figure 10, PC-3 cells treated with ligands

( )-38 or ( )-47 exhibited a rounded shape as the

predominant phenotype and showed actin skeleton reorgan-

ization. Similarly, cells treated with PSB603 showed similar

changes. In contrast, untreated PC-3 cells (control) showed

an elongated morphology and ellipsoid nuclei with eu- and

hetero-chromatin; actin ﬁlaments localized mainly beneath the

plasma membrane and carboxyﬂuorescein diacetate succini-

471

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 8. (A) Proposed binding mode of the 3,4-dihydropyrimidin-2-

one scaﬀold (left) and graphical analysis of the SAR from tables I

and IX (right). The bars represent the pK_ivalue for each of the R₅

substituents, for the three groups of R₄with the highest aﬃnity [2-

furyl (blue), 3-furyl (orange), and 3-thienyl (gray)]. Binding mode of

19 (B, in pink) and 157 (C, in blue), binding site waters (from apo

MD simulations) are depicted in spheres. Water 1 is replaced by

both compounds; however, waters 2 and 3 are not displaced by 19

(SYAF014). These waters are buried deeply in the binding site and

overlay with structural waters from the A_2AAR crystal structure

(PDB: 4EIY). It is likely that these waters are favorably

accommodated in the binding site and hence should not be

displaced by any compound, in agreement with the decrease in

aﬃnity of 157 compared to 19.

Figure 7. Binding mode of compounds 19 (subset I, orange) and 47

(subset II, blue), before and after the corresponding FEP

transformation in the A_2BAR model (top). It can be appreciated by

the shift in the binding orientation due to the heteroatom change.

Experimental and calculated binding free energies between this and

analogous compound pairs in subsets I and II are depicted in the bar

graph (bottom). *No detectable binding.

midyl ester (CFDA-SE) stained homogenously the cytoplasm

(Figure 9). The excellent antimigratory eﬀect of ( )-47 and

( )-38 supports the antimetastatic eﬀect observed. These

experiments additionally evidenced their superior eﬃcacy on

PC-3 cell lines and the higher antimetastatic proﬁle of ( )-47.

The outstanding antimigratory eﬀect documented herein for

optimized ligands suggests that they could be attractive

pharmacological tools to explore potential applications of

A_2BAR antagonists in the implementation of novel approaches

to cancer treatment.

insigniﬁcant inhibitory eﬀect at CYP3A4 and CYP2D6 and to

corroborate its microsomal stability. The antimetastatic eﬀect

of 3,4-dihydropyrimidin-2(1H)-ones 47 and 38 was prelimi-

narily validated in vitro in two androgen-insensitive (PC-3 and

DU145) and one androgen-sensitive (LNCaP) prostate cancer

cells. These results support a promising future for A_2BAR

antagonists in the context of emerging therapeutic approaches

for cancer treatment.

CONCLUSIONS

■

EXPERIMENTAL SECTION

■

In summary, we have disclosed a large library of 3,4-

dihydropyrimidin-2(1H)-ones as potent and selective A_2BAR

ligands and conﬁrmed its functional (antagonistic) behavior

through cAMP assays. The biological dataset enabled the

comprehensive examination of the most prominent features of

the SAR and SSR in this series. The SAR trends were

complemented and interpreted with a comprehensive

computational modeling analysis based on rigorous FEP

simulations, starting from the receptor-driven docking model

that initially guided the design of these series. The combined

use of preparative chiral HPLC and CD enabled to obtain

experimental evidences supporting the stereospeciﬁc inter-

action between the diﬀerent stereoisomers of the most

attractive hA_2BAR antagonists identiﬁed herein. An exploratory

ADME study of optimized A_2Bantagonists evidenced the

Chemistry. Unless otherwise indicated, all starting materials,

reagents, and solvents were purchased and used without further

puriﬁcation. After extraction from aqueous phases, the organic

solvents were dried over anhydrous sodium sulfate. The reactions

were monitored by thin-layer chromatography (TLC) on 2.5 mm

Merck silica gel GF 254 strips, and the puriﬁed compounds each

showed a single spot; unless stated otherwise, UV light and/or iodine

vapor were used to detect compounds. The Biginelli reactions were

performed in coated Kimble vials on a PLS (6 × 4) organic

synthesizer with orbital stirring. The purity and identity of all tested

compounds were established by a combination of HPLC, elemental

analysis, mass spectrometry, and NMR spectroscopy as described

below. Puriﬁcation of isolated products was carried out by column

chromatography (Kieselgel 0.040−0.063 mm, E. Merck) or medium

pressure liquid chromatography on a CombiFlash Companion

(Teledyne ISCO) with RediSep pre-packed normal-phase silica gel

472

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 10. Inhibition Data of ( )-38 and ( )-47 on CYP3A4 and CYP2D6

CYP3A4 (BFC) % inhibition

(1 μM) (%) (10 μM) (%)

15 44

13

CYP3A4 (DBF) % inhibition

(1 μM) (%) (10 μM) (%)

12

CYP2D6 % inhibition

(1 μM) (%) (10 μM) (%)

cmpd

( )-38

( )-47

4

15

22

12

1

3

1

a

Presented data are the mean SD of three (n = 3) experiments. The percentage of inhibition (at 1 and 10 μM) is described due to the negligible

eﬀect of the compounds at both cytochromes.

Figure 9. Migration of PC-3, DU145 and LNCaP cells. (A) A_2BAR antagonists ( )-38 and ( )-47 inhibited PC-3, LNCaP, and DU145 cell

migration, in comparison to the controls (CTL) and PSB603. Results are expressed as mean SE, from at least three separate experiments, each

▲

conducted in triplicates. *A_2BAR antagonists’ treatment was signiﬁcantly diﬀerent compared with control treatment (p < 0.05); A_2BAR

antagonist ( )-38 and A_2BAR antagonist ( )-47 were signiﬁcantly diﬀerent compared with A_2BAR antagonist PSB603 (p < 0.05). (B)

Representative microphotographs of PC-3 cells (40×) which migrated through the chamber membranes: (B₁) control, (B₂), A_2BAR antagonist

PSB603, (B₃) A_2BAR antagonist ( )-38, (B₄) A_2BAR antagonist ( )-47.

(35−60 μm) columns followed by recrystallization. Melting points

were determined on a Gallenkamp melting point apparatus and are

uncorrected. The NMR spectra were recorded on Bruker AM300 and

XM500 spectrometers. Chemical shifts are given as δ values against

tetramethylsilane as the internal standard, and J values are given in

Hz. Mass spectra were obtained on a Varian MAT-711 instrument.

High-resolution mass spectra were obtained on an Autospec

Micromass spectrometer. Analytical HPLC was performed on an

Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1 mm ×

150 mm, 5 μm column with gradient elution using the mobile phases

(A) H₂O containing 0.1% CF₃COOH and (B) MeCN and a ﬂow

rate of 1 mL/min. The purity of all tested compounds was

determined to be >95%. The structural and spectroscopic data

obtained for all compounds described is provided in the Supporting

Information.

473

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 10. Cytopathic changes of PC-3 cells observed by confocal laser scanning microscopy. Blue (DAPI), green (CFDA-SE), and red

(phalloidin−ATTO 647N) and merge of three channels. Cells treated with ( )-47 (10.2 nM) or ( )-38 (23.6 nM) showed predominantly

rounded shape phenotype with DNA condensation. Untreated PC-3 cells showed an elongated phenotype with cytoplasmic projections when

compared with A_B2AR antagonist-treated PC-3 cells.

The chiral resolution of selected racemic ligands [( )-38 and

( )-47] was performed using a Water Breeze 2 (binary pump 1525,

detector UV/visible 2489, 7725i Manual Injector Kit 1500 Series)

with a 250 mm × 10 mm Lux 5 μm amylose-2 (Phenomenex) using

a linear gradient of the mobile phase (n-hexane/i-propanol) at 25 °C.

