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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 October 15.
Published in final edited form as:
J Med Chem. 1994 September 30; 37(20): 3373–3382.
PMCID: PMC3471218
NIHMSID: NIHMS410767

Structure–Activity Relationships of 1,3-Dialkylxanthine Derivatives at Rat A3 Adenosine Receptors

Abstract

1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.

Introduction

Three major classes of adenosine receptors (A1, A2, and A3) have been characterized pharmacologically1,4 and through cloning.2,3,6 A1 receptors are coupled to the inhibition of adenylate cyclase through Gi proteins4,5 and have also been shown to couple to other second messenger systems, including inhibition or stimulation of phosphoinositol turnover and activation of ion channels.1,4 A2 receptors are further divided into two subtypes, A2a and A2b, at which adenosine agonists activate adenylate cyclase with high and low affinity, respectively. The A3 receptor sequence was first identified in a rat testes cDNA library,7 and this sequence, later cloned by homology to other G-protein coupled receptors from a rat brain cDNA library, was shown to correspond to a novel, functional adenosine receptor.6 A3 receptors are coupled to both inhibition of adenylate cyclase and activation of protein kinase C as second messengers. The rat A3 receptor was unlike the previously characterized adenosine receptors in lack of either displacement of radioligand binding or functional antagonism by the usual high affinity xanthine ligands. The A3 receptor was recently shown to be identical to an atypical adenosine receptor present in the RBL-2H3 rat mast cell line,5,8 the activation of which promotes degranulation.

In addition to possible involvement in the inflammatory response, activation of A3 receptors is associated with cardiovascular actions. Fozard and Carruthers34 have attributed a xanthine-insensitive component of the hypotensive effects of adenosine agonists in the rat to A3 receptor activation. Activation of A3 receptors has been suggested by Downey and colleagues11 to be involved in the cardioprotective effect of preconditioning by adenosine agonists. The occurrence of A3 receptors in the brain6,9,10 and testes7 also suggests that it may be important in regulation of central nervous system (CNS) function and reproduction.

We have studied in detail the structure–activity relationships for adenosine derivatives as agonists at A3 receptors.12,13 We recently reported9 that an N6- and 5′-substituted adenosine derivative, N6-(3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine (IB-MECA), is 50-fold selective for rat brain A3 receptors in binding experiments and is also selective in in vivo behavioral experiments.

We have reported that xanthines that are potent antagonists at rat, rabbit, and human A1 and A2 receptors only weakly displaced the binding of the radioligand [125I]APNEA (N6-[2-(4-amino-3-iodophenyl)-ethyl]adenosine) from cloned rat A3 receptors even at concentrations in the 10−4 M range.12 Linden et al.14 found that xanthines, particularly those containing anionic groups, bind appreciably to cloned sheep A3 receptors, but generally with less affinity than at A1 and A2 receptors in a variety of species.1 The human A3 receptor was recently cloned15 and found to resemble the sheep A3 receptor, in the respect that many potent xanthines bind in the micromolar range. Thus, for xanthines as antagonists,14 an unusually large species dependence of affinity at A3 receptors is apparent, perhaps even suggesting multiple subtypes of A3 receptors. Xanthines that are generally A3-selective across species are needed as pharmacological and biochemical probes to define more clearly the physiological role, distribution, and regulation of A3 adenosine receptors.

Results

Xanthine analogues modified with alkyl substituents at 1-, 3-, and 7-positions and with carboxylic acid groups on the 8-position substituent were synthesized (Schemes 14) and tested in radioligand binding assays (Tables 1 and and2)2) for affinity at rat brain A1, A2a, and A3 adenosine receptors. At A3 receptors affinity was measured in CHO cells stably expressing cloned rat brain A3 receptors.6 The radioligand used for binding to A3 receptors was the recently reported [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide).16 Affinity at A1 receptors was measured in rat cortical membranes using [3H]-(R)-N6-(phenylisopropyl)adenosine17 ([3H]PIA) and at A2a affinity in rat striatal membranes using [3H]CGS 21680.18

Scheme 1
Synthesis of 1,3-Dialkylxanthinesaa
Scheme 4
Synthesis of an Intermediate for Carboxylate Derivatization of Xanthinesa
Table 1
Affinities of Xanthine Derivatives in Radioligand Binding Assays at Rat Brain A1, A2a, and A3 Receptorsad with Ratios of Selectivity for A1 or A2 Receptors vs A3 Receptors
Table 2
Affinities of Xanthine Derivatives in Radioligand Binding Assays at Rat Brain A1, A2a, and A3 Receptorsad with Ratios of Selectivity for A1 or A2 Receptors vs A3 Receptors

Synthesis of 1,3-dibutylxanthine, 4, was performed by the classical method starting from N,N′-dibutylurea, 36a (Scheme 1). 6-Amino-1,3-dibutyluracil, 38a, was synthesized by condensation of N,N′-dibutylurea, 36a, and cyanoacetic acid in the presence of acetic anhydride followed by base-catalyzed cyclization.33 Nitrosation and Pd/C-catalyzed hydrogenation gave 5,6-diamino-1,3-dibutyluracil, 40a, which without purification was then condensed with formic acid in the presence of EDAC to yield 6-amino-5-formamido-1,3-dibutyluracil. 1,3-Dibutylxanthine, 4, was obtained by cyclization of 6-amino-5-formamino-1,3-dibutyluracil under basic conditions. Similarly, 3-benzyl-1-butylxanthine, 7, was prepared starting from N-benzyl-N′-butylurea.

8-Substituted xanthines were synthesized through the condensation of 5,6-diaminouracils, 40, with various anhydrides (Scheme 2) or an aldehyde, 45 (Scheme 3), followed by cyclization either in base or by the NaIO4 method,20 respectively. Detailed characterization of the compounds synthesized is listed in Table 3.

Scheme 2
Synthesis of 8-(Carboxyalkyl)-Substituted Xanthines
Scheme 3
Synthesis of 8-[[(Carboxymethyl)oxy]phenyl]-Substituted Xanthines
Table 3
Characterization of Xanthine Derivatives

8-Unsubstituted xanthines in which 1-, 3-, and 7-substituents were modified were compared (Table 1). A comparison of 1,3-dibutylxanthine, 4, and the corresponding 3-benzyl analogue, 7, indicated that for these simple xanthines, the presence of a bulky 3-position substituent only slightly increased A3 affinity (2-fold) and had no effect on A1 affinity, whereas A2 affinity was dramatically increased (24-fold). Limited aqueous solubility of the dibenzyl analogue, 6, prevented a precise comparison with 1,3-dibutylxanthine. A benzyl vs methyl group at the 7-position did not have a major effect on A2a or A3 affinity, but somewhat enhanced A1 affinity (5-fold).

