Development of Covalent, Clickable Probes for Adenosine A1and A3Receptors

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Development of Covalent, Clickable Probes for Adenosine A₁and A₃

Receptors

Phuc N. H. Trinh, Daniel J. W. Chong, Katie Leach, Stephen J. Hill, Joel D. A. Tyndall, Lauren T. May,*

Andrea J. Vernall,* and Karen J. Gregory *

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ABSTRACT: Adenosine receptors are attractive therapeutic

targets for multiple conditions, including ischemia-reperfusion

injury and neuropathic pain. Adenosine receptor drug discovery

eﬀorts would be facilitated by the development of appropriate tools

to assist in target validation and direct receptor visualization in

diﬀerent native environments. We report the development of the

ﬁrst bifunctional (chemoreactive and clickable) ligands for the

adenosine A₁receptor (A₁R) and adenosine A₃receptor (A₃R)

based on an orthosteric antagonist xanthine-based scaﬀold and on

an existing structure−activity relationship. Bifunctional ligands

were functional antagonists with nanomolar aﬃnity and irreversible

binding at the A₁R and A₃R. In-depth pharmacological proﬁling of

these bifunctional ligands showed moderate selectivity over A_2Aand A_2Badenosine receptors. Once bound to the receptor, ligands

were successfully “clicked” with a cyanine-5 ﬂuorophore containing the complementary “click” partner, enabling receptor detection.

These bifunctional ligands are expected to aid in the understanding of A₁R and A₃R localization and traﬃcking in native cells and

living systems.

compartments; however, GPCR-targeting antibodies notori-

ously lack speciﬁcity, and speciﬁc antibodies against the A₁R are

lacking. Chemical probes such as “preassembled” A₁R

ﬂuorescent ligands have been developed to interrogate A₁R

function.¹²⁻¹⁴However, large ﬂuorescent ligands also come

with challenges; for example, the ﬂuorescent moieties may

interfere with receptor aﬃnity or selectivity.¹⁵⁻¹⁷Furthermore,

the chemical synthesis and detailed characterization required for

each preassembled ﬂuorescent ligand limits the number of

diﬀerent ﬂuorophores that can be incorporated. Breaking up a

preassembled ﬂuorescent ligand into two parts with a

complementary “clickable” handle installed on each oﬀers an

alternative and advantageous “in situ labeling” solution to the

conventional binding of a single high-molecular weight

ﬂuorescent ligand. This strategy creates a bifunctional ligand

that requires a two-step labeling approach to detect GPCRs. The

ﬁrst step involves covalent attachment of either a chemoreactive

or photoreactive ligand to the receptor, followed by incorpo-

ration of a reporter (e.g., a ﬂuorophore or biotin) using a

INTRODUCTION

■

The adenosine receptor (AR) family consists of four (A₁, A_2A,

A_2B, and A₃) cell surface G protein-coupled receptor (GPCRs)

subtypes that are activated by adenosine, an endogenous purine

nucleoside present both intracellularly and extracellularly in

living cells.¹⁻³Among the AR subtypes, the A₁R remains a highly

pursued therapeutic target for a number of neurological,

cardiovascular, and renal pathologies. Activation of the A₁R

minimizes cardiac and renal ischemia-reperfusion injury, atrial

ﬁbrillation, and neuropathic pain, whereas A₁R inhibition can

enhance diuresis and cognition.⁴⁻⁸Despite signiﬁcant eﬀorts,

selective A₁R agonists or antagonists have not transitioned into

the clinic, typically due to a lack of on-target eﬃcacy or adverse

eﬀects.⁹In addition, traditional discovery pipelines have taken a

limited view of A₁R activity often relying on a single measure of

receptor activation to guide hit progression. However, we and

others have shown that biased agonism is operative at the A₁R,

raising the possibility that not all aspects of A₁R drug action have

been fully elucidated.⁹⁻¹¹One of the major challenges faced in

A₁R drug discovery is a need for speciﬁc tools for target

engagement and to study noncanonical measures of receptor

behavior that have gone unappreciated when assessing A₁R

ligands in traditional pipelines, such as receptor internalization

and localization.

Received: December 16, 2020

Published: June 14, 2021

Antibody-based approaches are commonly employed to

localize proteins of interest in diﬀerent tissues or subcellular

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bioorthogonal reaction (e.g., click chemistry). The feasibility of

this two-step labeling approach has been demonstrated for

multiple GPCRs, including metabotropic glutamate receptor

subtypes 2¹⁸and 5,¹⁹cannabinoid CB₂receptors,²⁰and

adenosine A_2Areceptors.²¹Importantly, relying on conjugation

between two functional groups that are foreign to the biological

system oﬀers exquisite selectivity. For example, bifunctional

cannabinoid CB₂receptor ligands enabled proﬁling of

endogenous receptor expression levels in peripheral blood

mononuclear cells by ﬂuorescence-activated cell sorting.²⁰

Combining bioorthogonal labeling with advanced imaging

approaches has the potential to yield great insights into

endogenous receptor localization and target engagement in

diﬀerent cell and tissue types. Our two-step approach also has

signiﬁcant advantages over a bespoke “preassembled” ﬂuores-

cent ligand in that diﬀerent ﬂuorophores can be tethered, akin to

the HaloTag and SNAP-tag technology, and ﬂuorophores

appropriate for a range of diﬀerent ﬂuorescent techniques,

including time-resolved ﬂuorescence resonance energy transfer

(TR-FRET) experiments, could be incorporated.

Fluorescent ligand design is often facilitated by detailed

structural information gained from X-ray crystallographic and

cryo-electron microscopic protein structures. In recent years, the

amount of structural information for GPCRs has increased

rapidly and is immensely helpful to interrogate ligand−receptor

interactions. The recently determined A₁R crystal structure

(PDB entry 5UEN)²²complexed with xanthine-based antago-

nist DU172 (1) (Figure 1) showed a covalent bond between the

with a C8-bicyclo[2.2.2]octyl that conferred A₁R selectivity.²⁴

A

xanthine N1-butyl (as opposed to propyl) alkyl chain was

hypothesized to imbue A₁R selectivity because of the deeper

hydrophobic pocket apparent in the A₁R (PDB entry 5UEN).²²

A bicyclo[2.2.2]octyl group was appended from the xanthine C8

with the aim of achieving excellent A₁R aﬃnity and selectivity as

has previously been reported. The bicyclo[2.2.2]octane-1,4-

dicarboxylic acid moiety was also advantageous over using a

cyclohexyl moiety with cis,trans isomer complications. Analo-

gous to DU172, a sulfonyl ﬂuoride warhead capable of

potentially covalently binding to Y271^7.36of the A₁R was

installed. A similarly positioned tyrosine is also present in the

A_2AR (Y271) and A₃R (Y265^7.36), but the use of the

bicyclo[2.2.2]octyl group, based on SAR, was hypothesized to

provide A₁R selectivity. The bifunctional ligands also required a

linker terminating in a “clickable” functional group. Linkers were

appended in an analogous position to the ethyl dimethylamine

of 3 for two reasons. (1) Variations in this part of the molecule

can retain A₁R aﬃnity,²⁴and (2) on the basis of the position of

DU172 in PDB entry 5UEN, it was envisaged that a linker

installed from this part of the ligand would be exposed to the

receptor surface and accessible for a “click” reaction. The

“clickable” handle design consisted of either an azide or an

alkyne, which could undergo a Huisgen 1,3-dipolar cyclo-

addition reaction with the complementary partner of, for

example, a ﬂuorophore, forming a stable triazole. Diﬀerent linker

lengths and types (alkyl vs PEG-like) were installed to elucidate

the optimal linker for binding, reaction with a tag, and

considering the overall solubility of the ligand.

Synthesis. Imide 4, prepared according to a literature²⁵

method, was alkylated using either bromobutane or bromopro-

pane to give 5 or the previously reported 6²⁵(Scheme 1). The

vinyl amine of substituted imidamides 5 and 6 was hydrolyzed to

give 7 and 8, respectively, which was then nitrated and reduced

to aﬀord diamines 9 and 10, respectively, in moderate yield.

With the 2,4-dimethoxybenzyl (DMB) protecting group in

place, diamines 9 and 10 were coupled with bicyclo[2.2.2]-

octane-1,4-dicarboxylic acid monomethyl ester using O-(7-

azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-

ﬂuorophosphate (HATU) to give the monoamidated product,

which subsequently underwent dehydrative ring closure and

methyl ester hydrolysis using KOH in isopropanol at reﬂux to

give carboxylic acids 11 and 12. The carboxylic acid was then

either methylated to give 13 or coupled via HATU-mediated

amide bond formation with a series of amino linkers containing a

terminal azide or alkyne to give 14−21.

The DMB group of 13−21 was removed, and the imide

alkylated using <1 equiv of an alkyl bromide at room

temperature to give UODC2, UODC5, UODC6, and

UODC9−UODC14 (Scheme 1). In all instances, one

regioisomer was produced, with alkylation of the imidazole

not detected. Potentially covalent sulfonyl ﬂuorides UODC6

and UODC2 were converted to sulfonic acids UODC7 and

UODC3, respectively, to allow comparison to analogues

without covalent capacity. Initially for all compounds, the

imidazole -NH was protected with a pivaloyloxymethyl (POM)

group but eventual removal of POM with a sulfonyl ﬂuoride

present proved to be troublesome with high conversion to the

sulfonic acid occurring under most conditions. To synthesize the

analogues without the sulfonyl ﬂuoride “covalent arm”, the

imidazole -NH of 14 and 15 was ﬁrst protected with POM to

give 22 and 23, respectively, followed by DMB cleavage and

reaction with >1 equiv of bromopropane at 50 °C to give 24 and

Figure 1. A₁R ligands DU172 (1), PSB36 (2), and 3.

DU172 sulfonyl moiety and Y271^7.36(Ballesteros−Weinstein

numbering in superscript) and the cyclohexyl group positioned

toward the top of the receptor surrounded by residues M177^5.35

,

L253^6.54, and T257^6.57. Subsequently, the co-crystal structure of

A₁R with orthosteric antagonist PSB36 (2) (Figure 1) (PDB

entry 5N2S)²³showed PSB36 resides deeper in the A₁R binding

pocket than in the A_2AR. In particular, the N1-butyl of PSB36

was directed toward a hydrophobic pocket of V87^3.32, L88^3.33

,

A91^3.36, M180^5.38, W247^6.48, and H251^6.52in the A₁R while this

region appeared to be less accessible in the A_2AR.²³There have

been many SAR studies of xanthine-based AR ligands. Notably,

xanthine C8-bicyclo[2.2.2]octyl derivatives with varying ex-

tensions from the bicyclo ring improved A₁R selectivity [for

example, 3 (Figure 1)].²⁴Herein, we describe the rational

design, synthesis, and evaluation of bifunctional (covalent and

clickable) ligands for the A₁R.

RESULTS AND DISCUSSION

Ligand Design. The design of the bifunctional ligands was

inspired by features of DU172 and A₁R antagonists such as 3

■

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a

Scheme 1

a

Reagents and conditions: (i) R¹-Br, Cs₂CO₃, TBAB, ACN, DMF, 60 °C, 3 h, 76−80%; (ii) 2 M KOH, MeOH, 50 °C, 1 h, 84−88%; (iii) NaNO₂,

50% AcOH, 70 °C, 1 h; (iv) Na₂S₂O₄, 25% aq. NH₄OH, MeOH, 80 °C, 4 h, 62−76% (over two steps); (v) bicyclo[2.2.2]octane-1,4-dicarboxylic

acid monomethyl ester, HATU, NEt₃, ACN, 1 h, rt; (vi) 1 M KOH, i-PrOH, reﬂux, 1 h, 59−73% (over two steps); (vii) H₂SO₄, MeOH, reﬂux, 3 h,

71%, or NH₂- “linker” (see the Supporting Information), HATU, DIPEA, DMF, 18 h, rt, 42−98%; (viii) TFA, 50 °C; (ix) 4-[(3-

bromopropyl)carbamoyl]benzene-1-sulfonyl ﬂuoride (see the Supporting Information), K₂CO₃, DMF, rt, 11−98% (over two steps); (x) 1 M

NaOH, MeOH, rt, 1 h, 88−95%; (xi) POM-Cl, K₂CO₃, DMF, 50 °C, 18 h, 52−57%; (xii) bromopropane, K₂CO₃, DMF, 50 °C, 18 h, 42−77%;

(xiii) 2 M NaOH, MeOH, rt, 1 h, 89−98%.

25, respectively. Finally, UODC4 and UODC1 were obtained

upon removal of the POM group with methanolic NaOH. One

example of a cyclohexyl moiety in place of the bicyclo-octyl was

synthesized using analogous reaction conditions to give

UODC8 (Scheme 2), the only diﬀerence to DU172 being an

N1-butyl instead of a propyl.

Pharmacology. We ﬁrst determined the apparent aﬃnity of

newly synthesized compounds for the hA₁R in membranes

derived from A₁R-FlpInCHO cells, based on displacement of the

antagonist radioligand, [³H]DPCPX (1 nM). All compounds,

except for UODC8, completely displaced the binding of

[³H]DPCPX, indicating a competitive interaction for the

orthosteric site (Figure 2 and Figure S1); the estimated aﬃnities

are listed in Table 1. The eﬀect of UODC8 was bell-shaped,

completely displacing [³H]DPCPX binding up to 1 μM;

however, at 10 μM, less displacement was observed. This bell-

shaped inhibition curve may indicate a bitopic mode of action²⁶

or alternatively nonspeciﬁc eﬀects or a lack of solubility at 10

μM. Thus, UODC8 was empirically ﬁtted to a one-site

inhibition mass action curve, except at 10 μM, to estimate

aﬃnity. The bicyclo[2.2.2]octyl variant of UODC8, UODC6,

had a similar aﬃnity for the A₁R, whereas substituting the

sulfonyl ﬂuoride for sulfonic acid (UODC7) decreased the

aﬃnity ∼10-fold. Given that incorporation of the sulfonyl

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a

compound being within 2-fold of that of UODC2. We next

sought to establish the tolerability of an alternative clickable

handle. Introducing a terminal alkyne to give UODC4 resulted

in 10-fold reduced aﬃnity when compared to that of the azido

equivalent (UODC1). Installing a terminal alkyne with the

sulfonyl ﬂuoride warhead (UODC5) yielded a bifunctional

probe with good aﬃnity, albeit 3-fold lower than that of

UODC2. Extending the linker between the amine and the

terminal alkyne to C4 resulted in the highest-aﬃnity bifunctional

A₁R probe (UODC14). UODC14 had a similar aﬃnity (within

3-fold) for the A₁R when compared with that of the previously

reported DU172 (1).²²Overall, it was pleasing that the

previously reported xanthine N3-sulfonyl ﬂuoride terminating

substituent^22,27was well tolerated on the UODC bifunctional

probes containing the C8-bicyclo[2.2.2]octyl clickable linkers.