A detailed description of the experimental protocols and relevant

parameters (retention times and stereochemical purities) is provided

in the Supporting Information. All single stereoisomers were isolated,

and their stereochemical purity was analyzed by chiral HPLC (>97%

for each stereoisomer) and then characterized by NMR in CDCl₃.

CD spectra were recorded on a Jasco-815 system equipped with a

Peltier-type thermostatic accessory (CDF-426S, Jasco). Measure-

ments were carried out at 20 °C using a 1 mm quartz cell in a

volume of 300−350 mL. Compounds (0.1 mg) were dissolved in

MeOH (1.0 mL). The instrument settings were bandwidth, 1.0 nm;

data pitch, 1.0 nm; speed, 500 nm/min; accumulation, 10; and

wavelengths, 400−190 nm.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (16−43) and 3,4-Dihydropyr-

imidin-2(1H)-thiones (44−71). A mixture of urea 13a or thiourea

13b (7.5 mmol), aldehyde 14a−d (5 mmol), the β-ketoester 15a−g

(5 mmol), and ZnCl₂(0.5 mmol) in 3 mL of tetrahydrofuran (THF)

in coated Kimble vials was stirred with orbital stirring at 80 °C for 12

h. After completion of the reaction, as indicated by TLC, the reaction

mixture was poured onto crushed ice and stirred for 5−10 min. The

solid separated was ﬁltered under suction, washed with ice-cold water

(20 mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (72−99). A mixture of methylurea

13c (7.5 mmol), aldehyde 14a−d (5 mmol), the β-ketoester 15a−g

(5 mmol), and H₃BO₃(0.5 mmol) in 3 mL of THF in coated

Kimble vials was stirred with orbital stirring at 80 °C for 12 h. After

completion of the reaction, as indicated by TLC, the reaction mixture

was poured onto crushed ice and stirred for 5−10 min. The solid

separated was ﬁltered under suction, washed with ice-cold water (20

mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (105−120). A mixture of the

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), the 1,3-diketone

100a−d (5 mmol), and AcOH (0.5 mmol) in 3 mL of THF in

coated Kimble vials was stirred with orbital stirring at 80 °C for 12 h.

After completion of the reaction, as indicated by TLC, the reaction

mixture was poured onto crushed ice and stirred for 5−10 min. The

solid separated was ﬁltered under suction, washed with ice-cold water

(20 mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (121−136). A mixture of the

474

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), the ketoamide

101a−d (5 mmol), and ZnCl₂(0.5 mmol) in 3 mL of THF in coated

Kimble vials was stirred with orbital stirring at 80 °C for 12 h. After

completion of the reaction, as indicated by TLC, the reaction mixture

was poured onto crushed ice and stirred for 5−10 min. The solid

separated was ﬁltered under suction, washed with ice-cold water (20

mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (137−144). A mixture of the

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), the ketothioester

102a−b (5 mmol) and CeCl₃(0.5 mmol) in 3 mL of THF in coated

Kimble vials was stirred with orbital stirring at 80 °C for 12 h. After

completion of the reaction, as indicated by TLC, the reaction mixture

was poured onto crushed ice and stirred for 5−10 min. The solid

separated was ﬁltered under suction, washed with ice-cold water (20

mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

of 100 μM R-PIA. The reaction mixture was incubated at 25 °C for

180 min. After the incubation time, membranes were washed and

ﬁltered, and radioactivity was detected in a MicroBeta Trilux reader

(PerkinElmer).

Functional Experiments. cAMP assays were performed at

human A_2BARs using a cAMP enzyme immunoassay kit (Amersham

Biosciences). HEK-293 cells were seeded (10,000 cells/well) in 96-

well culture plates and incubated at 37 °C in an atmosphere with 5%

CO₂in Eagle’s medium nutrient mixture F-12 (EMEM F-12),

containing 10% fetal calf serum and 1% ^L-glutamine. Cells were

washed 3× with 200 μL of assay medium (EMEM-F12 and 25 mM

N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid pH = 7.4) and

preincubated with assay medium containing 30 μM rolipram and test

compounds at 37 °C for 15 min. 10 μM NECA was incubated for 15

min at 37 °C (total incubation time 30 min). Reaction was stopped

with lysis buﬀer supplied in the kit, and the enzyme immunoassay

was carried out for detection of intracellular cAMP at 450 nm in an

Ultra Evolution detector (Tecan).

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (145−148). A mixture of the

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), diethyl (2-

oxopropyl)phosphonate 103 (5 mmol), and chloroacetic acid (0.5

mmol) in 3 mL of THF in coated Kimble vials was stirred with

orbital stirring at 90 °C for 12 h. After completion of the reaction, as

indicated by TLC, the reaction mixture was poured onto crushed ice

and stirred for 5−10 min. The solid separated was ﬁltered under

suction, washed with ice-cold water (20 mL), and then puriﬁed either

by recrystallization or column chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (149−152). A mixture of the

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), 1-(5-methylox-

azol-2-yl)propan-2-one 104 (5 mmol), and SnCl₂(0.5 mmol) in 3

mL of THF in coated Kimble vials was stirred with orbital stirring at

80 °C for 12 h. After completion of the reaction, as indicated by

TLC, the reaction mixture was poured onto crushed ice and stirred

for 5−10 min. The solid separated was ﬁltered under suction, washed

with ice-cold water (20 mL), and then puriﬁed either by

recrystallization or column chromatography on a silica gel.

General Procedure for the Biginelli Synthesis of 3,4-

Dihydropyrimidin-2(1H)-ones (153−180). A mixture of the

urea 13a (7.5 mmol), aldehyde 14a−d (5 mmol), the β-ketoester

15h−n (5 mmol), and ZnCl₂(0.5 mmol) in 3 mL of THF in coated

Kimble vials was stirred with orbital stirring at 80 °C for 12 h. After

completion of the reaction, as indicated by TLC, the reaction mixture

was poured onto crushed ice and stirred for 5−10 min. The solid

separated was ﬁltered under suction, washed with ice-cold water (20

mL), and then puriﬁed either by recrystallization or column

chromatography on a silica gel.

Pharmacological Characterization. Radioligand binding com-

petition assays were performed in vitro using hARs expressed in

transfected HeLa [hA_2AAR (9 pmol/mg protein) and hA₃AR (3

pmol/mg protein)], HEK-293 [hA_2BAR (1.5 pmol/mg protein)], and

CHO [hA₁AR (1.5 pmol/mg protein)] cells as described

previously.^41−43,49A brief description is given below. A₁AR

competition binding experiments were carried out in membranes

from CHO-A₁cells labeled with 1 nM [³H]DPCPX (K_D= 0.7 nM).

Nonspeciﬁc binding was determined in the presence of 10 μM R-

PIA. The reaction mixture was incubated at 25 °C for 60 min. A_2AAR

competition binding experiments were carried out in membranes

from HeLa-A_2Acells labeled with 3 nM [³H]ZM241385 (K_D= 2

nM). Nonspeciﬁc binding was determined in the presence of 50 μM

NECA. The reaction mixture was incubated at 25 °C for 30 min.

A_2BAR competition binding experiments were carried out in

membranes from HEK-293-A_2Bcells (Euroscreen, Gosselies,

Belgium) labeled with 25 nM [³H]DPCPX (K_D= 21 nM).

Nonspeciﬁc binding was determined in the presence of 400 μM

NECA. The reaction mixture was incubated at 25 °C for 30 min.

A₃AR competition binding experiments were carried out in

membranes from HeLa-A₃cells labeled with 10 nM [³H]NECA

(K_D= 8.7 nM). Nonspeciﬁc binding was determined in the presence

Data Analysis. IC₅₀values were obtained by ﬁtting the data with

nonlinear regression using Prism 5.0 software (GraphPad, San Diego,

CA). For those compounds that showed either little aﬃnity or poor

solubility, a percentage inhibition of speciﬁc binding is reported.

Results are the mean of three experiments (n = 3) each performed in

duplicates.

