Synthesis of Extended Uridine Phosphonates Derived from an Allosteric P2Y2 Receptor Ligand

In this study we report the synthesis of C5/C6-fused uridine phosphonates that are structurally related to earlier reported allosteric P2Y2 receptor ligands. A silyl-Hilbert-Johnson reaction of six quinazoline-2,4-(1H,3H)-dione-like base moieties with a suitable ribofuranosephosphonate afforded the desired analogues after full deprotection. In contrast to the parent 5-(4-fluoropheny)uridine phosphonate, the present extended-base uridine phosphonates essentially failed to modulate the P2Y2 receptor.

The P2Y 2 receptor (P2Y 2 R) is responsible for many physiological functions and therefore attracts considerable attention for developing therapeutics. For example, the P2Y 2 R agonist diquafosol, which stimulates the secretion of chloride and water by epithelial cells, is approved in Japan for treatment of dry eye disease [5]. P2Y 2 R activation protects cardiomyocytes from hypoxia in vitro and reduces postischemic myocardial damage in vivo [2]. Furthermore, the P2Y 2 R is a promising target for the treatment of neurodegenerative diseases, including Alzheimer's disease [6]. Recently, ATP activation of the P2Y 2 R was also shown to retard bone mineralization [1].
The development of selective P2Y 2 R agonists and antagonists is a challenging task. Introduction of a 2'-amino and a 2-thio modification in UTP afforded analogue 1 ( Figure 1) with enhanced potency and selectivity (versus P2Y 4 R) [7]. Unfortunately, most P2Y 2 R agonists contain a polyphosphate group, which is easily hydrolyzed by ecto-nucleotidases [4]. We recently reported a series of stable 5-substituted uridine phosphonate analogues, some of which (e.g., 2, Figure 1) showed promising allosteric partial agonistic activity at P2Y 2 R [8]. Given the interesting profile of the 5-aryluridine analogues we envisaged modifying this nucleobase to a more rigid structure as a way to potentially enhance affinity for the P2Y 2 R. In this light we opted for a ring transformation from a 5-aryluracil to a quinazoline-2,4-dione motif, analogous to the biphenyl-naphthalene ring transformations often encountered in medicinal chemistry [9]. The first series of 5,6-annulated uridine analogues were based on the aromatic Topliss-scheme [6-H (3a), Cl (3c), OMe (3e) and Me (3d)] [10]. Additionally the 6-fluorine derivative 3b was chosen based on the notable potency of parent compound 2 which bears a fluorine atom. Finally two nucleobases involving substitution of the benzene ring for a thiophene (3f) or a naphthalene core (3g) were included to gauge the effect of bioisosteric replacement or ring expansion of the nucleobase on the affinity for the P2Y 2 R. Figure 1. Structure of UTP, the selective P2Y 2 agonist 1, the allosteric partial agonist 2 and the target nucleoside phosphonates 3.

Synthesis
To gain access to the target C 5 /C 6 -fused uridine phosphonates we first prepared the required "base" moieties 4a-g by condensation of the appropriate ortho-amino aromatic carboxylic acids and urea at high temperature [11][12][13].
The synthesis of the appropriate glycosyl donor started from the known diol 6, which was obtained in two steps from the commercial 1,2:5,6-O-diisopropylidene-α-D-allofuranose (5) following a known procedure (Scheme 1) [14]. Cleavage of the resulting vicinal diol with sodium periodate gave the aldehyde [15], which was used without purification in the subsequent Wittig-type reaction. During formation of the diethylvinylphosphonate 7, C-4 may be prone to epimerization, which could be limited by performing the reaction at lower temperature. The yield for the conversion of 6 to 7 was significantly increased by using dichloromethane instead of ethanol for the periodate reaction. Hydrogenation with 10% Pd/C at atmospheric pressure allowed to simultaneous reduction of the double bond and removal of the benzyl group. One-pot deprotection of the isopropylidene group and acetylation of 8 afforded 9 (with an anomeric ratio of ca. 4:1 in favor of the  anomer), which was used in a silyl-Hilbert-Johnson reaction with the silylated benzo-or thieno[3,2-d]pyrimidine-2,4diones to give compounds 10a-g [16]. When HMDS was used as the silylating agent, careful removal of the silylating agents prior to the addition of the base and trimethylsilyltrifluoromethane sulfonate was required. Hence, the use of BSTFA for in situ silylation was preferred, despite the fact that it generally gave lower yields. Final deacetylation, followed by treatment with TMSBr/DCM afforded the desired nucleoside phosphonate analogues 3a-g. Scheme 1. Synthesis of the Target Extended Uridine-5'-methylenephosphonates 3a-g.
Compared to compound 2, the current extended uridine phosphonates were found to stimulate the P2Y 2 receptor to a lesser extent, indicating that rigidifying the base moiety is unfavorable for binding to a presumed allosteric binding site. It can be concluded that structural flexibility of the 5-substituent of the earlier discovered series of allosteric P2Y 2 agonists is crucial for allosteric agonist activity.

