Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2AReceptor

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Crystal Structure and Subsequent Ligand Design of a Nonriboside

Partial Agonist Bound to the Adenosine A_2AReceptor

Tasia Amelia, Jacobus P. D. van Veldhoven, Matteo Falsini, Rongfang Liu, Laura H. Heitman,

Gerard J. P. van Westen, Elena Segala, Gr é gory Verdon, Robert K. Y. Cheng, Robert M. Cooke,

Daan van der Es, and Adriaan P. IJzerman *

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ABSTRACT: In this study, we determined the crystal structure of an engineered

human adenosine A_2Areceptor bound to a partial agonist and compared it to structures

cocrystallized with either a full agonist or an antagonist/inverse agonist. The

interaction between the partial agonist, belonging to a class of dicyanopyridines, and

amino acids in the ligand binding pocket inspired us to develop a small library of

derivatives and assess their aﬃnity in radioligand binding studies and potency and

intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of

the derivatives retained the partial agonist proﬁle, whereas other ligands turned into

inverse agonists. We rationalized this remarkable behavior with additional computa-

tional docking studies.

studies to assess their aﬃnity for the (four) adenosine receptor

subtypes. We also tested these compounds in a label-free

impedance-based assay to determine their intrinsic activity on

the A_2Areceptor, revealing a huge variation in eﬃcacy, in

between agonism and inverse agonism. Further docking studies

shed light on the atomic/structural features responsible for this

behavior.

INTRODUCTION

■

Human adenosine receptors, of which there are four subtypes

(hA₁AR, hA_2AAR, hA_2BAR, and hA₃AR), belong to the large

superfamily of G protein-coupled receptors (GPCRs).¹The

hA_2AAR was one of the ﬁrst GPCRs crystallized;^2,3multiple

structures are available now, both with antagonists/inverse

agonists or (full) agonists bound.⁴Typically and unlike

prototypic antagonists such as ZM241385, agonists such as

adenosine itself bear a ribose moiety, which is deemed critical

for agonist activity (Figure 1).⁵The latter dogma was

challenged by a series of dicyanopyridines without a ribose

group, discovered, and further developed in the Bayer

laboratories as partial agonists predominantly for the

hA₁AR.^6,7A recent phase 2b clinical trial with one of these

compounds, neladenoson, failed to meet the primary endpoint

in patients with heart failure, however.⁸Depending on the

substitution pattern of these pyridine derivatives, aﬃnity for

RESULTS

■

Structure of the Compound 8−Receptor Complex. To

obtain crystals of the compound 8−receptor complex, we used

an existing thermostabilized receptor construct combined with

a bRIL fusion protein, coined A_2A-StaR2-bRIL,¹³and a

previously described approach in which crystals of A_2A-

StaR2-bRIL were formed with theophylline.¹⁴After soaking

the crystals with 8, they were exposed to synchrotron X-ray

radiation and diﬀraction data collected to 3.1 Å resolution

(Figure 2A and Figure S1A). Following initial rounds of

reﬁnement using the receptor only, both residual 2F_o− F_cand

F_o− F_celectron density maps (Figure S1B) revealed clearly

the entire pose of 8 and most surrounding side chains lining

the binding site.

A

_2Aand A_2Breceptors can be achieved as well. Colotta and co-

workers synthesized a number of derivatives with high aﬃnity

for hA_2BAR,⁹while BAY 60-6583 was reported to be a partial

agonist for A_2BAR.¹⁰We ourselves synthesized 8 (LUF5833)

and LUF5834 (Figure 1) with appreciable aﬃnity for the

adenosine A_2Areceptor and partial agonistic activity on this

receptor subtype.^11,12

Compound 8 adopts an extended pose in the orthosteric

binding site of hA_2AAR, engaging many of the residues

In this study, we present the crystal structure of 8 bound to

an engineered construct of the adenosine A_2Areceptor and

compare this structure with those of antagonist- and agonist-

bound receptors. We then synthesized a series of derivatives of

8 (Figure 1) inspired by the compound’s interaction with

speciﬁc amino acids in the binding pocket of the receptor.

These novel compounds were evaluated in radioligand binding

Received: November 11, 2020

Published: March 25, 2021

American Chemical Society

https://doi.org/10.1021/acs.jmedchem.0c01856

J. Med. Chem. 2021, 64, 3827−3842

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Figure 1. Chemical structures of adenosine, the nonriboside partial agonists 8 (LUF5833) and LUF5834, inverse agonist/antagonist ZM241385,

and the derivatives of 8 synthesized in this study (1−6).

previously described as interacting with agonists and

antagonists¹³(Figure 2B). The imidazole substituent lies

nearest the extracellular face in a pocket lined by the side

chains of Leu267^7.32, Met270^7.35, Tyr271^7.36, and Ile274^7.39

(superscript numbering according to Ballesteros and Wein-

stein¹⁵). The pyridine core is positioned similarly to the

adenine moiety in NECA, lying between Ile274^7.39and

Phe168^5.29, and the phenyl group lies deepest in the receptor,

similar to the ribose of adenosine, contacting Trp246 and

forming a π−π interaction with Phe168^5.29. One cyano

substituent forms a hydrogen bond with Asn253^6.55and the

the amino acids making up the binding pocket of ZM241385

do not include “agonistic” Ser277^7.42and His278^7.43, validating

the antagonistic nature of ZM241385 (Figures S2A and S3A).

Contrarily, the binding pocket around NECA’s ribose moiety

does include these two amino acids next to, e.g., Thr88^3.36

,

another amino acid known to be involved in full agonist

activation^16,19(Figures 2B and 3B). Although far from being

conclusive on the basis of 3D architecture alone, this

comparison between the three receptor structures suggests

that the binding pocket of 8 shares characteristics of both

agonist- and antagonist-occupied receptors. Hence, we decided

to synthesize six derivatives of 8 to shed further light on the

compound’s agonistic/antagonistic behavior.

Synthesis of Compound 8 Derivatives. In these

derivatives, the cyano groups and the number of nitrogen

atoms in the core were varied (compounds 1−6, Figure 1).

Due to the targeted change in the core scaﬀold, a diﬀerent

synthetic pathway was used for every analogue of 8. The

synthesis routes toward the pyrimidine and triazine derivatives

are shown in Scheme 1.

exocyclic amine interacts with Asn253^6.55and Glu169^EL2

.

Among the 19 amino acids close (<4 Å) to 8 (Figure 2C),

Ser277^7.42(Ala in A_2A-StaR2-bRIL; see Discussion and

Conclusions) and His278^7.43have been found crucial for full

agonist binding.^16,17For a further analysis of the latter, we

compared this structure with two reference A_2Areceptor

structures, one antagonist/inverse agonist (ZM241385)-bound

(PDB ID: 5IU4)¹³and the other agonist (NECA)-bound

(PDB ID: 2YDV)¹⁸(Figures S2 and S3). The overall

compound 8 and ZM241385 receptor structures were very

similar (protein−protein RMSD, 0.65 Å), not unexpectedly

since the same receptor construct was used in both. However,

Compound 1 was synthesized starting with a multi-

component reaction between benzaldehyde (9), malononitrile,

and thiourea, forming intermediate 10.²⁰Then, 2-bromome-

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Figure 2. (A) Crystal structure of stabilized hA_2AAR-STaR2-bRIL in complex with partial agonist 8 (color-coded according to protein domains;

TM: transmembrane domain; ECL: extracellular loop; Nt: N-terminus; Ct: C-terminus; bRIL structure omitted for clarity; compound 8 within TM

cavity); (B) ligand binding site of 8 (LUF5833) surrounded by amino acids (three-letter code) within 4 Å distance; (C) two-dimensional

representation of interacting amino acids and 8 docked into the hA_2AAR crystal structure with all nine thermostabilizing mutations reverted to wild-

type hA_2AAR, showing the ligand binding cavity within the 4 Å distance of 8 (green circle: hydrophobic; red circle: negatively charged; blue circle:

polar; gray circle: water; gradient gray circle: solvent exposed).

thylimidazole (7) was synthesized by reducing the commer-

cially available 2-imidazolecarboxaldehyde with sodium bor-

ohydride in absolute ethanol. Subsequent treatment with

hydrobromic acid (33% in acetic acid) furnished the

commonly used alkylating agent 7.^11,21Alkylation of

intermediate 10 with agent 7 gave cyanopyrimidine 1 in a

satisfactory 40% yield. The synthesis of 2 was started from the

commercially available cyanuric chloride 11. Grignard

alkylation with phenylmagnesium bromide provided 12 in a

relatively good yield of 52%. Amination was then performed

using 1.0 equiv of concentrated aqueous ammonia to give

crude 13.²²This was treated with an excess of sodium sulﬁde

nonahydrate to furnish the crystalline intermediate 14, which

was alkylated using 7 to give the ﬁnal compound 2. Although

compounds 1 and 3 are both cyanopyrimidine derivatives, the

synthesis of compound 3, in which the cyano group has an

opposed topology, was slightly more laborious. Commercially

available benzoyl acetonitrile (15) was treated with a strong

base to form the enolate, which was reacted with carbon

disulﬁde. The resulting dithiocarboxylic acid anions were

reacted with iodomethane to form ketene dithioacetal 16. The

following cyclization, using guanidine hydrochloride and an

excess base, provided the cyanopyrimidine scaﬀold.²³Sub-

sequent oxidation of the thioether gave sulfone 18, which was

subjected to a substitution-hydrolysis sequence using potas-

sium thioacetate followed by a strong base.²⁴The resulting free

thiol (19) was then alkylated using 7 to provide the ﬁnal

compound 3 with a yield of 46%.

The synthetic routes toward the pyridine derivatives are

shown in Scheme 2. The synthesis of compound 4 started with

a Knoevenagel condensation between acetophenone and

malononitrile.²⁵The thus formed intermediate 21 was reacted

with dimethyl cyanocarbonimidodithioate to establish the

cyanopyridine core.²⁶Similar to the synthesis route of

compound 3, treatment with meta-chloroperbenzoic acid

(mCPBA) provided the sulfone (23), which was converted

to free thiol 24. Alkylation of the free thiol using 7 gave the

ﬁnal compound 4. Similar to the synthesis of compound 2, the

synthesis of compound 5 started from a triple-halogen-

substituted heteroaromatic system. Commercially available

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a

Scheme 1. Synthesis Route toward Pyrimidine and Triazine Derivatives 1−3

a

Reagents and conditions: (a) malononitrile, thiourea, K₂CO₃, EtOH, reﬂux, 21% (for 10); (b) R-phenylmagnesium bromide, dry THF, N₂, rt,

52%; (c) NH₃(25%) in H₂O, CH₂Cl₂, rt, 66%; (d) Na₂S·9H₂O, DMF, 80 °C, 28% (for 14); (e) NaH, carbon disulﬁde, iodomethane, dry DMSO,

N₂, rt, 82%; (f) guanidine hydrochloride, TEA, DMF, reﬂux, 42%; (g) mCPBA,CH₂Cl₂, rt, 66%; (h) potassium thioacetate, DMF, rt, 53% (for 19);

(i) 7, Na₂CO₃, DMF, rt, 3−46%; (j) NaBH₄, absolute EtOH, rt, 75%; (k) 33% HBr in CH₃COOH, reﬂux, 65%.

