Fast, Efficient, and Versatile Synthesis of 6-amino-5-carboxamidouracils as Precursors for 8-Substituted Xanthines

Substituted xanthine derivatives are important bioactive molecules. Herein we report on a new, practical synthesis of 6-amino-5-carboxamidouracils, the main building blocks for the preparation of 8-substituted xanthines, by condensation of 5,6-diaminouracil derivatives and various carboxylic acids using the recently developed non-hazardous coupling reagent COMU (1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methanaminium hexafluorophosphate). Optimized reaction conditions led to the precipitation of pure products after only 5 to 10 min of reaction time. The method tolerates a variety of substituted 5,6-diaminouracil and carboxylic acid derivatives as starting compounds resulting in most cases in more than 80% isolated yield. Regioselectivity of the reaction yielding only the 5-carboxamido-, but not the 6-carboxamidouracil derivatives, was unambiguously confirmed by single X-ray crystallography and multidimensional NMR experiments. The described method represents a convenient, fast access to direct precursors of 8-substituted xanthines under mild conditions without the necessity of hazardous coupling or chlorinating reagents.

An established method for their preparation is the coupling of 5,6-diaminouracil derivatives with carboxylic acids in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) as a coupling reagent (Procedure B, Scheme 1) (Sauer et al., 2000;Hayallah et al., 2002;Hockemeyer et al., 2004;Basu et al., 2017). Another method requires the activation of the carboxylic acid by formation of the carboxylic acid chloride (Procedure C, Scheme 1) (Jacobson et al., 1989;Hockemeyer et al., 2004). Procedure C had been used to establish a multigram-scale synthesis of istradefylline (5). Drawbacks of this reaction are long reaction times (16 h) for the formation of the amide, only moderate yields (65%), and importantly, an additional step due to conversion of the acid into the corresponding acid chloride using hazardous chlorinating reagents. Furthermore, carboxylic acid chlorides are less stable than the corresponding carboxylic acids rendering storage and handling more demanding (Hockemeyer et al., 2004). Coupling reactions with the irritant and moisture-sensitive EDC-HCl also suffer from rather long reaction times, and typically provide moderate yields requiring tedious purification (Sauer et al., 2000;Hockemeyer et al., 2004).
All of these disadvantages motivated us to search for an alternative amide coupling procedure for the preparation of 6amino-5-carboxamidouracil derivatives being the most stable and easily storable xanthine precursors. Our aim was to develop a fast and effective coupling method applicable to a variety of diaminouracils and carboxylic acids that would allow simple work-up and straightforward isolation of the desired product (Scheme 3).
High resolution mass spectra (HR-MS) were recorded on a micrOTOF-Q mass spectrometer (Bruker), low resolution mass spectra (LR-MS) on an API 2000 (Applied Biosystems) mass spectrometer. 1 H NMR and 13 C NMR spectra were recorded in CDCl 3 or (CD 3 ) 2 SO on a Bruker Ascend 600 MHz NMR-spectrometer operating at 600.18 MHz ( 1 H), and 150.93 MHz ( 13 C). Chemical shifts (δ) are reported in ppm and are referenced to the chemical shifts of the residual solvent proton(s) present in chloroform δ [(CHCl 3 ) = 7.26 ppm for the 1 H NMR spectra and δ (CDCl 3 ) = 77.16 ppm for the 13 C NMR spectra] and in dimethylsulfoxide δ ((CH 3 ) 2 SO) = 2.50 ppm for the 1 H NMR spectra and δ ((CD 3 ) 2 SO) = 39.52 ppm for the 13 C NMR spectra. Multiplicity: s, singlet; d, doublet; q, quartet; m, multiplet. Coupling constants (J) are shown in Hertz (Hz). The infrared spectra were recorded as solid samples on an ALPHA-T (Bruker) with a Platinum ATR Module using the Opus software. The IR spectra were measured in the attenuated total reflection (ATR) mode in the region of 4,000-385 cm −1 (s, strong; m, medium; w, weak) and are reported in cm −1 .

