Article

[Link]/jmc

Synthesis of (E)‑8-(3-Chlorostyryl)caffeine Analogues Leading to

9‑Deazaxanthine Derivatives as Dual A2A Antagonists/MAO‑B
Inhibitors
Silvia Rivara,*,a Giovanni Piersanti,*,b Francesca Bartoccini,b Giuseppe Diamantini,b Daniele Pala,a
Teresa Riccioni,c Maria Antonietta Stasi,c Walter Cabri,c Franco Borsini,c Marco Mor,a Giorgio Tarzia,b
and Patrizia Minetti*,c
a
Dipartimento di Farmacia, Università degli Studi di Parma, Viale G.P. Usberti 27 A, I-43124 Parma, Italy
b
Department of Biomolecular Sciences, University of Urbino, Piazza Rinascimento 6, I-61029 Urbino (PU), Italy
c
Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040 Pomezia, Italy
Downloaded from [Link] by STOCKHOLM UNIV on 04/24/19. For personal use only.

*
S Supporting Information

ABSTRACT: A systematic modification of the caffeinyl core and

substituents of the reference compound (E)-8-(3-chlorostyryl)caffeine
led to the 9-deazaxanthine derivative (E)-6-(4-chlorostyryl)-1,3,5,-
trimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-(3H,5H)-dione (17f),
which acts as a dual human A2a antagonist/MAO-B inhibitor
J. Med. Chem. 2013.56:1247-1261.

(Ki(A2A) = 260 nM; IC50(MAO-B) = 200 nM; IC50(MAO-A) = 10

μM) and dose dependently counteracts haloperidol-induced catalepsy
in mice from 30 mg/kg by the oral route. The compound is the best
balanced A2A antagonist/MAO-B inhibitor reported to date, and it could be considered as a new lead in the field of anti-
Parkinson’s agents. A number of analogues of 17f were synthesized and qualitative SARs are discussed. Two analogues of 17f,
namely 18b and 19a, inhibit MAO-B with IC50 of 68 and 48 nM, respectively, being 5−7-fold more potent than the prototypical
MAO-B inhibitor deprenyl (IC50 = 334 nM).

■ INTRODUCTION
The complexity of the interactions among the several
distribution,15 and the dual activity of CSC as A2A antagonist
and MAO-B inhibitor,16 led to the proposal of jointly down-
neurotransmitters acting within the central nervous system regulating A2a and MAO-B as a novel approach for treating PD
(CNS) and the difficulty in obtaining acceptable therapeutic and to the design of several A2A/MAO-B dual-acting agents.17
options for the treatment of diseases such as depression, Studies on dual-acting A2A antagonists/MAO-B inhibitors are
Alzheimer’s (AD), and Parkinson’s (PD) diseases has led to a limited to compounds structurally related to caffeine and 8-
number of attempts to discover compounds acting simulta- styrylxanthine, and the subject has been recently reviewed.18
neously on more than one of the neurotransmitters known to CSC is a reference dual-acting agent with good A2A affinity (Ki
play a role in these disorders, particularly when their respective = 36 nM on receptors expressed on rat brain striatal
receptors are localized in identical or vicinal areas of the brain membranes)6 and MAO-B inhibitory potency (Ki = 235 nM
or when synergic actions among them have been shown.1 In the in tests performed on human liver mitochondria),16b effective
case of PD, the development of selective antagonists of the A2A in vivo in reversing the biochemical and behavioral
adenosine receptor (A2AR) initially led to the discovery of a modifications of 6-hydroxydopamine-lesioned rats.19 We
number of promising agents, such as (E)-8-(3-chlorostyryl)- decided therefore to examine whether it could be possible to
caffeine (CSC)2 istradefylline (KW6002),3 KF-17837,4 DMPX, obtain new dual A2A antagonists/MAO-B inhibitors with higher
and DPMTX,5 various derivative of 3,7-dimethylxanthines,6 and and more balanced potencies at the two targets. To select a
MSX-2 and its prodrugs,7 SCH 58261,8 ZM241385,9 CGS suitable scaffold, we ran a preliminary exploratory modification
15943,10 and ST153511 (Figure 1). In particular, the of the caffeinyl core of CSC to obtain derivatives belonging to
therapeutic potential of KW6002 for the treatment of PD has the different chemical classes reported in Figure 2. In particular,
been extensively investigated through a number of clinical trials, we evaluated the role of the nitrogen, carbonyl group, and
which showed that this compound significantly improved imidazole ring in the purine system by preparing compounds
motor impairment and reduced “off” time when coadministered 4a,b and the pyrimidinedione derivative 6. Bioisosteric
with levodopa.12 The therapeutic application of two irreversible replacement of the purine nitrogen atom in position 9 led to
inhibitors of monoamine oxidase B (MAO-B), (R)-deprenyl
and rasagiline, for alleviating the symptoms of PD,13 the age- Received: November 15, 2012
related increase in the expression of MAO-B,14 its cerebral Published: January 2, 2013

© 2013 American Chemical Society 1247 [Link]/10.1021/jm301686s | J. Med. Chem. 2013, 56, 1247−1261
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Figure 1. A2A receptor antagonists.

Figure 2. Chemical scaffolds of newly synthesized compounds.

9-deazaxanthines (17a−y, 18a−d, 19a−d, 20a,b, 21−23) and by N-alkylation with methyl iodide gave 4a,b in satisfactory
to their conformationally constrained tricyclic derivatives yields (Scheme 1).
(14a−c) (Figure 2, Schemes 1−9). Preliminary screening of Amide coupling of the scarcely reactive secondary enamine
a few prototypes for their A2A/MAO-B dual-activity allowed us 524 with trans-cinnamic acid was obtained using 1-ethyl-3-(3-
to identify the 9-deazaxanthine as the most promising scaffold dimethylaminopropyl) carbodiimide (EDCI) as a coupling
which was optimized by modifying the steric, electronic, and agent and afforded the desired amide (6) in modest yield.
lipophilic properties of the 8-substituent to give compound (Scheme 2).
17a−y, 18a−d, 19a−d, 20a,b, and 21−23. 1,3,5-Trimethyl-1H-pyrimido[5,4-b]indole-2,4(3H,5H)-

