Adenosine A2AR/A1R Antagonists Enabling Additional H3R Antagonism for the Treatment of Parkinson’s Disease

Antagonism for the Treatment of Parkinson ’s Disease

Article

Adenosine A R/A R Antagonists Enabling Additional H R

Stefanie Hagenow, Anna A ﬃ ni, Elsa Y. Pioli, Sonja Hinz, Yan Zhao, Gregory Porras,

Vigneshwaran Namasivayam, Christa E. Mu

ller, Jian-Sheng Lin, Erwan Bezard, and Holger Stark*

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sı Supporting Information

ABSTRACT: Adenosine A /A₂_Areceptors (A R/A R) represent targets in nondopaminergic treatment of motor disorders such as

Parkinson’s disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD

therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of

histamine H receptor (H R) antagonists in combination with the “caﬀeine-like eﬀects” of A R/A R antagonists, we designed A R/

A R/H R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H R pharmacophore was introduced with overlap into an

adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding proﬁles with overall nanomolar H₃R

aﬃnities (K < 55 nM). Compound 4 (ST-2001, K (A R) = 11.5 nM, K (A R) = 7.25 nM) and 12 (ST-1992, K (A R) = 11.2 nM,

K (A R) = 4.01 nM) were evaluated in vivo. L-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg

−

−1

kg , i.p. rats). Compound 12 (2 mg kg , p.o. mice) increased wakefulness representing novel pharmacological tools for PD

therapy.

INTRODUCTION

Already in the late 1990’s, A R/A R antagonists were

1 2A

■

suggested as nondopaminergic therapy for PD, one of the most

Adenosine receptors (ARs) are abundantly expressed in the

prominent motor disorders. PD patients suﬀer from distinct

dorsal and ventral striatum. The cortico-basal ganglia circuit,

which controls initiation and inhibition of body movement, is

modulated at the striatal level by dopaminergic input from the

substantia nigra pars compacta (SNpc). Heterodimerization of

ARs with dopaminergic receptors in striatum suggests that they

act as modulators for ﬁne-tuning of dopaminergic neuro-

motor abnormalities exempliﬁed by cardinal symptoms, such as

bradykinesia, rigidity, postural changes, and tremor. Pro-

gressive loss of dopaminergic neurons in the SNpc leads to

disruption of dopaminergic neurotransmission in the cortico-

basal ganglia circuit and consequently, a loss of controlling

−6

voluntary body movements. With dimerization of A Rs and

transmission,

and thus, they represent suitable targets for

dopamine D -like receptors, for example, dopamine D

the treatment of motor disorders such as Parkinson’s disease

receptors (D Rs), on striatal neurons, A R antagonists are

2 2A

(

PD). Adenosine can act via four distinct G-protein-coupled

receptors (GPCRs), namely, adenosine A , A , A , and A

assumed to modulate the “indirect pathway” of the cortico-

receptors (A R), showing either G -(A Rs and A Rs) or G -

coupling (A Rs and A Rs). A Rs and A Rs are highly

Received: January 25, 2021

Published: June 9, 2021

enriched in speciﬁc parts of the central nervous system (CNS),

while A Rs and A Rs display lower expression levels and are

mainly localized in peripheral tissues. In the CNS, A Rs are

widely distributed in several (sub)cortical areas, whereas A Rs

,10

are predominantly found in striatal areas.

2021 American Chemical Society

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Article

45,50

Figure 1. H R ligands UCL2190 (hH R K = 1.6 nM) and I, as well as potent adenosine antagonists (preladenant and JNJ-40255293)

taken as lead structures for the synthesis of MTLs (1−12).

,13,14

basal ganglia circuit.

This pathway attenuates body

high pathological complexity in CNS disorders, the develop-

ment of multitargeting ligands (MTLs) became an accepted

strategy to overcome the multiplicity of symptoms, with the

potential to challenge conventional combination therapies.

These MTLs may enable synergistic responses via simulta-

neous target modulation and bear several advantages compared

to comedications, that is, a uniﬁed and optimized pharmaco-

dynamic/−kinetic proﬁle. Separate drug evaluation and drug

regimen alignment prior to coapplication can be avoided with

movement controlled by dopamine acting via G -coupled D -

like receptors in a healthy brain. The motor-activating “direct

pathway” on the other hand is regulated by G -coupled D Rs,

which were found to form heterodimers with A Rs. In both

cases, stimulation of ARs, dimerized with either D R or D R,

results in decrease of dopamine binding to the respective

receptors. The impact on motor activity via A Rs has been

studied in numerous animal studies, proving the ability of A R

antagonists to stimulate locomotor activity in naive animals

and in parkinsonian animals, either alone or in combination

,10

30−33

MTLs.

During the last decades, a number of MTLs were

developed and investigated for the treatment of neuro-

10,15−17

with dopaminergic treatment.

Regarding contributing

degenerative diseases, while the histamine H₃receptor

eﬀects via A R, less is known in context of PD motor

(H R) has been suggested as a novel and suitable target. A

symptoms, while selective A R antagonists do not show motor

great variety of H R MTLs have been described, showing

18−20

improvement in PD models,

but they may provide a

biological activities at other neurotransmitter-regulating

therapeutic potential in PD-associated cognitive dysfunction

targets, for example, diﬀerent types of GPCRs, thus, proving

its overall utility in multitargeting drug design.

due to increase of cholinergic transmission via A R block-

1,22

ade.

Antagonists of the H R are able to modulate distinct

A small number of A R/A R and selective A R

neurotransmitters via presynaptic H R heteroreceptor block-

antagonists, respectively, reached advanced clinical trials for

motor disabilities in PD patients, such as the xanthine

derivatives caﬀeine and istradefylline, as well as the non-

ades. They are abundantly expressed in the basal ganglia and

currently under investigation for various CNS disorders, in

35,36

particular, cognitive and sleep disorders.

In 2016, pitolisant

xanthines tozadenant and preladenant (Figure 1). In Japan,

istradefylline received drug approval for therapy of PD motor

ﬂuctuations, evidencing a prospective utility of A R

was approved by the European Medicine Agency for the

treatment of narcolepsy with and without cataplexy (and in

2019 by FDA), while additional H R antagonists are currently

antagonists for treatment of movement disorders. Very

recently, istradefylline has also been approved by the Food and

Drug Administration (FDA) in the U.S. for the adjunct

treatment of PD. It was also suggested that istradefylline

improves postural abnormalities, which is a hardly treatable

in clinical trials for treatment of sleep disorders. Pitolisant is

also in clinical trial (phase III) for treatment of excessive

daytime sleepiness (EDS) in PD patients as well as for

cognitive impairment associated with schizophrenia. Thus,

H R antagonists may represent suitable tools against frequently

condition in mid- or late-stage PD patients. Caﬀeine, a mixed

observed nonmotor symptoms accompanying CNS movement

disorders such as PD.

A R/A R antagonist frequently examined in the context of

PD, is implemented in multiple clinical trials and investigated

on an epidemiological scale for early PD.

Furthermore, an additional pharmacological eﬀect in

regulation of basal ganglia function might be obtained via

25−27

37,38

The fact, that motor disorders such as PD are commonly

associated with multiple nonmotor complications such as

cognitive or sleep impairment, often demand application of

heterodimerization of H R not only with dopamine

but

Just recently, the existence of functional

A R-H R heteromers were conﬁrmed in recombinant over-

39,40

also with ARs.

28,29

comedications for therapeutic success.

Generally, with a

expressing cells as well as in the rat striatum. H R activation

247

J. Med. Chem. 2021, 64, 8246−8262

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Article

Scheme 1. Synthetic Route for the Design of MTLs 1−12

(

I) Benzyl bromide, K CO , dimethylformamide, r.t., 15 h; (II) 1-(3-chloropropyl)piperidine, K CO , KI, acetone, reﬂux, 15−24 h; (III)

appropriate benzaldehyde, NaOH, ethanol, r.t., 30 min; (IV) guanidine hydrochloride, NaOH, ethanol or methanol, N , reﬂux, 18−24 h; (V) Pd/C

(

wt 10%), ethanol, H , r.t., 18 h; (VI) 1-(3-chloropropyl)piperidine or 1-(3-chloropropyl)pyrrolidine, K CO , dimethylformamide, N , 80 °C, 4 h;

2 2 3 2

1 2

VII) NaOH, dimethylformamide, O , 80 °C, 2 h; Bn = benzyl; Y = 3-piperidinopropyloxy; Y = 3-pyrrolidinopropyloxy; 4 (ST-2001), 12 (ST-

992).

counteracts A R stimulation in rat globus pallidus on the

Promising A R/A R antagonists of this class, such as JNJ-

1 2A

striatal neuron terminals at the functional level of adenosine

40255293, were developed, which showed therapeutic

potential in multiple preclinical models of PD.

39,41

45−48

′,5′-cyclic monophosphate (cAMP) formation.

These

results suggest that A R/H R antagonists might be able to

control motor symptoms of PD via a connected pathway.

To obtain innovative MTLs for the treatment of PD, we

CHEMISTRY

■

A knowledge-based MTL design strategy was pursued by

merging a generally accepted H R pharmacophore, that is, a

cyclic aminopropyloxy-linked phenyl motif, with an A R/A R

antagonist arylindenopyrimidine core. Within this series, the

H R pharmacophore (referred as Y) was merged with the

introduced an H R antagonist pharmacophore with structural

merging into an A R/A R antagonist core motif. Pyrrolidino-/

piperidino(propyloxy)phenyl moieties were chosen as the

structural H R element, which can be found in numerous

H R antagonists/inverse agonists (e.g., UCL2190) and were

tricyclic core motif either as R and R of the indenyl moiety or

successfully applied in our recently described dual-targeting

as R ′ with the “outside” phenyl group. Furthermore, the

H R/monoamine oxidase B (MAO B) inhibitors (compound

merger of the H R pharmacophore with both an oxidized and a

I, Figure 1). Arylindenopyrimidines, structurally distinct

from traditional xanthine-based AR antagonists, were used as

A R/A R antagonist core motifs, which possess nanomolar

nonoxidized tricyclic arylindenopyrimidine core was con-

ducted, resulting in two subseries of MTLs.

4,45

receptor aﬃnities and functional antagonist eﬃcacies.

Great eﬀort has been invested in the design and optimization

of heterocycle-substituted arylindenopyrimidines.