We next sought to test if each probe retained A₁R antagonist

properties. The A₁R preferentially couples to G_i/oproteins,

resulting in adenylate cyclase inhibition and thereby reducing

cyclic adenosine monophosphate (cAMP) production. Thus, we

used the A₁R-mediated inhibition of forskolin-stimulated cAMP

accumulation assays in A₁R-FlpInCHO cells to test biological

eﬀects. Concentration−response curves for the nonselective

agonist NECA were generated in the absence and presence of

each compound at 1 μM (Figure 2 and Figure S1). Each

compound caused a rightward shift of the NECA curve, with no

suppression of the maximal response, consistent with each

compound acting as a competitive A₁R antagonist. Analysis of

these data yielded functional aﬃnity estimates (pK_b) that were

in good agreement with pK_iestimates from radioligand binding

Scheme 2

a

Reagents and conditions: (i) cyclohexanoic acid, HATU, NEt₃,

ACN, rt, 1 h; (ii) 1 N KOH, i-PrOH, reﬂux, 1 h, 88% (over two

steps); (iii) TFA, 50 °C; (iv) 4-[(3-bromopropyl)carbamoyl]-

benzene-1-sulfonyl ﬂuoride, K₂CO₃, DMF, rt, 18 h, 63% (over two

steps).

ﬂuoride warhead was tolerated, we next tested the impact of

incorporating an azide clickable handle to replace the ethyl

dimethylamine of 3 to generate the bifunctional probe, UODC2,

which had a high A₁R aﬃnity (sub-10 nM) equivalent to that

reported for 3 (K_i= 6 nM hA₁R). Notably, the reversible sulfonic

acid variant, UODC3, had an ∼100-fold lower aﬃnity compared

to that of UODC2. With regard to the N1 substituent, the N1-

propyl xanthine (UODC9) had the same aﬃnity relative to that

of N1-butyl (UODC2). Similarly, extending the azide linker

length to C4 (UODC10) or C8 (UODC11) or replacing the

carbon chain with a PEG2 (UODC12) or PEG3 (UODC13)

linker had little eﬀect on aﬃnity with the value for each

Figure 2. Pharmacological characterization of synthesized compounds in radioligand binding and cAMP inhibition assays. (A) Inhibition of binding of

[³H]DPCPX to membrane preparations from FlpInCHO-hA₁R. (B) NECA concentration−response curves for cAMP inhibition in the absence or

presence of test compounds (1 μM) and SLV320 (1 μM) in FlpInCHO-hA₁R cells. Data represent means SEM from at least three experiments

performed in duplicate (see Table 1 for exact numbers). Error bars not shown lie within the dimensions of the symbol.

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Table 1. Aﬃnity Estimates (pK_iand pK_B) of Test Compounds from [³H]DPCPX Competition Binding and cAMP Inhibition

a

Assays, Respectively, in FlpInCHO-hA₁R Cells

a

Data are expressed as means

SEM from the indicated number (n) of independent experiments performed in duplicate. n/a denotes not

b

available, and n/d denotes not determined. Negative logarithm of the equilibrium dissociation constant for antagonists determined from

c

displacement of [³H]DPCPX binding using the Cheng−Prusoﬀ equation. Negative logarithm of the equilibrium dissociation constant for

antagonists as determined from NECA concentration−response curves for cAMP inhibition in the absence and presence of the indicated

compound added 5 min prior to NECA addition using the Gaddum−Schild equations, assuming a Schild slope.

assays (Table 1). A high aﬃnity is desirable for covalent clickable

probes, as it allows suﬃcient receptor occupancy to ensure

negligible interactions with other unrelated targets.

To ascertain the irreversible interaction of sulfonyl ﬂuoride-

containing antagonists (after incubation for 3 h), radioligand

displacement assays were performed following extensive

washout (6 × 20 min).^19,27Preincubation with compounds

containing a sulfonyl ﬂuoride warhead (e.g., UODC2, UODC5,

UODC6, UODC9, and UODC14) completely abolished

[³H]DPCPX binding after the washing cycles, suggesting

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covalent and irreversible binding to the A₁R (representatives

shown in Figure 3, and all other covalent compounds are

extensive washing did not damage the membrane integrity or

alter membrane binding sites.

Subsequently, we tested sulfonyl ﬂuoride-containing clickable

antagonists UODC2, UODC5, and UODC9−UODC14 for

selectivity against the other adenosine receptor subtypes (A_2AR,

A_2BR, and A₃R). For this purpose, we employed a functional

response common to all four subtypes, namely, cAMP

accumulation, which is inhibited by the A₁R and A₃R but

stimulated by the A_2AR and A_2BR. Concentration−response

curves were generated in the absence or presence of a

submaximal EC₈₀concentration of NECA in parallel with a

reference selective antagonist for each subtype. With the

exception of UODC5 at the A_2AR and A_2BR, all compounds

inhibited the NECA response (Figure 4 and Figure S3). In

general, all compounds had similar aﬃnity (within 3-fold) for

the A₁R and A₃R (Table 2), the exception being UODC14,

which had a 10-fold higher aﬃnity for the A₁R than for the A₃R.

This was surprising given the excellent A₁R over A₃R selectivity

exhibited by previously reported bicyclo[2.2.2]octyl 1,3-

dipropylxanthine derivatives (e.g., compounds 17−22 in ref

24). However, an important diﬀerence is that our bicyclo[2.2.2]-

octyl xanthines contain a covalent warhead. Therefore, given the

similarly located Tyr in the A₁R and A₃R (Figure 5 and discussed

below), the covalent nature of these ligands may in part confer

A₃R aﬃnity. In addition, selectivity assessment of noncovalent

derivatives UODC1 and UODC4 also contradicted the earlier

SAR report, which relied on radioligand binding rather than

functional responses to determine the selectivity of

bicyclo[2.2.2]octyl xanthines.²⁴Importantly, each ligand

showed an ∼10-fold greater aﬃnity for the A₁R than for both

the A_2AR and the A_2BR (Table 2). The superior aﬃnity of the

UODC ligands for the A₁R and A₃R over the A_2AR and A_2BR is in

line with a previous xanthine SAR, in that longer alkyl and

Figure 3. A₁R clickable ligands with the ﬂuorosulfonyl warhead

irreversibly bind to the hA₁R. [³H]DPCPX binding after preincubation

with increasing concentrations of test compounds followed by a 6 × 20

min washout. Experiments were performed with ﬂuorosulfonyl-

containing ligands that bind irreversibly (UODC2, UODC5,

UODC6, UODC9, and UODC14) as well as sulfonic acid-containing

ligands that were reversible (UODC3 and UODC7). Data represent

the mean SEM from at least three experiments performed in duplicate

(see Table 1 for exact numbers). Error bars not shown lie within the

dimensions of the symbol. *p < 0.0001, from one-way analysis of

variance with Dunnett’s post-test compared to vehicle.

reported in Figure S2 and Table 1). Importantly, under the same

conditions, preincubation with sulfonic acid-containing com-

pounds UODC3 and UODC7, followed by washout, did not

displace the radioligand, demonstrating the extensive washout

steps successfully removed noncovalent compounds. In

addition, aﬃnity estimates for sulfonyl ﬂuoride-containing

compounds were unchanged with washout, suggesting the

Figure 4. Adenosine receptor subtype selectivity of A₁R antagonists, bearing a click handle (i.e., terminal alkyne and aliphatic azide) and ﬂuorosulfonyl

warhead. Functional cAMP concentration−response curves for NECA (blue line) or A₁R antagonists in the presence of a submaximal (EC₈₀) NECA

concentration at the indicated AR subtype stably expressed in FlpInCHO cells. Selective antagonists SLV320, SCH442416, PSB603, and MRS1220 for

the A₁R, A_2AR, A_2BR, and A₃R, respectively, were used in parallel as positive controls. Data represent means SEM from at least three experiments

performed in duplicate (see Table 2 for exact numbers). Error bars not shown lie within the dimensions of the symbol.

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Table 2. Adenosine Receptor Subtype Aﬃnity (pK_B) Values of Selected Test Compounds on cAMP Inhibition and Accumulation

a

in Human AR-Expressing FlpInCHO Cells

hA₁R

pK_B(nM)

hA_2A

R

hA_2B

R

hA₃R

pK_B(nM)

n

pK_B(nM)

n

pK_B(nM)

n

UODC2

UODC9

8.09 0.31 (8.1)

8.56 0.21 (2.8)

8.41 0.13 (3.9)

7.99 0.19 (10.2)

8.45 0.18 (3.5)

8.48 0.16 (3.3)

7.51 0.16 (30.9)

8.41 0.17 (3.9)

8.40 0.32

8

4

7

5

7

4

8

7.42 0.25 (38.0)

7.58 0.19 (26.3)

6.78 0.31 (166)

7

5

9

5

7

6

9

7.10 0.15 (79.4)

7.75 0.26 (17.8)

7.58 0.70 (26.3)

8.17 0.68 (6.7)

7.64 0.59 (22.9)

7.02 0.15 (95.5)

8

4

8

4

7

6

8

8.38 0.17 (4.2)

7.96 0.51 (11.0)

8.31 0.50 (4.9)

8.52 0.50 (3.0)

7.40 0.35 (39.8)

8.08 0.13 (8.3)

7.33 0.18 (46.8)

7.36 0.38 (43.7)

8.13 0.29

13

6

5

7

6

12

7

12

6

11

UODC10

UODC11

UODC12

UODC13

UODC5

UODC14

UODC1

UODC4

nd

7.50 0.26 (31.6)

7.44 0.34 (36.3)

nd

7.15 0.28 (70.8)

nd

9.47 0.20 (0.3)

nd

7.45 0.12 (35.4)

7.73 0.14

nd

8.87 0.23 (1.3)

7.01 0.66

9.44 0.43 (0.4)

7.45 0.36

8.30 0.34 (5.0)

b

reference antagonist

a

Data are expressed as means SEM from the indicated number (n) of independent experiments performed in duplicate. nd denotes not deﬁned

b

due to incomplete inhibition and a poor curve ﬁt. SLV320, SCH442416, PSB603, and MRS1220 were used as reference antagonists in A₁R-, A_2AR,

A_2BR-, and A₃R-FlpInCHO cells, respectively.

Molecular Modeling. Compound UODC2 was docked

into the A₁R crystal structure (PDB entry 5UEN)²²using a

covalent linkage to Y271^7.36. UODC2 binds to the A₁R in a

fashion similar to that of DU172 (ﬁve of the top six poses out of

10) with hydrogen bonds between N253^6.55and the xanthine

scaﬀold. A new hydrogen bond is formed with F171^EL2via the

amide carbonyl oxygen where the amide is ﬂipped compared

with DU172 (Figure 5). The ﬂexible terminal azide linker of

UODC2 was easily accommodated and oriented toward the

extracellular space, with hydrogen bonds seen between the azide

and the main chain carbonyl of K265^EL3. Some variation exists

between poses in the ﬂexible propylazido chain, which is to be

expected.

UODC2 was also docked into an A₃R homology model (using

the A₁R crystal structure, PDB entry 5UEN, as a template) using

a covalent linkage to Y265^7.36. The top-ranked pose is similar to

that of DU172 in the A₁R crystal structure (Figure 5), when

superimposed. A small cluster of three poses shows the xanthine

ring shifted toward a more central position; however, these

poses score much lower than the top-ranked pose and appear to

have close contacts between the phenyl ring of the covalent arm

and the bicyclo-octyl moiety. A key hydrogen bonding

interaction between N250^6.55and the xanthine scaﬀold was

maintained in the A₃R along with π-interactions with F168^EL2

.

Indeed, Yang et al.²⁸recently identiﬁed Y265^7.36forming a

covalent bond with the ﬂuorosulfonyl warhead of their

compound “17b”, which is structurally related to UODC2,

albeit with an ∼10-fold lower aﬃnity for the A₃R when

compared to that of UODC2. Residue Y265^7.36of the A₃R is in

the same position as Y271^7.36in the A₁R; therefore, UODC2 and

related compounds could covalently bind to the A₃R, as well.

Studies of the docking of UODC9 and UODC14 to the A₃R

showed both compounds could be accommodated within the

orthosteric site (data not shown). An equivalently positioned

Figure 5. Binding of UODC2 in (A) the crystal structure of the A₁R

(PDB entry 5UEN) and (B) the homology model of the A₃R. The A₁R

is shown as a green ribbon, A₃R as a white ribbon, and UODC2 as

purple and yellow sticks. Dashed yellow lines indicate hydrogen bonds.

UODC2 was docked covalently to Y271 and Y265.

Tyr is also present in the A_2AR (Y271)²⁹but not in the A_2BR.³⁰

A

bulkier moieties can tolerated at the N1 and N3 positions of the

A₁R and A₃R but not at those of the A_2AR and A_2BR (as discussed

by Cheng et al.²³). During ligand design, we hypothesized that a

xanthine N1-butyl (as opposed to propyl) alkyl chain might

imbue better A₁R selectivity over the other AR subtypes.

However, comparison between the one N1-propyl derivative

(UODC9) and the equivalent N1-butyl derivative (UODC2)

revealed little diﬀerence in activity across all AR subtypes (Table

2).

potential reason for the ∼10-fold greater aﬃnity for ligands

reported herein for the A₁R over the A_2AR and A_2BR could be

that both the A_2AR and the A_2BR contain a Met residue

(M270^7.35and M272^7.35, respectively) in the position occupied

by T270^7.35in the A₁R and L264^7.35in the A₃R, leading to Met−

cyclooctyl steric clashes. Docking of UODC2 to the A_2AR (PDB

entry 6LPK)³¹under the same covalent conditions produced no

poses binding in the orthosteric site (Figure S4).

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Given the high aﬃnity for the A₃R, we assessed the potential

for our covalent clickable compounds to irreversibly bind to the

hA₃R. For this purpose, we performed a NanoBRET displace-

ment assay in transiently transfected NLuc-tagged A₃R-

HEK293A cells using a washout protocol similar to that used

for the hA₁R (Figure 3). Antagonists UODC2, UODC6,

UODC5, and UODC14 weakened the binding of the

ﬂuorescent antagonist CA200645 measured by resonance

energy transfer, while sulfonic acid analogues UODC3 and

UODC7 were completely washed away (Figure 6). As such, our

the anti-V5 antibody was assessed in nontransfected FlpIn CHO

cells (Figure S5), which conﬁrmed these apparent bands are

A₁R-speciﬁc ﬂuorescent signals. As noted previously for the

CB₂R²⁰and A_2AR,²¹these multiple bands likely represent

diﬀerent post-translationally modiﬁed receptors. To demon-

strate the speciﬁcity of the aﬃnity-based probes, we investigated

whether a competitive antagonist could inhibit labeling. A₁R-

FlpInCHO cells were preincubated with a saturating concen-

tration (10 μM) of the irreversible competitor, UODC6, prior to

labeling and click chemistry to incorporate Cy5.5. In the

presence of UODC6, there was minimal Cy5.5 incorporation,

indicating the binding of each bifunctional probe was speciﬁcally

blocked (Figure 8). Similarly, there was no labeling of

nontransfected FlpInCHO cells in the presence of any of the

clickable probes (Figure 8). Collectively, these results show that

bifunctional compounds UODC2, UODC9, and UODC14 can

be employed as aﬃnity-based clickable probes for the A₁R.

CONCLUSION

■

In summary, we report the rational design and validation of

bifunctional A₁R and A₃R orthosteric antagonists that are both

covalent and clickable. The bicyclo[2.2.2]octyl xanthine-based

scaﬀold used herein showed similar aﬃnity (within 3-fold) for

the A₁R and A₃R, with moderate selectivity over A_2AR and A_2BR

subtypes. The ﬂuorosulfonyl moiety is predicted to form a

covalent bond with a Tyr^7.36at the top of TM7, which is

conserved between the A₁R and A₃R. Once bound to the

receptor, compounds UODC2, UODC9, and UODC14 were

conjugated via click chemistry with a ﬂuorophore. By

preincubation with a competitive ligand, we demonstrate that

probe-dependent labeling of the A₁R was inhibited. Therefore,

we propose that these ﬁrst clickable and covalent A₁R/A₃R

orthosteric antagonists present new tools to aid in the structural

determination of the A₃R, as well as receptor localization in

recombinant and native systems in which the A₁R and A₃R are

diﬀerentially expressed.

