Synthesis and Potency of Novel Uracil Nucleotides and Derivatives as P2Y2 and P2Y6 Receptor Agonists

Hyojin Ko; Rhonda L Carter; Liesbet Cosyn; Riccardo Petrelli; Sonia de Castro; Pedro Besada; Yixing Zhou; Loredana Cappellacci; Palmarisa Franchetti; Mario Grifantini; Serge Van Calenbergh; T Kendall Harden; Kenneth A Jacobson

doi:10.1016/j.bmc.2008.05.013

. Author manuscript; available in PMC: 2009 Jun 15.

Published in final edited form as: Bioorg Med Chem. 2008 May 9;16(12):6319–6332. doi: 10.1016/j.bmc.2008.05.013

Synthesis and Potency of Novel Uracil Nucleotides and Derivatives as P2Y₂ and P2Y₆ Receptor Agonists

Hyojin Ko ^a, Rhonda L Carter ^b, Liesbet Cosyn ^c, Riccardo Petrelli ^d, Sonia de Castro ^a, Pedro Besada ^a, Yixing Zhou ^b, Loredana Cappellacci ^d, Palmarisa Franchetti ^d, Mario Grifantini ^d, Serge Van Calenbergh ^c, T Kendall Harden ^b, Kenneth A Jacobson ^a

PMCID: PMC2483329 NIHMSID: NIHMS56542 PMID: 18514530

Abstract

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y₂, P2Y₄, and P2Y₆ receptors. The 2-thio modification, found previously to enhance P2Y₂ receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y₂ receptor, in the form of Up₄-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include: 9, α,β-methylene-UDP, a P2Y₆ receptor agonist; 30, Up₄-phenyl ester and 34, Up₄-[1]glucose, selective P2Y₂ receptor agonists; 43, the 2-thio analogue of INS37217 (P¹-(uridine 5′)-P⁴- (2′-deoxycytidine 5′) tetraphosphate), a potent and selective P2Y₂ receptor agonist.

Introduction

The P2Y receptor family consists of at least eight human subtypes that are activated by either or both adenine and uracil nucleotides.¹^,² P2Y₂ and P2Y₄ nucleotide receptors respond to uridine 5′-triphosphate (UTP, 1) and its analogues, and the P2Y₆ receptor responds to uridine 5′-diphosphate (UDP, 2) and analogues.³ However, this delineation of agonist selectivities is not absolute. For example, Müller and coworkers and Besada et al. reported that certain 5′-triphosphate derivatives potently activate the P2Y₆ receptor.⁴^,⁵ The conformational preference of the ribose moiety in binding to uridine nucleotide-activated P2Y receptors has been explored through substitution with the sterically constrained methanocarba (bicyclo[3.1.0]hexane) ring system. P2Y₂ and P2Y₄ receptors display a North conformational preference, while the P2Y₆ receptor prefers the South.⁶^–⁸

Several recent studies have explored structure activity relationships (SARs) at the P2Y₂, P2Y₄, and P2Y₆ receptors.⁴^–⁶^,⁹^,¹⁰^,²³ Key pharmacological probes introduced include the nonselective P2Y₂ receptor agonists 3 and 4, which have progressed to clinical studies for dry eye syndrome and pulmonary diseases, the potent and selective P2Y₂ receptor agonist 5,⁷ and the selective P2Y₆ receptor agonist 6.¹¹^,¹² Modification of the base moiety of UTP to form C-linked nucleotides is possible in P2Y₂ receptor agonists and results in enhanced stability.¹⁰ Various dinucleotides tend to activate P2Y₂ and P2Y₄ receptors (diuridine tetraphosphates) or P2Y₆ receptors (diuridine triphosphates) with moderate potency and with greater stability than analogues of UTP and UDP.⁹ Also, the pharmacological activity of diadenosine polyphosphates at both P2Y and P2X receptor subtypes has been characterized.¹^,² Diadenosine tetraphosphate is only 3-fold less potent than ATP at the human P2Y₂ receptor.

In the present study, we further investigated structure activity relationships at P2Y receptors through synthesis of molecules with additional substitutions of the uracil, ribose, and phosphates moieties and combinations thereof. These analogues of UDP, UTP and dinucleotides were assayed for capacity to promote P2Y₂, P2Y₄, and P2Y₆ receptor-mediated activation of phospholipase C (PLC).³ These novel derivatives incorporated groups such as 2-thio, found previously to enhance receptor potency.⁴^,⁷ Phosphonomethylene bridges and nonphosphate linkages were introduced to enhance stability of molecules against the action of ectonucleotidases.¹³ We also probed the effects of truncation of dinucleotide agonists of the P2Y₂ receptor by synthesizing and quantifying the activities of a series of Up₄-sugars.⁷

Results and Discussion

Chemical Synthesis

The synthetic routes to the novel nucleotide derivatives (Tables 1 – 3) are shown in Schemes 1 – 6. The potencies of five known reference compounds are listed in Table 1 (P2Y₆ agonist UDP 2), Table 2 (P2Y₂ agonists UTP 1 and MRS2698 5), and Table 3 (P2Y₂ agonists Up₄U 3 and INS37217 4). Types of modifications include: UDP analogues containing a methylene-bridged substitute for the diphosphate group (Scheme 1); UTP analogues containing a β,γ-dihalomethylene-bridge in the triphosphate group⁴ (Scheme 2); UTP analogues with modified uracil and ribose moieties⁷ (Scheme 3); a 5-iodo analogue of INS48823⁵^,¹² (Scheme 4); analogues of uridine 5′-tetraphosphate⁷ (Scheme 5) in which the terminal phosphate moiety was condensed with various alcohols, including sugars. Among the derivatives in Scheme 1, compounds 7 and 10 are new compounds, but 8 and 9 were previously reported.³²^,³³

Table 1.