The synthesized 5- and 6-hydroxy-1-indanones, P1−P2, and

the commercially available 1-indanone (P3) were used as the

starting material for the development of novel MTLs (Scheme

1). P1 and P2 were prepared from the corresponding methoxy

derivatives, which were demethylated with yields higher than

45,46

Multiple

variations were performed either on the basic heterocyclic

amine motif itself or its linkage (via amide, amino, or ether

groups), resulting in A R/A R antagonists with low toxicity,

90%.

The 1-(3-chloropropyl)piperidine and 1-(3-

suitable pharmacokinetics, and potent in vivo eﬃcacies.

chloropropyl)pyrrolidine precursors were synthesized in

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Journal of Medicinal Chemistry

Article

Table 1. Histamine H R and Adenosine (A R) Receptor Aﬃnities (K Values) of Designed MTLs

compound

R⁷

R⁸

R4′

hA₁R

hA R

hA₃R

hH₃R

K [nM] x ± SEM (or % inhibition, x ± SEM at 1 μM)

K [nM] x [95% CI]

Y¹

CH₂

CO

2510 ± 454

828 ± 166

>1000 (23 ± 6)

11.5 ± 1.6

>1000 (24 ± 6)

18.7 ± 1.3

717 ± 132

>1000 (40 ± 7)

941 ± 160

7.25 ± 1.53

>1000 (34 ± 3)

4.85 ± 0.90

35.5 ± 8.9

104 ± 18

>1000 (28 ±0)

>1000 (25 ±5)

>1000 (13 ±2)

36.9 ± 8.3

>1000 (34 ± 4)

58.1 ± 3.4

>1000 (24 ±4)

>1000 (35 ±11)

>1000 (50 ±5)

150 ± 10

>1000 (39 ± 4)

231 ± 38

>1000 (33 ± 4)

846 ± 117

>1000 (32 ± 8)

199 ± 9

0.49 [0.18, 1.34]

2.1 [1.1, 4.1]

0.60 [0.05, 7.4]

11 [0.45, 255]

2.3 [1.2, 4.1]

4.5 [1.7, 12]

0.28 [0.19, 0.42]

31 [14, 69]

0.31 [0.12, 0.80]

27 [3.0, 237]

36 [13, 100]

52 [15, 174]

n.d.

(ST-2001)

−H

133 ± 8

129 ± 7

331 ± 57

180 ± 18

≥1000 (44 ± 3)

12.6 ± 2.3

203 ± 80

>1000 (15 ± 1)

47.5 ± 3.4

6.24 ± 1.93

142 ± 28

367 ± 77

114 ± 20

>1000 (12 ± 3)

150 ± 13

430 ± 150

ST-1992 (12)

11.2 ± 0.7

4.01 ± 1.0

7.5 ± 4.9

43.5 ± 16.0

JNJ-40255293

Morph

48 ± 16

295 ± 10

230 ± 92

Preladenant

UCL2190

0.88 ± 0.23

≫ 1000 (−3%)

≫1000 (−1%)

>1000

n.d.

≫>1000 (−5%)

≫1000 (−6%)

≫1000 (0%)

≫1000 (8%)

≫1000 (−2%)

≫1000 (3%)

11 [3.5, 33]

224 [106, 475]

Ciproxifan

Data were obtained in radioligand competition binding studies (n ≥ 3), each performed at least in duplicate using [ H]CCPA. [ H]MSX-2.

α‑

[

H]PSB-603. [ H]PSB-11. [ H]N methylhistamine for A R, A R, A R, A R, and H R, respectively; K values are given as means either within

63 j

the 95% CI or with error (SEM) for hH R or hA Rs, respectively. Atack et al. Functional activity cAMP assay, EC . Burbiel et al. Aﬃni et

al.; Morph, 2-morpholinoethoxy; n.d. not determined.

quantitative yields by alkylation of piperidine or pyrrolidine

with 3-chloropropan-1-ol, followed by chlorination.

(yield <15%) but was not as eﬃcient as the palladium-

catalyzed hydrogenation of the benzyl moiety.

3−55

Depending on whether 3-piperidinopropyloxy or 3-pyrrolidi-

Following path 2, the synthesis of compounds 5 and 6

started with the alkylation of the hydroxy indanones P1 and P2

with 1-(3-chloropropyl)piperidine by the Williamson ether

nopropyloxy H R structural elements were introduced, two

diﬀerent reaction pathways were followed. For the synthesis of

MTLs 1−4 and 7−10, the H R pharmacophore was

reaction. The same procedure described for compounds P4

and P5 was used to convert, via aldol condensation,

compounds P6 and P7 into the corresponding 2-benzyli-

dene-1-indanone derivatives P12 and P13 with yields higher

than 80%. The latter compounds were then treated with

guanidine to obtain the corresponding arylindenopyrimidine

introduced as R or R (path 1), whereas for compounds 5

and 6 or 11 and 12, 1-(3-chloropropyl)piperidine was reacted

in the C5 or C6 position with the corresponding

hydroxyindanones (path 2), respectively.

Following path 1, the hydroxyindanones were protected

using the commercially available benzyl bromide to obtain

compounds 5 and 6 with a yield of 50 and 54%, respectively.

compounds P4−P5. Aldol condensation of the latter

compounds or 1-indanone (P3) with benzaldehyde or 4-

To identify the potential role of the carbonyl moiety, the

substituted arylindenopyrimidines, 1−4 and 5 and 6 were

oxidized via a radical mechanism to the corresponding ketones

7−12 by passing air through the solution (yields ranging from

(

benzyloxy)benzaldehyde was performed to obtain the

corresponding 2-benzylidene-1-indanone derivatives P8−P11

in good yields (80−90%). By reaction of the obtained

ketones with guanidine, benzyl ether pyrimidine intermediates

were formed, followed by deprotection using palladium-

catalyzed hydrogenation to give the key intermediates P14−

62 to 79%). Reaction time longer than 2 h caused further

oxidation and C−C cleavage to carboxylic acid.

The structures of ﬁnal compounds 1−12 were conﬁrmed by

H NMR, C NMR, and electrospray ionization high-

P17 with a yield ranging from 72 to 85%. Then, nucleophilic

resolution mass spectrometry (ESI-HRMS). The chemical

purity (>95%) was determined via high-performance liquid

chromatography (HPLC).

substitution was performed between the phenol derivatives and

-(3-chloropropyl)piperidine or 1-(3-chloropropyl)pyrrolidine,

respectively, to give compounds 1−4 (yields ranging from 65

Furthermore, some of the oxidized A R/A R/H R MTLs

to 70%). Synthesis of compounds P14−P17 was performed

analogously by using a methoxy group instead of the benzyl

ether moiety to protect the hydroxy indanones. Attempts to

deprotect methoxy pyrimidine intermediates using aluminum

chloride, lithium chloride, and sodium ethoxide were

possess potentially useful ﬂuorescence properties. As a

representative, 12 was investigated and the emission spectrum

showed a prominent peak at 546 nm with high Stokes shift

(λ_e_m− λ = 200 nm) (cf. Supporting Information). The

abs

emission spectra obtained for 12 were comparable to those of

previously reported ﬂuorescent C8-substituted indenopyrimi-

dine derivatives, used to stabilize quadruplex and duplex DNAs

2,58

unsuccessful.

However, a mixture of acetic and hydro-

bromic acid was eﬀective in removing the methoxy group

249

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Journal of Medicinal Chemistry

amidoadenosine (NECA) (Figure 2 ). Both compounds did not

Article

as possible anticancer therapy. These physicochemical

compounds bearing two H R pharmacophores, these ﬁndings

properties may be optimized to facilitate additional applica-

tions as possible pharmacological tools, for example, for

detection or labeling of H Rs, A Rs, and/or A Rs, similar to

may reﬂect a higher probability for entering the H R binding

pocket in either orientation, while these ligands may

demonstrate an unfavorable two-dimensional expansion for

ﬁtting the A R/A R binding pockets.

the recently described ﬂuorescence xanthine A R antagonist

used for binding assays.

Remarkably, in this small series, we were able to design

MTLs with distinct target aﬃnity proﬁles, despite using the

same structural elements. Major impacts, especially on AR

In Vitro AR/H R Receptor Binding Properties. The

designed AR/H R MTLs were evaluated for in vitro binding

aﬃnities in radioligand competition binding studies, as

subtype selectivity and also on H R aﬃnity could be

63−68

described previously using recombinant human ARs

and

Within this small series, submicromolar

AR aﬃnities were obtained, while some of the compounds (4,

, 10, and 12) showed the desired multitargeting properties

demonstrated, that is, MTL 5 as the pure H R ligand or 9 as

3,69

H R (Table 1).

A₂_AR-preferring MTL, stressing the importance of the diﬀerent

attachment sites for the H R pharmacophore. Particularly,

these ﬁndings provide deeper insights into the SAR of AR

subtypes and should aid the development of further

with aﬃnities in low nanomolar concentration ranges for both

types of receptors. These most potent MTLs preserved

comparable A R/A R aﬃnity to promising previously

pharmacological tools for ARs. In previous studies, A2A

preference of arylindenopyrimidines could be improved by

described arylindenopyrimidines, such as JNJ-40255293

para-ﬂuorination of the R ′ phenyl group or by replacement

7,48

(

Table 1),

but with additional aﬃnity for A Rs (K

of the phenyl with (un)substituted furan moieties. Distinct

substitutions at R ′ or scaﬀold variations in this position may

(

A R)/K (A R) = 0.08−0.19). Highest A R/A R aﬃnities

were observed for MTLs having the H R pharmacophore

attached as R , cf. 4, 6, 10, and 12, while attachment as R led

to more than 10- and 100-fold decrease in A R aﬃnity for

be a convenient strategy to increase AR subtype selectivity.

Functional screening at A2A receptors based on cAMP

accumulation has been performed for compounds 4 and 12

with the ribose-modiﬁed adenosine analogue N-ethylcarbox-

oxidized and nonoxidized/reduced MTLs, respectively (e.g.,

0 vs 8 and 4 vs 2). These observations conﬁrm previous

structure−activity relationships (SARs) for arylindenopyrimi-

dines, pointing out that substitution at C8 (as well as C9) led

to high aﬃnity at A R/A R, while substitution at C7 (as well

as C6) resulted in a decrease or abolishment of A R

4,47

binding.