Figure 6. Antagonists with a sulfonyl ﬂuoride warhead irreversibly bind

to hA₃R. Inhibition of CA200645 binding in HEK293A cells transiently

transfected with the Nluc-tagged A₃R. Competition binding after

preincubation for 3 h with a 6 × 20 min washout. Data represent means

SEM from at least three experiments performed in duplicate. *p <

0.01, one-way analysis of variance with Dunnett’s post-test compared to

the vehicle.

covalent clickable antagonists may be useful tools for hA₃R

structural elucidation, particularly given high-resolution struc-

tures that are currently lacking for the A₃R. Furthermore, despite

our clickable probes having high aﬃnities for the A₁R and A₃R,

their impact remains high with respect to visualizing receptor

subcellular distribution and traﬃcking in recombinant and

native cells. The A₁R and A₃R have diﬀerential tissue expression;

the A₁R is strongly expressed in the cerebral cortex, heart,

parathyroid gland, and pancreas, whereas the A₃R is strongly

expressed in granulocytes as per the Human Protein Atlas

(http://www.proteinatlas.org).³²

Fluorescent Labeling of the hA₁R. Having established the

selectivity and covalent binding properties of our novel A₁R

ligands, we next sought to conﬁrm the clickability of two azide-

containing (UODC2 and UODC9) and two alkyne-containing

(UODC5 and UODC14) probes. To accomplish this task, A₁R-

FlpInCHO cells were preincubated with increasing concen-

trations of probes to allow covalent binding, followed by click

chemistry to attach a complementary clickable near-infrared

ﬂuorophore, either Cy5.5-azide or Cy5.5-alkyne. Upon cell lysis,

probe-labeled proteins were separated by SDS−PAGE, and

ﬂuorescence was visualized and immunoblotting performed to

detect A₁R via a C-terminal V5 epitope tag (Figure 7). In the

absence of clickable probes, there was little Cy5.5 incorporation

in A₁R-FlpInCHO cells. Concentration-dependent click chem-

istry incorporation of Cy5.5 was observed in the presence of

UODC2, UODC9, and UODC14, but not UODC5, suggesting

a longer linker than N-propargyl is required to facilitate the

attachment of the ﬂuorophore. Importantly, multiple bands

were evident with molecular weights ranging from 37 to 50 kDa

in both Cy5.5 labeling and immunoblotting. The speciﬁcity of

EXPERIMENTAL SECTION

■

Chemistry. Chemicals were purchased from Sigma-Aldrich, AK

Scientiﬁc, Alfa Aesar, or Merck and used without further puriﬁcation.

Unless otherwise stated, anhydrous solvents and dry atmospheric

conditions were used in all reactions. Unless otherwise stated, all

reactions were carried out at room temperature (rt) under atmospheric

pressure. Thin layer chromatography (TLC) was carried out on silica

gel plates 60 F₂₅₄and visualized under UV light at 254 nm. Potassium

permanganate dip was used to visualize non-UV active compounds.

Flash column chromatography separations using 40−63 μm silica were

performed by the gradient elution method, and the elution solvent

system is given in each instance. Reverse-phase high-performance liquid

chromatography (RP-HPLC) was carried out on an Agilent 1260

Inﬁnity system, using a YMC-C8 5 μm (150 mm × 10 mm) column at a

rate of 3 mL/min for semipreparative RP-HPLC and a YMC-C8 5 μm

(150 mm × 4.6 mm) column at a rate of 1 mL/min for analytical RP-

HPLC. The following mobile phases were used A, H₂O (0.05% TFA),

and B, 9:1 ACN/H₂O (0.05% TFA). The standard analytical method

for analytical RP-HPLC (for which retention times of compounds are

given) was 5% B for 1 min and a linear gradient from 5% to 95% B from

1 to 27 min (followed by a 1 min hold at 95% B and then a 2 min linear

gradient from 95% to 5% B and a 4 min re-equilibration at 5% B). All

ﬁnal compounds were >95% RP-HPLC analytically pure as assessed by

peak integration at 245 nm. A typical semipreparative HPLC method

was carried out as described: 5% B for 1 min and a linear gradient from

5% to 95% B from 1 to 12 min (followed by a 2 min hold at 95% B and

then a 3 min linear gradient from 95% to 5%). High-resolution

electrospray ionization mass spectra (HRMS-ESI) were recorded on a

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Figure 7. Concentration-dependent incorporation of Cy5.5 is evident in hA₁R-FlpIn CHO cells by UODC2, UODC9, and UODC14 but not

UODC5. (A and B) Representative in-gel Cy5.5 ﬂuorescence images coupled with immunoblots for V5 (indicative of the A₁R) shown in gray scale and

merged Cy5.5 ﬂuorescence in red and anti-V5 in green. (C) Quantiﬁcation of ﬂuorescence intensity labeled with increasing concentrations of clickable

probes. Cy5.5 ﬂuorescence was corrected for relative A₁R level (based on V5 immunoblotting against the C-terminal V5 epitope tag), expressed as fold

over basal. Data represent means SEM from three experiments. Error bars not shown lie within the dimensions of the symbol.

microTOF_Qmass spectrometer. Proton (¹H), ﬂuorine (¹⁹F), and

carbon (¹³C) nuclear magnetic resonance (NMR) spectroscopy was

carried out using a 400 or 500 MHz Varian NMR spectrometer. Two-

dimensional NMR experiments, including COSY, HSQC, HMBC, and

NOESY, were used to assign chemical shifts and for some compounds

to assign a regioisomer. Chemical shifts are listed on the δ scale in parts

per million, referenced to CDCl₃(¹H NMR δ 7.26; ¹³C NMR δ 77.16)

or DMSO-d₆(¹H NMR δ 2.50; ¹³C NMR δ 39.52) with residual solvent

as the internal standard and coupling constants (J) recorded in hertz.

Note that not all magnetically non-equivalent carbons were observed in

the ¹³C NMR spectrum for all compounds. Signal multiplicities are

assigned as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd,

doublet of doublets; br, broad; m, multiplet.

General Procedure for the Alkylation of 4. To a stirred

suspension of 4 in acetonitrile and DMF were added Cs₂CO₃and tetra-

N-butylammonium bromide (TBAB). The bromoalkane was added

dropwise over 15 min, and the reaction mixture was stirred for 1 h at rt

and then for 3 h at 60 °C. Ice−water was added, and the mixture was

stirred for 30 min. The mixture was extracted with CH₂Cl₂and washed

with saturated aqueous NaHCO₃and brine. The pooled organic layers

were dried with MgSO₄, ﬁltered, and concentrated in vacuo. The solid

obtained was recrystallized with 50% aqueous methanol, ﬁltered,

washed with cold 50% aqueous methanol, and air-dried.

(3.25 g, 9.97 mmol), TBAB (20 mg, 0.06 mmol), and 1-bromobutane

(1.91 g, 1.51 mL, 13.93 mmol) gave 5 (2.75 g, 7.08 mmol, 76%) as oﬀ-

white crystals. ¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz,

3H), 1.25 (m, 2H), 1.47 (m, 2H), 2.83 (s, 3H), 3.04 (s, 3H), 3.71 (s,

3H), 3.76 (t, J = 7.2 Hz, 2H), 3.78 (s, 3H), 5.05 (s, 2H), 5.17 (s, 1H),

6.41 (dd, J = 2.4, 8.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.64 (d, J = 8.4

Hz, 1H), 8.04 (s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.72,

19.64, 29.60, 34.13, 39.46, 39.73, 39.92, 55.11, 55.35, 81.79, 98.13,

104.39, 118.19, 126.22, 151.77, 155.82, 157.03, 158.94, 159.20, 162.05.

+

HRMS (ESI) calcd for C₂₀H₂₉N₄O₄[M + H]⁺m/z 389.2183 and

C₂₀H₂₈N₄NaO₄⁺[M + Na]⁺m/z 411.2003, found m/z 389.2168 and

411.1981.

N′-{3-[(2,4-Dimethylphenyl)methyl]-2,6-dioxo-1-propyl-1,2,3,6-

tetrahydropyrimidin-4-yl}-N,N-dimethylmethanimidamide (6). 4

(1.00 g, 3.00 mmol), acetonitrile (3.5 mL), DMF (2 mL), Cs₂CO₃

(1.08 g, 3.30 mmol), TBAB (97 mg, 0.30 mmol), and 1-bromopropane

(0.56 g, 0.41 mL, 4.51 mmol) gave 6 (0.93 g, 2.39 mmol, 80%) as oﬀ-

white crystals. ¹H NMR (400 MHz, DMSO-d₆): δ 0.83 (t, J = 7.2 Hz,

3H), 1.51 (m, 2H), 2.83 (s, 3H), 3.04 (s, 3H), 3.71 (s, 3H), 3.72 (t, J =

7.2 Hz, 2H), 3.78 (s, 3H), 5.05 (s, 2H), 5.17 (s, 1H), 6.41 (dd, J = 2.4,

8.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 8.04 (s,

1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 11.19, 20.78, 34.15, 40.01,

40.20, 41.51, 55.12, 55.37, 81.79, 98.14, 104.39, 118.18, 126.22, 151.82,

155.85, 157.04, 158.94, 159.20, 162.10. HRMS (ESI) calcd for

N′-{1-Butyl-3-[(2,4-dimethylphenyl)methyl]-2,6-dioxo-1,2,3,6-

tetrahydropyrimidin-4-yl}-N,N-dimethylmethanimidamide (5). 4

(3.10 g, 9.32 mmol), acetonitrile (11 mL), DMF (6 mL), Cs₂CO₃

+

C₁₉H₂₆NaN₄O₄[M + Na]⁺m/z 397.1846 and C₃₈H₅₂N₈NaO₈[2M

+ Na]⁺,found m/z 397.1844 and 771.3760.

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Figure 8. Incorporation of Cy5.5 by UODC2, UODC9, and UODC14 is inhibited by preincubation with UODC6 in hA₁R-FlpIn CHO cells. (A and

B) Representative in-gel Cy5.5 ﬂuorescence images coupled with immunoblots for V5 (for hA₁R-FlpInCHO cells) or β-actin (for NT-FlpInCHO

cells) shown in gray scale. (C and D) Quantiﬁcation of ﬂuorescence intensity (prominent bands at 37−50 kDa) labeled by UODC2, UODC9, and

UODC14 in the absence and presence of an irreversible competitor in hA₁R-FlpInCHO (black) and NT-FlpInCHO (red) cells. Cy5.5 ﬂuorescence

was corrected for relative V5 level (for hA₁R-FlpInCHO) or β-actin level (for NT-FlpInCHO). Individual data points are shown with bars to represent

means SEM from three or four independent experiments.

General Procedure for the Synthesis of 7 and 8. To a stirred

suspension of formamidine-protected uracil in methanol was added 2

M aqueous KOH. The suspension was stirred at 50 °C for 1 h. The

reaction mixture was cooled to rt and neutralized to pH 7 with 2 M

aqueous HCl. After the mixture had been cooled to 5 °C and stirred for

2 h, a precipitate formed, which was collected by ﬁltration, washed with

water, and air-dried.

6-Amino-3-butyl-1-[(2,4-dimethylphenyl)methyl]-1,2,3,4-tetra-

hydropyrimidine-2,4-dione (7). 5 (3.48 g, 8.95 mmol), methanol (46

mL), and 2 M aqueous KOH (30 mL) gave 7 (2.58 g, 7.73 mmol, 86%)

as colorless ﬂuﬀy crystals. ¹H NMR (400 MHz, DMSO-d₆): δ 0.86 (t, J

= 7.2 Hz, 3H), 1.23 (m, 2H), 1.43 (m, 2H), 3.71 (t, J = 7.6 Hz, 2H),

3.73 (s, 3H), 3.82 (s, 3H), 4.72 (s, 1H), 4.90 (br s, 2H), 6.49 (dd, J =

2.4, 8.4 Hz), 6.59 (d, J = 2.0 Hz), 6.67 (br s, 2H), 6.71 (d, J = 8.4 Hz).

¹³C NMR (400 MHz, DMSO-d₆): δ 13.73, 19.61, 29.73, 39.31, 39.94,

J = 2.0 Hz), 6.67 (br s, 2H), 6.71 (d, J = 8.4 Hz). ¹³C NMR (400 MHz,

DMSO-d₆): δ 11.18, 20.87, 40.26, 41.38, 55.26, 55.49, 75.13, 98.45,

104.67, 116.24, 126.05, 151.37, 154.59, 157.21, 159.81, 161.23. HRMS

+

(ESI) calcd for C₁₆H₂₁N₃NaO₄[M + Na]⁺m/z 342.1424 and

C₃₂H₄₂N₆NaO₈⁺[2M + Na]⁺m/z 661.2956, found m/z 342.1413 and

661.2925.

General Procedure for the Synthesis of 9 and 10. The

aminouracil was dissolved in 50% aqueous acetic acid and heated to 70

°C. NaNO₂was added in three batches over 30 min, and the reaction

mixture was stirred for 1 h at 70 °C. The mixture was cooled to rt, and

the precipitate that formed was collected by ﬁltration, washed with

water and diethyl ether, and air-dried to give the nitro intermediate as a

pink solid. The solid was dissolved in 25% aqueous ammonia and

methanol and heated to 80 °C. Na₂S₂O₄was added in three batches

over 30 min, and the reaction mixture was stirred for 4 h at 80 °C. The

mixture was cooled to rt and stirred overnight. The crystals that formed

were collected by ﬁltration, washed with water and diethyl ether, and

air-dried. The product was used in the next reaction immediately

without further puriﬁcation due to instability.

55.25, 55.48, 75.16, 98.44, 104.69, 116.25, 126.09, 151.33, 154.56,

157.20, 159.80, 161.20. HRMS (ESI) calcd for C₁₇H₂₄N₃O₄⁺[M + H]⁺

m/z 334.1761 and C₁₇H₂₃N₃NaO₄⁺[M + Na]⁺m/z 356.1581, found

m/z 334.1746 and 356.1559.