8-Cyclopentyl-1,3-dipropylxanthine (CPX), 8, and 8-(3-chlorostyryl)caffeine (CSC), 9,21 two xanthines found previously to be highly selective for either A1 or A2a adenosine receptors,1,22 respectively, remained selective vs rat A3 receptors. Compound 9 was nearly inactive at A3 receptors. Among a related series of 8-styrylxanthines that showed selectivity for A2a receptors22 was a water soluble succinylamino derivative, 10. Compound 10 very weakly displaced [125I]AB-MECA binding in transfected CHO cells (Ki value 189 µM). An attempt to improve the A3 affinity by lengthening the alkyl carboxylate chain by one and two methylenes, e.g., 11 and 12b, was unsuccessful.

The susceptibility of 8-styrylxanthines to photoisomerization has been noted;23 thus binding assays of affinity of compounds 912b were carried out in the dark. Although the affinities at A3 receptors were minimal, the selectivity for A2a vs A1 receptors was maintained. Selectivity ratios (A2a vs A1) when the assays were performed in the dark were 1590 (10), 2300 (11), and 900 (12b).

A caffeine 8-acrylic acid derivative, 13, 1,3,7-trimethyl-8-(trans-2-carboxyvinyl)xanthine, which was reported to be moderately selective for rat A2a vs A1 receptors but not very potent,24 was found to be selective also for rat A3 vs A1 receptors. Lengthening the 1,3-dialkyl substituent, as in the corresponding dibutyl derivative, 16, greatly enhanced affinity at A1 receptors, and at A3 receptors affinity was unchanged, resulting in the loss of selectivity. The presence of a hydrogen at the 7-position, in 14, slightly enhanced affinity relative to 16 in parallel at A1, A2a, and A3 receptors. The methyl ester, 15, of the N7-H analogue was more potent at A1 and A2a receptors than the corresponding carboxylic acid, 14, as expected29 for a neutral vs an anionic 8-functionalized chain.

A series of 8-alkyl carboxylic acids (1719) indicated that optimal affinity at A3 relative to A1 and A2a receptors was observed for the 8-(3-carboxypropyl) derivative, 18, although none of these xanthines were truly selective for rat A3 receptors.

The 1,3-dimethyl analogue of 18 was prepared, since smaller 1,3-dialkyl substituents seemed to favor A3 selectivity (compare 13 vs 16), although not A3 affinity. Compound 20 was selective for A3 vs A1 receptors, although not very potent. Inclusion of the 2-thio modification in 21, 1,3-dimethyl-8-(carboxypropyl)-2-thioxanthine, resulted in enhanced affinity at all adenosine receptors subtypes and increased selectivity for A3 vs A2a receptors. The A3 receptor affinity for 21 vs 20 was enhanced by 10-fold.

The affinities of 8-arylxanthines at adenosine receptors were compared (Table 2). Linden et al.14 reported that at sheep A3 receptors both sulfonate- and carboxylate-containing 8-arylxanthines were among the most potent antagonists, many of which had Ki values in the submicromolar range. Thus we compared the affinities of a number of anionic derivatives of 1,3-dipropyl-8-phenylxanthine at rat A3 receptors. Both the p-carboxylate group, 23,25 and the p-acrylate group, 24 (BW1433),26 were more favorable in A3 affinity than the p-sulfonate group, 22. The affinities of 22 and 23 at both A1 and A2a receptors were nearly identical. The p-acrylate group in 24 resulted in enhanced (9-fold) A1 affinity, but the affinities of 22, 23, and 24 at A2a receptors were identical. A xanthine carboxylic congener, XCC, 26,19 containing a p-[(carboxymethyl)oxy] group was slightly less potent at A3 receptors than the corresponding 8-(4-carboxyphenyl)-1,3-dipropylxanthine, 23.25 On the basis of the finding that a carboxylate group is more desirable than a sulfonate group at rat A3 receptors, the affinities of additional carboxylate derivatives of 8-arylxanthines were compared.

The xanthine amine congener, XAC, 31,19 was highly selective for A1 and A2a receptors vs A3 receptors. The Ki value of XAC (29 µM) was similar to values for compounds 2224, indicating that rat A3 receptors, unlike A1 and A2a receptors, tolerate equally a distal carboxylate group and a distal amino group.

The iodinated xanthine derivative, 27, also known as I-ABOPX or BWA 577,27 had 65-fold enhanced affinity at A3 receptors in comparison to the parent XCC, 26. The enhancement of affinity by the 3-(3-iodo-4-aminobenzyl) group at A1 and A2a receptors was only 2–3-fold. Nevertheless, the affinity of 27 at rat A3 receptors was much less than at sheep A3 receptors,14 and it was not A3 selective.

Lengthening the 1,3-dialkyl substituents of XCC was found to increase affinity somewhat at A2a and A3 receptors, but A1 affinity was unaffected. The corresponding 1,3-dibutylxanthine carboxylic congener, 30, displayed a Ki value at A3 receptors of 28 µM, although the compound was still highly A1 selective. Introduction of unsaturation in the 1,3-dialkyl groups, 25, vs the corresponding 1,3-dipropyl analogue, 26, resulted in a 2-fold loss of affinity at A2a and A3 receptors and a 13-fold loss of affinity at A1 receptors.

A 2-thio vs 2-oxo substitution28 enhanced A3 affinity for 8-arylxanthines as well as for 8-alkylxanthines. The A3 affinity of 2-thioXCC, 28 (Ki value of 6.8 µM), was 11-fold greater than the affinity of XCC, 26.

The A3 affinity was not highly dependent on the distance of the carboxylate group from the xanthine pharmacophore. A glycine conjugate of XCC, 29,29 was equipotent to the parent carboxylate 26 at A1 and A3 receptors and 5-fold more potent at A2a receptors. Succinyl XAC, 32,30 although definitely not A3 selective, was nearly equipotent to XAC at A3 receptors and considerably less potent than XAC at A1 and A2a receptors, inconsistent with a proposed role of an anionic group in enhancing A3 affinity as noted for sheep A3 receptors.14 The A1, A2a, and A3 receptor affinities of this succinyl derivative, 32, were nearly the same as for the slightly shorter glycine conjugate, 29, and thus similar to the considerably shorter XCC, 26.

Introduction of multiple carboxylate groups on the 8-aryl substituent was accomplished by attaching a derivative of iminodiacetic acid to XAC followed by succinylation of the secondary amine (Scheme 4). However, the dicarboxylate, 35, did not display enhanced affinity at A3 receptors. The affinity at A1 and A2a receptors of 35 vs the monoanionic and Zwitterionic intermediates, 33 and 34, respectively, was greatly diminished.

We have shown that theophylline lacks functional antagonistic properties vs A3 agonist-elicited adenylate cyclase inhibition in A3-transfected CHO cells.12 We examined the antagonist properties of several carboxylic acid derivatives in a functional assay at A3 receptors, since these derivatives were more potent in binding than theophylline. Adenylate cyclase was inhibited by N6-benzyl-NECA in the concentration range of 10−9–10−4 M in transfected CHO cells (maximal degree of inhibition was 40%).12a Compounds 21 or 24 at concentrations as high as 20 µM (or 100 µM during a preincubation) showed no significant effect on potency or maximal effect of agonist inhibition of adenylate cyclase.

