Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1Receptor in Living Cells

pubs.acs.org/jmc

Article

Development and Application of Subtype-Selective Fluorescent

Antagonists for the Study of the Human Adenosine A₁Receptor in

Living Cells

Eleonora Comeo, Phuc Trinh, Anh T. Nguyen, Cameron J. Nowell, Nicholas D. Kindon, Mark Soave,

Leigh A. Stoddart, Jonathan M. White, Stephen J. Hill, Barrie Kellam, Michelle L. Halls,*

Lauren T. May,* and Peter J. Scammells *

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c02067

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: The adenosine A₁receptor (A₁AR) is a G-protein-coupled receptor

(GPCR) that provides important therapeutic opportunities for a number of

conditions including congestive heart failure, tachycardia, and neuropathic pain. The

development of A₁AR-selective ﬂuorescent ligands will enhance our understanding

of the subcellular mechanisms underlying A₁AR pharmacology facilitating the

development of more eﬃcacious and selective therapies. Herein, we report the

design, synthesis, and application of a novel series of A₁AR-selective ﬂuorescent

probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized

8-(adamant-1-yl) xanthine scaﬀolds. These ﬂuorescent conjugates allowed

quantiﬁcation of kinetic and equilibrium ligand binding parameters using

NanoBRET and visualization of speciﬁc receptor distribution patterns in living

cells by confocal imaging and total internal reﬂection ﬂuorescence (TIRF)

microscopy. As such, the novel A₁AR-selective ﬂuorescent antagonists described

herein can be applied in conjunction with a series of ﬂuorescence-based techniques

to foster understanding of A₁AR molecular pharmacology and signaling in living cells.

developing eﬃcacious and safe A₁AR-selective therapeutics is

that this same biological target also mediates the onset of

undesirable side eﬀects including bradycardia, dyspnoea, and

seizures.¹⁴⁻¹⁶As such, the clinical application of A₁AR ligands

has been hindered by the diﬃculty in separating on-target

beneﬁcial eﬀects from adverse eﬀects, coupled to the

widespread expression of A₁AR in multiple tissues.¹⁴⁻¹⁶

Alternative therapeutic approaches could be exploited to

address the aforementioned issues, which include more

selective targeting through A₁AR-biased agonism and alloster-

ism.¹⁷⁻¹⁹The development of more selective and eﬃcacious

therapies, however, heavily depends on our understanding of

how A₁AR pharmacology diﬀers across tissues, cell types, and

subcellular compartments.²⁰⁻²³Fluorescent ligands represent

valuable tools to enable real-time monitoring of receptor

pharmacology, including ligand−receptor interactions, recep-

tor signaling, and traﬃcking in diﬀerent cell types, in both

INTRODUCTION

■

Adenosine is an important endogenous nucleoside, which

exerts its modulatory role upon binding to four adenosine

receptor (AR) subtypes, namely, A₁, A_2A, A_2B, and A₃.^1,2

Adenosine receptors are class A G-protein-coupled receptors

(GPCRs)³and, due to their widespread distribution

throughout the human body, are implicated in modulating

many biological processes.⁴The adenosine A₁receptor (A₁AR)

exerts its regulatory role mainly upon coupling with the G_i/o

family of heterotrimeric G-proteins, resulting in adenylyl

cyclase inhibition and a subsequent reduction of cAMP

accumulation.¹A₁ARs are highly expressed in the brain and,

to a lesser extent, in peripheral tissues such as the heart, kidney,

and lungs,^1,5and represent an attractive drug target for the

development of therapeutic agents for CNS, cardiovascular,

renal, and respiratory disorders.^4,6,7For instance, adenosine

itself is administered to patients with paroxysmal supra-

ventricular tachycardia (PSVT) to slow heart rate and

contractility upon activation of the A₁AR.^4,8A₁AR activation

can also decrease ischemia−reperfusion injury and neuropathic

pain.^4,9,10On the other hand, inhibition of A₁AR function may

oﬀer therapeutic opportunities for the development of

potassium sparing diuretics,^4,10,11anti-asthmatic agents,¹²and

cognitive enhancers.^7,13However, a principal drawback in

Received: November 27, 2020

American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

A

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 1. Structures of representative ﬂuorescent ligands available to probe the A₁AR. Fluorescent ligand binding aﬃnity^a,d,e(pK_Dand pK_i)^28,42,44

and potency^b,c(pEC₅₀)^25,45,46quantiﬁed in cell lines stably expressing the human A₁AR (hA₁AR), rat A₁AR (rA₁AR), or human A₃AR (hA₃AR). NL

denotes the presence of an N-terminal NLuc tag.

health and disease.²⁴⁻²⁹Importantly, ﬂuorescent ligands are

amenable to quantifying receptor pharmacology at the single-

cell level, in real-time, and in unmodiﬁed, endogenously

expressing systems.³⁰⁻³²The ﬂuorescent ligand toolbox for the

adenosine receptors has rapidly expanded over the past 15

years.³³⁻³⁶Notably, the Jacobson group functionalized the

hA_2AAR selective antagonist SCH442416 with AlexaFluor 488,

resulting in a series of selective ﬂuorescent ligands that have

been successfully applied for ﬂuorescence polarization

(MRS5346)³⁷and ﬂow cytometry (MRS7416)³⁸studies.

Additionally, we have recently synthesized a novel series of

hA_2AAR selective ﬂuorescent probes based on preladenant,

which allowed the successful monitoring of ligand binding and

visualization of the hA_2AAR in living cells.³⁹Conjugation of a

green-emitting 4,4-diﬂuoro-4-bora-3a,4a-diaza-s-indacene

(BODIPY) ﬂuorophore with a 8-phenyl-1-propylxanthine

scaﬀold yielded the selective hA_2BAR probe (PSB-12105),

which could monitor ligand binding at the hA_2BAR by ﬂow

cytometry.⁴⁰There are also examples of selective ﬂuorescent

ligands that have been successfully developed and applied to

probe the hA₃AR.⁴¹⁻⁴³

The ﬂuorescent ligand toolkit available to study the hA₁AR

(Figure 1) includes probes prepared from the endogenous

A₁AR agonist, adenosine, labeled with ﬂuorescein isothiocya-

nate (FITC) and nitrobenzoxadiazole (NBD) (FITC-ADAC

and NBD-ADAC)⁴⁷or BODIPY-X-630/650 (ABA-X-

BY630).⁴⁸Additionally, probes prepared from the full agonist

NECA (dansyl-NECA,⁴⁹ABEA-BY630,⁴⁸and ABEA-G-(D)-

Ala-(D)-Ala-X-BY630²⁵) and antagonists based on a xanthine

amine congener (XAC)⁵⁰(XAC-X-BY630)²⁴and its analogue

with a polyamide linker (CA200645) are also available.^28,51

These probes have enabled researchers to gain important

insights into A₁AR spatio-temporal organization^24,48,52and

signaling in living cells,⁴⁴yet the ability of these probes to bind

other adenosine receptors limits their use to recombinant

expression systems. Interrogating A₁AR pharmacology in more

physiologically relevant models requires that the ﬂuorescent

probe engages with the targeted A₁AR with high selectivity as

other closely related adenosine receptors are often coex-

pressed.^5,32,53Eﬀorts to develop A₁AR-selective probes have

been undertaken. For example, Singh et al. designed

ﬂuorescent ligands based on a (benzimidazolyl)isoquinolinol

scaﬀold. However, these probes did not show measurable

B

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. (A) Sectional view of the hA₁AR Connolly surface to show the binding pose adopted by the co-crystallized irreversible ligand DU172

(compound 23a in Beauglehole et al.)⁶⁰(shown in grey sticks) with the 3-(4-ﬂuorosulfonylbenzamido)propyl group directed toward the

extracellular medium. Molecular docking of (B) tonapofylline (BG9928), (C) compound 16, and (D) congener 42 to the crystal structure of the

̈

hA₁AR (PDB: 5UEN) performed with Schrodinger’s glide (Schrodinger release 2019-2). Tonapofylline is rendered in brick-red sticks, while

compound 16 is shown in sticks (colored yellow) and congener 42 is rendered in pink sticks. The hA₁AR is shown in ribbon (colored marine blue).

The key residues are shown in sticks (violet) and are labeled. Hydrogen bonds are shown as yellow-dashed lines and the transmembrane (TM)

helices 1−3, 5, and 6 are labeled for clarity alongside extracellular loop (ECL) 2 and ECL3.

binding aﬃnity to the A₁AR.⁵⁴Therefore, there still remains a

need for ﬂuorescent probes with marked subtype selectivity for

the A₁AR.

community to further the understanding of the role of the

A₁AR in health and disease.

RESULTS AND DISCUSSION

In the present study, we report the design, synthesis, and

pharmacological evaluation of a novel series of subtype-

selective ﬂuorescent antagonists targeting the adenosine A₁AR

receptor, which are amenable for use in a variety of

ﬂuorescence-based techniques. Two diﬀerent positions of the

prototypical xanthine scaﬀold were assessed for ﬂuorophore/

linker functionalization. To this end, the desired ﬂuorescent

antagonists were prepared by conjugating 8-bicylo[2.2.2]-

octylxanthine and 3-functionalized 8-(adamant-1-yl)xanthine

scaﬀolds with a series of commercially available far-red

ﬂuorophores. We envisage that these novel probes will be

valuable pharmacological tools for the wider scientiﬁc

■

Fluorescent Ligand Design. The availability of crystal

structures for the hA₁AR^55,56allowed us to rationalize the

reported structure−activity relationships (SARs) relative to

xanthine-based A₁AR antagonists,^7,11,57,58thereby helping

guide our design strategy. We sought to explore two diﬀerent

positions of the prototypical xanthine scaﬀold, namely, the 3-

and 8-positions, for linker/ﬂuorophore attachment as struc-

tural data suggested that functionalization of either of these

positions could be harnessed for the development of chemical

probes (Figure 2). Tonapofylline (BG9928)^15,59and its

synthetic precursor, compound 16 (from Kiesman et al.)⁵⁹

with a carboxylate handle directly attached to the

C

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 1. Synthesis of the 8-Bicyclo[2.2.2]octylxanthine-Based Congeners

a

Reagents and conditions: (a) N-Boc-ethylenediamine, COMU, DIPEA, DMF, rt., 1 h, 58%; (b) 4 N HCl in dioxane, rt., 30 min, quantitative; (c)

N-Boc-8-amino-3,6-dioxaoctanoic acid or N-Boc-5-amino-3-oxapentanoic acid or N-Boc-β-alanine, COMU, DIPEA, rt., 1 h, 28−67%; (d) N-Boc-

β-alanine, COMU, DIPEA, rt., 1 h; (e) N¹-Boc-ethylenediamine or N-Boc-1,3-propanediamine, BOP, DIPEA, DMF, rt., 1 h, 78−86%; (f) β-alanine

methyl ester, BOP, DIPEA, DMF, rt., 56%; (g) LiOH·H₂O, MeOH/H₂O 1:1, rt., 40 min, 84%; (h) N-Boc-5-amino-3-oxapentanoic acid, COMU,

DIPEA, rt., 1 h.

bicyclo[2.2.2]octane group (Figure 2B,C), were selected as

parent molecules for the development of 8-position-function-

alized xanthine-based ﬂuorescent ligands. These two molecules

exhibit high binding aﬃnities for the hA₁AR (K_i= 7.4 nM and

K_i= 33 nM for tonapofylline and compound 16, respectively)

and good selectivity proﬁles with respect to binding other

adenosine receptor subtypes (hA_2AAR K_i= 6410 nM, hA_2BAR

K_i= 90 nM, hA₃AR K_i> 10,000 nM for tonapofylline, and

hA_2AAR K_i= 1070 nM, hA_2BAR (48%)^b, hA₃AR (100%)^b(^b: %

of speciﬁc radioligand binding) for compound 16).⁵⁹Addi-

tionally, the presence of a carboxylate group in both molecules

represented an attractive functional handle for subsequent

attachment of a linker and ﬂuorophore.

Molecular docking studies of tonapofylline and compound

16 to the 3.2 Å resolution crystal structure (PDB: 5UEN)⁵⁵of

the hA₁AR revealed that both ligands exhibited comparable

binding modes, with subtle diﬀerences in orientation of the 1-

and 3-propyl groups for each ligand (Figure 2B−D). The

pattern of key interactions established with the orthosteric site

of the receptor was in keeping with those observed for the

A₁AR crystal structure co-bound with xanthine-based ana-

logues, including hydrogen-bonding interactions with N254^6.55

and π-stacking interaction with F171^ECL2(ECL2 in super-

scripts refer to the Ballesteros−Weinstein numbering sys-

tem).^61,55,56For both molecules, the carboxylate group was

oriented toward the extracellular face of the A₁AR without

making direct interactions with neighboring residues. Struc-

ture−activity relationships (SAR) reported by Kiesman et al.,⁵⁹

suggested that receptor aﬃnity was maintained when the

carboxylate group was functionalized with amide linkers,

thereby further supporting our docking results. Consequently,

we set out to probe the area beyond the carboxylate group

using linkers of diﬀerent lengths and chemical compositions

with the aim to ﬁnd the optimal congener template upon

which to append a series of commercially available

ﬂuorophores.

In addition, we generated a second library of probes

featuring 3-functionalized xanthine-based ﬂuorescent ligands.

This second library displayed linkers with reduced length

compared to the ﬁrst library as structural data, complemented

with docking studies, suggested that shorter linkers would

provide suﬃcient spatial separation between the pharmaco-

phore and ﬂuorophore (Figure 2D). It was also of interest to

explore probes lacking the linker in which the ﬂuorophores

were directly conjugated to the orthosteric binding moiety as

previous studies from our laboratories suggested that such

modiﬁcation was advantageous for the development of A₁AR

ﬂuorescent probes.⁶²

The novel series of ﬂuorescent ligands reported here

included far-red emitting probes tethered with the 6-(((4,4-

diﬂuoro-5-(2-thien-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)-

styryloxy)acetyl)aminohexanoic acid (BODIPY630/650-X)

dye and the water-soluble sulfonated cyanine5 (Sulfo-Cy5)

dye. Far-red emitting ﬂuorophores were preferred as these

ﬂuorophores emit at wavelengths of 650−670 nm, where the

degree of overlap with background autoﬂuorescence from

living cells is minimized.⁶³Previous studies have demonstrated

that conjugation of the xanthine amine congener (XAC) with

green-emitting ﬂuorophores, such as 4,4-diﬂuoro-5,7-dimethyl-

4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL)

dye, generated probes with markedly reduced aﬃnity for the

hA₁AR and disadvantageous imaging properties due to

signiﬁcant membrane penetration.⁶⁴For these reasons,

BODIPY-FL ﬂuorophores were not included in the present

study. BODIPY 630/650 ﬂuorophores are amongst the

preferred candidates for ﬂuorescence derivatization due to

their advantageous spectral properties, including high

ﬂuorescence quantum yield (φ) and photochemical stabil-

ity.^65,66Moreover, BODIPY 630/650 ﬂuorophores are brighter

when located in lipid environments, such as the plasma

membrane, which is particularly advantageous when visualizing

speciﬁc cell surface ﬂuorescence in imaging studies.^45,64,67The

application of BODIPY dyes, however, may be limited by their

D

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 2. Synthesis of the 1-Propyl-3-(3-benzamidopropyl)-8-(3-adamantyl) Xanthine-Based Congeners

a

Reagents and conditions: (a) Na₂S₂O₄, 30% NH₄OH in H₂O, 85 °C, 92%; (b) (i) 1-adamantane carboxylic acid, COMU, DIPEA, DMF, rt., 15

+

−

min, 87% (ii) 1 M KOH/n-propanol 1:1, reﬂux, 1 h, 95%; (c) SEM-Cl, K₂CO₃, DMF, 4 h, 66%; (d) NH₄HCO₂, Pd/C, MeOH, 140 °C, 9 h,

67%; (e) N¹-Boc ethylenediamine, COMU, DIPEA, DMF, rt., 20 min, 75%; (f) 1 M NaOH, THF, rt., 3 h, 90%; (g) 3-bromopropylamine

hydrobromide, COMU, DIPEA, DMF, rt., 15 min, 61%; (h) 35 and 39, K₂CO₃, DMF, 25−40 °C, 24 h, 49%; (i) 4 M HCl in dioxane, rt., 30 min,

quantitative, then DIPEA; (j) Boc-β-Ala-OH, COMU, DIPEA, DMF, rt., 15 min, 47%.

intrinsic lipophilicity, which could lead to increased levels of

nonspeciﬁc binding⁶⁸and reduced stability in plasma. This

would thereby limit their utility in more physiologically

relevant models such as in vivo models.³⁰Fluorescent dyes

with improved water solubility, such as sulfonated cyanine5

dyes, may overcome these limitations as demonstrated by the

Cy5-labeled quinazoline-based probe developed by Ma et al.,

for the α₁-adrenoreceptor.⁶⁹In addition, to the best of our

knowledge, there are no reported examples of ﬂuorescent

hA₁AR antagonists integrating a Sulfo-Cy5 ﬂuorophore that

have been successfully utilized in studies of the hA₁AR in living

cells.

Chemistry: Synthesis of the 8-Bicyclo[2.2.2]-

octylxanthine-Based Congeners. The chemical synthesis

of the 8-bicyclo[2.2.2]octylxanthine-based amino-function-

alized congeners is outlined in Scheme 1. Tonapofylline

(BG9928) and 16 provided the framework for the develop-

ment of the series of ﬂuorescent ligands featuring linkers and

ﬂuorophores extending from the 8-position of the xanthine

scaﬀold. Tonapofylline (BG9928) and 16 were synthesized as

described in Keisman et al.⁵⁹(detailed procedures are reported

in the Supporting Information) and further functionalized with

a series of linker variants of diﬀerent lengths and chemical

compositions to aﬀord 10 ﬁnal N-Boc-protected amino-

functionalized congeners. Brieﬂy, Tonapofylline (BG9928)

w a s a c t i v a t e d w i t h ( 1 - c y a n o - 2 - e t h o x y - 2 -

oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexaﬂuorophosphate (COMU) in the presence of DIPEA and

subsequently coupled with N-Boc-enthylenediamine in DMF

to aﬀord the corresponding monoprotected amine intermedi-

ate (10). The acidolytic removal of the Boc-protecting group

aﬀorded the corresponding amine (11) as its HCl salt, which

was subsequently functionalized in the presence of DIPEA and

COMU with three linker variants of diﬀerent lengths and

chemical compositions: Boc-8-amino-3,6-dioxaoctanoic acid,

Boc-5-amino-3-oxapentanoic acid, and Boc-β-alanine to aﬀord

12, 13, and 14, respectively (Scheme 2).

Compound 16 was coupled with either N-Boc-ethylenedi-

amine or N-Boc-1,3-propanediamine to aﬀord the correspond-

ing monoprotected intermediates 17a and 17b, respectively.

Following acidolysis, N-Boc deprotection gave the correspond-

ing primary amines (18a and 18b) as their HCl salts. COMU-

mediated amide coupling of 18a and 18b with the N-protected

linkers Boc-8-amino-3,6-dioxaoctanoic acid, Boc-5-amino-3-

oxapentanoic acid, or Boc-β-alanine aﬀorded the Boc-

protected congeners 19−24. An additional chain-extended

congener was also synthesized to investigate the eﬀect of this

longer linker variant in modulating A₁AR binding aﬃnity. To

this end, 16 was activated with (benzotriazol-1-yloxy)tris-

(dimethylamino)phosphonium hexaﬂuorophosphate (BOP)

and coupled with β-alanine methyl ester to aﬀord 26.

Saponiﬁcation of the latter and subsequent amide coupling

with N-Boc-ethylenediamine yielded the corresponding Boc-

protected intermediate (28). Treatment of 28 with 4 M HCl in

E

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of the Fluorescent A₁AR Antagonists

a

Reagents and conditions: (i) 4 M HCl in dioxane, rt., 30 min, quantitative; (ii) BY630/650-X-NHS or Sulfo-Cy5-NHS, DIPEA, DMF, rt.,

exclusion of light, 39−100%.

dioxane aﬀorded the corresponding amine (29) as its HCl salt,

which was coupled with Boc-5-amino-3-oxapentanoic acid to

aﬀord the desired protected congener 30.

genation conditions using ammonium formate in the presence

of 10% palladium on carbon (Pd/C) in methanol at high

temperature,⁷⁴aﬀording the corresponding deprotected

xanthine 35 in good yield (67%). The aryl amide linker

variant was prepared starting from commercially available

monomethyl terephthalate (31), which was coupled with N-

Boc ethyelenediamine by means of COMU, in the presence of

DIPEA in DMF, aﬀording intermediate 37. The latter was then

treated with 1 M NaOH to generate the corresponding

carboxylic acid (38), which was subsequently coupled with 3-

bromopropylamine hydrobromide in the presence of DIPEA,

aﬀording intermediate 39 with good yield (61%). Alkylation of

the xanthine derivative 35 with 39 under basic conditions

yielded the corresponding 3-N-propyl-3-benzamide intermedi-

ate (40) in reasonable yield (49%). Concomitant SEM and

Boc removal was eﬀected by reacting 40 with 4 M HCl in

dioxane for 1 h, followed by treatment of the resulting HCl salt

with DIPEA to liberate the corresponding amine (41), which

was coupled with Boc-β-Ala-OH using COMU in DMF to

yield the ﬁnal Boc-protected congener (42).

Syntheses of the 1-Propyl-3-(3-benzamidopropyl)-8-

(3-adamantyl) Xanthine-Based Congeners. The synthetic

approach for the preparation of 3-functionalized analogues is

outlined in Scheme 2 and was adapted from procedures

reported by Beauglehole et al.⁶⁰6-Amino-1-benzyl-5-nitroso-3-

propylpyrimidine-2,4(1H,3H)-dione (31) was previously pre-

pared in our laboratory following reported literature

procedures.⁷⁰⁻⁷²Reduction of the nitroso group of 31 with

sodium dithionite aﬀorded the corresponding 5,6-diamino

uracil product (32), which was subsequently acylated with 1-

adamantane carboxylic acid in the presence of COMU⁷³to

aﬀord the corresponding 6-amino-5-carboxamido uracil

precursor. The latter then underwent cyclization in the

presence of 1 M KOH to the corresponding 8-adamantyl-

substituted xanthine (33) in excellent yield (95%). To

ultimately allow a selective alkylation of the unsubstituted 3-

position of the xanthine ring following its debenzylation, a 2-

(trimethylsilyl)ethoxymethyl (SEM) protecting group was

introduced at the 7-position to aﬀord the corresponding 7-

N-SEM-protected intermediate (34) in good yield (66%).

Subsequent debenzylation was achieved under transfer hydro-

Synthesis of Fluorescent A₁AR Antagonists. The 8-

bicyclo[2.2.2]octylxanthine-based congeners 18a and 25a and

the 8-adamantyl-3-propyl-3-benzamide-substituted congeners

41 and 42 were subsequently conjugated with a series of

F

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. Binding Aﬃnities of the Novel 8-Bicyclo[2.2.2]octylxanthine-Based Amino-Functionalized Congeners

a

pK_iSEM values were calculated from the negative logarithm of the equilibrium inhibitory constant (K_iin nM) determined at 4 °C using [³H]

DPCPX competition binding experiments in FlpIn-CHO cells stably expressing the hA₁AR. Values represent the mean

SEM from n = 3

experiments performed in duplicate. ** P < 0.01. compared to congeners lacking the dipeptide linker one-way ANOVA with Tukey’s multiple

comparison test.

commercially available far-red emitting ﬂuorophores (Scheme

3), namely, BODIPY630/650-X (BY630/650-X) and sulfo-

nated cyanine5 (Sulfo-Cy5) dyes, thereby yielding a focused

library of eight ﬂuorescent analogues (43a and 43b to 46a and

46b). The novel synthesized ﬂuorescent probes were puriﬁed

by reverse-phase high-performance liquid chromatography

(RP-HPLC), and the high purity of the ﬁnal probes was

conﬁrmed by analytical RP-HPLC with dual-wavelength

detection and measured as ≥96% homogeneity. Furthermore,

chemical identity of the ﬁnal probes was conﬁrmed by high-

resolution mass spectroscopy (HRMS) (TOF ES+ and ES−).