6-Amino-1-[(2,4-dimethylphenyl)methyl]-3-propyl-1,2,3,4-tetra-

hydropyrimidine-2,4-dione (8). 6 (0.92 g, 2.37 mmol), methanol (7.5

mL), and 2 M aqueous KOH (7.4 mL) gave 8 (0.66 g, 1.98 mmol, 84%)

as colorless ﬂuﬀy crystals. ¹H NMR (400 MHz, DMSO-d₆): δ 0.80 (t, J

= 7.4 Hz, 3H), 1.47 (m, 2H), 3.67 (t, J = 7.4 Hz, 2H), 3.73 (s, 3H), 3.82

(s, 3H), 4.72 (s, 1H), 4.90 (br s, 2H), 6.49 (dd, J = 2.4, 8.4 Hz), 6.59 (d,

5,6-Diamino-3-butyl-1-[(2,4-dimethylphenyl)methyl]-1,2,3,4-tet-

rahydropyrimidine-2,4-dione (9). 7 (2.33 g, 6.98 mmol), 50% aqueous

acetic acid (50 mL), and NaNO₂(0.97 g, 14.05 mmol) gave a pink solid

(2.33 g). Some of this nitro intermediate (0.70 g), 25% aqueous

ammonia (10 mL), methanol (10 mL), and Na₂S₂O₄(0.91 g, 5.22

mmol) gave 9 (0.56 g, 1.59 mmol, 76%) as gray-green crystals. ¹H NMR

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(400 MHz, DMSO-d₆): δ 0.87 (t, J = 7.4 Hz, 3H), 1.24 (m, 2H), 1.47

(m, 2H), 3.73 (s, 3H), 3.78 (t, J = 7.2 Hz, 2H), 3.83 (s, 3H), 4.96 (s,

2H), 6.47 (dd, J = 2.4, 8.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.73 (d, J =

8.4 Hz, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.74, 19.61, 29.73,

40.17, 40.33, 55.00, 55.20, 55.25, 96.13, 109.55, 116.53, 126.55, 144.28,

149.60, 157.20, 158.84, 159.82. HRMS (ESI) calcd for

¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.27 (m,

2H), 1.50 (m, 2H), 1.70−1.89 (m, 12H), 3.58 (s, 3H), 3.71 (s, 3H),

3.81 (s, 3H), 3.86 (t, J = 7.4 Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4

Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 13.00 (br s,

1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.71, 19.60, 27.58, 29.24,

29.68, 33.11, 38.16, 40.27, 41.07, 51.56, 55.17, 55.48, 98.30, 104.42,

106.52, 116.67, 126.90, 147.54, 150.58, 153.97, 157.40, 159.63, 160.04,

+

C₁₇H₂₄N₄NaO₄[M + H]⁺m/z 371.1690 and C₃₄H₄₈N₈NaO₈[2M

+ Na]⁺m/z 719.3487, found m/z 371.1701 and 719.3495.

+

176.97. HRMS (ESI) calcd for C₂₈H₃₆N₄NaO₆[M + Na]⁺m/z

5,6-Diamino-1-[(2,4-dimethylphenyl)methyl]-3-propyl-1,2,3,4-

tetrahydropyrimidine-2,4-dione (10). 8 (0.65 g, 2.03 mmol), 50%

aqueous acetic acid (15 mL), and NaNO₂(0.28 g, 4.06 mmol) gave a

pink solid (0.59 g). This nitro intermediate (0.59 g), 25% aqueous

ammonia (8.4 mL), methanol (8.4 mL), and Na₂S₂O₄(0.79 g, 4.54

mmol) gave 10 (0.42 g, 1.26 mmol, 62%) as gray-green crystals.

General Procedure for the Synthesis of Carboxylic Acids 11

and 12. To a suspension of bicyclo[2.2.2]octane-1,4-dicarboxylic acid

monomethyl ester in acetonitrile was added Et₃N, and the reaction

mixture was stirred for 5 min. HATU was added, and the reaction

mixture stirred for 15 min. The diaminouracil, dissolved in acetonitrile,

was added dropwise, and the reaction mixture stirred for 1 h. The

mixture was concentrated in vacuo, and 10% citric acid was added. The

aqueous solution was extracted three times with ethyl acetate, and then

the pooled organic layers were washed with saturated NaHCO₃and

brine and concentrated in vacuo. Isopropanol and 1 M aqueous KOH

were added, and the reaction mixture was heated at reﬂux for 1 h. The

mixture was cooled and concentrated in vacuo; water was added, and

the mixture was washed twice with CH₂Cl₂, discarding the organic

washings. The aqueous solution was acidiﬁed to pH 5 with 12 M

aqueous HCl. The mixture was refrigerated overnight, and the resultant

precipitate was collected by ﬁltration, rinsed with water, and oven-dried

at 80 °C.

547.2527, found m/z 547.2509.

General Procedure for the Synthesis of Carboxamides 14−

21. The carboxylic acid (11 or 12) was dissolved in DMF; then N,N-

diisopropylethylamine (DIPEA) was added, and the reaction mixture

stirred for 5 min. HATU was added, and the reaction mixture stirred for

15 min. The NH₂- “linker” (see the Supporting Information) was added

dropwise, and the reaction mixture stirred for 2.5 h. The mixture was

concentrated in vacuo, and then ethyl acetate and saturated aqueous

NaHCO₃were added. The aqueous layer was washed twice with ethyl

acetate, and the pooled organic phases were concentrated in vacuo. The

residue was puriﬁed using ﬂash column chromatography on silica gel.

4-{1-Butyl-3-[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-2,3,6,7-

tetrahydro-1H-purin-8-yl}-N-(prop-2-yn-1-yl)bicyclo[2.2.2]octane-

1-carboxamide (14). 11 (990 mg, 1.93 mmol), DMF (4 mL), DIPEA

(751 mg, 5.81 mmol), HATU (737 mg, 1.93 mmol), and propargyl

amine (138 mg, 2.50 mmol) following column elution with n-hexane/

ethyl acetate (6:4) gave 14 (807 mg, 1.47 mmol, 76%) as a colorless oil.

¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.27 (m,

2H), 1.50 (m, 2H), 1.66−1.87 (m, 12H), 3.02 (t, J = 2.4 Hz), 3.71 (s,

3H), 3.80 (m, 2H), 3.81 (s, 3H), 3.86 (t, J = 7.4 Hz, 2H), 5.03 (s, 2H),

6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.68 (d, J = 8.4

Hz, 1H), 7.89 (t, J = 5.6 Hz, 1H), 13.00 (br s, 1H). ¹³C NMR (400

MHz, DMSO-d₆): δ 13.76, 19.65, 27.70, 28.10, 29.59, 29.71, 33.23,

38.13, 38.08, 40.30, 41.13, 55.20, 55.51, 59.80, 72.32, 72.36, 81.79,

98.32, 104.43, 106.55, 116.68, 126.91, 150.61, 154.01, 157.42, 159.66,

176.20, 176.31. HRMS (ESI) calcd for C₃₀H₃₇N₅NaO5⁺[M + Na]⁺m/

z 570.2687 and C₆₀H₇₄N₁₀NaO₁₀⁺[2M + Na]⁺m/z 1117.5482, found

m/z 570.2598 and 1117.5312.

4-{1-Butyl-3-[(2,4-dimethylphenyl)methyl]-2,6-dioxo-2,3,6,9-tet-

rahydro-1H-purin-8-yl}bicyclo[2.2.2]octane-1-carboxylic Acid (11).

Bicyclo[2.2.2]octane-1,4-dicarboxylic acid monomethyl ester (49 mg,

0.23 mmol), acetonitrile (1.0 mL), Et₃N (90 mg, 124 μL, 0.89 mmol),

and HATU (88 mg, 0.23 mmol) and then 9 (81 mg, 0.23 mmol),

isopropanol (1.10 mL), and 1 M aqueous KOH (1.10 mL) gave 11 (87

mg, 0.17 mmol, 73%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆):

δ 0.88 (t, J = 7.2 Hz, 3H), 1.27 (m, 2H), 1.50 (m, 2H), 1.70−1.88 (m,

12H), 3.71 (s, 3H), 3.81 (s, 3H), 3.86 (t, J = 7.2 Hz, 2H), 5.04 (s, 2H),

6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.69 (d, J = 8.4

Hz, 1H), 12.07 (br s, 1H), 12.96 (s, 1H). ¹³C NMR (400 MHz, DMSO-

d₆): δ 13.72, 19.61, 27.44, 27.62, 29.38, 29.68, 33.19, 37.63, 37.72,

40.03, 40.27, 40.82, 55.18, 55.49, 98.31, 104.45, 106.49, 116.68, 126.94,

147.55, 150.60, 153.96, 157.42, 159.63, 160.22, 178.41, 178.45. HRMS

N-(3-Azidopropyl)-4-{1-butyl-3-[(2,4-dimethoxyphenyl)methyl]-

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

carboxamide (15). 11 (1.00 g, 1.95 mmol), DMF (4 mL), DIPEA (744

mg, 5.75 mmol), HATU (744 mg, 1.95 mmol), and 3-azidopropan-1-

amine (255 mg, 2.54 mmol) following column elution with CH₂Cl₂/

methanol (9.7:0.3) gave 15 (985 mg, 1.66 mmol, 85%) as a colorless

gummy liquid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz,

3H), 1.28 (m, 2H), 1.51 (m, 2H), 1.64 (m, 2H), 1.66−1.87 (m, 12H),

3.09 (q, J = 6.4 Hz, 2H), 3.30 (t, J = 6.8 Hz, 2H), 3.72 (s, 3H), 3.81 (s,

3H), 3.86 (t, J = 7.4 Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz,

1H), 6.57 (d, J = 2.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 5.8

Hz, 1H), 12.87 (br s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.70,

19.60, 27.82, 28.39, 29.63, 29.68, 33.21, 36.05, 38.08, 38.23, 40.26,

41.06, 48.47, 55.16, 55.48, 98.31, 104.43, 106.49, 116.71, 126.94,

147.54, 150.58, 153.96, 157.42, 159.64, 160.35, 176.46. HRMS (ESI)

+

(ESI) calcd for C₂₇H₃₅N₄O₆[M + H]⁺m/z 511.2551 and

C₂₇H₃₄N₄NaO₆⁺[M + Na]⁺m/z 533.2371, found m/z 511.2564 and

533.2385.

4-{3-[(2,4-Dimethoxyphenyl)methyl]-2,6-dioxo-1-propyl-2,3,6,7-

tetrahydro-1H-purin-8-yl}bicyclo[2.2.2]octane-1-carboxylic Acid

(12). Bicyclo[2.2.2]octane-1,4-dicarboxylic acid monomethyl ester

(0.30 g, 1.41 mmol), acetonitrile (5.5 mL), Et₃N (0.55 g, 0.76 mL,

5.43 mmol), and HATU (0.54 g, 1.41 mmol) and then 10 (0.47 g, 1.41

mmol), isopropanol (6 mL), and 1 M aqueous KOH (6 mL) gave 12

(0.42 g, 0.85 mmol, 59%) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆): δ 0.85 (t, J = 7.4 Hz, 3H), 1.54 (m, 2H), 1.68−1.88 (m, 12H), 3.71

(s, 3H), 3.81 (s, 3H), 3.83 (t, J = 7.2 Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J =

2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 12.10

(br s, 1H), 12.98 (s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 11.15,

20.84, 27.62, 29.38, 29.68, 33.18, 37.63, 37.72, 41.07, 42.05, 55.17,

55.49, 98.31, 104.44, 106.49, 116.67, 126.93, 147.55, 150.62, 153.99,

calcd for C₃₀H₄₁N₈O₅⁺[M + H]⁺m/z 593.3194 and C₃₀H₄₀N₈NaO₅

+

[M + Na]⁺m/z 615.3014, found m/z 593.3187 and 615.3014.

4-{1-Butyl-3-[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-2,3,6,9-

tetrahydro-1H-purin-8-yl}-N-(hex-5-yn-1-yl)bicyclo[2.2.2]octane-1-

carboxamide (16). 11 (100 mg, 0.19 mmol), DMF (1 mL), DIPEA (76

mg, 0.59 mmol), HATU (75 mg, 0.19 mmol), and 5-hexyn-1-amine (28

mg, 0.19 mmol) following column elution with 100% ethyl acetate gave

1

16 (58 mg, 0.09 mmol, 50%) as a colorless gummy liquid. H NMR

(400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.21−1.56 (m, 8H),

1.66−1.87 (m, 12H), 2.14 (t, J = 6.8 Hz, 2H), 2.74 (t, J = 2.8 Hz, 1 H),

3.03 (q, J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.81 (s, 3H), 3.863 (t, J = 7.4 Hz,

2H), 5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz,

1H), 6.70 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 5.8 Hz, 1H), 12.98 (s, 1H).

¹³C NMR (400 MHz, DMSO-d₆): δ 13.72, 17.40, 19.61, 25.34, 27.86,

28.26, 29.66, 33.23, 37.94, 38.06, 40.27, 41.07, 55.18, 55.49, 71.22,

84.47, 98.32, 104.45, 106.48, 116.70, 126.94, 147.55, 150.59, 153.96,

157.42, 159.64, 160.39, 162.29, 176.23. HRMS (ESI) calcd for

−

157.41, 159.63, 160.23, 178.40. HRMS (ESI) calcd for C₂₆H₃₁N₄O₆

[M − H]⁻m/z 495.2249, found m/z 495.2242.

Methyl 4-{1-Butyl-3-[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-

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

late (13). 11 (400 mg, 0.78 mmol) dissolved in methanol (20 mL) and

H₂SO₄(5 drops) were added, and the reaction mixture was stirred at

reﬂux for 3 h. The mixture was cooled to rt overnight, and the resultant

crystals were collected by ﬁltration, washed with water, and oven-dried

at 80 °C to give 13 (291 mg, 0.55 mmol, 71%) as white ﬂuﬀy crystals.

+

C₃₃H₄₄N₅O₅[M + H]⁺m/z 590.3337, C₃₃H₄₃NaN₅O₅[M + Na]⁺

8171

https://doi.org/10.1021/acs.jmedchem.0c02169

J. Med. Chem. 2021, 64, 8161−8178

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

+

m/z 612.3156, and C₆₆H₈₆NaN₁₀O₁₀[2M + Na]⁺m/z 1201.6421,

C₆₆H₉₂N₁₆NaO₁₄[2M + Na]⁺m/z 1355.6871, found m/z 689.3332

found m/z 590.3348, 612.3169, and 1201.6451.

and 1355.6796.

N-(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)-4-{1-butyl-3-

[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-2,3,6,9-tetrahydro-1H-

purin-8-yl}bicyclo[2.2.2]octane-1-carboxamide (21). 11 (200 mg,

0.39 mmol), DMF (1 mL), DIPEA (152 mg, 1.18 mmol), HATU (149

mg, 0.39 mmol), and {2-[2-(2-azidoethoxy)ethoxy]ethoxy}-

methanamine (85 mg, 0.39 mmol) following column elution with

ethyl acetate/methanol (9.5:0.5) gave 21 (200 mg, 0.29 mmol, 75%) as

a colorless gummy liquid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J

= 7.4 Hz, 3H), 1.27 (m, 2H), 1.50 (m, 2H), 1.64−1.89 (m, 12H), 3.18

(q, J = 6.2 Hz, 2H), 3.38 (t, J = 5.2 Hz, 4H), 3.45−3.57 (m, 8H), 3.59 (t,

J = 4.8 Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J = 2.2, 8.6 Hz, 1H), 6.57 (d, J =

2.0 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 5.6 Hz, 1H), 12.97 (s,

1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.72, 19.60, 27.81, 29.66,

29.70, 33.22, 35.78, 38.08, 38.59, 40.08, 40.83, 49.98, 55.18, 55.49,

68.87, 69.25, 69.57, 69.70, 69.75, 69.79, 98.31, 104.44, 116.73, 126.92,

147.53, 150.61, 153.98, 157.41, 159.62, 162.29, 176.51. HRMS (ESI)

N-(3-Azidopropyl)-4-{1-butyl-3-[(2,4-dimethoxyphenyl)methyl]-

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

carboxamide (17). 12 (100 mg, 0.19 mmol), DMF (1 mL), DIPEA (76

mg, 0.59 mmol), HATU (75 mg, 0.19 mmol), and 3-azidopropan-1-

amine (20 mg, 0.19 mmol) following column elution with 100% ethyl

acetate gave 17 (82 mg, 0.14 mmol, 70%) as a yellowish gummy liquid.