Relative potencies of UDP, 2, and UDP analogues for activation of the human P2Y₆ receptor. Unless noted: R¹ = OH; Inline graphic and R³ = H.


Compound	Modification	Structure	EC₅₀, µM
Compound	Modification	Structure	hP2Y₆ receptor^a
NUCLEOSIDES
7	Uridine-5′-malonate		NE
8	Uridine-5′-phosphonoacetate		NE
DIPHOSPHATES
2	UDP		0.30±0.06
9^b	Up-CH₂-p (α,β-methylene UDP)		0.66±0.11
10	Up-CF₂-p (α,β-difluoromethylene UDP)		NE
11	5-amino-UDP	R³ = NH₂	0.61±0.17
12	2′-deoxy-2′-ureido-UDP	R¹ = NHCONH₂	4.70±0.44

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Agonist potencies reflect stimulation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human P2Y₆ receptor. Potencies are presented in the form of EC₅₀ values, which represent the concentration of agonist at which 50% of the maximal effect is achieved. These values were determined using a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). The results are presented as mean ± standard error and are the average of three to six different experiments with each molecule.

9, MRS2782.

NE - no effect at 10 µM.

Table 3.

Relative potencies of dinucleotide derivatives for activation of the human P2Y₂, P2Y₄ and P2Y₆ receptors. Unless noted: R = Uridine; and X = O


Compound	Modification	Structure	EC₅₀, μM^a
Compound	Modification	Structure	hP2Y₂	hP2Y₄	hP2Y₆
DINUCLEOSIDE TRIPHOSPHATES (n = 1)
38^b	Up₃U		1.31±0.21	0.87±0.11	0.27±0.07
39^c	5-I-Up₃-(2′,3′-phenylethyl acetal)U		9.97±0.95	NE	5.49±0.68
DINUCLEOSIDE TETRAPHOSPHATES (n = 2)
3	(= Up₄U)		0.21±0.03	0.13±0.01	1.16±0.42
40	4-S-Up₄(4-S-U)	R¹ = R² = 4-thio-uridine	0.030±0.010	0.08±0.01	2.03±0.18
41	Up₂-CF₂-p₂-U (β,γ-difluoromethylene Up4U)	X = CF₂	2.27±1.39	>10^d	NE
42	Up₂-CCl₂-p₂-U (β,γ-dichloromethylene Up4U)	X = CCl₂	7.77±1.39	NE	NE
4	Up₄-2′-dC (INS37217)	R² = 2′-deoxy-cytidine	0.14±0.04	0.14±0.04	0.95±0.06
43	2-thio-Up₄-2′-dC	R¹ = 2-thio uridine R² = 2′-deoxy-cytidine	0.08±0.03	0.71±0.15	1.05±0.07
44	Up₄-2′-dG	R² = 2′-deoxy-guanosine	0.14±0.04	0.54±0.15	1.03±0.08

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Agonist potencies reflect stimulation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human P2Y₂, P2Y₄, or P2Y₆ receptor. Potencies are presented in the form of EC₅₀ values, which represent the concentration of agonist at which 50% of the maximal effect is achieved. These values were determined using a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). The results are presented as mean ± standard error and are the average of three to six different experiments with each molecule.

Reported in reference ⁹.

39, MRS2752; 43, MRS2657.

≤50% effect at 10 μM. Value of >10 μM was determined by extrapolation.

NE - no effect at 10 μM.

Scheme 1 — Synthesis of UDP analogues containing a methylene-bridged substitute for the diphosphate group. *Reagents and conditions*: (a) DCC, ROH, DMF, rt.

Scheme 6 — Synthesis of nucleoside intermediates. *Reagents and conditions*: A, benzotriazole-1-carboxamide, DMF; B, (a) silylated 2-thiouracil, SnCl₄, C₂H₄Cl₂; (b) Lawesson’s reagent, toluene, 80°C, overnight; (c) NaOMe, MeOH, reflux, 4 h; C, (a) H₂S, Et₃N, DMF, 200 psi, 2 days; D, (a) (i) 1,2-dichloroethane, hexamethyldisilazane, trimethylsilylchloride, 80°C, 4 h; (ii) SnCl₄, rt, 4 h; (b) NH₃/CH₃OH, rt, overnight.

Table 2.

Relative potencies of UTP, 1, and UTP analogues for activation of the human P2Y₂, P2Y₄ and P2Y₆ receptors. Unless noted: R¹, R² = OH; X = O; and R³, R⁴ = H.


