Synthesis and use of FSCPX, an irreversible adenosine A1 antagonist, as a 'receptor knock-down' tool

Article abstract of DOI:10.1016/S0960-894X(01)00069-5

A new preparative synthetic route for the irreversible adenosine A₁ antagonist 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N- propyl-xanthine (FSCPX, 1) is described. The availability of ample amounts of the irreversible antagonist FSCPX allowed us to use FSCPX as a research tool for adenosine A₁ receptors in in vivo experiments. After verification of the irreversible antagonistic function of FSCPX in in vitro experiments, FSCPX was used successfully as a 'receptor knock-down' tool in in vivo experiments on conscious rats.

Full text of DOI:10.1016/S0960-894X(01)00069-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 815±818
Synthesis and Use of FSCPX, an Irreversible Adenosine A1
Antagonist, as a `Receptor Knock-Down' Tool
Jacqueline E. van Muijlwijk-Koezen,a,* Henk Timmerman,a Richard P. van der Sluis,a
Andrea C. van de Stolpe,a Wiro M. P. B. Menge,a Margot W. Beukers,b
Piet H. van der Graaf,c Miriam de Grooteb and Adriaan P. IJzermanb
aLeiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry,
Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
bLeiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Gorlaeus Laboratories,
Universiteit Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
cP®zer Central Research, Discovery Biology, Sandwich, Kent CT13 9NJ, UK
Received 7 June 2000; revised 30 November 2000; accepted 26 January 2001
AbstractÐA new preparative synthetic route for the irreversible adenosine A1 antagonist 8-cyclopentyl-3-N-[3-((3-(4-¯uoro-
sulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, 1) isdescribed. The availability of ample amountsof the irrever-
sible antagonist FSCPX allowed us to use FSCPX as a research tool for adenosine A1 receptorsin in vivo experiment.s After
veri®cation of the irreversible antagonistic function of FSCPX in in vitro experiments, FSCPX was used successfully as a `receptor
knock-down' tool in in vivo experiments on conscious rats. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
meta-1,3-phenylenediisothiocyanate xanthine amine con-
gener(m-DITC-XAC)13 and 8-(3-isothiocyanatostyryl)
ca€eine (ISC),14 or via a p-(¯uorosulfonyl)benzoyl group
Adenosine is a purine nucleoside that is widely dis-
tributed throughout the mammalian body, modulating
a variety of physiological functions via extracellular
adenosine receptors. The currently known adenosine
receptors are classi®ed into four subtypes, called A1,
A2A, A2B and A3 receptors.1,2 Activation of the adeno-
sine A1 receptor has been shown to slow heart rate,3 to
reduce blood pressure,4 to inhibit lipolysis,5,6 and to
inhibit neurotransmitter release.7 Although the actions
of adenosine mediated by the adenosine A1 receptors
are well characterized, no selective ligands for this
receptor subtype are in clinical use yet. Due to the wide
distribution of adenosine A1 receptorsin the body,
many agonists show undesired e€ects.
in
zoyl)oxy)-propyl]-1-N-propylxanthine
8-cyclopentyl-3-N-[3-((3-(4-¯uorosulphonyl)-ben-
(FSCPX,
1)
(Fig. 1).15,16
The present study demonstrates that pretreatment of
animals with the irreversible antagonist FSCPX (1) is a
useful alternative or complement for knock-out animals
in in vivo experiments. When FSCPX is used as a
`receptor knock-down' tool, relatively large amountsof
FSCPX are needed. However, the available synthetic
Irreversible antagonists (photoanity as well as chemo-
reactive ligands) have been used to measure receptor
reserve,8À10 and to identify ligand binding sites.11,12
Chemoreactive compoundswere made through attach-
ment of reactive electrophilic groupsto xanthine deriva-
tives, for example with an isothiocyanate moiety in
*Corresponding author. Fax: +31-02-444-7610; e-mail: muylwyk@
chem.vu.nl
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00069-5