¹H NMR (400 MHz, DMSO-d₆): δ 0.85 (t, J = 7.4 Hz, 3H), 1.55 (m,

2H), 1.64 (m, 2H), 1.67−1.88 (m, 12H), 3.09 (q, J = 6.2 Hz, 2H), 3.30

(t, J = 6.8 Hz, 2H), 3.72 (s, 3H), 3.81 (s, 3H), 3.83 (t, J = 7.2 Hz, 2H),

5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H),

6.70 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 5.8 Hz, 1H), 12.98 (s, 1H). ¹³C

NMR (400 MHz, DMSO-d₆): δ 11.14, 20.84, 27.81, 28.38, 29.63,

30.76, 33.21, 35.77, 36.05, 38.08, 38.23, 41.06, 42.04, 48.46, 55.17,

55.49, 98.32, 104.45, 106.48, 116.69, 126.93, 147.55, 150.62, 153.98,

157.41, 159.63, 160.36, 162.28, 176.45.

N-(4-Azidobutyl)-4-{1-butyl-3-[(2,4-dimethoxyphenyl)methyl]-

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

carboxamide (18). 11 (100 mg, 0.19 mmol), DMF (1 mL), DIPEA (76

mg, 0.59 mmol), HATU (75 mg, 0.19 mmol), and 4-azidobutan-1-

amine (22 mg, 0.19 mmol) following column elution with 100% ethyl

acetate gave 18 (118 mg, 0.19 mmol, 98%) as a colorless gummy liquid.

¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.28 (m,

2H), 1.39−1.55 (m, 6H), 1.66−1.87 (m, 12H), 3.04 (q, J = 6.2 Hz,

2H), 3.31 (t, J = 6.4 Hz, 2H), 3.72 (s, 3H), 3.81 (s, 3H), 3.86 (t, J = 7.4

Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz,

1H), 6.70 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 5.8 Hz, 1H), 12.97 (s, 1H).

¹³C NMR (400 MHz, DMSO-d₆): δ 13.71, 19.60, 25.59, 26.32, 27.85,

+

calcd for C₃₅H₅₁N₈O₈⁺[M + H]⁺m/z 711.3824 and C₃₅H₅₀N₈NaO₈

[M + Na]⁺m/z 733.3644, found m/z 711.3839 and 733.3662.

General Procedure for the Synthesis of Sulfonyl Fluorides

UODC2, UODC5, UODC6, UODC9−UODC14. The DMB-protected

carboxamide was dissolved in neat TFA and stirred overnight at 50 °C.

The reaction mixture was cooled to rt and blown with compressed air

for 20 min to evaporate excess TFA. CH₂Cl₂was added, and the organic

solution was concentrated in vacuo and then dried overnight under high

vacuum. The residue was dissolved in DMF, K₂CO₃added, and the

reaction mixture stirred for 45 min. 4-[(3-Bromopropyl)carbamoyl]-

benzenesulfonyl ﬂuoride (in a solution of DMF) was added dropwise,

and the reaction mixture was stirred overnight. The reaction mixture

was concentrated in vacuo, and water was added. The aqueous solution

was extracted three times with ethyl acetate. The pooled organic layers

were dried with MgSO₄, ﬁltered, and concentrated in vacuo. The crude

product was puriﬁed by column chromatography and underwent

subsequent semipreparative RP-HPLC puriﬁcation.

29.65, 29.68, 30.76, 33.21, 38.06, 38.24, 40.26, 41.06, 50.34, 55.18,

55.49, 98.31, 104.45, 106.50, 116.70, 126.94, 147.55, 150.59, 153.97,

157.42, 159.64, 160.38, 176.29. HRMS (ESI) calcd for

C₃₁H₄₂NaN₈O₅⁺[M + Na]⁺m/z 629.3170, found m/z 629.3133.

N-(8-Azidooctyl)-4-{1-butyl-3-[(2,4-dimethoxyphenyl)methyl]-

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

carboxamide (19). 11 (100 mg, 0.19 mmol), DMF (1 mL), DIPEA (76

mg, 0.59 mmol), HATU (75 mg, 0.19 mmol), and 8-azidooctan-1-

amine hydrochloride (40 mg, 0.19 mmol) (preneutralized with 1 equiv

of DIPEA) following column elution with 100% ethyl acetate gave 19

(100 mg, 0.15 mmol, 77%) as a colorless gummy liquid. ¹H NMR (400

MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.14−1.41 (m, 12H),

1.45−1.56 (m, 4H), 1.64−1.88 (m, 12H), 3.01 (q, J = 6.6 Hz), 3.30 (t, J

= 6.8 Hz, 2H), 3.72 (s, 3H), 3.81 (s, 3H), 3.86 (t, J = 7.4 Hz, 2H), 5.04

(s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.70 (d,

J = 8.4 Hz, 1H), 7.35 (t, J = 5.8 Hz, 1H), 12.97 (s, 1H). ¹³C NMR (400

MHz, DMSO-d₆): δ 13.71, 19.59, 26.07, 26.17, 27.85, 28.21, 28.46,

28.58, 29.03, 29.66, 33.21, 38.02, 38.52, 39.98, 40.74, 40.25, 41.05,

50.61, 54.90, 55.17, 55.48, 98.31, 104.44, 106.48, 116.69, 126.94,

147.54, 150.58, 153.95, 157.41, 159.63, 160.38, 176.11. HRMS (ESI)

Methyl 4-[1-Butyl-3-(3-{[4-(ﬂuorosulfonyl)phenyl]formamido}-

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

octane-1-carboxylate (UODC6). 13 (290 mg, 0.55 mmol), TFA (2

mL), DMF (4 mL), K₂CO₃(77 mg, 0.55 mmol), and 4-[(3-

bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (270 mg, 0.83

mmol) (in a solution of DMF) following column elution with

CH₂Cl₂/(CH₃)₂CO (9:1) gave a white solid (218 mg). Some of this

white solid (68 mg) was further puriﬁed by semipreparative RP-HPLC

to give UODC6 (15 mg, 0.024 mmol, 15%) as white crystals. ¹H NMR

(500 MHz, DMSO-d₆): δ 0.88 (t, J = 7.3 Hz, 3H), 1.27 (m, 2H), 1.50

(m, 2H), 1.64−1.81 (m, 12H), 1.96 (m, 2H), 3.31 (q, J = 6.3 Hz, 2H),

3.58 (s, 3H), 3.85 (t, J = 7.3 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 8.17 (d, J

= 8.5 Hz, 2H), 8.27 (d, J = 8.5 Hz, 2H), 8.86 (t, J = 5.5 Hz, 1H), 12.96

(br s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ 13.71, 19.62, 27.44,

27.51, 29.16, 29.68, 33.02, 36.85, 38.10, 40.22, 40.43, 40.68, 51.53,

106.51, 128.60, 129.00, 133.41 (d), 141.54, 147.21, 150.59, 153.88,

157.55, 158.00, 159.92, 164.20, 176.89. ¹⁹F NMR (400 MHz, DMSO-

d₆): δ 66.13. HRMS (ESI) calcd for C₂₉H₃₆FN₅NaO₇S⁺[M + Na]⁺m/z

+

calcd for C₃₅H₅₀₊NaN₈O₅[M + Na]⁺m/z 685.3796 and

C₇₀H₁₀₀N₁₆NaO₁₆[2M + Na]⁺m/z 1347.7701, found m/z

685.3792 and 1347.7668.

+

640.2212 and C₅₈H₇₂F₂N₁₀NaO₁₄S₂[2M + Na]⁺m/z 1257.4531,

N-{2-[2-(2-Azidoethoxy)ethoxy]ethyl}-4-{1-butyl-3-[(2,4-

dimethoxyphenyl)methyl]-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-

8-yl}bicyclo[2.2.2]octane-1-carboxamide (20). 11 (200 mg, 0.39

mmol), DMF (1 mL), DIPEA (152 mg, 1.18 mmol), HATU (149 mg,

0.39 mmol), and 2-[2-(2-azidoethoxy)ethoxy]ethan-1-amine (68 mg,

0.39 mmol) following column elution with 100% ethyl acetate gave 20

(190 mg, 0.28 mmol, 72%) as a colorless gummy liquid. ¹H NMR (400

MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.27 (m, 2H), 1.51 (m,

2H), 1.62−1.90 (m, 12H), 3.18 (q, J = 6 Hz, 2H), 3.36−3.41 (m, 4H),

3.48−3.57 (m, 4H), 3.59 (t, J = 5.0 Hz, 2H), 3.72 (s, 3H), 3.81 (s, 3H),

3.86 (t, J = 7.4 Hz, 2H), 5.04 (s, 2H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57

(d, J = 2.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 5.8 Hz, 1H),

12.97 (br s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.71, 19.60,

27.78, 29.62, 29.68, 33.21, 38.07, 38.58, 40.08, 40.26, 41.06, 49.99,

55.18, 55.49, 68.91, 69.24, 69.59, 69.61, 98.31, 104.45, 106.48, 116.69,

126.93, 147.54, 150.59, 153.95, 157.41, 159.63, 160.35, 176.47. HRMS

found m/z 640.2177 and 1257.4438. HPLC: 19.28 min.

4-{[3-(1-Butyl-2,6-dioxo-8-{4-[(prop-2-yn-1-yl)carbamoyl]-

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

carbamoyl}benzene-1-sulfonyl Fluoride (UODC5). 14 (508 mg, 0.93

mmol), TFA (3 mL), DMF (4 mL), K₂CO₃(128 mg, 0.93 mmol), and

4-[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (270 mg, 0.83

mmol) (in a solution of DMF) following column elution with CH₂Cl₂/

(CH₃)₂CO (7:3) gave a white solid (300 mg). Some of this white solid

(101 mg) was further puriﬁed by semipreparative RP-HPLC to give

UODC5 (16 mg, 0.025 mmol, 12%) as white crystals. ¹H NMR (500

MHz, DMSO-d₆): δ 0.88 (t, J = 7.3 Hz, 3H), 1.28 (m, 2H), 1.50 (m,

2H), 1.77−1.88 (m, 12H), 1.97 (m, 2H), 2.24 (s, 1H), 2.24 (s, 2H),

3.31 (q, J = 6.5 Hz, 2H), 3.86 (t, J = 7.3 Hz, 2H), 4.08 (t, J = 7.0 Hz,

2H), 6.66 (m, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.26 (d, J = 9.0 Hz, 2H),

8.87 (t, J = 5.5 Hz, 1H), 12.98 (br s, 1H). ¹³C NMR (500 MHz, DMSO-

d₆): δ 10.49, 13.70, 19.62, 27.45, 29.05, 29.33, 29.68, 32.86, 33.11,

+

(ESI) calcd for C₃₃H₄₆N₈NaO₇[M + Na]⁺m/z 689.3382 and

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36.88, 40.23, 40.69, 106.52, 122.14, 128.60, 129.00, 133.32, 133.51,

141.54, 147.25, 147.74, 150.60, 153.88, 160.04, 164.20, 167.65. ¹⁹F

NMR (400 MHz, DMSO-d₆): δ 66.19. HRMS (ESI) calcd for

and 4-[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (48 mg,

0.15 mmol) (in a solution of DMF) following column elution with

100% ethyl acetate and then semipreparative RP-HPLC puriﬁcation

gave UODC10 (20 mg, 0.02 mmol, 19%) as white crystals. ¹H NMR

(500 MHz, DMSO-d₆): δ 0.88 (t, J = 7.3 Hz, 3H), 1.27 (m, 2H), 1.40−

1.55 (m, 6H), 1.60−1.84 (m, 12H), 1.96 (m, 2H), 3.05 (q, J = 6.2 Hz,

2H), 3.34 (q, J = overlap, 2H), 3.34 (t, J = overlap), 3.86 (t, J = 7.3 Hz,

2H), 4.06 (t, J = 7.0 Hz, 2H), 7.40 (t, J = 5.8 Hz, 1H), 8.16 (d, J = 8.5

Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H), 8.86 (t, J = 5.8 Hz, 1H), 12.94 (s,

1H). ¹⁹F NMR (400 MHz, DMSO-d₆): δ −73.72 (TFA salt), 66.15.

HRMS (ESI) calcd for C₃₂H₄₂FNaN₉O₆S⁺[M + Na]⁺m/z 722.2855,

found m/z 722.2849. HPLC: 19.31 min.

4-{[3-(8-{4-[(8-Azidooctyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-1-

butyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonyl Fluoride (UODC11). 19 (80 mg, 0.12

mmol), TFA (1 mL), DMF (2 mL), K₂CO₃(17 mg, 0.12 mmol), and 4-

[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (35 mg, 0.11

mmol) (in a solution of DMF) following column elution with 100%

ethyl acetate and then semipreparative RP-HPLC puriﬁcation gave

UODC11 (30 mg, 0.039 mmol, 36%) as white crystals. ¹H NMR (500

MHz, DMSO-d₆): δ 0.88 (t, J = 7.3 Hz, 3H), 1.16−1.41 (m, 12H),

1.46−1.56 (m, 4H), 1.61−1.81 (m, 12H), 1.96 (m, 2H), 3.00 (q, J = 6.5

Hz, 2H), 3.31 (q, J = overlap, 2H), 3.31 (t, J = overlap, 2H), 3.85 (t, J =

7.5 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 7.32 (t, J = 5.8 Hz, 1H), 8.15 (d, J

= 8.5 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.85 (t, J = 5.8 Hz, 1H), 12.93

(s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ 13.75, 19.64, 26.11, 26.20,

27.49, 27.82, 28.24, 28.48, 28.63, 29.07, 29.63, 29.71, 30.71, 33.18,

36.97, 38.02, 38.56, 40.25, 40.75, 50.65, 106.52, 128.62, 129.01, 133.41

(d), 141.57, 147.25, 150.64, 153.90, 157.60, 157.85, 158.10, 160.34,

164.28, 176.12. ¹⁹F NMR (400 MHz, DMSO-d₆): δ −73.75 (TFA salt),

66.18. HRMS (ESI) calcd for C₃₆H₅₁FN₉O₆S⁺[M + H]⁺m/z 756.3662

and C₃₆H₅₀FNaN₉O₆S⁺[M + Na]⁺m/z 778.3481, found m/z 756.3661

and 778.3473. HPLC: 21.75 min.

4-[(3-{8-[4-({2-[2-(2-Azidoethoxy)ethoxy]ethyl}carbamoyl)-

bicyclo[2.2.2]octan-1-yl]-1-butyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-

purin-3-yl}propyl)carbamoyl]benzene-1-sulfonyl Fluoride

(UODC12). 20 (43 mg, 0.06 mmol), TFA (1 mL), DMF (2 mL),

K₂CO₃(9 mg, 0.06 mmol), and 4-[(3-bromopropyl)carbamoyl]-

benzenesulfonyl ﬂuoride (19 mg, 0.05 mmol) (in a solution of DMF)

following column elution with 100% ethyl acetate and then semi-

preparative RP-HPLC puriﬁcation gave UODC12 (19 mg, 0.02 mmol,

43%) as white crystals. ¹H NMR (500 MHz, DMSO-d₆): δ 0.88 (t, J =

7.5 Hz, 3H), 1.27 (m, 2H), 1.50 (m, 2H), 1.62−1.82 (m, 12H), 1.96

(m, 2H), 3.18 (q, J = 6 Hz, 2H), 3.31 (q, J = 6.5 Hz, 2H), 3.35−3.62 (m,

8H), 3.60 (t, J = 4.8 Hz, 2H), 3.85 (t, J = 7.5 Hz, 2H), 4.06 (t, J = 7 Hz,

2H), 7.36 (t, J = 5.8 Hz, 1H), 8.15 (d, J = 8.5 Hz, 2H), 8.25 (d, J = 8.5

Hz, 2H), 8.85 (t, J = 5.8 Hz, 1H), 12.93 (s, 1H). ¹³C NMR (500 MHz,

DMSO-d₆): δ 13.74, 19.64, 27.52, 27.76, 29.59, 29.71, 30.71, 33.18,

37.00, 38.07, 38.60, 40.26, 40.77, 50.03, 68.94, 69.27, 69.64, 106.54,

106.91, 128.62, 129.01, 133.42 (d), 141.57, 147.24, 150.64, 153.64,

157.91, 158.17, 160.30, 164.28, 176.48. ¹⁹F NMR (400 MHz, DMSO-

d₆): δ −74.41 (TFA salt), 66.18. HRMS (ESI) calcd for

C₃₄H₄₇FN₉O₈S⁺[M + H]⁺m/z 760.3247 and C₃₄H₄₆FNaN₉O₈S⁺

[M + Na]⁺m/z 782.3066, found m/z 760.3246 and 782.3062. HPLC:

18.91 min.