As demonstrated previously, the carbonyl

moiety is neither necessary for AR binding nor signiﬁcantly

improving AR subtype selectivity comparing the most potent

MTLs within this series. Interestingly, introducing the H R

pharmacophore as R ′ is less tolerated by ARs, especially for

nonoxidized/reduced MTLs but may increase subtype

selectivity for A Rs over A R, as represented by oxidized

representatives 7 (K (A R)/K (A R) = 0.3) or 9 (K

(

A R)/ (A R) < 0.2). For MTLs having the H R

2A i 1 3

pharmacophore attached at unfavorable sites, that is, such as

R or R ′, oxidation status of the indenyl ring (X = −CH vs

Figure 2. cAMP accumulation experiments. Stimulation of cAMP

−

CO) led to signiﬁcant diﬀerences in AR aﬃnities (e.g., 1 vs

accumulation in CHO cells expressing the human adenosine A R

or 2 vs 11). Minor but signiﬁcant diﬀerences are observed

was induced by 0.3 μM of the agonist NECA (set at 100%). The one-

way ANOVA with Dunnett’s posthoc test indicated signiﬁcant

diﬀerences (***p < 0.001, ****p < 0.0001) between the eﬀect of

NECA in the absence and in the presence of A R antagonists (4, 12,

between ﬁve-membered (3-pyrrolidinopropyloxy, Y ) and six-

membered (3-piperidinopropyloxy, Y ) cyclic amine deriva-

tives (4 vs 6 and 10 vs 12), which is again in accordance with

previously designed arylindenopyrimidines.

and for comparison MSX-2 [3-(3-hydroxypropyl)-8-(m-methoxystyr-

The successful introduction of the H R pharmacophore into

yl)-7-methyl-1-propargylxanthine)].

arylindenopyrimidine AR antagonists with structural overlap at

the respective phenyl elements could be shown for all MTLs

show any agonist activity but were able to block the response

induced by the agonist NECA, similar to the eﬀect observed

having nanomolar H R aﬃnities. Once more, these ﬁndings

prove the high variation tolerance in H R ligands when

with the standard A R antagonist MSX-2 (3-(3-hydroxyprop-

maintaining the generally accepted H R blueprint. Negligible

yl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine).

impact on H R aﬃnities were observed by modiﬁcations in

Preclinical Proﬁling of Selected AR/H R MTLs. In vitro

either cyclic amine (2 vs 5; 8 vs 11) or the substitution position

binding aﬃnities at related histamine receptor subtypes (H₄R

of the indanone moiety (e.g., R vs R in 5 vs 6), while the

nonoxidized derivatives are around 10-fold more potent at H₃R

compared to oxidized ones (e.g., 2 vs 8 or 6 vs 12) with

exception of 4 versus 10, where no signiﬁcant diﬀerence was

and H R) and also additional PD-relevant targets (dopamine

D /D /D /D receptors, MAO A/B) were determined for the

most promising MTLs 4 (ST-2001) and 12 (ST-1992) (Table

2). Both MTLs showed low aﬃnity at H Rs as well as good to

found. K values in the subnanomolar concentration range for

excellent selectivity for H Rs over most homologous H Rs

H R could be obtained by attaching the H R pharmacophore

(ST-1992, K (H R)/K (H R) > 190; ST-2001, K (H R)/K

(H R) > 900), respectively.

i 4 i 3 i 4 i

to the phenyl moiety as R ′ (cf. 1 and 7). Also, MTL 9 and 3

bearing the H R pharmacophore in two positions (as R and

Binding aﬃnities at dopamine receptors were evaluated in

70,71

R ′) demonstrate excellent, subnanomolar H R bindings.

radioligand binding studies

since they are enriched in the

However, these compounds are the poorest AR ligands. For

SNpc, as part of the basal ganglia circuit and are aﬀected by

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Article

Table 2. Oﬀ-Target Aﬃnities of Most Promising A R/A R/H R MTLs

IC₅₀[nM] (% inhibition at 10 μM)

K [nM] x [95% CI]

x ± SD

MTL

hH₁R

hH₄R

hD₁R

hD₅R

hD R

hD₃R

hMAO A

hMAO B

ST-2001 (4)

ST-1992 (12)

>1000

>10 000

>1000

1246 [697−2228]

1586 [304−8266]

290 [272−309]

364 [175−759]

(7.4 ± 12)

(16 ± 3.6)

>1000 (57 ± 8.6)

(50 ± 16)

Oﬀ-target binding data were obtained in radioligand binding studies (n = 2−4) using [ H]pyrilamine, [ H]histamine, [ H]SCH23390, and

H]spiperone for hH R, hH R, D R/D R, and D R/D R, respectively. Spectrophotometric monoamine oxidase inhibition assay using

[

1 4 1 5 2S 3

−

−4

Kynuramine as the substrate and either 7−10 concentrations (between 10 and 10 M) or 10 μM of test ligand for IC₅₀determination or one-

point screening, respectively. K values are given as mean within the 95% CI, whereas MAO screening data are given as % inhibition mean ±

standard deviation (SD), n ≥ 3).

Table 3. Target Selectivity Indices (SI) for ST-2001 and ST-1992

SI calculated as K (b)/K (a) for ST-1992 and K (a)/K (b) for ST-2001, respectively. Desired “on-targets” are underlined. SI > 100 appear in

bold.

72,73

neurodegenerative processes in PD. Activated D -like

patients.

Some prominent dopamine agonists, that is,

receptors inhibit the “indirect pathway”, while activated D₁-

like receptors stimulate the “direct pathway”, leading to motor

cortex activation. Additional modulation of these receptors

would probably inﬂuence regulation of motor activity obtained

by the A R/A R blockade, especially due to evident

pramipexole, ropinirole, or dopamine itself, show a clear D₃R

preference over D Rs, prompting a therapeutic relevance of

73,74

D Rs.

Despite only moderate D R binding of MTLs, a

D R-mediated inﬂuence on in vivo activity cannot be ruled out.

In the most unfavorable case, as the designed AR/H R MTLs

,14

75−78

heterodimerization of these types of GPCRs.

However,

show rather structural overlap with D R antagonists,

their

also heterodimerization of H R with D R and D R receptors

desired motor-improving capacities might be counteracted via

D R blockade. Thus, design-out of any D R binding is

7,38

are described,

which further complicates the understanding

of therapeutic relevance of AR/H R MTLs in PD motor

mandatory in the future when designing AR/H R MTLs

symptoms. At D -like receptors (D R and D R), ST-2001 and

ST-1992 show low aﬃnity. However, investigations at PD-

using an arylindenopyrimidine core motif.

As especially MAO B participates in dopamine degradation

relevant D R and D R revealed moderate binding capabilities

after cellular reuptake, inhibitors of these enzymes represent

a suitable therapy for PD in early stages or as add-on therapy to

dopaminergic treatment (e.g., L-DOPA; 3,4-dihydroxypheny-

lalanine) in order to increase dopamine levels in the synaptic

with higher aﬃnities at D Rs for both MTLs. Still, selectivity

indices for ST-2001 and ST-1992 demonstrate 1−2 orders of

magnitude higher preference for the desired target receptors

80−82

(

Table 3). Overall, ST-2001 possesses more desirable

cleft.

Multitargeting MAO B inhibitors as innovative

selectivity with 10-fold and 100-fold higher for each target

strategy in PD therapy were previously described, such as our

receptor over D Rs and D Rs, respectively. While exact

mechanisms are hardly understood, there is no doubt about

recently reported series of structurally related indanone H R/

MAO B MTLs, which are synthetic precursor molecules or

involvement of mesolimbic D Rs in PD motor symptomology,

A₂_AR/MAO B dual acting phenylxanthine derivatives. Thus,

which are signiﬁcantly reduced by around 40% in PD

ST-2001 and ST-1992 were evaluated for potential MAO A/B

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Figure 3. (A−C) 3D docking model and (D−F) 2D schematic diagram of the docking model of ZM241385 (colored in green, A), preladenant

colored in magenta, B) and compound ST-1992 (colored in cyan, C) with the human A R. For modeling studies, the structure of the A R

(

cocrystallized with ZM241385 (PDB ID: 4EIY) was used, displayed in a cartoon representation (gray). Oxygen atoms are colored in red, nitrogen

atoms in blue, and hydrogen atoms in silver white.

inhibition, as described previously. At a concentration of 10

μM, negligible MAO inhibition (≤50%) could be found with

exception of ST-2001, where concentration-dependent eval-

uation at MAO B revealed an IC > 1000 nM. Thus, AR/H R

all calculated descriptors and properties were within the

expected threshold, suggesting a good oral absorption of these

MTLs. LE metrics of compounds 4 and 12 were calculated for

H R, A R, and A R. These MTLs showed acceptable LE

MTLs demonstrated weak MAO inhibition properties, whereas

a certain MAO B inhibition might have some beneﬁcial eﬀect

on synaptic dopamine availability.

Prior to in vivo testing, selected MTLs (ST-2001, 2, 6) were

tested for potential cytotoxic or antiproliferative eﬀects in

human neuroblastoma cells (SH-SY5Y). At a concentration of

values (LE ≥ 0.3) but with respective target speciﬁcity at a cost

of lipophilicity, as indicated by lipophilicity-corrected ligand

eﬃciency values, which were slightly outside the limits.

A R Docking Study. To validate results of in vitro

receptor binding assays, molecular docking studies were

performed using the C8-substituted arylindenopyrimidine

derivative ST-1992 ﬁtted into the A R binding pocket. ST-

000 nM, these compounds showed a negligible impact

(

remaining cell viability >75%) on cell viability after 24 h

1992, protonated at the piperidine nitrogen atom, modeled

incubation time (cf. Supporting Information ).

In order to evaluate drug-likeness of the synthetized

compounds, physicochemical properties and ligand e ﬃ ciency

into the crystal structure of the chimeric protein of A R-BRIL

104

(1.8 Å resolution),

was compared with ZM241385, a

structurally related A R inverse agonist with similar aﬃnity at

(

LE) metrics were calculated (cf. Supporting Information ).

this receptor subtype (K hA R = 1.6 nM and with

i 2A

Compounds 4 und 12 do not violate Lipinski’s rule of ﬁve as

preladenant (K hA R = 0.88 nM), both compounds

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Carbon atoms are colored in cyan, oxygen atoms in red, nitrogen atoms in blue, and hydrogen atoms on the amino group are shown in silver white.

Article

Figure 4. (A) 3D docking model and (B) 2D schematic diagram of compound ST-1992 using a homology model of the human H R (based on the

X-ray structure of the human H R, PDB ID: 3RZE). The homology model of the H R is displayed in a cartoon representation (light brown).