■ CHEMISTRY
The imidazopyridinones 4a,b were obtained by direct
dione derivatives 14a−c were prepared as described in Scheme
3. Compound 14a and 14c were obtained respectively from the
commercially available 7a and 9c. Compound 14b was obtained
condensation of 1a,b20 with trans-cinnamic acid or cinnamal- from 7b, which was synthesized by Suzuki reaction of 4-
dehyde to yield 2a,b that were oxidized to 3a,b with m- bromobenzaldehyde with 3-chlorophenylboronic acid.25 For-
chloroperoxybenzoic acid (m-CPBA).21 Rearrangement of 3a,b mation of ethyl 2-azidocinnamate (8a,b) by condensation of
with acetic anhydride22 or trifluoroacetic anhydride23 followed arylaldehyde (7a,b) with ethyl azidoacetate in the presence of
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Scheme 1. Synthesis of Imidazopyridinone Derivativesa sodium ethoxide at 0 °C and subsequently pyrolysis of 8a,b in
p-xylene a 150 °C gave the corresponding ethyl indole-2-
carboxylate (9a,b). Reaction of indole derivatives 9a−c and
benzenediazonium chloride provided azo compounds 10a−c.
Reduction with tin and hydrochloric acid, followed by
treatment with ethoxycarbonyl chloride, furnished carbamates
12a−c. Cyclization with methylamine and N-methylation with
methyl iodide provided 14a−c (Scheme 3).
1,3,5-Trimethyl-6-styryl-1H-pyrrolo[3,2-d]pyrimidine-2,4-
(3H,5H)diones 17a−w were obtained as described in Schemes
4 and 5. Compound 15a was synthesized as previously
reported,26 and the introduction of the styryl group was not
without problems. Synthesis of styryl derivatives with electron-
donating substituents was achieved by palladium cross-coupling
reaction under standard reaction condition,26 whereas the
procedure failed in the case of styryl containing electron-
a
Reagents and conditions: (i) for 2a, (E)-PhCHCHCO2H, POCl3, withdrawing substituents. The vinylation of 15a with vinyl
140 °C, 5 h, 44%; for 2b, MeOH, AcOH, (E)-PhCHCHCHO, rt, 1 boronate ester gave compound 16, which was used for Heck
h, 40%; (ii) for 3a, CH2Cl2, MeOH, m-CPBA, rt, 1 h, 68%; for 3b,
coupling with aryl bromides containing the electron with-
CHCl3, m-CPBA, rt, 30 min, 88%; (iii) (CH3CO)2O, 140 °C, 2 h,
56%; (iv) DMF, K2CO3, MeI, rt, 60 h, 28%; (v) DMF, (CF3CO)2O, rt, drawing groups. Compounds 17a−n were synthesized by path
16 h, 47%; (vi) NaOH 6 N, MeI, 50 °C, 12 h, 55%. (ii), whereas compounds 17o−t were prepared by path (iii) in
Scheme 4.
Scheme 2. Synthesis of Pyrimidines Derivative 6a Phenol derivatives 17u−w were obtained by demethylation
with BBr3 of the corresponding methoxy derivatives 17c,h,m
(compound 17m was not tested) (Scheme 5).
Ethynyl derivatives 18a−c were synthesized by Sonogashira
reaction as previously reported26 (Scheme 6).
The transition-metal-catalyzed formation of carbon-heter-
oatom bonds via cross-coupling reactions plays an important
role in the preparation of numerous products of pharmaceutical
a
Reagents and conditions: (i) MeOH, EDCI, PhCHCHCO2H, rt, interest,27 allowing the introduction of various nitrogen and
20 h, 37%. oxygen functions (amine, amide, urea, carbamate, alcohol,
phenol, thiol) onto aromatic or heteroaromatic cycles. The
bromo derivative 15a failed to give the desired N- or O-arylated

Scheme 3. Synthesis of 1,3,5-Trimethyl-1H-pyrimido[5,4-b]indole-2,4(3H,5H)-dione Derivativesa

a
Reagents and conditions: (i) EtOH, N3CH2CO2Et, EtONa, 0 °C, 2h, 55−58%; (ii) p-xylene, 150 °C, 2 h, 78−98%; (iii) (1) C6H5NH2, HCl 6 N,
NaNO2, 0 °C, 15 min; (2) DMF, Na2CO3 2 N, 0 °C, 1 h, 30−80%; (iv) (CH3)2CHOH, conc HCl, Sn, 85 °C, 2 h, 46−69%; (v) xylene, ClCOOEt,
140 °C, 2 h, 40−83%; (vi) (CH3)2CHOH, CH3NH2 40 wt % in H2O, 85 °C, 24 h, 15−52%; (vii) DMF, NaH, MeI, rt, 24 h, 46−86%.

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Scheme 4. Synthesis of 8-Styryl-9-deazaxanthine Derivativesa

a
Reagents and conditions: (i) DME, H2O, CH2CHBpin, TEA, PdCl2(PPh3)2, 80 °C, 16 h, 67%; (ii) DMF, KOAc, TBAB, molecular sieves,
styrene derivatives, Pd(OAc)2, 80 °C, 20 h, 20−86%; (iii) DMF, aryl bromide, TEA, P(o-tol)3, Pd(OAc)2, 80 °C, 2 h, 30−84%.

Scheme 5. Synthesis of Hydroxystyryl Derivativesa 2,2,6,6-tetramethyl-3,5-heptanedione and Cs2CO3 at 130 °C for

72 h.29 Copper(I) catalysts (bromide or iodide) allowed
coupling of amides and triazoles30 to give 21 and 22 in good
yield. Similar reaction conditions permitted also a C−C bond
formation by 2-C−H activation of the benzoxazole system to
give compound 23 (Scheme 7).
The reaction conditions reported above were employed for
the synthesis of different N-alkylated deazaxanthines (17x,y,
18d, 19d). In particular, compounds 17x,y were synthesized by
a
Reagents and conditions: (i) CH2Cl2, BBr3 in CH2Cl2, 0 °C, 1 h, 27− Heck coupling starting from 15c and 15d, respectively,
90%. compound 18d was synthesized by Sonogashira coupling
starting from 15d, while compound 19d was obtained by O-
Scheme 6. Synthesis of Alkynyl Derivativesa arylation of 15e (Scheme 8).
8-(p-Chlorostyryl)xanthine (25) was synthesized by a novel
Pd/Cu-catalyzed dehydrogenative Heck coupling that allows
direct alkenylation of caffeine (24) with 4-chlorostyrene
(Scheme 9), which eliminates the need of preparing the
heteroaryl halides.31