Discussion

In this study xanthine derivatives were examined for selectivity for rat A3 receptors. There is an uncommonly large species dependence of the affinity of xanthines at A3 receptors. Very few of the compounds studied were actually A3 selective in the rat. Compound 24 was 710-fold more potent at sheep A3 receptors14 than at rat A3 receptors. Compound 27 was 400-fold more potent at sheep A3 receptors14 than at rat A3 receptors. Certain xanthines were also found to be moderately potent in displacing radioligand from human A3 receptors.15 Thus, it is to be expected that some of these xanthine derivatives are potentially selective for A3 receptors in sheep and human tissue and possibly in other species.

A reported lead for enhancing A3 affinity at sheep and human A3 receptors was an anionic group on the 8-substituent.14,15 In this study the presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed29 an anionic group tended to diminish potency at A1 and A2a receptors.

One may speculate on which amino acid residues on the A3 receptor may interact with carboxylate group in sheep and human A3 receptors. Presumably the residue should be a positively charged residue that is conserved between sheep and human A3 receptor sequences but is absent in A1 or A2a receptors (across species) and in rat A3 receptors. In the vicinity of the transmembrane region, which is certainly but perhaps not exclusively involved in ligand binding,12 one residue which satisfies these conditions is the Arg173 residue (human) occurring approximately at the boundary of the fifth transmembrane helix and the second extracellular loop. The corresponding residues are Gly175 in the rat A3 receptor and Ser176/Pro173 in the human A1/A2a receptors. Site-directed mutagenesis and molecular modeling will be essential tools in locating the ligand binding site on the A3 receptor and determining if Arg173 plays a role. We recently proposed a molecular model for binding of nucleosides to A3 receptors based on the presence of a conserved His residue in the seventh transmembrane helix as a ribose-anchoring site.12 We demonstrated that 1,3-dialkylxanthine-7-ribosides displayed greatly enhanced affinity vs the parent xanthines at rat A3 receptors.

Several of the more potent derivatives in this study (compounds 21 and 24) were too weak to functionally antagonize the effects of an A3 agonist. Compound 27, BWA 522, was recently used as a weak antagonist in vivo at rat A3 receptors.31 Our findings suggest that although considerable affinity for A3 receptors is present (µM), it is still somewhat selective (32-fold) for A1 receptors.

In conclusion, the development of A3 receptor antagonists of general applicability across species remains a challenge. A3 receptor antagonists may prove useful as antiinflammatory agents. This study has identified several leads, such as 2-thio and 8-(3-carboxypropyl)-xanthines modifications. It is possible also that non-xanthine antagonists will also provide leads, although a screen of six non-xanthine A1 antagonists of diverse structure indicated negligible affinity at rat A3 receptors.12

Experimental Section

Chemistry

New compounds were characterized (and resonances assigned) by 300 MHz proton nuclear magnetic resonance spectroscopy using a Varian GEMINI-300 FT-NMR spectrometer. Unless noted, chemical shifts are expressed as ppm downfield from tetramethylsilane. Synthetic intermediates were characterized by chemical ionization mass spectrometry (NH3) on a JEOL SX102 mass spectrometer. In the EI mode accurate mass was determined using a VG7070F mass spectrometer. C, H, and N analyses were carried out by Atlantic Microlabs (Norcross, GA), and ±0.4% was acceptable. All xanthine derivatives were judged to be homogeneous using thin layer chromatography (silica, 0.25 mm, glass backed, Alltech Assoc., Deerfield, IL) following final purification. XAC, CPX, CSC, SPX, and 2-chloroadenosine were obtained from Research Biochemicals International (Natick, MA). Analytical TLC plates and silica gel (230–400 mesh) were purchased from VWR (Bridgeport, NJ). The following compounds were kindly provided by the sources stated: 23 (Dr. John Daly, NIDDK), 24 (Dr. Susan Daluge, Burroughs Wellcome), 27 (Dr. Joel Linden, University of Virginia, Charlottesville VA), 28 (Dr. John Neumeyer, Research Biochemicals International). Compound 13 was prepared as described in ref 24. Compound 25 was prepared as described in ref 20. Compound 26 was prepared as described in ref 19. Compound 29 was prepared as described in ref 29. Compound 32 was prepared as described in ref 30.

1,3-Di-n-butylxanthine (4)

A mixture of compound 39a (3.1 g, 11.6 mmol) and 5% Pd/C in dry DMF (30 mL) was hydrogenated at 50 psi until it become colorless solution (ca. 2 h). The catalyst was filtered off through Celite pad, and the filtrate was mixed with 88% formic acid (15 mL) and then was added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.1 g, 5.74 mmol) at 0 °C under nitrogen. The violet solution was stirred for 3 h at room temperature. DMF and formic acid were removed by rotary evaporation, and the solid residue was mixed with 2 N aqueous sodium hydroxide (50 mL, 100 mmol). The reaction mixture was heated for 1 h at reflux. The yellow solution was cooled and neutralized with 6 N hydrochloric acid to pH 3. The solid was collected by filtration, washed with water, and dried to give compound 4 (2.3 g, 75%): 1H NMR (DMSO-d6) δ 1.41–1.47 (m, 6 H, 2 × CH3), 1.61–1.68 (m, 4 H, 2 × CH2), 1.74–1.79 (m, 2 H, CH2), 1.82–1.85 (m, 2 H, CH2), 3.84 (t, J = 7.3 Hz, 2 H, N-CH2), 3.98 (t, J = 7.3 Hz, 2 H, N-CH2), 8.10 (s, 1 H, H-8), 13.7 (br s, 1 H, exchanged with D2O, NH).

3-Benzyl-1-butylxanthine (7)