Figure 5. Time-course panels reporting the eﬀect of best MTLs ST-2001 (4) (A−C) and ST-1992 (12) (D−F) and amantadine on either overall

ALO or axial AIM scores induced by L-DOPA (6 mg/kg, i.p.). AIM scores are expressed as individual data and median. Control (★), amantadine

(

●) 40 mg/kg (i.p.), L-DOPA + 4 (*) 0.1 mg/kg (i.p.), L-DOPA + 4 (☆) 1 mg/kg (i.p.), L-DOPA + 12 (▲) 0.1 mg/kg (i.p.), and L-DOPA + 12

▼) 1 mg/kg (i.p.). ST-2001 and ST-1992 testing followed a within-subject escalation dose design (5 male rats per compound). The data were

analyzed by the Friedman test, followed by Dunn’s multiple comparison test; *p < 0.05 and **p < 0.01 vs control (0 mg/kg).

representing analogues of adenine derivatives. The 3D and

D images of binding showed that the exocyclic amino group

Asn253 (Figure 3). In addition, due to the π-conjugated

system, the selective A R antagonist was able to form π−π

of ZM241385 and preladenant allows not only interactions

with Glu169 but also hydrogen bonding interactions with

stacking interactions with Phe168. The arylindenopyrimidine

moiety of the AR/H R ligand ST-1992 appears to share these

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Figure 6. Sleep−wake patterns following p.o. administration of compound ST-1992 (12) at 1 mg/kg (A) and 2 mg/kg (B) showing cumulative

indices after 4 h (a) and time-dependent eﬀects on wake (W) (b), SWS (c), and PS phases (d) (*p < 0.05 vs. control (black bar, 0 mg/kg);

Dunnett’s t-test after signiﬁcant ANOVA; n = 5).

interactions and could therefore constitute a good alternative

as a central aromatic core. The ligands demonstrated a similar

orientation in the binding pocket, where the phenyl moiety of

ST-1992 is likely to occupy the subpocket formed by Leu85,

Met177, His250, Trp246, Leu249, and Ile274 like the furan

ring in ZM241385 and in preladenant, as well as the basic 3-

OHDA-induced degeneration of dopaminergic neurons,

animals were primed with 6 mg/kg L-DOPA + 15 mg/kg

benzerazide (i.p.) once per day until AIM severity stabilized, as

previously described. The eﬀect of AR/H R MTLs ST-2001

and ST-1992 at two doses (0.1 and 1 mg/kg) in the presence

of L-DOPA (6 mg/kg) was investigated (Figure 5). Compared

to AIMs of control rats (L-DOPA alone), coapplication of both

MTLs tends to decrease LID either dose-dependently, as

shown for ST-2001, or at least for one dose, that is, 0.1 mg/kg

of ST-1992. Signiﬁcant diﬀerences could be demonstrated for

ST-2001 (1 mg/kg) in axial AIM scores (Figure 5C). Less

pronounced e ﬀ ects on limb and orolingual AIM scores were

observed (cf. Supporting Information ), explaining the missing

statistical signiﬁcance in the overall axial/limb/orolingual

(ALO) AIM score. Noteworthily, variations of AIMs in

control groups (0 mg/kg, n = 5) hampered statistical analysis.

Positive control groups treated with amantadine (Figure 5E,F)

do not always reach the level of signiﬁcance, suggesting a

limited validity of respective data sets. Observed tendencies in

AIMs might point out a slight antidyskinetic eﬃcacy of AR/

piperidinopropyloxy H R fragment is postulated to occupy the

same pocket as the phenol moiety of ZM241385 and the

piperazine moiety of preladenant. Overall, molecular docking

studies conﬁrmed the experimentally determined receptor

aﬃnities by demonstrating that arylindenopyrimidine-based

ligands bearing the H R pharmacophore in the C8-position,

including MTL 6, ST-2001, and ST-1992 almost perfectly ﬁt

into the A R binding pocket.

H R Docking Study. The dual ligand ST-1992 was

additionally docked into a homology model of the human

H R. The predicted binding pose of ST-1992 in the H R

binding pocket indicated that the exocyclic amino group at the

arylindenopyrimidine moiety forms a strong hydrogen bond

interaction with Asp114 (Figure 4). The protonated piperidine

may form hydrogen bond interactions with the side chain of

Tyr374 and the subpocket formed by Trp110, His187, Tyr374,

Phe398, and Trp402. The phenyl substituent on the

aminopyrimidine ring is predicted to occupy another

subpocket formed by Cys118, Ala122, Phe207, Phe367, and

Trp371. The predicted binding pose indicates that there is

space to insert additional substituents on the phenyl ring (as in

compounds 3 and 9). This double substitution might lock the

arylindenopyrimidine ring system into the binding pocket of

H R MTLs ST-2001 and ST-1992. Combination of L-DOPA

(6 mg/kg) with mixed A R/A R antagonists tends to mildly

improve LID, whereas selective A R antagonists were shown

to have no signiﬁcant eﬀect on LID, that is, preladenant or

istradefylline. This might indicate a beneﬁt by additional A₁R

antagonism for the treatment of dyskinesia as hypothesized

89,90

previously.

Although cotreatment of A R antagonists with

L-DOPA could not prevent the occurrence of dyskinesia, the

incidence of LID might be reduced via lowering the eﬀective

8,91

the H R, favoring the interaction of its amino group with

dose of L-DOPA.

Recently, it was reported that

Asp114. However, such bulky compounds do not ﬁt into the

binding pocket of the ARs due to limited space and therefore

show low AR aﬃnity.

preladenant, in combination with the serotonin receptor

agonist eltoprazine, attenuated LID without exacerbating the

PD disability score in MPTP-treated macaques when

-OHDA-Lesioned Rat Model of L-DOPA-Induced

administrated in a subthreshold dose of L-DOPA.

Dyskinesia. To assess the eﬀect of AR/H R MTLs on L-

Moderate in vitro aﬃnity of the tested MTLs at hD Rs were

DOPA-induced dyskinesia (LID), ST-2001 and ST-1992 were

evaluated in the rat analogue of LID, that is, the L-DOPA-

treated 6-hydroxydopamine (6-OHDA)-lesioned rats scoring

the abnormal involuntary movements (AIMs). In brief, after 6-

observed, that is, ST-2001 with D R SI (calculated as K (A R/

A R/H R)/K (D R)) of 0.03−0.04 and ST-1992 with SI =

0.01−0.14. Moderate D R binding might have aﬀected AIMs

in rats treated with ST-2001 and ST-1992. Previous studies

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J. Med. Chem. 2021, 64, 8246−8262

Journal of Medicinal Chemistry

Article

demonstrated that development of LID is modulated by D Rs,

suitable but so far moderate therapeutic features of A R/A R/

1 2A

H R MTLs for the treatment of PD.

where D R-selective partial agonists or antagonists were able to

improve LID without inﬂuencing the antiparkinsonian eﬀect of

3−95

L-DOPA.

As A R antagonists as an adjunct treatment extend or

EXPERIMENTAL SECTION

General Information. Reagent-grade chemicals and solvents were

obtained from various producers (Sigma-Aldrich, VWR Chemicals,

Fisher Scientiﬁc, Panreac AppliChem, Alfa Aesar, Chemsolute) and

■

improve the therapeutic eﬃcacy of L-DOPA, the anti-

parkinsonian capacity of designed AR/H R MTLs should be

were used without further puriﬁcations (unless stated otherwise). H

evaluated in future studies. In multiple studies, A R-selective

NMR and C NMR spectra were recorded on a Bruker AMX

spectrometer (Bruker, Germany) at 300 and 75 MHz, respectively,

where CDCl or DMSO-d was used as a solvent. Tetramethylsilane

but also mixed A R/A R antagonists were shown to enhance

96,97

L-DOPA-mediated contralateral rotation.

DOPA in 6-OHDA-lesioned rat was potentiated by cotreat-

ment with the structurally related A R/A R antagonist JNJ-

The eﬀect of L-

was used as the standard, and chemical shifts are reported in parts per

million (ppm). Spin multiplicities are given as s (singlet), d (doublet),

dd (doublet of doublets), t (triplet), q (quintet), or m (multiplet)

with approximate coupling constants (J) in Hertz (Hz); number and

assignment of protons: aip, arylindenopyrimidine; ax, axial; eq,

equatorial; ph, phenyl; prop, propyl; pip, piperidine; pyr, pyrrolidine.

ESI-HRMS was performed on an amaZon speed (Bruker, Germany)

with an ionization source operating in the positive mode. Data are

0255293 (1 mg/kg) or the A R-selective preladenant (0.1

48,98

mg/kg, p.o.).

EEG and Sleep−Wake Study in Mice. The MTL ST-

992 was investigated for its wake-promoting activity using

electroencephalogram (EEG) and polysomnographic studies in

mice (Figure 6). While ST-1992 administrated orally had no

clear eﬀects at a dose of 1 mg/kg, it signiﬁcantly increased the

time spent awake at a dose of 2 mg/kg, eﬀects being at the

expense of slow-wave sleep (SWS), which was considerably

reduced. Paradoxical sleep (PS), on the other hand, was barely

aﬀected. These results are in good accordance, ﬁrst, with the

wake-promoting eﬀects of H3R inverse agonists such as

pitolisant and, second, with previous ﬁndings for the

structurally related potent A R/A R antagonist JNJ-

listed as mass number ([M + H ]) and relative intensity (%). HRMS

was run in ESI mode. Melting points (mp, uncorrected) were

determined on a M-564 Buchi melting-point apparatus (Buchi,

̈ ̈

Germany). Preparative column chromatography was performed on

silica gel 60 M, 0.04−0.063 mm (Macherey-Nagel, Germany) and

thin-layer chromatography was carried out using precoated silica gel

0 with ﬂuorescence indicator at UV 254 nm (Macherey-Nagel,

Germany). Compound purity ≥95% was determined by HPLC

Knauer, Germany) with a TC-C18 column, an acetonitrile/water

(0.1% TFA) mobile phase, and a ﬂow rate of 1 mL/min. Detailed

experimental procedures for the synthesis of precursors are described

in the Supporting Information . The data below are for the ﬁnal MTLs

(

0255293. ST-1992 is eﬀective at a dosage of 2 mg/kg

(

p.o.) on waking and slow-wave sleeping state, but not on PS.

These studies prove per oral bioavailability. However, obtained

drug eﬃciency metrics suggest moderate drug-likeness of ST-

−12.