In Vitro Migration Assay. The eﬀects of ( )-38, ( )-47, and

PSB603 (23.6, 10.2, and 0.53 nM respectively) on PC-3, DU145,

and LNCaP cell migration tests were assessed by using 24-well

transwell cell culture chambers (6.5 mm diameter, 8.0 μm pore size,

polycarbonate membrane) (Sigma-Aldrich, Madrid, MD, Spain) with

Millicell Cell Culture Insert (Merck Millipore Madrid, MD, Spain).⁶⁷

In the upper chamber, 10⁵cells were seeded in 100 μL serum-free

fetal bovine serum (FBS), while the lower chamber was ﬁlled with

600 μL of complete medium with 10% FBS. After 24 h of incubation,

cells in the upper chamber were carefully removed with a cotton

swab and cells that had migrated through the membrane and had

stuck to the lower surface of the membrane were ﬁxed with 4%

paraformaldehyde (2 min) and stained with crystal violet stain

(Sigma-Aldrich, Madrid, MD, Spain) (15 min). Stained cells were

counted by photographing the membrane in ﬁve randomly selected

ﬁelds using a microscope equipped with a digital camera (Olympus,

Tokyo, Japan). At least three chambers from three diﬀerent

experiments were analyzed. Prostate adenocarcinoma cell lines PC-

3, DU145, and LNCaP. PCa cells were cultured in RPMI 1640

supplemented with 10% FBS (Life Technologies, Madrid, MD,

Spain), 100 U/mL penicillin, and 100 mg/mL streptomycin

(penicillin−streptomycin solution 30-2300; LGC Standards Barcelo-

na, CAT, Spain). All cells were maintained at 37 °C in a 5% CO₂

humidiﬁed incubator, grown to conﬂuence, and thereafter seeded

into well culture plates at assay-speciﬁc densities.

Migration Assay. As an indicator of the antimetastatic eﬀect, we

assessed PC-3, DU145, and LNCaP cell migration by using 24-well

transwell cell culture chambers (6.5 mm diameter, 8.0 μm pore size,

polycarbonate membrane) (cat. no. C6932; Sigma-Aldrich, Madrid,

MD, Spain) with Millicell Cell Culture Insert (cat. no. PI8P01250;

Merck Millipore Madrid, MD, Spain).⁶⁰In the upper chamber, 10⁵

cells were seeded in 100 μL serum-free FBS, while the lower chamber

was ﬁlled with 600 μL of complete medium with 10% FBS. After 24

h of incubation, cells in the upper chamber were carefully removed

with a cotton swab and cells that had migrated through the

membrane and had stuck to the lower surface of the membrane were

ﬁxed with 4% paraformaldehyde (2 min) and stained with crystal

violet stain (cat. no. C6158; Sigma-Aldrich, Madrid, MD, Spain) (15

min). Stained cells were counted by photographing the membrane in

ﬁve randomly selected ﬁelds using a microscope equipped with a

digital camera (Olympus, Tokyo, Japan). At least three chambers

from three diﬀerent experiments were analyzed. Migration was

evaluated after 72 h of cell culture without or with each of the

treatments tested.

Cell Morphological Changes by Scanning Confocal Micros-

copy. PC-3 cells were seeded in glass coverslips into incubation

475

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

chambers (24 h at 37 °C and 5% CO₂) in order to obtain full

adherence. Thereafter, the medium was replaced with fresh complete

medium containing antagonists 38 and 47, or vehicles and plates

were incubated for 72 h at 37 °C. Then, PC-3 cells were

ﬂuorescently stained for F-actin (phalloidin−ATTO 647N 1:500,

cat. no. 65906; Sigma-Aldrich, Madrid, MD, Spain), nuclei (4′,6-

diamidino-2-phenylindole, DAPI), and cytoplasm (CFDA-SE, cat.

no. 1351201EDU; Bio-Rad Laboratories, Madrid, MD, Spain). The

culture medium was replaced by PBS solution containing ﬂuorescent

dye CFDA-SE at 1 mM, and the cells were incubated 15 min.

Subsequently, CFDA-SE solution was removed; cells were washed

with PBS, ﬁxed with 70% ethanol for 5 min, washed again with PBS,

and ﬁnally, stained for with phalloidin−ATTO for 1 h. Then, the

cells were washed with 0.9% sodium chloride (NaCl, cat. no. S7653;

Sigma-Aldrich Madrid, MD, Spain) and stained with DAPI at 1 mg/

mL for 10 min. Finally, slides were embedded in a VectaShield

antifade mounting medium (cat. no. H-1000; Vector Laboratories,

Burlingame CA, U.S.A.). The cells were analyzed with a Leica

confocal microscope (Leica TCS SP5 II microscope).⁷⁹

Receptor Modeling and Ligand Docking. Our computational

strategy for the structure-based design of AR ligands involves a

combination of homology modeling, ligand−receptor docking, and

free energy calculations, as recently reviewed.⁸³Homology modeling

of and docking to the hA_2BAR: a 3D structure of the inactive form of

the receptor was generated at the beginning of this project.⁸⁴Brieﬂy,

the process consisted of the following sequential steps: (i) manual

curation of the sequence alignment with the template A_2AAR (PDB

code 3EML),⁶⁶(ii) generation and selection of homology models

and loop reﬁnement procedures with Modeler,⁸⁵(iii) assessment of

Asn/Gln/His rotamers and side chain protonation states with the

MolProbity web server (http://molprobity.biochem.duke.edu/), and

system was subject to 10 parallel replicates of unrestrained MD,

where the FEP protocol is applied for each ligand transformation.

Each of these MD replicates starts with a 0.25 ns unbiased

equilibration period, with diﬀerent initial velocities. Thereafter, the

FEP protocol follows, which consists of 21 FEP λ-windows,

distributed using a sigmoidal function and consisting of 10 ps each

for every investigated ligand pair. In order to fulﬁl a thermodynamic

cycle and calculate relative binding free energies, parallel FEP

transformations are run in a sphere of water for each ligand pair. In

these water simulations, the same parameters apply (i.e., sphere size,

simulation time, etc.), and the relative binding free energy diﬀerence

was estimated by solving the thermodynamic cycle utilizing the

Bennett acceptance ratio.⁹⁶

CYP3A4 and CYP2D6 Inhibition. The inhibitory activity of

ligands ( )-38 and ( )-47 was assessed by following a published

protocol.⁶⁸Incubations were conducted in a 200 μL of volume in 96

well microtiter plates (COSTAR 3915). Addition of the cofactor-

buﬀer mixture (KH₂PO₄buﬀer, 1.3 mM NADP, 3.3 mM MgCl₂, 3.3

mM glucose-6-phosphate, and 0.4 U/mL glucose-6-phosphate

dehydrogenase), supersomes control, and standard inhibitor

(ketoconazole from Sigma-Aldrich) previously diluted and com-

pounds to plates were carried out by a liquid handling station

(Zephyr Caliper). The plate was then preincubated at 37 °C for 5

min, and the reaction initiated by the addition of prewarmed

enzyme/substrate (E/S) mix. The E/S mix contained buﬀer

(KH₂PO₄), c-DNA-expressed P450 in insect cell microsomes,

substrate (DBF: dibenzylﬂuorescein), and other components to

give the ﬁnal assay concentrations in a reaction volume of 200 μL.

Reactions were terminated after various times (a speciﬁc time for

each cytochrome) by the addition of STOP solution [ACN/Tris-HCl

0.5 M 80:20 and NaOH 2 N for CYP3A4 (DBF)]. Fluorescence per

well was measured using a ﬂuorescence plate reader (Tecan Inﬁnite

M1000 Pro), and the percentage of inhibition was calculated.