4-{[3-(8-{4-[(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)-

carbamoyl]bicyclo[2.2.2]-octan-1-yl}-1-butyl-2,6-dioxo-2,3,6,9-tet-

rahydro-1H-purin-3-yl)propyl]carbamoyl}-benzene-1-sulfonyl Flu-

oride (UODC13). 21 (69 mg, 0.09 mmol), TFA (1 mL), DMF (2

mL), K₂CO₃(14 mg, 0.09 mmol), and 4-[(3-bromopropyl)-

carbamoyl]benzenesulfonyl ﬂuoride (28 mg, 0.08 mmol) (in a solution

of DMF) following column elution with 100% ethyl acetate and then

semipreparative RP-HPLC puriﬁcation gave UODC13 (19 mg, 0.023

mmol, 27%) as white crystals. ¹H NMR (500 MHz, DMSO-d₆): δ 0.88

(t, J = 7.5 Hz, 3H), 1.27 (m, 2H), 1.50 (m, 2H), 1.62−1.83 (m, 12H),

1.96 (m, 2H), 3.18 (q, J = 6 Hz, 2H), 3.31 (q, J = 6.5 Hz, 2H), 3.35−

3.62 (m, 12H), 3.60 (t, J = 5.0 Hz, 2H), 3.85 (t, J = 7.5 Hz, 2H), 4.06 (t,

J = 7 Hz, 2H), 7.37 (t, J = 5.5 Hz, 1H), 8.15 (d, J = 8.5 Hz, 2H), 8.25 (d,

J = 8.5 Hz, 2H), 8.85 (t, J = 5.8 Hz, 1H), 12.93 (s, 1H). ¹³C NMR (500

MHz, DMSO-d₆): δ 13.72, 19.63, 27.51, 27.75, 29.58, 29.69, 33.16,

36.99, 38.06, 38.60, 40.24, 40.75, 50.00, 68.90, 69.27, 69.60, 69.71,

C_{3 1}H_{3 7}FN₆NaO₆S⁺[M

+

Na]⁺m/z 663.2372 and

C₆₂H₇₄F₂N₁₂NaO₁₂S⁺[2M + Na]⁺m/z 1303.4851, found m/z

663.2321 and 1303.4685. HPLC: 19.24 min.

4-{[3-(8-{4-[(3-Azidopropyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-

1-butyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonyl Fluoride (UODC2). 15 (469 mg, 0.79

mmol), TFA (2.5 mL), DMF (4 mL), K₂CO₃(109 mg, 0.79 mmol),

and 4-[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (230 mg,

0.71 mmol) (in a solution of DMF) following column elution with

CH₂Cl₂/(CH₃)₂CO (7:3) gave a white solid (190 mg). Some of this

white solid (76 mg) was further puriﬁed by semipreparative RP-HPLC

to give UODC2 (22 mg, 0.032 mmol, 11%) as white crystals. ¹H NMR

(500 MHz, DMSO-d₆): δ 0.80 (t, J = 7.3 Hz, 3H), 1.27 (m, 2H), 1.50

(m, 2H), 1.65 (m, 2H), 1.65−1.81 (m, 12H), 1.96 (m, 2H), 3.09 (q, J =

6.3 Hz, 2H), 3.31 (t, J = 6.8 Hz, 2H), 3.31 (q, J = 6.5 Hz, 2H), 3.86 (t, J

= 7.5 Hz, 2H), 4.06 (t, J = 7 Hz, 2H), 7.45 (t, J = 5.8 Hz, 1H), 8.16 (d, J

= 8.5 Hz, 2H), 8.25 (d, J = 9.0 Hz, 2H), 8.85 (t, J = 5.5 Hz, 1H), 12.94

(s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ 13.71, 19.62, 27.49, 27.77,

28.40, 29.58, 29.68, 33.15, 36.04, 36.95, 38.05, 40.22, 40.73, 48.47,

106.53, 128.59, 128.98, 133.41 (d), 141.54, 147.20, 150.61, 153.88,

157.97, 158.25, 160.25, 164.23, 176.40. ¹⁹F NMR (400 MHz, DMSO-

d₆): δ 66.18. HRMS (ESI) calcd for C₃₁H₄₀FN₉NaO₆S⁺[M + Na]⁺m/z

+

708.2699 and C₆₂H₈₀F₂N₁₈NaO₁₂S₂[2M + Na]⁺m/z 1393.5505,

found m/z 708.2656 and 1393.5440. HPLC: 18.49 min.

4-{[3-(1-Butyl-8-{4-[(hex-5-yn-1-yl)carbamoyl]bicyclo[2.2.2]-

octan-1-yl}-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonyl Fluoride (UODC14). 16 (100 mg,

0.17 mmol), TFA (1 mL), DMF (2 mL), K₂CO₃(23 mg, 0.17 mmol),

and 4-[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (49 mg,

0.15 mmol) (in a solution of DMF) following column elution with

100% ethyl acetate and then semipreparative RP-HPLC puriﬁcation of

all material gave UODC14 (35 mg, 0.051 mmol, 33%) as white crystals.

¹H NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.4 Hz, 3H), 1.27 (m,

2H), 1.35−1.54 (m, 8H), 1.61−1.83 (m, 12H), 1.96 (m, 2H), 2.15 (t, J

= 6.2 Hz, 2H), 2.74 (t, J = 2.4 Hz, 1H), 3.03 (q, J = 6.4 Hz, 2H), 3.31 (q,

J = 6.6 Hz, 2H), 3.85 (t, J = 7.5 Hz, 2H), 4.06 (t, J = 7 Hz, 2H), 7.39 (t, J

= 5.8 Hz, 1H), 8.15 (d, J = 8.4 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 8.85 (t,

J = 5.6 Hz, 1H), 12.94 (s, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ

13.73, 17.41, 19.64, 25.34, 27.51, 27.82, 27.96, 28.27, 29.62, 29.69,

36.48, 37.95, 38.04, 40.24, 40.74, 71.22, 84.47, 106.51, 128.61, 128.99,

133.41 (d), 141.54, 147.23, 150.62, 153.88, 160.33, 160.91, 164.23,

176.18. ¹⁹F NMR (400 MHz, DMSO-d₆): δ −69.31, −71.20 (TFA

salt), 66.13. HRMS (ESI) calcd for C₃₄H₄₄FN₆O₆S⁺[M + H]⁺m/z

683.3022 and C₃₄H₄₃FNaN₆O₆S⁺[M + Na]⁺m/z 705.2841, found m/z

683.3035 and 705.2856. HPLC: 19.39 min.

4-{[3-(8-{4-[(3-Azidopropyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-

2,6-dioxo-1-propyl-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonyl Fluoride (UODC9). 17 (50 mg, 0.08

mmol), TFA (1 mL), DMF (2 mL), K₂CO₃(12 mg, 0.08 mmol), and 4-

[(3-bromopropyl)carbamoyl]benzenesulfonyl ﬂuoride (25 mg, 0.07

mmol) (in a solution of DMF) following column elution with 100%

ethyl acetate gave a white solid (107 mg). Some of this solid (39 mg)

was further puriﬁed by semipreparative RP-HPLC to give UODC9 (11

mg, 0.016 mmol, 71%) as white crystals. ¹H NMR (500 MHz, DMSO-

d₆): δ 0.85 (t, J = 7.3 Hz, 3H), 1.54 (m, 2H), 1.65 (m, 2H), 1.62−1.81

(m, 12H), 1.96 (m, 2H), 3.09 (q, J = 6.3 Hz, 2H), 3.31 (t, J = 6.8 Hz,

2H), 3.82 (t, J = 7.5 Hz, 2H), 4.06 (t, J = 7.0 Hz, 2H), 7.45 (t, J = 5.8 Hz,

2H), 8.16 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H), 8.86 (t, J = 5.5

Hz, 1H), 12.94 (s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ 11.20,

20.86, 27.51, 27.79, 28.41, 29.60, 33.18, 36.08, 36.97, 38.07, 40.75,

42.04, 48.49, 106.54, 128.63, 129.02, 133.43 (d), 141.57, 147.24,

150.68, 153.94, 157.89, 158.14, 160.32, 164.30, 176.47. ¹⁹F NMR (400

MHz, DMSO-d₆): δ 66.15. HRMS (ESI) calcd for C₃₀H₃₈FNaN₉O₆S⁺

[M + Na]⁺m/z 694.2542, found m/z 694.2537. HPLC: 18.17 min.

4-{[3-(8-{4-[(4-Azidobutyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-1-

butyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonyl Fluoride (UODC10). 18 (100 mg,

0.16 mmol), TFA (1 mL), DMF (2 mL), K₂CO₃(22 mg, 0.16 mmol),

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69.77, 69.80, 106.53, 128.60, 128.99, 133.41 (d), 141.55, 147.21,

150.62, 153.89, 157.88, 158.14, 160.28, 164.24, 176.44. ¹⁹F NMR (400

MHz, DMSO-d₆): δ −74.10 (TFA salt), 66.16. HRMS (ESI) calcd for

C₃₆H₅₀FNaN₉O₉S⁺[M + Na]⁺m/z 826.3328, found m/z 826.3326.

HPLC: 19.00 min.

General Procedure for the Synthesis of Sulfonic Acids

UODC7 and UODC3. The sulfonyl ﬂuoride was dissolved in 1 M

methanolic NaOH and stirred for 1 h. The reaction mixture was

acidiﬁed to pH 4 with 12 M HCl and concentrated in vacuo. Ethyl

acetate was added, and then the organic layer was washed with saturated

aqueous NaHCO₃and concentrated in vacuo.

mg, 0.80 mmol), DMF (4 mL), K₂CO₃(166 mg, 1.20 mmol), and

pivaloxymethyl chloride (157 mg, 1.04 mmol) following column

elution with n-hexane/ethyl acetate (1:1) gave 23 (325 mg, 0.46 mmol,

57%) as a gummy liquid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.86 (t, J =

7.4 Hz, 3H), 1.12 (s, 9H), 1.25 (m, 2H), 1.48 (m, 2H), 1.69−2.00 (m,

12H), 3.09 (q, 2H), 3.31 (t, J = 6.8 Hz), 3.72 (s, 3H), 3.79 (s, 3H), 3.85

(t, J = 7.4 Hz, 2H), 5.06 (s, 2H), 6.33 (br s, 2H), 6.42 (dd, J = 2.4, 8.4

Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.46 (t, J =

5.6 Hz, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.66, 19.50, 26.54,

27.60, 28.36, 29.34, 29.53, 34.55, 36.03, 37.86, 38.16, 39.95, 40.91,

48.45, 55.17, 55.45, 69.03, 98.31, 104.49, 106.66, 116.44, 127.72,

146.79, 150.27, 153.83, 157.54, 159.79, 159.95, 175.99, 176.31. HRMS

4-[(3-{1-Butyl-8-[4-(methoxycarbonyl)bicyclo[2.2.2]octan-1-yl]-

2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl}propyl)carbamoyl]-

benzene-1-sulfonic Acid (UODC7). UODC6 (24 mg, 0.039 mmol)

and 1 M methanolic NaOH (2 mL) following semipreparative RP-

HPLC puriﬁcation gave UODC7 (21 mg, 0.034 mmol, 88%) as a white

solid. ¹H NMR (500 MHz, DMSO-d₆): δ 0.89 (t, J = 7.3 Hz, 3H), 1.28

(m, 2H), 1.51 (m, 2H), 1.73−1.90 (m, 12H), 1.92 (m, 2H), 3.27 (q, J =

6.8 Hz, 2H), 3.59 (s, 3H), 3.86 (t, J = 7.3 Hz, 2H), 4.04 (t, J = 7 Hz, 2H),

6.54 (br s, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8 Hz, 2H), 8.44 (t, J

= 5.8 Hz, 1H), 12.98 (s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ

13.72, 19.62, 27.58, 27.85, 29.25, 29.68, 33.11, 36.78, 38.17, 40.24,

40.86, 51.57, 106.53, 125.34, 126.63, 134.44, 147.24, 150.61, 150.73,

153.87, 157.64, 157.89, 160.04, 165.79, 176.98. HRMS (ESI) calcd for

C₂₉H₃₇N₅O₈S⁻[M − H]⁻m/z 614.2290, found m/z 614.2249. HPLC:

14.29 min.

4-{[3-(8-{4-[(3-Azidopropyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-

1-butyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-purin-3-yl)propyl]-

carbamoyl}benzene-1-sulfonic Acid (UODC3). UODC2 (21 mg,

0.031 mmol) and 1 M methanolic NaOH (2 mL) following

semipreparative RP-HPLC puriﬁcation gave UODC3 (19 mg, 0.028

mmol, 95%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆): δ 0.89

(t, J = 7.3 Hz, 3H), 1.28 (m, 2H), 1.51 (m, 2H), 1.57−1.73 (m, 12H),

1.68 (m, 2H), 1.93 (m, 2H), 3.10 (q, J = 6.3 Hz, 2H), 3.26 (q, J = 6.3 Hz,

2H), 3.32 (t, J = 7.0 Hz, 2H), 3.86 (t, J = 7.5 Hz, 2H), 4.06 (t, J = 7.0 Hz,

2H), 4.99 (br s, 1H), 7.51 (t, J = 5.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H),

7.81 (d, J = 8.5 Hz, 2H), 8.46 (t, J = 5.8 Hz, 1H), 12.85 (br s, 1H). ¹³C

NMR (500 MHz, DMSO-d₆): δ 13.74, 19.63, 27.58, 27.76, 28.36,

29.50, 29.70, 33.10, 36.10, 36.26, 38.00, 40.19, 40.68, 40.43, 48.59,

106.42, 125.40, 126.79, 134.67, 147.27, 150.44, 150.61, 153.89, 158.18,

158.48, 160.26, 165.83, 176.43. HRMS (ESI) calcd for C₃₁H₄₀N₉O₇S⁻

[M − H]⁻m/z 682.2777, found m/z 682.2761. HPLC: 14.04 min.

General Procedure for POM Protection to Give 22 and 23.

The xanthine was dissolved in DMF, K₂CO₃added, and the reaction

mixture stirred for 45 min at 50 °C. Pivaloxymethyl chloride was added

dropwise, and the reaction mixture was stirred for 5 h at 50 °C. The

reaction mixture was cooled to rt and concentrated in vacuo. Water was

added, and the aqueous solution was extracted twice with ethyl acetate.

The pooled organic layers were dried over MgSO₄, ﬁltered, and

concentrated in vacuo.