General Procedure for the Synthesis of 3-Piperidino/

pyrrolidinopropyloxy Arylindenopyrimidine Derivatives (1−

1992, what possibly explains the diﬀerences in eﬀective dosing

). Compounds 1−4. Under a N atmosphere, the corresponding

compared to that of JNJ-40255293.

hydroxy arylindenopyrimidine derivatives (P14−P17, 1 equiv),

K CO (1.5 equiv), potassium iodide (1 equiv), and 1-(3-

The herein designed mixed AR/H R MTLs, a simultaneous

blockade of A R/A Rs and H Rs, might provide a modulation

chloropropyl)piperidine or -pyrrolidine (1.2) were dissolved in

dimethylformamide and heated to 80 °C. After 4 h, the mixture

was cooled down to room temperature and the inorganic salts were

ﬁltered oﬀ. The ﬁltrate was concentrated to dryness and resuspended

in dichloromethane. The crude product was taken up in methylene

chloride and 2 N NaOH solution. The organic phase was then washed

with saturated NaCl solution and water, dried over magnesium

sulfate, and concentrated under vacuum. Crude products were

puriﬁed with column chromatography (methylene chloride/NH₃

methanol = 9.5:0.5).

of the waking state in a synergistic manner. While the wake-

promoting capacity of H R antagonists/inverse agonists is

beyond any doubt, a certain role of A Rs in regulation of the

sleep-wake cycle is suggested in preclinical studies. In a study

with A R and A R knockout (KO) mice, it was conﬁrmed that

only A R-competent mice showed increased waking after

application of the mixed A R/A R antagonist caﬀeine. This

suggests that A Rs plays a negligible role in sleep−wake

00,101

regulation.

However, caﬀeine failed to improve EDS in

Compounds 5−6. A solution of guanidine hydrochloride (1

PD patients. On the contrary, recent studies reported that

the A R-selective antagonist istradefylline improved daytime

equiv) in ethanol was treated with powder NaOH (1 equiv). After 30

min, the free base was added to a solution of P12 and P13 (0.4 equiv)

in ethanol and the reaction mixture was reﬂuxed for 18 h. The crude

product was puriﬁed by column chromatography (methylene

sleepiness in PD patients. Thus, A R/A R/H R MTLs

might represent an innovative pharmacotherapy to simulta-

neously improve motor and nonmotor symptoms.

chloride/NH methanol = 97:3) and recrystallized from methanol

or ethanol.

-(4-(3-(Piperidin-1-yl)propoxy)phenyl)-5H-indeno[1,2-d]-

CONCLUSIONS

pyrimidin-2-amine (1). Yield: 69%; mp 166.1 °C. H NMR (300

■

MHz, DMSO-d ): δ 8.14−8.05 (m, 2H, ph -2′,6′H), 7.94−7.87 (d,

Within this study, multitargeting A R/A R/H R ligands were

described for the ﬁrst time by applying a knowledge-based

MTL design approach. The H R fragments 3-piperidinopro-

pyloxy or the pyrrolidino analogue were attached to an

(

H, J = 7.8, ph -3′H), 7.72−7.64 (d, 1H, J = 7.1, ph -5′H), 7.59−7.44

m, 2H, aip-6,7H), 7.13−7.03 (m, 2H, aip-8,9H), 6.60 (s, 2H, aip-

NH ), 4.12 (s, 2H, aip-5H ), 4.11−4.02 (t, 2H, J = 5.9, prop-1″H ),

2.44−2.37 (t, 2H, J = 6.9, prop-3″H ), 2.38−2.23 (m, 4H, pip-5″,

aromatic A R/A R arylindenopyrimidine scaﬀold in three

9″H

), 1.97−1.82 (q, 2H, J = 6.4, 1.9, prop-2″H

), 1.58−1.44 (m, 4H,

pip-6″, 8″H ), 1.43−1.30 (m, 2H, pip-7″H ). C NMR (75 MHz,

diﬀerent positions. Within this series of compounds, those

DMSO-d ): δ 162.2, 163.5, 159.9, 159.2, 145.8, 138.9, 130.0, 129.8,

bearing the H R pharmacophore such as R showed the most

6 C

27.1, 125.4, 121.0, 117.9, 114.2, 66.0, 55.0, 53.9, 34.3, 26.0, 25.4,

3.0. ESI-HRMS m/z: calcd for C H N O (MH ), 401.2341; found,

promising multitargeting features with aﬃnities in nanomolar

concentration ranges at the desired targets. With these

innovative in vitro proﬁles, ST-2001 (1 mg/kg, i.p.) and ST-

992 (2 mg/kg, p.o.) improved LID and increased wakefulness

in rodents, respectively. These initial studies demonstrate

01.2333. HPLC: 97.3%.

-Phenyl-7-(3-(pyrrolidin-1-yl)propoxy)-5H-indeno[1,2 d]-

pyrimidin-2-amine (2). Yield: 65%; mp 141.6 °C. H NMR (300

MHz, DMSO-d ): δ 8.10−8.02 (m, 2H, ph -2′,6′H), 7.84−7.77 (d,

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J. Med. Chem. 2021, 64, 8246−8262

Journal of Medicinal Chemistry

Article

(

H, J = 7.8, aip-9H), 7.58−7.45 (m, 3H, aip-6H, ph -3′,5′H), 7.26−

sulfate, and concentrated in vacuum. The obtained solid was puriﬁed

.20 (d, 1H, J = 7.2, ph -4′H), 7.08−7.0 (dd, 1H, J = 7.1, aip-8H),

by column chromatography (methylene chloride/NH methanol =

.58 (s, 2H, aip-NH ), 4.16−4.07 (t, 2H, J = 6.3, prop-1″H ), 4.01 (s,

95:5) and washed one night in hexane.

H, aip-5H ), 2.62−2.53 (t, 2H, J = 6.7, prop-3″H ), 2.49−2.37 (m,

2-Amino-4-(4-(3-(piperidin-1-yl)propoxy)phenyl)-5H-indeno[1,2-

H, pyr-5″, 8″H ), 1.99−1.85 (q, 2H, J = 5.6, prop-2″H ), 1.76−1.59

d]pyrimidin-5-one (7). Yield: 73%; mp 100.5 °C. H NMR (300

m, 4H, pyr-6″, 7″H ). C NMR (75 MHz, DMSO-d ): δ 168.9,

MHz, DMSO-d

aip-NH

aip-8,9H), 4.18−4.04 (t, 2H, J = 6.4, prop-1″H

= 5.8, prop-3″H ), 2.38−2.20 (m, 4H, pip-5″, 9″H

2H, J = 6.0, prop-2″H ), 1.60−1.45 (m, 4H, pip-6″, 8″H

): δ

8.18−8.08 (m, 2H, ph -2′,6′H), 7.93 (s, 2H,

63.6, 160.9, 158.8, 148.3, 138.0, 131.4, 129.5, 128.3, 128.1, 122.2,

), 7.83−7.61 (m, 4H, aip-6,7H, ph -3′,5′), 7.10−7.0 (m, 2H,

), 2.48−2.39 (t, 2H, J

), 1.96−1.82 (q,

), 1.44−1.32

187.6, 175.8,

18.1, 114.3, 110.9, 66.1, 55.0, 54.1, 34.09, 28.09, 23.07. ESI-HRMS

m/z: calcd for C H N O (MH ), 387.2185; found, 387.2177.

HPLC: 96.3%.

-(3-(Piperidin-1-yl)propoxy)-4-(4-(3-(piperidin-1-yl)propoxy)-

phenyl)-5H-indeno[1,2-d]pyrimidin-2-amine (3). Yield: 66%; mp

(m, 2H, pip-7″H ). C NMR (75 MHz, DMSO-d ): δ

2 6 C

164.7, 164.0, 161.0, 139.4, 136.2, 134.0, 132.8, 131.4, 127.8, 122.9,

65.3 °C. H NMR (300 MHz, DMSO-d ): δ 8.16−8.05 (m, 2H,

120.7, 113.4, 110.1, 66.1, 55.0, 54.0, 26.2, 25.5, 24.1. ESI-HRMS m/z:

ph -2′,6′H), 7.60−7.51 (m, 1H, aip-9H), 7.42−7.32 (d, 1H, J = 7.4,

calcd for C H N O (MH ), 415.2134; found, 415.2132. HPLC:

aip-6H), 7.18−7.03 (m, 3H, ph -3′,5′H, aip-8H), 6.58 (s, 2H, aip-

96.3%.

NH ), 4.18−4.05 (t, 4H, J = 6.5, prop/prop -1″H ), 4.03 (s, 2H, aip-

2-Amino-7-(3-(pyrrolidin-1-yl)propoxy)-4-phenyl-5H-indeno[1,2-

H ), 2.48−2.23 (m, 12H, prop/prop -3″H , pip/pip -5″, 9″H ),

d]pyrimidin-6-one (8). Yield: 76%; mp 145.3 °C. H NMR (300

.83−1.61 (m, 4H, prop/prop -2″H ), 1.44−1.30 (m, 12H, pip/pip -

MHz, DMSO-d

aip-NH

): δ

= 8.05−7.96 (m, 2H, ph -2′,6′H), 7.94 (s, 2H,

″, 7″, 8″H ). C NMR (75 MHz, CDCl ): δ 169.7, 163.0, 160.3,

), 7.73−7.64 (d, 1H, J = 7.7, aip-9H), 7.60.7.44 (m, 3H, aip-

60.2, 158.7, 140.0, 138.1, 129.9, 128.5, 124.9, 119.0, 114.4, 106.3,

6H, ph -3′,5′H), 7.24−7.18 (dd, 1H, J = 7.2, ph -4′H), 7.18−7.11 (d,

1H, J = 7.2, aip-8H), 4.22−4.06 (t, 2H, J = 6.4, prop-1″H ), 2.64−

2.53 (t, 2H, J = 5.3, prop-3″H ), 2.49−2.41 (m, 4H, pyr-5″, 8″H ),

2.01−1.85 (q, 2H, J = 6.7, prop-2″H ), 1.79−1.58 (m, 4H, pyr-6″,

). C NMR (75 MHz, DMSO-d ): δ 187.0, 175.7, 164.9,

05.6, 66.6, 55.8, 54.4, 54.3, 34.1, 26.1, 25.2, 23.9. ESI-HRMS m/z:

calcd for C H N O (M2H ), 543.3573; found, 543.3574. HPLC:

96.7%.

-Phenyl-8-(3-(pyrrolidin-1-yl)propoxy)-5H-indeno[1,2-d]-

7″H

pyrimidin-2-amine (ST2001). Yield: 70%; mp 140.5 °C. H NMR

164.3, 162.7, 138.7, 135.7, 131.3, 130.6, 129.3, 127.6, 122.5, 119.6,

(

300 MHz, DMSO-d ): δ 8.14−8.04 (m, 2H, ph -2′,6′H), 7.60−

110.6, 108.8, 66.6, 53.5, 51.9, 27.8, 23.0. ESI-HRMS m/z: calcd for

.47 (m, 4H, aip-6,9H, ph -3′,5′H), 7.42−7.36 (d, 1H, J = 7.4, ph -

C H N O (MH ), 401.1978; found, 401.1976. HPLC: 97.5%.