2,6-dichloro-4-iodopyridine (25) was selectively alkylated on

the 4-position using a Suzuki cross-coupling reaction to give

intermediate 26 in quantitative yield.²⁷Subsequent sequential

nucleophilic aromatic substitution of the two chloro-groups

using equimolar tert-butylthiol and tert-butyl carbamate,

respectively, gave intermediate 28.^28,29Treatment with

triﬂuoroacetic acid (TFA), followed by treatment with conc.

HCl, yielded the deprotected intermediate 30 in two steps. It

was later found that this deprotection sequence could also be

performed in one step by directly using hydrochloric acid. The

ﬁnal compound 5 was obtained in a yield of 33% by alkylation

of 30 with 7 using the same procedure as mentioned before.

Last, toward compound 6, commercially available ethylbenzoyl

acetate (31) was reacted with 2-cyanoacetamide to give the

cyanopyridine intermediate 32, which was subjected to double

chlorination using phosphorus oxychloride to yield the

asymmetric cyanopyridine dichloride 33.^30,31In an initial

attempt to directly substitute one of the chlorides, tert-

butylthiol was added to intermediate 33. However, even after

varying the reaction temperature and substitution on the thiol,

inseparable mixtures of 2-(tert-alkylthio)-6-chloro-substituted

(desired compound), 6-(tert-alkylthio)-2-chloro-substituted,

and disubstituted 2,6-bis(tert-alkylthio)-4-phenylnicotinonitrile

were obtained. Instead, Buchwald−Hartwig amination using

tert-butyl carbamate was attempted. Gratifyingly, using this

procedure, intermediate 34 was obtained with the correct

substitution and as a single product. Subsequent addition of

tert-butylthiol followed by one-step deprotection of both

protective groups using hydrochloric acid gave intermediate

36, which was alkylated using the procedure mentioned

previously to produce the ﬁnal compound 6 in 8% yield.

Pharmacological Assessment of the Library of Com-

pound 8 Derivatives. We ﬁrst assessed the compounds’

aﬃnities for all adenosine receptor subtypes in radiolabeled

antagonist¹⁰binding studies on cell membranes expressing the

individual receptors (A₁: [³H]DPCPX; A_2A: [³H]ZM241385;

A_2B: [³H]PSB-603; A₃: [³H]PSB-11). Single-point displace-

ment assays at 1 μM on all four human adenosine receptor

subtypes were performed as a preliminary assay for all

compounds. Only compounds that showed greater than 50%

radioligand displacement were evaluated in full-range concen-

tration-dependent displacement assays to determine their

aﬃnities (Table 1 and Figure S4).

Of all compounds tested, the parent compound 8 displayed

the highest aﬃnity toward all human adenosine receptor

subtypes, being slightly selective for the hA₁AR with a K_ivalue

of 3 nM. Modiﬁcation of 8 by changing one cyano group at the

thiol side for a nitrogen atom in the pyridine core yielded

compound 1 with a signiﬁcant, 10−30 fold, decreased aﬃnity

toward all adenosine receptor subtypes. Changing the other

cyano group in 8 in the same way at the other, “left-hand” side

provided compound 3 with similar aﬃnity to 8 on hA_2AAR (K_i

= 10 nM), while it showed approximately 25-fold lower aﬃnity

at hA₁AR and hA₃AR, rendering this compound the more

selective derivative at hA_2AR. The non-nitrile triazine

compound 2 displayed lower aﬃnity at all adenosine receptor

subtypes with minimal aﬃnity toward hA_2BAR (only 2%

displacement of radioligand binding). The mono-cyano

pyridine derivative 4 with structural resemblance to 1 displayed

lower aﬃnity than 1 on all adenosine receptor subtypes except

hA_2BAR, where it had a K_ivalue of 370 nM, rendering it the

more nonselective derivative over all four adenosine receptor

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a

Scheme 2. Synthesis Route toward Pyridine Derivatives 4−6²⁵

a

Reagents and conditions: (a) phenylboronic acid pinacol esther, Pd(PPh₃)Cl₂, Na₂CO₃, H₂O/MeCN, N₂, 70 °C, 92%; (b) 2-methyl-

propanethiol, Cs₂CO₃, DMF, 80 °C, quantitative yield; (c) Pd(OAc)₂, Xantphos, Cs₂CO₃, t-butyl carbamate, dry 1,4-dioxane, N₂, 110 °C, 27%;

(d) TFA, CH₂Cl₂, reﬂux, 75%; (e) 37% HCl, 100 °C; (f) malononitrile, ammonium acetate, toluene, reﬂux in Dean−Stark apparatus, 70%; (g) (i)

dimethyl cyanocarbonimidodithioate, K₂CO₃, DMF, rt, (ii) piperidine, 80 °C, 62%; (h) mCPBA,CH₂Cl₂, rt, 56%; (i) potassium thioacetate, DMF,

rt, 61% (for 24); (k) 2-cyanoacetamide, KOH, EtOH, reﬂux, 34%; (m) POCl₃in an autoclave, 180 °C, 70%; (n) Pd(OAc)₂, Xantphos, Cs₂CO₃, t-

butyl carbamate, dry 1,4-dioxane, N₂, 40 °C, 33%; (o) 2-methyl-propanethiol, Cs₂CO₃, DMF, 90 °C, 89%; (p) 37% HCl, 100 °C, 37% (for 36);

(q) 7, NaHCO₃, DMF, rt, 8−57%.

Table 1. Aﬃnities of 8 and Derivatives (1−6) in

Radioligand Binding Assays on the Human Adenosine

Receptors

of radioligand binding in the preliminary single-concentration

assays (1 μM). Apparently, the number of nitrogen atoms in or

around the scaﬀold is an important determinant of receptor

binding, especially for hA_2AAR.

a

pK_ior % displacement

Next, we examined the compounds’ eﬃcacy/intrinsic activity

in a label-free impedance-based assay on cells expressing

hA_2AAR. As the parent compound 8 had been identiﬁed as a

partial agonist,^11,12we sought to characterize the new

derivatives for their functional behavior along the agonist/

antagonist spectrum. Intrinsic activities of 8 and its derivatives

at hA_2AAR were assessed using a label-free whole-cell assay

with a real-time cell analysis (RTCA) system by measuring the

ﬂow of electrons transmitted between two gold electrodes at

the bottom of a well in a 96-well plate. Upon receptor

activation, the HEK293hA_2AR cell morphology changes and

covers more of the electrodes, which impedes the electron

ﬂow. The impedance produced is displayed as a unit-less

number, the so-called cell index (CI).³²A reference full agonist

of hA_2AAR, ribose-containing CGS21680, was used to deﬁne

the highest CI observed, and the peak values obtained were

used to determine EC₅₀values. Concentration−response

curves of CGS21680 and the compounds in the library are

displayed in Figure 3. In this study, the parent compound 8

showed a lower potency than CGS21680 and acted as a partial

agonist of the receptor, with an intrinsic activity (E_maxvalue) of

66 5% (Table 2). A similar observation had been reported in

a previous study by Beukers et al.,¹¹focusing on cAMP

production as a signaling readout.

b

c

d

e

compound

A₁AR

A_2AAR

A_2BAR

A₃AR

8

1

2

3

4

5

6

8.52 0.04

7.07 0.01

6.54 0.05

7.16 0.02

6.38 0.06

8.13 0.05

7.17 0.08

6.25 0.08

8.00 0.05

6.42 0.01

7.40 0.05

7.38 0.01

6.46 0.06

5.42 0.06

5.94 0.05

5.57 0.05

f

51%

2%

50%

f

6.43 0.03

g

f

g

12%

−17%

−12%

37%

46%

f

6.95 0.08

5.99 0.12

5.87 0.10

a

Data are expressed as mean SEM of three separate experiments

b

each performed in duplicate, unless indicated otherwise. Displace-

ment of [³H]DPCPX binding in CHO cells expressing hA₁AR.

c

Displacement of [³H]ZM241385 binding in HEK293 cells expressing

d

hA_2AAR. Displacement of [³H]PSB-603 binding in CHO cells

e

expressing hA_2BAR. Displacement of [³H]PSB-11 binding in CHO

f

cells expressing hA₃AR. Percent displacement (n = 2) of speciﬁc

[³H]PSB-603 binding in CHO cells expressing hA_2BAR at 1 μM

g

ligand concentrations. Percent displacement (n = 2) of speciﬁc

radioligand binding at 10 μM ligand concentrations.

subtypes. The mono-cyano pyridine derivative 6 with the same

cyano position as in pyrimidine 3 showed a signiﬁcant, 100-

fold lower aﬃnity to hA_2AAR compared to 3, although it had

similar aﬃnity to the other subtypes. Pyridine 5, lacking both

cyano groups, was almost devoid of aﬃnity at all adenosine

receptor subtypes, as it showed no or negligible displacement

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Figure 3. Concentration−response curves for CGS21680 and nonriboside compounds at HEK293hA_2AR derived from the peak analysis of cell

index (CI) changes obtained in the xCELLigence RTCA system. The cellular responses were normalized and shown as % of the maximal CI by 1

μM CGS21680. Representative graphs from one experiment performed in duplicate.

patterns while having appreciable aﬃnity for the adenosine A_2A

receptor. To prepare the receptor structure for this analysis,

the bRIL fusion partner was removed from the crystal structure

and the third intracellular loop (ICL3) was implemented

again.³³Mutated amino acids used to thermostabilize the

structure in the crystallization process, such as M1P, A54L,

T88A, R107A, K122A, N154A, L235A, and V239A, were

changed back to the wild-type amino acids. Missing water

molecules were retrieved from a previous high-resolution

crystal structure (PDB ID: 4EIY)²to provide stability of the

receptor−ligand interaction in the docking simulation. First, as

a control experiment, compound 8 was docked into this wild-

type hA_2AAR model. Induced ﬂexible docking allowed the

imidazole moiety to adopt slightly diﬀerent positions, not

surprisingly in view of its orientation toward the more ﬂexible

extracellular loops. The dicyanopyridine scaﬀold, however, did

hardly move (Figure S5). Next, compounds 1 and 3 were

ﬂexibly docked³⁴into the binding pocket with the binding pose

of 8 as a reference (Figures 4−6, respectively). The partial

agonist 1 showed a similar binding pose as 8 after convergence

of the docking procedure, with two hydrogen bonds with

Asn253^6.55, one hydrogen bond with Glu169^ECL2, a π−π

stacking interaction with Phe168^5.29, and its phenyl moiety

surrounded by Ser277^7.42and His278^7.43(Figures 4 and 6).

The inverse agonist 3 took a slightly diﬀerent pose due to the

absence of the cyano substituent on the “left-hand” side of the

molecule. The ligand moved closer to Asn253^6.55as to

maintain hydrogen bonding, now to the nitrogen atom in

the ligand’s core. As a result, it also forms two hydrogen bonds

with Asn253^6.55, one hydrogen bond with Glu169^ECL2, and a

π−π stacking interaction between its pyrimidine core and

Phe168^5.29. However, this movement also caused a shift of the

phenyl moiety away from Ser277^7.42and His278^7.43, bringing

this moiety closer to His250^6.52(Figures 5 and 6). A similar

alignment is also present in the binding pocket of antagonist/

inverse agonist ZM241385. Its furan ring forms a π−π stacking

interaction with His250^6.52(Figure S2A).

Table 2. Compound Potency and Intrinsic Activity Derived

from the Label-Free Whole-Cell Assay Performed on

HEK293-hA_2AR Cells

a

compound

pEC₅₀

E_max, %

CGS21680

8.85 0.08

8.01 0.22

6.57 0.37

6.09 0.23

7.35 0.28

5.78 0.38

N.D.

