Various 8-substituted xanthine and thioxanthine derivatives were synthesized via 1,3-dialkyl-5,6-diaminouracils as shown in . The substituted uracil and 2-thiouracil intermediates were prepared via an optimized Traube synthesis.6,7a
1,3-Dimethyl- and 1,3-di-n
-propyl-5,6-diaminouracil and their 2-thio derivatives were obtained by condensation of the corresponding dialkyl urea (4a
) or thiourea (4c
) with ethyl cyanoacetate (5
). The products after ring closure, substituted 6-aminouracil derivatives 6
, were then nitrosated at the 5-position. The nitroso group was reduced through chemical reduction or catalytic hydrogenation to form the intermediate 1,3-dialkyl-5,6-diaminouracil derivatives 8 in good yield.
The next step of the synthesis was to form the imidazole ring of the purine nucleus, resulting in the xanthine derivatives, as listed in (compounds 1, 2, and 13–28). The more nucleophilic 5-amino group of compound 8 was acylated by using a carboxylic acid chloride 9, forming the 1,3-dialkyl-5-(acylamino)-6-aminouracil derivatives 10. 1,3-Dialkyl-5-(acylamino)-6-aminouracils derived from thiophene-2-carboxylic acid and -3-carboxylic acid chlorides and from cyclopentanecarboxylic acid chloride were isolated and characterized (). The various 1,3-dialkyl-5-(acylamino)-6-aminouracil derivatives were then cyclized to the corresponding xanthine and 2-thioxanthine derivatives () by treatment with aqueous sodium hydroxide.
Potencies of Xanthine Derivatives at Adenosine A1 and A2 Receptors in Nanomolar Concentration Unitsa,b
Synthesis and Characterization of 1,3-Dialkyl-5-(acylamino)-6-aminouracils
Synthesis and Characterization of Xanthine Derivatives
-[(carboxymethyl)oxy]phenyl]xanthine derivatives related to a xanthine amine congener, compound 3
, an alternate route was used to form the imidazole ring. 1,3-Dipropyl-5,6-diaminouracil (8b
) or the corresponding 2-thiouracil (8d
) was condensed with [(p
-formylphenyl)-oxy]acetic acid, forming the imine 12
. Upon oxidation, the carboxylic acid congeners 27
(XCC) and 28
were obtained. The xanthine carboxylic acid derivatives were then esterified, giving the ethyl esters 29
, respectively, which were treated with neat ethylenediamine as previously reported7
to give the amine derivatives 3
. Since the A2
potency of compound 31
was enhanced over the oxygen analogue (see below), compound 3
, we synthesized other 8-aryl-2-thioxanthine derivatives in an effort to increase A2
potency. Other amine derivatives were synthesized through aminolysis reactions (compounds 32–35
) or by carbodiimide coupling (compounds 36–39
) as reported.10b
Lysyl conjugates 38–43
were prepared as described.10c
-hydroxysuccinimide ester derivative, 44
, was reported to be an irreversible inhibitor of A1
-adenosine receptors at concentrations greater than 50 nM.10a
If shown to be a potent and nonselective adenosine antagonist, this xanthine may be a potential inhibitor of both A1
- and A2
-adenosine receptors. Certain isothiocyanate-containing xanthines related to compound 3
also have been shown to be chemically reactive with A1
Efforts to synthesize analogous xanthine–isothiocyanates containing the 2-thio substitution were unsuccessful, likely due to side reactions involving the more reactive thio group.
A thiation reaction was used to generate 6-thioxanthine derivatives from the corresponding oxygen analogues. It is known11
that xanthine derivatives are preferentially thiated at the 6-position with P4
. Dioxane was the favored reaction medium to give high yields of the anticipated 6-thio- and 2,6-dithioxanthines (compounds 52–57
). For example, CPX was converted to 8-cyclopentyl-1,3-dipropyl-6-thioxanthine (55
) by using this thiation reaction.
Iodinated xanthine derivatives, synthesized by using a prosthetic group12
or by classical methodology, have been introduced as high-affinity radioligands for adenosine receptors.12,13
We have explored the use of a 2-thienyl substituent as a site for selective iodination, via mercuration (). These substituted thiophene derivatives, such as 59
, undergo regioselective mono-mercuration at the unsubstituted 2-position, rapidly and at ambient temperature, in the presence of stoichiometric quantities of mercury salts such as mercuric acetate.14
The 2-mercuriothiophene salt 60
is then exposed to elemental iodine, resulting in the corresponding 2-iodothiophene derivative 61
Use of 2-Thienyl Derivatives as Prosthetic Groups for Mercuration and Subsequent Iodinationa
New prosthetic groups designed for facile radiodination of functionalized drugs and peptides are still being sought.15,16
We have used thiophene-2-acetic acid (as its reactive N
-hydroxysuccinimde ester, 58
) and thiophene-2-methanamine (62
) as prosthetic groups for iodination, via mercuration.