Compound	Modification	Structure	EC₅₀, μM^a
Compound	Modification	Structure	hP2Y₂	hP2Y₄	hP2Y₆
TRIPHOSPHATES (n = 2)
1^b	(= UTP)		0.060±0.00	0.090±0.01	>10^d
13^b	2′-deoxy-2′-amino-UTP	R¹ = NH₂	0.062±0.008	1.2±0.3	NE
14^b	2-thio-UTP	X¹ = S	0.035±0.004	0.35±0.10	~1.5^f
5^b, ^c	2-thio-2′-deoxy-2′-amino-UTP	X¹ = S, R¹ = NH₂	0.008±0.002	2.4±0.8	NE
15	2-thio-β,γ-difluoromethylene-UTP	X¹ = S, X³= CF₂	1.63±0.36	8.11±0.69	5.15±0.69
16^c	2-thio-β,γ-dichloromethylene-UTP	X¹ = S, X³=CCl₂	2.51±0.65	NE	NE
17	2-thio-4-methylthio-UTP	X¹ = S, X²= SCH₃	0.91±0.06	5.35±1.20	>10^d
18	2′-deoxy-2′-ureido-UTP	R¹ = NHCONH₂	1.74±0.25	4.64±2.05	>10^d
19	2-thio-arabino-UTP	R¹ = H, R³ = OH	0.14±0.01	7.93±0.81	NE
20	2′-methyl-UTP	R³ = CH₃	1.45±0.26	1.26± 0.14	NE
21	3′-methyl-UTP	R⁴ = CH₃	NE	NE	NE
TETRA- (n = 3) AND PENTA- (n = 4) PHOSPHATES
22	Up₄		2.61±1.39	4.64±2.05	7.56±1.07
23	2-thio-Up₄	X¹ = S	0.60±0.20	5.52±1.75	6.83±1.74
24	4-thio-Up₄	X² = S	0.070± 0.01	0.28±0.06	6.46±0.41
25	2-thio-Up₅	X¹ = S, n = 4	0.57±0.16	5.27±1.24	7.33±0.66
26	Up₄-OMe	R²= OCH₃	3.95±0.51	2.70±0.43	>10^d
27	Up₄-δ-Me- phosphonate	R² = CH₃	4.18±0.43	2.53±0.57	8.16±0.74
28	Up₄-O(CH₂)₂CN	R² = (CH₂)₂CN	1.70±0.22	1.96±0.53	>10^d
29	Up₄-OCH₂CHOHCH₂OH	R ² = OCH₂ CHOH-CH₂OH	1.87±0.15	1.12±0.04	8.19±0.41
30^c	Up₄-OC₆H₅		1.89±1.07	NE	NE
31	Up₄-OC₆H₁₁		5.86±0.33	>10^d	>10^d
TETRAPHOSPHATE SUGARS (N=3)
32	Up₄-[5]ribose		1.88±0.03	4.78±0.40	>10^d
33	Up₄-[6]fructose		3.33±0.42	6.30±0.75	>10^d
34^c	Up₄-[1]glucose	R² =	0.30±0.13	2.06±0.18	7.83±0.17
35	Up₄-[1]galactose		4.85±2.07	1.77±0.41	8.19±0.73
36	Up₄-[6]mannose		>10^d	>10^d	>10^d
37	Up₄-[6]2′-deoxyglucose		3.54±0.96	4.32±1.50	>10^d

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Agonist potencies from reference ⁷.

5, MRS2698; 16, MRS2725; 30, MRS2768; 34, MRS2732.

≤50% effect at 10 µM. Values of >10 µM were determined by extrapolation.

NE - no effect at 10 µM.

Scheme 2 — Synthesis of UTP analogues containing a β,γ-dihalomethylene-bridge in the triphosphate group. *Reagents and conditions*: (a) PO(OH)₂CX₂PO(OH)₂, DMF, rt.

Scheme 3 — Synthesis of UTP analogues with modified uracil and ribose moieties. *Reagents and conditions*: (a) (i) POCl₃, proton sponge, PO(OMe)₃, 0°C; (ii) (Bu₃NH)₂H₂P₂O₇, Bu₃N, DMF, 0°C; (iii) TEAB 0.2M rt.; (b) (i) POCl₃, PO(OMe)₃, 4 h, 0°C; (ii) conc. NH₄OH; (iii) Bu₃N, CDI, DMF, 48; (iv) (Bu₃NH)₂H₂P₂O₇; TEAB 1M for 19; (c) (i) POCl₃, PO(OMe)₃, 49 or 50, 0°C; (ii) conc. NH₄OH; (iii) CDI, DMF, rt; (iv) (Bu₃NH)₂H₂P₂O₇, DMF, rt for 20 or 21. Note that the UDP analogue 6 (not shown in scheme) was prepared by a similar method to (a), except for use of a phosphoric acid salt in the second step: (ii) (Bu₃NH)₂H₂PO₄, Bu₃N, DMF, 0°C.

Scheme 4 — Synthesis of a 5-iodo analogue of INS48823. *Reagents and conditions*: (a) phenylacetaldehyde dimethylacetal, TFA, rt; (b) 5-iodo-uridine-5′-diphosphoimidazolidate, DMF, rt.

Scheme 5 — Synthesis of 5′-tetraphosphate analogues, including Up₄-sugars and dinucleotides. The 2′-deoxyguanosine derivative 44 was prepared in the same manner as 43. *Reagents and conditions*: (a) (i) DCC, DMF, rt; (ii) RPO(OH)₂, DMF, rt.

The synthesis of UTP analogues 12 and 17 – 25 from the corresponding nucleosides was by standard methods of di-, triphosphate formation.⁶ In each case the unprotected nucleoside was first treated with phosphorous oxychloride (Scheme 3). The reaction mixture was either treated immediately with bis(tri-n-butylammonium) pyrophosphate (phosphoric acid for 12) or the isolated 5′-monophosphate was activated with 1,1′-carbonyldiimidazole (CDI) followed by the pyrophosphate salt. An attempt to synthesize 2,4-dithio-UTP led to isolation only of the 4-methylthio analogue 17. Identification of nucleotide compounds was confirmed by NMR (¹H and ¹³P) and by high-resolution mass spectrometry (HRMS), and purity was demonstrated with high-performance liquid chromatography (HPLC) in two different solvent systems.