100

66

41

−48

−50

37

2

5

8

1

2

3

4

6

7

N.D.

a

Data are shown as mean S.E.M. of three independent experiments

performed in duplicate. Log potency (pEC₅₀) and eﬃcacy (E_max) were

calculated from concentration−response curves derived from the peak

analysis of CI changes. N.D., not determined.

Various responses were shown by the nonriboside

derivatives. The pyrimidine compound 1 behaved as a partial

agonist as well (E_max= 41

5%), although it had lower

potency than 8 (pEC₅₀of 6.57 0.37 vs 8.01 0.22). The

pyridine derivative 4 was a partial agonist too (E_max= 37

7%) but with the lowest measurable potency in the series

(pEC₅₀= 5.78

0.38). Remarkably, compound 3, the

derivative with the highest selectivity for hA_2AAR, had an

opposite activity compared to 8. Figure 3 shows that this

cyanopyrimidine compound acted as an inverse agonist at the

receptor (E_max= −50 5%) with a potency quite comparable

to 8 (pEC₅₀= 7.35 0.28). The absence of both cyano groups

in the triazine compound 2 also led to inverse agonism at

hA_2AAR with similar eﬃcacy (−48

5%) to 3, albeit with

signiﬁcantly lower potency (pEC₅₀= 6.09 0.23 vs 7.35

0.28). Compound 6 did not show any measurable degree of

intrinsic activity, as it neither activated nor inactivated the

receptor (Figure 3). These results show that modiﬁcation/

deletion of the cyano groups alters the functional character-

istics of the compounds. Keeping the cyano group at the

amino, “left-hand” side maintained agonism, while the absence

of this group abolished this feature, resulting in inverse

agonism for 2 and 3 and, possibly, neutral antagonism for 6.

Molecular Modeling. Intrigued by the ﬁndings in the

pharmacological experiments, we decided to perform induced-

ﬁt docking studies of compounds 1 and 3, the two compounds

with the most obvious diﬀerences in receptor activation

DISCUSSION AND CONCLUSIONS

■

The ligand binding pocket of 8 encompasses amino acids that

have been subjected to a mutagenesis study by Lane et al. in

which the reference agonist CGS21680 and LUF5834 (Figure

1), a close analogue of 8, were compared.¹⁶In fact, within the

scope of the present study, we also attempted to obtain a

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Figure 5. (A) Proposed binding mode of compound 3 in the hA_2AAR

crystal structure with all nine thermostabilizing mutations reverted to

wild-type hA_2AAR, obtained by induced-ﬁt docking. Compared to

Figure 4 (compound 1), the hydrogen bond interaction between the

nitrogen atom in the pyrimidine ring and Asn253^6.55causes a shift of

3’s position in the binding cavity. A part of helix 7 was omitted for

better visualization. (B) Two-dimensional representation of the

interaction between 3 and hA_2AAR in the ligand binding cavity

within a 4 Å distance (green circle: hydrophobic; red circle: negatively

charged; blue circle: polar; gray circle: water; gradient gray circle:

solvent exposed). Most key interactions for binding to the receptor

are maintained, but the phenyl moiety is moved away from Ser277^7.42

Figure 4. (A) Proposed binding mode of compound 1 in the hA_2AAR

crystal structure with all nine thermostabilizing mutations reverted to

wild-type hA_2AAR, obtained by induced-ﬁt docking. It shows the

interactions formed between 1 and the receptor, in particular three

hydrogen bond interactions with Asn253^6.55and Glu169^5.30, and two

π−π stacking interactions with Phe168^5.29. A part of helix 7 was

omitted for better visualization. (B) Two-dimensional representation

of the interaction between 1 and hA_2AAR in the ligand binding cavity

within a 4 Å distance (green circle: hydrophobic; red circle: negatively

charged; blue circle: polar; gray circle: water; gradient gray circle:

solvent exposed). The phenyl moiety is surrounded by Ser277^7.42and

His278^7.43, key residues in receptor activation.

and His278^7.43

.

LUF5834’s potency and aﬃnity increased somewhat and the

compound turned into a full agonist.

receptor crystal structure with LUF5834 but were unsuccessful.

The authors performed cAMP measurements to demonstrate

that the potencies of both CGS21680 and LUF5834 were

dramatically (>100-fold) reduced in the Phe168^5.29alanine

mutant, in accordance with the pivotal role this amino acid

plays in coordinating the scaﬀold and phenyl substituent of 8

in the crystal structure. The Asn253^6.55alanine mutation

negatively aﬀected CGS2160 potency and LUF5834 intrinsic

activity, with the latter losing its partial agonist activity and

turning into an antagonist. The Ala mutant of Glu169^EL2on

the contrary slightly increased the potency and intrinsic activity

of LUF5834 with no eﬀect on CGS21680. This amino acid has

been shown to interact with a histidine (His264^6.66) residue to

form a lid above the binding pocket of the receptor, aﬀecting

the residence time of AR ligands.¹³Alanine mutation of

Ser277^7.42, as present in the crystal structure, reduced the

potency of CGS21680 by two orders of magnitude, while

Rather than reiterating this mutagenesis study to include 8,

we decided to synthesize and test six derivatives of the

compound for a further analysis of its agonistic characteristics.

The decoration of the scaﬀold was altered in a one-by-one step

fashion to compare the eﬀect of relatively minor changes on

both potency and intrinsic activity. These structural alterations

caused changes in aﬃnity, as assessed on all four adenosine

receptor subtypes through radioligand binding studies. All

compounds were less active than 8, in particular compound 5,

bearing the pyridine scaﬀold without cyano substituents.

Interestingly, compound 3 was the most selective for

hA_2AAR; it kept its aﬃnity on this receptor subtype, while its

aﬃnity for the other adenosine receptor subtypes dropped

substantially (Table 1).

We then examined the functional characteristics of the

compounds in label-free functional experiments on intact

HEK293 cells overexpressing hA_2AAR. We had reported

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Figure 6. Superimposition of compounds 1 (orange), 3 (pink), and 8 (green) in the hA_2AAR crystal structure with all nine thermostabilizing

mutations reverted to wild-type hA_2AAR, obtained by induced-ﬁt docking. For reasons of clarity, only ﬁve helices and the amino acid Asn253^6.55are

shown.

previously that the endogenous expression of hA_2BAR on these

cells does not compromise the functional evaluation of hA_2AAR

occupancy.¹⁶Hence, we were able to record the pEC₅₀values

of all compounds in this cell system (Table 2 and Figure 4)

and compared these with the radioligand binding data. The pK_i

and pEC₅₀values were strongly correlated (r²= 0.91, P = 0.01,

Figure S6) yet another indication of the unequivocal

involvement of hA_2AAR. It also suggests there is no or

negligible receptor reserve, in line with the partial nature of the

compounds’ eﬃcacy.

In conclusion, in this study, we elucidated the structure of

the adenosine A_2Areceptor bound to a chemically distinct

partial agonist, compound 8. The interaction proﬁle between

the ligand and the receptor included amino acids that had been

shown to be involved in receptor activation in earlier, e.g.,

mutagenesis studies. The synthesis and molecular docking of a

series of derivatives of the partial agonist allowed us to further

investigate the requirements for receptor activation. With this

newly solved structure, we signiﬁcantly expand the repertoire

of A_2Areceptor structures, allowing a more elaborate analysis

of the ligand binding pocket than in any other GPCR.

We were quite surprised to record huge changes in intrinsic

activity. Here, some of the compounds (2 and 3) were turned

into inverse agonists instead of (partial) agonists. Only

compounds 1 and 4, both retaining the cyano substituent on

the “left-hand” side of the molecule, kept the partial agonist

proﬁle as in 8. Induced-ﬁt docking studies helped interpret our

pharmacological ﬁndings. Subtle ligand movements in the

binding pocket carried some ligands away from “agonistic”

amino acids to negate their activation potential, while other

ligands kept a partial agonistic proﬁle. These ligand movements

may also be dependent on the presence of explicit water

molecules. In the hA_2AAR-ZM241385 crystal structure, three

water molecules occupy a region next to the furan ring of

ZM241385,^2,3which is taken by the riboside moiety in an

hA_2AAR-agonist structure.³⁵It is entirely plausible that 8 and

derivatives take intermediate positions with varying amounts of

water molecules, reﬂecting their antagonistic vs agonistic

nature. In a previous study focusing on the hA₁AR, we had

synthesized a series of quite diﬀerent monocyanopyrimidines

but also with the cyano substituent on either the “left hand” or

“right hand” side of the scaﬀold.³⁶These compounds were all

inverse agonists, however, suggesting that depending on the

hAR subtype, subtle changes at various parts of these

molecules can alter the activation proﬁle dramatically and

diﬀerently.

EXPERIMENTAL SECTION

■

Crystal Structure Determination. A_2A-Star2 bRIL Construct.

The A_2A-StaR2 construct consists of nine thermostabilizing

mutations: A54L^2.52, T88A^3.36, R107A^3.55, K122A^4.43, N154A,

L202A^5.63, L235A^6.37, V239A^6.41, and S277A^7.42. The ﬁrst three

amino acids at the N-terminus were deleted and the protein had a

C-terminal truncation after L315 where 3 additional alanines and 10

histidines were added. The apocytochrome b 562 RIL (bRIL) was

inserted into the third intracellular loop (ICL3) between residues

L208 and G218.

Expression. The A_2A-StaR2-bRIL was expressed using the

baculovirus system. Tni PRO cells were grown in suspension in

ﬂasks up to a maximum volume of 500 mL in 2 L roller bottles at 27

°C with shaking. Cells were grown in ESF921 (Expression Systems)

medium supplemented with 5% (v/v) FBS and 1% (v/v) penicillin/

streptomycin. Cells were infected at a density of 2.6 × 10⁶cells/mL

with recombinant virus at an approximate multiplicity of infection of

1. Cells were harvested by centrifugation 48 h post infection.

Membranes were prepared from a pellet of 2 L cells resuspended in

40 mM Tris−HCl (pH 7.6), 1 mM EDTA, and Complete EDTA-free

protease inhibitor cocktail tablets (Roche). Cells were disrupted

through a microﬂuidizer (processor M-110L Pneumatic, Micro-

ﬂuidics) cooled down with ice (lysis pressure, ∼15,000 psi) and

membranes were pelleted by centrifugation at 200,000g for 50 min.

Membranes were washed with 40 mM Tris−HCl (pH 7.6), 1 M

NaCl, and Complete EDTA-free protease inhibitor cocktail tablets

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and centrifuged at 200,000g for 50 min. After removal of the

supernatant, membranes were resuspended in 50 mL with 40 mM

Tris−HCl (pH 7.6) and Complete EDTA-free protease inhibitor

cocktail tablets and frozen at −80 °C.

Table 3. Data Collection and Reﬁnement Statistics

A_2A-LUF5833 (compound 8)

data collection

number of crystals

Puriﬁcation and Lipidic Cubic Phase Crystallization. Cells from 2

L cultures were resuspended and disrupted using a microﬂuidizer

(Processor M-110L Pneumatic, Microﬂuidics). Membranes pelleted

by ultracentrifugation were subjected to a high-salt wash in a buﬀer

containing 1 M NaCl before being resuspended in 50 mL of 40 mM

Tris (pH 7.6) supplemented with Complete EDTA-free protease

inhibitor cocktail tablets and stored at −80 °C until further use.