+

(ESI) calcd for C₃₊₆H₅₀N₈NaO₇[M + Na]⁺m/z 729.3695,

C₇₂H₁₀₀N₁₆NaO₁₄[2M

C₁₀₈H₁₅₀N₂₄NaO₂₁m/z 2142.1300, found m/z 729.3659,

+

Na]⁺m/z 1435.7497, and

+

1435.7399, and 2143.1233.

General Procedure for DMB Deprotection and Alkylation to

Give 24 and 25. The DMB-protected xanthine was dissolved in neat

TFA and stirred overnight at 50 °C. The reaction mixture was cooled to

rt and blown with compressed air for 20 min to evaporate excess TFA.

CH₂Cl₂was added, and the organic solution was concentrated in vacuo

and then dried overnight under high vacuum. The residue was dissolved

in DMF, K₂CO₃added, and the reaction mixture stirred for 45 min at 50

°C. 1-Bromopropane was added dropwise, and the reaction mixture was

stirred overnight at 50 °C. The reaction mixture was cooled to rt and

concentrated in vacuo. Water was added, and the aqueous solution was

extracted twice with ethyl acetate. The pooled organic layers were dried

over MgSO₄, ﬁltered, and concentrated in vacuo.

(1-Butyl-2,6-dioxo-8-{4-[(prop-2-yn-1-yl)carbamoyl]bicyclo-

[2.2.2]octan-1-yl}-3-propyl-2,3,6,7-tetrahydro-1H-purin-7-yl)methyl

2,2-Dimethylpropanoate (24). 22 (370 mg, 0.56 mmol), TFA (2.5

mL), DMF (4 mL), K₂CO₃(154 mg, 1.11 mmol), and 1-

bromopropane (136 mg, 1.11 mmol) following column elution with

n-hexane/ethyl acetate (7:3) gave 24 (239 mg, 0.43 mmol, 77%) as a

gummy liquid. ¹H NMR (400 MHz, CDCl₃): δ 0.92 (t, J = 7.4 Hz, 3H),

0.97 (t, J = 7.6 Hz, 3H), 1.20 (s, 9H), 1.36 (m, 2H), 1.60 (m, 2H), 1.79

(m, 2H), 2.01−2.17 (m, 12H), 2.29 (s, 1H), 2.29 (s, 2H), 3.98 (t, J =

7.4 Hz, 2H), 4.05 (t, J = 7.2 Hz, 2H), 6.37 (br s, 2H), 6.65 (m, 1H). ¹³C

NMR (400 MHz, CDCl₃): δ 11.25, 11.61, 14.17, 20.55, 21.84, 27.28,

29.57, 30.13, 30.47, 33.64, 35.39, 39.14, 41.49, 45.17, 69.27, 77.56,

107.89, 122.02, 147.59, 148.86, 151.45, 154.96, 159.87, 168.63, 177.44.

+

HRMS (ESI) calcd for C₃₀H₄₃N₅NaO₅[M + Na]⁺m/z 576.3156,

found m/z 576.3132.

(8-{4-[(3-Azidopropyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-1-

butyl-2,6-dioxo-3-propyl-2,3,6,9-tetrahydro-1H-purin-9-yl)methyl

2,2-Dimethylpropanoate (25). 23 (288 mg, 0.41 mmol), TFA (2 mL),

DMF (4 mL), K₂CO₃(113 mg, 0.81 mmol), and 1-bromopropane (75

mg, 0.61 mmol) following column elution with n-hexane/ethyl acetate

1

(8:2) gave 25 (102 mg, 0.17 mmol, 42%) as a white solid. H NMR

(400 MHz, DMSO-d₆): δ 0.86 (t, J = 7.4 Hz, 3H), 0.86 (t, J = 7.4 Hz,

3H), 1.11 (s, 9H), 1.25 (m, 2H), 1.48 (m, 2H), 1.65−2.02 (m, 16H),

3.10 (q, 2H), 3.31 (t, J = 6.8 Hz), 3.84 (t, J = 7.2 Hz, 2H), 3.93 (t, J = 6.4

Hz, 2H), 6.32 (s, 2H), 7.48 (t, J = 5.4 Hz, 1H). ¹³C NMR (400 MHz,

DMSO-d₆): δ 10.91 (d), 13.56 (d), 26.42 (d), 27.52 (d), 28.27 (d),

29.25 (d), 29.46 (d), 34.44 (d), 35.94 (d), 37.79 (d), 38.17, 43.96 (d),

48.36 (d), 56.02 (d), 68.88 (d), 106.53 (d), 109.31, 128.34 (d), 146.47

(d), 150.17 (d), 153.66 (d), 159.88 (d), 175.89 (d), 176.23 (d). HRMS

(1-Butyl-3-[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-8-{4-[(prop-

2-yn-1-yl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-2,3,6,7-tetrahydro-

1H-purin-7-yl)methyl 2,2-Dimethylpropanoate (22). 14 (600 mg,

1.10 mmol), DMF (5 mL), K₂CO₃(227 mg, 1.64 mmol), and

pivaloxymethyl chloride (214 mg, 1.42 mmol) following column

elution with n-hexane/ethyl acetate (1:1) gave 22 (384 mg, 0.58 mmol,

52%) as a gummy liquid. ¹H NMR (400 MHz, DMSO-d₆): δ 0.86 (t, J =

7.4 Hz, 3H), 1.12 (s, 9H), 1.25 (m, 2H), 1.48 (m, 2H), 1.69−2.00 (m,

12H), 3.72 (s, 3H), 3.79 (s, 3H), 3.81 (t, J = 2.4 Hz, 2H), 3.85 (t, J = 7.4

Hz, 2H), 5.06 (s, 2H), 6.33 (br s, 2H), 6.42 (dd, J = 2.4, 8.4 Hz, 1H),

6.57 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 5.6 Hz,

1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 13.66, 19.50, 26.54, 27.45,

28.05, 29.27, 29.53, 34.55, 37.88, 38.16, 39.95, 40.92, 54.88, 55.17,

55.45, 77.29, 81.70, 98.32, 104.49, 106.65, 109.52, 116.43, 127.65,

146.79, 150.27, 153.84, 157.53, 159.78, 159.91, 176.00. HRMS (ESI)

+

(ESI) calcd for C₃₀H₄₆N₈NaO₅[M + Na]⁺m/z 621.3483 and

+

C₆₀H₉₂N₁₆NaO₁₀[2M + Na]⁺m/z 1219.7075, found m/z 621.3456

and 1219.6990.

General Procedure for POM Deprotection to give UODC4

and UODC1. The POM-protected xanthine was dissolved in 2 M

methanolic NaOH and stirred for 1 h. The reaction mixture was

neutralized with AcOH to pH 4 and cooled overnight, and the

precipitate that formed was collected by ﬁltration, washed with water,

and air-dried.

+

calcd for C₃₆H₄₇NaN₅O₇[M + Na]⁺m/z 684.3368 and

+

C₇₂H₉₄NaN₁₀O₁₄[2M + Na]⁺m/z 1345.6843, found m/z 684.3323

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

N-(prop-2-yn-1-yl)bicyclo[2.2.2]octane-1-carboxamide (UODC4).

24 (110 mg, 0.20 mmol) and NaOH (2 mL) gave UODC4 (85 mg,

0.19 mmol, 98%) as a white ﬂuﬀy solid. ¹H NMR (500 MHz, DMSO-

and 1345.6739.

(8-{4-[(3-Azidopropyl)carbamoyl]bicyclo[2.2.2]octan-1-yl}-1-

butyl-3-[(2,4-dimethoxyphenyl)methyl]-2,6-dioxo-2,3,6,9-tetrahy-

dro-1H-purin-9-yl)methyl 2,2-Dimethylpropanoate (23). 15 (475

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d₆): δ 1.11 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7 Hz, 3H), 1.53 (m, 2H), 1.76

(m, 2H), 1.93 (m, 2H), 2.11−2.25 (m, 12H), 2.24 (s, 2H), 2.25 (s, 1H),

3.86 (t, J = 7.3 Hz, 2H), 3.93 (t, J = 7.3 Hz, 2H), 6.67 (s, 1H), 12.97 (s,

1H). ¹³C NMR (500 MHz, DMSO-d₆): δ 10.52, 11.00, 13.70, 19.61,

20.86, 29.13, 29.42, 29.68, 32.93, 33.18, 39.90, 40.17, 44.22, 106.48,

122.16, 147.33, 147.78, 150.58, 153.88, 160.10, 167.75. HRMS (ESI)

were added to both the A₁R crystal structure and the A₃R homology

model prior to docking. DU172 was successfully docked into the ligand-

free receptor from the A₁R crystal structure (PDB entry 5UEN) using

covalent docking protocols as a positive control.

Biology. All novel compounds, including all novel assayed

compounds, were screened for PAINS using https://www.cbligand.

org/PAINS/search_struct.php, and all compounds passed the ﬁlter.

Dulbecco’s modiﬁed Eagle’s medium (DMEM), fetal bovine serum

(FBS), trypsin, and the Pierce BCA protein assay kit were purchased

from Invitrogen (Carlsbad, CA). Adenosine deaminase (ADA) and

hygromycin B were purchased from Roche Diagnostics (Mannheim,

Germany). Ultima Gold scintillation cocktail, MicroScint-O,

[³H]DPCPX {8-cyclopentyl-1,3-dipropylxanthine, [dipropyl-2,3-3H-

(N)]}, and the LANCE cAMP kit were purchased from PerkinElmer

Life Sciences (Glen Waverley, Australia). Adenosine receptor ligands,

8-{4-[4-(4-chlorophenyl)piperazide-1-sulfonyl]phenyl}-1-propylxan-

thine (PSB-603), N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]-

quinazolin-5-yl]benzene acetamide (MRS1220), 2-(2-furanyl)-7-[3-

(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-

pyrimidin-5-amine (SCH442416), and trans-4-[(2-phenyl-7H-pyrrolo-

[2,3-d]pyrimidin-4-yl)amino]cyclohexanol (SLV320) were purchased

from Tocris Bioscience (Bristol, U.K.). CA200645, the ﬂuorescent A₃

antagonist, was from Hello Bio (Bristol, U.K.), while furimazine was

from Promega (Alexandria, Australia). Unless stated otherwise, all

other reagents ordered from Sigma-Aldrich (Castle Hill, Australia)

were of analytical quality.

Cell Culture and Membrane Preparation. Nontransfected

HEK293 adherent (NT-HEK293A) and nontransfected FlpIn Chinese

hamster ovary (NT-FlpInCHO) cells were maintained in DMEM

supplemented with 5% FBS and 16 mM HEPES. FlpInCHO cells stably

transfected with the human A₁, A_2A, A_2B, or A₃adenosine receptor

(A₁R-FlpInCHO, A_2AR-FlpInCHO, A_2BR-FlpInCHO, or A₃R-FlpIn-

CHO, respectively) were grown in DMEM supplemented with 5% FBS,

16 mM HEPES, and hygromycin B (500 μg/mL). All cells were

maintained at 37 °C in a 5% CO₂humidiﬁed incubator, grown to

conﬂuence, and then seeded in 96-well culture plates at assay-speciﬁc

densities. Membrane preparation was performed as previously

described.³⁶The protein content was determined using a Pierce BCA

protein assay kit according to the manufacturer’s instructions.

+

calcd for C₂₄H₃₃N₅NaO₃[M + Na]⁺m/z 462.2476 and

+

C₄₈H₆₆N₁₀NaO₆[2M + Na] m/z 901.5059, found m/z 462.2436

and 901.4979. HPLC: 19.08 min.

N-(3-Azidopropyl)-4-(1-butyl-2,6-dioxo-3-propyl-2,3,6,9-tetrahy-

dro-1H-purin-8-yl)bicyclo[2.2.2]octane-1-carboxamide (UODC1).

25 (11 mg, 0.018 mmol) and NaOH (200 μL) following column

elution with n-hexane/ethyl acetate (3:7) and then semipreparative RP-

HPLC puriﬁcation gave UODC1 (8 mg, 0.016 mmol, 89%) as a white

solid. ¹H NMR (500 MHz, DMSO-d₆): δ 0.85 (t, J = 7.5 Hz, 3H), 0.89

(t, J = 7.3 Hz, 3H), 1.27 (m, 2H), 1.51 (m, 2H), 1.61−1.91 (m, 16H),

3.10 (q, J = 6.2 Hz, 2H), 3.31 (t, J = 6.8 Hz, 2H), 3.86 (t, J = 7.5 Hz,

2H), 3.92 (t, J = 7.5 Hz, 2H), 7.48 (t, J = 5.8 Hz, 1H), 12.92 (br s, 1H).

¹³C NMR (500 MHz, DMSO-d₆): δ 11.01, 13.72, 13.94, 19.60, 20.86,

27.83, 28.39, 29.65, 29.68, 33.21, 36.06, 38.10, 39.45, 44.21, 48.47,

106.43, 147.30, 150.59, 153.88, 160.31, 176.48. HRMS (ESI) calcd for

C₂₄H₃₆N₈NaO₃⁺[M + Na]⁺m/z 507.2803 and C₄₈H₇₂N₁₆NaO₆⁺[2M

+ Na]⁺m/z 991.5713, found m/z 507.2804 and 991.5728. HPLC:

17.76 min.

1-Butyl-8-cyclohexyl-3-[(2,4-dimethoxyphenyl)methyl]-2,3,6,9-

tetrahydro-1H-purine-2,6-dione (26). According to the procedure

described for 11, cyclohexanoic acid (129 mg, 1.00 mmol), Et₃N (392

mg, 3.87 mmol), HATU (383 mg, 1.00 mmol), and 9 (351 mg, 1.00

mmol) gave 26 (391 mg, 0.88 mmol, 88%) as a white solid. ¹H NMR

(400 MHz, DMSO-d₆): δ 0.88 (t, J = 7.2 Hz), 1.10−1.90 (m, 14H),

1.72 (m, 2H), 2.66 (m, 1H), 3.71 (s, 3H), 3.82 (s, 3H), 3.86 (t, J = 7.4

Hz), 5.04 (s, 1H), 6.39 (dd, J = 2.4, 8.4 Hz, 1H), 6.57 (d, J = 2.4 Hz),

6.66 (d, J = 8.4 Hz), 13.10 (br s, 1H). ¹³C NMR (400 MHz, DMSO-d₆):

δ 13.73, 19.63, 25.26, 25.33, 29.70, 30.95, 37.59, 40.28, 41.12, 55.19,

55.48, 98.31, 104.42, 106.05, 109.56, 116.65, 126.68, 147.79, 150.65,

+

153.96, 157.37, 158.25, 159.63. HRMS (ESI) calcd for C₂₄H₃₃N₄O₄

+

[M + H]⁺m/z 441.2496 and C₂₄H₃₂N₄NaO₄[M + Na]⁺m/z

463.2316, found m/z 441.2496 and 463.2318.

4-{[3-(1-Butyl-8-cyclohexyl-2,6-dioxo-2,3,6,9-tetrahydro-1H-

purin-3-yl)propyl]carbamoyl}benzene-1-sulfonyl Fluoride

(UODC8). According to the procedure described for UODC6, 26

(332 mg, 0.75 mmol), TFA (2.5 mL), and 4-[(3-bromopropyl)-

carbamoyl]benzenesulfonyl ﬂuoride (370 mg, 1.14 mmol) following

column chromatography eluting with n-hexane/ethyl acetate (4:6) gave

cAMP Accumulation. The A₁R-, A_2AR-, A_2BR-, and A₃R-

FlpInCHO cells were seeded into 96-well culture plates at a density

of 30000 cells/well and incubated overnight at 37 °C in a 5% CO₂

incubator. The cAMP assays were performed as described previously.³⁷

Ligand concentration−response curves were normalized to the baseline

response (0%) mediated by 3 μM forskolin (A₁R- and A₃R-FlpInCHO)

or buﬀer (A_2AR- and A_2BR-FlpInCHO) and the maximal response

(100%) mediated by buﬀer (A₁R- and A₃R-FlpInCHO) or 3 μM

forskolin (A_2AR- and A_2BR-FlpInCHO).