The preferred method of synthesis of 2′-MeUTP (20) and 3′-MeUTP (21) was through isolation of the monophosphate. 2′-C-Methyl-uridine-5′-mophosphate¹⁴ and 3′-C-methyl-uridine-5′-monophosphate¹⁵ were obtained as ammonium salts following the Yoshikawa procedure¹⁹ starting from nucleosides 2′-C-methyl-uridine (49) and 3′-C-methyl-uridine (50). The nucleotides also were prepared by the one-pot method using a sequential reaction of 49 and 50 with phosphorus oxychloride and pyrophosphoric acid tributylammonium salt, but the yields were lower.

Most of the required nucleoside precursors were readily available, with several exceptions. 2′-Ureido-2′-deoxyuridine 46 was prepared by a one step method from 2′-amino-2′-deoxyuridine 57 (Scheme 6A).¹⁷ The synthesis of 2,4-dithiouridine 47 is depicted in Scheme 6. The commercially available β-D-ribofuranose 1,2,3,5-tetraacetate 58 was coupled with silylated 2-thiouracil under SnCl₄-catalyzed Vorbrüggen conditions.¹⁸ 4-Thionation of the resulting 2′,3′,5′-tri-O-acetyl-2-thiouridine 59 was performed using Lawesson’s reagent.¹⁹ Subsequent sugar deprotection of 60 afforded 2,4-dithiouridine 47 in 59% overall yield. The nucleoside 1-(β-D-arabinofuranosyl)-2-thio(1H) pyrimidin-4-one 48 was obtained via opening of 2,2′-O-anhydrouridine 61 with H₂S and triethylamine in anhydrous DMF (Scheme 6).²⁰ The synthetic routes to the nucleosides 2′-C-methyl-uridine 49 and 3′-C-methyl-uridine 50, synthesized using the strategy reported by Wolfe & Harry-O’kuru²¹ and Mikhailov et al.^22a with some pmodifications, are outlined in Scheme 6D.

Pharmacological Activity

Activation of PLC by a range of concentrations of each nucleotide derivative (7 – 44) was studied in [³H]inositol-labeled 1321N1 human astrocytoma cells stably expressing the human P2Y₂, P2Y₄, or P2Y₆ receptors (Tables 1 – 3) by methodology (see Experimental Section) we have described previously in detail.¹^,³^,⁶^,²⁴

Table 1 illustrates UDP analogues that were designed for possible interaction with the P2Y₆ receptor. Compounds 7 and 8 are derivatives substituted with an anionic carboxylic acid or phosphonate acetyl ester moiety with the goal of approximating the charge and electronic characteristics of the diphosphate moiety for interaction with cationic residues in the ligand binding pocket⁸. These molecules were inactive at the P2Y₆ receptor. Introduction of an α,β-methylene 9 substitution only slightly reduced potency at the P2Y₆ receptor (Figure 1), while the α,β-difluoromethylene analogue 10 was strikingly inactive. Nucleobase substitution was also examined in the UDP series. We previously reported that the higher homologue of 11, 5-amino-UTP, displays increased potency relative to UTP as a P2Y₆ receptor agonist⁷. In contrast, the corresponding diphosphate 11 was ~50-fold less potent than UDP. The introduction of a polar ureido group on the ribose moiety of adenosine derivatives provided tailored analogues for selective recognition by adenosine neoceptors.²⁷ Therefore, we replaced the 2′-hydroxy group of UDP with a highly H bonding ureido group. This molecule exhibited a 360-fold reduction of potency at the P2Y₆ receptor compared to UDP.

Activity of compounds 2 (the native agonist, UDP) and 9 (α,β-methylene-UDP) at the P2Y₆ receptor as indicated by activation of PLC in stably transfected astrocytoma cells.

Table 2 illustrates composite data for UTP analogues designed to activate the P2Y₂ and P2Y₄ receptors. We previously illustrated that substitution of a 2-thio group enhances potency and/or selectivity of UTP analogues (e.g. 5 and 14) at the P2Y₂ receptor.⁷ The combination of the 2-thio modification with a β,γ-difluoromethylene 15 or β,γ-dichloromethylene 16 substitution of the triphosphate moiety or 4-methylthio 17 group resulted in analogues that were 50 – 100-fold weaker than 14. A report of another UTP analogue,4 5-bromo-β,γ-dichloromethylene-UTP, that displayed submicromolar P2Y₂ receptor potency had suggested the possibility of greater potency in 15 and 16 than we observed experimentally in the current study. Nevertheless, the dichloromethylene group of 16 provides moderate selectivity for the P2Y₂ receptor, while the equipotent difluoromethylene derivative 15 was only marginally selective. Replacement of the 2′-hydroxy group of UTP with a ureido group 18 resulted in a 36-fold reduction of potency at the P2Y₂ receptor and >100-fold reduction at the P2Y₄ receptor.