Pharmacology. To assess which linker functionality could

be harnessed for the development of the ﬁrst library of

ﬂuorescent probes, the 10 newly synthesized Boc-protected 8-

bicyclo[2.2.2]octylxanthine congeners 12, 13, 15, 19, 20, 22,

23, 25a, 25b, and 30 (Scheme 1) were characterized using

[³H] DPCPX binding in FlpIn-CHO cells stably expressing the

hA₁AR (A₁AR-FlpInCHO cells). All congeners were able to

bind the hA₁AR with good aﬃnity (7.65−8.36 pK_irange, Table

1), demonstrating that all linker combinations were well

tolerated. Comparison of the reported binding aﬃnities⁵⁹of

tonapofylline (BG9928) and 16 with our newly synthesized

congeners corroborated the original observation that function-

alization of the carboxylate handle of either tonapofylline or 16

could be exploited for linker conjugation. Despite the similar

aﬃnity of these ligands, subtle diﬀerences in SARs were

observed. There was a general trend for congeners integrating

the β-Ala-β-Ala linker moiety to display higher hA₁AR aﬃnities

relative to congeners lacking the dipeptide linker (15, 25a, and

25b; Table 1. ** P < 0.01 one-way ANOVA with the Tukey’s

multiple comparison test). Likewise, congeners with an

ethylenediamine spacer seemed to be better tolerated than

those with a propane-1,3-diamine. Based on these initial

results, compound 25a (Table 1 and Scheme 1) was therefore

selected as our model-functionalized congener for the

development of the 8-bicyclo[2.2.2]octylxanthine-based ﬂuo-

G

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

rescent ligands. Given the greater A₁AR aﬃnity observed with

congeners conjugated with β-alanine, we also used the β-

alanine linker for the development of the 1-propyl-3-(3-

benzamidopropyl)-8-(3-noradamantyl)xanthine-based ﬂuores-

cent ligands. Furthermore, structural data suggested that a

single β-alanine linker, coupled with an ethylenediamine

spacer, should provide the optimal linker length for

conjugating the 1-propyl-3-(3-benzamidopropyl)-8-(3-norada-

mantyl) xanthine-based scaﬀold with the desired ﬂuorophore.

The novel-synthesized 8-bicyclo[2.2.2]octylxanthine- and 1-

propyl-3-(3-benzamidopropyl)-8-(3-noradamantyl) xanthine-

based ﬂuorescent ligands (Scheme 3) were characterized in a

variety of pharmacological assays. First, the ﬂuorescent ligand

binding was quantiﬁed using [³H] DPCPX competition

binding in A₁AR-FlpInCHO cells (Figure 3 and Table 2).

Table 2. Binding Aﬃnities of the Novel 8-

Bicyclo[2.2.2]octylxanthine-Based (43a, 43b, 44a, and 44b)

and 1-Propyl-3-(3-benzamidopropyl)-8-(3-noradamantyl)

Xanthine-Based (45a, 45b, 46a, and 46b) Fluorescent

Antagonists

a

cmpd

linker

ﬂuorophore

pK_i(Log M) SEM

SLV320

43a

43b

44a

44b

45a

45b

46a

8.77 0.05

7.69 0.06^####

6.07 0.09

9.54 0.05****

6.70 0.06

8.23 0.07

<5

BY630/650-X

Sulfo-Cy5

BY630/650-X

Sulfo-Cy5

BY630/650-X

Sulfo-Cy5

BY630/650-X

Sulfo-Cy5

β-Ala-β-Ala

β-Ala

8.01 0.03

<5

46b

a

pK_ivalues were calculated from the negative logarithm of the

equilibrium inhibitory constant (K_iin nM) measured at 37 °C by

radioligand competition binding employing [³H] DPCPX as the

radiotracer in FlpIn-CHO cells stably expressing the hA₁AR. Values

represent the mean

triplicate (statistical signiﬁcance was deﬁned as ****p < 0.0001 one-

way ANOVA with the Tukey’s multiple comparison test comparing

the pK_iof 44a vs 44b.

Tukey’s multiple comparison test comparing the pK_iof 44a vs 43a).

SEM from n = 5 experiments performed in

Figure 3. Inhibition of [³H] DPCPX speciﬁc binding (1 nM) in the

presence of increasing concentrations of ﬂuorescent antagonist (43a

and 43b to 46a and 46b) at 37 °C in hA₁AR-FlpInCHO cells. TB

stands for total radioligand binding. Each data point represents the

####

p < 0.0001 one-way ANOVA with the

combined mean

triplicate.

SEM from n = 5 experiments performed in

This suggests that there may be a synergistic eﬀect of both

linker and ﬂuorophore in inﬂuencing the binding aﬃnity of the

ﬁnal ﬂuorescent conjugates. The introduction of the Sulfo-Cy5

ﬂuorophore to the 1-propyl-3-(3-benzamidopropyl)-8-(3-nor-

adamantyl) xanthine scaﬀold (probes 45b and 46b) eﬀectively

abolished hA₁AR aﬃnity (pK_i< 5). In contrast, conjugation

with BY630/650-X ﬂuorophore yielded ﬂuorescent analogues

45a and 46a with good hA₁AR aﬃnity (pK_i= 8.23 0.07 and

pK_i= 8.01 0.03, respectively). These results supported our

original hypothesis, whereby ﬂuorophore derivatization could

be achieved at both the 8- and 3-positions of the prototypical

xanthine scaﬀold.

There was a broad variability of the binding aﬃnities

measured across the novel ﬂuorescent ligands synthesized

(Table 2). These results are consistent with previous studies,

which demonstrated a signiﬁcant contribution of the

ﬂuorophore in modulating the pharmacology of the ﬁnal

conjugates.^39,41,64Indeed, there was a statistical diﬀerence for

the BY630/650-X-labeled compounds (43a−46a) to display

the highest aﬃnities of the series compared to the

corresponding Sulfo-Cy5-labeled analogues (43b−46b)

(Table 2). Overall, conjugation with the Sulfo-Cy5 probe

was detrimental with regard to binding to the hA₁AR.

Fluorescent analogue 44a displayed the highest aﬃnity of the

series at the hA₁AR (pK_i= 9.54 0.05). The addition of the

BY630/650-X ﬂuorophore resulted in a circa 16-fold increase

of hA₁AR aﬃnity compared to the Boc-protected congener

25a. Replacement of the BY630/650-X ﬂuorophore with Sulfo-

Cy5 yielded ﬂuorescent antagonist 44b (pK_i= 6.70 0.06),

which resulted in a signiﬁcant reduction in hA₁AR binding

aﬃnity (690-fold) compared to the BY630/650-X-labeled

analogue. The ﬂuorophore moiety alone, however, was not the

only driver of hA₁AR aﬃnity as demonstrated by compounds

43a and 43b, which had lower aﬃnity compared to 44a and

44b. These two ﬂuorescent analogues lacked the dipeptide β-

Ala-β-Ala linker and were synthesized to explore the eﬀect of

moving the ﬂuorophore closer to the 8-bicyclo[2.2.2]-

octylxanthine scaﬀold. Despite the reasonable hA₁AR aﬃnity

displayed by compounds 43a and 43b, the removal of the

dipeptide β-Ala-β-Ala linker proved to be disadvantageous.

The SAR of these novel ﬂuorescent ligands could be

rationalized in the context of A₁AR structural information. For

example, crystal structures for the hA₁AR^55,56,75revealed the

presence of a secondary hydrophobic pocket deﬁned by

residues F171^ECL2, E170^ECL2,V83^3.28, I69^2.64, and L65^2.60

.

Molecular dynamics simulation (MD) studies suggest that

this pocket provides an important recognition mechanism for

ligand binding, with A₁AR ligand aﬃnity and selectivity being

inﬂuenced by their ability to engage with this accessory

hydrophobic site.^76,77The importance of hydrophobic pockets

in inﬂuencing ligand binding has been suggested for various

GPCRs, including the A_2AAR and the C−C chemokine

receptor type 5 (CCR5).^78,79Accordingly, the lipophilic nature

of the BODIPY probes may enable the boron dipyrromethene

scaﬀold to establish favorable interactions with this accessory

hydrophobic site with concomitant displacement of weakly

associated water molecules from this pocket, thereby further

H

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3. Binding Aﬃnities of Fluorescent 8-Bicyclo[2.2.2]octylxanthine-Based Conjugates 44a and 44b and Fluorescent 1-

Propyl-3-(3-benzamidopropyl)-8-(3-noradamantyl) Xanthine-Based Conjugate 46a at NanoLuc-hA₁AR, NanoLuc-hA_2AAR,

NanoLuc-hA_2BAR, and NanoLuc-hA₃AR

cmpd

binding

NLuc-hA₁AR

NLuc-hA_2AAR

7.55 0.09

NLuc-hA_2BAR

8.11 0.08

NLuc-hA₃AR

6.87 0.04

a

44a

pK_D(Log M)

B_max

pK_D(Log M)

B_max

pK_D(Log M)

B_max

8.93 0.02

0.018 0.002

8.50 0.07

0.019 0.0005

7.35 0.09

0.035 0.001

7.30 0.15

0.003 0.0003****

<6

0.016 0.003

7.26 0.08

0.004 0.0004****

<6

0.012 0.001

7.23 0.09

0.001 0.00007****

<6

46a

44b

b

0.030 0.006

N.D.

a

pK_Dvalues were calculated from the negative logarithm of the equilibrium dissociation constant (K_D) measured at 37 °C by NanoBRET

saturation binding using increasing concentration of the ﬂuorescent ligand in the absence or presence (to deﬁne nonspeciﬁc binding) of selective

unlabeled AR antagonist (1 μM SLV320 for NanoLuc-A₁AR, 1 μM ZM241385 for NanoLuc-A_2AAR, 1 μM PSB603 for NanoLuc-A_2BAR, and 1 μM

MRS1220 for NanoLuc-A₃AR). Data are represented as mean SEM of n = 3 experiments, where each experiment was performed in duplicate.

b

N.D. = not determined. **** P < 0.0001. compared to NanoLucA₁B_max, one-way ANOVA with the Tukey’s multiple comparison test.

Figure 4. NanoBRET saturation ligand binding curves (a)−(c) measured in HEK293A cells transiently expressing the NanoLuc-hA₁AR for (a)

44a, (b) 46a, and (c) 44b in the presence (open circles) and absence (closed circles) of 1 μM SLV320. Graphs (d)−(f) depict the selectivity

proﬁles of the probes tested against each subtype of adenosine receptors. Speciﬁc binding (d)−(f) of (d) 44a, (e) 46a, and (f) 44b at each subtype

of adenosine receptor was measured from saturation binding curves (a)−(c) after subtraction of the nonspeciﬁc binding component from the total

binding. Nonspeciﬁc binding was determined in the presence of 1 μM of ZM241385 for NanoLuc-hA_2AAR, 1 μM PSB603 for NanoLuc-hA_2BAR,

and 1 μM MRS1220 for NanoLuc-hA₃AR. Data points represent the combined mean SEM from n = 3 experiments performed in duplicate.

enhancing the overall free energy of binding for the probe

bound to the A₁AR. In contrast, molecular interactions of this

type could not be established with the corresponding water-

soluble Sulfo-Cy5 analogues as, due to their hydrophilic nature,

the interaction with the solvent could be more favorable.

One of the principal objectives of this study was to develop

subtype-selective probes that can selectively bind A₁ARs in

cells where other adenosine receptors are co-expressed. To this

end, the selectivity proﬁle of the newly synthesized probes was

quantiﬁed using a bioluminescent resonance energy transfer

(BRET)^29,80,81ligand binding assay, a proximity assay (<10

nm), which relies upon the energy transfer between a donor

bioluminescent protein (NanoLuc)⁸²tagged on the N-

terminus of the receptor of interest (NanoLuc-hA₁AR), and

an acceptor ﬂuorophore. The binding aﬃnity of the most

promising ﬂuorescent ligands of both BODIPY- and Sulfo-

Cy5-bearing probes (44a, 44b, and 46a; Scheme 3), selected

based on [³H]DPCPX binding results (Table 2), was

quantiﬁed at each adenosine receptor subtype using Nano-

BRET saturation ligand binding assay in HEK293A cells

transiently expressing either the NanoLuc-A₁AR, NanoLuc-

A_2AAR, NanoLuc-A_2BAR, or the NanoLuc-A₃AR. A robust,

speciﬁc, and saturable BRET signal was detected at the

NanoLuc-A₁AR for each ﬂuorescent analogue investigated

(44a, 44b, and 46a), with low levels of nonspeciﬁc binding for

all concentrations tested. Fluorescent analogue 44b showed

reasonable NanoLuc-hA₁AR aﬃnity (pK_D= 7.35 0.09, n =

3) and the most promising selectivity proﬁle among the

ﬂuorescent probes tested in the NanoBRET assay (Table 3 and

Figure 4f). Indeed, minimal BRET signal was detected at either

NanoLuc-hA_2AAR, NanoLuc-hA_2BAR, or NanoLuc-hA₃AR at

500 nM-1 μM 44b (Figure 4f). A concentration-dependent

BRET signal was observed for ﬂuorescent analogues 44a and

46a at the NanoLuc-hA_2AAR, NanoLuc-hA_2BAR, and Nano-

Luc-hA₃AR (Figure 4e), allowing estimation of their

equilibrium dissociation constant (K_D) (Table 3). 44a

I

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 5. Eﬀects of antagonists 44a (a), 46a (b), and 44b (c) on the NECA-mediated inhibition of forskolin-stimulated cAMP accumulation.

hA₁AR-FIpInCHO cells were stimulated with increasing concentrations of NECA in the absence and presence of increasing concentrations of the

ﬂuorescent antagonist. Data have been presented as percentage of cAMP accumulation in response to 3 μM forskolin (0%) or buﬀer (100%) alone.

Each data point represents the combined mean SEM from n separate experiments (n = 3 for 44a, n = 3 for 46a, and n = 4 for 44b) performed in

duplicate.

exhibited high aﬃnity for the NanoLuc-hA₁AR (pK_D= 8.93

0.02, n = 3), which was 25-fold, 7-fold, and 117-fold greater

than its aﬃnity for the NanoLuc-hA_2AAR, NanoLuc-hA_2BAR,

and NanoLuc-hA₃AR, respectively. Fluorescent analogue 46a

rightward shift of the NECA concentration response curve

only at the highest concentration tested (1 μM) (Figure S9),

with no detectable eﬀect at the hA₃AR. Therefore, the

estimated aﬃnity of 46a for each of the adenosine receptor

subtypes, quantiﬁed using the Gaddum equation as previously

described, was relatively low (pK_DhA_2AAR = 6.11 0.08, pK_D

hA_2BAR = 6.16 0.07, pK_DhA₃AR <6, n = 3)^41,88compared

to that observed from the NanoBRET assay (Figure 4 and

Table 3). Therefore, the small BRET signal detected with 46a

in HEK293 cells expressing NanoLuc-hA_2AAR and NanoLuc-

hA_2BAR may reﬂect BRET between 46a bound to endogenous

hA₁ARs and the transfected NanoLuc-tagged A₂AR receptors

as a result of oligomerization. The lower binding aﬃnity

obtained for 46a in cells expressing NanoLuc-hA_2AAR and

NanoLuc-hA_2BAR, compared to NanoLuc-hA₁AR, would be

consistent with negative allosterism across oligomeric receptor

interfaces.⁸⁹

The ability of probes 44a, 46a, and 44b (Scheme 3) to

retain functional antagonism at the hA₁AR was also

investigated. This was performed in FlpIn-CHO cells stably

expressing the hA₁AR. All three ﬂuorescent probes proved to

be competitive antagonists at the A₁AR, causing a parallel

rightward shift of the NECA response curve in a concen-

tration-dependent manner (Figure 5). From this data, we

performed Schild regression analysis and the pK_Bvalue for

each of the compounds tested was calculated (Table 4). All

three ﬂuorescent ligands 44a, 46a, and 44b retained a

functional antagonism activities at the hA₁AR comparable to

the parent Tonapofylline⁵⁹and DU172.⁵⁵Moreover, the

functional aﬃnity values (pK_B) measured for each of the

ﬂuorescent ligands were in agreement with the binding

also displayed high aﬃnity at the NanoLuc-hA₁AR (pK_D

=

8.50 0.07, n = 3), while the aﬃnities measured at the other

adenosine receptor subtypes were appreciably lower, displaying

approximately 20-fold greater aﬃnity for the NanoLuc-hA₁AR

compared to the NanoLuc-hA_2AAR, NanoLuc-hA_2BAR, and

NanoLuc-hA₃AR.

The maximal binding capacity (B_max) value of probe 46a was

signiﬁcantly higher at the NanoLuc-hA₁AR compared to

binding to either of the NanoLuc-hA_2AAR, NanoLuc-

hA_2BAR, and NanoLuc-hA₃AR receptor subtypes (maximum

BRET ratios of 0.003, 0.004, and 0.001, respectively) (Table

3). They were also substantially diﬀerent to the B_maxvalues

obtained at the NanoLuc-hA_2AAR, NanoLuc-hA_2BAR, and

NanoLuc-hA₃AR receptor subtypes with 44a (0.035, 0.016,

and 0.012, respectively). These ﬁndings are unlikely to reﬂect a

lack of receptor expression for A_2AAR, A_2BAR, and A₃AR when

assessing 46a, as these assays were performed in parallel with

44a, which exhibited robust expression for all adenosine

receptor subtypes. We therefore hypothesized that the BRET

signal resulting from probe 46a binding to the NanoLuc-

hA_2AAR, NanoLuc-hA_2BAR, and NanoLuc-hA₃AR could be a

result of the so-called “bystander” BRET,⁸³⁻⁸⁵that is, non-

speciﬁc enrichment of probe 46a at the plasma membrane of

cells expressing hA_2AAR, hA_2BAR, or hA₃AR receptors could

produce a measurable BRET signal owing to the close

proximity between the probe and bioluminescent protein

NanoLuc. Alternatively, HEK293 cells are known to express

hA₁AR at very low levels^86,87and it is therefore possible that

the low BRET signal detected with 46a at the NanoLuc-

hA_2AAR and NanoLuc-hA_2BAR is a result of close proximity

between the endogenous hA₁AR and NanoLuc-hA_2AAR or

NanoLuc-hA_2BAR receptors (such as might occur in

oligomeric complexes).

Table 4. pK_BValues of Fluorescent Analogues 44a, 46a, and

44b

a

cmpd

hA₁AR pK_B

n

44a (BY630/650)

46a (BY630/650)

44b (Sulfo-Cy5)

9.75 0.11

8.72 0.02

7.12 0.12

3

4

To further assess its subtype selectivity, probe 46a was

therefore evaluated for its ability to antagonize NECA-

mediated cAMP accumulation in FlpIn-CHO cells expressing

either of hA_2AAR, hA_2BAR, or hA₃AR. If the aﬃnity of 46a for

the other AR subtypes measured in the NanoBRET saturation

assay was “true aﬃnity”, one would expect to measure

comparable aﬃnity values in this functional assay. At the

hA_2AAR and hA_2BAR, 46a caused a small but signiﬁcant

a

Aﬃnities of the ﬂuorescently-labeled antagonists 44a, 46a, and 44b

measured from the antagonism of NECA-mediated inhibition of

forskolin-stimulated cAMP accumulation in hA₁AR-FIpInCHO cells.

Schild slopes were 1.14, 1.22, and 0.89 for 44a, 46a, and 44b,

respectively. Data represent mean value

SEM of n separate

experiments, where each experiment was performed in duplicate.

J

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 6. NanoBRET association binding kinetic curves of speciﬁc 44a (a), 46a (b), and 44b (c) binding to NanoLuc-hA₁AR at 37 °C. HEK293A

cells transiently expressing the NanoLuc-hA₁AR were treated with the indicated concentration of the ﬂuorescent ligand, and the NanoBRET signal

was monitored every 30 s for 1 h. Nonspeciﬁc binding was determined in the presence of 1 μM SLV320. Each data point represents the combined

mean value SEM of n = 3 separate experiments performed in triplicate.

a

Table 5. Kinetic Parameters of Fluorescent Analogues 44a, 44b, and 46a Binding to the NanoLuc-hA₁AR

cmpd

k_on(× 10⁶M⁻¹min⁻¹

)

k_off(min⁻¹

)

RT (1/k_off) (min)

kinetic pK_D(Log M)

saturation pK_D(Log M)

44a

44b

46a

17.90 3.69

0.86 0.13

3.60 0.90

0.054 0.009

0.117 0.016

0.046 0.008

19.7 3.7

9.0 1.0

23.8 3.9

8.51 0.18

6.86 0.03*

7.88 0.04*

8.93 0.02

7.35 0.09

8.50 0.07

a

The kinetic parameters k_on, k_off, and pK_Dwere calculated by monitoring the speciﬁc BRET signal of four concentrations of the ﬂuorescent ligand

every 30 s for 1 h at 37 °C. The residence time (RT) was calculated from the reciprocal of the k_offvalue, and the pK_Dwas derived from the negative

logarithm of the kinetic K_D. The values listed represent the mean SEM of n = 3 performed in triplicate. *p < 0.05, kinetically derived pK_Dversus

equilibrium saturation derived pK_Dvalue, Student’s unpaired t-test.

Figure 7. Displacement of 3 nM 46a (a) and 40 nM 44b (b) by unlabeled A₁AR antagonists SLV320 and DPCPX and agonist NECA in HEK293A

cells transiently expressing the NanoLuc-hA₁AR. The reduction of BRET signal was monitored as a function of increasing concentration of the

competing ligand. Data were normalized to maximal BRET signal in the absence of the unlabeled competing ligand (total binding, TB). Each data

point represents the combined mean SEM from n = 3 experiments performed in duplicate.

aﬃnities measured in the same cell line (hA₁AR-FIpInCHO)

by radioligand binding experiments.

concentrations of the ﬂuorescent probe over a time frame of 1

h. The speciﬁc BRET signal for each ﬂuorescent probe was

calculated after subtraction of the nonspeciﬁc binding, deﬁned

using 1 μM of unlabeled antagonist SLV320, from the total

binding component at each time point (Figure 6). The kinetic

parameters k_on, k_off, and pK_D, along with the calculated

residence time (1/k_off), were measured by globally ﬁtting the

resulting BRET data (Table 5).

The kinetics of ligand−receptor binding is emerging as a

powerful pharmacological paradigm for predicting the eﬃcacy

of drug candidates in vivo.^90,91The availability of probes and

techniques, which allow real-time monitoring of the rate at

which a drug binds to its biological target (k_on) and dissociates

from it (k_off), are therefore of great utility.^32,92,93It has been

recently observed with the A₃AR that the kinetics of the

labeled probe can inﬂuence the kinetic parameters of the

unlabeled ligands under investigation, suggesting that a labeled

probe should be chosen with regard to its adequate kinetic

proﬁle for the given study.⁴³In this regard, the ligand binding

kinetics of ﬂuorescent analogues 44a, 44b, and 46a were

monitored by the NanoBRET ligand binding kinetic assay in

HEK293A cells transiently expressing the NanoLuc-hA₁AR.

The kinetic parameters of each ﬂuorescent ligand were derived

by measuring the observed association constant (k_obs) at four

The results obtained showed that all three ﬂuorescent

ligands displayed relatively fast kinetics. Additionally, for probe

44a, the pK_Dvalues measured from the kinetic analyses were in

agreement with that obtained from saturation ligand binding

assays, while for 44b and 46a, the kinetic pK_Dvalues were

signiﬁcantly lower compared to the saturation pK_D. There

were, however, large diﬀerences in the measured association

rates (k_on) between the compounds. In particular, the BY630/

650-X-labeled probe 44a displayed k_ontwo orders of

magnitude faster (1.79

3.69 × 10⁷M⁻¹min⁻¹) than its

K

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Sulfo-Cy5-labeled analogue 44b (8.6

0.13 × 10⁵M⁻¹

the plasma membrane of living cells. To be suitable for imaging

studies, a ﬂuorescent ligand must possess adequate properties,

showing speciﬁc binding of the target protein at the cell surface

coupled with both low levels of nonspeciﬁc membrane

incorporation and diﬀusion into the cell cytosol. To this end,

confocal microscopy studies of ﬂuorescent probes 44a, 46a,

and 44b were carried out in HEK293A cells transiently

transfected with the engineered SNAP-tagged human A₁AR.