Membranes were thawed, resuspended in a total volume of 150 mL,

and incubated with 3 mM theophylline (Sigma-Aldrich) for 2 h at

room temperature. Membranes were then solubilized by addition of

1.5% n-decyl-β-^D-maltopyranoside (DM, Anatrace) and incubation for

2 h at 4 °C, followed by centrifugation at 145000g for 60 min to

harvest a solubilized material. The solubilized material was applied to

a 5 mL Ni-NTA (nickel-nitrilotriacetic acid) Superﬂow cartridge

(Qiagen) and the protein was eluted with a buﬀer containing 1 mM

theophylline. Collected fractions were pooled, concentrated, and

applied to a Superdex200 size exclusion column (GE Healthcare).

Fractions containing the protein were then concentrated to ∼35 mg/

mL and subjected to an ultracentrifugation at 436,000g prior to

crystallization. Protein concentrations were measured using the

detergent compatible (DC) protein assay (Bio-Rad).

The A_2A-StaR2-bRIL was crystallized ﬁrst in complex with

theophylline in the lipidic cubic phase at 20 °C. The ﬁnal protein/

lipid ratio was 40:60 (w/w), and 40 nL of boli was dispensed using a

Mosquito LCP crystallization robot (TTP Labtech) and overlaid with

800 nL of precipitant solution. Crystals grew within 2 weeks in 0.1 M

trisodium citrate (pH 5.3−5.4), 0.05 M sodium thiocyanate, 29−32%

PEG400, 2% (v/v) 2,5-hexanediol, and 0.5 mM theophylline. Then,

incisions were made into the Laminex cover over base wells

containing crystals, and wells were ﬂooded with 10 μL of mother

liquor supplemented by compound 8 (1 mM). Flooded wells were

then resealed and plates were incubated for 24 h at 20 °C. Single

crystals were mounted in LithoLoops (Molecular Dimensions Ltd.)

and ﬂash-frozen in liquid nitrogen without the addition of further

cryoprotectant.

1

space group

cell dimensions

a, b, c (Å)

α, β, γ (°)

resolution (Å)

no. of reﬂections

R_pim

C222₁

39.54, 181.03, 140.85

90, 90, 90

43.09−3.12 (3.31−3.12)

7990 (399)

0.134 (0.934)

a

I/σ(I)

CC_1/2

5.2 (1.1)

0.992 (0.309)

a

completeness (%)

spherical

ellipsoidal

a

84.5 (26.3)

a

87.2 (32.5)

a

redundancy

3.9 (4.0)

reﬁnement

resolution (Å)

_work/R_free

43.09−3.12

0.223/0.254

R

no. of atoms

protein

ligand

2937

24

other

69

B factors

protein

ligand

other

77.21

62.56

67.69

r.m.s. deviations

bond lengths (Å)

bond angles (°)

0.007

0.81

a

Values in parentheses are for the highest-resolution shell.

Data Collection and Structure Solution. X-ray diﬀraction data

(0.25°/frame; ∼500 frames) were collected on an Eiger 16M detector

at beamline X06SA (Swiss Light Source) in a single swipe from one

crystal. Integration was carried out using XDS³⁷and scaled using aP

scale³⁸(Global Phasing, Cambridge, U.K.). The structure of the A_2A-

StaR2-bRIL complex with compound 8 was solved by molecular

replacement (MR) with Phaser³⁹within the CCP4 interface using the

previously reported A_2A-StaR2-bRIL-theophylline complex structure

as the search model (PDB code: 5MZJ). Model reﬁnement was

performed initially using phenix.reﬁne⁴⁰and then using BUSTER⁴¹

(Global Phasing), including TLS reﬁnement for two groups

corresponding to the receptor and bRIL562, respectively. The

crystallographic data reduction and reﬁnement statistics are presented

in Table 3.

1.3 mL/min with a three-component system of H₂O/MeCN/1% TFA

in H₂O. The elution method was set up as follows: in a 1−4 min

isocratic system of H₂O/MeCN/1% TFA in H₂O, 80:10:10, from the

4th min, a gradient was applied from 80:10:10 to 0:90:10 within 9

min, followed by 1 min of equilibration at 0:90:10 and 1 min at

80:10:10. All ﬁnal compounds showed a single peak at the designated

retention time and were thus considered at least 95% pure. Liquid

chromatography−mass spectrometry (LC−MS) analyses were

performed on a Thermo Finnigan Surveyor−LCQ Advantage Max

LC−MS system, Shimadzu LCMS-2020 system, and Phenomenex

Gemini C18 110A columns (50 × 4.6 mm, 3 μm; 50 × 3 mm, 3 μm).

The sample preparation was the same as for HPLC analysis. The

compounds were eluted from the column within 15 min after

injection, with a three-component system of H₂O/MeCN + 0.1% FA,

while decreasing the polarity of the solvent mixture in time from

90:10 to 10:90. Puriﬁcation by column chromatography was achieved

by use of Grace Davison Davisil silica column material (LC60A 30−

200 micron). Solutions were concentrated using a Heidolph Laborota

W8 2000 eﬃcient rotary evaporation apparatus. All reactions in the

synthetic routes were performed without a nitrogen atmosphere,

unless stated otherwise.

Chemistry. All solvents and reagents were of analytical grade and

purchased from commercial sources. Demineralized water was simply

referred to as H₂O and used in all cases, unless stated otherwise (i.e.,

brine). Thin-layer chromatography (TLC), aluminum-coated Merck

silica gel F254 plates were used to monitor the progression of the

1

reactions. The H NMR spectra were recorded on a Bruker AV400

liquid spectrometer (¹H NMR, 400 MHz; ¹³C NMR, 100 MHz) at

ambient temperature. Chemical shifts are reported in parts per million

(ppm), designated by δ and downﬁeld to the internal standard

tetramethylsilane (TMS) in CDCl₃. Coupling constants are reported

in Hz and designated as J. The analytical purity of the ﬁnal

compounds was determined by high-performance liquid chromatog-

raphy (HPLC) with a Phenomenex Gemini 3 μm C18 110 Å column

(50 × 4.6 mm, 3 μm), measuring UV absorbance at 254 nm. Sample

preparation and HPLC method were as follows: 0.3−1.0 mg of

compound was dissolved in 1 mL of a 1:1:1 mixture of MeCN/H₂O/

tBuOH and eluted from the column within 15 min at a ﬂow rate of

(1H-Imidazol-2-yl)methanol (37).¹¹To a suspension of NaBH₄

(0.787 g, 20.8 mmol, 2.0 equiv) in absolute EtOH (15 mL) was added

commercially available 1H-imidazole-2-carbaldehyde (1 g, 10.4 mmol,

1.0 equiv) portion-wise. The mixture was stirred at room temperature

for 3 h. After the completion of the reaction, the mixture was

quenched with water and concentrated in vacuo. The crude

compound was puriﬁed by ﬂash column chromatography (10%

MeOH in CHCl₃) to obtain 37 as a pale yellow solid (769 mg, 7.85

mmol, 75%). ¹H NMR (400 MHz, MeOD): δ 6.97 (s, 2H,

imidazoyl), 4.61 (s, 2H, CH₂).

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2-(Bromomethyl)-1H-imidazole hydrobromide (7).¹¹A suspen-

sion of 37 (0.55 g, 5.6 mmol) in HBr 33% (w/v) acetic acid solution

(10 mL) was heated at 100 °C for 5 h. The mixture was concentrated

in vacuo and treated with 25% PET in diethyl ether. The suspended

solid was collected by ﬁltration and dried to give 7 as a pale brown

solid (885 mg, 3.66 mmol, 65%). H NMR (400 MHz, MeOD): δ

7.60 (s, 2H, imidazolyl), 4.82 (s, 2H, CH₂). HNMR is consistent with

the reported literature data.

solid was collected by ﬁltration and extracted with boiling EtOH (20

mL × 5). The collected organic layers were concentrated in vacuo and

washed with warm DCM (15 mL × 3) to aﬀord a pale yellow solid

(190 mg, 0.92 mmol, 28%). ¹H NMR (400 MHz, DMSO) δ 12.84 (br

s, 1H, SH), 8.22 (d, J = 7.6 Hz, 2H, Ph), 8.20 (br s, 1H, 0.5 NH₂),

7.77 (br s, 1H, 0.5 NH₂), 7.59 (t, J = 7.6 Hz, 1H, Ph), 7.51 (t, J = 8.0

Hz, 2H, Ph). MS (ESI) calcd for C₉H₈N₄S [M + H]⁺, 205.05; found,

205.10.

1

4-Amino-2-sulfanyl-6-phenylpyrimidine-5-carbonitrile (10).²⁰

To malononitrile (0.99 g, 15 mmol, 1.0 equiv) dissolved in ethanol

(7.5 mL) were added benzaldehyde (1.528 mL, 15 mmol, 1.0 equiv),

thiourea (1.14 g, 15 mmol, 1.0 equiv), and K₂CO₃(2.07 g, 15 mmol,

1.0 equiv). The mixture was stirred at reﬂux for 5 h. Upon the

completion of the reaction, the precipitate formed in the mixture was

ﬁltered and then stirred with warm water (50 °C). The ﬁltrate was

acidiﬁed with acetic acid to pH below 7. The precipitate formed was

ﬁltrated and dried in vacuo to obtain 10 as a white solid (716 mg, 3.14

mmol, 21%). ¹H NMR (400 MHz, DMSO): δ 13.07 (br s, 1H, SH),

8.45 (br s, 1H, 0.5 NH₂), 7.93 (br s, 1H, 0.5 NH₂), 7.68 (d, J = 6.8

Hz, 2H, Ph), 7.63−7.53 (m, 3H, Ph).

4-(1H-Imidazol-2-yl)methylthio-6-phenyl-1,3,5-triazin-2-amine

(2). Similar procedure as synthesis of 1 using 14 (0.148 g, 0.725

mmol, 1.0 equiv) as a starting material. The ﬁnal compound 2 was

obtained as a white solid (7 mg; 0.02 mmol; yield, 3%). Mp 209 °C.

¹H NMR (400 MHz, MeOD): δ 8.34 (dd, J = 8.4, 1.2 Hz, 2H, Ph),

7.53 (tt, J = 7.6, 1.2 Hz, 1H, Ph), 7.45 (t, J = 7.6 Hz, 2H, Ph), 6.97 (br

s, 2H, imidazolyl), 4.49 (s, 2H, CH₂). MS (ESI): calcd for C₁₃H₁₂N₆S

[M + H]⁺, 285.08; found, 285.00. HPLC: 6.906 min; purity, 98%.

2-Benzoyl-3,3-bis(methylthio)acrylonitrile (16).⁴⁵A solution of

benzoylacetonitrile 15 (1.189 g, 8.2 mmol, 1.0 equiv) in 16 mL of dry

DMSO was added dropwise to a stirred suspension of 60% NaH

dispersion in mineral oil (0.656 g, 16.4 mmol, 2.0 equiv) in 16 mL of

dry DMSO under the N₂condition at room temperature. Carbon

disulﬁde (0.49 mL, 8.2 mmol, 1.0 equiv) was added dropwise under

external water bath cooling, and the mixture was stirred for 2 h.

Iodomethane (1.0 mL, 16.4 mmol, 2.0 equiv) was then added to the

mixture dropwise under external water bath cooling, and the reaction

was stirred overnight. The reaction progress was monitored by TLC.

After the completion of the reaction, the mixture was poured into 600

mL of ice-cold water, and the precipitate formed was ﬁltered and

dried in vacuo to provide 16 in good yield (1.677 g, 6.72 mmol, 82%).