Human Microsomal Stability. The human microsomes

employed were purchased from Tebu-Xenotech. The A_2Bantagonists

[( )-38 and ( )-47] were incubated with the microsomes at 37 °C

in a 50 mM phosphate buﬀer (pH = 7.4) containing 3 mM MgCl₂, 1

mM NADP, 10 mM glucose-6-phosphate, and 1 U/mL glucose-6-

phosphate-dehydrogenase. Samples (75 μL) were taken from each

well at 0, 10, 20, 40, and 60 min and transferred to a plate containing

4 °C 75 μL acetonitrile, and 30 μL of 0.5% formic acid in water was

added for improving the chromatographic conditions. The plate was

centrifuged (46,000g, 30 min), and supernatants were taken and

analyzed in an UPLC−MS/MS (Xevo-TQD, Waters) by employing a

BEH C18 column and an isocratic gradient of 0.1% formic acid in

water/0.1% formic acid acetonitrile (60:40). The metabolic stability

of the compounds was calculated from the logarithm of the

remaining compounds at each of the time points studied.

̈

(iv) use of tools from the Schrodinger Suite for energetic structural

reﬁnements.⁸³The previously described binding mode of SYAF014⁶⁴

in the A_2BAR model was used as a starting point to manually dock all

reported compounds within the series.

MD and FEP Calculations. The hA_2BAR model obtained in the

previous stage was inserted in the membrane and equilibrated under

periodic boundary conditions using the PyMemDyn protocol

described elsewhere.⁸⁶Shortly, the starting structure is automatically

embedded in a pre-equilibrated membrane consisting of 1-palmitoyl-

2-oleoyl phosphatidylcholine lipids, with the TM bundle aligned to

its vertical axis. This hexagonal-prism shaped box is then soaked with

bulk water and energy minimized with GROMACS 4.6.⁸⁷using the

OPLS-AA force ﬁeld⁸⁸for protein and ligands, combined with the

Berger parameters for the lipids.⁸⁹The same setup is used for a 2.5

ns MD equilibration, where initial restraints on protein and ligand

atoms are gradually released as described in detail in our original

protocol.⁸⁶The equilibrated binding site is then transferred to the

MD software Q⁹⁰for FEP calculations under spherical boundary

conditions, using the automated QligFEP protocol.⁹¹A 25 Å sphere

centered on the center of geometry of the ligand is considered for

these MD simulations. Protein atoms in the boundary of the sphere

(22−25 Å outer shell) had a positional restraint of 20 kcal/mol/Å²,

while solvent atoms were subject to polarization and radial restrains

using the surface constrained all-atom solvent^90,92model to mimic

the properties of bulk water at the sphere surface. Atoms lying

outside the simulation sphere are tightly constrained (200 kcal/mol/

Å²force constant) and excluded from the calculation of nonbonded

interactions. Long range electrostatic interactions beyond a 10 Å cut

oﬀ were treated with the local reaction ﬁeld method,⁹³except for the

atoms undergoing the FEP transformation where no cut-oﬀ was

applied. Solvent bonds and angles were constrained using the

SHAKE algorithm.⁹⁴All titratable residues outside the sphere were

neutralized, and histidine residues were assigned a hydrogen atom on

the δ nitrogen. Residue parameters were translated from the OPLS-

AA/M force ﬁeld,⁹⁵and the parameters for the ligand and lipids were

inherited from the previous MD stage. The simulation sphere was

warmed up from 0.1 to 298 K, during a ﬁrst equilibration period of

0.61 ns, where an initial restraint of 25 kcal/mol/Å²imposed on all

heavy atoms was slowly released for all complexes. Thereafter, the

ASSOCIATED CONTENT

* Supporting Information

■

sı

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01431.

Molecular formula strings (CSV)

Peak summary report (PDF)

Computational chemistry; general information; general

procedure for Biginelli synthesis; spectroscopic and

analytical data for all compounds described; and

analytical HPLC chromatograms of AF80 (38) and

AF80 (47) (PDF)

AUTHOR INFORMATION

Corresponding Authors

■

́

Jose Brea − Centro Singular de Investigación en Medicina

Molecular y Enfermedades Crónicas (CIMUS), Universidade

de Santiago de Compostela, 15782 Santiago de Compostela,

476

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Spain; Phone: +34 881815459; Email: pepo.brea@usc.es;

Fax: +34-8818115474

Santiago de Compostela, 15782 Santiago de Compostela,

Spain

́

Hugo Gutierrez-de-Teran − Department of Cell and

Molecular Biology, Uppsala University, SE-75124 Uppsala,

Sweden; orcid.org/0000-0003-0459-3491; Phone: +46

18 471 5056; Email: hugo.gutierrez@icm.uu.se; Fax: +46

18 536971

Carlos Rodríguez-García − Centro Singular de Investigación

en Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Eddy Sotelo − Centro Singular de Investigación en Química

Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain; orcid.org/0000-0001-

5571-2812; Phone: +34 881815732; Email: e.sotelo@

usc.es; Fax: +34-881815704

Xerardo García-Mera − Departamento de Química Orgánica,

Facultade de Farmacia, Universidade de Santiago de

Compostela, 15782 Santiago de Compostela, Spain

̃

Olga Caamano − Departamento de Química Orgánica,

Facultade de Farmacia, Universidade de Santiago de

Compostela, 15782 Santiago de Compostela, Spain

Christian Fernandez-Masaguer − Departamento de Química

Orgánica, Facultade de Farmacia, Universidade de Santiago

de Compostela, 15782 Santiago de Compostela, Spain

Javier F. Sardina − Centro Singular de Investigación en

Química Biolóxica y Materiais Moleculares (CIQUS),

Universidade de Santiago de Compostela, 15782 Santiago de

Compostela, Spain; orcid.org/0000-0002-6758-0068

Angela Stefanachi − Dipartimento di Farmacia-Scienze del

Farmaco, Università degli Studi di Bari ALDO MORO,

70125 Bari, Italy; orcid.org/0000-0002-9430-7972

Abdelaziz El Maatougui − Centro Singular de Investigación

en Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Authors

María Majellaro − Centro Singular de Investigación en

Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Willem Jespers − Department of Cell and Molecular Biology,

Uppsala University, SE-75124 Uppsala, Sweden;

orcid.org/0000-0002-4951-9220

Abel Crespo − Centro Singular de Investigación en Química

Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Ana Mallo-Abreu − Centro Singular de Investigación en

Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Johan Åqvist − Department of Cell and Molecular Biology,

Uppsala University, SE-75124 Uppsala, Sweden;

orcid.org/0000-0003-2091-0610

́

̃

María J. Nunez − SNL, Departamento de Farmacología,

Facultade de Medicina, Universidade de Santiago de

Compostela, 15782 Santiago de Compostela, Spain

Silvia Novio − SNL, Departamento de Farmacología,

Facultade de Medicina, Universidade de Santiago de

Compostela, 15782 Santiago de Compostela, Spain

Jhonny Azuaje − Centro Singular de Investigación en

Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01431

Author Contributions

^¶M.M., W.J., and A.C. contributed equally to this work.

́

Ruben Prieto-Díaz − Centro Singular de Investigación en

Química Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain; orcid.org/0000-0003-

2539-3106

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was ﬁnancially supported by the Consellería de

Cultura, Educacion e Ordenacion Universitaria of the Galician

Government: (grant: ED431B2017/70), Centro singular de

■

́

Claudia Gioe − Centro Singular de Investigación en Química

́

Biolóxica y Materiais Moleculares (CIQUS) and

Departamento de Química Orgánica, Facultade de

Farmacia, Universidade de Santiago de Compostela, 15782

Santiago de Compostela, Spain

́

Investigacion de Galicia accreditation 2016−2019 (ED431G/

09), Xunta de Galicia (ED431C 2018/21) and the European

Regional Development Fund (ERDF), the Swedish Research

Council (grant: 521-2014-2118), and by the Swedish strategic

research program eSSENCE. The computations were

performed on resources provided by the Swedish National

Infrastructure for Computing (SNIC).