The equatorial images acquired with the confocal micro-

scope (Figure 8) revealed high levels of ﬂuorescent labeling of

the SNAP-tagged-hA₁AR localized at the cell membrane for all

the three red-shifted ﬂuorescent probes tested. Moreover, pre-

incubation of the cells with 10 μM unlabeled A₁AR antagonist

SLV320 prevented the binding of the ﬂuorescent probes to the

SNAP-tagged hA₁AR causing a reduction of ﬂuorescence. This

demonstrated that the binding of the probes was speciﬁc at the

SNAP-tagged-hA₁AR localized at the cell membrane. Fur-

thermore, when nontransfected (NT) HEK293 cells were

treated with probes 44a, 46a, and 44b, under the same

experimental conditions as the SNAP-tagged-hA₁AR HEK293

cells, no ﬂuorescence signal was detected, further conﬁrming

the speciﬁcity of these ﬂuorescent ligands for binding to the

hA₁AR and their low levels of nonspeciﬁc binding (Figure

S11).

Finally, we investigated the suitability of the most hA₁AR

selective probes reported here for high resolution ﬂuorescence

microscopy. To this end, the Sulfo-Cy5-labeled ligand 44b and

BODIPY-630/650-X-labeled ligand 46a were used in total

internal reﬂection ﬂuorescence microscopy (TIRF-M) to

probe receptor distribution at the plasma membrane of living

HEK293 cells transiently expressing the SNAP-tagged hA₁AR.

TIRF-M is a wide-ﬁeld imaging method that allows for the

visualization of molecule localization and organization on the

surface of living cells with high spatial and temporal

resolution.^103−105Imaging living cells with probe 44b allowed

for the detection of the SNAP-hA₁AR as single red ﬂuorescent

spots localized at the plasma membrane (Figure 9a). The

location of the red ﬂuorescent spots correlated with the green

ﬂuorescent spots of AF488-labeled SNAP-hA₁AR and is shown

in Figure 9a. Additionally, pre-incubation of the cells with 10

μM SLV320 prevented the visualization of SNAP-hA₁ARs by

probe 44b, further validating the speciﬁcity of probe 44b

(bottom frames, Figure 9a). While we found that the

ﬂuorescent ligand 44b had a high signal-to-noise ratio for

high resolution microscopy applications, ﬂuorescent ligand 46a

also did not perform in this way. Indeed, when 46a was used in

TIRF-M experiments, it was not possible to distinguish the

same localization patterns as the AF488-labeled SNAP-hA₁AR

(Figure S12). The BY630/650 moiety increases the lip-

ophilicity of the ﬂuorescent conjugate and might allow probe

46a to stick either to the plasma membrane of the cell or to the

glass coverslip surface, resulting in higher background signal in

this particular microscopy approach.

min⁻¹). These two ﬂuorescent analogues diﬀer only by the

attached ﬂuorophore motif, demonstrating the potential of the

ﬂuorescent pharmacophore to establish diﬀerent interactions

with the receptor and nearby environment in a way that aﬀects

the observed binding kinetics. This would be consistent with

observations described recently for the A₃AR.⁴³Moreover, in

an elegant study with the β₂-adrenoreceptor, Sykes et al.,

showed that the lipophilic nature of some structural motifs in a

molecule can inﬂuence the observed binding kinetics, depend-

ent on direct interactions between these motifs and the plasma

membrane.⁹⁴This could therefore be the case for the BODIPY

moiety in our ﬂuorescent analogues.⁹⁵

After having thoroughly characterized the pharmacology of

the synthesized ﬂuorescent A₁AR antagonists, we then

investigated their utilities in a broad range of ﬂuorescence-

based techniques. For example, ﬂuorescent analogues 44b and

46a were subsequently applied to monitor the ligand binding

of unlabeled ligands to the hA₁AR. This was achieved through

the NanoBRET competition ligand binding assay in HEK293A

cells transiently expressing the NanoLuc-hA₁AR. Cells were

incubated with the indicated concentration of ﬂuorescent

ligand and increasing concentrations of hA₁AR antagonists

SLV320, DPCPX, and the nonselective agonist NECA. A clear

concentration dependent inhibition of the BRET signal was

observed for all the compounds tested (Figure 7), allowing for

the relative half-maximal inhibitory concentrations (IC₅₀) to be

measured. These values were converted to the corresponding

inhibitory constant (K_i) by means of the Cheng−Prusoﬀ

equation and are listed in Table 6.

Table 6. Binding Aﬃnities of A₁AR Ligands Measured by

the NanoBRET Competition Ligand Binding Assay and

Aﬃnities Reported in Literature

d

pK_i(Log M) SEM

cmpd

44b (Sulfo-Cy5)

46a (BY630/650)

pK_i(literature)

a

SLV320

DPCPX

NECA

9.15 0.07

8.13 0.05

6.40 0.10

8.85 0.07

7.68 0.01

5.96 0.06

9

b

7.4−9.2

c

5.3−8.2

a

Reported binding aﬃnities.²Value determined in radioligand

binding experiments in hA₁AR-CHO-K1 cells using [³H] DPCPX.⁹⁶

b

Values determined in radioligand binding experiments in A₁AR-

COS-7⁹⁷or hA₁AR-CHO-K1 cell membranes^57,98or whole cells⁹⁹

c

using [³H] DPCPX. Values determined in radioligand binding

experiments in cells expressing the wild-type hA₁AR using [³H]

DPCPX^57,100,101or [³H]R-PIA.¹⁰²Measured pK_iin NanoBRET

d

competition binding experiments in whole NanoLuc-

hA₁ARHEK293A cells using the ﬂuorescent ligand indicated. Values

are mean SEM of n = 3 experiments performed in duplicate.

NanoBRET competition binding assays with ﬂuorescent

ligands 46a and 44b and the unlabeled competing A₁AR

ligands yielded pK_ivalues that were comparable to the

reported literature values measured by radioligand binding

assays (R²= 0.95 using 46a, R²= 0.94 using 44b; Figure

S10a,b) and comparable to those measured in NanoBRET

binding assays by Stoddart et al.²⁹and Cooper et al.⁴⁴Overall,

the pK_ivalues measured using both ﬂuorescent probes were

also comparable (R²= 0.99 44b vs 46a; Figure S10c).

CONCLUSIONS

■

In the study presented herein, we have reported the successful

design, synthesis, and pharmacological evaluation of a series of

novel, high aﬃnity, and subtype-selective ﬂuorescent probes

for the study of human A₁AR in living cells. We have

demonstrated that both positions of the prototypical xanthine

scaﬀold, namely, the 3- and 8-positions, could be harnessed for

linker/ﬂuorophore functionalization. Additionally, modiﬁca-

tions at both positions yielded ﬂuorescent probes that retained

To further expand the utility of the novel synthesized

probes, we assessed whether ﬂuorescent analogues 44a, 46a,

and 44b would allow visualization of the hA₁AR expressed at

L

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Furthermore, ﬂuorescent conjugates 44b and 46a were

successfully applied to monitor ligand binding of known A₁AR

ligands by the recently described NanoBRET ligand binding

assay, demonstrating that these probes can be used as

alternatives to radioligands. Confocal microscopy studies

showed that ﬂuorescent analogues 44a, 46a, and 44b could

be applied to visualize the hA₁AR expressed at the membrane

of living cells as demonstrated by the high degree of

ﬂuorescence intensity observed at the plasma membrane.

The binding of the probes could be prevented by incubating

the cells with a high concentration of nonﬂuorescent A₁AR

antagonist, demonstrating the speciﬁcity of the binding of the

probes to the SNAP-tagged-hA₁AR with very little nonspeciﬁc

cellular uptake detected across all the compounds tested.

Finally, the Sulfo-Cy5-labeled probe 44b was successfully

combined with TIRF microscopy to map hA₁AR distribution

at the plasma membrane of HEK293 cells, suggesting that

probe 44b could be coupled with advanced microscopy

techniques for the study of the spatial organization of A₁ARs at

the plasma membrane of living cells. Remarkably, despite the

introduction of the Sulfo-Cy5 ﬂuorescent tag causing a reduced

aﬃnity for the hA₁AR compared to the corresponding BY630/

650-labeled analogues, we found that the Sulfo-Cy5-labeled

probe (44b) proved as speciﬁc and eﬀective as the BY630/

650-labeled analogues (44a and 46a) and possessed even

superior physicochemical properties for single-molecule

microscopy applications. These results highlight the impor-

tance of developing small-molecule probes labeled with dyes of

diﬀerent physicochemical properties and that the character-

ization and selection of these probes should not be merely

based on their apparent binding aﬃnity for the target protein

under investigation, but, principally, for the speciﬁc biological

application.

In summary, we have expanded the available ﬂuorescent

A₁AR ligand toolbox. The novel probes reported herein were

thoroughly characterized, displayed improved A₁AR pharma-

cology, and were amenable to a broad range of ﬂuorescence-

based techniques. We envisage that these novel A₁AR

ﬂuorescent probes will be valuable pharmacological tools to

the wider scientiﬁc community to aid in understanding the

molecular mechanisms underlying A₁AR pharmacology in

health and disease.

EXPERIMENTAL SECTION

■

Figure 8. Live-cell confocal imaging studies of ﬂuorescent analogues

44a (5 nM) (a), 46a (10 nM) (b), and 44b (50 nM) (c) binding to

SNAP-tagged human A₁AR expressed in HEK293 cells in the absence

(top frames) or presence (bottom frames) of 10 μM unlabeled A₁AR

antagonist SLV320. Cells were treated with BG-Alexa Fluor 488 for

30 min at 37 °C to label the SNAP-tag on the N-terminus of the

A₁AR (left hand frames). Middle frames show ﬂuorescent labeling of

SNAP-tagged-hA₁AR by the red-shifted ﬂuorescent analogues tested

at the given concentration. The images presented here are

representative of images captured in three independent experiments.

Scale bar = 15 μm

Chemistry: Materials and Methods. Chemicals and solvents

were purchased from commercial suppliers and used without further

puriﬁcation. Davis silica gel (40−63 mm) was supplied by Grace

Davison Discovery Science (Victoria, Australia), and a deuterated

solvent was purchased from Cambridge Isotope Laboratories (USA,

distributed by Novachem Pty. Ltd., Victoria, Australia). Reactions

were monitored by thin-layer chromatography on commercially

available precoated silica aluminium-backed plates (Merck Kieselgel

60F₂₅₄). Visualization was by examination under UV light (254 and

366 nm), followed by staining with ninhydrin or KMnO₄dips. All

organic extracts collected after aqueous workup procedures were dried

over anhydrous MgSO₄, before gravity ﬁltering and evaporation to

dryness. Organic solvents were evaporated in vacuo at 40 °C (water

bath temperature). NMR spectra were recorded on a Bruker Avance

Nanobay III 400 MHz Ultrashield Plus spectrometer. ¹HNMR

spectra were recorded at 400.13, and¹³CNMR were recorded at

101.62 MHz. Chemical shifts (δ) are recorded in parts per million

(ppm) with reference to the chemical shift of the deuterated solvent

used, and coupling constants are recorded in Hz. The following

abbreviation are used to describe the signal shape and multiplicities:

singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br),

binding aﬃnity and functional activity at the hA₁AR. Overall, a

good selectivity proﬁle for the probes synthesized was

observed, with the Sulfo-Cy5-bearing probe 44b and BY630/

650-X-bearing probe 46a displaying the best selectivity proﬁle

for the hA₁AR compared to the other adenosine receptors. To

the best of our knowledge, probes 44b and 46a represent the

ﬁrst examples of subtype-selective ﬂuorescent hA₁AR antago-

nists reported to date.

M

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 9. Fluorescent ligand 44b (50 nM) allowed the visualization of the organization of SNAP-tagged-hA₁AR localized at the plasma membrane

of living HEK293 cells by total internal reﬂection ﬂuorescence microscopy (TIRF-M) in the absence (top frames) or presence (bottom frames) of

10 μM unlabeled A₁AR selective antagonist SLV320. (a) Cells were labeled with 0.2 μM BG-Alexa Fluor 488 dye for 30 min at 37 °C (left hand

frames). Top middle frame shows the labeling of SNAP-hA₁AR by probe 44b (Sulfo-Cy5) at the given concentration. Bottom middle frame shows

that pretreatment of the cells with high concentration (10 μM) of A₁AR selective antagonist SLV320 prevents the binding of ﬂuorescent ligand 44b

to the hA₁AR. Right hand frames represent merged green (AF488) and red (Sulfo-Cy5) channels. (b) Plot of ﬂuorescence intensities of Sulfo-Cy5-

labeled (44b) spots vs AF448-labeled spots, in the absence (blue) or presence (cyan) of 10 μM SLV320. (c) Distribution of the nearest neighbor

distances between single SNAP-hA₁ARs labeled with probe 44b (red) and the BG-Alexa Fluor 488 (cyan). The images reported here are

representative TIRF-M images from three independent experiments, each of which included the analysis of 14−18 cells. The data reported in

graphs (b) and (c) is the average of data analyzed from 14 to 18 cells in a single representative experiment of three independent experiments. Scale

bar = 12 μm

doublet of doublets (dd), doublet of triplets (dt), triplet of doublets

(td), and multiplet (m). Processing of the NMR data was carried out

using NMR software Mnova 12.0.4. LC−MS spectra were recorded

on an Agilent 6100 series single quad coupled to an Agilent 1200

series HPLC instrument using the following buﬀers: buﬀer A, 0.1%

formic acid in H₂O; buﬀer B, 0.1% formic acid in CH₃CN. The

following gradient was used with a Poroshell 120 EC-C18 3.0 ×

50mm, 2.7 μm column a ﬂow rate of 0.5 mL/min and a total run time

of 5 min: 0−1 min 95% buﬀer A and 5% buﬀer B, 1−2.5 min 0%

buﬀer A and 100% buﬀer B, 2.5−3.8 min 0% buﬀer A and 100%

buﬀer B, 3.8−4 min 95% buﬀer A and 5% buﬀer B, 4−5 min 95%

buﬀer A and 5% buﬀer B (method A). Mass spectra were acquired in

positive and negative ion modes with a scan range of 100−1000 m/z,

and UV detection was monitored at 254 and 214 nm. All high-

resolution mass spectra (HRMS) were recorded on an Agilent 6624

TOF LC−MS spectrometer coupled to an Agilent 1290 Inﬁnity

system (Agilent, Palo Alto, CA). All data were acquired, and reference

mass corrected via a dual-spray electrospray ionization (ESI) source.

Acquisition was performed using Agilent Mass Hunter Data

Acquisition software version B.05.00 Build 5.0.5042.2, and analysis

was performed using Mass Hunter Qualitative Analysis version

B.05.00 Build 5.0.519.13. Preparative HPLC was performed on an

Agilent 1260 inﬁnity system coupled with a binary preparative pump

and Agilent 1260 FC-PS fraction collector using an Altima C8, 250 ×

22 mm, 5 μm column. The following conditions were used for this

system: buﬀer A, H₂O and buﬀer B, CH₃CN with a gradient of 30−

100% buﬀer B over 11 min, with a ﬂow rate of 20 mL/min (method

B). Spectra were analyzed using Agilent OpenLAB CDS software.

Purity was veriﬁed on an analytical reverse-phase Agilent HPLC

system coupled directly to a photodiode array detector and using a

Zorbax Eclipse Plus C18 Rapid resolution 4.6 × 100 mm, 3.5 μm

column using the following solvent system: buﬀer A, 0.1% TFA in

H₂O; buﬀer B, 0.1% TFA in CH₃CN. The elution was performed on a

gradient of 95% buﬀer A and 5% buﬀer A to 0% buﬀer A and 100%

buﬀer B over 12 min at a ﬂow rate of 1 mL/min (method C). Final

products were one single peak and >95% purity.

General Procedure A: Amide Coupling. To a solution of the

respective carboxylic acid (1.0 equiv) in DMF (1−2 mL) were added

N

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

DIPEA (1.10 equiv) and COMU or BOP (1.10 equiv). After stirring

for 5 min at rt., the respective amine (1.10 equiv) was added to the

reaction mixture and the resulting solution was stirred at rt. for 15−60

min. Upon completion of the reaction, checked by LC/MS and TLC,

the reaction mixture was evaporated to dryness. The residue was

taken up in EtOAc and washed with 1 M aq. HCl, sat. NaHCO₃, and

brine. The organic layer was collected, dried over anhydrous MgSO₄,

ﬁltered, and evaporated to dryness. The resulting residue was puriﬁed

by ﬂash column chromatography as indicated.

General Procedure B: Deprotection of tert-Butyl Carbamate

(Boc-Group). To the respective Boc-protected amine (1 equiv) was

added an excess of 4 M HCl in dioxane (1 mL). The resulting mixture

was stirred at rt. for 30 min. Upon completion of the reaction,

monitored by TLC and LC/MS, the solvent was evaporated to

dryness to give the corresponding amine as its HCl salt, which was

used for the subsequent step without further puriﬁcation.

71.2, 70.5, 70.1, 45.3, 43.2, 40.6, 38.9, 36.8, 34.0, 31.5, 30.6, 30.6,

28.5, 21.5, 21.5, 11.7, 11.3. Analytical RP-HPLC t_R= 5.77 min, purity

>99% (method C).

tert-Butyl(2-(2-((2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahy-

dro-1H-purin-8-yl)bicyclo[2.2.2]octan-1-yl)propanamido)ethyl)-

amino)-2-oxoethoxy)ethyl)carbamate (13). Following general pro-

cedure A, to a solution of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)-

acetic acid (linker B) (12 mg, 55 μmol, 1.0 equiv) in DMF (0.5 mL)

were added DIPEA (38 μL, 219 μmol, 4.0 equiv) and COMU (25.80

mg, 60 μmol, 1.10 equiv), followed by a solution of 11 (28 mg, 60

μmol, 1.10 equiv) in DMF (0.5 mL). Puriﬁcation by ﬂash column

chromatography using a gradient of 96:4 to 94:6 DCM/MeOH gave

the title compound as colorless oil (10 mg, 28%). LC/MS m/z calcd

for C₃₃H₅₂N₇O₇[MH]⁻: 658.20, found 658.40 t_R= 3.70 min (method

A). HRMS (TOF ES⁺) calcd for C₃₃H₅₄N₇O₇660.4079, found

1

660.4096; calcd for C₃₃H₅₄N₇O₇Na 682.3899, found 682.3917. H

NMR (CDCl₃) δ 7.43 (s, 1H), 6.44 (s, 1H), 5.57 (s, 1H), 4.09 (t, J =

6.9 Hz, 2H), 4.00 (t, J = 6.9 Hz, 2H), 3.95 (s, 2H), 3.56 (t, J = 5.0 Hz,

2H), 3.45−3.40 (m, 4H), 3.40−3.33 (m, 2H), 2.17−2.11 (m, 2H),

2.02−1.95 (m, 6H), 1.78 (h, J = 7.4 Hz, 2H), 1.69 (h, J = 7.0 Hz,

2H), 1.55−1.47 (m, 8H), 1.44 (s, 9H), 0.95 (t, J = 7.4 Hz, 6H). ¹³C

NMR (CDCl₃) δ 174.9, 170.8, 161.7, 156.5, 155.4, 151.2, 148.8,

106.8, 79.4, 71.3, 70.4, 70.4, 45.4, 43.2, 40.0, 36.8, 34.0, 31.5, 30.6,

30.6, 28.6, 21.5, 21.5, 11.6, 11.3. Analytical RP-HPLC t_R= 5.77 min,

purity >99% (method C).

General Procedure C: Syntheses of the Fluorescent

Ligands. The respective amine congener (as HCl salt) (1.1 equiv)

was dissolved in DMF (0.5 mL), treated with DIPEA (3 equiv), and

reacted with BODIPY 630/650-X-NHS (0.5 mg, 1 equiv) or Sulfo-

Cy5-NHS (0.5 mg, 1 equiv). The resulting mixture was stirred at rt.

under the exclusion of light for 15−60 min. Upon completion of the

reaction, monitored by TLC and LC/MS, the solvent was removed in

vacuo and the residue was dissolved in a mixture of 1:1 CH₃CN/H₂O.

Puriﬁcation by reverse-phase preparative HPLC (followed by

preparative TLC with DCM/MeOH 80:20 for the Sulfo-Cy5 probes)

and subsequent lyophilization yielded the pure ﬂuorescent ligand as a

bright blue solid.

tert-Butyl(3-((2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octan-1-yl)propanamido)ethyl)amino)-

3-oxopropyl)carbamate (14). Following general procedure A, to a

solution of 3-((tert-butoxycarbonyl)amino)propanoic acid (Boc-β-

Ala-OH) (10 mg, 53 μmol, 1.0 equiv) in DMF (0.5 mL) were added

DIPEA (37 μL, 211 μmol, 4.0 equiv) and COMU (25 mg, 58 μmol,

1.10 equiv), followed by a solution of 11 (27 mg, 58 μmol, 1.10

equiv) in DMF (0.5 mL). Puriﬁcation by ﬂash column chromatog-

raphy using a gradient of 96:4 to 94:6 DCM/MeOH gave the title

compound as an oﬀ-white solid (29 mg, 78%). LC/MS m/z calcd for

C₃₂H₅₂N₇O₆[MH]⁺: 630.20, found 630.40 t_R= 3.69 min (method

tert-Butyl(2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl) bicyclo[2.2.2]octan-1-yl)propanamido)ethyl)carbamate

(10). Following general procedure A, to a solution of 3-(4-(2,6-dioxo-

1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octan-1-

yl)propanoic acid, tonapofylline (120 mg, 288 μmol 1.0 equiv) in

DMF (1 mL), were added DIPEA (55 μL, 317 μmol, 1.10 equiv) and

COMU (136 mg, 317 μmol 1.10 equiv) followed by a solution of tert-

butyl (2-aminoethyl)carbamate (51 mg, 317 μmol 1.10 equiv) in

DMF (0.5 mL). Puriﬁcation by ﬂash column chromatography using a

gradient of 97:3 to 94:6 DCM/MeOH gave the title compound as

colorless oil (93 mg, 58%). LC/MS m/z calcd for C₂₉H₄₇N₆O₅

1

A). H NMR (CD₃OD) δ 4.07 (t, J = 6.3 Hz, 2H), 3.95 (t, J = 6.8,

2H), 3.29 (br s, 4H), 2.37 (t, J = 6.8 Hz, 2H), 2.20−2.14 (m, 2H),

2.00−1.93 (m, 6H), 1.78 (h, J = 6.8 Hz, 2H), 1.66 (h, J = 6.8 Hz,

2H), 1.59−1.48 (m, 8H), 1.45 (s, 9H), 0.95 (t, J = 7.5 Hz, 6H). ¹³C

NMR (CD₃OD) δ 176.9, 174.1, 162.7, 155.8, 152.6, 149.3, 107.8,

79.9, 45.8, 43.6, 39.8, 37.9, 37.3, 34.9, 31.8, 31.4, 31.4, 31.2, 28.5,

22.2, 22.1, 11.3, 11.1. Analytical RP-HPLC t_R= 5.65 min, purity >99%

(method C).

