Azolo substitution into the purine scaffold in nucleoside cyclic 3',5'-phosphorothioates

Azolation in the 8-position in the purine scaffold of cAMP (adenosine 3′,5′-cyclic monophosphate) and cAMPS (adenosine 3′,5′-cyclic monophosphorothioate) provided derivatives with an azole ring directly attached to the purine via an annular azole nitrogen. Electrophilic bromination in the 8-position was followed by nucleophilic substitution with metalated azoles to afford 8-imidazo and 8-triazolo derivatives. The substrates were appropriately protected (Sp)-3′,5′-cyclic N-benzylphosphoramidate. A subsequent carbon disulfide promoted thiation reaction afforded corresponding (Rp)-8-azolo-3′.5′-cAMPS products. The reactions were stereoselective. The products as tri-n-butylammonium salts were soluble in organic solvents and were purified by chromatography. The ammonium salts were converted to sodium salts.


Introduction
The heterocyclic purine framework is widely incorporated into essential biomolecular systems and constitutes an important part of medicinal chemistry. Adenine and guanine are common purine nucleobases in such frameworks as exemplified by adenosine in Fig. 1. In this report the emphasis is on development of methodology for introduction or exchange of substituents in the purine 8-position [1]. The 8-position in the purine scaffold is active in both electrophilic and nucleophilic substitution reactions [2]. 8-Bromides or 8-chlorides are useful substrates for nucleophilic substitutions affording 8-aza, 8-oxa, or 8-thia derivatives [3][4][5]. More recently, 8-nitro analogues have become available by nucleophilic displacements [6]. 8-Carbylation is achieved by Pd-promoted organometallic cross-coupling reactions with metalated carbocyclic and heterocyclic arenes [7]. Even heteroatom substituents can be introduced by cross-coupling procedures [8]. In this manner a number of 8-substituted purine skeletons have become available.
In (R P )-adenosine-3′,5′-cyclic phosphorothioic acid (cAMPS), one of the oxygen atoms pendant from the phosphorus atom in (R P )-adenosine-3′,5′-cyclic phosphoric acid (cAMP) has been replaced by a sulfur atom in a stereoselective manner. The resulting (R p )-and (S p )-isomers of cAMPS are stereochemically stable and useful for various bioscreening programs associated with cyclic adenosine and cyclic guanosine monophosphates (cGMP). Both cAMP and cGMP are important secondary messenger in regulating a wide range of cell functions in response to specific hormones [9,10]. We have reported a method for stereocontrolled preparation of 8-substituted (R P )-adenosine-3′,5′-cyclic phosphorothioic acids that possess protein kinase antagonistic activity and has a stimulating effect on the immune system [2]. In this report, we describe sp 2 -hybridized aminonitrogen hinged to the 8-position in the purine skeleton by substitution reactions from the corresponding bromides or chlorides. Iodides will react in a similar manner but are less readily available intermediates. The products were cAMPS derivatives with sp 2 -hybridized azole nitrogen in the form of imidazole and triazoles attached to the 8-position in the nucleotide. The nucleophiles were metalated azoles. The triazolo heterocycles are π-electron deficient, and both the 1,2,3-triazoles and 1,2,4-triazoles possess low basicity. In contrast imidazole behaves as a base and nucleophile. Acyclic 8-azido derivatives were included as a less polarized species.