¹H NMR (400 MHz, DMSO) δ 7.85 (d, J = 7.2 Hz, 2H, Ph), 7.67 (t,

2-(1H-Imidazol-2-yl)methylthio-4-amino-6-phenylpyrimidine-5-

carbonitrile (1). Alkylation was performed according to previously

reported procedures.¹¹To a solution of free thiol 10 (0.684 g, 3

mmol, 1.0 equiv) in DMF (12 mL) were added 2-bromomethylimi-

dazole 7 (0.483 g, 3 mmol, 1.0 equiv) and Na₂CO₃(0.318 g, 3 mmol,

1.0 equiv). The reaction was stirred at 50 °C for 4 h. Water (60 mL)

was added to force the crude product to precipitate, which was

collected by ﬁltration. The precipitate was washed with DMF (6 mL)

and EtOAc (6 mL), and water (30 mL) was added to cause

precipitation, which was ﬁltered and dried in vacuo to obtain the

desired product as a white powder (370 mg; 1.20 mmol; yield, 40%).

J = 7.6 Hz, 1H, Ph), 7.55 (t, J = 7.6 Hz, 2H, Ph), 2.80 (s, 3H, CH₃),

2.53 (s, 3H, CH₃).

1

Mp 237 °C. H NMR (850 MHz, DMSO): δ 11.85 (br s, 1H, NH),

8.30 (br s, 1H, imidazolyl), 7.86−7.84 (m, 2H, Ph), 7.80 (br s, 1H,

imidazolyl), 7.58 (tt, J = 7.6, 1.7 Hz, 1H, Ph), 7.54 (tt, J = 6.8, 1.7 Hz,

2H, Ph), 7.10−6.80 (m, 2H, NH₂), 4.43 (s, 2H, CH₂). MS (ESI):

calcd for C₁₅H₁₂N₆S [M + H]⁺, 309.08; found: 309.00. HPLC: 3.107

min; purity: 99%.

2-Amino-4-(methylthio)-6-phenylpyrimidine-5-carbonitrile

(17).⁴⁶A solution of 16 (0.168 g, 6.73 mmol, 1.0 equiv), guanidine

hydrochloride (0.643 g, 6.73 mmol, 1.0 equiv), and triethylamine

(2.34 mL, 16.8 mmol, 2.5 equiv) in 21 mL of DMF was heated at

reﬂux for 6 h. The reaction progress was monitored by TLC. After the

completion of the reaction, the mixture was cooled down to room

temperature and 32 mL of water was added to cause precipitation.

The precipitate was collected by ﬁltration and washed with water and

methanol to give compound 17 as a pale yellow solid (680 mg, 2.83

2,4-Dichloro-6-phenyl-1,3,5-triazine (12).⁴²To a stirred suspen-

sion of commercially available cyanuric chloride 11 (0.5 g, 2.71 mmol,

1.0 equiv) in dry THF (20 mL) at 0 °C under a nitrogen atmosphere

was added a 3 M solution of phenylmagnesium bromide (0.993 mL,

2.98 mmol, 1.1 equiv) in THF (10 mL) dropwise (over 30 min). The

reaction mixture was stirred at room temperature for 6 h. Upon the

completion of the reaction (monitored by TLC), the mixture was

treated with 10% aqueous HCl (50 mL) and extracted with EtOAc

(40 mL × 3). The combined organic layer was washed with water (30

mL), dried on MgSO₄, and evaporated under reduced pressure to give

the intermediate as a brown solid (318 mg, 1.42 mmol, 52%). The

crude product was used for the next reaction without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.50 (dd, J = 8.0, 1.6 Hz,

2H, Ph), 7.66 (tt, J = 7.6, 1.2 Hz, 1H, Ph), 7.54 (t, J = 8.0 Hz, 2H,

Ph).

1

mmol, 42%). H NMR (400 MHz, DMSO) δ 7.88 (br s, 1H, 0.5

NH₂), 7.81 (br s, 1H, 0.5 NH₂) 7.80−7.78 (m, 2H, Ph), 7.57−7.51

(m, 3H, Ph), 2.58 (s, 3H, CH₃).

2-Amino-4-(methylsulﬁnyl)-6-phenylpyrimidine-5-carbonitrile

(18).⁴⁶Intermediate 17 (0.706 g, 2.92 mmol, 1.0 equiv) was dissolved

in 10 mL of CH₂Cl₂at room temperature, and mCPBA (0.808 g, 4.68

mmol, 1.6 equiv) was added to the mixture. The mixture was stirred

at room temperature for 3.5 h and monitored by TLC. The precipitate

formed in the reaction was ﬁltered, washed by CH₂Cl₂, and dried

1

under reduced pressure to obtain 18 (495 mg, 1.93 mmol, 66%). H

NMR (400 MHz, DMSO) δ 8.39 (br s, 1H, 0.5 NH₂), 8.32 (br s, 1H,

0.5 NH₂), 7.84 (d, J = 7.2 Hz, 2H, Ph), 7.64−7.54 (m, 3H, Ph), 2.93

(s, 3H, CH₃).

4-Chloro-6-phenyl-1,3,5-triazine-2-amine (13).⁴³Aqueous am-

monia (25%) was added dropwise (0.1 mL, 1.327 mmol, 1.0 equiv) to

a stirred solution of 12 (0.3 g, 1.327 mmol, 1.0 equiv) in CH₂Cl₂(5

mL). The resulting mixture was stirred at room temperature for 8 h.

The reaction progress was monitored by TLC. The precipitate formed

was ﬁltered, rinsed with CH₂Cl₂(30 mL), and dried under reduced

pressure, aﬀording a pale orange solid of 13 (180 mg, 0.87 mmol,

66%). The compound was used in the next reaction without further

puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ 8.40 (d, J = 8.0, 1.6 Hz,

2H, Ph), 7.57 (tt, J = 7.6, 1.6 Hz, 1H, Ph), 7.51 (t, J = 8.0 Hz, 2H,

Ph), 5.71 (br s, 2H, NH₂). MS (ESI) calcd for C₉H₇ClN₄[M + H]⁺:

207.04; found, 207.10.

2-Amino-4-sulfanyl-6-phenylpirimidine-5-carbonitrile (19).⁴⁷To

a stirred suspension of 18 (0.495 g, 1.92 mmol, 1.0 equiv) in 17 mL of

DMF was added potassium thioacetate (0.376 g, 3.84 mmol, 2.0

equiv), and the mixture was stirred at room temperature for 19 h. The

reaction progress was monitored by TLC. After the completion of the

reaction, the mixture was cooled down in an external ice water bath,

and 17 mL of 2 M aqueous NaOH was added to the cold solution.

The mixture was stirred for another 1.5 h and diluted with 17 mL of

H₂O, followed by pH adjustment to 5−6 with the addition of 1 M

HCl ( 50 mL). The mixture was diluted with 70 mL of H₂O and

stirred for 1 h at room temperature. The precipitate formed in the

mixture was collected by ﬁltration, washed with H₂O (25 mL × 3),

and dried under reduced pressure to give 19 in good yield (232 mg,

4-Amino-6-phenyl-1,3,5-triazine-2-thiol (14).⁴⁴A suspension of

13 (0.68 g, 3.29 mmol, 1.0 equiv) and sodium sulﬁde nonahydrate

(1.185 g, 4.93 mmol, 1.5 equiv) in DMF (3 mL) was stirred at 80 °C

for 3 h. The reaction progress was monitored by TLC. DMF was

evaporated under reduced pressure at 80 °C. The resulting residue

was treated carefully with HCl in EtOAc (10 mL). The suspended

1

1.02 mmol, 53%). H NMR (400 MHz, DMSO) δ 12.91 (br s, 1H,

SH), 8.67 (br s, 1H, 0.5 NH₂), 7.78 (d, J = 7.6 Hz, 2H, Ph), 7.61−

7.48 (m, 3H, Ph), 6.95 (br s, 1H, 0.5 NH₂).

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Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4-((1H-Imidazol-2-yl)methylthio)-2-amino-6-phenylpyrimidine-

5-carbonitrile (3). Similar procedure as synthesis of 1 using 19 (0.187

g, 0.82 mmol, 1.0 equiv) as a starting material to aﬀord a light yellow

mmol, 1 equiv), and NaHCO₃(0.166 g, 1.98 mmol, 2 equiv) in DMF

(3 mL) was stirred at 50 °C for 4 h. After the completion of the

reaction as shown by TLC, the mixture was concentrated in vacuo and

dissolved in EtOAc followed by washing three times with water and

brine. The organic phase was dried over MgSO₄, concentrated in

vacuo to give a crude yellow oil, and puriﬁed by dry-loaded column

chromatography (5% MeOH in CH₂Cl₂) as a column chromatog-

raphy eluent. Compound 4 was obtained as a white solid (0.115 g;

0.376 mmol; yield, 57%). Mp 220 °C. ¹H NMR (400 MHz, DMSO)

δ 11.86 (br s, 1H, NH), 7.587.53 (m, 2H, Ph), 7.52−7.49 (m, 3H,

Ph), 7.17 (br s, 2H, NH₂), 7.05 (br s, 1H, imidazolyl), 6.82 (br s, 1H,

imidazolyl), 6.68 (s, 1H, pyridinyl), 4.39 (s, 2H, CH₂). MS (ESI):

calcd for C₁₆H₁₃N₅S [M + H]⁺, 308.09; found, 308.05. HPLC: 6.33

min; purity: 99%.

1

solid (116 mg; 0.38 mmol; yield, 46%). Mp 224 °C. H NMR (400

MHz, DMSO) δ 11.86 (br s, 1H, NH), 7.97 (m, 2H, NH₂), 7.79 (d, J

= 6.8 Hz, 2H, Ph), 7.60−7.50 (m, 3H, Ph), 7.08 (br s, 1H,

imidazolyl), 6.85 (br s, 1H, imidazolyl), 4.53 (s, 2H, CH₂). MS (ESI):

calcd for C₁₅H₁₂N₆S [M + H]⁺, 309.08; found, 309.00. HPLC: 4.377

min; purity: 99%.

2-(1-Phenylethylidene)malononitrile (21).²⁵The synthesis of this

compound was adapted from the conditions previously described by

Longstreet et al.²⁵A mixture of acetophenone 20 (2.80 mL, 24 mmol,

1.0 equiv), malononitrile (1.586 g, 24.00 mmol, 1 equiv), and

ammonium acetate (0.370 g, 4.80 mmol, 0.2 equiv) in toluene (20

mL) was heated at reﬂux under Dean−Stark conditions for 5 h. The

reaction progress was monitored by TLC. After the completion of the

reaction, the mixture was cooled down to room temperature. The

reaction was diluted with EtOAc (50 mL), washed with saturated

NaHCO₃solution (50 mL × 3) followed by water (50 mL), dried

over MgSO₄, and concentrated in vacuo to obtain a brown oil, which

solidiﬁed at room temperature after 30 min. Puriﬁcation was

performed by column chromatography (CH₂Cl₂) to obtain 21 as a

white solid (2.82 g, 16.8 mmol, 70%). ¹H NMR (400 MHz, CDCl₃) δ

7.58−7.26 (m, 5H, Ph), 2.64 (s, 3H, CH₃). HNMR is consistent with

the reported literature data.