A solution of 6-amino-1-benzyl-3-butyl-5-nitrosouracil and 6-amino-3-benzyl-1-butyl-5-nitrosouracil (39b, 10 g, 33 mmol) in DMF (100 mL) was hydrogenated using 5% Pd/C (0.5 g) catalyst until the pink color disappear to give a mixture of 5,6-diaminouracil derivatives. After removal of catalyst by filtration through Celite pad, the filtrate was mixed with 88% formic acid (25 mL) and then 1-[3-(dimethylamino)propyl]-1-ethylcarbodiimide (3.5 g, 18 mmol). The reaction mixture was stirred for 2.5 h at room temperature. It was concentrated to dryness, and 2 N NaOH (100 mL) was added. The mixture was refluxed for 1 h, cooled, neutralized with 6 N HCl, and extracted with ethyl acetate (200 mL). The organic layer was washed with brine (50 mL), dried over anhydrous MgSO4, filtered, and concentrated to give a brown solid which was purified on silica gel column chromatography (CHCl3–MeOH, 50:1 → 20:1) to give a mixture of 1-benzyl-3-butylxanthine and 3-benzyl-1-butylxanthine (6.76 g, 68.5%) as a colorless solid: 1H NMR (DMSO-d6) δ 0.89 (t, J = 7.3 Hz, 3 H, CH3), 1.23–1.68 (m, 4 H, CH2CH2), 3.88, 4.00 [2 × t, J = 7.3 Hz, 2 H (2.2:1), N-CH2], 5.08, 5.18 [2 × s, 2 H (1:2.4), N-CH2Ph], 7.20–7.35 (m, 5 H, Ph), 8.02, 8.04 [2 × s, 1 H (2.4:1), H-8, 13.60 (br s, 1 H, NH). In order to separate two isomers, the solid was recrystallized in ethyl acetate to yield compound 7: 1H NMR (CDCl3) δ 0.96 (t, J = 7.3 Hz, 3 H, CH3), 1.40 (sextet, J = 7.5 Hz, 2 H, CH2), 1.65 (m, 2 H, CH2), 4.07 (pseudo t, J = 7.6 and 7.4 Hz, N-CH2), 5.33 (s, 2 H, NCH2Ph), 7.28–7.53 (m, 5 H, Ph), 7.77 (s, 1 H, H-8), 11.85 (br s, 1 H, exchanged with D2O, NH).

1,3,7-Trimethyl-8-[3-[[(3-carboxypropyl)carbonyl]amino]styryl]xanthine (11)

was prepared by acylation of the corresponding arylamine using glutaric anhydride by a procedure similar to that reported for compound 10 in ref 22: 1H NMR (DMSO-d6) δ 1.83 (m, 2 H, CH2), 2.28 and 2.38 (each: m, 2 H, COCH2), 3.23 (s, 3 H, N3-CH3), 3.47 (s, 3 H, N1-CH3), 4.03 (s, 3 H, N7-CH3), 7.2–7.6 (m, 3 H, Ar), 7.24 and 7.61 (each: d, J = 15.8 Hz, 1 H, vinyl), 7.90 (m, 1 H, Ar).

1,3,7-Trimethyl-8-[3-[[[4-(methyloxycarbonyl)butyl]-carbonyl]amino]styryl]xanthine (12a)

1,3,7-Trimethyl-8-(3-aminostyryl)xanthine22 (33.1 mg, 106 µmol), adipic acid monomethyl ester (38 mg, 0.24 mmol), and EDAC (47 mg, 0.24 mmol) were suspended in DMF (1 mL) and sonicated for 5 min. The mixture was treated with DMAP (3 mg), and sonicated for 30 min. Upon addition of ice water, a fine yellow precipitate (27 mg, 57%) formed: 1H NMR (DMSO-d6) δ 1.60 (br s, 4 H, 2 × CH2), 2.35 (br s, 4 H, 2 × CH2), 3.23 (s, 3 H, N3-CH3), 3.47 (s, 3 H, N1-CH3), 3.59 (s, 3 H, COOCH3), 4.03 (s, 3 H, N7-CH3), 7.21–7.89 (m, 3 H, Ar), 7.24 and 7.61 (each: d, J = 15.8 Hz, 1 H, vinyl), 9.98 (m, 1 H, Ar).

1,3,7-Trimethyl-8-[3-[[(4-carboxybutyl)carbonyl]amino]styryl]xanthine (12b)

Compound 12a (14 mg, 45 µmol) was dissolved in methanol (1 mL) and treated with 0.2 mL of 1 N NaOH. The solution was heated to 50 °C and cooled. The reaction mixture was acidified with 4 N hydrochloric acid, and the precipitate was collected and dried to give compound 12b (12.7 mg, 66%): 1H NMR (DMSO-d6) δ 1.59 (m, 4 H, 2 × CH2), 2.25 and 2.34 (m, 4 H, 2 × CH2), 3.23 (s, 3 H, N3-CH3), 3.47 (s, 3 H, N1-CH3), 4.03 (s, 3 H, N7-CH3), 7.21–7.89 (m, 3 H, Ar), 7.24 and 7.61 (each: d, J = 15.8 Hz, 1 H, vinyl), 9.98 (m, 1 H, Ar).

1,3-Dibutyl-8-(trans-2-carboxyvinyl)xanthine (14)

A solution of 39a (1.95 g, 7.28 mmol) and 5% Pd/C (200 mg) in dry DMF (30 mL) was hydrogenated for 3 h. The catalyst was removed by filtration through Celite, and the filtrate was divided for synthesis of several compounds. An aliquot of the filtrate (12 mL) was mixed with monomethyl fumarate (0.258 g, 1.79 mmol), DMAP (50 mg, 0.41 mmol), imidazole (100 mg, 1.47 mmol), and EDAC (0.4 g, 1.73 mmol) and sonicated for 3 h. DMF was removed by rotary evaporation, and the residue was mixed with 2 N NaOH (40 mL). After the reaction mixture was heated for 1 h, it was cooled, filtered, and neutralized with 6 N HCl. The solid was collected by filtration and dried to give compound 14 (0.17 g, 32%): 1H NMR (DMSO-d6) δ 0.87–0.93 (m, 6 H, 2 × CH3), 1.26–1.34 (m, 4 H, 2 × CH2), 1.51–1.66 (m, 4 H, 2 × CH2), 3.88 (pseudo t, J = 7.4 and 7.2 Hz, 2 H, N-CH2), 4.00 (pseudo t, J = 7.2 and 7.0 Hz, 2 H, N-CH2), 6.76 and 7.29 (each: d, J = 15.9 Hz, 1 H, vinyl).

1,3-Dibutyl-7-methyl-8-[trans-2-(methyloxycarbonyl)-vinyl]xanthine (15)

Compound 14 (78 mg, 0.23 mmol) was dissolved in DMF (2 mL) and treated with methyl iodide (0.1 mL, 1.6 mmol) and potassium carbonate (0.1 g). After heating at 50 °C for 30 min, ice water was added to precipitate the product (77 mg, 91%): 1H NMR (DMSO-d6) δ 0.87–0.93 (m, 6 H, 2 × CH3), 1.25–1.34 (m, 4 H, 2 × CH2), 1.49–1.66 (m, 4 H, 2 × CH2), 3.34 (s, 3 H, COOCH3), 3.87 (pseudo t, J = 7.4 and 6.9 Hz, 2 H, N-CH2), 3.99 (pseudo t, J = 7.2 and 6.9 Hz, 2 H, N-CH2), 4.04 (s, 3 H, N7-CH3), 6.80 and 7.62 (each: d, J = 15.5 Hz, 1 H, vinyl).