816
J. E. van Muijlwijk-Koezen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 815±818
Scheme 1. Reagents: (i) NaOEt; (ii) cyanoacetic ethyl ester, ~; (iii) NaNO2, acetic acid , 6 M HCl; (iv) Na2S2O4, 50% NH4OH; (v) cyclopentane-
carboxylic acid, EDCI, DMAP, DMF; (vi) 2 N NaOH, ~; (vii) p-methoxybenzylchloride, K2CO3, DMF, ~; (viii) K2CO3, n-propyl iodide, DMF,
~; (ix) BBr3, À78 ꢀC, CH2Cl2; (x) TFA, anisole, H2SO4 (conc); (xi) 4-(¯uorosulfonyl)benzyl chloride, dioxane, ~.
method15 for FSCPX hasa very low overall yield and
comprises two preparative HPLC separations in the last
steps preventing the scaling up of this synthetic method.
To obtain FSCPX on a preparative scale we developed
a new, eight-step, synthesis route for FSCPX (Scheme 1)
with an overall yield of 10%. In thispaper, we describe
the synthesis of FSCPX and the measurement of its
irreversible binding to adenosine A1 receptorsin bind-
ing studies. Subsequently, we describe the `knock-down'
approach which wasveri®ed in in vitro (cAMP produc-
tion in time), and in vivo (heart rate in conscious rats)
studies.
gave a mixture of the expected N3-3-hydroxypropyl
derivative and the N3-3-bromopropyl derivative. This
mixture wasdeprotected at the N7 position to give pure
xanthine 9 in 51% yield. Esteri®cation of the N3-3-
hydroxypropyl group gave FSCPX (1) in 10% overall
yield.23
Biology, Results and Discussion
FSCPX (1) was tested for its anity at, and irreversible
binding to rat brain adenosine A1 receptors. Cortical
membraneswere preincubated with or without (control)
10 nM FSCPX for 1 h at 37 ꢀC. Membraneswere
washed 6 times by centrifugation for 10 min at 12,000 g
and were resuspended in 50 mM Tris/HCl bu€er
(pH=7.4). In subsequent radioligand binding studies
the percentage binding to the membranesof the radio-
labelled antagonist [3H]-DPCPX wasmeausred at dif-
Chemistry
FSCPX (1) wassynthesized according to Scheme 1. N-3-
Methoxypropylureum (2)17 wascondensed with cyano-
acetic ethylester to give uracil 3 (64%).18 At thisstage,
we attempted a selective alkylation of the N3 of the
uracil 5 by silylation with HMDS followed by reaction
with propylbromide according to the procedure of
Muller et al.19 However, starting material remained
present after the silylation step even after prolonged
heating. We tried therefore a direct alkylation of the
uracil with propyl iodide using several solvents, bases
and reaction times, but the expected product, besides di-
and trialkylated products, could only be detected (1H
NMR, N3-CH2 d=3.7 ppm, 25% in a mixture) after
ferent concentrationsof the reference A
agonist N6-cyclopentyladenosine (CPA).
receptor
1
Figure 2 shows that FSCPX binds irreversibly to the rat
adenosine A1 receptors. The maximum level of [3H]-
DPCPX binding wasdecreaesd, indicating that even
after the extensive washing procedure receptors were
still occupied by FSCPX. In Table 1, the anity of CPA
for the adenosine A1 receptor ispresented. CPA recog-
nized a high and a low anity site with anities of
9.2 and 322 nM, respectively. After pretreatment with
10 nM FSCPX, CPA still recognized these sites
(Ki=11.5 nM and Ki=230 nM, respectively). Moreover,
the percentage of high anity sites had not been a€ec-
ted by the pretreatment with FSCPX. These data indi-
cate that the remaining receptorshad unchanged
binding characteristics.
reaction with sodium ethoxide. Likewise, in EtOH with
20
KOH asbaes
low amountsof the product could be
measured. Isolation of the pure product was tedious and
gave only low yieldsof product. We therefore decided to
alkylate thisnitrogen atom in the xanthine tsage after
selective protection of the N7 position. Standard nitro-
sation with sodium nitrite and reduction with sodium
dithionite, followed by condensation of the resulting
diaminouracil 5 with cyclopentanecarboxylic acid using
EDCI and DMAP a€orded 3-(3-methoxypropyl)-8-
cyclopentylxanthine 6 in a good yield (78%). After
protection of N7 with 4-methoxybenzyl chloride, the N1
position was alkylated with propylbromide.21 Depro-
The `knock-down' approach wastetsed in vitro in a
study on cAMP production in time. CHO cells, stably
expressing the human adenosine A1 receptor, were trea-
ted with rolipram (50 mM), cilostamide (50 mM) and
adenosine deaminase (10 IU/mL) during 40 min, fol-
lowed by 15 min incubation with 10 mM forskolin and
tection of the methoxy groupswith BBr at À78 ꢀC22
3