1

[MH]⁺: 559.20, found 559.20 t_R= 3.78 min (method A). H NMR

(CDCl₃) δ 12.13 (s, 1H), 6.33 (s, 1H), 5.03 (s, 1H), 4.08 (t, J = 7.0

Hz, 2H), 4.00 (t, J = 7.3 Hz, 2H), 3.35 (q, J = 5.4 Hz, 2H), 3.26 (q, J

= 5.7 Hz, 2H), 2.16−2.08 (m, 2H), 2.03−1.93 (m, 6H), 1.79 (h, J =

7.4 Hz, 2H), 1.70 (h, J = 6.7, 6.0 Hz, 2H), 1.54−1.38 (m, 17H), 0.95

(t, J = 6.5 Hz, 6H). ¹³C NMR (CDCl₃) δ 174.2, 161.9, 157.2, 155.5,

151.2, 148.9, 106.9, 79.8, 45.3, 43.2, 41.1, 40.3, 36.8, 34.0, 31.5, 30.6,

30.6, 28.5, 21.5, 11.7, 11.3.

tert-Butyl(3-((3-((2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahy-

dro-1H-purin-8-yl)bicyclo[2.2.2]octan-1-yl)propanamido)ethyl)-

amino)-3-oxopropyl)amino)-3-oxopropyl)carbamate (15). Follow-

ing general procedure B, compound 14 (27 mg, 1.0 equiv) was

reacted with 4 M HCl in dioxane (1 mL), and after the removal of the

solvent in vacuo, the resulting HCl salt was used for the subsequent

step without further puriﬁcation. The synthesis of 15 was carried out

as described for xanthine 14, following general procedure A, reacting a

solution of the 3-((2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octan-1-yl)propanamido)ethyl)amino)-

3-oxopropan-1-aminium chloride intermediate (22 mg, 41 μmol, 1.10

equiv) in DMF (0.5 mL) with a solution of 3-((tert-Butoxycarbonyl)-

amino)propanoic acid (Boc-β-Ala-OH) (7 mg, 37 μmol, 1.0 equiv),

DIPEA (32 μL, 185 μmol, 5 equiv), COMU (17.43 mg, 41 μmol, 1.10

equiv) in DMF (0.5 mL). Puriﬁcation by ﬂash column chromatog-

raphy using a gradient of 96:4 to 93:7 DCM/MeOH gave the title

compound as an oﬀ-white solid (12 mg, 46%). LC/MS m/z calcd for

C₃₅H₅₇N₈O₇[MH]⁺: 701.20, found 701.40 t_R= 3.57 min (method

A). HRMS (TOF ES⁺) calcd for C₃₅H₅₇N₈O₇701.4350, found

tert-Butyl(15-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octan-1-yl)-8,13-dioxo-3,6-dioxa-9,12-

diazapentadecyl)carbamate (12). Following general procedure B,

compound 11 (as HCl salt) was synthesized (75 mg, quantitative)

and was used for the next steps without further puriﬁcation. LC/MS

m/z calcd for C₂₄H₃₉N₆O₃[MH]⁺: 459.20, found 459.20 t_R= 3.07

min (method A). Following general procedure A, to a solution of 2,2-

dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (linker A) (15

mg, 57 μmol, 1.0 equiv) in DMF (0.5 mL) was added DIPEA (40 μL,

228 μmol, 4.0 equiv), followed by COMU (26.84 mg, 63 mmol, 1.10

equiv) and a solution of compound 11 (29 mg, 63 μmol, 1.10 equiv)

in DMF (0.5 mL). Puriﬁcation by ﬂash column chromatography using

a gradient of 97:3 to 95:5 DCM/MeOH gave the title compound as

colorless oil (15 mg, 38%). LC/MS m/z calcd for C₃₅H₅₆N₇O₈

[MH]⁻: 702.20, found 702.20 t_R= 3.63 min (method A). HRMS

(TOF ES⁺) calcd for C₃₅H₅₈N₇O₈704.4341, found 704.4356; calcd

1

for C₃₅H₅₈N₇O₈Na 726.4161, found 726.4178. H NMR (CDCl₃) δ

701.4360; calcd for C₃₅H₅₇N₈O₇Na 723.4164, found 723.4184. H

11.86 (br s, 1H), 7.33 (t, J = 5.7 Hz, 1H), 6.42 (s, 1H), 5.19 (s, 1H),

4.09 (t, J = 6.7 Hz, 2H), 4.00 (m, 4H), 3.68−3.61 (m, 4H), 3.57 (t, J

= 5.3 Hz, 2H), 3.47−3.38 (m, 4H), 3.36−3.29 (m, 2H), 2.15−2.07

(m, 2H) 2.02−1.95 (m, 6H), 1.79 (h, J = 7.4 Hz, 2H), 1.69 (h, J = 7.1

Hz, 2H), 1.54−1.46 (m, 8H), 1.43 (s, 9H,), 0.95 (t, J = 7.4 Hz, 6H).

¹³C NMR (CDCl₃) δ 174.2, 161.7, 156.3, 155.1, 151.3, 148.8, 106.9,

NMR (CD₃OD) δ 4.07 (t, J = 6.3 Hz, 2H), 3.95 (t, J = 6.3 Hz, 2H),

3.45 (t, J = 6.8 Hz, 2H), 3.33−3.31 (m, 2H), 3.30 (br s, 4H), 2.38 (dt,

J = 9.0, 6.8 Hz, 4H), 2.20−2.14 (m, 2H), 2.00−1.92 (m, 6H), 1.78 (h,

J = 7.5 Hz, 2H), 1.66 (h, J = 7.0 Hz, 2H), 1.59−1.49 (m, 8H), 1.44 (s,

9H), 0.95 (t, J = 7.4, 6H). ¹³C NMR (CD₃OD) δ 177.1, 174.1, 162.7,

158.3, 156.0, 152.9, 149.5, 80.1, 45.9, 43.8, 40.2, 40.0, 38.1, 37.4, 37.1,

O

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

35.1, 32.1, 31.6, 31.4, 28.8, 22.4, 22.3, 11.5, 11.3. Analytical RP-HPLC

t_R= 5.52 min, purity >99% (method C).

general procedure A. Compound 18a (20 mg, 46 μmol, 1.1 equiv)

was added to a solution of 2-(2-((tert-butoxycarbonyl)amino)-

ethoxy)acetic acid (10 mg, 46 μmol, 1.0 equiv), BOP (30 mg, 68

μmol, 1.50 equiv), and DIPEA (32 mL, 182 μmol, 4.0 equiv) in DMF

(1 mL). Puriﬁcation by ﬂash column chromatography using a gradient

of 97:3 to 96:4 DCM/MeOH gave the title compound as colorless oil,

which slightly solidiﬁed upon cooling (10 mg, 36%). LC/MS m/z

calcd for C₃₁H₅₀N₇O₇[MH]⁺: 632.20, found 632.40 t_R= 3.53 min

(method A). HRMS (TOF ES⁺) calcd for C₃₁H₅₀N₇O₇632.3766,

found 632.3781; calcd for C₃₁H₅₀N₇O₇Na 654.3586, found 654.3601.

¹H NMR (CDCl₃) δ 11.86 (s, 1H), 7.38 (s, 1H), 6.57 (s, 1H), 5.46

(s, 1H), 4.09 (t, J = 6.4 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.96 (s,

2H), 3.57 (t, J = 5.0 Hz, 2H), 3.47−3.41 (m, 4H), 3.40−3.33 (m,

2H), 2.09−2.01 (m, 6H), 1.92−1.85 (m, 6H), 1.79 (h, J = 6.9 Hz,

2H), 1.69 (h, J = 6.9 Hz, 2H), 1.45 (s, 9H), 0.95 (t, J = 7.5 Hz, 6H).

¹³C NMR (CDCl₃) δ 178.5, 171.1, 156.4, 155.5, 151.2, 106.9, 79.7,

tert-Butyl(2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl) bicyclo[2.2.2]octane-1-carboxamido)ethyl)carbamate

(17a). Following general procedure A, to a solution of (2,6-dioxo-

1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl) bicyclo [2.2.2] octane-

1-carboxylic acid (compound 16 from Kiesman et al.)⁵⁹(80 mg, 206

μmol, 1.0 equiv), tert-butyl (2-aminoethyl) carbamate (33 mg, 206

μmol, 1.0 equiv), and BOP (137 mg, 309 μmol, 1.50 equiv) in dry

DMF (2 mL) was added DIPEA (143 μL, 824 μmol, 4.0 equiv).

Puriﬁcation by ﬂash column chromatography using a gradient of 97:3

to 96:4 DCM/MeOH gave the title compound as oﬀ-white solid (85

mg, 78%). LC/MS m/z calcd for C₂₇H₄₃N₆O₅[MH]⁺: 531.30, found

1

531.30 t_R= 3.61 min (method A). H NMR (CDCl₃) δ 11.67 (br s,

1H), 6.53 (br s, 1H), 4.90 (br s, 1H), 4.09 (t, J = 6.3 Hz, 2H), 4.02 (t,

J = 6.6 Hz, 2H), 3.37−3.26 (m, 4H), 2.08−2.01 (m, 6H), 1.93−1.87

(m, 6H), 1.80 (h, J = 6.5 Hz, 2H), 1.70 (h, J = 6.5 Hz, 2H), 1.45 (s,

9H), 0.96 (t, J = 7.5 Hz, 6H). ¹³C NMR (CDCl₃) δ 176.1, 155.5,

152.2, 151.3, 148.9, 106.9, 30.2, 28.5, 21.5, 11.6, 11.3.

70.4, 69.5, 43.3, 40.7, 39.9, 39.0, 33.9, 30.1, 29.8, 28.6, 28.5, 21.5,

21.5, 11.6, 11.3. Analytical RP-HPLC t_R= 5.87 min, purity >97%

(method C).

tert-Butyl(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propyl)carbamate

(17b). The synthesis of the title compound 17b was carried out as

described for xanthine (17a), following general procedure A. To a

stirred solution of (2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl) bicyclo [2.2.2] octane-1-carboxylic acid (compound 16

from Kiesman et al.) (110 mg, 283 μmol, 1 equiv), tert-butyl (3-

aminopropyl) carbamate (49 mg, 283 μmol, 1.0 equiv), and BOP

(188 mg, 425 μmol, 1.50 equiv) in dry DMF (2 mL) was added

DIPEA (197 μL, 1.13 μmol, 4.0 equiv). Puriﬁcation by ﬂash column

chromatography using a gradient of 97:3 to 96:4 DCM/MeOH gave

the title pure compound as an oﬀ-white solid (132 mg, 86%). LC/MS

m/z calcd for C₂₈H₄₅N₆O₅[MH]⁺: 545.30, found 545.40 t_R= 3.66

tert-Butyl(3-((2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)ethyl)amino)-3-

oxopropyl)carbamate (21). Compound 18a (22 mg, 53 μmol, 1.0

equiv) was reacted with Boc-β-Ala-OH (10 mg, 53 μmol, 1.0 equiv)

in the presence of BOP (35 mg, 79 μmol, 1.50 equiv) and DIPEA (37

μL, 211 μmol, 4.0 equiv) in DMF (2 mL) according to general

procedure A. Puriﬁcation by ﬂash column chromatography using a

gradient of 97:3 to 96:4 DCM/MeOH gave the title compound as

colorless oil, which slightly solidiﬁed upon cooling (12 mg, 38%). LC/

MS m/z calcd for C₃₀H₄₈N₇O₆[MH]⁺: 602.20, found 602.40 t_R

=

1

3.48 min (method A). H NMR (CD₃OD) δ 4.07 (t, J = 6.9, 2H),

3.95 (t, J = 6.9, 2H), 3.32 (s, 2H), 3.30 (s, 4H), 2.37 (t, J = 6.8 Hz,

2H), 2.05−1.98 (m, 6H), 1.95−1.87 (m, 6H), 1.77 (h, J = 7.5, 2H),

1.66 (h, J = 7.5, 2H), 1.45 (s, 9H), 0.96 (t, J = 7.4, 6H).

1

min (method A). H NMR (CDCl₃) δ 12.20 (br s, 1H), 6.55 (br s,

1H), 4.91 (br s, 1H), 4.08 (t, J = 6.9, Hz, 2H), 4.02 (t, J = 6.2 Hz,

2H), 3.30 (q, J = 4.6 Hz, 2H), 3.16 (q, J = 4.6 Hz, 2H), 2.10−2.04

(m, 6H), 1.94−1.88 (m, 6H), 1.78 (h, J = 6.5 Hz, 2H), 1.70 (h, J =

6.5 Hz, 2H), 1.62−1.54 (m, 2H), 1.43 (s, 9H), 0.95 (t, J = 7.0 Hz,

6H). ¹³C NMR (CDCl₃) δ 177.6, 161.4, 156.9, 155.6, 151.2, 149.0,

107.0, 79.6, 45.3, 43.2, 39.1, 36.9, 35.6, 33.9, 30.2, 28.5, 21.5, 11.6,

11.3.

tert-Butyl(3-((3-((2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)ethyl)amino)-3-

oxopropyl)amino)-3-oxopropyl)carbamate (25a). Compound 21

(12 mg, 1.0 equiv) was treated with 4 M HCl in dioxane following

general procedure B to yield the corresponding 3-((2-(4-(2,6-dioxo-

1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-

carboxamido)ethyl)amino)-3-oxopropan-1-aminium chloride inter-

mediate (9 mg, quantitative), which was used for the next step

without any further puriﬁcation. The synthesis of the title compound

25a was carried out as described for 8-bicyclo[2.2.2]octylxanthine 21,

treating the 3-((2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)ethyl)amino)-3-oxo-

propan-1-aminium chloride intermediate (5 mg, 1.10 equiv) with

DIPEA (31 μL, 186 μmol, 10.0 equiv), Boc-β-Ala-OH (4 mg, 19

μmol, 1.0 equiv), and BOP (12 mg, 28 μmol, 1.50 equiv) in DMF (1

mL). Puriﬁcation by ﬂash column chromatography using a gradient of

96:4 to 94:6 DCM/MeOH gave the title compound as colorless oil,

which solidiﬁed upon cooling (8 mg, 67%). LC/MS m/z calcd for

C₃₃H₅₃N₈O₇[MH]⁺: 673.20, found 673.40 t_R= 3.42 min (method

A). HRMS (TOF ES⁺) calcd for C₃₃H₅₃N₈O₇673.4032, found

tert-Butyl(1-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octan-1-yl)-1,6-dioxo-8,11-dioxa-2,5-dia-

zatridecan-13-yl)carbamate (19). Following general procedure B,

compound 17a (85 mg, 206 μmol, 1.0 equiv) was treated with 4 M

HCl in dioxane (1 mL) to aﬀord compound 18a (68 mg,

quantitative) as HCl salt, which was used in the next step without

further puriﬁcation. LC/MS m/z calcd for C₂₂H₃₅N₆O₃[MH]⁺:

431.34, found 431.30 t_R= 3.10 min. Compound 19 was synthesized

according to general procedure A, reacting a solution of compound

18a (25 mg, 57 μmol, 1.1 equiv) in DMF (0.5 mL) with 2,2-dimethyl-

4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (15 mg, 57 μmol, 1.0

equiv), DIPEA (40 mL, 228 μmol, 4.0 equiv), and BOP (38 mg, 85

μmol, 1.50 equiv). Puriﬁcation by ﬂash column chromatography using

a gradient of 98:2 to 96:4 DCM/MeOH gave the title compound as

colorless oil (15 mg, 38%). LC/MS m/z calcd for C₃₃H₅₄N₇O₈

[MH]⁺: 676.20, found 676.40 t_R= 3.50 min (method A). HRMS

(TOF ES⁺) calcd for C₃₃H₅₄N₇O₈676.4028, found 676.4045; calcd

for C₃₃H₅₄N₇O₈Na 698.3848, found 698.3869. ¹H NMR (CD₃OD) δ

4.07 (t, J = 6.9, Hz, 2H), 4.01 (s, 2H), 3.95 (t, J = 7.1 Hz, 2H), 3.72−

3.66 (m, 4H), 3.56 (t, J = 5.7 Hz, 2H), 3.41−3.37 (m, 2H), 3.37−

3.34 (m, 2H), 3.27 (td, J = 5.7, 4.4 Hz, 2H), 2.05−1.98 (m, 6H),

1.93−1.86 (m, 6H), 1.79 (h, J = 7.3 Hz, 2H), 1.65 (h, J = 7.3 Hz,

2H), 1.44 (s, 9H), 0.96 (t, J = 7.4, 6.3 Hz, 6H). ¹³C NMR (CD₃OD)

δ 180.5, 173.3, 160.9, 156.0, 152.9, 151.6, 149.5, 108.2, 80.1, 72.0,

71.3, 71.2, 71.1, 49.6, 45.9, 43.8, 41.3, 40.5, 40.5, 40.1, 39.7, 34.9,

31.0, 29.2, 28.8, 22.4, 22.3, 11.5, 11.4. Analytical RP-HPLC t_R= 5.85

min, purity >99% (method C).

1

673.4043; calcd for C₃₃H₅₂N₈O₇Na 695.3851, found 695.3866. H

NMR (CD₃OD) δ 4.07 (t, J = 6.3 Hz, 2H), 3.95 (t, J = 5.7 Hz, 2H),

3.45 (t, J = 6.8 Hz, 2H), 3.30 (s, 4H), 2.38 (t, J = 6.8 Hz, 4H), 2.04−

1.98 (m, 6H), 1.93−1.87 (m, 6H), 1.77 (h, J = 7.4 Hz, 2H), 1.66 (h, J

= 6.4 Hz, 2H), 1.43 (s, 9H), 0.96 (t, J = 7.2 Hz, 6H). ¹³C NMR

(CD₃OD) δ 179.1, 172.9, 161.5, 160.8, 151.5, 95.1, 78.7, 46.8, 42.5,

39.1, 38.7, 35.7, 35.5, 33.6, 33.6, 33.3, 29.6, 27.8, 27.4, 20.9, 20.9,

10.1, 9.9. Analytical RP-HPLC t_R= 5.58 min, purity >97% (method

C).

Methyl 3-(4-(2,6-Dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl) bicyclo[2.2.2]octane-1-carboxamido)propanoate (26).

To a solution of 4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxylic acid (compound 16

from Kiesman et al.) (80 mg, 206 μmol, 1.0 equiv) in DMF (1

mL) were added DIPEA (0.143 mL, 824 μmol, 4.0 equiv), β-Ala-

methyl ester (29 mg, 206 μmol, 1.0 equiv), and BOP (136 mg, 309

μmol, 1.50 equiv). The resulting mixture was stirred at rt. for 1 h.

tert-Butyl(2-(2-((2-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)ethyl)amino)-2-

oxoethoxy)ethyl)carbamate (20). The synthesis of 20 was carried

out as described for 8-bicyclo[2.2.2]octylxanthine 19, following

P

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Upon completion of the reaction, checked by TLC and LC/MS,

DMF was evaporated to dryness and the resulting residue was taken

up in water (20 mL) and extracted with EtOAc. The organic layer was

collected and washed with 1 M HCl, sat. NaHCO₃, and brine. The

organic layer was then dried over anhydrous Na₂SO₄, ﬁltered, and

evaporated to dryness to give the crude as pale yellow oil. Puriﬁcation

by ﬂash column chromatography using a gradient of 96:4 to 94:6

DCM/MeOH gave the title compound as an oﬀ-white solid (76 mg,

56%). LC/MS m/z calcd for C₂₄H₃₆N₅O₅[MH]⁺: 474.20, found

474.30 t_R= 3.57 min (method A). ¹H NMR (CDCl₃) δ 12.25 (s, 1H),

6.33 (t, J = 6.0 Hz, 1H), 4.08 (t, J = 7.6, 6.1 Hz, 2H), 4.01 (t, J = 6.7

Hz, 2H), 3.69 (s, 3H), 3.51 (q, J = 6.0 Hz, 2H), 2.52 (t, J = 6.2 Hz,

2H), 2.10−2.02 (m, 6H), 1.87 (m, 6H), 1.79 (h, J = 7.4 Hz, 2H),

1.74−1.64 (h, J = 7.4 Hz, 2H), 0.95 (t, J = 7.4 Hz, 6H). ¹³C NMR

(CDCl₃) δ 177.1, 173.5, 161.2, 155.5, 151.2, 149.0, 107.1, 51.9, 45.3,

43.3, 38.9, 34.9, 33.9, 33.7, 30.1, 28.5, 21.5, 11.6, 11.3.

3-(4-(2,6-Dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl)-

bicyclo[2.2.2]octane-1-carboxamido)propanoic acid (27). Methyl

3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-purin-8-yl)-

bicyclo[2.2.2]octane-1-carboxamido)propanoate (26) (76 mg, 160

mmol, 1.0 equiv) was dissolved in MeOH (5 mL). To the mixture

was added a solution of LiOH·H₂O (33.67 mg, 802 μmol, 5.0 equiv)

in water (5 mL). The resulting mixture was stirred at rt. for 40 min.

By that time, TLC and LC/MS showed that the reaction was

completed. MeOH was then removed under reduced pressure, and

the water layer was acidiﬁed with 2 M HCl to pH = 1. The water layer

was extracted with EtOAc (2 × 20 mL). The organic layer was

separated, dried over anhydrous MgSO₄, ﬁltered, and evaporated to

dryness to give the title compound as an oﬀ-white solid (61 mg, 84%).

¹H NMR (CDCl₃) δ 12.10 (s, 1H), 6.45 (t, J = 6.0 Hz, 1H), 4.08 (t, J

(method A). HRMS (TOF ES⁺) calcd for C₃₄H₅₅N₈O₈703.4137,

found 703.4155; calcd for C₃₄H₅₅N₈O₈Na 725.3957, found 725.3974.

¹H NMR (CDCl₃) δ 12.05 (s, 1H), 7.57 (s, 1H), 7.10 (s, 1H), 6.71

(s, 1H), 5.62 (s, 1H), 4.07 (t, J = 6.1 Hz, 2H), 4.01−3.93 (m, 4H),

3.59−3.50 (m, 4H), 3.43−3.38 (m, 4H), 3.38−3.32 (m, 2H), 2.56−

2.42 (m, 2H), 2.07−1.98 (m, 6H), 1.90−1.82 (m, 6H), 1.78 (h, J =

7.4 Hz, 2H), 1.67 (h, J = 7.4 Hz, 2H), 1.43 (s, 9H), 0.94 (t, J = 7.5

Hz, 6H). ¹³C NMR (CDCl₃) δ 161.5, 160.9, 156.5, 155.4, 151.2,

148.8, 107.0, 101.7, 79.9, 70.5, 45.3, 43.2, 39.9, 39.0, 35.9, 35.8, 33.9,

30.1, 28.6, 28.4, 21.5, 11.6, 11.3. Analytical RP-HPLC t_R= 5.62 min,

purity >98% (method C).

tert-Butyl(1-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octan-1-yl)-1,7-dioxo-9,12-dioxa-2,6-dia-

zatetradecan-14-yl)carbamate (22). The title compound was

synthesized as described for 8-bicyclo[2.2.2]octylxanthine 19,

following general procedure A: to a solution of 2,2-dimethyl-4-oxo-

3,8,11-trioxa-5-azatridecan-13-oic acid (11 mg, 42 μmol, 1.0 equiv) in

DMF (0.7 mL) was added DIPEA (15 μL, 85 μmol, 4.0 equiv)

followed by COMU (20 mg, 46 μmol, 1.10 equiv) and intermediate

18b (21 mg, 46 μmol, 1.10 equiv) previously dissolved in DMF (0.5

mL). Puriﬁcation by ﬂash column chromatography using a gradient of

97:3 to 95:5 DCM/MeOH gave the title compound as colorless oil

(15 mg, 39%). LC/MS m/z calcd for C₃₄H₅₆N₇O₈[MH]⁺: 690.20,

found 690.40 t_R= 3.57 min (method A). HRMS (TOF ES⁺) calcd for

C₃₄H₅₆N₇O₈690.4185, found 690.4199; calcd for C₃₄H₅₆N₇O₈Na

1

712.4004, found 712.4020. H NMR (CDCl₃) δ 11.86 (s, 1H), 7.17

(t, J = 6.6 Hz, 1H), 6.86 (t, J = 6.8 Hz, 1H), 4.92 (br s, 1H), 4.10 (t, J

= 6.5 Hz, 2H), 4.04−3.99 (m, 4H), 3.71−3.66 (m, 2H), 3.66−3.63

(m, 2H), 3.57 (t, J = 5.3 Hz, 2H), 3.35 (dt, J = 10.5, 5.7 Hz, 4H), 3.26

(q, J = 6.1 Hz, 2H), 2.10−2.03 (m, 6H), 1.97−1.90 (m, 6H), 1.79 (m,

2H), 1.74−1.67 (m, 2H), 1.67−1.60 (m, 2H), 1.44 (s, 9H), 0.96 (t, J

= 7.5 Hz, 6H). ¹³C NMR (CDCl₃) δ 177.5, 171.2, 166.7, 156.1, 155.5,

151.2, 148.7, 106.9, 75.4, 71.2, 70.5, 70.5, 70.5, 70.1, 43.3, 39.1, 35.4,

35.3, 33.9, 30.2, 29.7, 28.6, 28.5, 21.5, 21.5, 11.6, 11.3. Analytical RP-

HPLC t_R= 5.92 min, purity >99% (method C).