1,3-Dibutyl-7-methyl-8-(trans-2-carboxyvinyl)xanthine (16)

Compound 15 (66 mg, 0.18 mmol) was dissolved in DMF (2 mL) and treated with 1 mL of 1 N NaOH. After warming to 40 °C for 10 min, the solution was acidified with HCl. The precipitate was collected and dried (42 mg, 66%): 1H NMR (DMSO-d6) δ 0.90 (m, 6 H, 2 × CH3), 1.30 (m, 4 H, 2 × CH2), 1.54 (m, 4 H, 2 × CH2), 1.66 (m, 4 H, -CH2CH2), 3.87 (t, 2 H, N-CH2), 4.00 (t, 2 H, CH2), 6.76 and 7.54 (each: d, J = 15.8 Hz, 1 H, vinyl).

4-(1,3-Dibutylxanth-8-yl)propionic Acid (17)

An aliqout (7 mL) of the solution of hydrogenated compound 39a (see procedure for 14) was treated with succinic anhydride (0.19 g, 0.19 mmol) overnight. Half saturated NaCl was added, and the slowly forming insoluble residue was separated by decantation. This residue was dissolved in a 1:1 mixture of methanol and 4 N NaOH and heated to reflux for 30 min. Upon acidification, a precipitate formed, which was collected and dried (159 mg, 45% overall): 1H NMR (DMSO-d6) δ 0.89 (m, 6 H, 2 × CH3), 1.27 (m, 4 H, 2 × CH2), 1.50 and 1.63 (each: m, 2 H, CH2), 2.71 (pseudo t, J = 7.7 and 6.8 Hz, 2 H, CH2), 2.91 (pseudo t, J = 7.2 and 7.0 Hz, 2 H, CH2), 3.86 (pseudo t, J = 7.4 and 6.9 Hz, 2 H, N-CH2), 3.95 (t, J = 7.2 Hz, 3 H, CH2).

4-(1,3-Dibutylxanth-8-yl)butyric acid (18)

was prepared by a similar procedure using glutaric anhydride: 1H NMR (DMSO-d6) δ 0.89 (pseudo t, J = 7.3 and 7.2 Hz, 6 H, 2 × CH3), 1.24–1.31 (m, 4 H, 2 × CH2), 1.50 and 1.62 (each: m, 2 H, CH2), 1.90 (m, 2 H, aliphatic CH2), 2.27 (t, J = 7.3 Hz, 2 H, CH2), 2.71 (pseudo t, J = 7.4 and 7.3 Hz, 2 H, CH2), 3.86 (t, J = 7.3 Hz, 2 H, N-CH2), 3.96 (t, J = 7.3 and 6.8 Hz, 2 H, CH2). MS (CI) 368, 351, 333.

4-(1,3-Dibutylxanth-8-yl)pentanoic Acid (19)

A solution of 39a (0.31 g, 1.15 mmol) and 5% Pd/C (26 mg) in dry DMF (20 mL) was hydrogenated for 15 h. After filtration of Pd/C through a Celite pad, the filtrate treated with DMAP (37.6 mg, 0.03 mmol) and EDAC (332 mg, 1.73 mmol) for 3.3 h at room temperature. DMF was removed by rotary evaporation, and the residue was mixed with 2 N NaOH (20 mL). After the reaction mixture was heated for 2 h, it was cooled, filtered, and neutralized with 6 N HCl. The solid was collected by filtration and dried to give compound 19 (31.5 mg, 13%). Analytical sample was prepared by dissolving 19 in DMF/concentrated NH4OH, filtering, neutralizing with formic acid, and crystallizing with water: 1H NMR (DMSO-d6) δ 0.89 (m, 6 H, 2 × CH3), 1.28 (m, 4 H, 2 × CH2), 1.51 (m, 4 H, 2 × CH2), 1.65 (m, 4 H, CH2CH2), 2.23 (pseudo t, J = 7.3 and 7.2 Hz, 2 H, CH2), 2.28 (pseudo t, J = 7.3 and 7.2 Hz, 2 H, CH2), 3.86 (pseudo t, J = 7.5 and 7.2 Hz, 2 H, N-CH2), 3.96 (pseudo t, J = 7.2 and 7.1 Hz, 2 H, CH2), 11.98 (s, 1 H, exchanged with D2O, COOH), 13.14 (s, 1 H, exchanged with D2O, NH).

General Procedure for Scheme 2. 4-(1,3-Dimethylxanth-8-y1)butyric Acid (20)

Glutaric anhydride (0.5 g, 4.38 mmol) was added to a red solution of 5,6-diamino-1,3-dimethyluracil (0.5 g, 2.94 mmol, purchased from Aldrich Chemical Co.) in anhydrous DMF (10 mL), and the reaction mixture was stirred at room temperature for 17 h. The yellow solution was concentrated to dryness to give a yellow solid, which was mixed with 2 N NaOH (15 mL). After heating for 1.5 h at 75 °C, the yellow solution was cooled and neutralized with 6 N HCl. The solid was collected by suction and washed with small quantity of water and dried to give compound 20 (0.436 g, 56%) as a colorless solid. 1H NMR (DMSO-d6) δ 1.94 (sextet, J = 7.4 Hz, 2 H, CH2CH2CH2), 2.27 (t, J = 7.3 Hz, 2 H, CH2), 2.72 (t, J = 7.5 Hz, 2 H, CH2), 3.33, 3.42 (each: s, 3H, CH3), 12.10 (s, 1 H, exchanged with D2O, COOH), 13.20 (s, 1 H, exchanged with D2O, NH).

4-(1,3-Dimethyl-2-thioxanth-8-yl)butyric Acid (21)

A solution of 6-amino-1,3-dimethyl-5-nitroso-2-thiouracil (39c, 0.5 g, 2.5 mmol) in anhydrous DMF (100 mL) was hydrogenated for 2 days in the presence of 5% Pd/C (0.1 g), and the catalyst was removed by filtration through Celite pad. The filtrate was treated with glutaric anhydride (0.43 g, 3.77 mmol) for 18 h at room temperature. After DMF was removed in vacuo, the solid was dissolved in 2 N NaOH (16 mL), heated for 1.5 h at 80 °C, and neutralized with 6 N HCl. The solid formed was filtered and dried to give 21 (0.337 g, 48%): 1H NMR (DMSO-d6) δ 1.94 (quintet, J = 7.3 Hz, 2 H, CH2CH2-CH2), 2.29 (pseudo t, J = 7.3 and 7.2 Hz, 2 H, CH2), 2.78 (t, J = 7.5 Hz, 2 H, CH2), 3.68, 3.87 (2 × s, 2 × 3 H, 2 × CH3), 12.10 (s, 1 H, exchanged with D2O, COOH), 13.60 (s, 1 H, exchanged with D2O, NH).