Belma Alispahic − Department of Cell and Molecular

Biology, Uppsala University, SE-75124 Uppsala, Sweden

María I. Loza − Centro Singular de Investigación en Medicina

Molecular y Enfermedades Crónicas (CIMUS), Universidade

de Santiago de Compostela, 15782 Santiago de Compostela,

Spain

Manuel Freire-Garabal − SNL, Departamento de

Farmacología, Facultade de Medicina, Universidade de

Santiago de Compostela, 15782 Santiago de Compostela,

Spain

ABBREVIATIONS

■

ARs, adenosine receptors; hA_2BR, human A_2Badenosine

receptors; hA_2AR, human A_2Aadenosine receptors; CHO

cells, Chinese hamster ovary cells; c-AMP, cyclic adenosine

monophosphate; FEP, free energy perturbation; HPLC, high

performance liquid chromatography; MAE, mean absolute

Carlota Garcia-Santiago − SNL, Departamento de

Farmacología, Facultade de Medicina, Universidade de

477

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(17) Addi, A. B.; Lefort, A.; Hua, X.; Libert, F.; Communi, D.;

Ledent, C.; Macours, P.; Tilley, S. L.; Boeynaems, J.-M.; Robaye, B.

Modulation of Murine Dendritic Cell Function by Adenine

Nucleotides and Adenosine: Involvement of the A2B Receptor.

Eur. J. Immunol. 2008, 38, 1610−1620.

(18) Xaus, J.; Mirabet, M.; Lloberas, J.; Soler, C.; Lluis, C.; Franco,

R.; Celada, A. IFN-γ Up-Regulates the A2B Adenosine Receptor

Expression in Macrophages: A Mechanism of Macrophage

Deactivation. J. Immunol. 1999, 162, 3607−3614.

(19) Kong, T.; Westerman, K. A.; Faigle, M.; Eltzschig, H. K.;

Colgan, S. P. HIF-dependent Induction of Adenosine A2B Receptor

in Hypoxia. FASEB J. 2006, 20, 2242−2250.

error; MD, molecular dynamics; MPLC, medium pressure

liquid chromatography; NMR, nuclear magnetic resonance

spectroscopy; PCa, prostate cancer; PC-3 and DU145,

androgen-independent human prostate cancer cell line cell

lines; PDB, Protein Data Bank; LNCaP, androgen-dependent

human prostate cancer cell line cell line; SAR, structure−

activity relationships; SEM, standard error of the mean;

SCAAS, surface constrained all-atom solvent

REFERENCES

■

(1) Rivkees, S. A.; Reppert, S. M. RFL9 Encodes an A2b-Adenosine

Receptor. Mol. Endocrinol. 1992, 6, 1598−1604.

(20) Hart, M. L.; Jacobi, B.; Schittenhelm, J.; Henn, M.; Eltzschig,

H. K. Cutting Edge: A2B Adenosine Receptor Signaling Provides

Potent Protection during Intestinal Ischemia/Reperfusion Injury. J.

Immunol. 2009, 182, 3965−3968.

(2) Pierce, K. D.; Furlong, T. J.; Selbie, L. A.; Shine, J. Molecular

Cloning and Expression of an Adenosine A2b Receptor from Human

Brain. Biochem. Biophys. Res. Commun. 1992, 187, 86−93.

(3) Beukers, M. W.; Den Dulk, H.; Van Tilburg, E. W.; Brouwer, J.;

Ijzerman, A. P. Why Are A2B Receptors Low-Affinity Adenosine

Receptors? Mutation of Asn273 to Tyr Increases Affinity of Human

A2B Receptor for 2-(1-Hexynyl)adenosine. Mol. Pharmacol. 2000,

58, 1349−1356.

(4) Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G. Comparison of

the Potency of Adenosine as an Agonist at Human Adenosine

Receptors Expressed in Chinese Hamster Ovary Cells. Biochem.

Pharmacol. 2001, 61, 443−448.

(5) Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Linden, J.;

̈

Muller, C. E. International Union of Basic and Clinical Pharmacol-

ogy. LXXXI. Nomenclature and Classification of Adenosine

Receptors-An Update. Pharmacol. Rev. 2011, 63, 1−34.

(6) Yang, D.; Chen, H.; Koupenova, M.; Carroll, S. H.; Eliades, A.;

Freedman, J. E.; Toselli, P.; Ravid, K. A New Role for the A2b

Adenosine Receptor in Regulating Platelet Function. J. Thromb.

Haemostasis 2010, 8, 817−827.

(7) Yang, D.; Koupenova, M.; McCrann, D. J.; Kopeikina, K. J.;

Kagan, H. M.; Schreiber, B. M.; Ravid, K. The A2b Adenosine

Receptor Protects against Vascular Injury. Proc. Natl. Acad. Sci. U.S.A.

2008, 105, 792−796.

(8) Yang, D.; Zhang, Y.; Nguyen, H. G.; Koupenova, M.; Chauhan,

A. K.; Makitalo, M.; Jones, M. R.; St. Hilaire, C.; Seldin, D. C.;

Toselli, P.; Lamperti, E.; Schreiber, B. M.; Gavras, H.; Wagner, D. D.;

Ravid, K. The A2B Adenosine Receptor Protects against Inflamma-

tion and Excessive Vascular Adhesion. J. Clin. Invest. 2006, 116,

1913−1923.

(9) Morello, S.; Miele, L. Targeting the Adenosine A2b Receptor in

the Tumor Microenvironment Overcomes Local Immunosuppression

by Myeloid-Derived Suppressor Cells. Oncoimmunology 2014, 3,

No. e27989.

(10) Vecchio, E. A.; Tan, C. Y. R.; Gregory, K. J.; Christopoulos,

A.; White, P. J.; May, L. T. Ligand-Independent Adenosine A2B

Receptor Constitutive Activity as a Promoter of Prostate Cancer Cell

Proliferation. J. Pharmacol. Exp. Ther. 2016, 357, 36−44.

(11) Johnston-Cox, H.; Eisenstein, A. S.; Koupenova, M.; Carroll,

S.; Ravid, K. The Macrophage A2b Adenosine Receptor Regulates

Tissue Insulin Sensitivity. PLoS One 2014, 9, No. e98775.

(12) Sun, Y.; Huang, P. Adenosine A2B Receptor: From Cell

Biology to Human Diseases. Front. Chem. 2016, 4, 37.

(13) Corset, V.; Nguyen-Ba-Charvet, K. T.; Forcet, C.; Moyse, E.;

́

(21) Hasko, G.; Csoka, B.; Nemeth, Z. H.; Vizi, E. S.; Pacher, P.

A2B Adenosine Receptors in Immunity and Inflammation. Trends

Immunol. 2009, 30, 263−270.

(22) Aherne, C. M.; Kewley, E. M.; Eltzschig, H. K. The

Resurgence of A2B Adenosine Receptor Signaling. Biochim. Biophys.

Acta, Biomembr. 2011, 1808, 1329−1339.

(23) Mittal, D.; Sinha, D.; Barkauskas, D.; Young, A.; Kalimutho,

M.; Stannard, K.; Caramia, F.; Haibe-Kains, B.; Stagg, J.; Khanna, K.

K.; Loi, S.; Smyth, M. J. Adenosine 2B Receptor Expression on

Cancer Cells Promotes Metastasis. Cancer Res. 2016, 76, 4372−4382.

(24) Iannone, R.; Miele, L.; Maiolino, P.; Pinto, A.; Morello, S.

Blockade of A2b Adenosine Receptor Reduces Tumor Growth and

Immune Suppression Mediated by Myeloid-Derived Suppressor Cells

in a Mouse Model of Melanoma. Neoplasia 2013, 15, 1400−1409.

(25) Gao, Z.-G.; Jacobson, K. A. A2B Adenosine Receptor and

Cancer. Int. J. Mol. Sci. 2019, 20, 5139.

(26) Mousavi, S.; Panjehpour, M.; Izadpanahi, M. H.; Aghaei, M.

Expression of Adenosine Receptor Subclasses in Malignant and

Adjacent Normal Human Prostate Tissues. Prostate 2015, 75, 735−

747.