= 6.6 Hz, 2H), 3.98 (t, J = 7.0 Hz, 2H), 3.58 (q, J = 6.0 Hz, 2H), 2.66

(t, J = 4.9 Hz, 2H), 2.07−2.00 (m, 6H), 1.96−1.91 (m, 6H), 1.78 (h,

J = 7.0 Hz, 2H), 1.65 (h, J = 7.0 Hz, 2H), 0.94 (t, J = 7.4, 6H). ¹³C

NMR (CDCl₃) δ 178.2, 176.6, 161.4, 156.4, 150.8, 149.7, 106.5, 45.5,

43.6, 38.9, 34.6, 33.8, 30.2, 28.5, 21.4, 11.4, 11.3.

tert-Butyl(2-(2-((3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propyl)amino)-

2-oxoethoxy)ethyl)carbamate (23). The synthesis of 23 was carried

out as described for 8-bicyclo[2.2.2]octylxanthine 20, following

general procedure A: to a solution of 2-(2-((tert-butoxycarbonyl)-

amino)ethoxy)acetic acid (13 mg, 59.30 μmol, 1.0 equiv) in DMF

(0.7 mL) was added DIPEA (21 mL, 118.6 μmol, 4.0 equiv), followed

by COMU (27.93 mg, 65 μmol, 1.10 equiv) and intermediate 18b

(29 mg, 65.23 μmol, 1.10 equiv) previously dissolved in DMF (0.5

mL). Puriﬁcation by ﬂash column chromatography using a gradient of

96:4 to 95:5 DCM/MeOH gave the title compound as colorless oil

(14 mg, 37%). LC/MS m/z calcd for C₃₂H₅₂N₇O₇[MH]⁺: 646.20,

found 646.40 t_R= 2.82 min (method A). HRMS (TOF ES⁺) calcd for

C₃₂H₅₂N₇O₇646.3923, found 646.3937; calcd for C₃₂H₅₂N₇O₇Na

tert-Butyl(2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propanamido)-

ethyl)carbamate (28). Following general procedure A, to a solution

of compound 27 (61.00 mg, 133 μmol, 1 equiv) in DMF (1 mL) were

added DIPEA (92 μL, 531 μmol, 4.0 equiv), tert-butyl (2-

aminoethyl)carbamate (21.27 mg, 133 μmol, 1.0 equiv), and BOP

(88 mg, 199 mmol, 1.50 equiv). Puriﬁcation by ﬂash column

chromatography using a gradient of 96:4 to 94:6 DCM/MeOH gave

the title compound as an oﬀ-white solid (62 mg, 70%). LC/MS m/z

calcd for C₃₀H₄₈N₇O₆[MH]⁺: 602.20, found 602.40 t_R= 3.62 min

1

(Method A). H NMR (CDCl₃) δ 6.86 (br s, 1H), 6.65 (br s, 1H),

5.02 (br s, 1H), 4.10 (t, J = 6.4 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H),

3.57−3.48 (m, 2H), 3.39−3.32 (m, 2H), 3.31−3.22 (m, 2H), 2.46−

2.37 (m, 2H), 2.10−2.00 (m, 6H), 1.95−1.85 (m, 6H), 1.79 (h, J =

7.3 Hz, 2H), 1.68 (h, J = 7.4 Hz, 2H), 1.44 (s, 9H), 0.95 (t, J = 7.5

Hz, 6H).

1

668.3742, found 668.3758. H NMR (CDCl₃) δ 12.08 (s, 1H) 7.43

(br s, 1H), 6.52 (br s, 1H), 5.54 (br s, 1H), 4.09 (t, J = 6.0 Hz, 2H),

4.01 (t, J = 6.3 Hz, 2H), 3.98 (s, 2H), 3.58 (t, J = 5.0 Hz, 2H), 3.40−

3.35 (m, 2H), 3.34−3.27 (m, 4H), 2.11−2.04 (m, 6H), 1.96−1.90

(m, 6H), 1.79 (h, J = 6.5 Hz, 2H), 1.71 (h, J = 6.5 Hz, 2H), 1.66−

1.57 (m, 2H), 1.43 (s, 9H), 0.95 (t, J = 7.5 Hz, 6H). ¹³C NMR

(CDCl₃) δ 178.1, 170.5, 161.1, 156.4, 155.5, 151.2, 148.8, 106.9, 79.4,

71.4, 70.5, 45.4, 43.3, 40.7, 39.2, 35.4, 34.9, 33.9, 30.2, 29.7, 28.6,

21.5, 21.5, 11.6, 11.3. Analytical RP-HPLC t_R= 5.95 min, purity >99%

(method C).

tert-Butyl(3-((3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propyl)amino)-3-

oxopropyl)carbamate (24). The synthesis of compound 24 was

carried out as described for 8-bicyclo[2.2.2]octylxanthine 21,

following general procedure A: to a solution of Boc-β-Ala-OH (12

mg, 61 μmol, 1.0 equiv) in DMF (0.5 mL) was added DIPEA (21 μL,

122 μmol, 2.0 equiv) followed by COMU (29 mg, 67 μmol, 1.10

equiv) and intermediate 18b (30 mg, μmol, 1.10 equiv) previously

dissolved in DMF (0.5 mL). Puriﬁcation by ﬂash column

chromatography using a gradient of 96:4 to 94:6 DCM/MeOH

gave the title compound as an oﬀ-white solid (36 mg, 92%). LC/MS

tert-Butyl(1-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octan-1-yl)-1,5,10-trioxo-12-oxa-2,6,9-tria-

zatetradecan-14-yl)carbamate (30). Following general procedure B,

compound 2-(3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-1H-

purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propanamido)ethan-

1-aminium chloride (29) was synthesized reacting 28 (64 mg, 1.0

equiv) with 4 M HCl in dioxane (1 mL). The resulting intermediate

29 was used for the subsequent step without further puriﬁcation. The

synthesis of the title compound 30 was carried out following general

procedure A, reacting a solution of 2-(3-(4-(2,6-dioxo-1,3-dipropyl-

2,3,6,9-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-

carboxamido)propanamido)ethan-1-aminium chloride (29) (54 mg,

100 μmol, 1.10 equiv) in DMF (0.5 mL) with a solution of 2-(2-

((tert-butoxycarbonyl)amino)ethoxy)acetic acid (20 mg, 91 μmol, 1.0

equiv), DIPEA (64 μL, 365 μmol, 4.0 equiv), and COMU (43 mg,

100 μmol, 1.10 equiv) in DMF (0.5 mL). Puriﬁcation by ﬂash column

chromatography using a gradient of 97:3 to 94:6 DCM/MeOH gave

the title compound as an oﬀ-white solid (37 mg, 58%). LC/MS m/z

calcd for C₃₄H₅₅N₈O₈[MH]⁺: 703.20, found 703.40 t_R= 3.37 min

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

m/z calcd for C₃₁H₅₀N₇O₆[MH]⁺: 616.37, found 616.40 t_R= 3.43

min (method A). H NMR (CDCl₃) δ 6.58−6.49 (m, 2H), 5.20 (s,

1H), 4.09 (t, J = 6.5 Hz, 2H), 4.02 (t, J = 6.7 Hz, 2H), 3.41 (t, J = 5.3

Hz, 2H), 3.31−3.23 (m, 4H), 2.45 (t, J = 6.1 Hz, 2H), 2.11−2.04 (m,

6H), 1.96−1.89 (m, 6H), 1.79 (h, J = 6.5 Hz, 2H), 1.70 (h, J = 6.4

Hz, 2H), 1.65−1.56 (m, 2H), 1.42 (s, 9H), 0.96 (t, J = 7.4 Hz, 6H).

¹³C NMR (CDCl₃) δ 177.9, 161.1, 157.9, 156.4, 155.5, 151.2, 148.8,

δ 177.9, 159.0, 151.5, 150.7, 136.6, 128.6, 127.3, 126.4, 88.3, 45.2,

42.1, 36.3, 27.8, 20.9, 11.3. (1R,3R,5S)-N-(6-Amino-1-benzyl-2,4-

dioxo-3-propyl-1,2,3,4-tetrahydropyrimidin-5-yl)adamantane-1-car-

boxamide (0.254 g, 0.58 mmol, 1.0 equiv) was dissolved in propan-2-

ol (7 mL). To the resulting mixture was added 1 M KOH (7 mL),

and the resulting solution was heated to reﬂux for 1 h. Upon

completion of the reaction, the solvent was removed under reduced

pressure and the residue was taken up in water and extracted with

DCM (2 × 20 mL). The organic layers were combined, dried over

anhydrous MgSO₄, ﬁltered, and evaporated to dryness to give the title

compound as a pale yellow solid. Subsequent recrystallization from

hot ethanol and water gave the title compound as a ﬂuﬀy oﬀ-white

solid (0.232 g, 95%). LC/MS m/z calcd for C₂₅H₃₁N₄O₂[MH]⁺:

419.20, found 419.20 t_R= 4.73 min (method A). ¹H NMR (CDCl3)

δ 12.10 (s, 1H), 7.60−7.56 (m, 2H), 7.34−7.24 (m, 3H), 5.32 (s,

2H), 4.04 (t, J = 6.3 Hz, 2H), 2.19−2.09 (m, 9H), 1.90−1.77 (m,

6H), 1.69 (h, J = 6.5 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR

(CDCl₃) δ 162.8, 155.5, 151.3, 148.9, 136.9, 129.3, 128.5, 127.8,

106.7, 46.9, 43.3, 41.0, 36.6, 35.9, 28.3, 21.4, 11.4.

Methyl 4-((2-((tert-Butoxycarbonyl)amino)ethyl)carbamoyl)-

benzoate (37). 4-(Methoxycarbonyl) benzoic acid 36 (0.200 g,

1.11 mmol, 1.0 equiv) was dissolved in DMF (2 mL). To the resulting

solution were added DIPEA (0.21 mL, 1.22 mmol, 1.10 equiv),

COMU (0.523 g, 1.22 mmol, 1.10 equiv), and tert-butyl (2-

aminoethyl) carbamate (0.19 mL, 1.22 mmol, 1.10 equiv). The

reaction was stirred at rt. for 20 min. Upon completion of reaction,

checked by LC−MS and TLC, cold water (7 mL) was added and a

pale pink precipitate was developed, which was collected by suction

ﬁltration and washed with cold water. The title compound was

obtained after air drying (0.270 g, 75%). LC/MS m/z calcd for

C₁₆H₂₂N₂O₅[MH]⁺: 223.15, found 223.10 (−Boc); t_R= 2.69 min

(method A). ¹H NMR (CDCl₃)) δ 8.08 (d, J = 8.0 Hz, 2H), 7.89 (d, J

= 7.3 Hz, 2H), 7.43 (br s, 1H), 5.00 (br s, 1H), 3.94 (s, 3H), 3.56 (dt,

J = 6.5, 4.8 Hz, 2H), 3.45−3.39 (m, 2H), 1.43 (s, 9H). ¹³C NMR

(CDCl₃) δ 166.9, 166.5, 155.6, 138.2, 132.8, 129.9, 127.2, 80.4, 52.5,

28.5.

1

106.9, 47.7, 45.4, 43.3, 39.2, 36.8, 35.8, 35.5, 33.9, 30.2, 29.8, 28.6,

28.5, 21.5, 21.5, 11.6, 11.3.

tert-Butyl(3-((3-((3-(4-(2,6-dioxo-1,3-dipropyl-2,3,6,9-tetrahydro-

1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamido)propyl)amino)-

3-oxopropyl)amino)-3-oxopropyl)carbamate (25b). Following gen-

eral procedure B, the intermediate 3-((3-(4-(2,6-dioxo-1,3-dipropyl-

2,3,6,7-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-

carboxamido)propyl)amino)-3-oxopropan-1-aminium chloride was

synthesized and used for the subsequent step without further

puriﬁcation. The synthesis of the title compound 25b was carried

out as described for xanthine 24, following general procedure A: to a

solution of Boc-β-Ala-OH (11 mg, 58 μmol, 1.0 equiv), DIPEA (41

μL, 233 μmol, 4.0 equiv), and COMU (22 mg, 64 μmol, 1.10 equiv)

in DMF (0.5 mL) was added a solution of 3-((3-(4-(2,6-dioxo-1,3-

dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-

carboxamido)propyl)amino)-3-oxopropan-1-aminium chloride (33

mg, 64 μmol, 1.10 equiv) in DMF (0.5 mL). Puriﬁcation by ﬂash

column chromatography using a gradient of 96:4 to 93:7 DCM/

MeOH gave the title compound as an oﬀ-white solid (12 mg, 31%).

LC/MS m/z calcd for C₃₄H₅₅N₈O₇[MH]⁺: 687.20, found 687.40 t_R=

3.38 min (method A). HRMS (TOF ES⁺) calcd for C₃₄H₅₅N₈O₇

687.4188, found 687.4202; calcd for C₃₄H₅₅N₈O₇Na 709.4008, found

709.4026. ¹H NMR (CD₃OD) δ 4.07 (t, J = 6.9 Hz, 2H), 3.95 (t, J =

6.9 Hz, 2H), 3.46 (t, J = 6.7 Hz, 2H), 3.31 (s, 2H), 3.22 (dt, J = 6.7,

4.2 Hz, 4H), 2.39 (dt, J = 15.7, 6.8 Hz, 4H), 2.06−1.99 (m, 6H),

1.95−1.89 (m, 6H), 1.78 (h, J = 6.9 Hz, 2H), 1.72−1.61 (m, 4H),

1.44 (s, 9H), 0.96 (t, J = 7.3 Hz, 6H). ¹³C NMR (CD₃OD) δ 190.9,

181.9, 159.9, 156.0, 153.9, 143.9, 109.1, 108.3, 83.8, 82.3, 73.34, 57.9,

40.2, 36.9, 34.9, 31.0, 29.3, 28.8, 22.4, 22.3, 19.4, 11.5, 11.3. Analytical

RP-HPLC t_R= 5.30 min, purity >99% (method C).

4-((2-((tert-Butoxycarbonyl)amino)ethyl)carbamoyl)benzoic

Acid (38). Methyl 4-((2-((tert-butoxycarbonyl)amino)ethyl)-

carbamoyl)benzoate (37) (0.260 g, 0.81 mmol, 1.0 equiv) was

dissolved in THF (10 mL). To the resulting mixture was added 1 M

NaOH (8 mL), and the solution was stirred at rt. for 3 h. Upon

completion of the reaction checked by TLC and LCMS, the organic

layer was removed under reduced pressure. The dropwise addition of

conc. HCl gave the title compound as white solid, which was obtained

after suction ﬁltration and vacuum drying (0.223, 90%). LC/MS m/z

calcd for C₁₅H₂₁N₂O₅[MH]⁺: 209.14, found 209.10 (−Boc); calcd

for C₁₅H₂₁N₂O₅Na [MH]⁺331.14, found 331.10; t_R= 3.18 min.

(method A). ¹H NMR (CD₃OD) δ 8.08 (d, 2H), 7.89 (d, J = 8.3 Hz,

2H), 3.46 (t, J = 5.6 Hz, 2H), 3.28 (t, J = 6.1 Hz, 2H), 1.41 (s, 9H).

¹³C NMR (CD₃OD) δ 169.0, 158.8, 139.6, 134.8, 130.8, 128.4, 80.2,

5,6-Diamino-1-benzyl-3-propylpyrimidine-2,4(1H,3H)-dione

(32).⁷²A solution of 6-amino-1-benzyl-5-nitroso-3-propylpyrimidine-

2,4(1H,3H)-dione (31) (0.250 g, 0.87 mmol, 1 equiv) in water (25

mL) and 30% NH₃OH (10 mL) was heated to 85 °C, and sodium

dithionite (3.0 equiv) was added in small portions until the color

disappeared. The reaction was cooled to rt., and the mixture was

extracted with EtOAc. The organic phase was dried over anhydrous

MgSO₄, ﬁltered, and evaporated to dryness to give the title compound

as yellow solid (0.218 g, 92%). LC/MS m/z calcd for C₁₄H₁₉N₄O₂

1

[MH]⁺: 275.14, found 275.10 t_R= 2.90 min (method A). H NMR

(DMSO-d₆) δ 7.36−7.30 (m, 2H), 7.27−7.22 (m, 1H), 7.21−7.15

(m, 2H), 6.13 (s, 2H), 5.13 (s, 2H), 3.76 (t, J = 6.2 Hz, 2H), 2.95 (br

s, 2H), 1.51 (h, J = 7.3 Hz, 2H), 0.82 (t, J = 7.5 Hz, 3H). ¹³C NMR

(DMSO-d₆) δ 158.9, 149.8, 144.1, 136.9, 128.4, 127.1, 126.4, 96.3,

44.9, 42.0, 20.9, 11.2.

41.5, 40.8, 28.7.

tert-Butyl(2-(4-((3-bromopropyl)carbamoyl)benzamido)ethyl)-

carbamate (39). To a solution of 4-((2-((tert-Butoxycarbonyl)-

amino)ethyl)carbamoyl)benzoic acid 38 (0.100 g, 0.32 mmol, 1.0

equiv) in DMF (2 mL) were added COMU (0.153 g, 0.36 mmol, 1.10

equiv) and 3-bromopropyl amine hydrobromide (0.092 g, 0.42 mmol,

1.30 equiv). DIPEA (0.112 mL, 0.65 mmol, 2.0 equiv) was

subsequently added to the reaction mixture dropwise. The resulting

mixture was stirred at rt. for 15 min. Completion of the reaction was

checked by TLC (R_f0.35 in DCM/MeOH 95:5) and LCMS. The

resulting mixture was diluted with water and extracted with EtOAc.

The organic layer was subsequently washed with 1 M HCl (10 mL),

dried over MgSO₄, ﬁltered, and concentrated to dryness to aﬀord the

crude product as orange oil. Puriﬁcation by ﬂash column

chromatography on a gradient of 97:3 to 94:6% DCM/MeOH

8-((1R,3R,5S)-Adamantan-1-yl)-3-benzyl-1-propyl-3,7-dihydro-

1H-purine-2,6-dione (33). To a solution of xanthine 32 (0.201 g, 0.73

mmol, 1.0 equiv) in DMF (1 mL) was added DIPEA (0.13 mL, 0.73

mmol, 1.10 equiv). The resulting mixture was added to a solution of

1-adamantane carboxylic acid (0.120 g, 0.67 mmol, 1 equiv) and

COMU (0.314 g, 0.73 mmol, 1.10 equiv) in DMF (1 mL). The

resulting solution was stirred at rt. for 15 min. Upon completion of

the reaction (LC−MS), cold water was added to the reaction mixture

and subsequent precipitation of a white solid occurred. The solid was

ﬁltrated, washed with cold water, and dried. The title intermediate

(1R,3R,5S)-N-(6-amino-1-benzyl-2,4-dioxo-3-propyl-1,2,3,4-tetrahy-

dropyrimidin-5-yl)adamantane-1-carboxamide was obtained as an oﬀ-

white solid (0.254 g, 87%).⁷³LC/MS m/z calcd for C₂₅H₃₃N₄O₄

1

[MH]⁺: 437.14, found 437.20 t_R= 2.99 min (method A). H NMR

1

aﬀorded the title product as an oﬀ-white solid (0.085 g, 61%). H

(DMSO-d₆) δ 7.83 (s, 1H), 7.37−7.31 (m, 2H), 7.27−7.22 (m, 1H),

7.23−7.17 (m, 2H), 6.28 (s, 2H), 5.16 (s, 2H), 3.71 (t, J = 7.2, 2H),

2.00−1.92 (m, 3H), 1.89−1.86 (m, 5Hl), 1.71−1.60 (m, 6H), 1.49

(h, J = 7.3 Hz, 2H), 0.82 (t, J = 7.5 Hz, 3H). ¹³C NMR (DMSO-d₆))

NMR (DMSO-d₆) δ 8.70 (t, J = 5.6 Hz, 1H), 8.55 (t, J = 5.3 Hz, 1H),

7.83 (s, 4H), 3.51 (t, J = 6.6 Hz, 2H), 3.40−3.33 (m, 2H), 3.31−3.26

(m, 2H), 3.10 (t, J = 6.1 Hz, 2H), 2.05 (p, J = 6.7 Hz, 2H), 1.31 (s,

R

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

9H). ¹³C NMR (DMSO-d₆) δ 167.6, 155.1, 129.9, 128.2, 36.8, 36.0,

34.3, 33.1, 28.8 (two quaternary carbon not observed).

mL). Puriﬁcation by ﬂash column chromatography using a gradient of

96:4 to 93:7 DCM/MeOH gave the title compound as an oﬀ-white

solid (18 mg, 47%). LC/MS m/z calcd for C₃₉H₅₅N₈O₇[MH]⁺:

747.40, found 747.40; t_R= 3.71 min (method A). ¹H NMR (CD₃OD)

δ 7.93 (s, 4H), 4.59 (s, 1H), 4.26 (t, J = 6.6 Hz, 2H), 3.95 (t, J = 6.3

Hz, 2H), 3.52 (t, J = 5.8 Hz, 2H), 3.48−3.39 (m, 4H), 3.36 (s, 2H),

2.37 (t, J = 6.7 Hz, 2H), 2.10 (q, J = 6.5 Hz, 2H), 2.02−1.96 (m, 9H),

1.81−1.70 (m, 6H), 1.69−1.62 (m, 2H), 1.42 (s, 9H), 0.94 (t, J = 7.5

Hz, 3H). ¹³C NMR (CD₃OD) δ 187.4, 181.9, 169.2, 163.6, 156.0,

152.9, 149.5, 138.7, 138.2, 128.6, 128.5, 107.9, 80.2, 43.9, 42.2, 41.9,

40.9, 40.1, 38.0, 37.6, 37.4, 36.9, 29.5, 28.9, 28.7, 22.3, 11.6.

(E)-N-(2-(6-(2-(4-(2-(5,5-Diﬂuoro-7-(thiophen-2-yl)-5H-4l⁴,5l⁴-

dipyrrolo[1,2-c:2′,1′-f ][1,3,2]diazaborinin-3-yl)vinyl)phenoxy)-

acetamido)hexanamido)ethyl)-4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-

tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamide

(43a). Following general procedure C, amine congener 18a (0.5 mg,

1.10 equiv) was converted to the BODIPY 630/650 conjugate.

Puriﬁcation by RP-HPLC (method B) gave, after lyophilization, the

title compound as a blue solid (0.4 mg, 39%). LC/MS m/z calcd for

C₅₁H₆₁BF₂N₉0₆S [MH]⁺: 976.30, found 976.30 t_R= 3.98 min

(method A). HRMS (TOF ES⁺) calcd for C₅₁H₆₁BF₂N₉0₆S [MH]⁺:

976.4530, found 976.4530; calcd for C₅₁H₆₁BF₂N₉0₆SNa [M + Na]⁺:

998.4349, found 998.4335. Analytical RP-HPLC t_R= 6.86 min, purity

>99% (method C).

1-(6-((2-(4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-

8-yl)bicyclo[2.2.2]octane-1-carboxamido)ethyl)amino)-6-oxohex-

yl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-

ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate (43b).