Methyl groups were placed on the ribose ring of UTP at the 2′ and 3′ positions in 20 and 21, respectively. Like the methanocarba modification of ribose,⁶^,²⁸ this approach is a means of conformational control of the ribose ring that has proven effective for achieving selectivity in A₁ adenosine receptor agonists.²⁹^,³⁰ Compound 20 maintains a North conformation of the ribose ring, and as is the case with UTP, activated the P2Y₂ and P2Y₄ receptors nearly equipotently. However, a 20–30-fold decrease in potency relative to UTP was observed. In contrast, 21, which maintains a South conformation of the ribose ring, was inactive at both P2Y₂ and P2Y₄ receptors. These results are consistent with previously reported conformational preferences of these receptors deduced from our studies with methanocarba-derivatives of UTP.⁶

No previous studies have reported the potency of nucleoside 5′-tetraphosphates at the P2Y₂ receptor, although adenosine 5′-tetraphosphate (Ap₄) was reported to activate a presumed P2X receptor.³¹ Therefore, we synthesized and evaluated the activities of 5′-tetraphosphate and pentaphosphate derivatives 22–25. The potency of uridine 5′-tetraphosphate 22 (Up₄) was greatly reduced in comparison to UTP 1. The high P2Y₂ receptor potency of 2-thio-UTP 14 was remarkably preserved in the corresponding 4-thio-5′-tetraphosphate analogue 24; in contrast, the potency of the 2-thio analogue 23 was reduced. Nevertheless, these results illustrate that 2- or 4-thio substitution of Up₄ results in marked increases of potency in tetraphosphate molecules since both 23 and 24 were more potent at the P2Y₂ receptor than Up₄ 22. Homologation of 23 to the pentaphosphate 25 had no effect on potency at the P2Y₂ receptor.

Although dinucleoside tetraphosphates, such as 3 and 4, are known to activate P2Y₂ and P2Y₄ receptors with moderate potency,⁹ the SAR of the terminal nucleoside moiety mainly has been explored for uridine and other nucleoside units. Therefore, we investigated the effects of substitution of the terminal nucleoside moiety with small organic moieties 26 – 31 or to sugars alone 32 – 37. Most of these modifications led to P2Y₂ receptor potencies in the micromolar range. A terminal methyl phosphoester 26 and the corresponding phosphonate 27 were identical in potency as relatively weak P2Y₂ receptor agonists, with EC₅₀ values of 4 µM. A terminal cyclohexyl ester 31 was significantly less potent than the corresponding terminal phenyl ester 30, suggesting that aromatic or hydrophilic groups are more favored than simple hydrophobic groups in this region. Curiously, substitution of an acyclic alkyl phosphate or phosphonate at the terminal position (26–29) did not favor selective interaction with the P2Y₂ versus P2Y₄ receptors, while an aryl phosphate ester (30) substitution resulted in high selectivity for the P2Y₂ receptor (Figure 2).

Activity of compound 30 (uridine 5′-tetraphosphate δ-phenyl ester) at P2Y₂, P2Y₄, and P2Y₆ receptors as indicated by activation of PLC in stably transfected astrocytoma cells. The effect of UTP corresponds to 100%.

Among tetraphosphate sugar derivatives, Up₄-[1]glucose 34 was the most potent with an EC₅₀ of 0.3 µM (Figure 3). The P2Y₂ receptor selectivity of 34 in comparison to P2Y₄ and P2Y₆ receptors was 7- and 26-fold, respectively. Inversion of the chirality of the 4′-hydroxyl group in the sugar, i.e. galactose rather than glucose, in 35 reduced potency at the P2Y₂ receptor (16-fold), but not at the P2Y₄ receptor. The [6]mannose adduct 36 of Up₄ was essentially inactive at the P2Y₂ receptor. Removal of a single hydroxyl group of 36 to form 37 increased the potency at both P2Y₂ and P2Y₄ receptors. These results are consistent with our recent P2Y₂ receptor modeling study, which proposes specific interactions in the region of the distally-binding uridine moiety of Up₄U.⁷

Activity of compounds 34 – 37 (uridine 5′-tetraphosphate δ-sugars) at the P2Y₂ receptor as indicated by activation of PLC in stably transfected astrocytoma cells.

The SAR of dinucleotides at the P2Y₂ and P2Y₆ receptors also was further explored (Table 3). An analogue of a known P2Y₆ receptor agonist⁹, Up₃U 38, was designed to combine multiple reported favorable modifications of uracil nucleotides directed toward activation of the P2Y₆ receptor based on the agonist INS48823 6.¹² The resulting hybrid compound 39, containing 5-iodo substitution and a 2′,3′-phenylmethylacetyl group on opposite uridine moieties of Up₃U, was a nonselective agonist of the P2Y₆ receptor.

Several new dinucleoside tetraphosphate derivatives 40 – 44 were synthesized, and their activities were compared to previously studied compounds 3, 4.⁹^,¹¹ The inclusion of a β,γ-difluoromethylene 41 or dichloromethylene 42 bridge in the tetraphosphate moiety resulted in analogues that were at least an order of magnitude weaker at the P2Y₂ receptor than Up₄U 3. The difluoro analogue 41, which exhibited an EC₅₀ of ~2 µM at the P2Y₂ receptor, apparently provides a better mimic of the phosphate ester group than does the dichloro substitution in 42. These results are consistent with electronic effects of the difluoromethylene group noted for other phosphonates.²⁵ The P2Y₂ receptor agonist INS37217 4 also was prepared for comparison.¹¹ Substitution of a 2-thio group in 43 resulted in enhanced potency (EC₅₀ ~80 nM) and selectivity for the P2Y₂ receptor (9-fold in comparison to P2Y₄). A 2′-deoxyguanosine analogue 44 also exhibited moderate potency at the P2Y₂ receptor, as reported.¹¹

Many of the analogues synthesized and studied including Up₄-sugars retained moderate potency at the P2Y₄ receptor. However, our work to date has not revealed molecules that exhibit notable selectivity for the P2Y₄ receptor subtype over the other uridine nucleotide activated receptors.