′H), 7.14−7.06 (dd, 1H, J = 7.1, aip-7H), 6.66 (s, 2H, aip-NH ),

2-Amino-7-(3-(piperidin-1-yl)propoxy)-4-(4-(3-(piperidin-1-yl)-

.16−4.08 (t, 2H, J = 6.4, prop-1″H ), 4.02 (s, 2H, aip-5H ), 2.65−

propoxy)phenyl)-5H-indeno[1,2-d]pyrimidin-5-one (9). Yield: 62%;

.55 (t, 2H, J = 6.8, prop-3″H ), 2.55−2.39 (m, 4H, pyr-5″, 8″H ),

mp 116.5 °C. H NMR (300 MHz, DMSO-d ): δ 8.16−8.06 (m,

6 H

.01−1.85 (q, 2H, J = 5.9, prop-2″H ), 1.75−1.61 (m, 4H, pyr-6″,

2H, ph -2′,6′H), 7.80 (s, 1H, aip-NH ), 7.63−7.55 (d, 1H, J = 7.6,

″H ). C NMR (75 MHz, DMSO-d ): δ 168.9, 163.6, 159.6,

aip-9H), 7.25−7.19 (d, 1H, J = 7.2, aip-6H), 7.15−7.07 (dd, 1H, J =

58.2, 140.1, 138.0, 137.8, 129.7, 128.4, 128.2, 126.2, 119.6, 118.4,

7.1, aip-8H), 7.06−6.96 (m, 2H, ph -3′,5′H), 4.24−4.12 (t, 2H, J =

04.9, 66.1, 53.5), 52.1, 33.3, 28.0, 23.0. ESI-HRMS m/z: calcd for

C H N O (MH ), 387.2185; found, 387.2186. HPLC: 97.3%.

6.5, prop -1″H

), 4.12−4.00 (t, 2H, J = 6.5, prop-1″H ), 2.47−2.38

(m, 4H, prop/prop -3″H

2.02−1.81 (m, 4H, prop/prop -2″H

6″, 8″H ), 1.43−1.27 (m, 4H, pip/pip -7H ″). C NMR (75 MHz,

), 2.38−2.17 (m, 8H, pip/pip -5″, 9″H

-Phenyl-7-(3-(piperidin-1-yl)propoxy)-5H-indeno[1,2-d]-

), 1.58−1.44 (m, 8H, pip/pip -

1 13

pyrimidin-2-amine (5). Yield: 50%; mp 176.3 °C. H NMR (300

MHz, DMSO-d ): δ 8.17−8.08 (dd, 2H, J = 8.1, ph -2′,6′H), 7.90−

DMSO-d ): δ 187.0, 174.9, 164.6, 163.6, 163.4, 160.9, 142.1, 131.5,

6 C

.82 (d, 1H, J = 7.8, aip-9H), 7.66−7.51 (m, 3H, aip-6H, ph -3′,5′H),

128.5, 127.8, 124.9, 118.2, 113.4, 111.0, 106.2, 67.1, 66.0, 55.0, 54.8,

.31−7.24 (d, 1H, J = 7.3, ph -4′H), 7.15−7.05 (dd, 1H, J = 7.1, aip-

54.0, 26.1, 25.9, 25.4, 24.0. ESI-HRMS m/z: calcd for C H N O

(MH ), 556.3288; found, 556.3279. HPLC: 97.2%.

H), 6.63 (s, 2H, aip-NH ), 4.20−4.13 (t, 2H, J = 6.2, prop-1″H ),

.12 (s, 2H, aip-5H ), 2.51−2.42 (t, 2H, J = 6.9, prop-3″H ), 2.41−

2-Amino-8-(3-(pyrrolidin-1-yl)propoxy)-4-phenyl-5H-indeno[1,2-

.28 (m, 4H, pip-5″, 9″H ), 2.02−1.88 (q, 2H, J = 5.9, prop-2″H ),

d]pyrimidin-5-one (10). Yield: 75%; mp 144.5 °C. H NMR (300

.61−1.49 (m, 4H, pip-6″, 8″H ), 1.48−1.37 (m, 2H, pip-7″H ).

MHz, DMSO-d

aip-NH

): δ

= 8.05−7.97 (m, 2H, ph -2′, 6′H), 7.90 (s, 2H,

NMR (75 MHz, DMSO-d ): δ 168.9, 163.6, 160.9, 158.8, 148.3,

), 7.62−7.56 (d, 1H, J = 7.6, aip-6H), 7.56−7.45 (m, 3H, aip-

38.0, 131.3, 129.5, 128.3, 128.1, 122.2, 118.1, 114.3, 110.9, 66.2,

5.0, 54.0, 34.0, 26.2, 25.5, 24.1. ESI-HRMS m/z: calcd for

C H N O (M2H ), 402.2420; found, 402.2412. HPLC: 96.5%.

-Phenyl-8-(3-(piperidin-1-yl)propoxy-5H-indeno[1,2-d]-

pyrimidin-2-amine (6). Yield: 44%; mp 142.1 °C. H NMR (300

9H, ph -3′, 5′H), 7.27−7.22 (d, 1H, J = 7.2, ph -4′H), 7.16−7.09 (dd,

1H, J = 7.1, aip-7H), 4.25−4.11 (t, 2H, J = 6.4, prop-1″H ), 2.59−

2.51 (t, 2H, J = 6.8, prop-3″H ), 2.48−2.38 (m, 4H, pyr-5″, 8″H ),

1.99−1.86 (q, 2H, J = 5,8, prop-2″H ), 1.75−1.61 (m, 4H, pyr-6″,

7″H ). C NMR (75 MHz, DMSO-d ): δ 186.5, 174.8, 164.8,

MHz, DMSO-d ): δ 8.15−8.03 (m, 2H, ph -2′,6′H), 7.61−7.47 (m,

164.0, 163.6, 142.1, 135.6, 130.6, 129.5, 128.6, 128.2, 124.9, 118.3,

H, aip-6,9H, ph -3′,5′H), 7.41−7.35 (d, 1H, J = 7.4, ph -4′H), 7.16−

111.5, 106.3, 66.8, 53.5, 52.0, 29.9, 23.0. ESI-HRMS m/z: calcd for

.05 (dd, 1H, J = 7.1, aip-7H), 6.66 (s, 2H, aip-NH ), 4.12−4.05 (t,

C H N O (MH ), 401.1978; found, 401.1972. HPLC: 97.1%.

24 24

H, J = 6.4, prop-1″H ), 4.03 (s, 2H, aip-5H ), 2.45−2.38 (t, 2H, J =

2-Amino-7-(3-(piperidin-1-yl)propoxy)-4-phenyl-5H-indeno[1,2-

.8, prop-3″H ), 2,36−2.25 (m, 4H, pip-5″, 9″H ), 1.97−1.84 (q, 2H,

d]pyrimidin-6-one (11). Yield: 79%; mp 182.2 °C. H NMR (300

J = 5.8, prop-2″H ), 1.57−1.43 (m, 4H, pip-6″, 8″H ), 1.43−1.31 (m,

MHz, DMSO-d

aip-NH

): δ

= 8.02−7.96 (m, 2H, ph -2′, 6′H), 7.96 (s, 2H,

H, pip-7″H ). C NMR (75 MHz, DMSO-d ): δ 168.9, 163.6,

), 7.71−7.65 (d, 1H, J = 7.7, aip-9H), 7.58−7.45 (m, 3H, aip-

59.6, 158.0, 140.1, 137.9, 137.8, 129.7, 128.4, 128.2, 126.2, 119.6,

6H, ph -3′, 5′H), 7.23−7.16 (dd, 1H, J = 7.2, ph -4′H), 7.16−7.12 (d,

1H, J = 7.2, aip-8H), 4.18−4.07 (t, 2H, J = 6.4, prop-1″H ), 2.47−

2.25 (m, 6H, prop-3″H , pip-5″, 9″H ), 1.96−1.82 (q, 2H, J = 5.8,

prop-2″H ), 1.55−1.44 (m, 4H, pip-6″, 8″H ), 1.43−1.31 (m, 2H,

): δ 187.0, 175.7, 164.9,

18.4, 104.9, 66.2, 55.1, 54.0, 33.3, 26.2, 25.6, 24.1. ESI-HRMS m/z:

calcd for C H N O (MH ), 401.2341; found, 401.2339. HPLC:

7.1%.

General Procedure for the Synthesis of 3-Piperidino/

Pyrrolidinopropyloxy Carbonyl Arylindenopyrimidine Deriv-

Derivatives 1−6 (1 equiv) and sodium hydroxide

1.2 equiv) were suspended in dimethylformamide. The resulting

pip-7″H

). C NMR (75 MHz, DMSO-d

164.3, 162.7, 138.7, 135.7, 131.3, 130.6, 129.3, 127.6, 122.4, 119.6,

42,43

atives (7−12).