Following general procedure C, amine congener 18a (0.5 mg, 1.10

equiv) was converted to the Sulfo-Cy5 conjugate 43b. Puriﬁcation by

RP-HPLC (method B) gave after lyophilization the title compound as

a bright blue solid (0.5 mg, 56%). LC/MS m/z calcd for

C₅₄H₇₁N₈0₁₀S₂[MH]⁺: 1055.30, found 1055.30; t_R= 3.09 min

(method A). HRMS (TOF ES⁺) calcd for C₅₄H₇₁N₈0₁₀S₂[MH]⁺:

1055.4729, found 1055.4705; calcd for C₅₄H₇₁N₈0₁₀S₂Na [M + Na]⁺:

1077.4549, found 1077.4527. Analytical RP-HPLC t_R= 4.93 min,

purity >99% (method C).

8-((1R,3R,5S)-Adamantan-1-yl)-3-benzyl-1-propyl-7-((2-

(trimethylsilyl)ethoxy)methyl)-3,7-dihydro-1H-purine-2,6-dione

(34). Potassium carbonate was added to a solution of xanthine 33

(0.232 g, 0.55 mmol, 1.0 equiv) in DMF (3 mL), and the resulting

mixture was stirred at rt. for 2 h. (2-(Chloromethoxy)ethyl)-

trimethylsilane (0.110 g, 0.67 mmol, 1.20 equiv) was added dropwise,

and the solution was stirred at rt. for additional 2 h. TLC showed

complete consumption of the starting material. The reaction was then

evaporated to dryness, and the residue was adsorbed on isolute.

Puriﬁcation by ﬂash column chromatography using a gradient of

100:0 to 80:20 PE/EtOAc gave the title compound as colorless oil,

1

which solidiﬁed upon cooling (0.200 g, 66%). H NMR (CDCl₃) δ

7.61−7.56 (m, 2H), 7.34−7.24 (m, 3H), 5.91 (s, 2H), 5.26 (s, 2H),

3.94 (t, J = 6.9 Hz, 2H), 3.70 (t, J = 6.9 Hz, 2H), 2.23−2.17 (m, 6H),

2.15−2.09 (m, 3H), 1.85−1.78 (m, 6H), 1.64 (h, J = 7.2 Hz, 2H),

0.96−0.90 (m, 5H), −0.02 (s, 9H). ¹³C NMR (CDCl₃) δ 161.6,

155.3, 151.3, 147.0, 137.0, 129.5, 128.5, 127.9, 107.6, 73.6, 66.5, 46.5,

43.1, 40.9, 37.9, 36.6, 28.5, 21.5, 18.2, 11.5, −1.3.

8-((1R,3R,5S)-Adamantan-1-yl)-1-propyl-7-((2-(trimethylsilyl)-

ethoxy)methyl)-3,7-dihydro-1H-purine-2,6-dione (35).¹⁰⁶Xanthine

34 (0.188 g, 0.34 mmol, 1.0 equiv) was dissolved in methanol, and to

the solution was added 10% Pd/C (100 mg), followed by ammonium

formate (3.53 mmol, 15 equiv). The ﬂask was equipped with a

condenser with a balloon with N₂at the top and heated to 140 °C for

9 h. The completion of the reaction was monitored by TLC. The

reaction mixture was then cooled to rt. and ﬁltered through Celite.

The pad was washed with MeOH and DCM. The colorless solution

was concentrated to dryness to give the title compound as an oﬀ-

white solid (0.105 g, 67%). ¹H NMR (CDCl₃) δ 5.93 (s, 2H), 3.95 (t,

J = 7.2, 5.4 Hz, 2H), 3.71 (t, J = 7.2 Hz, 2H), 2.19 (s, 6H), 2.09 (s,

3H), 1.78 (s, 6H), 1.66 (h, J = 7.6 Hz, 2H), 0.98−0.89 (m, 5H),

−0.02 (s, 9H). ¹³C NMR (CDCl₃) δ 162.1, 155.9, 107.7, 73.6, 66.5,

42.5, 40.8, 37.9, 36.5, 28.4, 21.5, 18.2, 11.5, −1.3.

tert-Butyl(2-(4-((3-(8-((3R,5R,7R)-adamantan-1-yl)-2,6-dioxo-1-

propyl-7-((2-(trimethylsilyl)ethoxy)methyl)-1,2,6,7-tetrahydro-3H-

purin-3-yl)propyl)carbamoyl)benzamido)ethyl)carbamate (40). To

a solution of xanthine 35 (105 mg, 1.0 equiv) in DMF (2 mL) was

added K₂CO₃(42 mg, 1.3 equiv). The resulting mixture was stirred at

rt. for 1 h. After that time, a solution of tert-butyl (2-(4-((3-

bromopropyl)carbamoyl)benzamido)ethyl)carbamate (39) (113 mg,

1.15 equiv) in DMF (1 mL) was added to the reaction mixture, which

was stirred at 40 °C for 23 h. The reaction was subsequently ﬁltered

and concentrated to dryness. The crude material was adsorbed on

isolute and puriﬁed by ﬂash column chromatography using a gradient

of 50:50 to 20:80 PE/EtOAc to give the title compound as oﬀ-white

foam (90 mg, 49%). ¹H NMR (CDCl₃) δ 7.90 (s, 4H), 7.78 (t, J = 6.1

Hz, 1H), 7.47 (s, 1H), 5.92 (s, 2H), 5.14 (br s, 1H), 4.26 (t, J = 5.6

Hz, 2H), 3.98 (t, J = 7.3 Hz, 2H), 3.71 (t, J = 8.0 Hz, 2H), 3.56 (q, J =

4.8 Hz, 2H), 3.45−3.37 (m, 4H), 2.13−2.08 (m, 6H), 2.08−1.97 (m,

5H), 1.76−1.60 (m, 8H), 1.42 (s, 9H), 0.93 (m, 5H), −0.03 (s, 9H).

¹³C NMR (CDCl₃) δ 166.9, 161.9, 155.1, 151.8, 147.0, 137.7, 136.7,

(E)-N-(20-(4-(2-(5,5-Diﬂuoro-7-(thiophen-2-yl)-5H-4l⁴,5l⁴-

dipyrrolo[1,2-c:2′,1′-f ][1,3,2]diazaborinin-3-yl)vinyl)phenoxy)-

4,8,12,19-tetraoxo-3,7,11,18-tetraazaicosyl)-4-(2,6-dioxo-1,3-di-

propyl-2,3,6,9-tetrahydro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-car-

boxamide (44a). Following general procedure B, compound 25a (5

mg, 1.0 equiv) was subjected to acidolysis, and after the removal of

the solvent, the resulting intermediate (as HCl salt) was used for the

next step without further puriﬁcation (4 mg, quantitative). LC−MS

m/z calcd for C₂₈H₄₅N₈O₅[MH]⁺: 573.34, found 573.30 t_R= 3.13

min. Following general procedure C, the resulting amine congener

was converted to the BODIPY 630/650 conjugate. Puriﬁcation by

RP-HPLC (method B) gave, after lyophilization, the title compound

as a blue solid (0.9 mg, 70%). LC/MS m/z calcd for

C₅₇H₇₁BF₂N₁₁0₈S [MH]⁺: 1118.30, found 1118.40 t_R= 3.78 min

(method A). HRMS (TOF ES⁺) calcd for C₅₇H₇₁BF₂N₁₁0₈S [MH]⁺:

1118.5273 found 1118.5252; calcd for C₅₇H₇₁BF₂N₁₁0₈SNa [M +

Na]⁺: 1140.5092, found 1140.506. Analytical RP-HPLC t_R= 6.73

min, purity >99% (method C).

107.7, 80.2, 73.7, 66.6, 43.1, 40.9, 40.4, 37.9, 36.4, 35.9, 28.5, 28.3,

21.5, 18.2, 11.5, −1.3.

N¹-(3-(8-((3R,5R,7R)-Adamantan-1-yl)-2,6-dioxo-1-propyl-

1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)-N⁴-(2-aminoethyl)-

terephthalamide (41). The synthesis of the title compound 41 was

achieved by following general procedure B. The title compound 31

was used for the subsequent step without any further puriﬁcation (29

mg, quantitative). LC−MS m/z calcd for C₃₁H₄₂N₇O₄[MH]⁺:

576.20, found 576.40; t_R= 3.16 min (method A).

1-(1-(4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-

yl)bicyclo[2.2.2]octan-1-yl)-1,6,10,14-tetraoxo-2,5,9,13-tetraazano-

nadecan-19-yl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sul-

foindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfo-

nate (44b). Following Boc-deprotection of 25a, the resulting amine

congener (0.5 mg, 1.10 equiv) was converted to the Sulfo-Cy5

conjugate 44b by following general procedure C. Puriﬁcation by RP-

HPLC (method B) gave, after lyophilization, the title compound as a

bright blue solid (1.1 mg, quantitative). LC/MS m/z calcd for

C₆₀H₇₉N₁₀0₁₂S₂[MH]⁻: 1195.54, found 1195.60; t_R= 3.05 min

(method A). HRMS (TOF ES⁺) calcd for C₆₀H₈₁N₁₀0₁₂S₂[MH]⁺:

1197.5471, found 1197.5450; calcd for C₆₀H₈₁N₁₀0₁₂S₂Na [M +

Na]⁺: 1219.5291, found 1219.5255. Analytical RP-HPLC t_R= 4.88

min, purity >99% (method C).

tert-Butyl(3-((2-(4-((3-(8-((3R,5R,7R)-adamantan-1-yl)-2,6-dioxo-

1-propyl-1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)carbamoyl)-

benzamido)ethyl)amino)-3-oxopropyl)carbamate (42). The title

compound 42 was synthesized by following general procedure A,

reacting a solution of Boc-β-Ala-OH (10 mg, 1.0 equiv), DIPEA (18

μL, 2.0 equiv) and COMU (24.90 mg, 1.10 equiv) in DMF (0.5 mL)

with a solution of N1-(3-(8-((1R,3R,5S)-adamantan-1-yl)-2,6-dioxo-

1-propyl-1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)-N-4-(2-

aminoethyl)terephthalamide (41) (34 mg, 1.10 equiv) in DMF (0.5

N¹-(3-(8-((3R,5R,7R)-Adamantan-1-yl)-2,6-dioxo-1-propyl-

1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)-N⁴-(2-(6-(2-(4-((E)-2-(5,5-

S

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

diﬂuoro-7-(thiophen-2-yl)-5H-4l⁴,5l⁴-dipyrrolo[1,2-c:2′,1′-f ][1,3,2]-

diazaborinin-3-yl)vinyl)phenoxy)acetamido)hexanamido)ethyl)-

terephthalamide (45a). Following general procedure C, amine

congener 41 (0.7 mg, 1.10 equiv) was converted to the BODIPY 630/

650 conjugate. Puriﬁcation by RP-HPLC (method B) gave, after

lyophilization, the title compound as a blue solid (0.7 mg, 59%). LC/

MS m/z calcd for C₆₀H₆₈BF₂N₁₀0₇S [MH]⁺: 1121.30, found 1121.30

t_R= 4.05 min (method A). HRMS (TOF ES⁺) calcd for

C₆₀H₆₈BF₂N₁₀0₇S [MH]⁺: 1121.5059, found 1121.5060; calcd for

C₆₀H₆₈BF₂N₁₀0₇SNa [M + Na]⁺: 1143.4878, found 1143.4855.

Analytical RP-HPLC t_R= 7.00 min, purity >99% (method C).

1-(6-((2-(4-((3-(8-((3R,5R,7R)-Adamantan-1-yl)-2,6-dioxo-1-prop-

yl-1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)carbamoyl)benzamido)-

ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-tri-

methyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-

ium-5-sulfonate (45b). Following general procedure C, amine

congener 41 (0.6 mg, 1.10 equiv) was converted to the Sulfo-Cy5

conjugate 45b. Puriﬁcation by RP-HPLC (method B), followed by

preparative TLC using 80:20 DCM/MeOH as an eluent, after

lyophilization, the title compound as a bright blue solid (0.9 mg,

83%). LC/MS m/z calcd for C₆₃H₇₈N₉0₁₁S₂[M-2]⁺: 600.76, found

600.8; t_R= 3.19 min (method A). HRMS (TOF ES⁺) calcd for

C₆₃H₇₈N₉0₁₁S₂[MH]⁺: 1200.5257, found 1200.5220; [M-

2]⁺:600.7665, found 600.7680. Analytical RP-HPLC t_R= 5.19 min,

purity >99% (method C).

optimized using PROPKA at pH = 7.0. The structure of the protein

was energy minimized using an OPLS3 force ﬁeld. The docking site

was deﬁned using Glide Grid generation with the barycenter of the

co-crystallized DU172 representing the center of the grid. Tonapofyl-

line 16 and congener 42 were prepared for docking using a LigPrep

tool. Molecular docking of these two ligands was performed using

Glide with XP (extra precision) mode and ﬂexible ligand sampling

with no restriction applied. For both ligands, the highest docking

scoring pose was selected and depicted with PyMOL to include key

binding interactions and distance measurements.

Pharmacology: Materials and Methods. Dulbecco’s modiﬁed

Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin

were purchased from Invitrogen (Carlsbad, CA). Adenosine

deaminase (ADA) and hygromycin B were purchased from Roche

Diagnostics (Mannheim, Germany). Ultima Gold scintillation cock-

tail, [³H] DPCPX (8-cyclopentyl-1,3-dipropylxanthine, [dipropyl-

2,3-³H(N)], speciﬁc activity 137 Ci/mmol), and LANCE→ cAMP kit

were purchased from PerkinElmer Life Sciences (Glen Waverley,

Australia). All plates were obtained from Corning Costar (Corning

Incorporated, Corning, NY, USA). Furimazine was purchased from

Promega (Alexandria, Australia). Adenosine receptor ligands, 8-(4-(4-

(4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine

(PSB-603), N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]-

quinazolin-5-yl]benzene acetamide (MRS1220), 4-(2-[7-Amino-2-

(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol

(ZM241385), and trans-4-((2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-

yl)amino)cyclohexanol (SLV320) were purchased from Tocris

Bioscience (Bristol, UK). 8-Cyclopentyl-1,3-dipropylxanthine

(DPCPX), 5′-(N-ethylcarboxamido)adenosine (NECA), and all

other reagents were purchased from Sigma-Aldrich (Castel Hill,

Australia) and were of analytical quality.

Cell Culture. Chinese Hamster Ovary FlpIn (FlpIn-CHO) cells

stably expressing the human A₁AR were generated as described

previously.¹⁰⁷A₁AR-FlpIn-CHO cell lines were cultured in DMEM,

supplemented with 10% (v/v) FBS and the selection antibiotic

hygromycin B (500 μg/mL) to maintain adenosine receptor

expression. Nontransfected HEK293 adherent (HEK293A) cells

were cultured in DMEM, supplemented with 10% (v/v) FBS. All

cells were cultured at 37 °C in a humidiﬁed incubator containing 5%

CO₂, grown to conﬂuence, and then seeded into 96-well culture plates

at assay-speciﬁc densities. All tissue culture procedures were

performed in a class II laminar ﬂow hood using sterile conditions.

Transfection of NanoLuc-tagged human A₁AR, A_2AAR, A_2BAR, and

A₃AR and SNAP-tagged-hA₁AR in HEK293A cells was performed

using polyethylamine (PEI) at the 4:1 ratio of PEI/DNA. Cells were

transfected with 3 μg of DNA in 10 mm × 20 mm style dishes at a

density of 5 × 10⁶cells per dish and used 48 h following transfection.

Experiments: [³H] DPCPX Binding Assay. A₁AR-FlpIn-CHO cells

were seeded into white 96-well isoplates at 40,000 cells/well and

incubated overnight. Before the experiment, DMEM was replaced

with binding buﬀer (10 mM HEPES, 10 mM ^D-glucose, 145 mM

NaCl, 5 mM KCl, 1 mM MgSO₄, 1.5 mM NaHCO₃, 2 mM CaCl₂, 1

U/mL ADA, pH 7.4), and cells were incubated with increasing

concentrations of competing ligands, followed by the addition of 1

nM [³H] DPCPX (the precise concentration in each experiment was

determined by β-counting). The cells were incubated for 4 h in

binding buﬀer at 37 °C with 5% CO₂. Buﬀer was removed, and cells

were washed three times with ice-cold phosphate buﬀer saline (PBS,

10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl)

and then dissolved in Ultima Gold scintillation liquid (100 μL/well).

Isoplates were sealed and allowed to incubate at rt. for 1 h before

radioactivity was determined by b-counting using a MicroBeta 2

LumiJET microplate counter (PerkinElmer). In all radioligand

binding experiments, nonspeciﬁc binding was determined in the

presence of 1 mM SLV320.

N¹-(3-(8-((3R,5R,7R)-Adamantan-1-yl)-2,6-dioxo-1-propyl-

1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)-N⁴-(2-(3-(6-(2-(4-((E)-2-

(5,5-diﬂuoro-7-(thiophen-2-yl)-5H-4l⁴,5l⁴-dipyrrolo[1,2-c:2′,1′-f ]-

[1,3,2]diazaborinin-3-yl)vinyl)phenoxy)acetamido)hexanamido)-

propanamido)ethyl)terephthalamide (46a). Following general

procedure B, compound 42 (5 mg, 1.0 equiv) was converted to the

corresponding HCl salt intermediate, which, after the removal of the

solvent in vacuo, was used for the subsequent step without further

puriﬁcation. LC/MS m/z calcd for C₃₄H₄₇N₈O₈[MH]⁺: 647.34,

found 647.40 t_R= 3.18 min. Following general procedure C, the

resulting amine congener (0.7 mg, 1.10 equiv) was converted to the

BODIPY 630/650 conjugate. Puriﬁcation by RP-HPLC (method B)

gave the title compound as a blue solid (0.7 mg, 56%). LC−MS m/z

calcd for C₆₃H₇₃BF₂N₁₁0₈S [MH]⁺: 1192.40, found 1192.40 t_R= 3.95

min (method A). HRMS (TOF ES⁺) calcd for C₆₃H₇₃BF₂N₁₁0₈S

[MH]⁺: 1192.5430, found 1192.5433; calcd for C₆₃H₇₃BF₂N₁₁0₈SNa

[M + Na]⁺: 1214.5250, found 1214.5229. Analytical RP-HPLC t_R=

6.98 min, purity >97% (method C).

1-(6-((3-((2-(4-((3-(8-((3R,5R,7R)-Adamantan-1-yl)-2,6-dioxo-1-

propyl-1,2,6,7-tetrahydro-3H-purin-3-yl)propyl)carbamoyl)-

benzamido)ethyl)amino)-3-oxopropyl)amino)-6-oxohexyl)-3,3-di-

methyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)-

penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate (46b). Following

general procedure C, the amine congener (0.6 mg, 1.10 equiv) was

converted to the Sulfo-Cy5 conjugate 46b. Puriﬁcation by RP-HPLC

(method B), followed by preparative TLC using 80:20 DCM/MeOH,

after lyophilization, was isolated the title compound as a bright blue

solid (0.9 mg, 79%). LC−MS m/z calcd for C₆₆H₈₃N₁₀0₁₂S₂[M-2]⁺:

636.3, found 636.3; t_R= 3.17 min (method A). HRMS (TOF ES⁺)

calcd for C₆₆H₈₃N₁₀0₁₂S₂[MH]⁺: 1271.5628, found 1271.5571; calcd

for C₆₆H₈₃N₁₀0₁₂S₂Na [M + Na]⁺: 1293.5447, found 1293.5364; [M-

2]⁺:636.2850, found 636.2861. Analytical RP-HPLC t_R= 5.25 min,

purity >99% (method C).

Molecular Modeling of Tonapofylline (BG9928) and

Compound 16. Molecular docking simulation of tonapofylline

(BG9928) and 16 to the 3.2 Å resolution A₁AR crystal structure was

̈

performed using Schrodinger software suite (release 2019-2). The 3.2

Å A₁AR crystal structure in complex with the irreversible ligand

DU172 was retrieved from the Protein Data Bank (PDB: 5UEN)

depository and was ﬁrstly prepared in PyMOL (2.2.0) as follows: one

copy of the A₁AR-dimer crystal structure was removed and the

covalent bond between the Y271 and irreversible antagonist DU172

was broken to facilitate the deﬁnition of docking site during the Grid

generation step. This structure was subsequently imported on

Maestro and was prepared with the Protein Preparation Wizard

tool. Hydrogen atoms were added, and the H-bonding network was

NanoBRET Binding Assays. HEK293A cells were transiently

transfected to express either the NanoLuc-tagged human A₁AR,

A_2AAR, A_2BAR, and A₃AR. 24 h following transfection, cells were

seeded into 96-well poly-^D-lysine-coated solid white bottom plates at

40,000 cells/well and incubated overnight in serum-free DMEM.

T

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Before the experiment, DMEM was replaced with binding buﬀer. For

saturation and competition assays, the required concentration of

ﬂuorescent ligand, and competing ligands, was added at the same time

and the plates were then incubated at 37 °C for 1 h. After 50 min

incubation, 1 μM furimazine was added to each well and plates were

incubated for additional 10 min (1 h total) at 37 °C. For association

kinetic experiments, 1 μM furimazine was added to each well and

incubated at 37 °C for 15 min in the dark allowing the luminescence

signal to reach equilibrium. The required concentration of the

ﬂuorescent ligand in the presence or absence of 1 μM SLV320 (to

deﬁne nonspeciﬁc binding) was added simultaneously. The plates

were then read immediately, with each well read once every 30 s for

60 min. Bioluminescence emission at two diﬀerent wavelengths was

measured at room temperature (for saturation and competition

binding experiments) or at 37 °C (for kinetic binding assays) using a

PheraSTAR Omega plate reader (BMG Labtech) using 460 nm (80

nm bandpass; donor NanoLuc emission) and >610 nm (long pass

ﬁlter; ﬂuorescent ligand emission). The raw BRET ratio was

calculated by dividing the >610 nm emission by the 420 nm

emission. In all NanoBRET binding experiments, nonspeciﬁc binding

was determined in the presence of 1 μM SLV320.

Total Internal Reﬂection Fluorescence Microscopy (TIRF-M).

HEK293A cells were seeded into 10 mm × 20 mm style dishes at a

density of 5 × 10⁶cells per dish in complete DMEM. After 12 h, cells

were transiently transfected with 2 μg cDNA of SNAP-hA₁AR using

the 1:4 ratio of DNA/PEI to achieve lower receptor expression levels.

After 12 h, transiently transfected SNAP-hA₁AR HEK293 cells were

seeded onto a poly-^D-lysine-precoated FluoroDish (35 mm, 23 mm

well) to 70−80% conﬂuency. On the day of the experiment (24 h

following transfection), DMEM was removed and the cells were

incubated with 0.2 μM BG-AF488 in binding buﬀer (10 mM HEPES,

10 mM ^D-glucose, 145 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1.5 mM

NaHCO₃, 2 mM CaCl₂, 1 U/mL ADA, pH 7.4) for 30 min at 37 °C.

The binding buﬀer was removed, and the cells were washed twice

with warm binding buﬀer to remove any unbound BG-AF488.

Subsequently, the cells were treated with or without 10 μM SLV320

for 30 min at 37 °C. After that time, the cells were treated with the

required concentration of ﬂuorescent ligand under investigation.

TIRF Imaging. Images were captured on a Leica DMI6000 3D

GSD TIRF microscope running LAS X version 1.9 using a HC Plan

Apo 100× NA 1.43 TIRF Objective. Excitation was provided from a

633 nm laser for the BODIPY-630/650 and Sulfo-Cy5 ﬂuorophores.