In conclusion, we have synthesized novel uracil nucleotide derivatives that are directed toward activation of the P2Y₂ or P2Y₆ receptor. Key compounds reported in this study include: 34, Up₄-[1]glucose, which displayed submicromolar potency at the P2Y₂ receptor; 43, the 2-thio analogue of INS37217, which was a potent and selective P2Y₂ receptor agonist. Thus, the 2-thio modification, found previously to enhance P2Y₂ receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y₂ receptor, in the form of Up₄-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule.

Experimental Section

Chemical Synthesis

¹H NMR spectra were obtained with a Varian Gemini 300 or Varian Mercury 400 spectrometers using D₂O, CDCl₃ or DMSO-d₆ as a solvent. The chemical shifts are expressed as relative ppm from HOD (4.80 ppm). ¹³P NMR spectra were recorded at rt (rt) by use of Varian XL 300 (121.42 MHz) or Varian Mercury 400 (162.10 MHz) spectrometers; orthophosphoric acid (85%) was used as an external standard. In several cases the signal of the terminal phosphate moiety was not visible due to high dilution.

Purity of compounds was checked using a Hewlett–Packard 1100 HPLC equipped with a Zorbax Eclipse 5 μm XDB-C18 analytical column (250 × 4.6 mm; Agilent Technologies Inc, Palo Alto, CA). Mobile phase: linear gradient solvent system: 5 mM TBAP (tetrabutylammonium dihydrogenphosphate)-CH₃CN from 80:20 to 40:60 in 20 min; the flow rate was 1 mL/min. Peaks were detected by UV absorption with a diode array detector at 254, 275, and 280 nm. All derivatives tested for biological activity showed >99% purity by HPLC analysis (detection at 254 nm).

High-resolution mass measurements were performed on Micromass/Waters LCT Premier Electrospray Time of Flight (TOF) mass spectrometer coupled with a Waters HPLC system, unless noted. Purification of the nucleotide analogues for biological testing was carried out on (diethylamino)ethyl (DEAE)-A25 Sephadex columns with a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase. Compounds 16, 20, 21, 39, and 54 were purified by Sephadex alone (and isolated in the ammonium salt form), and all other compounds were additionally purified by HPLC with a Luna 5 μ RP-C18(2) semipreparative column (250 × 10.0 mm; Phenomenex, Torrance, CA) and using the following conditions: flow rate of 2 mL/min; 10 mM triethylammonium acetate (TEAA)-CH₃CN from 100:0 to 95:5 (or up to 99:1 to 92:8) in 30 min (and isolated in the triethylammonium salt form).

All reagents were of analytical grade. 2′-Amino-2′-deoxyuridine was from Metkinen Chemistry (Kuusisto, Finland). 2,2′-O-Anhydrouridine 61 was obtained from Wako Chemicals. 5-Amino-UDP was purchased from ALT, Inc. (Lexington, KY). Other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO).

General Procedure for the Preparation of Nucleoside Triphosphates (e.g. 14 and 17), Tetraphosphates (22 – 24), Pentaphosphate (25). Procedure A

To a solution of nucleoside (0.054 mmol) and Proton Sponge (23 mg, 0.11 mmol) in trimethyl phosphate (1 mL) was added phosphorous oxychloride (0.01 mL, 0.11 mmol) at 0°C. The reaction mixture was stirred for 2 h at 0°C and tributylamine (0.03 mL, 0.12 mmol) was added. Tributylammonium pyrophosphate (1.6 moles C₁₂H₂₇N per mole H₄P₂O₇, 110 mg, 0.23 mmol) in DMF (0.3 mL) was added at once to the reaction mixture. After 10 min, 0.2 M triethylammonium bicarbonate solution (2 mL) was added, and the clear solution was stirred at rt for 1 h. The latter was lyophilized overnight, and the resulting residue was purified by ion-exchange column chromatography using a Sephadex-DEAE A-25 resin with a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase to obtain the corresponding nucleotides as the ammonium salts. The collected portions were purified by HPLC again as described above.

General Procedure for the Preparation of Nucleosides Tetraphosphates (26 – 31), Nucleoside Tetraphosphate Sugars (32 – 37), and Dinucleoside Tetraphosphates (40 – 44). Procedure B

Uridine triphosphate trisodium salt (15 mg, 0.027 mmol or UTP analogues for compounds 40 – 44) and the corresponding monophosphate (0.109 mmol) were converted to the tributylammonium salts by treatment with ion-exchange resin (DOWEX 50WX2-200 (H)) and tributylamine. After removal of the water, the obtained tributylammonium salts were dried under high-vacuum overnight. To a solution of uridine triphosphate tributylammonium salt (0.027 mmol) in DMF (2 mL) was added N,N′-dicyclohexylcarbodiimide (DCC, 14 mg, 0.07 mmol). After stirring the reaction mixture at rt for 1 h, a solution of the corresponding monophosphate tributylammonium salt (0.109 mmol) in DMF (1 mL) was added. The reaction mixture was stirred at rt for 48 h. After removal of the solvent, the residue was purified by ion-exchange column chromatography with a Sephadex-DEAE A-25 resin, followed by a semi preparative HPLC purification as described above. Free nucleoside 5′-tetra- and pentaphosphates were not stable upon prolonged storage at 4°C, but were stable at −20°C for at least 3 weeks. Dinucleoside tetraphosphates were stable for several months at −20°C and later showed signs of gradual decomposition.