2,6-Dichloro-4-phenylpyridine (26).⁴⁹A stirred suspension of 2,6-

dichloro-4-iodopyridine 25 (1 g, 3.65 mmol, 1.0 equiv), Na₂CO₃

(1.16 g, 10.95 mmol, 3.0 equiv), phenylboronic acid pinacol ester

(0.745 g, 3.65 mmol, 1.0 equiv), and Pd(PPh₃)₂Cl₂(0.128 g, 0.183

mmol, 0.05 equiv) in a mixture of acetonitrile (12 mL) and H₂O (8

mL) under a N₂atmosphere was heated at 70 °C for 16 h. The

reaction progress was monitored by TLC (2% EtOAc in PET) and

HPLC. The mixture was diluted with EtOAc (50 mL) and washed

with brine (30 mL × 3). The organic layer was dried over MgSO₄and

evaporated under reduced pressure. The resulting brown oil was

puriﬁed by column chromatography (2% EtOAc in PET) to aﬀord a

white solid (750 mg, 3.36 mmol, 92%). ¹H NMR (400 MHz, CDCl₃)

δ 7.61−7.57 (m, 2H, Ph), 7.53−7.48 (m, 3H, Ph), 7.47 (s, 2H,

pyridinyl). HNMR in accordance with the literature.

2-Amino-6-(methylthio)-4-phenylnicotinonitrile (22).⁴⁸A mix-

ture of 21 (2.5 g, 14.86 mmol, 1.0 equiv), dimethyl cyanocarboni-

midodithioate (3.69 g, 25.3 mmol, 1.7 equiv), and potassium

carbonate (2.465 g, 17.84 mmol, 1.2 equiv) in DMF (30 mL) was

stirred at room temperature for 6 h. After the completion of the

reaction, piperidine (2.4 mL, 23.78 mmol, 1.6 equiv) was added and

the mixture was stirred at 80 °C overnight. The reaction progress was

monitored by TLC. The mixture was then concentrated in vacuo,

diluted with water, and extracted three times with CH₂Cl₂. The

organic layer was dried over MgSO₄and concentrated in vacuo. The

brown oil obtained was puriﬁed by column chromatography

(CH₂Cl₂), yielding 22 as a yellow solid (2.21 g, 9.21 mmol, 62%).

¹H NMR (400 MHz, CDCl₃) δ 7.56−7.52 (m, 2H, Ph), 7.49−7.47

(m, 3H, Ph), 6.64 (s, 1H, pyridinyl), 5.27 (br s, 2H, NH₂), 2.54 (s,

3H, CH₃).

2-(tert-Butylthio)-6-chloro-4-phenylpyridine (27). A suspension

of 2,6-dichloro-4-phenylpyridine 26 (0.2 g, 0.89 mmol, 1.0 equiv),

Cs₂CO₃(0.58 g, 1.78 mmol, 2.0 equiv), and 2-methylpropane-2-thiol

(0.084 g, 0.93 mmol, 1.04 equiv) in DMF was heated at 80 °C

overnight. The reaction progress was monitored by TLC (10% EtOAc

in PET) and HPLC. The mixture was diluted with EtOAc (50 mL)

and washed with brine (30 mL × 5). The organic phase was dried on

MgSO₄and evaporated to aﬀord a pale yellow oil (249 mg, 0.89

mmol, quantitative yield). ¹H NMR (400 MHz, CDCl₃) δ 7.69−7.54

(m, 2H, Ph), 7.50−7.43 (m, 3H, Ph), 7.37 (d, J = 1.2 Hz, 1H,

pyridinyl), 7.26 (d, J = 1.2 Hz, 1H, pyridinyl) 1.59 (s, 9H, t-butyl).

MS (ESI): calcd for C₁₅H₁₆ClNS [M + H]⁺, 278.07; found, 277.90.

tert-Butyl-(6-(tert-butylthio)-4-phenylpyridin-2-yl)-carbamate

(28). A suspension of 2-(tert-butylthio)-6-chloro-4-phenylpyridine 27

(0.23 g, 0.83 mmol, 1.0 equiv), Cs₂CO₃(0.54 g, 1.66 mmol, 2.0

equiv), Xantphos (0,144 g, 0.25 mmol, 0.3 equiv), Pd(OAc)₂(0.028

g, 0.124 mmol, 0.15 equiv), and t-butyl carbamate (0.097 g, 0.83

mmol, 1.0 equiv) in dry 1,4-dioxane (2.8 mL) under a N₂atmosphere

was heated at 110 °C overnight. The reaction progress was monitored

by TLC (2% EtOAc in PET) and HPLC. The mixture was treated

with warm acetone (25 mL) and ﬁltered. The organic layer was

evaporated and puriﬁed by column chromatography (20% EtOAc in

2-Amino-6-(methylsulfonyl)-4-phenylnicotinonitrile (23). A mix-

ture of 22 (1 g, 4.14 mmol, 1 equiv) and mCPBA (1.114 g, 4.97

mmol, 1 equiv) in 120 mL of CH₂Cl₂was stirred at room temperature

for 5 min. The mixture was then washed twice with NaHCO₃solution

followed by water, dried over MgSO₄, and concentrated in vacuo to

yield a mixture of sulfoxide and sulfone product. Without further

separation, it was then reacted with 77 wt % mCPBA (0.674 g, 3.90

mmol, 0.94 equiv) in CH₂Cl₂(70 mL) and stirred at room

temperature for another 5 min. The mixture was washed twice with

NaHCO₃solution followed by water, dried over MgSO₄, and

concentrated in vacuo. Puriﬁcation was performed by column

chromatography (2% MeOH in CH₂Cl₂), yielding 23 as a yellow

1

PET) to aﬀord 28 as a yellow solid (80 mg, 0.22 mmol, 27%). H

1

NMR (400 MHz, DMSO) δ 9.96 (s, 1H, NH), 7.84 (d, J = 1.6 Hz,

1H), 7.67 (d, J = 6.8 Hz, 2H, Ph), 7.54−7.44 (m, 3H, Ph), 7.15 (d, J

= 1.6 Hz, 1H), 1.53 (s, 9H), 1.50 (s, 9H). MS (ESI): calcd for

C₂₀H₂₆N₂O₂S [M + H]⁺, 359.17; found, 358.9.

solid (0.631 g, 2.31 mmol, 56%). H NMR (400 MHz, DMSO) δ

7.68−7.65 (m, 4H, NH₂+ Ph), 7.59−7.57 (m, 3H, Ph), 7.17 (s, 1H,

pyridinyl), 3.27 (s, 3H, CH₃).

2-Amino-6-sulfanyl-4-phenylnicotinonitrile (24). A mixture of 23

(0.631 g, 2.31 mmol, 1 equiv) and potassium ethanethioate (0.527 g,

4.62 mmol, 2 equiv) in DMF (8 mL) was stirred at 80 °C for 4 h.

After the completion of the reaction as shown by TLC, 2 M NaOH

solution was added and the mixture was stirred at room temperature

for another 2 h. The mixture was then diluted with water (80 mL)

and 1 M HCl solution was added to adjust pH to 1. The precipitate

formed was ﬁltered and dried in vacuo to obtain a brown crude solid.

Puriﬁcation was performed by column chromatography (3% MeOH

in CH₂Cl₂) to obtain the product as a yellow solid (0.321 g, 1.41

6-(tert-Butylthio)-4-phenylpyridin-2-amine (29). To a solution of

tert-butyl (6-(tert-butylthio)-4-phenylpyridin-2-yl)-carbamate 28

(0.25 g, 0.697 mmol, 1.0 equiv) in CH₂Cl₂(5 mL) was added TFA

(0.266 mL, 3.48 mmol, 5.0 equiv). The mixture was reﬂuxed

overnight. The reaction progress was monitored by TLC (6% MeOH

in CH₂Cl₂) and HPLC. The mixture was diluted with EtOAc (50

mL) and washed with H₂O (25 mL × 4). The organic phase was

dried on MgSO₄and evaporated to aﬀord a brown oil. The crude

compound was puriﬁed by liquid chromatography (50% EtOAc in

PET) to give 29 as a pale yellow oil (135 mg, 0.523 mmol, 75%). ¹H

NMR (400 MHz, DMSO) δ 7.66 (dd, J = 6.8, 2.0 Hz, 2H, Ph), 7.54−

7.47 (m, 3H, Ph), 6.96 (s br, 1H, 0.5 NH₂), 6.76 (s br, 1H, 0.5 NH₂),

1.46 (s, 9H, t-butyl). MS (ESI): calcd for C₁₅H₁₈N₂S [M + H]⁺,

259.12; found, 259.0 [M + H]⁺.

1

mmol, 61%). H NMR (400 MHz, DMSO) δ 12.58 (s, 1H, SH),

7.58−7.48 (m, 6H), 7.31 (br s, 2H, NH₂), 6.47 (s, 1H). MS (ESI):

calcd for C₁₂H₉N₃S [M + H]⁺, 228.05; found, 228.13.

6-(((1H-Imidazol-2-yl)methyl)thio)-2-amino-4-phenylnicotinoni-

trile (4). A mixture of 24 (0.12 g, 0.66 mmol, 1 equiv), 7 (0.16 g, 0.66

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J. Med. Chem. 2021, 64, 3827−3842

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

6-Amino-4-phenylpyridine-2-thiol (30). A stirred solution of 6-

(tert-butylthio)-4-phenylpyridin-2-amine 29 (0.445 g, 1.722 mmol) in

37% HCl (15 mL) was heated at 100 °C for 10 h. The reaction

progress was monitored by HPLC. The mixture was carefully treated

with NaHCO₃saturated solution to pH = 7 and extracted with EtOAc

(40 mL × 5). The organic phase was dried over MgSO₄and

evaporated to aﬀord an orange solid (135 mg, mixture of the desired

compound and the corresponding dimer). The compound was used

for the next reaction without further puriﬁcation. ¹H NMR (400

MHz, DMSO) δ 12.25 (s br, 1H, SH), 7.59−7.55 (m, 2H, Ph), 7.49−

7.43 (m, 3H, Ph), 6.85 (s br, 1H, 0.5 NH₂), 6.40 (s br, 1H, 0.5 NH₂).

MS (ESI): calcd for C₁₁H₁₀N₂S [M + H]⁺, 203.06; found, 203.1.

6-(((1H-Imidazol-2-yl)methyl)thio)-4-phenylpyridin-2-amine (5).

To a suspension of 6-amino-4-phenylpyridine-2-thiol 30 (0.145 g,

0.716 mmol, 1.0 equiv) and NaHCO₃(0.061 g, 0.57 mmol, 0.8 equiv)

in dry DMF (2 mL) was added 7 (0.225 g, 0.931 mmol, 1.3 equiv).

The mixture was stirred at room temperature for 23 h. The reaction

progress was monitored by HPLC. DMF was evaporated under

reduced pressure (water bath at 70 °C). The resulting residue was

treated with H₂O (20 mL) and extracted with EtOAc (30 mL × 4).

The organic phase was dried over MgSO₄and evaporated to aﬀord an

orange oil. The compound was puriﬁed by liquid chromatography

(10% MeOH in CH₂Cl₂) to obtain pure 5 as a pale red solid (67 mg;

0.24 mmol; yield, 33%). Mp 109 °C. ¹H NMR (400 MHz, MeOD) δ

7.55−7.50 (m, 2H, Ph), 7.46−7.32 (m, 3H, Ph), 6.94 (s, 2H,

pyridinyl), 6.70 (d, J = 1.2 Hz, 1H, imidazolyl), 6.50 (d, J = 1.2 Hz,

1H, imidazolyl), 4.39 (s, 2H, CH₂). MS (ESI): calcd for C₁₅H₁₄N₄S

[M + H]⁺, 283.09; found, 283.00. HPLC: 3.722 min; purity: 97%.