J. E. van Muijlwijk-Koezen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 815±818
817
cAMP production. A decreased inhibition of cAMP
production wasperceived, mots probably due to
FSCPX binding irreversibly overtime to the adenosine
A1 receptors. In other words, FSCPX `knocked-down'
the adenosine A1 receptors.
A series of experiments were performed to demonstrate
that FSCPX also acts as an irreversible adenosine A1
receptor antagonist in vivo. First, even at the lowest
(0.3 mg/kg iv) dose tested, FSCPX directly and com-
pletely antagonised the decrease in heart rate induced by
a 15 min iv infusion of 0.2 mg/kg of the selective ade-
nosine A1 receptor agonist, CPA (data not shown).
Subsequently, the irreversible nature of the antagonism
by FSCPX wasdemontsrated by pretreating ratswith
di€erent doses of FSCPX (0, 1, 3 and 5 mg/kg given as
an iv infusion over 5 min) 20 h prior to administration
of CPA. FSCPX had no e€ect on basal heart rate but
the CPA-mediated bradycardia wasmarkedly atte-
nuated 20 h after the treatment in a dose-dependent
manner, asdetermined by the area under the time±e€ect
relationship (Fig. 4). Remarkably, in some rats of the 5
mg/kg treatment group, CPA did not display any
bradycardiac e€ect at all, while basal heart rate was
unchanged. The antagonism by FSCPX was not due to
a nonspeci®c e€ect, because the decrease in heart rate
produced by the cholinergic agonist, carbachol, was not
a€ected by FSCPX pretreatment (data not shown).
Figure 2. Displacement by CPA of [3H]-DPCPX binding to FSCPX
pretreated and non-treated rat cortical membranes. The data in Table
1 were calculated using a curve ®tting procedure according to a two-
state receptor model (H=high anity state, L=low anity state).
Table 1.
Control
+10 nM FSCPX
Ki,H
Ki,L
fhigh
9.2Æ0.4 nM
322Æ227 nM
72Æ10%
11.5Æ7.3 nM
230Æ91 nM
61Æ14%
10 mM CPA. The antagonist FSCPX (5 mM) or 8-cyclo-
pentyltheophylline (CPT, 50 mM) wasadded and at dif-
ferent time pointscAMP formation wastsopped by
aspiration of the medium and addition of 200 mL 0.1 N
HCl. The amount of cAMP wasdetermined in a com-
petition binding assay with [3H]cAMP and protein
kinase A (PKA). The results are given in Figure 3
(FSCPX (&), reversible antagonist CPT (*)).
FSCPX was tested for stability in rat whole blood by a
method described by Pavan and IJzerman24 and ana-
lyzed using TLC. We observed a rapid degradation at
37 ꢀC, that isafter 2.5 min only the hydrolysed product
wasdetected. Therefore, it isvery unlikely that the los
of e€ect of CPA was due to the presence of signi®cant
concentrationsof FSCPX in the circulation during the
time of the experiments. The short half-life makes this
compound useful as `knock-down' tool in in vivo stud-
iesby iv infusion. 25 Overall, therefore, these experiments
show that speci®c and gradual `knock-down' of adeno-
sine A1 receptor-mediated responses can also be
achieved in vivo with FSCPX.
The adenosine A1 receptor agonist CPA almost fully
inhibited the forskolin-stimulated cAMP production
(data not shown). Treatment with the reversible
antagonist CPT reduced this decrease slightly, and after
10 min the cAMP production was stabilized because of
the settled equilibrium between the agonist- and
antagonist-bound receptors. However, treatment of the
cellswith FSCPX counteracted the induced decrease in
Figure 4. Dose-dependent in vivo pharmacological knockdown of
cardiac adenosine A1 receptors. In vivo e€ect on heart rate in con-
scious rats (n=6±9) of a 15 min infusion of the selective adenosine A1
receptor agonist, CPA (0.2 mg/kg iv) administered 20 h after pretreat-
ment with 0, 1, 3 and 5 mg/kg FSCPX (5 min iv infusion). The inserted
graph shows the corresponding relationship between dose of FSCPX
and area under the CPA time±e€ect curve (AUC).
Figure 3. Time course for inhibition of the CPA-induced decrease of
cAMP production in CHO cells expressing the human adenosine A1
receptor.

818
J. E. van Muijlwijk-Koezen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 815±818
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