a
■ RESULTS AND DISCUSSION
The compounds were tested for their binding affinity for
Reagents and conditions: (i) dioxane, TEA, CuI, (PPh3)2PdCl2,
alkynyl derivatives, 100 °C, 20 h, 54−62%.
human A2A (hA2A) receptors and inhibitory potency on human
MAO-B (hMAO-B) (Tables 1−3). Selectivity versus MAO-A
was also evaluated for the compounds displaying MAO-B IC50
products under variously modified reaction conditions (solvent, values lower than 1400 nM. Investigation on the structural
base, palladium and copper precursor, ancillary coligand), determinants for the interaction with A2A and MAO-B was
whereas the more reactive iodo derivative 15b, prepared from initially focused on the pyrimidinedione moiety of the
15a by copper-catalyzed halogen exchange reaction using dechloro-CSC. Replacement by 1-methyl-2-pyridinone ring
potassium iodide and N,N′-dimethylethylendiamine ligand28 led to compound 4a, which was inactive at both targets, and the
(Scheme 7), underwent copper or palladium-catalyzed coupling same result was obtained with its regioisomer 4b (Table 1).
with different heteronucleophiles. Ether derivatives 19a−c Deletion of the imidazole ring of the xanthine scaffold and
could be obtained in acceptable yield by copper-catalyzed insertion of an amide group on the pyrimidinedione ring (6) to
reaction using 1,10-phenanthroline as a ligand at 110 °C for maintain the coplanarity of the styryl substituent was also
prolonged time. The same reaction condition did not work with detrimental for the interaction with both targets. A more
phenols as substrates, and the diarylethers 20a,b were obtained conservative modification, yielding the 9-deazaxanthine 17a,
by using ferric chloride as a catalyst in DMF in the presence of was tolerated by both targets, with only a limited decrease of
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Scheme 7. Synthesis of 8-Functionalized Deazaxanthine Derivativesa

a
Reagents and conditions: (i) CuI, KI, N,N′-dimethylethylendiamine, n-BuOH, 120 °C, 36 h, 72%; (ii) toluene, benzylalcohol derivatives, CuI, 1,10-
phenanthroline, Cs2CO3, 100 °C, 5 h, 38−43%; (iii) DMF, phenol derivatives, FeCl3, 2,2,6,6-tetramethyl-3,5-heptanedione, Cs2CO3,130 °C, 72 h,
37−43%; (iv) dioxane, K2CO3, CuI, trans-N,N′-dimethyl cyclohexan-1,2-diamine, 4-ClC6H4CONH2, 100 °C, 20 h, 35%; (v) DMSO/H2O 9:1,
NaN3, 4-ClC6H4CCH, CuSO4·H2O, sodium ascorbate, L-proline, 60 °C, 18 h, 39%; (vi) DMF, 6-chlorobenzo[d]oxazole, Pd(OAc)2, P(t-Bu)3,
Cs2CO3, CuBr, 150 °C, 2 h, 39%.

Scheme 8. Synthesis of 8-Functionalized Deazaxanthine Derivativesa

a
Reagents and conditions: (i) DMF, KOAc, TBAB, molecular sieves, styrene derivatives, Pd(OAc)2, 80 °C, 20 h, 43−45%; (ii) dioxane, TEA, CuI,
(PPh3)2PdCl2, alkynyl derivatives, 100 °C, 20 h, 45%; (iii) toluene, benzylalcohol derivatives, CuI, 1,10-phenanthroline, Cs2CO3, 100 °C, 5 h, 40%.

Scheme 9. Synthesis of 8-Functionalized Xanthine MAO-B inhibitory potency is described. Crystal structures of
Derivativea the A2A receptor in complex with caffeine and XAC (N-(2-
aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-
1H-purin-8-yl)phenoxy]-acetamide) (where no water mole-
cules were resolved)33 showed that the N9 of the xanthine
nucleus does not directly interact with residues of the binding
site (Figure 3A). On the other hand, superposition of the
a
Reagents and conditions: (i) 4-ClC6H4CHCH2, Cu(OAc)2, crystal structures of the A2A−XAC complex with the recently
Pd(OAc)2, Ag2CO3, t-BuCO2H, 90 °C, 72 h, 60%. solved A2A−ZM241385 complex, where several water mole-
cules are present,34 showed that N9 of XAC is roughly
potency (Table 1). The chlorine atom in position meta slightly superposed to the N4 of ZM241385, forming a hydrogen bond
decreased A2A binding affinity, compared to the unsubstituted with a conserved water molecule belonging to a cluster located
derivative 17b, and had a negligible effect on MAO-B inhibitory inside the binding site (Figure 3B). Lack of interaction with this
potency. 8-Styryl-9-deazaxanthines have already been reported cluster may explain the limited loss of binding affinity of the 9-
as A2 receptor ligands,32 but this is the first time that their deaza derivative 17a compared to CSC.
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Table 1. A2A Adenosine Receptor Affinities (Ki) and MAO-B and MAO-A Inhibitory Potencies (IC50) for the Synthesized
Compounds

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Table 1. continued

a
Ki values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff,42 and are the mean of four
determinations performed in duplicate.

No crystal structure is available for either xanthine or 9- Glide software,35 and Figure 4A depicts the best docking
deazaxanthine derivatives within the MAO-B catalytic site. We solution. CSC occupies both the substrate and the entrance
therefore docked CSC into the MAO-B crystal structure using cavities, with the xanthine moiety near the FAD cofactor and
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Figure 3. (A) Superposition of the crystal structures of A2A receptor in complex with caffeine (green carbons, PDB code: 3RFM) and XAC (yellow
carbons, PDB code: 3REY). (B) Superposition of the crystal structures of A2A receptor in complex with ZM241385 (orange carbons, PDB code:
4EIY) and XAC (yellow carbons). Water molecules are represented with red spheres. (C) Best docking poses for compounds 17f (orange carbons)
and 17q (cyan carbons) within the A2A receptor structure built from 3REY. XAC molecule is depicted with yellow carbons.