Results and discussion
The generally low solubility of nucleosides and nucleotides in organic solvents may be partly overcome by initial conversions to amidates [2]. A solution of the (S p )-8-bromo amidate 1 and the sodium salts of the azoles in DMF afforded the 8-azolo products. We have previously described methodology for stereoselective preparation of amidates [2]. Substitution of intermediate amidates with imidazole as sodium salt in DMF proceeded readily at elevated temperature to afford the imidazo derivative 2 (Scheme 1). 1,2,4-Triazole was reacted similarly, but the heating time was increased to 20 h because of lower reaction rate. Exclusive formation of the N-1 product 3 was observed. The proton NMR spectra were used for assignment of structures to the regioisomers. Substitution at the annular 4-nitrogen atom will yield a symmetrical triazole derivative with the same chemical shifts for the two annular triazolo protons. Different chemical shifts for the two hydrogen atoms in the azole ring in the product were observed. The unsymmetrical structure 3 was assigned to the product.
Two regioisomers were formed with 1,2,3-triazole as a reactant, viz. the symmetrical structure 4 and its isomer 5 (i) (S p )-1, imidazole, NaH, DMF, rt (30 min), 80 °C, 4-20 h; or (ii) (S p )-1, 1,2,4-triazole, DMF, 60 °C, 20 h. in the ratio 3:2 (Scheme 2). The isomers were separated by flash chromatography. The symmetrical structure has the same chemical shift for both triazolo protons in the 1 H NMR spectra whereas the second isomer showed two different chemical shifts for the annular triazolo protons. 1,2,3-Triazoles carrying a methyl (6) or a chloro substituent (7) in the 4-position (Scheme 2) were reacted similarly with sodium hydride as base in DMF. The 4-methyltriazole afforded the regioisomers 8 and 10 in the ratio 6:1. The 4-chlorotriazole afforded a 3:1 ratio of the regioisomers 9 and 11 that were separated by flash chromatography. The 1 H shift in the methyl triazole isomers differed significantly, and isomer structures could be assigned. The major isomer was fluorescent in UV light. The chemical shifts for the triazolo proton in the two chloro isomers were almost the same. The major isomer was fluorescent and was tentatively assigned structure 8. The 5-methyl and 5-chloro triazole reactants 6 and 7 were available by literature procedures from 1-SEM-protected 1,2,3-triazol [11]. Finally, the acyclic 8-azido derivative was prepared by heating the bromide 1 with sodium azide in DMF to afford the 8-azido derivative 12. The latter is also a potential intermediate for 8-amino derived products.
Scheme 3 illustrates adaption of the Stec thiylation reaction for generation of thiophosphoric acids [12]. Initial proton abstraction from the benzylic amino group in the phosphoramidates 2-5, 8, 9 by LDA in THF at − 70 to − 40 °C afforded a lithium ylide that reacted with carbon disulfide. A sulfur atom in the intermediate becomes a nucleophile and a cyclization reaction occurs. Cleavage of the P-N bond occurs with retention of the true configuration at the phosphorus atom. The hydrophobic nature of the bulky TBDMS-protecting group attached to the 2′-hydroxy group leads to precipitation of the thioic acid from the aqueous mixture. The product was desilylated by ammonium fluoride in methanol at 45 °C to afford the target compounds (Scheme 3). The 8-azido derivative 12 was thiated in the same manner.
The deprotected products were isolated as tributylammonium salts after addition of tributylamine to the acid. The tributylammonium salts 15, 16, 20-22, 23, 24 of the products were soluble in polar organic solvents and could be purified by recrystallization or by flash chromatography on silica gel using CH 2 Cl 2 :MeOH:NBu 3 . The azido amidate 12 reacted in the same manner to furnish the phosphorothioic acid 24. The tributylammonium salts were converted into sodium salts 25-31 by dissolution of the ammonium salts in methanolic sodium hydroxide. Precipitation of the salts was by addition of diethyl ether (Scheme 4).
The products were subjected to T cell proliferation assays by established methodologies [2,9,13]. Comparison with previously reported systems indicate low specific effect by the nature of the annular heteroatom(s) in the five-membered hetarene in the purine 8-position.

Conclusion
Methods for preparation of novel 8-imidazolo and 8-triazolo derivatives of cAMP and cAMPS have been developed. Azolation in the 8-position in the purine scaffold of cAMP and cAMPS provides derivatives with annular sp 2 -hybridized azole amino-nitrogen attached directly to the purine ring. In the process, an appropriately silyl protected (S p )-8-bromoadenosine 3′,5′-cyclic N-benzylphosphoramidate is aminated with a metalated azole as nucleophile, followed by stereoselective thiation at the phosphorus atom to deliver adenosine 3′,5′-cyclic phosphorothioates with retention of the configuration at the phosphorus atom. The (R p ).8-azolo-cAMPS products are analogues of cAMP that regulates a broad range of essential cellular functions.