(27) Novio, S.; Nunez-Iglesias, M. J.; Freire-Garabal, M. Adenosine

̃

Signaling Pathways as Potential Therapeutic Targets in Prostate

Cancer Disease. Molecular Oncology: Underlying Mechanisms and

Translational Advancements; Springer International Publishing:

Madrid, 2017; pp 93−107.

(28) Allard, D.; Turcotte, M.; Stagg, J. Targeting A2 Adenosine

Receptors in Cancer. Immunol. Cell Biol. 2017, 95, 333−339.

(29) Sorrentino, C.; Morello, S. Role of Adenosine in Tumor

Progression: Focus on A2B Receptor as Potential Therapeutic

Target. J. Cancer Metastasis Treat. 2017, 3, 127.

́

(30) Sepulveda, C.; Palomo, I.; Fuentes, E. Role of Adenosine A2b

Receptor Overexpression in Tumor Progression. Life Sci. 2016, 166,

92−99.

(31) Morin, F.; Beauregard, J.-M.; Bergeron, M.; Nguile Makao, M.;

Lacombe, L.; Fradet, V.; Fradet, Y.; Pouliot, F. Metabolic Imaging of

Prostate Cancer Reveals Intrapatient Intermetastasis Response

Heterogeneity to Systemic Therapy. Eur. Urol. Focus 2017, 3,

639−642.

(32) Wei, Q.; Costanzi, S.; Balasubramanian, R.; Gao, Z.-G.;

Jacobson, K. A. A2B Adenosine Receptor Blockade Inhibits Growth

of Prostate Cancer Cells. Purinergic Signalling 2013, 9, 271−280.

(33) Kaji, W.; Tanaka, S.; Tsukimoto, M.; Kojima, S. Adenosine

A2B Receptor Antagonist PSB603 Suppresses Tumor Growth and

Metastasis by Inhibiting Induction of Regulatory T Cells. J. Toxicol.

Sci. 2014, 39, 191−198.

́

Chedotal, A.; Mehlen, P. Netrin-1-Mediated Axon Outgrowth and

CAMP Production Requires Interaction with Adenosine A2b

Receptor. Nature 2000, 407, 747−750.

(14) Peakman, M.-C.; Hill, S. J. Adenosine A_2B-receptor-mediated

Cyclic AMP Accumulation in Primary Rat Astrocytes. Br. J.

Pharmacol. 1994, 111, 191−198.

(34) Borrmann, T.; Hinz, S.; Bertarelli, D. C. G.; Li, W.; Florin, N.

̈

C.; Scheiff, A. B.; Muller, C. E. 1-Alkyl-8-(Piperazine-1-Sulfonyl)-

Phenylxanthines: Development and Characterization of Adenosine

A2B Receptor Antagonists and a New Radioligand with Sub-

nanomolar Affinity and Subtype Specificity. J. Med. Chem. 2009, 52,

3994−4006.

(15) Hua, X.; Kovarova, M.; Chason, K. D.; Nguyen, M.; Koller, B.

H.; Tilley, S. L. Enhanced Mast Cell Activation in Mice Deficient in

the A2b Adenosine Receptor. J. Exp. Med. 2007, 204, 117−128.

(16) Eckle, T.; Faigle, M.; Grenz, A.; Laucher, S.; Thompson, L. F.;

Eltzschig, H. K. A2B Adenosine Receptor Dampens Hypoxia-Induced

Vascular Leak. Blood 2008, 111, 2024−2035.

(35) Jiang, J.; Seel, C. J.; Temirak, A.; Namasivayam, V.; Arridu, A.;

̈

Schabikowski, J.; Baqi, Y.; Hinz, S.; Hockemeyer, J.; Muller, C. E.

478

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

A2B Adenosine Receptor Antagonists with Picomolar Potency. J.

Med. Chem. 2019, 62, 4032−4055.

Adenosine Receptor Antagonists. Chem. Heterocycl. Compd. 2017, 53,

316−321.

(36) Hayallah, A. M.; Sandoval-Ramírez, J.; Reith, U.; Schobert, U.;

(51) Kappe, C. O. Biologically Active Dihydropyrimidones of the

Biginelli-Type - A Literature Survey. Eur. J. Med. Chem. 2000, 35,

1043−1052.

Preiss, B.; Schumacher, B.; Daly, J. W.; Muller, C. E. 1,8-

̈

Disubstituted Xanthine Derivatives: Synthesis of Potent A2B-

Selective Adenosine Receptor Antagonists. J. Med. Chem. 2002, 45,

1500−1510.

(52) Kappe, C. O. Recent Advances in the Biginelli Dihydropyr-

imidine Synthesis. New Tricks from an Old Dog. Acc. Chem. Res.

2000, 33, 879−888.

(37) Kim, Y.-C.; Ji, X.-d.; Melman, N.; Linden, J.; Jacobson, K. A.

Anilide Derivatives of an 8-Phenylxanthine Carboxylic Congener Are

Highly Potent and Selective Antagonists at Human A(2B) Adenosine

Receptors. J. Med. Chem. 2000, 43, 1165−1172.

(53) Bose, D. S.; Fatima, L.; Mereyala, H. B. Green Chemistry

Approaches to the Synthesis of 5-Alkoxycarbonyl-4-aryl-3,4- dihy-

dropyrimidin-2(1H)-ones by a Three-Component Coupling of One-

Pot Condensation Reaction: Comparison of Ethanol, Water, and

Solvent-free Conditions. J. Org. Chem. 2003, 68, 587−590.

(54) Mobaraki, A.; Movassagh, B.; Karimi, B. Hydrophobicity-

Enhanced Magnetic Solid Sulfonic Acid: A Simple Approach to

Improve the Mass Transfer of Reaction Partners on the Surface of

the Heterogeneous Catalyst in Water-Generating Reactions. Appl.

Catal., A 2014, 472, 123−133.

(38) Stewart, M.; Steinig, A. G.; Ma, C.; Song, J.-P.; McKibben, B.;

Castelhano, A. L.; MacLennan, S. J. [3H]OSIP339391, a Selective,

Novel, and High Affinity Antagonist Radioligand for Adenosine A2B

Receptors. Biochem. Pharmacol. 2004, 68, 305−312.

(39) Taliani, S.; Pugliesi, I.; Barresi, E.; Simorini, F.; Salerno, S.; La

Motta, C.; Marini, A. M.; Cosimelli, B.; Cosconati, S.; Di Maro, S.;

Marinelli, L.; Daniele, S.; Trincavelli, M. L.; Greco, G.; Novellino, E.;

Martini, C.; Da Settimo, F. 3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-

4(10H)-one: A Novel Template for the Design of Highly Selective

A2BAdenosine Receptor Antagonists. J. Med. Chem. 2012, 55, 1490−

1499.

(55) Ghasemi, Z.; Farshbaf Orafa, F.; Pirouzmand, M.; Zarrini, G.;

Nikzad Kojanag, B.; Salehi, R. Zn²⁺/MCM-41 Catalyzed Biginelli

Reaction of Heteroaryl Aldehydes and Evaluation of the Antimicro-

bial Activity and Cytotoxicity of the Pyrimidone Products.

Tetrahedron Lett. 2015, 56, 6393−6396.

(56) Agarwal, S.; Desai, R. Novel compounds for treatment of

diabetes, obesity or related disorders. WO 2013102929 A1, 2013.

(57) Ali, F.; Khan, K. M.; Salar, U.; Iqbal, S.; Taha, M.; Ismail, N.

H.; Perveen, S.; Wadood, A.; Ghufran, M.; Ali, B. Dihydropyr-

imidones: As novel class of β -glucuronidase inhibitors. Bioorg. Med.

Chem. 2016, 24, 3624−3635.

(58) Zabihollahi, R.; Fassihi, A.; Aghasadeghi, M. R.; Memarian, H.