General Methods and Materials
All reagents were from standard commercial sources and of analytic grade. All reaction were performed under argon atmosphere except specified otherwise. Dry solvents were directly acquired from commercial sources. Precoated Merck Silica Gel F254 plates were used for TLC. Spots were examined under ultraviolet light at 254 nm and further visualized by sulfuric acid-anisaldehyde spray. Column chromatography was performed on silica gel (200-400 mm, 60 Å). 1 H, 13 C, and 31 P-NMR spectra were recorded in CDCl 3 , DMSO-d 6 , or D 2 O on a Varian Mercury 300 MHz spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are given in parts per million (ppm δ), δ relative to residual solvent peak or TMS for 1 H and 13 C and to external D 3 PO 4 for 31 P. Structural assignment was confirmed with COSY, HSQC and HMBC. Exact mass measurements were performed on a LCT Premier XE orthogonal time-of flight spectrometer with API-ES source (Waters, Zellik, Belgium) Waters LCT Premier XETM Time of flight (TOF) instrument. Samples were infused in a CH 3 CN/H 2 O (1:1) mixture at 10 mL/min. (7): NaIO 4 (0.52 g, 2.22 mmol) was added to a stirred solution of compound 6 in CH 2 Cl 2 (15 mL) and H 2 O (10 mL) and the reaction mixture was stirred for 0.5 h at rt. The mixture was then filtrated and the filtrate was extracted with CH 2 Cl 2 . The combined organic layers were concentrated in vacuo to give the crude aldehyde, which was used in the next step without further purification. To a stirred suspension of NaH (0.31 g, 7.81 mmol) in dry THF (10 mL) was added tetraethyl methylene bisphosphonate (1.41 g, 4.88 mmol) under argon. After 0.5 h the mixture was cooled in an ice bath and a solution of the crude aldehyde in THF (10 mL) was added. The reaction mixture was allowed to warm to rt and stirred overnight. The crude mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried (Na 2 SO 4 ) and concentrated   When the starting material was consumed completely, the reaction mixture was evaporated in vacuo and co-evaporated twice with AcCN. The residue was dissolved in dry pyridine (12 mL) at 0 °C under argon followed by dropwise addition of acetic anhydride (1.56 mL, 16.52 mmol). The reaction mixture was slowly warmed to rt and stirred overnight. Then it was concentrated and co-evaporated with toluene. The residue was purified by silica gel column chromatography (CH 2 Cl 2 /MeOH 95:5) to give 9 (0.47 g, 83.2%) as a low melting solid (α:β anomeric ratio = 41:9).
Method 2: To a suspension of appropriate quinazoline-2,4-(1H,3H)-dione (1.5 eq.) in hexamethyldisilazane (50 eq.) was added trimethylsilyl chloride (0.7 eq.) and pyridine (10 eq.) under argon. The mixture was stirred at 130 °C overnight. The reaction mixture was evaporated to dryness under high vacuum. To the obtained residue a solution of triacetate 9 (1 eq.) in dry acetonitrile (50 eq.) was added under nitrogen followed by trimethylsilyl triflate (1.5 eq.). The solution was stirred for 2 h at rt. and the work up was similar as described in method 1.

General Procedure for the Deprotection of 10a-g to 3a-g
The appropriate phosphonic ester 10 (1 eq.) was dissolved in dry CH 2 Cl 2 (50 eq.) and treated with TMSBr (20 eq.) at 0 °C under argon. After stirring overnight at rt, the mixture was quenched with 7 N NH 3 /MeOH (20 eq.) and evaporated. The residue was dissolved in H 2 O (15 mL for 0.1 mmol phosphonic ester) and washed with CH 2 Cl 2 (3 × 10 mL for 0.1 mmol phosphonic ester). The aqueous layer was evaporated and purified with RP high-performance liquid chromatography (HPLC, Phenomenex Luna C-18, H 2 O/0.1% HCOOH in CH 3 CN, 90:10→0:100 in 23 min, flow 17.5 mL/min) to give the desired product after lyophilization of the appropriate fractions.