and prompted us to further explore the SARs for this class of

compounds. We thus focused on the role of substituents in
position 1, 3, 7, and 8. Substituents with different size, shape,
lipophilicity, and electronic properties were introduced in meta
or para position of the 8-styryl radical. Meta substituents did
not lead to an increase of binding affinity for the A2A receptor.
Indeed, a chlorine (17a) or a methoxy group (17c) slightly
reduced binding affinity, while trifluoromethyl (17d), hydroxyl
Figure 4. (A) Best docking poses for CSC (yellow carbons) and 14b (17u), acetyl (17o), or cyano (17p) substitution led to inactive
(orange carbons) within the MAO-B active site. The cocrystallized compounds. Tests performed on rat A2 receptors demonstrated
safinamide is depicted with green transparent carbons and the FAD that a m-chlorine doubled the binding affinity and that a m-
cofactor in ball-and-sticks with white carbons. (B) Chemical structures methoxy and a m-trifluoromethyl were tolerated by the
of MAO-B inhibitors safinamide and coumarin derivative. corresponding 8-styrylxanthine analogues, highlighting a differ-
ent SAR profile.2 Meta-substituted compounds carrying lip-
the 6-carbonyl group interacting with Tyr435 (not shown in ophilic groups had the highest MAO-B inhibitory potencies,
Figure 4A). The purine core fits between Tyr398 and Leu171 with the best results obtained for the methoxy (17c) and the
on one side and Tyr435 and Gln206 on the other one. This trifluoromethyl (17d) derivatives. Hydrophilic substituents
accommodation is very similar to that described for some 8- were not tolerated (17u, 17p). Compound 17c was the best
benzyloxycaffeine analogues.36 The 3-chlorostyryl protrudes meta-substituted derivative, endowed with balanced potencies
into the entrance cavity in s-cis conformation, favored by the at the two targets, even if with limited MAO-B/MAO-A
lower steric hindrance of N9 compared to the N7-methyl selectivity. On the 8-styrylxanthine series, electron-withdrawing
group. In this pose, the m-chlorine atom occupies the same groups showed a more pronounced effect. Indeed, m-chlorine
region as the m-fluorine of safinamide and m-chlorine of some and m-trifluoromethyl increased inhibitory potencies about 10-
coumarin inhibitors (Figure 4B) in their crystallographic fold compared to the 4-fold observed for the 8-styryl-9-
complexes.37 The nitrogen atom in position 9 of CSC does deazaxanthine analogues.17c
not make polar interactions with the amino acids in the A larger exploration was performed on the para position, but
catalytic site. The best docking solution obtained for 17a was only the acetyl substituent (17q) led to a modest increase of
strictly superimposable on that described for CSC (not shown). A2A binding affinity compared to the unsubstituted compound
To test the reliability of these poses, we constrained the s-cis 17b. Lipophilic substituents produced a limited decrease of
geometry of the styryl group in the 9-deaza series into a binding affinity. Indeed, fluorine (17e), chlorine (17f),
pyrimido[4,5-b]indole nucleus (14a−c). This tricyclic ring trifluoromethyl (17g), and methoxy (17h) groups were
could be docked within the MAO-B active site with the meta- tolerated, as well as the longer n-propyloxy substituent (17i),
chlorine of 14b occupying the same position as that of 3-fluoro while the bromine derivative 17j completely lost A2A binding
of safinamide (Figure 4A). Consistently with what had been affinity. Hydrophilic methylsolfonyl (17r), cyano (17s), and
observed in the xanthine series,17c removal of the m-chlorine hydroxyl (17v) groups were not tolerated. Lipophilic, electron-
(14a) led to a decrease of inhibitory potency for MAO-B. withdrawing groups, such as halogens (17e, 17f, and 17j) and
Limited MAO-B inhibitory potency was also observed for trifluoromethyl (17g), produced the greatest increase in MAO-
compound 14c, having a more flexible benzyloxy substituent in B inhibitory potency. A p-chlorine and a p-trifluoromethyl
position 8. Unfortunately, the tricyclic compounds were devoid substituent increased inhibitory potencies in the 8-styrylxan-
of binding affinity for the A2A receptor. For this reason, we thine series as well, while fluorine had a negligible effect.17c
decided to abandon the exploration of pyrimido[4,5-b]indole Summarizing, different compounds with good potencies at both
scaffold and went back to the 9-deazaxanthine series. 8-Styryl-9- targets were obtained within the 8-(para-substituted-styryl)-9-
deazaxanthines did not strictly follow the same structure− deazaxanthines series, such as the acetyl (17q), fluorine (17e),
activity relationships (SARs) as 8-styrylxanthines, lacking the trifluoromethyl (17g) and, in particular, the chlorine derivative
positive effect of the m-chlorine group. This might be attributed 17f. Compound 17f (ST3564) is characterized by the best
to different equilibria between s-cis and s-trans conformations combination of A2A binding affinity (Ki = 260 nM) and MAO-B
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Table 2. A2A Adenosine Receptor Affinities (Ki) and MAO-B and MAO-A Inhibitory Potencies (IC50) for the Synthesized
Compounds

a
Ki values were calculated from IC50 values, obtained from competition curves by the method of Cheng and Prusoff,42 and are the mean of four
determinations performed in duplicate.