R.; Soleimani, M.; Majidzadeh-A, K. Inhibitory Effect and Structure-

Activity Relationship of Some Biginelli-Type Pyrimidines against

HSV-1. Med. Chem. Res. 2013, 22, 1270−1276.

(59) Abdel-Fattah, A. A. Expedient Synthesis of Biginelli-Type

Dihydropyrimidinones Using α -(Benzotriazolyl)alkyl Urea Deriva-

tives. Synthesis 2003, 2358−2362.

(60) Shamim, S.; Khan, K. M.; Salar, U.; Ali, F.; Lodhi, M. A.;

Taha, M.; Khan, F. A.; Ashraf, S.; Ul-Haq, Z.; Ali, M.; Perveen, S. 5-

Acetyl-6-Methyl-4-Aryl-3,4-Dihydropyrimidin-2(1H)-Ones: As Po-

tent Urease Inhibitors; Synthesis, in Vitro Screening, and Molecular

Modeling Study. Bioorg. Chem. 2018, 76, 37−52.

(61) Lacotte, P.; Buisson, D.-A.; Ambroise, Y. Synthesis, Evaluation

and Absolute Configuration Assignment of Novel Dihydropyrimidin-

2-Ones as Picomolar Sodium Iodide Symporter Inhibitors. Eur. J.

Med. Chem. 2013, 62, 722−727.

(62) Uray, G.; Verdino, P.; Belaj, F.; Kappe, C. O.; Fabian, W. M.

F. Absolute Configuration in 4-Alkyl- and 4-Aryl-3,4-Dihydro-2(1H)-

Pyrimidones: A Combined Theoretical and Experimental Inves-

tigation. J. Org. Chem. 2001, 66, 6685−6694.

(40) Vidal, B.; Nueda, A.; Esteve, C.; Domenech, T.; Benito, S.;

́

Reinoso, R. F.; Pont, M.; Calbet, M.; Lopez, R.; Cadavid, M. I.; Loza,

́

M. I.; Cardenas, A.; Godessart, N.; Beleta, J.; Warrellow, G.; Ryder,

H. Discovery and Characterization of 4 ′-(2-Furyl)-N-Pyridin-3-yl-4,

5 ′-Bipyrimidin-2 ′-Amine (LAS38096), a Potent, Selective, and

Efficacious A2B Adenosine Receptor Antagonist. J. Med. Chem.

2007, 50, 2732−2736.

́

(41) El Maatougui, A.; Azuaje, J.; Gonzalez-Gomez, M.; Miguez, G.;

Crespo, A.; Carbajales, C.; Escalante, L.; García-Mera, X.; Gutierrez-

de-Teran, H.; Sotelo, E. Discovery of Potent and Highly Selective

A2B Adenosine Receptor Antagonist Chemotypes. J. Med. Chem.

2016, 59, 1967−1983.

(42) Carbajales, C.; Azuaje, J.; Oliveira, A.; Loza, M. I.; Brea, J.;

́

Cadavid, M. I.; Masaguer, C. F.; García-Mera, X.; Gutierrez-de-

́

Teran, H.; Sotelo, E. Enantiospecific Recognition at the A2B

Adenosine Receptor by Alkyl 2-Cyanoimino-4-Substituted-6-Methyl-

1,2,3,4-Tetrahydropyrimidine-5-Carboxylates. J. Med. Chem. 2017,

60, 3372−3382.

(43) Crespo, A.; El Maatougui, A.; Biagini, P.; Azuaje, J.; Coelho,

́

A.; Brea, J.; Loza, M. I.; Cadavid, M. I.; García-Mera, X.; Gutierrez-

́

de-Teran, H.; Sotelo, E. Discovery of 3,4-Dihydropyrimidin-2(1H)-

ones As a Novel Class of Potent and Selective A2B Adenosine

Receptor Antagonists. ACS Med. Chem. Lett. 2013, 4, 1031−1036.

(44) Wilson, C. N.; Jamal, M. S. Handbook of Experimental

Pharmacology: Adenosine Receptors in Health and Disease; Springer:

Berlin, 2009.

(45) Baraldi, P. G.; Tabrizi, M. A.; Gessi, S.; Borea, P. A. Adenosine

Receptor Antagonists: Translating Medicinal Chemistry and

Pharmacology into Clinical Utility. Chem. Rev. 2008, 108, 238−263.

(46) Jacobson, K. A.; Tosh, D. K.; Jain, S.; Gao, Z.-G. Historical

and Current Adenosine Receptor Agonists in Preclinical and Clinical

Development. Front. Cell. Neurosci. 2019, 13, 124.

(63) Krenn, W.; Verdino, P.; Uray, G.; Faber, K.; Kappe, C. O.

Determination of Absolute Configuration in 4-aryl-3,4-dihydro-

2(1H)-pyrimidones by High Performance Liquid Chromatography

and CD Spectroscopy. Chirality 1999, 11, 659−662.

(64) Crespo, A.; El Maatougui, A.; Biagini, P.; Azuaje, J.; Coelho,

(47) Muller, C. E.; Baqi, Y.; Hinz, S.; Namasivayam, V. Medicinal

̈

́

A.; Brea, J.; Loza, M. I.; Cadavid, M. I.; García-Mera, X.; Gutierrez-

Chemistry of A2B Adenosine Receptors. Receptors 2018, 34, 137−

́

de-Teran, H.; Sotelo, E. Discovery of 3,4-Dihydropyrimidin-2(1H)-

168.

ones As a Novel Class of Potent and Selective A2B Adenosine

Receptor Antagonists. ACS Med. Chem. Lett. 2013, 4, 1031−1036.

(65) Liu, W.; Chun, E.; Thompson, A. A.; Chubukov, P.; Xu, F.;

Katritch, V.; Han, G. W.; Roth, C. B.; Heitman, L. H.; Ijzerman, A.

P.; Cherezov, V.; Stevens, R. C. Structural Basis for Allosteric

Regulation of GPCRS by Sodium Ions. Science 2012, 337, 232−236.

(66) Jaakola, V.-P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.;

Chien, E. Y. T.; Lane, J. R.; Ijzerman, A. P.; Stevens, R. C. The 2.6 Å

Crystal Structure of a Human A2A Adenosine Receptor Bound to an

Antagonist. Science 2008, 322, 1211−1217.

(48) Kalla, R. V.; Zablocki, J. Progress in the Discovery of Selective,

High Affinity A2B Adenosine Receptor Antagonists as Clinical

Candidates. Purinergic Signalling 2009, 5, 21−29.

(49) Mallo-Abreu, A.; Majellaro, M.; Jespers, W.; Azuaje, J.;

́

Caamano, O.; García-Mera, X.; Brea, J. M.; Loza, M. I.; Gutierrez-de-

̃

́

Teran, H.; Sotelo, E. Trifluorinated Pyrimidine-Based A2B

Antagonists: Optimization and Evidence of Stereospecific Recog-

nition. J. Med. Chem. 2019, 62, 9315−9330.

(50) Crespo, A.; El Maatougui, A.; Azuaje, J.; Escalante, L.;

́

Majellaro, M.; Loza, M. I.; Brea, J.; Cadavid, M. I.; Gutierrez-de-

́

(67) Bortolato, A.; Tehan, B. G.; Bodnarchuk, M. S.; Essex, J. W.;

Mason, J. S. Water Network Perturbation in Ligand Binding:

Teran, H.; Sotelo, E. Exploring the Influence of the Substituent at

Position 4 in a Series of 3,4-Dihydropyrimidin-2(1H)-One A2B

479

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Adenosine A2A Antagonists as a Case Study. J. Chem. Inf. Model.

2013, 53, 1700−1713.

(68) Nayadu, S.; Behera, D.; Sharma, M.; Kaur, G.; Gudi, G.

Fluorescent Probe Based CYP Inhibition Assay: A High Throughput

Tool for Early Drug Discovery Screening. Int. J. Pharm. Sci. 2013, 5,

303−307.