General Procedure for the Preparation of Compounds (7 – 10). Procedure C

To a solution of uridine (25 mg, 0.1 mmol) and DCC (62 mg, 0.3 mmol) in DMF (1.5 mL) was added the appropriate carboxylic acid or phosphonic acid (0.15 mmol): malonic acid for 7, phosphonoacetic acid for 8, methylene diphosphonic acid for 9, difluoromethylene diphosphonic acid for 10. After stirring the reaction mixture at rt for 24 h to 48 h, the solvent was removed. The residue was purified by ion-exchange column chromatography with a Sephadex-DEAE A-25 resin, followed by a semi preparative HPLC purification as described above.

Uridine-5′-phosphonoacetate triethylammonium salt (8). Procedure C

Compound 8 (13.4 mg, 36%) was obtained as a white solid from uridine (25 mg, 0.1 mmol). ¹H NMR (D₂O) δ 7.81 (d, J = 8.1 Hz, 1H), 5.94 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 4.5 Hz, 1H), 4.41 (m, 3H), 4.33 (m, 2H), 2.89 (d, J = 20.4 Hz, 2H); ¹³P NMR (D₂O) δ 12.23; ¹³C NMR (D₂O) δ (169.27, 169.19), 164.92, 150.34, 140.53, 101.18, 88.21, 80.14, 72.06, 68.06, 62.87, (36.28, 34.73); HRMS-EI found 367.0658 (M−H)⁻. C₁₁H₁₆N₂O₁₀P requires 367.0543; purity > 99% by HPLC (retention time: 5.9 min).

Uridine-5′-α,β-methylene-diphosphate triethylammonium salt (9). Procedure C

Compound 9 (14 mg, 33%) was obtained as a white solid from uridine (25 mg, 0.1 mmol). ¹H NMR (D₂O) δ 8.03 (d, J = 8.1 Hz, 1H), 5.98 (m, 2H), 4.41 (m, 2H), 4.28 (m, 1H), 4.19 (m, 2H), 2.18 (t, J = 19.5 Hz, 2H); ¹³P NMR (D₂O) δ 19.37 (m), 14.44 (m); HRMS-EI found 403.0245 (M+H)⁻. C₁₀H₁₇N₂O₁₁P₂ requires 403.0308; purity > 99% by HPLC (retention time: 12.6 min).

Uridine-5′-α,β-difluoromethylenediphosphate triethylammonium salt (10). Procedure C

Compound 10 (14 mg, 33%) was obtained as a white solid from uridine (25 mg, 0.1 mmol). ¹H NMR (D₂O) δ 8.02 (d, J = 8.1 Hz, 1H), 5.98 (m, 2H), 4.40 (m, 2H), 4.26 (m, 1H), 4.19 (m, 2H); ¹³P NMR (D₂O) δ 4.69 (m), 3.48 (m); HRMS-EI found 436.9977 (M−H)⁻. C₁₀H₁₃N₂O₁₁F₂P₂ requires 436.9963; purity > 99% by HPLC (retention time: 14.1 min).

2′-Deoxy-2′-ureido-uridine-5′-diphosphate triethylammonium salt (12)

A solution of the compound 46 (38 mg, 0.13 mmol) and Proton Sponge (43 mg, 0.20 mmol) in trimethyl phosphate (1 mL) was stirred for 10 min at 0°C. Then phosphorous oxychloride (25 μL, 0.27 mmol) was added dropwise, and the reaction mixture was stirred for 2 h at 0°C. A mixture of tributylamine (0.25 mL, 1.05 mmol) and a solution 0.35 M of bis(tributylammonium) salt of phosphoric acid in DMF (2.28 mL) was added at once. This salt was prepared by mixing tributylamine (0.4 mL, 1.65 mmol) and phosphoric acid (85 mg, 0.87 mmol) in DMF (2.5 mL). After 10 min, 0.2 M triethylammonium bicarbonate (TEAB) solution (3 mL) was added, and the clear solution was stirred at rt for 30 min. The latter was lyophilized overnight. The residue was purified by Sephadex-DEAE A-25 resin ion-exchange column chromatography, followed by semipreparative HPLC as described above to obtain 12 (8 mg, 8%) as a white solid. ¹H NMR (D₂O) δ 7.98 (d, J = 8.1 Hz, 1H), 6.02 (m, 2H), 4.51 (m, 1H), 4.34 (m, 2H), 4.20 (m, 2H); ¹³P NMR (D₂O) δ −7.24, −10.14 (d, J = 22.0 Hz); HRMS-EI found 445.0172 (M−H)⁻. C₁₀H₁₅N₄O₁₂P₂ requires 445.0162; purity > 99% by HPLC (retention time: 12.7 min).

2-Thio-uridine-5′-β,γ-difluoromethylene-triphosphate triethylammonium salt (15)

To a solution of 2-thio-uridine 5′-monophosphate morpholidate 4-morpholine-N,N dicyclohexylcarboxamidine salt, 45²⁶ (10 mg, 0.014 mmol) in DMF (2 mL), difluoromethylene diphosphonate tributylammonium salt (20 mg, 0.021 mmol) was added. After being stirred 3 days at rt, the reaction mixture was evaporated to remove the solvent and purified by Sephadex-DEAE A-25 resin followed by HPLC purification to give 15 (4 mg, 31%). ¹H NMR (D₂O) δ 8.18 (d, J = 8.1 Hz, 1H), 6.69 (d, J = 3.0 Hz, 1H), 6.28 (d, J = 8.1 Hz, 1H), 4.31–4.47 (m, 5H); ¹³P NMR (D₂O) δ 3.47, −3.80 (m), −10.69 (d, J = 31.2 Hz); HRMS-EI found 532.9375 (M−H)⁻. C₁₀H₁₄N₂O₁₃F₂P₃S requires 532.9398; purity > 99% by HPLC (retention time: 18.1 min).