2,6-Dihydroxy-4-phenylnicotinonitrile (32).³⁰A suspension of

commercially available ethylbenzoyl acetate 31 (3 g, 15.6 mmol, 1.0

equiv), 2-cyanoacetamide (1.31 g, 15.6 mmol, 1.0 equiv), and KOH

(0.96 g, 15.6 mmol, 1.0 equiv) in EtOH (20 mL) was reﬂuxed for 24

h. The reaction progress was monitored by HPLC and TLC (20%

MeOH in CH₂Cl₂). After the completion of the reaction, the mixture

was cooled down to 0 °C. The suspended solid was collected by

ﬁltration and subsequently dissolved in warm water (60 mL). The

alkaline solution was carefully treated with 37% HCl solution to adjust

the pH to 1. The precipitate was collected by ﬁltration and dried to

2-methyl-propanethiol (0.03 mL, 0.273 mmol, 1.0 equiv), and

Cs₂CO₃(0.178 g, 0.56 mmol, 2 equiv) in DMF (2 mL) was heated

at 90 °C for 20 h. The reaction progress was monitored by HPLC.

The mixture was cooled down to room temperature, diluted with

EtOAc (50 mL), and washed with brine (25 mL × 5). The organic

layer was dried on MgSO₄and evaporated to aﬀord an orange oil (93

mg, 0.211 mmol, 89%). The compound was used for the next reaction

1

without further puriﬁcation. H NMR (400 MHz, CDCl₃) δ 7.81 (s,

1H, pyridinyl), 7.59−7.55 (m, 2H, Ph), 7.48−7.44 (m, 3H, Ph), 7.32

(s br, 1H, NH), 1.65 (s, 9H, t-butyl), 1.54 (s, 9H, t-butyl). MS (ESI):

calcd for C₂₁H₂₅N₃O₂S [M + H]⁺, 384.17; found, 284.00.

6-Amino-2-sulfanyl-4-phenylnicotinonitrile (36). A suspension of

35 (0.7 g, 1.825 mmol) in 37% HCl (7 mL) was heated at 100 °C for

2 h. The reaction progress was monitored by HPLC. The mixture was

cooled down to room temperature and diluted with NaHCO₃

saturated solution (7 mL). The resulting suspension was treated

carefully with solid NaHCO₃to pH 7 and extracted with EtOAc (30

mL × 5). The organic layer was dried over MgSO₄and evaporated to

aﬀord an orange solid, which was puriﬁed by column chromatography

(40% EtOAc in PET and 10% MeOH in CH₂Cl₂). Intermediate 36

1

was obtained as a pale yellow solid (156 mg, 0.675 mmol, 37%). H

NMR (400 MHz, MeOD) δ 7.56−7.51 (m, 2H, Ph), 7.51−7.47 (m,

3H, Ph), 6.05 (s, 1H, pyridinyl). MS (ESI): calcd for C₁₂H₉N₃S [M +

H]⁺, 228.05; found, 228.08.

2-(((1H-Imidazol-2-yl)methyl)thio)-6-amino-4-phenylnicotinoni-

trile (6). A suspension of 36 (0.096 g, 0.422 mmol, 1.0 equiv),

NaHCO₃(0.020 g, 0.422 mmol, 1.0 equiv), and 7 in dry DMF (2.5

mL) was stirred at room temperature for 4 h. The reaction progress

was monitored by TLC (10% MeOH in CH₂Cl₂). DMF was

evaporated under reduced pressure (water bath at 70 °C). The

resulting residue was treated with water (10 mL) and extracted with

EtOAc (30 mL × 5). The organic layer was dried over MgSO₄and

evaporated to aﬀord a pale brown oil. The ﬁnal compound 6 was

obtained after puriﬁcation by column chromatography (PET/EtOAc/

MeOH, 1:7.8:1.2) and recrystallized using diethyl ether and MeOH

to yield 6 as a pale yellow solid (10 mg; 0.032 mmol; yield, 8%). Mp

222 °C. ¹H NMR (400 MHz, MeOD) δ 7.53−7.45 (m, 5H, Ph), 6.97

(s, 2H, imidazolyl), 6.28 (s, 1H, pyridinyl), 4.53 (s, 2H, CH₂). MS

(ESI): calcd for C₁₆H₁₃N₅S [M + H]⁺, 308.09; found, 308.00. HPLC:

7.490 min; purity: 99%.

1

give 32 in good yield (1.3 g, 5.3 mmol, 34%). H NMR (400 MHz,

DMSO) δ 7.52 (s, 5H, Ph), 5.80 (s, 1H, pyridinyl). MS (ESI): calcd

for C₁₂H₈N₂O2 [M + H]⁺, 213.06; found, 213.1. HNMR is consistent

with the reported literature data.

Pharmacology. Cell Lines and Chemicals. Chinese hamster

ovary (CHO) cells stably expressing the human adenosine A₁

receptor (CHOhA₁R) were kindly provided by Prof. S. J. Hill

(University of Nottingham, U.K.), human embryonic kidney 293 cells

stably expressing the human adenosine A_2Areceptor (HEK₂₉₃hA_2AR)

were kindly provided by Dr. J. Wang (Biogen/IDEC, Cambridge,

MA), CHO-spap cells stably expressing the wild-type (WT) hA_2B

receptor (CHO-spap-hA_2BR) were kindly provided by S. J. Dowell

(GlaxoSmithKline, U.K.), and CHO cells stably expressing the human

adenosine A₃receptor (CHOhA₃R) were a gift from Dr. K.-N. Klotz

2,6-Dichloro-4-phenylnicotinonitrile (33). A suspension of 2,6-

dihydroxy-4-phenylnicotinonitrile 32 (1.2 g, 5.657 mmol, 1.0 equiv)

in POCl₃(5.3 mL, 56.6 mmol, 10.0 equiv) was heated at 180 °C in an

autoclave for 16 h. The reaction progress was monitored by HPLC.

The mixture was cooled down to 0 °C and treated with crushed ice.

The suspended solid was collected by ﬁltration, rinsed with petroleum

ether (30 mL), and dried under reduced pressure to aﬀord 33 as a

gray solid (0.990 g, 3.96 mmol, 70%). ¹H NMR (400 MHz, CDCl₃) δ

7.63−7.60 (m, 5H, Ph), 7.45 (s, 1H, pyridinyl). HNMR is consistent

with the reported literature data.

(University of Wu

̈

rzburg, Germany). [³H]1,3-Dipropyl-8-cyclopentyl-

xanthine ([³H]DPCPX; speciﬁc activity, 120 Ci/mmol) was

purchased from ARC, Inc. (St. Louis, USA), [³H]4-(2-[7-amino-2-

(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl) phe-

nol ([³H]-ZM241385; speciﬁc activity, 50 Ci/mmol) was purchased

from ARC, Inc. (St. Louis, USA), [³H]8-(4-(4-(4-chlorophenyl)-

piperazide-1-sulfonyl)phenyl)-1-propylxanthine ([³H]PSB-603; spe-

ciﬁc activity, 79 Ci/mmol) was purchased from Quotient Bioresearch

(Waltham, MA), and [³H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-

tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([³H]PSB-11; speciﬁc ac-

tivity, 56 Ci/mmol) was obtained with the kind help of Prof. C. E.

tert-Butyl-(6-chloro-5-cyano-4-phenylpyridin-2-yl)-carbamate

(34). To a suspension of 2,6-dichloro-4-phenylnicotinonitrile 33 (0.2

g, 0.806 mmol, 1.0 equiv), t-butyl carbamate (0.095 g, 0.806 mmol,

1.0 equiv), Cs₂CO₃(0.54 g, 1.66 mmol, 2.06 equiv), and Xantphos

(0.139 g, 0.242 mmol, 0.3 equiv) in dry 1,4-dioxane (2.7 mL) under a

N₂atmosphere was added Pd(OAc)₂(0.027 g, 0.121 mmol, 0.15

equiv). The mixture was heated at 40 °C for 23 h. The reaction

progress was monitored by TLC (2% EtOAc in PET) and HPLC.

The mixture was then treated with warm acetone (25 mL) and

ﬁltered. The organic layer was evaporated to aﬀord a pale brown solid

(90 mg, 0.26 mmol, 33%). The product was used for the next reaction

Muller (University of Bonn, Germany). 5′-N-Ethylcarboxamidoade-

̈

nosine (NECA), N⁶-cyclopentyladenosine (CPA), and adenosine

deaminase (ADA) were purchased from Sigma-Aldrich (Steinheim,

Germany). Unlabeled 4-(2-[7-amino-2-(furan-2-yl)-[1,2,4]triazolo-

[1,5-a][1,3,5]triazin-5-ylamino)ethyl) phenol (ZM241385) was a

kind gift from Dr. S. M. Poucher (Astra Zeneca, Manchester, U.K.).

3-[4-[2-[[6-Amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihy-

droxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl] propanoic acid

(CGS21680) was purchased from Ascent Scientiﬁc (Bristol, U.K.).

1

without further puriﬁcation. H NMR (300 MHz, CDCl₃) δ 8.07 (s,

1H, pyridinyl), 7.64−7.58 (m, 2H, Ph), 7.55−7.47 (m, 4H, Ph +

NH), 1.53 (s, 9H, t-butyl). MS (ESI): calcd for C₁₇H₁₆ClN₃O₂[M +

H]⁺, 330.09; found, 329.92.

tert-Butyl (6-(tert-butylthio)-5-cyano-4-phenylpyridin-2-yl)-car-

bamate (35). A suspension of tert-butyl (6-chloro-5-cyano-4-

phenylpyridin-2-yl)-carbamate 34 (0.09 g, 0.273 mmol, 1.0 equiv),

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Article

Bicinchoninic acid (BCA) protein assay reagents were obtained from

Pierce Chemical Company (Rockford, IL, USA). All other chemicals

were of analytical grade and were obtained from standard commercial

sources.

Label-Free Impedance-Based Assay. Label-free whole-cell assays

were performed on the xCELLigence real-time cell analyzer (RTCA)

system.^51,52In this system, the cells are adhered to arrayed gold

electrodes embedded at the bottom of microelectronic E-plate 96

́

(Bioke, Leiden, NL), which is compatible with the xCELLigence

Cell Culture and Membrane Preparation. CHOhA₁R and

CHOhA₃R cells were grown in Dulbecco’s modiﬁed Eagle’s medium

(DMEM) and Ham’s F12 medium (1:1) supplemented with 10% (v/

v) newborn calf serum, 50 μg·mL⁻¹streptomycin, 50 IU·mL⁻¹

penicillin, and 200 μg·mL⁻¹G418 at 37 °C and 5% CO₂. CHOhA₁R

cells were subcultured twice a week at a ratio of 1:20 on 10 cm Ø

plates and 15 cm Ø plates. CHOhA₃cells were subcultured twice a

week at a ratio of 1:8 on 10 cm Ø plates and 15 cm Ø plates.

HEK₂₉₃hA_2AR cells were grown in a culture medium consisting of

Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with

10% newborn calf serum, 50 μg·mL⁻¹streptomycin, 50 IU·mL⁻¹

penicillin, and 500 μg·mL⁻¹G418 at 37 °C and 7% CO₂. Cells were

subcultured twice a week at a ratio of 1:8 on 10 cm Ø plates and 15

cm Ø plates. CHO-spap-hA_2BR cells were grown in Dulbecco’s

modiﬁed Eagle’s medium (DMEM) and Ham’s F12 medium (1:1)

supplemented with 10% (v/v) newborn calf serum, 100 μg·mL⁻¹

streptomycin, 100 IU·mL⁻¹penicillin, 1 mg·mL⁻¹G418, and 0.4 mg·

mL⁻¹hygromycin at 37 °C and 5% CO₂. Cells were subcultured at a

ratio of 1:20 twice weekly.