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inhibitory potency (IC50 = 200 nM), and it is selective versus highest MAO-B inhibitory potency (IC50 = 48 nM) in the
MAO-A (IC50 = 10000 nM). It is more potent on MAO-B than whole series, about 4-fold higher than that of the styryl
CSC and the corresponding (E)-8-(4-chlorostyryl)caffeine (25) analogue 17f and with a good selectivity versus MAO-A (Table
that we prepared and tested in our experimental conditions. 2). Unfortunately, it did not interact with the A2A receptor. The
Other structural modifications were attempted on the 8- presence of an additional m-trifluoromethyl group (19b) led to
styryl-9-deazaxanthine scaffold. An o-hydroxyl group (17w) a 10-fold decrease of MAO-B inhibitory potency with no effect
reduced the potency on both targets, as already observed for on A2A binding affinity, while an increase in the size of the
the same substituent in meta (17u) and para (17v) positions. substituent in the β-naphthylmethyloxy derivative 19c led to a
The dichloro-derivative 17k was the most potent MAO-B completely inactive compound. Shortening the 8-methyloxy
inhibitor in the 8-styryl-9-deazaxanthine series (IC50 = 133 linker to an oxygen atom, as in the m-chlorophenyloxy (20a)
nM), but it did not bind to A2A receptors. A similar behavior and m-biphenyloxy (20b) derivatives, was also detrimental for
was shown by compound 17l, with a methylenedioxy portion the ability to interact with both targets. On the other hand,
bridging positions meta and para, which was tolerated at the replacement of the styryl double bond with a triple one led to
MAO-B binding site only. The lack of A2A binding affinity of the m-chlorophenyl- and p-chlorophenyl-ethynyl derivatives
17l represents another difference from the xanthine series. 18a and 18b having MAO-B inhibitory potencies higher than
Indeed, 8-(3,4-dimethoxystyryl)caffeine had Ki = 18 nM and 1- the corresponding styryl analogues. However, neither one
propargyl-3,7-dimethyl-8-[3′,4′-(methylenedioxy)styryl]- showed affinity for the A2A receptor. Differently, the 1-
xanthine had Ki = 35 nM on rat striatum A2A receptors.6 propargyl-3,7-dimethylxanthine carrying an 8-phenylethynyl
Replacement of the benzene ring in the styryl portion with a substituent displayed Ki = 300 nM on A2A receptors of rat
more hydrophilic and basic ring was detrimental for MAO-B striatum.38 Insertion of a methylene spacer between the phenyl
inhibitory potency, as both the 4-pyridyl (17t) and 3-amino-2- ring and the triple bond led to compound 18c, which was
pyridyl (17n) derivatives were inactive. Compound 17n inactive at both targets. Replacement of the double bond with
showed modest affinity for A2A receptors, about five times an amide group (21) or insertion of a 6-chlorobenzo[d]oxazol-
lower than that of its benzene analogue 17b. 2-yl substituent (23) in position 8 was not tolerated by either
We prepared (E)-8-(4-chlorostyryl)caffeine (25) to compare targets. When a 1,2,3-triazolyl ring replaced the ethylene linker
m- and p-chlorine-substituted compounds. For both xanthine (22), a good MAO-B inhibitory potency was maintained.
and 9-deazaxanthine series, para substitution resulted in higher SARs for A2A xanthine antagonists showed that longer alkyl
hA2A binding affinity and hMAO-B inhibitory potency than substituents on the nitrogen atoms led to higher A2A binding
those observed for meta substitution. affinity than the corresponding methyl analogues.2,38 Therefore
As already stated, SARs for our 8-styryl-9-deazaxanthine we inserted ethyl groups in positions 1, 3, or 7 of the 9-
derivatives are different from those reported on rat A2A deazaxanthine nucleus. Contrary to expectations, ethyl groups
receptors for 8-styryl-xanthines, where meta substitution and had a negative effect on potency, irrespective of the nature of
meta−para disubstitution were favorable, contrary to what we the substituent in position 8. Inactive compounds at both
observed on human receptor. On the other hand, para targets were obtained with 8-(p-chlorostyryl) (17y, 17x), 8-(p-
substitution with a chlorine atom gave higher hA2A binding chlorophenylethynyl) (18d), and 8-(p-chlorobenzyloxy) (19d)
affinity in both series (17f and 25 for 8-styryl-9-deazaxanthine substituents (Table 3). This further confirms the different SAR
and 8-styryl-xanthine, respectively). To investigate if different profile observed for xanthine and 9-deazaxanthine derivatives.
binding modes could be supposed for the two series, we docked Compound 17f, having balanced A2A /MAO-B activity, was
the most potent para-substituted 8-styryl-9-deazaxanthine tested against a panel of 53 receptors, ion channels, and
derivatives 17f and 17q into the binding site of the A2A transporters.40 At the concentration of 10 μM, it produced 59%
receptor, cocrystallized with the xanthine derivative XAC. In displacement of specific ligand at the A1 receptor, 60% at the A3
their best docking poses, the two compounds showed an receptor, and 91% at the A2A receptor. Compound 17f appears
accommodation inside the binding site very close to that of therefore not selective for the A2A subtype, having also affinity
XAC, undertaking interactions with the same amino acids for the A1 and A3 subtypes. However, interaction with the A1
(Figure 3C), and 8-styryl-xanthines docked in the same pose as receptor could be a positive element due to the ability of A1
well (not shown). Interestingly, the acetyl group of 17q had its antagonists to facilitate dopamine release and to counteract PD-
carbonyl superposed to the amide group of XAC. Therefore, no related symptoms.41 Compound 17f did not significantly bind
significant differences can be seen among the poses of the two to other receptors or ion channels and transporters at 10 μM,
classes, with the exception of the already cited possibility to with the exception of NK2 and 5-HT2B receptors and
undertake interactions with the cluster of water molecules norepinephrine transporter, where it showed 75%, 72%, and
within the binding site, and differences with SARs reported for 57% displacement of specific ligands, respectively (Supporting
8-styryl-xanthines should be ascribed to different receptor Information Table S2). General behavioral response and
origins. antagonism of haloperidol-induced catalepsy were evaluated
The styryl substituent of (E)-8-styrylxanthines is known to in vivo for compound 17f. In the Irwin test, compound 17f
undergo light-induced isomerization to the less potent Z induced striatal (biting and licking) and limbic (sniffing)
isomer.16b,38 We therefore investigated if it was possible to stereotypes, after 90 min post treatment at the doses of 30 and
replace the 8-styryl group with substituents lacking the double 100 mg/kg. The dose of 100 mg/kg presented licking behavior
bond while maintaining the ability to interact with both targets. after 240 min post treatment. No stereotypes were observed at
The benzyloxy group is typical of numerous MAO-B inhibitors, the dose of 10 mg/kg. All the animals showed catalepsy before
such as safinamide,39 coumarin derivatives, and also xanthine compound administration. Compound 17f significantly antag-
inhibitors.37 We therefore prepared the 8-(p-chlorobenzyloxy)- onized haloperidol-induced catalepsy at doses of 30 and 100
9-deazacaffeine 19a, as the p-chlorine derivative 17f was the mg/kg (Figure 5, p < 0.05), while it was inactive at a dose of 10
best one in the styryl series. Compound 19a showed the mg/kg.
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Journal of Medicinal Chemistry Article

Table 3. A2A Adenosine Receptor Affinities (Ki) and MAO-B reported for xanthines. The observed variance may be due
and MAO-A Inhibitory Potencies (IC50) for the Synthesized either to differences in the structure of the two classes or in the
Compounds biological tests that were used to characterize the compounds.
Indeed, data for xanthines were mainly obtained from rat A2/
A2A receptors and baboon or rat MAO-B, while our data came
from human proteins.