[4-(1,3-Dibutylxanth-8-yl)phenoxy]acetic Acid (30)

A solution of 6-amino-1,3-dibutyl-5-nitrosouracil (39a, 2.68 g, 10 mmol) in methanol (27 mL) was hydrogenated in the presence of 5% Pd/C (0.2 g). Catalyst was removed by suction through Celite pad, and the filtrate was mixed with (4-formylphenoxy)-acetic acid (1.8 g, 10 mmol). The reaction mixture was stirred for 1 h on a stream of steam bath. It was cooled, and the solid was collected by suction and dried to give [4-[[(6-amino-1,3-dibutyluracil-5-yl)imino]methyl]phenoxy]acetic acid (46c, 3.24 g, 78%): 1H NMR (DMSO-d6) δ 0.86–0.93 (m, 5 H, 2 × CH3), 1.24–1.36 (sextet, J = 7.5 Hz, 4 H, 2 × CH2), 1.45–1.58 (m, 4 H, 2 × CH2), 3.80 (pseudo t, J = 7.5 and 6.9 Hz, 2 H, N-CH2), 3.95 (pseudo t, J = 7.5 and 7.2 Hz, 2 H, N-CH2), 4.72 (s, 2 H, CH2COOH), 6.93 (d, J = 8.6 Hz, 2 H, Ph), 7.28 (s, 2 H, exchanged with D2O, NH2), 7.32 (d, J = 8.7 Hz, 2 H, Ph), 9.67 (s, 1 H, N=CH), 12.99 (s, 1 H, exchanged with D2O, COOH). Anal. Calcd for C21H27N4O5: C, 60.71; H, 6.55; N, 13.49. Found: C, 60.60; H, 6.72; N, 13.45.

To a solution of 46c (2.9 g, 7.0 mmol) in DMF (50 mL) was added sodium periodate (1.5 g, 7 mmol) in water (8 mL), and the mixture became clear immediately. While the reaction mixture was stirring for 3 h, a solid formed and it was filtered (1.1 g), and the filtrate was concentrated to dryness. The residue was triturated with ether–methanol, and the solid was filtered by suction (2.4 g). The combined solid was recrystallized from 4 N NaOH–AcOH to yield compound 30 (1.66 g, 58%) as a colorless solid. Analytical sample of 30 was prepared by dissolving in DMF/concentrated NH4OH and acidifying with formic acid. The solid was filtered and dried in vacuo overnight: 1H NMR (DMSO-d6) δ 0.87–0.94 (m, 5 H, 2 × CH3), 1.26–1.36 (sextet, J = 7.5 Hz, 4 H, 2 × CH2), 1.49–1.59 (m, 2 H, CH2), 1.65–1.74 (m, 2 H, CH2), 3.89 (pseudo t, J = 7.5 and 7.1 Hz, 2 H, N-CH2), 4.05 (pseudo t, J = 7.1 and 6.9 Hz, 2 H, N-CH2), 4.77 (s, 2 H, CH2COOH), 7.04 (d, J = 9.3 Hz, 2 H, Ph), 8.04 (d, J = 8.6 Hz, 2 H, Ph), 13.02 (s, 1 H, exchanged with D2O, COOH), 13.61 (s, 1 H, exchanged with D2O, NH).

Compound 33

Compound 49 (33 mg, 0.10 mmol) was dissolved in 4 mL of an equivolume mixture of dimethylformamide and ethyl acetate. XAC (compound 31, 36 mg, 83 µmol) was added, and the mixture was warmed (50 °C) for 1 hour. The volume was reduced by half by evaporation, and sodium bisulfate (1 M) was added. The precipitate was collected and dried in vacuo to give 50 mg (78% yield) of product 33. This could be recrystallized from ethyl acetate/petroleum ether to give pure 33: 1H NMR (DMSO-d6) δ 0.85–0.92 (m, 6 H, 2 × CH3), 1.60 and 1.72 (each: m, 2 H, 2 × CH2), 3.41 (m, 2 H), 3.86 (pseudo t J = 7.4 and 7.2 Hz, 4 H), 4.00 (s, 4 H), 4.21 (s, 4 H), 4.53 (d, J = 8.7 Hz, 2 H), 7.08 (pseudo t, J = 8.3 and 7.7 Hz, 2 H), 7.29 (psudo t, J = 7.4 and 6.8 Hz, 2 H), 7.40 (pseudo t, J = 7.3 and 6.9 Hz, 2 H), 7.60 (dd, J = 7.3 and 3.9 Hz, 2 H), 7.87 (d, J = 6.8 Hz, 2 H), 8.07 (m, 2 H), 8.21 and 8.23 (each: br s, 1 H).

8-[4[[[[[2-[[[[(Carboxymethyl)amino]methyl]carbonyl]-amino]ethyl]amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine (34)

Compound 33 (12.1 mg, 15.8 µmol) was dissolved in DMF (0.5 mL), treated with diethylamine (0.2 mL), and stirred for 1 h. The solvent was evaporated, and the residue was washed with ether and dried in vacuo: 1H NMR (DMSO-d6) δ 0.88 (m, 6 H, 2 × CH3), 1.58 and 1.74 (each: m, 2 H, CH2), 3.21 and 3.29 (m, 8 H, COCH2, CH2-COOH, and NHCH2), 3.87 (pseudo t, J = 8.3 and 6.4 Hz, 2 H, N-CH2), 4.02 (t, J = 7.3 and 6.2 Hz, 2 H, N-CH2), 4.55 (m, 2 H, OCH2CO), 7.10 and 8.08 (each: d, J = 8.8 Hz, 1 H, vinyl H), 8.11 and 8.23 (each: br s, 1 H, NH).

8-[4-[[[[[2-[[[[(3-Carboxypropanoyl)(carboxymethyl)-amino]methyl]carbonyl]amino]ethyl]amino]carbonyl]-methyl]oxy]phenyl]-1,3-dipropylxanthine (35)

Compound 34 (5.4 mg, 10 µmol) was dissolved in DMF and treated with succinic anhydride (10 mg, 100 µmol) and diisopropylethylamine (10 µL) and sonicated for 1 h. Dilute HCl was added to acidify and the solvent was evaporated. Upon addition of methanol and ether a precipitate formed. The precipitate was collected and dried in vacuo: 1H NMR (DMSO-d6) δ 0.89 (m, 6 H, 2 × CH3), 1.57 and 1.74 (each: m, 2 H, CH2), 2.42 [m, 4 H, HO2C(CH2)2CO], 3.23 and 3.59 (m, 4 H, 2 × NCH2), 3.87 and 4.00 (m, 8 H, COCH2, CH2COOH, NCH2), 4.55 (m, 2 H, OCH2CO), 7.11 and 8.08 (each: d, J = 9.0 Hz, 1 H, vinyl H), 8.23 and 8.38 (each: br s, 1 H, NH).