(

110.6, 108.8, 66.7, 54.8, 53.9, 25.9, 25.4, 23.9. ESI-HRMS m/z: calcd

for C H N O (MH ), 415.2134; found, 415.2126. HPLC: 96.5%.

mixture was heated to 80 °C and air was bubbled through the solution

using a steel needle. After 4 h, the reaction was cooled to room

temperature, and the crude product was taken up in dichloromethane

and sodium carbonate. The organic phase was then washed several

times with saturated NaCl solution and water, dried over magnesium

2-Amino-8-(3-(piperidin-1-yl)propoxy)-4-phenyl-5H-indeno[1,2-

d]pyrimidin-5-one (ST-1992). Yield: 74%; mp 176.6 °C. H NMR

(300 MHz, DMSO-d ): δ 8.05−7.97 (m, 2H, ph -2, 6H), 7.96−7.81

(s, 2H, pyrim-NH ), 7.62−7.57 (d, 1H, J = 7.6, ph-6H), 7.57−7.45

(m, 3H, ph-9H, ph -3, 5H), 7.26−7.22 (d, 1H, J = 7.2, ph -4H),

256

J. Med. Chem. 2021, 64, 8246−8262

Journal of Medicinal Chemistry

Article

.16−7.09 (dd, 1H, J = 7.1, ph-7H), 4.21−4.11 (t, 2H, J = 6.4, prop-

cAMP Accumulation Experiments. cAMP experiments in

recombinant Chinese hamster ovary (CHO) cells expressing the

human adenosine A R (CHO-A cells) were conducted according

to De Filippo et al. with modiﬁcations. Diﬀerent dilutions of the

antagonists were added to the CHO-A₂_Acells and incubated for 20

H ), 2.47−2.38 (t, 2H, J = 6.7, prop-3H ), 2.37−2.23 (m, 4H, pip-2,

H ), 1.98−1.84 (q, 2H, J = 5.6, prop-2H ), 1.57−1.45 (m, 4H, pip-3,

109

H ), 1.44−1.31 (m, 2H, pip-4H ). C NMR (75 MHz, DMSO-d ):

δ 186.5, 174.8, 164.8, 164.1, 163.6, 142.1, 135.6, 130.6, 129.5, 128.6,

27.6, 125.0, 118.3, 111.5, 106.3, 66.0, 54.8, 54.0, 26.0, 25.5, 24.0.

min at 37 °C and 5% CO . Then, the agonist NECA was added and

ESI-HRMS m/z: calcd for C H N O (MH ), 415.2134; found,

incubated for 15 min under the same conditions. The supernatant was

removed, and 500 μL of hot lysis buﬀer (90 °C, 4 mM EDTA, 0.01%

Triton X-100, pH 7.4) was added. After homogenization, the cAMP

amount in the lysates was determined by radioligand competition

binding experiments. Three independent experiments were performed

each in duplicates. The cAMP amount in the lysates was calculated

based on a calibration curve of known cAMP concentrations and

normalized to the eﬀect induced by 0.3 μM of the A R agonist

15.2129. HPLC = 97.0%.

Docking Studies. The X-ray structure of the human A R with

the antagonist ZM241385 was obtained from the Protein Data

Bank.

apocytochrome bRIL was removed, and the receptor was prepared

using the protein preparation tool implemented in Molecular

Operating Environment (MOE 2018.01). A homology model of

103

From the downloaded crystal structure, the cocrystallized

104

the H R was downloaded from the GPCR database (https://www.

NECA (∼EC ) set at 100%. Data analysis and statistics (one-way

105

gpcrdb.org).

The model was generated using a multitemplate

ANOVA followed by Dunnett’s post hoc test) were performed with

GraphPad Prism version 8.0.2.

approach with the human H R (PDB ID: 3RZE) as the main

template, showing a sequence similarity of 38%. For both receptors,

the hydrogen atoms were assigned according to the Protonate3D

module in MOE 2018.01. The prepared receptors were applied for

ﬂexible ligand docking using AutoDock 4.2, and during the docking

simulations, the ligands were fully ﬂexible and the residues of the

Oﬀ-Target Aﬃnities at Dopamine and Histamine Receptor

Subtypes. Radioligand depletion studies using crude membrane

preparations of either HEK-293 (D

(D2SR, D R, and H R) stably expressing or Sf9 cells (H

R and D

R) and CHO cells

R) transiently

expressing the human recombinant receptors were performed as

106

receptors were treated as rigid.₁The atomic partial charges were

described recently. Oﬀ-target aﬃnities were determined using at

added using AutoDockTools.

The three-dimensional energy

least ﬁve diﬀerent concentrations of test ligands.

scoring grids for a box of 60 × 70 × 60 points with a grid spacing

of 0.375 Å were computed, and the grids were centered based on the

cocrystallized ligand ZM241385 in case of A R. Fifty independent

MAO A/B Inhibition. Monoamine oxidase (MAO) A/B inhibition

was assessed in a continuous spectrophotometric assay as described

previously, using human recombinant MAO A (900 units/mL) or

MAO B (375 units/mL) and Kynuramine (Sigma-Aldrich, St. Louis,

docking simulations were calculated using the varCPSO-ls algorithm

from PSO@Autodock implemented in AutoDock 4.2.

The

MO) as the substrate for both isoforms. In brief, test ligands were

parameters of the varCPSO-ls algorithm, the cognitive and social

coeﬃcients c1 and c2, were set to 6.05 with 60 individual particles as

the swarm size, and the simulations were terminated after 50,000

evaluation steps. All other parameters of the algorithm were set at

their default values. The possible binding modes of the compounds

were explored by visual inspection of the poses resulting from docking

simulations.

incubated with Kynuramine (2 × K ) and MAO in a total volume of

0.1 mL potassium phosphate buﬀer (50 mM). Product formation was

observed spectrophotometrically at 316 nm over a period of at least

−

30 min. Initial enzyme conversion rates (as mAbs min ) were plotted

relative to control (no inhibitor) or, in case of IC50 determinations,

versus -log concentrations of test compounds and ﬁtted by nonlinear

regression “log (inhibitor) versus response (three parameters)”.

In Vivo Characterization. 6-OHDA PD Model. To produce

neurodegeneration of dopaminergic neurons, rats received a unilateral

6-OHDA injection in the medial forebrain (MFB) by stereotactic

delivery. Five male Sprague−Dawley rats (Janvier, France) weighed

125−150 g before surgery and were housed in a temperature-

controlled room under a 12 h light/dark cycle, with free access to

food and water. All experiments were carried out in accordance with

the European Committee Council Directive of 24 November 1986

(86/609/EEC) and were approved by the Animal Care and Use

Committee (Bordeaux) under no 5012099-A.

In Vitro Characterization. Radioligand Competition Binding

Studies. The aﬃnities (K values) of the designed compounds were

determined in radioligand receptor binding studies. Stock solutions of

the test compounds were prepared in dimethyl sulfoxide (DMSO).

Membrane preparations expressing the respective receptor were

incubated with the radioligand and competitor. Membranes with the

bound radioligand were harvested by ﬁltration and thereby separated

from the free radioligand and subsequently analyzed by liquid

scintillation counting. Speciﬁc counts per minute (cpm) were plotted

versus −log concentrations of test ligands and analyzed via nonlinear

regression ﬁt using GraphPad Prism (version 4 or 6, San Diego, CA,

Buprenorphine (0.05 mg/kg; 0.03 mg/mL) was given as an

analgesic before and after the surgery. Thirty minutes prior to surgery,

male Sprague−Dawley rats received the noradrenaline uptake

inhibitor desipramine (25 mg/kg; i.p.) to protect noradrenergic

neurons. Each animal was placed in an anesthetic chamber supplied

with a continuous ﬂow of oxygen (1.5 L/min) and 5% isoﬂurane.

Following loss of consciousness, animals were placed in a stereotactic

frame (Kopf), and 6-OHDA (5 mg/mL dissolved in sterile 0.1%

ascorbic acid) was injected into the right MFB bundle at 0.4 μL/min

over a 10 min period using a Hamilton 1701N syringe (30Ga/15

mm/PST3) coupled to an automatic injector UMP-3 linked to a

Micro-4 controller (WPI). The injection site was located according to

USA). The resulting IC₅₀values were transformed to K values using

the Cheng−Prusoﬀ equation based on previously described K

values. For histamine receptor binding assays, statistical analysis

was applied on −log K values of at least three independent

experiments, each performed at least in duplicate, while mean values

and conﬁdence intervals (CI) (95%) were converted to nanomolar

concentrations.

Adenosine Receptor Aﬃnity. Crude membrane preparations of

CHO cells stably expressing human recombinant ARs were obtained

as described previously. Radioligand competition binding studies

were performed as previously described using [ H]CCPA (0.5 nM,

K_D= 0.61 nM), [ H]MSX-2 (1 nM, K = 8 nM), [ H]PSB-603

the following coordinates relative to bregma: −2.2 mm rostral, 1.6

103

(

0.3 nM, K = 0.41 nM), and [ H]PSB-11 (0.5 nM, K = 4.9 nM)

mm lateral and 8.5 mm below the skull.

Two weeks postlesion,

for determination of A R, A R, A R, and A R aﬃnities, respectively.

At least three separate experiments were performed. Data were

expressed as means ± standard error of the mean (SEM).

Histamine H₃Receptor Aﬃnity. Radioligand depletion studies

using crude membrane preparations of HEK-293 cells stably

prior to L-DOPA treatment, rats were injected subcutaneously with

0.05 mg/kg apomorphine (dissolved in 0.9% saline) and placed in

rotometer bowls, where movements were observed for 15 min to

assess the extent of the lesion. Animals responding with rotations were

included in the subsequent study. After 3 weeks of recovery, animals

were primed by administration of 6 mg/kg L-DOPA + 15 mg/kg

benzerazide (i.p.) once a day until AIM severity stabilization (up to 21

consecutive days). ST-2001 and ST-1992 at two concentrations (0.1,

1 mg/kg i.p.) were administered just before the L-DOPA injection,

whereas amantadine (40 mg/kg, s.c.) was administered 100 min

expressing the human recombinant H R were performed as described

43,71

recently.

with test compounds (10 to 10 M) and [ H]N -methylhistamine

2 nM, K_D= 3.08 nM, PerkinElmer, Waltham, MA) in the binding

buﬀer (total volume 0.2 mL) for 90 min under continuous shaking.

In brief, crude membrane preparations were incubated

−

−4

(

257

J. Med. Chem. 2021, 64, 8246−8262

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

before L-DOPA injection. The eﬀects of the test items in combination

with L-DOPA (6 mg/kg, i.p.) were evaluated via AIM scores. Brieﬂy,

animals were placed in clear Perspex boxes (22 cm × 34 cm × 20 cm).

Each rat was observed for 1 min at 30 min intervals following the L-

DOPA administration over a 2 h time period. Three subtypes of AIMs

were assessed including axial (A), limb (L), and orolingual (O). The

severity of AIMs was scored between 1 and 4 depending on the

duration of AIMs throughout the observational minute [(1) present

for less than 30 s, (2) present for more than 30 s, (3) present

throughout the minute but suppressed by external stimuli, (4) present

throughout the minute but not suppressible by external stimuli].

Statistical analysis was performed using Prism 7.0 software (GraphPad

Inc., La Jolla, California, USA). AIM data were subjected to a

nonparametric one-way analysis of variance (ANOVA) using

Friedman’s test with repeated measures for the overall treatment

comparison, followed by Dunn’s multiple comparison test for the

pairwise comparisons (vs L-DOPA + vehicle), where p < 0.05 was

considered as statistically signiﬁcant.

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00914.