All cells were grown to 80−90% conﬂuency and detached from

plates by scraping them into 5 mL of PBS. Detached cells were

collected and centrifuged at 200g for 5 min. Pellets derived from 100

15 cm Ø plates were pooled and resuspended in 70 mL of ice-cold 50

mM Tris−HCl buﬀer (pH 7.4). A Heidolph Diax 900 homogenizer

was used to homogenize the cell suspension. Membranes and the

cytosolic fraction were separated by centrifugation at 100,000g in a

Beckman Optima LE-80K ultracentrifuge (Beckman Coulter, Full-

erton, CA) at 4 °C for 20 min. The pellet was resuspended in 35 mL

of Tris−HCl buﬀer, and the homogenization and centrifugation steps

were repeated. Tris−HCl buﬀer (25 mL) was used to resuspend the

pellet, and ADA was added (0.8 U/mL) to break down endogenous

adenosine. Membranes were stored in 250 and 500 μL aliquots at −80

°C. Total protein concentrations were measured using the BCA

method.⁵⁰

Radioligand Binding Studies. Membrane aliquots containing 5 μg

(CHOhA₁R) or 30 μg (HEK₂₉₃hA_2AR) of total protein were

incubated in a total volume of 100 μL assay buﬀer (50 mM Tris−

HCl, pH 7.4) at 25 °C for 1 h. Membrane aliquots containing 30 μg

(CHO-spap-hA_2BR) of total protein were incubated in a total volume

of 100 μL assay buﬀer (0.1% CHAPS in 50 mM Tris−HCl, pH 7.4) at

25 °C for 2 h, or those containing 15 μg (CHOhA₃R) of total protein

were incubated in a total volume of 100 μL assay buﬀer (50 mM

Tris−HCl, pH 8.0, supplemented with 10 mM MgCl₂, 1 mM EDTA,

and 0.01% (w/v) CHAPS) at 25 °C for 2 h. Radioligand displacement

experiments were performed using six concentrations of the

competing ligand in the presence of 1.6 nM [³H]DPCPX for

CHOhA₁R, 5.5 nM [³H]ZM241385 for HEK₂₉₃hA_2AR, 1.5 nM

[³H]PSB-603 for CHO-spap-hA_2BR, and 10 nM [³H]PSB-11 for

CHOhA₃R. At these concentrations, the total radioligand binding did

not exceed 10% of that added to prevent ligand depletion. Nonspeciﬁc

binding was determined in the presence of 100 μM CPA for

CHOhA₁R, 100 μM NECA for HEK₂₉₃hA_2AR and CHOhA₃R, and 10

μM ZM241385 for CHO-spap-hA_2BR. Incubations were terminated

by rapid vacuum ﬁltration to separate the bound and free radioligands

through prewetted 96-well GF/B ﬁlter plates using a PerkinElmer

Filtermate-harvester (PerkinElmer, Groningen, The Netherlands).

Filters were subsequently washed 12 times with ice-cold wash buﬀer:

50 mM Tris−HCl (pH 7.4) for CHOhA₁R and HEK₂₉₃hA_2AR, 0.1%

BSA in 50 mM Tris−HCl (pH 7.4) for CHO-spap-hA_2BR, and 50

mM Tris−HCl supplemented with 10 mM MgCl₂and 1 mM EDTA

(pH 8.0) for CHOhA₃R. The plates were dried at 55 °C after which

MicroScint-20 cocktail was added (PerkinElmer, Groningen, The

Netherlands). After 3 h, the ﬁlter-bound radioactivity was determined

by scintillation spectrometry using a 2450 MicroBeta Microplate

Counter (PerkinElmer, Groningen, The Netherlands).

RTCA system (ACEA Bioscience, San Diego, CA, USA). Upon

activation of GPCR-mediated signaling, the cell morphology changes

and therefore aﬀects the local ionic environment at the cell−electrode

interface. This leads to an increased electronic readout of cell-sensor

impedance (Z), which is displayed in real time as the cell index (CI).

Speciﬁcally, the CI value at each time point is deﬁned as (Z_i− Z₀)Ω/

15Ω, where Z_iis the impedance at each individual time point, and Z₀

is the impedance derived from the electrode/solution interface in the

absence of cells prior to the start of the experiment. HEK293hA_2AR

cells were cultured as a monolayer on 10 cm Ø culture plates to 80−

90% conﬂuency and subsequently harvested and centrifuged two

times at 1000 rpm for 5 min. Initially, 45 μL of culture media was

added to wells in E-plates 96 to obtain background readings (Z₀)

followed by the addition of 50 μL of cell suspension containing

20,000 cells/well. The E-plate containing the cells was left at room

temperature for 30 min before being placed on the recording device

station in the incubator at 37 °C in 7% CO₂. Afterward, cell

attachment, spreading, and proliferation were continuously monitored

every 15 min. The cells were cultured until the end of log phase

(∼18−20 h) to obtain an optimal assay window. Prior to agonist

application (either the reference compound CGS21680, 8, or newly

synthesized compounds), the interval between two measurements was

adjusted to 15 s. Subsequently, 5 μL of compound solution (ﬁnal

concentration of 0.1% DMSO) or vehicle control (0.1% DMSO) was

added to each well, after which the CI was recorded for 60 min.

Molecular Modeling. All preparations and simulations were

performed using Schrodinger Suite.⁵³The crystal structure of hA_2AAR

̈

in complex with compound 8 was obtained in-house (vide supra) and

used for the in silico experiment. All thermostabilizing mutations were

reverted to wild-type hA_2AAR, the bRIL fusion protein was removed

from the crystal structure, and the third intracellular loop (ICL3) was

ﬁlled in using Prime.^3,33,54Missing water molecules in the binding

pocket of the crystal structure were added from 4EIY.³The protein

was prepared by the “Protein Preparation Wizard” at default settings.

The compounds (1 and 3) were built and prepared using LigPrep.⁵⁵

Induced-ﬁt docking³⁴was performed ﬂexibly with the crystal ligand 8

as the center of workspace in the standard protocol and OPLS3 as the

force ﬁeld. Figures were rendered using PyMol.⁵⁶

Data Analysis. All experimental data were analyzed using the

nonlinear regression curve ﬁtting program GraphPad Prism 7.0

(GraphPad Software Inc., San Diego, CA). IC₅₀values obtained from

competition displacement binding data were converted into K_ivalues

using the Cheng−Prusoﬀ equation.⁵⁷The K_Dvalue of [³H]DPCPX

(1.6 nM) at CHOhA₁R membranes was taken from Kourounakis et

al.⁵⁸The K_Dvalue (1.0 nM) of [³H]ZM241385 at hA_2AR membranes,

the K_Dvalue (1.71 nM) of [³H]PSB-603 at CHO-spap-hA_2BR

membranes, and the K_Dvalue (17.3 nM) of [³H]PSB-11 at

CHOhA₃R membranes were taken from in-house determinations.

Eﬃcacy data from xCELLigence were obtained using RTCA software

1.2 (Roche, Germany) by normalizing CI traces to deﬁne peak

responses within 60 min after compound addition. Peak values were

used further for the determination of concentration−eﬀect curves,

yielding EC₅₀(concentration causing a half-maximum eﬀect) and E_max

(maximum eﬀect relative to CGS21680) values.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01856.

Additional ﬁgures of crystal structure and computational

models (Figures S1−S3 and S5), graph of displacement

curves (Figure S4), linear correlation plot (Figure S6),

3839

https://doi.org/10.1021/acs.jmedchem.0c01856

J. Med. Chem. 2021, 64, 3827−3842

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(PDB ID) 7ARO. Receptor models with compounds 1 and 3

are available as well. The authors will release the atomic

coordinates and experimental data upon article publication.

and HNMR and LCMS analyses of compounds 1−6

(PDF)

Molecular formula strings (CSV)

ACKNOWLEDGMENTS

■

AUTHOR INFORMATION

Corresponding Author

Adriaan P. IJzerman − Division of Drug Discovery and Safety,

Leiden Academic Centre for Drug Research, Leiden

University, 2333 CC Leiden, The Netherlands; orcid.org/

0000-0002-1182-2259; Phone: +31-71-5274651;

Email: ijzerman@lacdr.leidenuniv.nl

■

T.A. acknowledges ﬁnancial support by the Indonesia Endow-

ment Fund for Education (LPDP). Eelke B. Lenselink is

acknowledged for his help with the computational aspects of

the study.

ABBREVIATIONS USED

■

bRIL, thermostabilized apocytochrome b562RIL; GPCR, G

protein-coupled receptor; hAR, human adenosine receptor;

mCPBA, meta-chloroperoxybenzoic acid; RTCA, real-time cell

analysis; StaR, (thermo)stabilized receptor; TFA, triﬂuoro-

acetic acid

Authors

Tasia Amelia − Division of Drug Discovery and Safety, Leiden

Academic Centre for Drug Research, Leiden University, 2333

CC Leiden, The Netherlands; School of Pharmacy, Bandung

Institute of Technology, 40132 Bandung, Indonesia;

orcid.org/0000-0002-7414-9428

Jacobus P. D. van Veldhoven − Division of Drug Discovery

and Safety, Leiden Academic Centre for Drug Research,

Leiden University, 2333 CC Leiden, The Netherlands

Matteo Falsini − Division of Drug Discovery and Safety,

Leiden Academic Centre for Drug Research, Leiden

University, 2333 CC Leiden, The Netherlands

Rongfang Liu − Division of Drug Discovery and Safety, Leiden

Academic Centre for Drug Research, Leiden University, 2333

CC Leiden, The Netherlands

Laura H. Heitman − Division of Drug Discovery and Safety,

Leiden Academic Centre for Drug Research, Leiden

University, 2333 CC Leiden, The Netherlands; orcid.org/

0000-0003-3662-8177

Gerard J. P. van Westen − Division of Drug Discovery and

Safety, Leiden Academic Centre for Drug Research, Leiden

University, 2333 CC Leiden, The Netherlands; orcid.org/

0000-0003-0717-1817

Elena Segala − Sosei Heptares, Cambridge CB21 6DG, United

Kingdom

Grégory Verdon − Sosei Heptares, Cambridge CB21 6DG,

United Kingdom

Robert K. Y. Cheng − Sosei Heptares, Cambridge CB21 6DG,

United Kingdom

Robert M. Cooke − Sosei Heptares, Cambridge CB21 6DG,

United Kingdom

Daan van der Es − Division of Drug Discovery and Safety,

Leiden Academic Centre for Drug Research, Leiden

University, 2333 CC Leiden, The Netherlands

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Author Contributions

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Notes

The authors declare no competing ﬁnancial interest.

The 8−hA_2AAR complex structure coordinates and structure

factors are available via the Protein Data Bank, accession code

3840

https://doi.org/10.1021/acs.jmedchem.0c01856

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https://doi.org/10.1021/acs.jmedchem.0c01856

J. Med. Chem. 2021, 64, 3827−3842

Article Doi

Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A2AReceptor

DOI: 10.1021/acs.jmedchem.0c01856

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Article abstract of DOI:10.1021/acs.jmedchem.0c01856

Full text of DOI:10.1021/acs.jmedchem.0c01856

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