N-Benzyl-N′-butylurea (36b)

A solution of butyl isocyanate (10.1 mL, 97.6 mmol) in chloroform (10 mL) was added dropwise to a solution of benzylamine (10 mL, 91.6 mmol) in chloroform (10 mL) at 0 °C. The reaction mixture was stirred for 1 h at room temperature, and solvent was removed in vacuo to give N-benzyl-N′-butylurea [36b, Rf = 0.75 (CHCl3–MeOH, 8.5:1.5), 18.2 g, 96%] as a colorless solid: mp 98 °C; MS 206 (M+); 1H NMR (DMSO-d6) δ 0.85 (pseudo t, J = 7.1 and 6.9 Hz, 3 H, CH3), 1.2–1.4 (m, 4 H, CH2CH2), 2.98 (m, 2 H, NH-CH2), 4.2 (d, J = 5.0 Hz, 2 H, CH2Ph), 5.85, 6.26 (2 × br s, 2 × 1 H, exchanged with D2O, NH), 7.18–7.25 (m, 5 H, Ph).

1,3-Di-n-butyl-6-aminouracil (38a)

N,N-Di-n-butylurea (36a, 34.45 g, 0.2 mol), cyanoacetic acid (19.56 g, 0.23 mol), and acetic anhydride (81.66 mL, 0.8 mol) was heated at 80 °C for 2 h under nitrogen, and the reaction mixture was concentrated to dryness to give crude compound 37a. The crude mixture was dissolved in methanol (100 mL), and 4 N sodium hydroxide (100 mL) was added. The reaction mixture was stirred for 30 min with cooling, and the solid formed was filtered and dried to give compound 38a (47.04 g, 99.5%): mp 90.1–92.4 °C; 1H NMR (DMSO-d6) δ 1.72–1.93 (m, 6 H, 2 × CH3), 1.18–1.33 (m, 4 H, 2 × CH2), 1.39–1.52 (m, 4 H, 2 × CH2), 3.70 (t, 2 H, N-CH2), 3.76 (t, 2 H, N-CH2), 4.64 (s, 1 H, H-4), 6.77 (br s, exchangeable with D2O, 2 H, NH2).

6-Amino-1-benzyl-3-butyluracil and 6-Amino-3-benzyl-1-butyluracil (38b)

A mixture of N-benzyl-N′-butylurea (36b, 18 g, 87.3 mmol), cyanoacetic acid (8.2 g, 96.1 mmol), and acetic anhydride (30 mL) was heated for 2 h at 80 °C under nitrogen. After the solution was cooled, it was concentrated to dryness to give 37b [Rf = 0.5 (Hx–EtOAc, 3:1)]. It was dissolved in methanol (20 mL), and 20% NaOH (10 mL) was added. The reaction mixture was stirred for 0.7 h at 0 °C and for 10 min at room temperature. It was concentrated to half volume, and the resulting yellow solid was filtered and dried to give a mixture of 6-amino-1-benzyl-3-butyluracil and 6-amino-3-benzyl-1-butyluracil [38b, Rf = 0.22 (Hx–EtOAc, 1:3), 23.5 g, 98%]: mp 100–103 °C; 1H NMR (DMSO-d6) δ 0.81–0.90 (m, 3 H, CH3), 1.17–1.62 (m, 4 H, CH2CH2), 3.69–3.80 (m, 2 H, N-CH2), 4.70 (s, 1 H, H-4), 5.07 (s, 2 H, CH2Ph), 6.80 (br s, 2 H, exchanged with D2O, NH2), 7.15–7.38 (m, 5 H, Ph).

6-Amino-1,3-dimethyl-2-thiouracil (38c)

A mixture of dimethylthiourea (purchased from Aldrich Chemical Co., 10.4 g, 0.1 mol), cyanoacetic acid (9.3g, 0.11 mol), and acetic anhydride (40 mL) was heated for 2 h at 80 °C under nitrogen. The dark solution was concentrated in vacuo, and methanol (45 mL) and 4 N NaOH (45 mL) were added. After the reaction mixture was stirred for 10 min, the solid was collected by suction and dried for 0.5 h at 60 °C to give compound 38c as a slightly yellow solid (9.02 g, 53%): mp 277–279 °C; 1H NMR (DMSO-d6) δ 3.31 (s, 3 H, CH3), 3.52 (s, 3 H, CH3), 5.03 (s, 1 H, CH=C), 7.02 (br s, 2 H, exchanged with D2O, NH2).

6-Amino-1,3-di-n-butyl-5-nitrosouracil (39a)

To a mixture of compound 38a (27 g, 112.8 mmol), glacial acetic acid (10 mL), and 6 N hydrochloric acid (18.8 mL) in water (500 mL) was added dropwise a solution of sodium nitrite (7.78 g, 112.8 mmol) in water (30 mL) on ice bath. The reaction mixture was stirred for 30 min. The violet solid was collected by filtration, washed with water (2 × 20 mL), and dried for 2 days at 60 °C in a vacuum oven to give compound 39a (24.99 g, 82.6%): mp 199–205 °C; 1H NMR (CDCl3) δ 0.97–1.01 (m, 6 H, 2 × CH3), 1.36–1.49 (m, 4 H, 2 × CH2), 1.62–1.74 (m, 4 H, 2 × CH2), 3.94 (t, J = 7.8 Hz, 2 H, N-CH2), 4.08 (t, J = 7.5 Hz, 2 H, N-CH2), 6.46 (br s, 2 H, NH2). Anal. Calcd for C12H20N4O3: C, 53.72; H, 7.51; N, 20.88. Found: C, 54.37; H, 7.67; N, 20.65.

6-Amino-1-benzyl-3-butyl-5-nitrosouracil and 6-Amino-3-benzyl-1-butyl-5-nitrosouracil (39b)

A mixture of 6-amino1-benzyl-3-butyluracil and 6-amino-3-benzyl-1-butyluracil (38b, 23.5 g, 86 mmol) was nitrosated to give a mixture of 6-amino-1-benzyl-3-butyl-5-nitrosouracil and 6-amino-3-benzyl-1-butyl-5-nitrosouracil (39b, 25.5 g, 98%) as a pink solid using the same method for 6-amino-1,3-dibutyl-5-nitrosouracil (39a): mp 178–9 °C; 1H NMR (DMSO-d6) δ 0.87–0.98 (m, 3 H, CH3), 1.22–1.60 (m, 4 H, CH2CH2), 3.80, 3.92 (t, J = 7.8 Hz, 2 H, N-CH2), 5.40, 5.60 (2 × s, 2 H, N-CH2Ph), 7.22–7.40 (m, 5 H, Ph), 9.70, 13.20 (2 × br s, 2 H, exchanged with D2O, NH2).

6-Amino-1,3-dimethyl-5-nitroso-2-thiouracil (39c)

A solution of sodium nitrite (3.63 g, 52.6 mmol) in water (15 mL) was added to a suspension of 6-amino-1,3-dimethyl-2-thiouracil (38c, 9 g, 52.6 mmol) in water (850 mL) and the 6 N HCl (8.8 mL) was added dropwise. The reaction mixture was stirred well for 20 min, and the blue solid was collected by suction and dried to give compound 39c (10.12 g, 96%): mp 228 °C; 1H NMR (DMSO-d6) δ 3.71, 3.74 (2 × s, 2 × 3 H, 2 × CH3), 9.11, 12.88 (2 × br s, s × 1H, exchanged with D2O, NH2).