Compound 58 reacted with XAC (3) to form an amide, compound 46. This xanthine bearing a 2-alkylthienyl prosthetic group was readily mercurated to give 47.
Iodination via 2-mercuriothiophene intermediates as in may be carried out selectively in the presence of other susceptible aromatic groups, such as phenols. Compound 58 reacted with L-tyrosylglycine to form an amide [compound 59, in which R3 = CH(CH2C6H4OH)-CONHCH2COOH]. Upon sequential treatment with mercuric acetate and iodine (1 equiv), the corresponding monoiodinated peptide derivative, 61 [R4 = CONHCH-(CH2C6H4OH)CONHCH2COOH], was obtained in high yield.
The N-succinoyl derivative [63a; R3 = (CH2)2COOH] of thiophene-2-methanamine was mercurated to form an internal salt, 60b [R4 = NHCO(CH2)2COOH] which precipitated from methanol. Upon treatment with iodine an immediate reaction occurred. This reaction was followed by NMR in DMSO-d6. The complete reaction of the 2-mercuriothiophene derivative was indicated by shifts of the thiophene aromatic signals to 6.68 and 7.13 ppm from TMS, corresponding to the 2-iodo derivative 61b.
Affinity at A1
- and A2
-adenosine receptors was measured in competitive binding assays, using as radioligands [3
(with rat cerebral cortical membranes) and [3
-ethylcarbamoyl)adenosine (with rat striatal membranes),18
A sulfur substitution at the 2-position carbonyl group of 1,3-dialkylxanthines usually did not decrease the affinity of the xanthines for A1- or A2-adenosine receptors. In the case of the 2-thio analogue of CPX, compound 14, the A1 affinity was enhanced by the thio substitution. The 2-thioxanthine amine congener, compound 30, bound with greater affinity at A2 receptors and with less affinity at A1 receptors than the corresponding oxygen analogue, compound 3. Potency at A2 receptors was enhanced 7-fold by the 2-thio substitution in the case of a carboxylic acid congener (compounds 27 and 28).
N-Methylated analogues (32–37) of compound 31 were prepared. As in the 2-oxo series, the secondary N-methylamine derivative 33 was the most potent at A2 receptors with a Ki value of 6.8 nM. Thus, the combination of two modifications of compound 3 enhanced its A2 affinity 10-fold.
A sulfur substitution at the 6-position carbonyl group of 1,3-dialkylxanthines was not well tolerated at either A1 or A2 binding sites. Thus, the 6-thio analogue of CPX 55 was 17-fold less potent than CPX at A1 receptors. The 6-thio analogue of 1,3-diethyl-8-phenylxanthine (57) was 23-fold less potent than DPX (24) at A1 receptors and 12-fold less potent at A2 receptors. 2,6-Dithio analogues, such as 53, were intermediate in potency between the corresponding 2-thio and 6-thio analogues.
Substitutions of thienyl and furyl groups at the 8-position of xanthines have approximately equivalent effects on affinity at both receptor subtypes. Both substitutions are generally slightly less potent than the corresponding 8-phenylxanthine analogues. For both thienyl and furyl derivatives (several of which were reported previously, by Bruns et al.18
), attachment at the 3-position relative to the heteroatom (sulfur or oxygen, respectively) results in greater potency at adenosine receptors than attachment at the 2-position. Preference for 3-thienyl derivatives was evident, particularly at the A2
subtype (cf. 18
1,3-Dipropyl-8-(2-thienyl)-2-thioxanthine (22) did not bind measurably to A2 receptors at its limit of aqueous solubility. At pH 7.7 in Tris buffer this concentration was 5.8 μM, determined with a log ε for absorption in methanol at 320 nm of 4.38. At this concentration there was not even a partial displacement of tritiated [3H]-5′-(N-ethylcarbamoyl)adenosine from striatal membranes. Thus, compound 22 was > 142-fold A1 selective in these binding assays. Compound 30 is > 740-fold A1 selective, but low solubility may limit its usefulness. The aqueous solubilities of A1-selective xanthines 26a and 30 are 9.0 and 5.4 μM, respectively.