Detection was through either a 540−620 nm emission ﬁlter or 640-

700 nm emission ﬁlter. The TIRF ﬁeld penetration depth was kept at

80 nm for all wavelengths and samples. Images were captured with an

Andor iXon3 897 Ultra EM-CCD camera with exposure times and

gain settings being consistent across all images.

cAMP Assay. A₁AR-FlpIn-CHO cells were seeded into 96-well

culture plates at 20,000 cells/well and incubated overnight. The G_i/o

-

protein-mediated inhibition of cAMP accumulation was determined

as described previously.¹⁰⁸Brieﬂy, media were removed and cells were

incubated with stimulation buﬀer (140 mM NaCl, 5.4 mM KCl, 0.8

μM MgSO₄, 0.2 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂,

5.6 mM ^D-glucose, 5 mM HEPES, 0.1% bovine serum albumin (BSA),

0.1 U/mL ADA, and 10 μM rolipram, pH 7.4) and incubated at 37 °C

for 30 min. Inhibition of cAMP was assessed by pre-incubation with

increasing concentrations of antagonists for 10 min. NECA

concentration response curves were performed by subsequent

incubation of increasing concentrations of NECA in the presence of

3 μM forskolin for additional 30 min at 37 °C. The reactions were

terminated by the addition of 50 μL ice-cold 100% ethanol. The lysis

buﬀer (0.1% BSA, 0.3% tween-20, 5 mM HEPES, pH 7.4) was added

to the cells after ethanol evaporation. The level of cAMP was detected

using LANCE cAMP 384 kits (PerkinElmer) following the

manufacturer’s protocol, and ﬂuorescence was measured with an

EnVision plate reader (PerkinElmer). Ligand concentration-response

curves were normalized to the response mediated by 3 μM forskolin

(0%) or buﬀer (100%) alone. All experiments were performed in

duplicate.

TIRF Image Analysis. Analysis of TIRF images were carried out

using a custom macro written in the Fiji distribution of ImageJ

(Schindelin et al., 2012). In brief, the user was asked to manually

outline the cell of interest. Both image channels were then ﬁltered to

remove noise (Gaussian ﬁlter-Sigma = 1) and discrete spots were

extracted from the red channel using the ﬁnd maxima command

(prominence = 3) and displaying the same as single-binary points.

The extracted binary points were used to measure the intensity in

both the red and far red channel for each discrete spot.

Data Analysis. All data are represented as mean

SEM of n

experiments performed either in duplicate or triplicate. n refers to the

number of separate experiments. A separate experiment required a

separate ﬂask of cells and a separate drug dilution used throughout the

experiment. The data were presented and analyzed using Prism

software (GraphPad Prism 8.0, San Diego, CA) and Excel.

[³H] DPCPX Binding Assay and NanoBRET Competition Binding

Assays. The competition binding curves were ﬁt to the following

equation:

Confocal Imaging. Transiently transfected SNAP-tagged human

A₁AR HEK293 cells were grown to 80−90% conﬂuence in a μ-Slide 8

100 × [A]

uninhibited specific binding = 100 −

[A] + (IC₅₀)

̈

well-chambered coverslip (Ibidi GmbH, Grafelﬁng, Germany)

precoated with poly-^D-lysine. On the day of the experiment, culture

media were removed and the cells were labeled for 30 min at 37 °C

with a 0.2 μM SNAP AlexaFluor488 label as required in binding

buﬀer (10 mM HEPES, 10 mM ^D-glucose, 145 mM NaCl, 5 mM KCl,

1 mM MgSO₄, 1.5 mM NaHCO₃, 2 mM CaCl₂, 1 U/mL ADA, pH

7.4). After washing twice with binding buﬀer, the cells were treated

with or without 10 μM SLV320 for 30 min at 37 °C. After that time,

the cells were treated with the required concentration of ﬂuorescent

ligand under investigation. Live cell imaging was performed at 37 °C

on a Leica TCS SP8 X confocal running system ﬁtted with an HC PL

APO CS2 40x/1.1 NA water immersion objective. A diode 633 laser

was used for the excitation of BODIPY-630/650 and Sulfo-Cy5

ﬂuorophores and a beam splitter TD 488/552/638 and the emission

was collected using a hybrid detector (HyD) (emission ﬁlter range

642−782 nm). A 488 optically pumped semiconductor laser (OPLS)

was used for the excitation of the SNAP AF488 label, and the

emission was detected using a hybrid detector (HyD) (emission ﬁlter

range 493−572 nm). The pinhole diameter (1 Airy Unit; 1.1 μm

optical slice), laser power, and gain remained constant in all the

experiments. The images were captured using 1024 × 1024 pixel

resolution, 16-bit depth, and a scan speed of 200 Hz with a line

averaging of 6. Images were processed in FIJI (ImageJ)¹⁰⁹version

2.0.0 software, and linear adjustments to the brightness and contrast

have been applied equally across all the images.

where [A] is the concentration of competing drug and the IC₅₀is the

molar concentration of ligand required to inhibit 50% of the speciﬁc

binding of speciﬁed concentrations of ﬂuorescent ligands or 1 nM

[³H] DPCPX.

The Cheng−Prusoﬀ equation was used to correct ﬁtted IC₅₀values

to K_ivalues:

IC₅₀

K_i=

L

1 +

K_D

where L is the concentration of ﬂuorescent ligand or [³H] DPCPX in

nM and K_Dis the dissociation constant of ﬂuorescent ligand or [³H]

DPCPX in nM. The K_Dvalues used were calculated from the

saturation binding experiments. The concentration in the assay of

[³H] DPCPX was directly determined by measuring DPM counts

with a scintillation counter using the following equation:

DPM

[l] =

SA × 2.2 × 10⁶

volume

where SA is the speciﬁc activity of the radioligand in Ci/mmol and

volume is the ﬁnal volume of the assay in liter.

U

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

NanoBRET Saturation Binding Assay. Total and nonspeciﬁc

saturation binding curves were ﬁt simultaneously using the following

equation:

AUTHOR INFORMATION

■

Corresponding Authors

Michelle L. Halls − Drug Discovery Biology, Monash Institute

of Pharmaceutical Sciences, Monash University, Parkville,

Victoria 3052, Australia; Phone: +613 9903 9094;

Email: michelle.halls@monash.edu

Lauren T. May − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, Monash University, Parkville,

Victoria 3052, Australia; Phone: +613 9903 9095;

Email: lauren.may@monash.edu

Peter J. Scammells − Medicinal Chemistry, Monash

University, Parkville, Victoria 3052, Australia; orcid.org/

0000-0003-2930-895X; Phone: +613 9903 9542;

Email: peter.scammells@monash.edu

B_max[B]

B + (K_D)

BRET ratio =

+ M(B) + C

where B_maxis the maximal response, [B] is the concentration of

ﬂuorescent ligand in nM, K_Dis the equilibrium dissociation constant

in nM, M is the slope of the nonspeciﬁc binding component, and C is

the intercept with the y axis.

cAMP Assay. Antagonism by ﬂuorescent ligands of the NECA-

induced inhibition of cAMP accumulation in FlpIn-CHO cells

expressing the human A₁AR was analyzed. For each concentration

of the antagonists, the ratio (DR, dose ratio) of NECA concentrations

required to produce the same magnitude of response in the presence

and absence of the antagonist was determined. To estimate antagonist

aﬃnity values, functional interaction studies between NECA and

single concentration of each antagonist in the cAMP assays were ﬁtted

to the following form of Schild regression analysis:

Authors

Eleonora Comeo − Medicinal Chemistry, Monash University,

Parkville, Victoria 3052, Australia; Division of Biomolecular

Sciences and Medicinal Chemistry, School of Pharmacy,

Biodiscovery Institute, University of Nottingham, Nottingham

NG7 2RD, United Kingdom; Centre of Membrane Proteins

and Receptors (COMPARE), University of Birmingham, B15

2TT and University of Nottingham, Birmingham NG7 2UH,

United Kingdom

Phuc Trinh − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, Monash University, Parkville,

Victoria 3052, Australia

Anh T. Nguyen − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, Monash University, Parkville,

Victoria 3052, Australia

Cameron J. Nowell − Drug Discovery Biology, Monash

Institute of Pharmaceutical Sciences, Monash University,

Parkville, Victoria 3052, Australia

Nicholas D. Kindon − Division of Biomolecular Sciences and

Medicinal Chemistry, School of Pharmacy, Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

United Kingdom; Centre of Membrane Proteins and

Receptors (COMPARE), University of Birmingham, B15

2TT and University of Nottingham, Birmingham NG7 2UH,

United Kingdom

(E_max− bottom)

response = bottom +

Ä

Å

É

Ñ

HillSlope

Ä

Å

É

S

Ñ

i

j

_Ñy

[B]

−pEC

50

Ñ

_Ñz

10

1 +

_Ñz

−pA

Ñ

_Ñz

2

10

Å

Ç

^Öz

Å

Ç

Ñ

j

^Öz

z

1 +

z

[A]

j

z

j

z

j

z

k

{

where pEC₅₀is the negative logarithm of the EC₅₀of NECA (A) in

the absence of antagonist (B). HillSlope is the slope of the agonist

curve, S is the Schild slope, and pA₂is the negative logarithm of the

molar concentration of antagonist necessary to shift the agonist EC₅₀

by a factor of two. The Schild slope parameter, S, was constrained to

1, and therefore, the estimated pA₂values for each antagonist are

equal to the pK_B(negative logarithm of the antagonist equilibrium

dissociation constant).

NanoBRET Kinetic Binding Assay. For association binding kinetics,

nonspeciﬁc binding was determined at each concentration of the

ﬂuorescent ligands at each time point by adding 1 μM SLV320, and

this was subtracted from total binding to obtain speciﬁc binding

measurements. The association rate (k_on) in M⁻¹min⁻¹and

dissociation rate (k_off) min⁻¹constants were calculated from speciﬁc

binding using the following equation:

k_obs− k_off

k_on

=

[L]

Mark Soave − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queens Medical Centre,

University of Nottingham, Nottingham NG7 2UH, United

Kingdom; Centre of Membrane Proteins and Receptors

(COMPARE), University of Birmingham, B15 2TT and

University of Nottingham, Birmingham NG7 2UH, United

Kingdom

Leigh A. Stoddart − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queens Medical Centre,

University of Nottingham, Nottingham NG7 2UH, United

Kingdom; Centre of Membrane Proteins and Receptors

(COMPARE), University of Birmingham, B15 2TT and

University of Nottingham, Birmingham NG7 2UH, United

Kingdom

where [L] is the concentration of the ﬂuorescent conjugate in M and

k_obsis calculated from the global ﬁtting of the data to the following

exponential association function:

_obst

Y = Y_max(1 − e^−k

)

where Y is the speciﬁc binding at time t, Y_maxrepresents the speciﬁc

binding at inﬁnite time (t), and k_obsis the rate constant for the

observed rate of association.

The kinetic equilibrium dissociation constant (K_D) was determined

from the data using the following equation:

k_off

k_on

K_D

=

Jonathan M. White − School of Chemistry and the Bio21

Molecular Science and Biotechnology Institute, The University

of Melbourne, Melbourne, Victoria 3010, Australia;

orcid.org/0000-0002-0707-6257

Stephen J. Hill − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queens Medical Centre,

University of Nottingham, Nottingham NG7 2UH, United

Kingdom; Centre of Membrane Proteins and Receptors

(COMPARE), University of Birmingham, B15 2TT and

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02067.

■

sı

Additional detailed chemistry methods, analytical HPLC

chromatograms, and pharmacological data of the ﬁnal

ﬂuorescent ligands (PDF)

Molecular formula strings of tested compounds (CSV)

V

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(6) Deb, P. K.; Deka, S.; Borah, P.; Abed, S. N.; Klotz, K.-N.

Medicinal Chemistry and Therapeutic Potential of Agonists,

Antagonists and Allosteric Modulators of A₁Adenosine Receptor:

Current Status and Perspectives. Curr. Pharm. Des. 2019, 25, 2697−

2715.

(7) Gao, Z.; Tosh, D. K.; Jain, S.; Yu, J.; Suresh, R. R. A₁Adenosine

Receptor Agonists, Antagonists and Allosteric Modulators. In The

Adenosine Receptors, The Receptors; Borea, P., Varani, K., Gessi, S.,

Merighi S, V. F. (eds), Ed.; Humana Press, New York, NY, 2018; 59−

89.

University of Nottingham, Birmingham NG7 2UH, United

Kingdom; orcid.org/0000-0002-4424-239X

Barrie Kellam − Division of Biomolecular Sciences and

Medicinal Chemistry, School of Pharmacy, Biodiscovery

Institute, University of Nottingham, Nottingham NG7 2RD,

United Kingdom; Centre of Membrane Proteins and

Receptors (COMPARE), University of Birmingham, B15

2TT and University of Nottingham, Birmingham NG7 2UH,

United Kingdom; orcid.org/0000-0003-0030-9908

(8) Delacretaz, E. Supraventricular Tachycardia. N. Engl. J. Med.

2006, 28, 109−111.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02067

(9) Linden, J. Adenosine in Tissue Protection and Tissue

Regeneration. Mol. Pharmacol. 2005, 67, 1385−1387.

(10) Borea, P. A.; Gessi, S.; Merighi, S.; Vincenzi, F.; Varani, K.

Pharmacology of Adenosine Receptors: The State of the Art. Physiol.

Rev. 2018, 98, 1591−1625.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

(11) Muller, C. E.; Jacobson, K. A. Recent Developments in

̈

■

Adenosine Receptor Ligands and Their Potential as Novel Drugs.

Biochim. Biophys. Acta, Biomembr. 2011, 1808, 1290−1308.

(12) Gao, Z. G.; Jacobson, K. A. Purinergic Signaling in Mast Cell

Degranulation and Asthma. Front. Pharmacol. 2017, 8, 1−14.

(13) Mendiola-Precoma, J.; Padilla, K.; Rodríguez-Cruz, A.;

Berumen, L. C.; Miledi, R.; García-Alcocer, G. Theobromine-Induced

Changes in A₁Purinergic Receptor Gene Expression and Distribution

in a Rat Brain Alzheimer ’s Disease Model. J. Alzheimer’s Dis. 2017, 55,

1273−1283.

The work was funded by the Medical Research Council (Grant

number MR/N020081/1 to B.K. and S.J.H.), the Australian

Research Council (ARC Discovery Project Grant DP19010145

to P.J.S.), the National Health and Medical Research Council

of Australia (NHMRC) (Grant number APP1145420 to

L.T.M. and S.J.H.; and APP1147291 to L.T.M.), the Monash

Fellowship (from the faculty of Pharmacy and Pharmaceutical

Sciences and Monash University to M.L.H.), and the Centre of

Membrane Proteins and Receptors (COMPARE). This work

was supported by the Nottingham-Monash Joint Doctoral

Training Centre program. We thank Dr. Jason Dang for

assistance with the spectroscopy work. L.T.M. is an Australian

Heart Foundation Future Leader Fellow.

(14) Massie, B. M.; O’Connor, C. M.; Metra, M.; Ponikowski, P.;

Teerlink, J. R.; Cotter, G.; Weatherley, B. D.; Cleland, J. G. F.;

Givertz, M. M.; Voors, A.; DeLucca, P.; Mansoor, G. A.; Salerno, C.

M.; Bloomfield, D. M.; Dittrich, H. C. Rolofylline, an Adenosine A₁-

Receptor Antagonist, in Acute Heart Failure. N. Engl. J. Med. 2010,

363, 1419−1428.

(15) Ensor, C. R.; Russell, S. D. Tonapofylline: A Selective

Adenosine-1 Receptor Antagonist for the Treatment of Heart Failure.

Expert Opin. Pharmacother. 2010, 11, 2405−2415.

(16) Voors, A. A.; Bax, J. J.; Hernandez, A. F.; Wirtz, A. B.; Pap, A.

F.; Ferreira, A. C.; Senni, M.; van der Laan, M.; Butler, J. Safety and

Efficacy of the Partial Adenosine A₁Receptor Agonist Neladenoson

Bialanate in Patients with Chronic Heart Failure with Reduced

Ejection Fraction: A Phase IIb, Randomized, Double-Blind, Placebo-

Controlled Trial. Eur. J. Heart Fail. 2019, 21, 1426−1433.

(17) Valant, C.; May, L. T.; Aurelio, L.; Chuo, C. H.; White, P. J.;

Baltos, J.-A.; Sexton, P. M.; Scammells, P. J.; Christopoulos, A.

Separation of On-Target Efficacy from Adverse Effects through

Rational Design of a Bitopic Adenosine Receptor Agonist. Proc. Natl.

Acad. Sci. U. S. A. 2014, 111, 4614−4619.

(18) Aurelio, L.; Baltos, J. A.; Ford, L.; Nguyen, A. T. N.; Jörg, M.;

Devine, S. M.; Valant, C.; White, P. J.; Christopoulos, A.; May, L. T.;

Scammells, P. J. A Structure-Activity Relationship Study of Bitopic

N⁶-Substituted Adenosine Derivatives as Biased Adenosine A₁

Receptor Agonists. J. Med. Chem. 2018, 61, 2087−2103.

(19) May, L. T.; Leach, K.; Sexton, P. M.; Christopoulos, A.

Allosteric Modulation of G-Protein Coupled Receptors. Annu. Rev.

Pharmacol. Toxicol. 2007, 47, 1−51.

(20) Bunnage, M. E.; Chekler, E. L. P.; Jones, L. H. Target

Validation Using Chemical Probes. Nat. Chem. Biol. 2013, 9, 195−

199.

(21) Ralevic, V.; Burnstock, G. Receptors for Purines and

Pyrimidines. Pharmacol. Rev. 1998, 50, 413−492.

ABBREVIATIONS

■

AR, adenosine receptor; BODIPY, 4,4-diﬂuoro-4-bora-31,4a-

diaza-s-indacene; BOP, (benzotriazol-1-yloxy)tris-

(dimethylamino)phosphonium hexaﬂuorophosphate; CHO,

chinese hamster ovary; COMU, (1-cyano-2-ethoxy-2-

oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexaﬂuorophosphate; Cy5, cyanine5; DIPEA, diisopropylethyl-

amine; ESI, electrospray ionization; GPCR, G-protein-coupled

receptor; HEK, human embryonic kidney; HPLC, high-

performance liquid chromatography; NanoBRET, nanolucifer-

ase-bioluminescence-energy transfer; NanoLuc, nanoluciferase;

NMR, nuclear magnetic resonance; RP, reverse phase; SAR,

structure−activity relationship

REFERENCES

■

(1) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.;

Linden, J. International Union of Pharmacology. XXV. Nomenclature

and Classification of Adenosine Receptors. Pharmacol. Rev. 2001, 53,

527−552.

(2) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.

International Union of Pharmacology. XXV. Nomenclature and

Classification of Adenosine Receptors-An Update. Pharmacol. Rev.

2011, 63, 527−552.

(3) Alexander, S. P.; Christopoulos, A.; Davenport, A. P.; Kelly, E.;

Marrion, N. V.; Peters, J. A.; Faccenda, E.; Harding, S. D.; Pawson, A.

J.; Sharman, J. L.; Southan, C.; Davies, J. A. The Concise Guide To

Pharmacology 2017 / 18: G Protein-Coupled Receptors. Br. J.

Pharmacol. 2017, 174, S17−S129.

(4) Jacobson, K. A.; Gao, Z.-G. Adenosine Receptors as Therapeutic

Targets. Nat. Rev. Drug Discov. 2006, 49, 247−264.

(22) Peakman, M.-C.; Hill, S. J. Adenosine A₁Receptor-mediated

Changes in Basal and Histamine-stimulated Levels of Intracellular

Calcium in Primary Rat Astrocytes. Br. J. Pharmacol. 1995, 115, 801−

810.

(23) Ginés, S.; Ciruela, F.; Burgueno, J.; Casadó, V.; Canela, E. I.;

̃

Mallol, J.; Lluís, C.; Franco, R. Involvement of Caveolin in Ligand-

Induced Recruitment and Internalization of A₁Adenosine Receptor

and Adenosine Deaminase in an Epithelial Cell Line. Mol. Pharmacol.

2001, 59, 1314−1323.

(5) Fredholm, B. B.; Arslan, G.; Halldner, L.; Kull, B.; Schulte, G.;

Wasserman, W. Structure and Function of Adenosine Receptors and

Their Genes. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2000, 362,

364−374.

W

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(24) Briddon, S. J.; Middleton, R. J.; Cordeaux, Y.; Flavin, F. M.;

Weinstein, J. A.; George, M. W.; Kellam, B.; Hill, S. J. Quantitative

Analysis of the Formation and Diffusion of A₁-Adenosine Receptor −

Antagonist Complexes in Single Living Cells. Proc. Natl. Acad. Sci. U.

S. A. 2004, 101, 4673−4678.

(25) Stoddart, L. A.; Vernall, A. J.; Briddon, S. J.; Kellam, B.; Hill, S.

J. Direct Visualisation of Internalization of the Adenosine A3

Receptor and Localization with Arrestin3 Using a Fluorescent

Agonist. Neuropharmacology 2015, 98, 68−77.

(26) Schembri, L. S.; Stoddart, L. A.; Briddon, S. J.; Kellam, B.;

Canals, M.; Graham, B.; Scammells, P. J. Synthesis, Biological

Evaluation, and Utility of Fluorescent Ligands Targeting the μ -Opioid

Receptor. J. Med. Chem. 2015, 58, 9754−9767.

(27) Conroy, S.; Kindon, N. D.; Glenn, J.; Stoddart, L. A.; Lewis, R.

J.; Hill, S. J.; Kellam, B.; Stocks, M. J. Synthesis and Evaluation of the

First Fluorescent Antagonists of the Human P2Y ₂Receptor Based on

AR-C118925. J. Med. Chem. 2018, 61, 3089−3113.

(28) Stoddart, L. A.; Vernall, A. J.; Denman, J. L.; Briddon, S. J.;

Kellam, B.; Hill, S. J. Fragment Screening at Adenosine-A3 Receptors

in Living Cells Using a Fluorescence-Based Binding Assay. Chem. Biol.

2012, 19, 1105−1115.

(29) Stoddart, L. A.; Johnstone, E. K. M.; Wheal, A. J.; Goulding, J.;

Robers, M. B.; MacHleidt, T.; Wood, K. V.; Hill, S. J.; Pfleger, K. D.

G. Application of BRET to Monitor Ligand Binding to GPCRs. Nat.

Methods 2015, 12, 661−663.

(30) Alcobia, D. C.; Ziegler, A. I.; Kondrashov, A.; Comeo, E.;

Mistry, S.; Kellam, B.; Chang, A.; Woolard, J.; Hill, S. J.; Sloan, E. K.

Visualizing Ligand-Binding to a GPCR In Vivo Using NanoBRET.

iScience 2018, 6, 280−288.

(31) Arruda, M. A.; Stoddart, L. A.; Gherbi, K.; Briddon, S. J.;

Kellam, B.; Hill, S. J. A Non-Imaging High Throughput Approach to

Chemical Library Screening at the Unmodified Adenosine-A 3

Receptor in Living Cells. Front. Pharmacol. 2017, 8, 1−13.

(32) Soave, M.; Briddon, S. J.; Hill, S. J.; Stoddart, L. A. Fluorescent

Ligands: Bringing Light to Emerging G Protein-coupled Receptor

Paradigms. Br. J. Pharmacol. 2019, 1−14.

(33) Vernall, A. J.; Hill, S. J.; Kellam, B. The Evolving Small-

Molecule Fluorescent-Conjugate Toolbox for Class A GPCRs. Br. J.

Pharmacol. 2014, 171, 1073−1084.

(34) Federico, S.; Lassiani, L.; Spalluto, G. Chemical Probes for the

Adenosine Receptors. Pharmaceuticals 2019, 12, 1−21.

(35) Kozma, E.; Suresh Jayasekara, P.; Squarcialupi, L.; Paoletta, S.;

Moro, S.; Federico, S.; Spalluto, G.; Jacobson, K. A. Fluorescent

Ligands for Adenosine Receptors. Bioorg. Med. Chem. Lett. 2013, 23,

26−36.

(36) Soave, M.; Goulding, J.; Markus, R.; Hill, S. J.; Stoddart, L. A.

Application of Fluorescent Purinoceptor Antagonists for Bio-

luminescence Resonance Energy Transfer Assays and Fluorescent

Microscopy. In Purinergic Signalling: Methods in Molecular Biology, Vol.

2041; Walker, J.M.; Pelegrin, P., Eds.; Humana Press: New York,

2020; pp163−181.

(37) Kecskés, M.; Kumar, T. S.; Yoo, L.; Gao, Z. G.; Jacobson, K. A.