■ EXPERIMENTAL SECTION
Chemistry. General Methods. All reactions were run in air except
when differently noted. Column chromatography purifications were
performed in flash conditions using Merck 230−400 Mesh silica gel.
Analytical thin layer chromatography (TLC) was carried out on Merck
silica gel plates (Silica Gel 60 F254), that were visualized by exposure
to ultraviolet light. 1H NMR and 13C NMR spectra were recorded on a
Bruker Avance 200 spectrometer, using CDCl3 or DMSO-d6 as
solvent. Chemical shifts (δ scale) are reported in parts per million
(ppm) relative to the central peak of the solvent. Coupling constants
(J values) are given in hertz (Hz). EI-MS spectra (70 eV) were taken
on a Fison Trio 1000, and molecular ions (M+ or M−) are given. ESI-
MS spectra were taken on a Waters Micromass ZQ instrument, and
only molecular ions (M + 1 or M − 1) are given. IR spectra were
obtained on a Nicolet Avatar 360 FT-IR spectrometer, absorbance are
reported in cm−1. Melting points were determined on a Buchi SMP-
510 capillary melting point apparatus and are uncorrected. Elemental
analyses were performed on a Carlo Erba analyzer, and the results are
within ±0.4 of the theoretical values (C,H,N). Purity of tested
a
Ki values were calculated from IC50 values, obtained from compounds was greater than 95%.
competition curves by the method of Cheng and Prusoff,39 and are General Procedure A for the Heck Coupling of 8-Halogen-9-
the mean of four determinations performed in duplicate. deazaxanthine and Styrene Derivatives (17a−n,x,y). A flame-dried
Schlenk tube was charged with potassium acetate (73 mg, 0.74 mmol),
tetrabutylammonium bromide (119 mg, 0.37 mmol), powder 3 Å
molecular sieves (74 mg), and dry DMF (0.4 mL), and the mixture
was stirred for 15 min. The appropriate 6-bromo-1,3,5-alkyl-1H-
pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (15a,c,d) (0.37 mmol)
and the opportune styrene (0.74 mmol) were successively added, and
the suspension was stirred for another 15 min before addition of
Pd(OAc)2 (4 mg, 0.019 mmol). The mixture was stirred at 80 °C for
20 h and then cooled at room temperature, diluted with CH2Cl2 (3
mL), filtered over Celite, and washed with CH2Cl2 (3 × 10 mL). The
solvent was evaporated under reduced pressure, and the residue
obtained was purified by flash chromatography (cyclohexane/ethyl
acetate 7:3).
For a Representative Example. (E)-6-(3-Chlorostyryl)-1,3,5-
trimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (17a). Or-
ange solid; (67 mg, 55%). MS (ESI): 330−332 [M + H]+. 1H NMR
(CDCl3): δ 3.42 (s, 3H), 3.49 (s, 3H), 4.11 (s, 3H), 6.19 (s, 1H), 6.90
(d, 1H, J = 16.2 Hz), 7.08 (d, 1H, J = 16.2 Hz), 7.29−7.37 (m, 3H),
Figure 5. Dose−response curve of compound 17f on haloperidol- 7.50 (s, 1H). 13C NMR (CDCl3): δ 27.9, 31.7, 31.8, 90.9, 111.4, 116.1,
induced catalepsy in mice (AUC). Columns represent mean + SEM of 124.9, 126.3, 128.5, 130.1, 131.4, 134.9, 135.6, 138.1, 139.5, 151.5,
155.8. FTIR (nujol, cm−1): 1647, 1685, 2854, 2924. Melting point:
eight mice. Haloperidol (2 mg/kg) was administered ip 2.5 h before
decomposition with color change starting from 200 °C (ethanol).
compound 17f. AUC was calculated throughout 3 h of recording. One
Anal. (C17H16ClN3O2) C, H, N.
way Anova: F3,28 = 4.9; p < 0.01; Dunnett’s test: * p < 0.05 vs 0 mg/kg.
General Procedure B for the Heck Coupling of 8-Vinyl-9-

■
deazaxanthine and Aryl Bromide (17o−t). A flame-dried Schlenk
CONCLUSIONS tube was charged with 1,3,5-trimethyl-6-vinyl-1H-pyrrolo[3,2-d]-
pyrimidine-2,4(3H,5H)-dione (16) (80 mg, 0.36 mmol), the
Structural modulation of CSC allowed us to obtain 9- opportune aryl bromide (0.73 mmol), TEA (0.24 mL, 1.69 mmol),
deazaxanthine derivatives acting as A2A antagonists/MAO-B P(o-tol)3 (22 mg, 0.072 mmol), Pd(OAc)2 (8 mg, 0.036 mmol), and
inhibitors. Chemical exploration in position 8 of the 9- dry DMF (3.6 mL). The mixture was stirred at 80 °C for 2 h under N2
deazaxanthine scaffold provided the p-chlorostyryl derivative and then cooled at room temperature, diluted with CH2Cl2 (15 mL),
17f characterized by balanced potencies at the two targets and and saturated aqueous sodium chloride solution (15 mL). The phases
by reversing haloperidol-induced catalepsy in mice. Replace- were separated, and the aqueous phase was extracted with further
ment of the 8-styryl portion of CSC with either a benzyloxy CH2Cl2 (15 mL). The combined organic phases were dried over
anhydrous Na2SO4, the solvent was evaporated under reduced
(19a) or a phenylalkynyl (18b) substituent led to compounds
pressure, and the residue obtained was purified by flash chromatog-
with remarkable MAO-B inhibitory potencies, higher than raphy (gradient from cyclohexane/ethyl acetate 7:3 to ethyl acetate/
those of CSC and deprenyl. Structural requirements for A2A methanol 9:1).
binding were stringent and could be met by 8-styryl derivatives For a Representative Example. (E)-6-(4-Acetylstyryl)-1,3,5-tri-
only. SARs for 9-deazaxanthines were different from those methyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (17q). Yellow