1

UODC8 (343 mg, 0.64 mmol, 85%) as a white solid. H NMR (500

MHz, DMSO-d₆): δ 0.88 (t, J = 7.3 Hz, 3H), 1.12−2.00 (m, 16H), 2.62

(m, 1H), 3.31 (q, J = 6.5 Hz, 2H), 3.85 (t, J = 7.3 Hz, 2H), 4.07 (t, J =

6.8 Hz, 2H), 8.17 (d, J = 8.5 Hz, 2H), 8.26 (d, J = 8.5 Hz, 2H), 8.87 (t, J

= 5.5 Hz, 1H), 13.04 (br s, 1H). ¹³C NMR (500 MHz, DMSO-d₆): δ

13.70, 19.62, 25.18, 25.28, 25.34, 27.44, 29.68, 30.87, 36.82, 37.54,

40.20, 40.70, 106.01, 128.59, 128.99, 192.23 (d), 141.62, 147.51,

150.60, 150.93, 153.86, 158.13, 164.24. ¹⁹F NMR (400 MHz, DMSO-

d₆): δ 66.04. HRMS (ESI) calcd for C₂₅H₃₁FN₅O₅S⁻[M − H]⁻m/z

532.2035, found m/z 532.2068. HPLC: 19.52 min.

Molecular Modeling and Docking. The three-dimensional

structure of UODC2 was generated using Avogadro 1.2.0³³and

minimized using the universal force ﬁeld (UFF). Ligand docking was

performed using GOLD 5.8.1 (CCDC Software)³⁴centered on

residues F171 (A₁R) and F168 (A₃R homology model) with a distance

extension of 15 Å. The covalent binding mode was used, and the

covalent link was deﬁned between position 4 of Y271^7.36(A₁R) or

Y265^7.36(A₃R homology model) and the oxygen atom of the sulfonyl

moiety (SO₃). The highest-ranked pose at each receptor is presented.

The docking results were visualized in PyMOL (The PyMOL

[³H]DPCPX Binding Assay Using A₁R-FlpInCHO Membranes

and Whole Cells. Radioligand binding experiments were performed 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, and 1 unit/

mL ADA (pH 7.4)]. Membrane homogenates of A₁AR-FlpInCHO (15

μg) were incubated with diﬀerent concentrations of competing ligands

in the presence of approximately 1 nM [³H]DPCPX (precise

concentrations in each experiment were determined by β-counting)

for 4 h at 25 °C. Incubation was terminated by rapid ﬁltration through

UniFilter-96 GF/C (Whatman) microplates using a Filtermate 96

harvester (Packard). Filter plates were washed three times with ice-cold

0.9% NaCl, dried overnight before the addition of MicroScint-O

scintillation cocktail (40 μL/well), and allowed to stand for at least 1 h

before radioactivity was determined by β-counting using a MicroBeta 2

LumiJET microplate counter (PerkinElmer).

To assess the irreversible binding of selected clickable probes, A₁R-

FlpInCHO cells were plated in 96-well poly-^D-lysine isoplates at a

density of 40000 cells/well and incubated overnight at 37 °C in a 5%

CO₂incubator. After removal of cell media, cells were incubated with

increasing concentrations of competing ligands for 3 h in binding buﬀer,

followed by extensive washing steps (6 × 20 min, total of 2 h) at room

temperature. Cells were incubated with approximately 1 nM

̈

Molecular Graphics System, version 2.3.2, Schrodinger, LLC). The

A₃R homology model was generated using Modeler 9.22³⁵based on the

antagonist-bound DU172-A₁R crystal structure (PDB entry 5UEN),²²

which includes the command “a.md_level = reﬁne.slow” that performs a

thorough MD optimization. All structures were protonated appropri-

ately prior to docking via either PyMOL or Hermes. Hydrogen atoms

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[³H]DPCPX (precise concentrations in each experiment were

determined by β-counting) for 4 h at 4 °C. Upon removal of the

radioligand, cells were washed ﬁve times with ice-cold phosphate-

buﬀered saline (PBS) (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM

NaCl, and 2.7 mM KCl) and dissolved in Ultima Gold scintillation

liquid (100 μL/well). Isoplates were sealed and allowed to incubate for

at least 1 h before radioactivity was determined by β-counting using a

MicroBeta 2 LumiJET microplate counter (PerkinElmer). For all

radioligand binding experiments, nonspeciﬁc binding was assessed

using 1 μM SLV320, a selective A₁R antagonist. After subtraction of the

nonspeciﬁc binding, competition binding data were normalized to

radioligand binding values in the absence of competitors and ﬁtted to

the one-site competition binding equation in Prism 8.0 (GraphPad) to

derive log K_ivalues.

NanoBRET Binding Displacement Assay. Nontransfected

HEK293A cells were transiently transfected with 3 μg of cDNA of N-

terminal NanoLuc (Nluc)-tagged A₃R using polyethylenimine (PEI) at

a 4:1 ratio in 10 cm dishes. Twenty-four hours post-transfection, cells

were seeded into 96-well poly-^D-lysine solid white bottom plates at a

density of 40000 cells/well and incubated overnight at 37 °C in a 5%

CO₂incubator in serum-free DMEM. On the day of the assay, 48 h

post-transfection, cells were incubated with increasing concentrations

of competing ligands for 3 h, prepared in a binding buﬀer. After ligand

incubation, 5 nM CA200645 was added for 60 min at room

temperature and then the NLuc substrate furimazine was added to a

ﬁnal concentration of 10 μM. Sequential emission measurements were

taken using a PHERAStar Omega plate reader (BMG Labtech) using

460 nm (80 nm bandpass, donor NLuc emission) and 610 nm (long-

pass ﬁlter, ﬂuorescent ligand emission) ﬁlters at 25 °C. Raw BRET

ratios were calculated by dividing the 610 nm emission (acceptor) by

the 460 nm emission (donor). To assess irreversible binding of selected

clickable probes, 6 × 20 min (total of 2 h) extensive washing steps were

added before the addition of a ﬂuorescent antagonist and furimazine. In

all NanoBRET binding experiments, nonspeciﬁc binding was assessed

using 1 μM MRS1220, a selective A₃R antagonist. After subtraction of

the nonspeciﬁc binding, competition binding data were normalized to

total binding values in the presence of a vehicle and ﬁtted to the one-site

competition binding equation in Prism 8.0 (GraphPad) yielding log K_i

values.

Following three washes with PBST, membranes were imaged using the

Typhoon system (Amersham). The signal intensities of the Cy5.5 click

tag and secondary antibodies were quantiﬁed using ImageStudio

(LiCOR). The Cy5.5 ﬂuorescence was normalized to the relative V5

level (for A₁R-FlpInCHO cells) or β-actin level (for nontransfected

FlpIn CHO cells) in each sample. To assess nonspeciﬁc protein

labeling, prior to the two-step labeling experiment, A₁R-FlpInCHO

cells were preincubated with 10 μM UODC6 or a vehicle control (0.1%

DMSO) for 90 min, followed by labeling with either 1 or 10 μM

clickable probes for an additional 90 min. Samples were then subjected

to the click chemistry procedure using the protocol described above.

Data Analysis. Nonlinear regression curve ﬁtting and statistical

analyses were performed using Prism 8.0 (GraphPad Software Inc., San

Diego, CA). To derive potency estimates, agonist concentration−

response data were ﬁtted to a three-parameter Hill equation as

described previously.¹⁰Radioligand inhibition binding data with

competitive ligands were ﬁtted to a one-site inhibition mass action

curve. Resulting IC₅₀values were converted to dissociation constants

(K_ivalues) using the Cheng−Prusoﬀ equation.³⁸To estimate

antagonist aﬃnity values, functional interaction studies between

NECA and multiple concentrations of each antagonist in the cAMP

assays were ﬁtted to the competitive model as described previously.²²

All results were expressed as means SEM.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02169.

■

sı

Molecular formula strings of the prepared compounds

(CSV)

A₃AR homology model (PDB)

Additional synthetic details such as the concentration−

response curves for data reported in Tables 1 and 2

(Figures S1−S3), docking of UODC2 into the A_2AR

(PDB entry 6LPK) (Figure S4), and an immunoblotting

control experiment (Figure S5) (PDF)

Whole Cell Aﬃnity Labeling and SDS−PAGE. Nontransfected

FlpInCHO cells and A₁R-FlpInCHO cells were incubated with

clickable probes at indicated concentrations (0.1, 1, and 10 μM) or a

vehicle control (0.1% DMSO) in a binding buﬀer for 90 min. Cells were

washed twice with PBS (10 min/wash, gentle shaking), lifted from the

plate, and centrifuged (300g for 5 min at rt). Then, cell pellets were

resuspended in a click chemistry conjugation reaction mixture for 1 h at

rt with intermittent vortexing. The click reagents were added in the

following sequence: 1 μM ﬂuorescent Cy5.5 azide or Cy5.5 alkyne

(Lumiprobe), 1 mM ascorbic acid (made immediately prior to use),

TBTA/tert-butanol, and 1 mM CuSO₄. Following click chemistry,

unbound click reagents were washed oﬀ by centrifugation of the

samples (300g for 5 min at rt) and resuspending the material in PBS

once before resuspending the cells in a radioimmunoprecipitation assay

(RIPA) lysis buﬀer (10 mM Tris, 2 mM EDTA, 20 mM β-

glycerophosphate, 0.1% NP40, 100 mM NaCl, and 12 mM sodium

deoxycholate) for 10 min at 4 °C. Cell lysates were then centrifuged

(16000g for 10 min at 4 °C), and the protein concentration of the

supernatant was determined by the Pierce BCA protein assay. After

normalization of the protein concentration, 4× SDS loading buﬀer was

added and the protein material denatured for 30 min at 37 °C. Proteins

were separated by SDS−PAGE on 10% polyacrylamide gels (200 V for

50 min at rt). In-gel ﬂuorescence was detected using a Typhoon system

(Amersham). Proteins were subsequently transferred to nitrocellulose

membranes (20 V overnight at 4 °C), and membranes were blocked

with Odyssey blocking buﬀer and incubated with the anti-V5 (1:2500,

mouse monoclonal, Sigma) or anti-β-actin (1:2500, rabbit polyclonal,

Abcam) antibody overnight at 4 °C. Thereafter, membranes were

washed three times (20 min/wash) with PBS-T (PBS with 0.1% Tween

20) and incubated with the donkey anti-mouse 488 (1:10000, Abcam)

or donkey anti-rabbit 488 (1:10000, Abcam) antibody for 2h at rt.

AUTHOR INFORMATION

Corresponding Authors

■

Lauren T. May − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, and Department of Pharmacology,

Monash University, Parkville, VIC 3052, Australia;

Email: lauren.may@monash.edu

Andrea J. Vernall − Department of Chemistry, University of

Otago, Dunedin 9016, New Zealand; orcid.org/0000-

0001-8056-0726; Email: andrea.vernall@otago.ac.nz

Karen J. Gregory − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, and Department of Pharmacology,

Monash University, Parkville, VIC 3052, Australia;

orcid.org/0000-0002-3833-2137;

Email: karen.gregory@monash.edu

Authors

Phuc N. H. Trinh − Drug Discovery Biology, Monash Institute

of Pharmaceutical Sciences, and Department of Pharmacology,

Monash University, Parkville, VIC 3052, Australia

Daniel J. W. Chong − School of Pharmacy, University of Otago,

Dunedin 9016, New Zealand

Katie Leach − Drug Discovery Biology, Monash Institute of

Pharmaceutical Sciences, and Department of Pharmacology,

Monash University, Parkville, VIC 3052, Australia;

orcid.org/0000-0002-9280-1803

Stephen J. Hill − Centre of Membrane Proteins and Receptors

(COMPARE), University of Birmingham and University of

Nottingham, The Midlands NG7 2UH, U.K.; Division of

8176

https://doi.org/10.1021/acs.jmedchem.0c02169

J. Med. Chem. 2021, 64, 8161−8178

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Physiology, Pharmacology and Neuroscience, School of Life

Sciences, University of Nottingham, Nottingham NG7 2UH,

U.K.; orcid.org/0000-0002-4424-239X

Joel D. A. Tyndall − School of Pharmacy, University of Otago,

Dunedin 9016, New Zealand; orcid.org/0000-0003-

0783-1635

REFERENCES

■

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c02169

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P.N.H.T. and D.J.W.C. contributed equally to this work. K.L.,

J.D.A.T., L.T.M., A.J.V., and K.J.G. participated in study design.

D.J.W.C., J.D.A.T., and A.J.V. conducted and/or analyzed

medicinal chemistry and computational experiments. P.N.H.T.

and K.J.G. performed pharmacology and biochemical experi-

ments. P.N.H.T., L.T.M., and K.J.G. performed data analysis and

interpretations of pharmacological experiments. S.J.H. con-

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

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This work was supported by National Health and Medical

Research Council of Australia (NHMRC) Project Grants

APP1123722 (K.J.G., L.T.M., and A.J.V.), APP1145420

(L.T.M. and S.J.H.), and APP1147291 (L.T.M.). L.T.M. is an

Australian National Heart Foundation Future Leaders Fellow.

K.J.G. and K.L are supported by Australian Research Council

Future Fellowships (FT160100075 and FT170100392).

D.J.W.C. was supported by a University of Otago Doctoral

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ABBREVIATIONS USED

■

A₁R, adenosine A₁receptor; A_2AR, adenosine A_2AR receptor;

A_2BR, adenosine A_2BR receptor; A₃R, adenosine A₃receptor;

ACN, acetonitrile; AcOH, acetic acid; AR, adenosine receptor;

br, broad; CCDC, Cambridge Crystallographic Data Centre;

CHO, Chinese hamster ovary; COSY, homonuclear correlation

spectroscopy; DIPEA, diisopropylethylamine; DMB, 3,4-

dimethoxybenzyl; DMF, dimethylformamide; DMSO, dimethyl

sulfoxide; GPCR, G protein-coupled receptor; HATU, O-(7-

azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-

ﬂuorophosphate; HEK, human embryonic kidney; HMBC,

heteronuclear multiple-bond correlation; HPLC, high-perform-

ance liquid chromatography; HRMS, high-resolution mass

spectrometry; HSQC, heteronuclear single-quantum coher-

ence; NMR, nuclear magnetic resonance; NOESY, nuclear

Overhauser eﬀect spectroscopy; PDB, Protein Data Bank; PEG,

polyethylene glycol; POM, pivaloyloxymethyl; POM-Cl, chlor-

omethyl pivalate; RP-HPLC, reverse-phase HPLC; SAR,

structure−activity relationship; SDS−PAGE, sodium dodecyl

sulfate−polyacrylamide gel electrophoresis; SEM, standard

error of the mean; TBAB, tetra-n-butylammonium bromide;

TFA, triﬂuoroacetic acid; TLC, thin layer chromatography; TR-

FRET, time-resolved ﬂuorescence resonance energy transfer;

UFF, universal force ﬁeld

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Development of Covalent, Clickable Probes for Adenosine A1and A3Receptors

DOI: 10.1021/acs.jmedchem.0c02169

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