General Amide Formation Procedure
To a solution of the respective carboxylic acid (1.0 equiv.) and COMU (1.1 equiv.) dissolved in a minimum of dimethylformamide (DMF), a mixture of diaminouracil (1.1 equiv.) and N,N-diisopropylethylamine (DIPEA) (1.1 equiv.) dissolved in a minimum DMF was added dropwise. The reaction was stirred for 5-10 min at room temperature, and water was added. The resulting precipitate was filtered off, washed with water and dried under reduced pressure. Most of the reactions were performed using 300 mg of the respective diaminouracil and 4 ml of DMF. The product was precipitated using 20 ml of water and washed with small portions of water (10 ml). The reaction generally performed well from 60 mg up to 1.5 g of diaminouracil as a precursor. For the 1.5 g scale 8 ml of DMF were used for dissolution, and 40 ml of water for precipiation, and 20 ml for the subsequent washing step. All other conditions were identical, and virtually the same percentage of yield as obtained independent of the scale of the reaction.

RESULTS AND DISCUSSION
Disadvantages of irritant and hazardous coupling procedures, long reaction times and moderate yields encouraged us to search for a new method to yield the desired 6-amino-5carboxamidouracil derivatives. After initial experiments with various procedures, the coupling reagent COMU showed the most promising results. COMU, which was developed in 2009, does not contain a potentially explosive benzotriazole moiety, and is therefore safer than classical coupling reagents such as, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU). COMU shows high solubility, is stable in typically used solvents, can be easily removed due to the water-solubility of its products, and may be used for a broad range of carboxylic acids and amines yielding the corresponding amides ((El-Faham et al., 2009;Albericio, 2010, 2011);Hjørringgaard et al., 2012).
The synthetic procedure which led to differently substituted 6-amino-5-carboxamidouracils is shown in Scheme 2. Diaminouracil derivatives and carboxylic acids were used as starting materials and subjected to amide coupling using COMU. N1-mono-and N1,N3-disubstituted 5,6-diaminouracil derivatives (14-20, Figure 2) were individually prepared (for details see Supporting Material Data Sheet 1) according to previously described procedures and (Maxwell and Salivar, 1952;Müller et al., 1993;Hockemeyer et al., 2004), while the employed carboxylic acid derivatives were in most cases commercially available.