1257 [Link]/10.1021/jm301686s | J. Med. Chem. 2013, 56, 1247−1261

Journal of Medicinal Chemistry Article

solid; (73 mg, 60%). MS (EI): 337 (M)+. 1H NMR (CDCl3): δ 2.62 the Italian Ministry of Health. Experiments were performed according
(s, 3H), 3.42 (s, 3H), 3.49 (s, 3H), 4.12 (s, 3H), 6.23 (s, 1H), 7.14 (s, to a randomized schedule. Compound 17f (ST3564) was suspended
2H), 7.58 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz, 2H). 1H NMR in a solution containing 0.1% Tween 80 in 0.5 carboxymethylcellulose
(acetone-d6): δ 2.59 (s, 3H), 3.29 (s, 3H), 3.45 (s, 3H), 4.15 (s, 3H), CMC (medium viscosity) (Sigma-Aldrich, Milan) that was used as
6.61 (s, 1H), 7.40 (d, J = 16.2 Hz, 1H), 7.54 (d, J = 16.2 Hz, 1H), 7.79 vehicle. The drug was administered in a volume of 10 mL/kg in mice.
(d, J = 8.5 Hz, 2H), 8.01 (d, J = 8.5 Hz, 2H). 13C NMR (CDCl3): δ Male CD-1 mice (Charles River, Calco, Italy), 5−6 wk old, were kept
26.5, 27.8, 31.6, 31.8, 91.2, 111.6, 117.2, 126.6, 129.0, 131.5, 135.6, for 1 week at 22 + 2 °C, at 50 + 15% relative humidity, with 15−20 air-
136.7, 139.4, 140.7, 151.5, 155.8, 197.1. FTIR (nujol, cm−1): 1693, volume/h changes, and 12 h light/dark cycle (lights on at 07:00 h).
1674, 1655. Melting point: decomposition with color change starting Animals were group-housed (n = 10) in Makrolon R cages (42.5 cm ×
from 250 °C (ethanol). Anal. (C19H19N3O3) C, H, N. 26.6 cm × 16 high cm) with standard food pellets and water ad
Pharmacology. Inhibitory Potency on Monoamine Oxidase. libitum.
Monoamine oxidase (MAO) activity was evaluated by a commercial Irwin Test. The test was performed at 60 and 240 min after oral 10,
kit (MAO-Glo Assay, Promega). The kit provides a homogeneous 30, and 100 mg/kg administration of compound 17f. Mortality was
luminescent method for measuring MAO activity and the effects on it recorded during the 24 h postdose injection. General parameters
of test compounds. The assay was performed by incubating the MAO (salivation, lacrimation, diarrhea, ptosis, tremors, convulsions,
enzyme with a luminogenic MAO substrate, which is a derivative of piloerection, Straub tail, aggressiveness, and stereotypes) and specific
beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thia- observations (loss of reflexes, catalepsy, motor activity, hot plate, body
zolecarboxylic acid) converted by MAO to methyl ester luciferin. After temperature, and acute death) were recorded.
the MAO reaction has been performed, the “Luciferin Detection Catalepsy. Animals were gently placed by their forepaws on a small
Reagent” was added to simultaneously stop the MAO reaction, convert metal bar at a height of 4.5 cm. Catalepsy was induced by haloperidol
the methyl ester derivative to luciferin, and produce light. The amount (2 mg/kg) injected intraperitoneally 2.5 h before oral administration
of light produced is directly proportional to MAO activity. The of compound 17f (10, 30, 100 mg/kg) or vehicle. At time 0 min,
experiments was performed in duplicate in 96-well plates at room successful induction of catalepsy in all animals was checked before
temperature. In each well, the reaction mixture was composed of 12.5 compound administration, then catalepsy was scored every 60 min for
μL of MAO substrate at its Km values, corresponding to a final 3 h. The catalepsy was measured as the time necessary for the animal
concentration of 40 μM for reaction with MAO-A and 4 μM for to step down with at least one forepaw with a cut off time for each
reaction with MAO-B, 12.5 μL of test compounds, and 25 μL of either animal of 60 s; after this time, the mouse was gently removed from the
human MAO-A or MAO-B (Sigma) at a final concentration of 1 μg of wire. Catalepsy was recorded using a video camera and by an observer
protein/well. After 1 h of incubation, 50 μL/well of “Luciferin who was unaware of the treatment. Area under the curve (AUC)
Detection Reagent” was added to each well and, after an additional 20 throughout 3 h and One-Way Anova followed by Dunnett’s test were
min of incubation to generate and stabilize the luminescent signal, the calculated. Basal time was not included because this time point was
plate was read at the luminometer (Wallac Victor). The net signal used only to check that catalepsy was successfully induced in all
from reactions of substrate and MAO enzyme in the absence of animals.
inhibitors represents the positive control (total MAO activity), Molecular Modeling. Molecular modeling was performed using
whereas the signal from reaction of substrate and inhibitors without the Schrodinger software suite. Receptor and ligand structures were
MAO enzyme represents the negative control. Compounds were first prepared in Maestro 9.243 and refined using Macromodel 9.9.44
examined at fixed concentrations of 10 and 1 μmol/L. Then, if active Docking studies were carried out with Glide 5.735 using the SP scoring
at these concentrations, a concentration−response curve was function. Default settings were used unless stated otherwise.
performed. Results were expressed as percent of control values and, Docking Studies into MAO-B. The crystal structure of human
in the case of multiple concentrations, as IC50 (concentration causing a MAO-B in complex with safinamide (PDB code: 2V5Z)37 was selected
half maximal inhibition of control values). Four experiments were for docking studies. After the correction of valences of the FAD
performed in duplicate. cofactor and of the cocrystallized ligand, hydrogen atoms were added
A2A Receptor Binding Affinity. Membranes from human embryonic to the structure and protonation states for ionizable side chains were
kidney (HEK) 293 cells, stably transfected with the human adenosine chosen to be consistent with physiological pH. The all-atoms receptor
A2A receptor gene, were used in the radioligand binding experiments. structure was submitted to an energy minimization procedure using
Competition binding experiments were performed incubating the MMFFs force field45 to a convergence threshold of 0.05 kJ mol−1
membranes (5−10 μg of protein/sample) with a single concentration Å−1, holding all heavy atoms fixed. Starting structures of compound
of the A2A antagonist [3H]ZM241385 (Biotrend, Cologne, Germany) 14b and CSC were minimized with the MMFFs force field to a
(2 nmol/L) in the presence of fixed concentrations of test compounds convergence threshold of 0.05 kJ mol−1 Å−1. Glide grids were centered
in 96-well filter plates (MultiScreen system, cat. MAFBN0B10, on the cocrystallized safinamide, setting the enclosing box and
Millipore, Billerica, MA, USA) for 1 h at 4 °C in a total volume of bounding box dimensions to 30 Å and 10 Å, respectively. Twenty
200 μL/well of appropriate buffer (50 mmol/L Tris-HCl, pH 7.4, 10 docking poses were collected for each ligand. The top-ranked poses
mmol/L MgCl2). Some compounds were retested at increasing according to the GlideScore value obtained for compound 14b and
concentrations ranging from 0.01 nmol/L to 10 μmol/L. Nonspecific CSC were merged into the MAO-B crystal structure, and the resulting
binding was determined in the presence of 10 μmol/L cold ZM241385 complexes were energy-minimized using the MMFFs force field to a
(Tocris, Ellisville, MO, USA). At the end of incubation, bound and convergence threshold of 5 kJ mol−1 Å−1, keeping the protein
free radioligands were separated by filtering the 96-well filter plates backbone fixed.
using a Millipore filtration apparatus (MultiscreenHTS vacuum Docking Studies into A2A Receptor. The crystal structure of human
manifold). Filter plates were then washed several times with ice-cold A2A receptor in complex with XAC (PDB: 3REY)33 was retrieved from
buffer (50 mmol/L Tris-HCl, pH 7.4) and filter-bound radioactivity the Protein Data Bank and processed using the Protein Preparation
measured using a MicroBeta counter (PerkinElmer) after addition of Wizard. After the reconstruction of the missing Lys150-Gln157
30 μL/well of OptiPhase SuperMix scintillation cocktail (PerkinElm- sequence with Prime 3.0,46 the protonation states for all ionizable side
er). Four experiments were performed in duplicate. IC50 values were chains were assigned consistent with physiological pH. The overall
determined by nonlinear fitting strategies with the program GraphPad hydrogen bonding network was optimized by adjusting the
Prism. The Ki values were calculated from the IC50 values in tautomerization states of histidine residues and by sampling the
accordance with the Cheng−Prusoff equation.42 orientation of hydroxy and thiol groups as well as the side chain
In Vivo Experiments. All experiments were conducted in amides of asparagine and glutamine residues. Protein C- and N-termini
accordance with the guidelines for care and use of experimental were capped with acetyl and methylamino groups, respectively. The
animals of the European Communities Directive (86/609 EEC; all-atoms receptor structure was submitted to a restrained
27.01.1992, no.116) and approved by the company veterinarian and minimization procedure to the rmsd of 0.3 Å by applying the

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Journal of Medicinal Chemistry Article