■

Spectroscopic and analytical data; general LC−MS

analytics; general synthesis, ﬂuorescence characterization

of compound 12 (ST-1992); drug-likeness of synthe-

tized MTLs 4 (ST-2001) and 12 (ST-1992); cell

viability of human neuroblastoma cells treated with

MTLs 2, 4 (ST-2001) , and 6; in vitro radioligand

binding experiments for compounds 4 (ST-2001) and

12 (ST-1992); in vivo studies of compound 4 (ST-

2001); additional in vivo evaluation of compound 12

(ST-1992); and experimental part with references

(PDF)

Molecular formula strings (CSV)

Modeling structure of A R with preladenant (PDB)

Sleep Studies in Mice. All experiments followed the European

Ethics Community Directive (86/609/EEC), and every eﬀort was

made to minimize the number of animals used and their suﬀering.

Modeling structure of A R with ST-1992 (PDB)

110

The general protocol

of chronic sleep−wake recordings and

AUTHOR INFORMATION

Corresponding Author

■

pharmacological studies in mice was approved by the Ethic

Committee on Animal Experimentation of Claude Bernard University

Holger Stark − Institute of Pharmaceutical and Medicinal

Chemistry, Heinrich Heine University Duesseldorf, 40225

Duesseldorf, Germany; orcid.org/0000-0003-3336-1710;

Phone: +49 211 8110478; Email: stark@hhu.de

(no DR2013-50).

At the age of 11−14 weeks, male C57BL/6 mice weighing 25−30

mg were chronically implanted with four cortical electrodes (gold-

plated tinned copper wire, Ø = 0.4 mm; Filotex, Draveil, France) and

three muscle electrodes (ﬂuorocarbon-coated gold-plated stainless

steel wire, Ø = 0.03 mm; Cooner Wire, Chatsworth, CA), under deep

isoﬂurane anesthesia (2% in oxygen with gas anesthesia machine,

TEM, France), in order to record the EEG and electromyogram

Authors

Stefanie Hagenow − Institute of Pharmaceutical and

Medicinal Chemistry, Heinrich Heine University Duesseldorf,

40225 Duesseldorf, Germany

111

(

EMG) and to monitor the sleep−wake cycle. All electrodes were

Anna Aﬃni − Institute of Pharmaceutical and Medicinal

Chemistry, Heinrich Heine University Duesseldorf, 40225

Duesseldorf, Germany

Elsa Y. Pioli − Motac Neuroscience Limited, SK10 4TF

Macclesﬁeld, U.K.

Sonja Hinz − PharmaCenter Bonn, Pharmaceutical Institute,

Pharmaceutical & Medicinal Chemistry, University of Bonn,

previously soldered to a multichannel electrical connector, and each

was separately insulated with a covering of heat-shrinkable polyoleﬁn/

polyester tubing. The cortical electrodes were inserted into the dura

through two pairs of holes (Ø = 0.5 mm) made in the skull, located in

the frontal (1 mm lateral and anterior to the bregma) and parietal (1

mm lateral to the midline at the midpoint between the bregma and

lambda) cortices. EMG electrodes were inserted into the neck muscle.

Finally, the electrode assembly was anchored and aﬃxed to the skull

with Super-Bond (Sun Medical Co., Shiga, Japan) and dental cement.

After surgery, the animals were housed individually in transparent

barrels (Ø = 20 cm, height 30 cm, with open ﬁeld) placed in an

insulated, sound-proof recording room maintained at an ambient

temperature of 23 ± 1 °C and on a 12 h light/dark cycle (light on at 7

a.m.), food and water being available ad libitum. After a recovery

period of 2 weeks, the mice were habituated to the recording cable for

one more week before starting polysomnographic recording.

3121 Bonn, Germany; Institute of Pharmacology and

Toxicology, School of Medicine, University of Witten/

Herdecke, Center for Biomedical Education and Research

(

ZBAF), Faculty of Health, 58448 Witten, Germany

Yan Zhao − Laboratory of Integrative Physiology of the Brain

Arousal Systems, Lyon Neuroscience Research Center,

INSERM UI028, CNRS UMR 5292, Claude Bernard

University, 69373 Lyon, France

Gregory Porras − Motac Neuroscience Limited, SK10 4TF

Macclesﬁeld, U.K.

Vigneshwaran Namasivayam − PharmaCenter Bonn,

Pharmaceutical Institute, Pharmaceutical & Medicinal

Chemistry, University of Bonn, 53121 Bonn, Germany;

orcid.org/0000-0003-3031-3377

After a 2-day baseline recording, mice were subjected to the cortical

EEG and sleep−wake recordings following oral administrations of

placebo or ST-1992 (1 and 2 mg/kg) with a washout of 7 days.

Polysomnographic recordings subsequent to administration were for

12 h. Cortical EEG (ipsilateral frontoparietal leads) and EMG signals

Christa E. Muller − PharmaCenter Bonn, Pharmaceutical

were ampliﬁed and digitized with a resolution of 256 Hz using AM

system ampliﬁers assisted by SleepSign software for Animal (Kissei).

With the assistance of EEG spectral analysis, polysomnographic

records were visually scored by 10 s epochs for W, SWS, and

Institute, Pharmaceutical & Medicinal Chemistry, University

of Bonn, 53121 Bonn, Germany; orcid.org/0000-0002-

0013-6624

110,112

Jian-Sheng Lin − Laboratory of Integrative Physiology of the

Brain Arousal Systems, Lyon Neuroscience Research Center,

INSERM UI028, CNRS UMR 5292, Claude Bernard

University, 69373 Lyon, France

Erwan Bezard − Motac Neuroscience Limited, SK10 4TF

Macclesﬁeld, U.K.; Univ. de Bordeaux, Institut des Maladies

Neurodégénératives, UMR 5293, 33000 Bordeaux, France;

PS.

The EEG and sleep-wake parameters obtained after all admin-

istrations were quantiﬁed and compared between the experimental

conditions. Each mouse served as its own control. One way and

multiple factorial ANOVA and the post hoc Student’s t-test (two

tailed) were used to compare and evaluate any obtained diﬀerence

110−112

using Excel or/and Statgrafy softwares.

258

J. Med. Chem. 2021, 64, 8246−8262

Journal of Medicinal Chemistry

(7) Soliman, A. M.; Fathalla, A. M.; Moustafa, A. A. Adenosine role

Article

CNRS, Institut des Maladies Neurodégénératives, UMR

in brain functions: Pathophysiological influence on Parkinson ’s

disease and other brain disorders. Pharmacol. Rep. 2018 , 70 , 661 −667.

293, 33000 Bordeaux, France

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00914

8) Pinna, A.; Serra, M.; Morelli, M.; Simola, N. Role of adenosine

A2A receptors in motor control: Relevance to Parkinson ’s disease and

dyskinesia. J. Neural. Transm. 2018 , 125 , 1273 −1286.

Author Contributions

(9) Burnstock, G. Purinergic signalling: From discovery to current

S.H. and A.A. have contributed equally to this work. A.A. and

S.H. wrote the main manuscript. A.A. and H.S. performed the

synthesis and analyses of the compounds. S.H. and H.S.

developments. Exp. Physiol. 2014, 99, 16−34.

(10) Glaser, T.; Andrejew, R.; Oliveira-Giacomelli, A.; Ribeiro, D.

E.; Bonfim Marques, L.; Ye, Q.; Ren, W.-J.; Semyanov, A.; Illes, P.;

Tang, Y.; Ulrich, H. Purinergic receptors in basal ganglia diseases:

Shared molecular mechanisms between Huntington ’s and Parkinson ’s

disease. Neurosci. Bull. 2020, 36, 1299−1314.

performed H R aﬃnity studies, selectivity studies (dopamine

receptors, histamine receptors, and monoamine oxidases), and

cytotoxicity assays. S.H. and C.E.M. investigated AR aﬃnities

and functional properties. V.N. performed molecular docking

studies supervised by C.E.M. E.B., E.Y.P., G.P., as well as Y.Z.

and J.-S.L. conducted in vivo EEG and sleep studies,

respectively. H.S. designed and supervised the project and

revised the manuscript. All authors contributed to writing the

manuscript and approved the ﬁnal version.

(11) Smith, Y.; Wichmann, T.; Factor, S. A.; DeLong, M. R.

Parkinson ’s disease therapeutics: New developments and challenges

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Funding

This work was kindly supported by COST Actions CM1103,

CA15135, CA18133, and CA18240, as well as DFG INST

(13) Ferré, S.; Fuxe, K.; B. Fredholm, B.; Morelli, M.; Popoli, P.

Adenosine-dopamine receptor-receptor interactions as an integrative

mechanism in the basal ganglia. Trends Neurosci. 1997, 20 , 482 −487.

208/664e1 FUGG (Germany) and Inserm U 628.

(14) Fuxe, K.; Marcellino, D.; Genedani, S.; Agnati, L. Adenosine

Notes

A2A receptors, dopamine D2 receptors and their interactions in

The authors declare no competing ﬁnancial interest.

Parkinson ’s disease. Mov. Disord. 2007, 22, 1990 −2017.

(15) Xu, K.; Bastia, E.; Schwarzschild, M. Therapeutic potential of

ABBREVIATIONS

■

adenosine A2A receptor antagonists in Parkinson ’s disease. Pharmacol.

Ther. 2005, 105, 267−310.

AIMs, abnormal involuntary movements; ARs, adenosine

receptors; ALO, axial/lingual/oral; CNS, central nervous

system; CPM, counts per minute; DMSO, dimethyl sulfoxide;

EDS, excessive daytime sleepiness; EEG, electroencephalo-

gram; EKG, electrocardiogram; EMA, European Medicines

Agency; EMG, electromyogram; ESI, electrospray ionization;

GPCRs, G-protein-coupled receptors; HPLC, high-perform-

ance liquid chromatography; HRMS, high-resolution mass

spectrometry; KO, knockout; LID, L-DOPA-induced dyskine-

sia; MAO B, monoamine oxidase B; MFB, medial forebrain;

MS, mass spectrometry; MTL, multi-targeting ligand; PD,

Parkinson’s disease; PS, paradoxical sleep; REM, rapid eye

movement; SI, selectivity; SNpc, substantia nigra pars

compacta; SWS, slow-wave sleep; TLC, thin-layer chromatog-

raphy; W, waking

(

16) Armentero, M. T.; Pinna, A.; Ferré, S.; Lanciego, J. L.; Muller,

C. E.; Franco, R. Past, present and future of A2A adenosine receptor

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Tsukada, H.; Ishiwata, K.; Luurtsema, G.; de Vries, E. F. J.; Elsinga, P.

H. Potential therapeutic applications of adenosine A2A receptor

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