Fmoc-iminodiacetic Acid (48)

Iminodiacetic acid (47, 2.19 g, 16.5 mmol, Sigma Chemical Co., St. Louis, MO) was dissolved in a mixture of 1 M sodium bicarbonate (40 mL) and acetone (30 mL). A solution of 9-fluorenylmethyl chloroformate (2.0 g, 7.7 mmol) in acetone (10 mL) was added to the above solution. After 6 h, the mixture was diluted with saline, acidified with 1 N HCl, and extracted with ethyl acetate. Drying and evaporation of the organic layer afforded 2.38 g (87%) of 48: mp 213–215.5 °C; 1H NMR (DMSO-d6) δ 3.99 and 4.07 (each: s, 2 H, CH2), 4.24 (s, 3 H), 7.32 and 7.42 (each: pseudo t, J = 7.5 and 7.3 Hz, 2 H, Ph), 7.63 and 7.90 (each: d, J = 7.5 Hz, 2 H, Ph), 12.75 and 12.77 (each: br s, 1 H, COOH); the mass spectrum (CI-NH3) showed a peak at 373 corresponding to M + 1 + NH3. Anal. Calcd for C19H17N1O6: C, 64.22; H, 4.82; N, 3.94. Found C, 64.15; H, 4.83; N, 3.90.

4-Fmoc-morpholine-2,6-dione (49)

Fmoc-iminodiacetic acid, compound 48 (0.40 g, 1.13 mmol), was suspended in 30 mL of ethyl acetate, treated with 1 mL of trifluoroacetic anhydride, and stirred for 1 h. Evaporation of most of the solvent, addition of petroleum ether, and evaporation resulted in a solid residue which was dried in vacuo at room temperature. The yield was 0.37 g (97%); mp 180–185 °C. This compound tended to hydrolyze upon long term storage. Anal. Calcd for C19H15NO5•0.8H2O: C, 64.88; H, 4.76; N, 3.98. Found: C, 64.58; H, 4.77; N, 3.90.

Biological Methods. Materials

F-12 (Ham’s) medium, fetal bovine serum (FBS), and penicillin/streptomycin were from Gibco BRL (Gaithersburg, MD). [125I]AB-MECA was prepared as described.16 [3H]PIA was from Amersham (Arlington Heights, IL), and [3H]CGS 21680 was from DuPont NEN (Boston, MA). Adenosine deaminase (ADA) was from Boehringer Mannheim (Indianapolis, IN). Composition of lysis buffer: 10 mM Tris/5 mM EDTA, pH 7.4 at 5 °C. 50/10/1 buffer: 50 mM Tris; 10 mM MgCl2; 1 mM EDTA, pH 8.26 at 5 °C. All other materials were from standard local sources and of the highest grade commercially available.

Cell Culture and Membrane Preparation

CHO cells stably expressing the A3 receptor6 were grown in F-12 medium containing 10% FBS and penicillin/streptomycin (100 units/mL and 100 µg/mL, respectively) at 37 °C in a 5% CO2 atmosphere. When cells had reached confluence, they were washed twice with Dulbecco’s phosphate buffer solution before dislodging after addition of 3 mL of trypsin–EDTA. For the final passage cells were grown in 150 × 50 mm tissue culture dishes. Cells were washed twice with 10 mL of lysis buffer. After addition of 5 mL of lysis buffer, cells were mechanically scraped and homogenized in an ice-cold Dounce homogenizer (20 strokes by hand). The suspension was centrifuged at 43000g for 10 min. The pellet was resuspended in the minimum volume of ice-cold 50/10/1 buffer required for the binding assay and homogenized in a Dounce homogenizer. Typically, 6–8 175-cm2 flasks were used for a 48-tube assay. Adenosine deaminase was added to a final concentration of 3 units/mL, and the suspension was incubated at 37 °C for 15 min; the membrane suspension was subsequently kept on ice until use. When large batches (ca. 100 flasks) were processed, homogenization was performed with a Polytron (Brinkman, Luzern, Switzerland), and further workup was as described above. The preparation was stored at −70 °C and retained its [125I]AB-MECA binding properties for at least 1 month.

Receptor Binding

Binding of [125I]AB-MECA to CHO cells stably transfected with the A3 receptor clone was performed essentially as described.16 Assays were performed in 50/10/1 buffer in glass tubes and contained 100 µL of the membrane suspension, 50 µL of [125I]AB-MECA (final concentration 0.3 nM), and 50 µL of inhibitor. Inhibitors were routinely dissolved in DMSO, while the final DMSO concentration never exceeded 1%. Incubations were carried out in duplicate for 1 h at 37 °C and were terminated by rapid filtration over Whatman GF/B filters using a Brandell cell harvester (Brandell, Gaithersburg, MD). Tubes were washed three times with 3 mL of buffer. Radioactivity was determined in a Beckman 5500B γ-counter. Nonspecific binding was determined in the presence of 200 µM NECA. Ki values were calculated according to Cheng–Prusoff,32 assuming a Kd for [125I]AB-MECA of 1.48 nM.28

Binding of [3H]PIA to A1 receptors from rat brain membranes and of [3H]CGS 21680 to A2 receptors from rat striatal membranes was performed as described previously.17,18 Rat cerebral cortical membranes and striatal membranes were prepared18 and treated with adenosine deaminase (2 units/mL) for 30 min at 37 °C prior to storage at −70 °C. Solid samples of the adenosine derivatives were dissolved in DMSO and stored in the dark at −20 °C. The stock solutions were diluted with DMSO to a concentration of ≤0.1 mM prior to adding to the aqueous medium. The final concentration of DMSO in the assay medium was generally 2%, and adenosine deaminase was not added.

At least six different concentrations spanning 3 orders of magnitude, adjusted appropriately for the IC50 of each compound, were used. IC50 values, computer-generated using a nonlinear regression formula on the InPlot program (Graph-pad, San Diego, CA), were converted to apparent Ki values using Kd value12 of 1.0 and 15.5 nM for [3H]PIA and [3H]CGS 21680 binding, respectively, and the Cheng–Prusoff equation.32

Adenylate cyclase in transfected CHO cell membranes was measured as previously described.6,12

Abbreviations

SPX
1,3-dipropyl-8-(4-sulfophenyl)xanthine
DMF
N,N-dimethylformamide
DMSO
dimethyl sulfoxide
EDAC
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
Fmoc
9-fluorenylmethyloxycarbonyl
NECA
5′-(N-ethylcarboxamido)adenosine
Tris
tris(hydroxymethyl)-aminomethane
XAC
8-[4-[[[[(2-aminoethyl)amino]carbonyl]-methyl]oxy]phenyl]-1,3-dipropylxanthine
XCC
8-[4-[(carboxymethyl)oxy]phenyl]-1,3-dipropylxanthine

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