OPLS2005 force field.47 A loop refinement procedure was then Actions of Adenosine A2A Receptor Antagonist KW 6002 on Drug-
performed with Prime46 on the Pro149-Gly158 sequence to optimize Induced Catalepsy and Hypokinesia Caused by Reserpine or MPTP.
the geometry of the newly inserted residues. Ten different Psychopharmacology 1999, 147, 90−95. (c) Kase, H. The adenosine
conformations were generated for the Pro149-Gly158 loop, and the A2A receptor selective antagonist KW6002: Research toward a novel
structure having the lowest energy was selected. Initial conformations non-dopaminergic therapy for Parkinson’s disease. Neurology 2003, 61,
of compounds 17f and 17q were energy-minimized by applying a S97−S100.
convergence threshold of 0.05 kJ mol−1 Å−1 using the OPLS2005 force (4) (a) Shimada, J.; Suzuki, F.; Nonaka, H.; Ishii, A.; Ichikawa, S. (E)-
field.47 Glide grids were centered on the cocrystallized XAC, setting 1,3-Dialkyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanthines: Potent and
the dimensions of enclosing and bounding boxes to 30 and 10 Å, Selective Adenosine A2 Antagonists. J. Med. Chem. 1992, 35, 2342−
respectively. Twenty docking poses were retained for each ligand and 2345. (b) Nonaka, Y.; Shimada, J.; Nonaka, H.; Koike, N.; Aoki, N.;
subsequently ranked according to their GlideScore value. The top- Kobayashi, H.; Kase, H.; Yamaguchi, K.; Suzuki, F. Photoisomerization
ranked poses for compounds 17f and 17q were selected and are of a Potent and Selective Adenosine A2 Antagonist, (E)-1,3-Dipropyl-
depicted in Figure 3C.

■
8-(3,4-dimethoxystyryl)-7-methylxanthine. J. Med. Chem. 1993, 36,
3731−3733. (c) Suzuki, F.; Shimada, J.; Ishii, A.; Nonaka, H.; Kosaka,
ASSOCIATED CONTENT N.; Ichikawa, S. Xanthine Derivatives. PCT Patent WO 92/06976,
*
S Supporting Information 1992. (d) Correa, M.; Wisniecki, A.; Betz, A.; Dobson, D. R.; O’Neill,
Experimental details and characterizations for compounds 2a,b, M. F.; O’Neill, M. J.; Salamone, J. D. The adenosine A2A antagonist
3a,b, 4a,b, 6, 8a,b, 9a,b, 10a−c, 11a−c, 12a−c, 13a−c, 14a−c, KF17837 reverses the locomotor suppression and tremulous jaw
movements induced by haloperidol in rats: possible relevance to
17b−p,r−w, 18a−d, 19a−d, 20a,b, 21−23, and 25; selectivity
parkinsonism. Behav. Brain Res. 2004, 148, 47−54.
profile of compound 17f. This material is available free of (5) Seale, T. W.; Abla, K. A.; Shamim, M. T.; Carney, J. M.; Daly, J.
charge via the Internet at [Link]

■
W. 3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo
antagonist of adenosine analogs. Life Sci. 1988, 43, 1671−1684.
AUTHOR INFORMATION (6) Müller, C. E.; Geis, U.; Hipp, J.; Schobert, U.; Frobenius, W.;
Corresponding Author Pawłowski, M.; Suzuki, F.; Sandoval-Ramìrez, J. Synthesis and
*For S.R.: phone, +390521905061; fax, +390521905006; E- structure-activity relationships of 3,7-dimethyl-1-propargylxanthine
mail, [Link]@[Link]. For G.P.: phone, +390722303313; derivatives, A2A-selective adenosine receptor antagonists. J. Med.
fax, +390722303320; E-mail, [Link]@[Link]. For Chem. 1997, 40, 4396−4405.
(7) (a) Sauer, R.; Maurinsh, J.; Reith, U.; Fülle, F.; Klotz, K.-N.;
P.M.: phone, +390691393906; fax, +390691393638; E-mail,
Müller, C. E. Water soluble phosphate prodrugs of 1-propargyl-8-
[Link]@[Link]. styrylxanthine derivatives, A2A selective adenosine receptor antago-
Notes nists. J. Med. Chem. 2000, 43, 440−448. (b) Hockemeyer, J.; Burbiel, J.
The authors declare no competing financial interest. C.; Muller, C. E. Multigram-scale syntheses, stability, and photo-

■ ACKNOWLEDGMENTS
We thank Anna Maria Russo (Sigma-Tau) for the excellent
reactions of A2A adenosine receptor antagonists with 8-styrylxanthine
structure: potential drugs for Parkinson’s disease. J. Org. Chem. 2004,
69, 3308−3318. (c) Vollmann, K.; Qurishi, R.; Hockemeyer, J.; Muller,
C. E.. Synthesis and properties of a new water-soluble prodrug of the
technical work. The authors from the universities of Urbino and adenosine A2A receptor antagonist MSX-2. Molecules 2008, 13, 348−
Parma gratefully acknowledge financial support to the project 359. (d) Yang, M.; Soohoo, D.; Soelaiman, S.; Kalla, R.; Zablocki, J.;
from Sigma-Tau.

■
Chu, N.; Leung, K.; Yao, L.; Diamond, I.; Belardinelli, L.; Shryock, J.
C. Characterization of the potency, selectivity, and pharmacokinetic
ABBREVIATIONS USED profile for six adenosine A2A receptor antagonists. Naunyn
AD, Alzheimer’s disease; CNS, central nervous system; m- Schmiedeberg’s Arch. Pharmacol. 2007, 375, 133−144.
CPBA, meta-chloroperoxybenzoic acid; CSC, (E)-8-(3- (8) (a) Baraldi, P. G.; Manfredini, S.; Simoni, D.; Zappaterra, L.;
chlorostyryl)caffeine; DME, 1,2-dimethoxyethane; DMF, dime- Zocchi, C.; Dionisotti, S.; Ongini, E. Synthesis of New Pyrazolo-[4,3-
e]1,2,4-triazolo[1,5-c]pyrimidine and 1,2,3-triazolo[4,5-e]1,2,4-
thylformamide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
triazolo[1,5-c]pyrimidine Displaying Potent and Selective Activity as
carbodiimide; MAO-B, monoamine oxidase B; PD, Parkinson’s A2A Adenosine Receptor Antagonists. Bioorg. Med. Chem. Lett. 1994,
disease; SAR, structure−activity relationship; TBAB, tetrabuty- 4, 2539−2544. (b) Zocchi, C.; Ongini, E.; Conti, A.; Monopoli, A.;
lammonium bromide; TEA, triethylamine

■
Negretti, A.; Baraldi, P. G.; Dionisotti, S. The Non-Xanthine
Heterocyclic Compound SCH 58261 is a New Potent and Selective
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