Experimental
1 H NMR spectra were recorded in CDCl 3 or MeOH-d 4 at 200 and 300 MHz. The 13 C NMR spectra were recorded at 75 and 100 MHz. Chemical shifts are reported in ppm using CHCl 3 (7.24 ppm) and CDCl 3 (77 ppm) as references, and in MeOH-d 4 , 3.30 ppm in 1 H NMR and 49.0 ppm in 13 C NMR. The 31 P spectra were recorded in CDCl 3 , MeOH-d 4 at 81 MHz or 121 MHz with a Bruker DPX 200 or 300 instrument with 85% H 3 PO 4 as an external reference. Mass spectra were recorded at 70 eV with a Fisons VG Prospectrometer. The spectra are presented as m/z (% relative intensity). Electrospray spectra were obtained with a Micromass QTOF 2 W spectrometer with electrospray ionisation quadrupole time of flight. Merck silica gel 60 (230-400) was used for flash chromatography.

(S p )-2′O-(tert-Butyldimethylsilyl)-8-(imidazol-1-yl)adenosine-3′,5′-cyclic N-benzylphosphoramidate (2, C 26 H 35 N 8 O 5 PSi)
A solution of 143 mg imidazole (2.1 mmol) in 5 cm 3 dry DMF was added slowly via a syringe to an oven-dried flask containing 84 mg powdered NaH (60% dispersion in mineral oil, 2.1 mmol) and 8 cm 3 anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.22 g (S p )-8bromoadenosine-2-O′-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.0 mmol) in 8 cm 3 DMF was added. The mixture was stirred at room temperature for 30 min and finally at 80 °C for 4 h. The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 10% methanol in CH 2 Cl 2 . Yield: 586 mg (49%) of a white solid material; 1  1 mmol) in 5 cm 3 dry DMF was added slowly via a syringe to an oven-dried flask containing 84 mg powdered NaH (60% dispersion in mineral oil, 2.1 mmol) and 8 cm 3 anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.22 g (S p )-8-bromoadenosine-2′O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.0 mmol) in 8 cm 3 DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was distilled off at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 8% methanol in CH 2 Cl 2 to afford 659 mg (55%) of the triazole product 3 as a white solid. 1 3 dry DMF was added slowly via a syringe to an oven-dried flask containing 122 mg powdered NaH (60% dispersion in mineral oil, 3.06 mmol) and 10 cm 3 anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 1.70 g (S p )-8-bromoadenosine-2'O-(tert-butyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 2.78 mmol) in 10 cm 3 DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 8% MeOH in CH 2 Cl 2 to furnish the white product as a mixture (4 + 5), yield 880 mg (53%) in ratio 3:2 by 1 H NMR spectroscopy analysis. The isomers were subjected twice to separation by flash chromatography using 8% methanol in CH 2   4-chloro-1,2,3-triazole (1.2 mmol) in 5 cm 3 dry DMF was added slowly via a syringe to an oven-dried flask containing 48 mg powdered NaH (60% dispersion in mineral oil, 1.2 mmol) and 3 cm 3 anhydrous DMF under argon. The mixture was stirred at room temperature for 30 min before a solution of 611 mg (S p )-8-bromoadenosine-2'O-(tertbutyldimethylsilyl)-3′,5′-cyclic N-benzylphosphoramidate (1, 1.0 mmol) in 5 cm 3 DMF was added. The mixture was stirred at room temperature for 30 min and finally at 85 °C overnight (20 h). The solvent was removed at reduced pressure, and the residual material subjected to flash chromatography on silica gel using 5% MeOH in CH 2 Cl 2 to afford 350 mg (55%) of (9 + 11) as a white solid mixture in the ratio 3:1 ( 1 H NMR). Pure 9 was isolated after flash chromatography twice. Yield: 215 mg; 1