(69) McDonnell, A. M.; Dang, C. H. Basic Review of the

Cytochrome P450 System. J. Adv. Pract. Oncol. 2013, 4, 263−268.

(70) Guengerich, F. P.; Waterman, M. R.; Egli, M. Recent

Structural Insights into Cytochrome P450 Function. Trends

Pharmacol. Sci. 2016, 37, 625−640.

(71) Guengerich, F. P. Cytochrome P450 and Chemical

Toxicology. Chem. Res. Toxicol. 2008, 21, 70−83.

(72) Virtanen, S. S.; Kukkonen-Macchi, A.; Vainio, M.; Elima, K.;

(87) Hess, B.; Kutzner, C.; Van Der Spoel, D.; Lindahl, E.

GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and

Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4,

435−447.

(88) Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen,

W. L. Evaluation and Reparametrization of the OPLS-AA Force Field

for Proteins via Comparison with Accurate Quantum Chemical

Calculations on Peptides. J. Phys. Chem. B 2001, 105, 6474−6487.

̈

(89) Berger, O.; Edholm, O.; Jahnig, F. Molecular Dynamics

Simulations of a Fluid Bilayer of Dipalmitoylphosphatidylcholine at

Full Hydration, Constant Pressure, and Constant Temperature.

Biophys. J. 1997, 72, 2002−2013.

(90) Marelius, J.; Kolmodin, K.; Feierberg, I.; Åqvist, J. Q: A

Molecular Dynamics Program for Free Energy Calculations and

Empirical Valence Bond Simulations in Biomolecular Systems. J. Mol.

Graphics Modell. 1998, 16, 213−225.

̈

Harkonen, P. L.; Jalkanen, S.; Yegutkin, G. G. Adenosine Inhibits

Tumor Cell Invasion via Receptor-Independent Mechanisms. Mol.

Cancer Res. 2014, 12, 1863−1874.

́

(91) Jespers, W.; Esguerra, M.; Åqvist, J.; Gutierrez-de-Teran, H.

Qligfep: An Automated Workflow for Small Molecule Free Energy

Calculations in Q. J. Cheminf. 2019, 11, 1−16.

(73) Vijayan, D.; Young, A.; Teng, M. W. L.; Smyth, M. J.

Targeting Immunosuppressive Adenosine in Cancer. Nat. Rev. Cancer

2017, 17, 709−724.

(92) King, G.; Warshel, A. A Surface Constrained All-Atom Solvent

Model for Effective Simulations of Polar Solutions. J. Chem. Phys.

1989, 91, 3647−3661.

(93) Lee, F. S.; Warshel, A. A Local Reaction Field Method for Fast

Evaluation of Long-Range Electrostatic Interactions in Molecular

Simulations. J. Chem. Phys. 1992, 97, 3100−3107.

(74) Chen, S.; Akdemir, I.; Fan, J.; Linden, J.; Zhang, B.; Cekic, C.

The Expression of Adenosine A2B Receptor on Antigen-Presenting

Cells Suppresses CD8+ T-cell Responses and Promotes Tumor

Growth. Cancer Immunol. Res. 2020, 8, 1064−1074.

(75) Bahreyni, A.; Samani, S. S.; Rahmani, F.; Behnam-Rassouli, R.;

Khazaei, M.; Ryzhikov, M.; Parizadeh, M. R.; Avan, A.; Hassanian, S.

M. Role of Adenosine Signaling in the Pathogenesis of Breast Cancer.

J. Cell. Physiol. 2018, 233, 1836−1843.

(94) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical

Integration of the Cartesian Equations of Motion of a System with

Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys.

1977, 23, 327−341.

(95) Robertson, M. J.; Tirado-Rives, J.; Jorgensen, W. L. Improved

Peptide and Protein Torsional Energetics with the OPLS-AA Force

Field. J. Chem. Theory Comput. 2015, 11, 3499−3509.

(96) Bennett, C. H. Efficient Estimation of Free Energy Differences

from Monte Carlo Data. J. Comput. Phys. 1976, 22, 245−268.

(76) Dhar, S.; Gu, F. X.; Langer, R.; Farokhzad, O. C.; Lippard, S. J.

Targeted Delivery of Cisplatin to Prostate Cancer Cells by Aptamer

Functionalized Pt(IV) Prodrug-PLGA −PEG Nanoparticles. Proc.

Natl. Acad. Sci. U.S.A. 2008, 105, 17356−17361.

́

̃

(77) Nunez-Iglesias, M. J.; Novio, S.; García-Santiago, C.; Cartea,

M. E.; Soengas, P.; Velasco, P.; Freire-Garabal, M. Effects of 3-

Butenyl Isothiocyanate on Phenotypically Different Prostate Cancer

Cells. Int. J. Oncol. 2018, 53, 2213−2223.

(78) Mao, L.; Yang, C.; Wang, J.; Li, W.; Wen, R.; Chen, J.; Zheng,

J. SATB1 Is Overexpressed in Metastatic Prostate Cancer and

Promotes Prostate Cancer Cell Growth and Invasion. J. Transl. Med.

2013, 11, 111.

(79) Hejchman, E.; Taciak, P.; Kowalski, S.; Maciejewska, D.;

́

Czajkowska, A.; Borowska, J.; Sladowski, D.; Młynarczuk-Biały, I.

Synthesis and Anticancer Activity of 7-Hydroxycoumarinyl Gallates.

Pharmacol. Rep. 2015, 67, 236−244.

̈

(80) Harma, V.; Haavikko, R.; Virtanen, J.; Ahonen, I.; Schukov,

H.-P.; Alakurtti, S.; Purev, E.; Rischer, H.; Yli-Kauhaluoma, J.;

Moreira, V. M.; Nees, M.; Oksman-Caldentey, K.-M. Optimization of

Invasion-Speci ﬁ c E ﬀ ects of Betulin Derivatives on Prostate Cancer

Cells through Lead Development. PLoS One 2015, 10 ().

DOI: 10.1371/journal.pone.0126111.

(81) Bai, S.; Herrera-Abreu, M.; Rohn, J. L.; Racine, V.; Tajadura,

V.; Suryavanshi, N.; Bechtel, S.; Wiemann, S.; Baum, B.; Ridley, A. J.

Identification and Characterization of a Set of Conserved and New

Regulators of Cytoskeletal Organization, Cell Morphology and

Migration. BMC Biol. 2011, 9, 54.

(82) Jackson, S. J. T.; Singletary, K. W.; Venema, R. C.

Sulforaphane Suppresses Angiogenesis and Disrupts Endothelial

Mitotic Progression and Microtubule Polymerization. Vasc. Pharma-

col. 2007, 46, 77−84.

̈

(83) Schrodinger; Schrodinger LLC: New York, 2009.

́

́ ́

(84) Rodríguez, D.; Pineiro, A.; Gutierrez-De-Teran, H. Molecular

̃

Dynamics Simulations Reveal Insights into Key Structural Elements

of Adenosine Receptors. Biochemistry 2011, 50, 4194−4208.

̌

(85) Sali, A.; Blundell, T. L. Comparative Protein Modelling by

Satisfaction of Spatial Restraints. J. Mol. Biol. 1993, 234, 779−815.

́

(86) Gutierrez-de-Teran, H.; Bello, X.; Rodríguez, D. Character-

ization of the Dynamic Events of GPCRs by Automated Computa-

tional Simulations. Biochem. Soc. Trans. 2013, 41, 205−212.

480

https://dx.doi.org/10.1021/acs.jmedchem.0c01431

J. Med. Chem. 2021, 64, 458−480

Article Doi

3,4-Dihydropyrimidin-2(1 H)-ones as Antagonists of the Human A2BAdenosine Receptor: Optimization, Structure-Activity Relationship Studies, and Enantiospecific Recognition

DOI: 10.1021/acs.jmedchem.0c01431

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01431

Full text of DOI:10.1021/acs.jmedchem.0c01431

Products guided by the article

R&D Labs maybe for 568568-16-1

Relevant to this article

Hot Product