Amide Coupling Reaction
Amide formation with the coupling reagent required the adjustment of different parameters, including solvent, reaction time, temperature and base. With DMF, DIPEA and COMU the optimal conditions were found (Scheme 2). The reaction may also be performed in other solvents, such as CH 2 Cl 2 , ethyl acetate or tetrahydrofurane (MacMillan et al., 2013), however, DMF is preferred resulting in short reaction times, and, importantly, the product can easily be precipitated in high purity by the addition of water. This renders a tedious isolation and purification procedure dispensable.
Scheme 3 depicts the proposed reaction mechanism, which is based on the mechanism proposed for the synthesis of esters using COMU (Twibanire and Grindley, 2011). The first step is the nucleophilic attack of the carboxylic acid (A) at the uronium moiety of COMU (B) resulting in intermediate C.
Decomposition of C, followed by addition of the resulting anion E to the carbonyl group of D and subsequent elimination of the urea derivative F leads to the activated carboxylic acid G. Finally, the corresponding amide derivative is formed by nucleophilic attack of an amine and elimination of the watersoluble side product H.
According to the proposed reaction mechanism, the carboxylic acid was converted to its active ester after dissolving it (1.0 equiv) together with COMU (1.1 equiv) in a minimum of DMF (mixture A, Scheme 2). Then, a solution of the 5,6diaminouracil derivative (1.2 equiv) and diisopropylethylamine (DIPEA, 1.1 equiv) as a base dissolved in a minimum of DMF (mixture B) was added, followed by 5-10 min of stirring at room temperature (Scheme 2). Upon addition of cold water, the product precipitated. It was filtered off, washed with cold water, and dried under reduced pressure yielding the target compounds 21-39 (Table 1) in high purity and with yields ranging from 62 to 99%. Due to our interest in AR antagonists, we prepared various precursors for 8-substituted xanthines, which we could obtain in high yields and isolate by simple precipitation as shown for various examples (22-29). The 1,3-dipropyl derivatives 23 and 30 were formed in 87 and 85% yield, with 98 and 99% purity, respectively. Compound 23 is a precursor of the dual-acting A 1 AR-opioid receptor ligands, such as 9.
Compound 22 was obtained in 78% yield and provides access to the A 3 AR antagonists PSB-11 (8b). Compound 24, the key compound for the synthesis of highly potent and selective A 2B AR antagonists, was successfully condensed and precipitated. The carboxylic acid for the synthesis of 24 was not commercially available and was therefore prepared according to a literature procedure (Borrmann et al., 2009). To gain a purity of over 95% for 24, an additional chromatographic purification procedure was required. Compound 25, the precursor of the A 1 AR antagonist rolofylline (4), which contains an 8-noradamantanyl substituent, and propyl residues on N1 and N3, precipitated in high purity (99%); fractional precipitation after cooling to 0 • C was required to give a final yield of 79%. The less bulky and less hydrophobic cyclopentanecarboxylic acid was reacted with 5,6-diamino-3-methyluracil to obtain amide 26 as a precursor for 8-cyclopentyltheophylline (CPX), and was isolated in 69% yield with 99% purity. The additional substituent on N1 can be easily introduced subsequently by alkylation according to literature procedures (Hockemeyer et al., 2004). The precursor 29 of the A 1 AR antagonist bamifylline (11), with methyl groups at both uracil nitrogen atoms, precipitated immediately in 85% yield and 99% purity. Compound 27, the precursor of the   (Borrmann et al., 2009); c (Moore et al., 1999); d (Rabasseda et al., 2001); e (Hockemeyer et al., 2004); f (Daly et al., 1985); g (Rodríguez-Borges et al., 2010).
A 2A AR antagonist and anti-Parkinson drug istradefylline (5), precipitated in 70% yield with 97% purity. Amide formation with 3-methoxycinnamic acid, carrying the styrene moiety, which is required for the preparation of the potent and selective A 2A AR antagonists of the MSX series (6a-c), gave the 6amino-5-carboxamidouracil precursor 28 in 83% isolated yield after precipiation. To investigate the impact of different carboxylic acid derivatives regarding precipitation of the product, we used 3-ethyldiaminouracil and various carboxylic acids as a test system for the formation of differently substituted 6amino-5-carboxamidouracils (Table 1). Compound 32, with a phenylpropionyl residue, was isolated in 90% yield. The analogous compound 33 containing a rigidified cyclopropyl ring gave a similar yield of 89%, as did the ether analog 34. The presence of an α-methyl group in compound 35 resulted in quantitative product formation and precipitation. The 6-amino-5-carboxamidouracil 38 bearing an alkyl residue was isolated in 81% yield with 99% purity.
Comparing all reactions, we observed the following trends: 1,3-disubstituted uracils could be formed best in case of a bulky, hydrophobic carboxylic acid derivative, which favors precipitation from the DMF/H 2 O solution. Reactions of N1-unsubstituted diaminouracils generally gave higher product yields, and the products were easily precipitated. The melting points of those products were high indicating the formation of intermolecular hydrogen bonds in the solid state, which was confirmed by the crystal structure of 32 (see below).
In literature, the product of the first reaction step has been described as a 5-nitroso derivative. Based on our NMR experiments, the 5-(hydroxyimino)-6-imino derivative is the tautomer that is present in chloroform employed as a solvent SCHEME 4 | NMR signals of 6-aminouracil derivatives with various substituents in the 5-position, and NOESY cross correlation for structure/tautomer analysis determined in chloroform-d 1 . (Scheme 4). The chemical shift of the 5-amino group in compound 16 indicates a magnetic shielding of the hydrogen atoms giving the nitrogen atom a more nucleophilic character, which is in accordance with our regioselectivity studies.
Finally, we tried to obtain a crystal structure of 25. Different crystallization experiments were performed but the crystallization of 25 has not been successful. Fortunately, compound 32, crystallized from DMSO solution at room temperature, yielding a crystal of the size 0.4 × 0.2 × 0.08 mm.
Measurement and analysis of the resulting crystal structure using a Bruker X8-KappaApexII instrument showed a monocline crystal system within the space group P2 1 . In accordance with the NMR experiment of 25 the crystal structure of 32 confirmed a regioselective amide coupling of the carboxylic acid with the 5,6-diaminouracil derivative in position 5. The crystal is mainly formed by intermolecular hydrogen bonds. π-Stacking or interaction with the solvent could not be observed. The most important intermolecular hydrogen bonds are summarized in Figure 3. All NH groups showed a donor functionalization and all oxygen atoms showed acceptor properties to surrounding molecules. Figure 3 visualizes these intermolecular interactions. The surrounding molecules are shaded while the intermolecular interactions are shown in turquoise. All bond lengths were in the expected range.