Novel Alexa Fluor-488 Labeled Antagonist of the A2A Adenosine

Receptor: Application to a Fluorescence Polarization-Based Receptor

Binding Assay. Biochem. Pharmacol. 2010, 80, 506−511.

(38) Duroux, R.; Ciancetta, A.; Mannes, P.; Yu, J.; Boyapati, S.;

Gizewski, E.; Yous, S.; Ciruela, F.; Auchampach, J. A.; Gao, Z.-G.;

Jacobson, K. A. Bitopic Fluorescent Antagonists of the A _2AAdenosine

Receptor Based on Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidin-5-

Amine Functionalized Congeners. MedChemComm. 2017, 8, 1659−

1667.

(39) Comeo, E.; Kindon, N. D.; Soave, M.; Stoddart, L. A.;

Kilpatrick, L. E.; Scammells, P. J.; Hill, S. J.; Kellam, B. Subtype-

Selective Fluorescent Ligands as Pharmacological Research Tools for

the Human Adenosine A_2AReceptor. J. Med. Chem. 2020, 63, 2656−

2672.

Antagonist Enables Competition Binding Assay by Flow Cytometry. J.

Med. Chem. 2018, 61, 4301−4316.

(41) Vernall, A. J.; Stoddart, L. A.; Briddon, S. J.; Hill, S. J.; Kellam,

B. Highly Potent and Selective Fluorescent Antagonists of the Human

Adenosine A3 Receptor Based on the 1,2,4-Triazolo[4,3-a]-

Quinoxalin-1-One Scaffold. J. Med. Chem. 2012, 55, 1771−1782.

(42) Vernall, A. J.; Stoddart, L. A.; Briddon, S. J.; Ng, H. W.;

Laughton, C. A.; Doughty, S. W.; Hill, S. J.; Kellam, B. Conversion of

a Non-Selective Adenosine Receptor Antagonist into A 3-Selective

High Affinity Fluorescent Probes Using Peptide-Based Linkers. Org.

Biomol. Chem. 2013, 11, 5673−5682.

(43) Bouzo-Lorenzo; Stoddart, L. A.; Xia, L.; IJzerman, A. P.;

Heitman, L. H.; Briddon, S. J.; Hill, S. J. A Live Cell NanoBRET

Binding Assay Allows the Study of Ligand-Binding Kinetics to the

Adenosine A₃Receptor. Purinergic Signal. 2019, 15, 139−153.

(44) Cooper, S. L.; Soave, M.; Jörg, M.; Scammells, P. J.; Woolard,

J.; Hill, S. J. Probe Dependence of Allosteric Enhancers on the

Binding Affinity of Adenosine A₁-Receptor Agonists at Rat and

Human A₁-Receptors Measured Using NanoBRET. Br. J. Pharmacol.

2019, 176, 864−878.

(45) May, L. T.; Self, T. J.; Briddon, S. J.; Hill, S. J. The Effect of

Allosteric Modulators on the Kinetics of Agonist-G Protein-Coupled

Receptor Interactions in Single Living Cells. Mol. Pharmacol. 2010,

78, 511−523.

(46) Cordeaux, Y.; Briddon, S. J.; Alexander, S. P. H.; Kellam, B.;

Hill, S. J. Agonist-Occupied A3 Adenosine Receptors Exist within

Heterogeneous Complexes in Membrane Microdomains of Individual

Living Cells. FASEB J. 2007, 22, 850−860.

(47) Jacobson, K. A.; Ukena, D.; Padgett, W.; Kirk, K. L.; Daly, J. W.

Molecular Probes for Extracellular Adenosine Receptors. Biochem.

Pharmacol. 1987, 36, 1697−1707.

(48) Middleton, R. J.; Briddon, S. J.; Cordeaux, Y.; Yates, A. S.; Dale,

C. L.; George, M. W.; Baker, J. G.; Hill, S. J.; Kellam, B. New

Fluorescent Adenosine A₁-Receptor Agonists That Allow Quantifica-

tion of Ligand - Receptor Interactions in Microdomains of Single

Living Cells. J. Med. Chem. 2007, 50, 782−793.

(49) Macchia, M.; Salvetti, F.; Barontini, S.; Calvani, F.; Gesi, M.;

Hamdan, M.; Lucacchini, A.; Pellegrini, A.; Soldani, P.; Martini, C.

Fluorescent Probes for Adenosine Receptors: Synthesis and Biology

of N⁶-Dansylaminoalkyl-Substituted Neca Derivatives. Bioorg. Med.

Chem. Lett. 1998, 8, 3223−3228.

(50) Jacobson, K. A.; Ukena, D.; Padgett, W.; Daly, J. W.; Kirk, K. L.

Xanthine Functionalized Congeners as Potent Ligands at A2-

Adenosine Receptors. J. Med. Chem. 1987, 30, 211−214.

(51) Corriden, R.; Kilpatrick, L. E.; Kellam, B.; Briddon, S. J.; Hill, S.

J. Kinetic Analysis of Antagonist-Occupied Adenosine-A3 Receptors

within Membrane Microdomains of Individual Cells Provides

Evidence of Receptor Dimerization and Allosterism. FASEB J. 2014,

28, 4211−4222.

(52) Briddon, S. J.; Middleton, R. J.; Yates, A. S.; George, M. W.;

Kellam, B.; Hill, S. J. Application of Fluorescence Correlation

Spectroscopy to the Measurement of Agonist Binding to a G-Protein

Coupled Receptor at the Single Cell Level. Faraday Discuss. 2004,

126, 197−207.

(53) Simon, G. M.; Niphakis, M. J.; Cravatt, B. F. Determining

Target Engagement in Living Systems. Nat. Chem. Biol. 2013, 9, 200−

205.

(54) Singh, S.; Cooper, S. L.; Glenn, J. R.; Beresford, J.; Percival, L.

R.; Tyndall, J. D. A.; Hill, S. J.; Kilpatrick, L. E.; Vernall, A. J.

Synthesis of Novel (Benzimidazolyl)Isoquinolinols and Evaluation as

Adenosine A₁Receptor Tools. RSC Adv. 2018, 8, 16362−16369.

(55) Glukhova, A.; Thal, D. M.; Nguyen, A. T.; May, L. T.; Sexton,

P. M.; Christopoulos, A.; Scammells, P. J. Structure of the Adenosine

A₁Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168,

867−877.e13.

(56) Cheng, R. K. Y.; Segala, E.; Robertson, N.; Marshall, F. H.;

Cooke, R. M.; Errey, J. C.; Marshall, F. H.; Cooke, R. M. Structures of

Human A₁and A_2AAdenosine Receptors with Xanthines Reveal

Determinants of Selectivity. Structure 2017, 25, 1275−1285.e4.

(40) Köse, M.; Gollos, S.; Karcz, T.; Fiene, A.; Heisig, F.;

Behrenswerth, A.; Kiec-Kononowicz, K. J.; Namasivayam, V.;

̈

Muller, C. E. Fluorescent-Labeled Selective Adenosine A2B Receptor

X

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(57) Weyler, S.; Fu

̈

lle, F.; Diekmann, M.; Schumacher, B.; Hinz, S.;

(75) Draper-joyce, C. J.; Khoshouei, M.; Thal, D. M.; Liang, Y.-L.;

Nguyen, A. T. N.; Furness, S. G. B.; Venugopal, H.; Baltos, J.; Plitzko,

J. M.; Danev, R.; Baumeister, W.; May, L. T.; Wootten, D.; Sexton, P.

M.; Glukhova, A.; Christopoulos, A. Structure of the Adenosine-

Bound Human Adenosine A₁Receptor−Gi Complex. Nature 2018,

558, 559−563.

(76) Mattedi, G.; Deflorian, F.; Mason, J. S.; de Graaf, C.; Gervasio,

F. L. Understanding Ligand Binding Selectivity in a Prototypical

GPCR Family. J. Chem. Inf. Model. 2019, 59, 2830−2836.

(77) Bolcato, G.; Bissaro, M.; Deganutti, G.; Sturlese, M.; Moro, S.

New Insights into Key Determinants for Adenosine 1 Receptor

Antagonists Selectivity Using Supervised Molecular Dynamics

Simulations. Biomolecules 2020, 10, 1−11.

Klotz, K. N.; Muller, C. E. Improving Potency, Selectivity, and Water

̈

Solubility of Adenosine A1 Receptor Antagonists: Xanthines Modified

at Position 3 and Related Pyrimido[1,2,3-Cd]Purinediones. Chem-

MedChem 2006, 1, 891−902.

(58) 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.

(59) Kiesman, W. F.; Zhao, J.; Conlon, P. R.; Dowling, J. E.; Petter,

R. C.; Lutterodt, F.; Jin, X.; Smits, G.; Fure, M.; Jayaraj, A.; Kim, J.;

Sullivan, G.; Linden, J. Potent and Orally Bioavailable 8-

Bicyclo[2.2.2]Octylxanthines as Adenosine A₁Receptor Antagonists.

J. Med. Chem. 2006, 49, 7119−7131.

(60) Beauglehole, A. R.; Baker, S. P.; Scammells, P. J.

Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antag-

onists for the A₁-Adenosine Receptor. J. Med. Chem. 2000, 43, 4973−

4980.

(61) Ballesteros, J. A.; Weinstein, H. Integrated Methods for the

Construction of Three-Dimensional Models and Computational

Probing of Structure-Function Relations in G Protein-Coupled.

Methods Neurosci. 1995, 25, 366−428.

(62) Dale, C. L.; Hill, S. J.; Kellam, B. New Potent, Short-Linker

BODIPY-630/650 Labelled Fluorescent Adenosine Receptor Ago-

nists. MedChemComm 2012, 3, 333−338.

(63) Pansare, V. J.; Hejazi, S.; Faenza, W. J.; Prud’homme, R. K.

Review of Long-Wavelength Optical and NIR Imaging Materials:

Contrast Agents, Fluorophores, and Multifunctional Nano Carriers.

Chem. Mater. 2012, 24, 812−827.

(64) Baker, J. G.; Middleton, R.; Adams, L.; May, L. T.; Briddon, S.

J.; Kellam, B.; Hill, S. J. Influence of Fluorophore and Linker

Composition on the Pharmacology of Fluorescent Adenosine A₁

Receptor Ligands: Themed Section: Imaging in Pharmacology

Research Paper. Br. J. Pharmacol. 2010, 159, 772−786.

(65) Karolin, J.; Johansson, L. B. A.; Strandberg, L.; Ny, T.

Fluorescence and Absorption Spectroscopic Properties of Dipyrro-

metheneboron Difluoride (BODIPY) Derivatives in Liquids, Lipid

Membranes, and Proteins. J. Am. Chem. Soc. 1994, 116, 7801−7806.

(66) Ulrich, G.; Ziessel, R.; Harriman, A. The Chemistry of

Fluorescent Bodipy Dyes: Versatility Unsurpassed. Angew. Chem., Int.

Ed. 2008, 47, 1184−1201.

(67) Hill, S. J.; May, L. T.; Kellam, B.; Woolard, J. Allosteric

Interactions at Adenosine A₁and A₃Receptors: New Insights into the

Role of Small Molecules and Receptor Dimerization. Br. J. Pharmacol.

2014, 171, 1102−1113.

(78) Mason, J. S.; Bortolato, A.; Weiss, D. R.; Deflorian, F.; Tehan,

B.; Marshall, F. H. High End GPCR Design: Crafted Ligand Design

and Druggability Analysis Using Protein Structure, Lipophilic

Hotspots and Explicit Water Networks. Silico Pharmacol. 2013, 1,

1−12.

(79) Mason, J. S.; Bortolato, A.; Congreve, M.; Marshall, F. H. New

Insights from Structural Biology into the Druggability of G Protein-

Coupled Receptors. Trends Pharmacol. Sci. 2012, 33, 249−260.

(80) Stoddart, L. A.; Kilpatrick, L. E.; Hill, S. J. NanoBRET

Approaches to Study Ligand Binding to GPCRs and RTKs. Trends

Pharmacol. Sci. 2018, 39, 136−147.

(81) Dale, N. C.; Johnstone, E. K. M.; White, C. W.; Pfleger, K. D.

G. NanoBRET: The Bright Future of Proximity-Based Assays. Front.

Bioeng. Biotechnol. 2019, 7, 1−13.

(82) Hall, M. P.; Unch, J.; Binkowski, B. F.; Valley, M. P.; Butler, B.

L.; Wood, M. G.; Otto, P.; Zimmerman, K.; Vidugiris, G.; MacHleidt,

T.; Robers, M. B.; Benink, H. A.; Eggers, C. T.; Slater, M. R.;

Meisenheimer, P. L.; Klaubert, D. H.; Fan, F.; Encell, L. P.; Wood, K.

V. Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing

a Novel Imidazopyrazinone Substrate. ACS Chem. Biol. 2012, 7,

1848−1857.

(83) Mercier, J. F.; Salahpour, A.; Angers, S.; Breit, A.; Bouvier, M.

Quantitative Assessment of Β 1- and Β 2-Adrenergic Receptor Homo-

and Heterodimerization by Bioluminescence Resonance Energy

Transfer. J. Biol. Chem. 2002, 277, 44925−44931.

(84) Marullo, S.; Bouvier, M. Resonance Energy Transfer

Approaches in Molecular Pharmacology and Beyond. Trends

Pharmacol. Sci. 2007, 28, 362−365.

(85) Pfleger, K. D. G.; Eidne, K. A. Illuminating Insights into

Protein-Protein Interactions Using Bioluminescence Resonance

Energy Transfer (BRET). Nat. Methods 2006, 3, 165−174.

(86) Uhlén, M.; Fagerberg, L.; Hallström, B. M.; Lindskog, C.;

Oksvold, P.; Mardinoglu, A.; Sivertsson, Å.; Kampf, C.; Sjöstedt, E.;

Asplund, A.; Olsson, I.; Edlund, K.; Lundberg, E.; Navani, S.; Al-

Khalili Szigyarto, C.; Odeberg, J.; Djureinovic, D.; Ottosson Takanen,

J.; Hober, S.; Alm, T.; Edqvist, P. H.; Berling, H.; Tegel, H.; Mulder,

J.; Rockberg, J.; Nilsson, P.; Schwenk, J. M.; Hamsten, M.; von

Feilitzen, K.; Forsberg, M.; Persson, L.; Johansson, F.; Zwahlen, M.;

von Heijne, G.; Nielsen, J.; Pontén, F. Tissue-Based Map of the

Human Proteome. Science 2015, 347, 394.

(68) Rose, R. H.; Briddon, S. J.; Hill, S. J. A Novel Fluorescent

Histamine H(1) Receptor Antagonist Demonstrates the Advantage of

Using Fluorescence Correlation Spectroscopy to Study the Binding of

Lipophilic Ligands. Br. J. Pharmacol. 2012, 165, 1789−1800.

(69) Ma, Z.; Lin, Y.; Cheng, Y.; Wu, W.; Cai, R.; Chen, S.; Shi, B.;

Han, B.; Shi, X.; Zhou, Y.; Lupei, D.; Li, M. Discovery of the First

Environment-Sensitive Near-Infrared (NIR) Fluorogenic Ligand for

α₁-Adrenergic Receptors Imaging in Vivo. J. Med. Chem. 2016, 59,

2151−2162.

̈

ller, C. E. Synthesis of 3-Substituted 6-Aminouracils.

(87) The Human Protein Atlas https://www.proteinatlas.org/

ENSG00000163485-ADORA1/cell (accessed Nov 1st, 2020).

(88) Schwehm, C.; Kellam, B.; Garces, A. E.; Hill, P. S. J.; Kindon,

N. D.; Tracey, D.; Li, J.; Macdonald, S. J. F.; Rowedder, J. E.;

Stoddart, L. A.; Stocks, M. J. Design and Elaboration of a Tractable

Tricyclic Scaffold to Synthesize Drug-like Inhibitors of Dipeptidyl

Peptidase-4 (DPP-4), Antagonists of the C-C Chemokine Receptor

Type 5 (CCR5), and Highly Potent and Selective Phosphoinositol-3

Kinase δ (PI3K δ ) Inhibitors. J. Med. Chem. 2017, 60, 1534−1554.

(89) May, L. T.; Bridge, L. J.; Stoddart, L. A.; Briddon, S. J.; Hill, S.

J. Allosteric Interactions across Native Adenosine-A₃Receptor

Homodimers: Quantification Using Single-Cell Ligand-Binding

Kinetics. FASEB J. 2011, 25, 3465−3476.

(70) Mu

Tetrahedron Lett. 1991, 32, 6539−6540.

(71) Muller, C. E.; Sandoval-Ramirez, J. A New Versatile Synthesis

̈

of Xanthines with Variable Substituents in the 1-, 3-, 7- and 8-

Positions. Synthesis 1995, 10, 1295−1299.

(72) Baraldi, P. G.; Borea, P. A.; Preti, D.; Tabrizi, M. A. Novel

Adenosine A₃Receptor Modulators. WO 2006/083916 A2, Aug 10,

2006.

(73) Marx, D.; Wingen, L. M.; Schnakenburg, G.; Muller, C. E.;

̈

Scholz, M. S. Fast, Efficient, and Versatile Synthesis of 6-Amino-5-

Carboxamidouracils as Precursors for 8-Substituted Xanthines. Front.

Chem. 2019, 7, 1−15.

(74) Olsson, R. A.; Holschbach, M. H.; Schrader, J. Adenosine

Receptor Antagonists With Improved Bioactivity. WO 99/31101, Jun

24, 1999.

(90) Hoffmann, C.; Castro, M.; Rinken, A.; Leurs, R.; Hill, S. J.;

Vischer, H. F. Ligand Residence Time at G-Protein-Coupled

Y

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Receptors - Why We Should Take Our Time to Study It. Mol.

Pharmacol. 2015, 88, 552−560.

(108) Nguyen, A. T. N.; Baltos, J. A.; Thomas, T.; Nguyen, T. D.;

Munoz, L. L.; Gregory, K. J.; White, P. J.; Sexton, P. M.;

Christopoulos, A.; May, L. T. Extracellular Loop 2 of the Adenosine

A₁Receptor Has a Key Role in Orthosteric Ligand Affinity and

Agonist Efficacy. Mol. Pharmacol. 2016, 90, 703−714.

(109) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.;

Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.;

Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.;

Tomancak, P.; Cardona, A. Fiji: An Open-Source Platform for

Biological-Image Analysis. Nat. Methods 2012, 9, 676−682.

̃

(91) Guo, D.; Heitman, L. H.; Ijzerman, A. P. Kinetic Aspects of the

Interaction between Ligand and g Proteincoupled Receptor: The

Case of the Adenosine Receptors. Chem. Rev. 2017, 117, 38−66.

(92) Sykes, D. A.; Stoddart, L. A.; Kilpatrick, L. E.; Hill, S. J. Binding

Kinetics of Ligands Acting at GPCRs. Mol. Cell. Endocrinol. 2019, 485,

9−19.

(93) Stoddart, L. A.; Vernall, A. J.; Bouzo-Lorenzo, M.; Bosma, R.;

Kooistra, A. J.; De Graaf, C.; Vischer, H. F.; Leurs, R.; Briddon, S. J.;

Kellam, B.; Hill, S. J. Development of Novel Fluorescent Histamine

H₁-Receptor Antagonists to Study Ligand-Binding Kinetics in Living

Cells. Sci. Rep. 2018, 8, 1−19.

(94) Sykes, D. A.; Parry, C.; Reilly, J.; Wright, P.; Fairhurst, R. A.;

Charlton, S. J. Observed Drug-Receptor Association Rates Are

Governed by Membrane Affinity: The Importance of Establishing

‘Micro-Pharmacokinetic/Pharmacodynamic Relationships ’ at the β₂-

Adrenoceptor. Mol. Pharmacol. 2014, 85, 608−617.

(95) Gherbi, K.; Briddon, S. J.; Charlton, S. J. Micro-

Pharmacokinetics: Quantifying Local Drug Concentration at Live

Cell Membranes. Sci. Rep. 2018, 8, 1−8.

(96) Kalk, P.; Eggert, B.; Relle, K.; Godes, M.; Heiden, S.;

Sharkovska, Y.; Fischer, Y.; Ziegler, D.; Bielenberg, G. W.; Hocher, B.

The Adenosine A₁Receptor Antagonist SLV320 Reduces Myocardial

Fibrosis in Rats with 5/6 Nephrectomy without Affecting Blood

Pressure. Br. J. Pharmacol. 2007, 151, 1025−1032.

(97) De Ligt, R. A. F.; Rivkees, S. A.; Lorenzen, A.; Leurs, R.;

Ijzerman, A. P. A ‘Locked-on,’ Constitutively Active Mutant of the

Adenosine A₁Receptor. Eur. J. Pharmacol. 2005, 510, 1−8.

(98) Iredale, P. A.; Alexander, S. P. H.; Hill, S. J. Coupling of a

Transfected Human Brain A₁Adenosine Receptor in CHO-K1 Cells

to Calcium Mobilisation via a Pertussis Toxin-sensitive Mechanism.

Br. J. Pharmacol. 1994, 111, 1252−1256.

(99) Baker, J. G.; Hill, S. J. A Comparison of the Antagonist

Affinities for the Gi- and Gs- Coupled States of the Human Adenosine

A₁-Receptor. J. Pharmacol. Exp. Ther. 2007, 320, 218−228.

(100) Townsend-Nicholson, A.; Schofield, P. R. A Threonine

Residue in the Seventh Transmembrane Domain of the Human A₁

Adenosine Receptor Mediates Specific Agonist Binding. J. Biol. Chem.

1994, 269, 2373−2376.

(101) Rivkees, S. A.; Barbhaiya, H.; IJzerman, A. P. Identification of

the Adenine Binding Site of the Human A₁Adenosine Receptor. J.

Biol. Chem. 1999, 274, 3617−3621.

(102) Gao, Z. G.; Mamedova, L. K.; Chen, P.; Jacobson, K. A. 2-

Substituted Adenosine Derivatives: Affinity and Efficacy at Four

Subtypes of Human Adenosine Receptors. Biochem. Pharmacol. 2004,

68, 1985−1993.

(103) Steyer, J. A.; Almers, W. A Real-Time View of Life within 100

NM of the Plasma Membrane. Nat. Rev. Mol. Cell Biol. 2001, 2, 268−

275.

(104) Calebiro, D.; Rieken, F.; Wagner, J.; Sungkaworn, T.; Zabel,

̈

U.; Borzi, A.; Cocucci, E.; Zurn, A.; Lohse, M. J. Single-Molecule

Analysis of Fluorescently Labeled G-Protein-Coupled Receptors

Reveals Complexes with Distinct Dynamics and Organization. Proc.

Natl. Acad. Sci. U. S. A. 2013, 110, 743−748.

(105) Halls, M. L.; Yeatman, H. R.; Nowell, C. J.; Thompson, G. L.;

Gondin, A. B.; Civciristov, S.; Bunnett, N. W.; Lambert, N. A.; Poole,

D. P.; Canals, M. Plasma Membrane Localization of the μ-Opioid

Receptor Controls Spatiotemporal Signaling. Sci. Signaling 2016, 9,

1−14.

(106) Lin, K.-C.; Vu, C. Condensed Purine Derivatived as A₁

Adenosine Receptor Antagonists. WO 02/44182 A1, Jun 6, 2002.

(107) Stewart, G. D.; Valant, C.; Dowell, S. J.; Mijaljica, D.;

Devenish, R. J.; Scammells, P. J.; Sexton, P. M.; Christopoulos, A.

Determination of Adenosine A₁Receptor Agonist and Antagonist

Pharmacology Using Saccharomyces Cerevisiae: Implications for

Ligand Screening and Functional Selectivity. J. Pharmacol. Exp. Ther.

2009, 331, 277−286.

Z

https://dx.doi.org/10.1021/acs.jmedchem.0c02067

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1Receptor in Living Cells

DOI: 10.1021/acs.jmedchem.0c02067

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c02067

Full text of DOI:10.1021/acs.jmedchem.0c02067

Products guided by the article

R&D Labs maybe for 676524-06-4

Relevant to this article

Hot Product