2-Thio-uridine-5′-β, γ-dichloromethylene-triphosphate ammonium salt (16)

To a solution of 2-thio-uridine 5′-monophosphate morpholidate 4-morpholine-N,N dicyclohexylcarboxamidine salt, 45²⁶ (7 mg, 0.01 mmol) in DMF (2 mL), dichloromethylene diphosphonate tributylammonium salt (25 mg, 0.025 mmol) was added. After being stirred 3 days at rt, the reaction mixture was evaporated to remove the solvent and purified by Sephadex-DEAE A-25 resin to give 16 (5 mg, 80%). ¹H NMR (D₂O) δ 8.20 (d, J = 8.1 Hz, 1H), 6.69 (d, J = 2.7 Hz, 1H), 6.29 (d, J = 8.4 Hz, 1H), 4.45 (m, 2H), 4.39 (m, 2H), 4.34 (m, 1H); ¹³P NMR (D₂O) δ 8.08 (d, J = 17.7 Hz), 1.17 (dd, J =17.7, 31.2 Hz), −10.60 (d, J = 31.2 Hz); HRMS-EI found 564.8817 (M−H)⁻. C₁₀H₁₄N₂O₁₃Cl₂P₃S requires 564.8807; purity > 99% by HPLC (retention time: 19.5 min).

2-Thio-4-methylthio-uridine-5′-triphosphate triethylammonium salt (17). Procedure A

Compound 17 (3.2 mg, 6.4%) was obtained as a white solid from 2,4-dithio-uridine, 47 (15 mg, 0.054 mmol). ¹H NMR (D₂O) δ 8.51 (d, J = 7.5 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 6.53 (br s, 1H), 4.45 (m, 2H), 4.38 (m, 3H), 2.60 (s, 3H); ¹³P NMR (D₂O) δ −11.00 (d, J = 19.2 Hz), −21.02 (m); HRMS-EI found 528.9315 (M−H)⁻. C₁₀H₁₆N₂O₁₃P₃S₂ requires 528.9307; purity > 99% by HPLC (retention time: 17.1 min).

2′-Deoxy-2′-ureido uridine-5′-triphosphate triethylammonium salt (18). Procedure A

A solution of the compound 46 (38 mg, 0.13 mmol) and Proton Sponge (43 mg, 0.20 mmol) in trimethyl phosphate (1 mL) was stirred for 10 min at 0°C. Phosphorous oxychloride (25 μL, 0.27 mmol) was then added dropwise, and the reaction mixture was stirred for 2 h at 0°C. A solution of tributylammonium pyrophosphate (377 mg, 0.80 mmol) and tributylamine (0.13 mL, 0.53 mmol) in DMF (1 mL) was added and stirring was continued at 0°C for additional 10 min. Triethylammonium bicarbonate solution (TEAB, 3 mL of 0.2 M) was added, and the reaction mixture was stirred at rt for 30 min. The latter was lyophilized overnight. The residue was purified by Sephadex-DEAE A-25 resin ion-exchange column chromatography, followed by semipreparative HPLC as described above to obtain 18 (9 mg, 7%) as a white solid. ¹H NMR (D₂O) δ 7.95 (d, J = 8.4 Hz, 1H), 6.05 (d, J = 7.8 Hz, 1H), 6.01 (d, J = 8.4 Hz, 1H), 4.51 (m, 1H), 4.35 (m, 2H), 4.25 (m, 2H); ¹³P NMR (D₂O) δ −10.15, −12.15 (d, J = 19.5 Hz), −23.6; HRMS-EI found 524.9812 (M−H)⁻. C₁₀H₁₆N₄O₁₅P₃ requires 524.9825; purity > 99% by HPLC (retention time: 16.6 min).

1-(β-D-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one 5′-triphosphate triethyl ammonium salt (19)

Solution of 48 (150 mg, 0.58 mmol) in trimethyl phosphate (5.8 mL) was cooled to 0°C, POCl₃ (342 μL, 3.8 mmol) was added dropwise and the mixture was stirred for 4 h at 0°C and for 30 min at rt. The mixture was poured into ice-water (10 mL), neutralized with concentrated NH₄OH and evaporated to dryness. The resulting residue was purified by column chromatography (i-PrOH:NH₄OH:H₂O 60:30:5). After lyophilization of the collected pure fractions, the 5′-monophosphate of 48 was obtained as a white solid (124 mg, 60%). ¹H NMR (D₂O) δ 7.97 (d, J = 8.2 Hz, 1H), 6.78 (d, J = 5.0 Hz, 1H), 6.07 (d, J = 8.2 Hz, 1H), 4.48 (app t, J = 4.8 Hz, 1H), 4.12 (app t, J = 4.8 Hz, 1H), 3.90–4.01 (m, 3H); ¹³P NMR (D₂O) δ 3.42; HRMS-EI found 363.0271 [M+Na]⁺. C₉H₁₂N₂O₈P₁S₁Na requires 363.0281. To a solution of 5′-monophosphate of 48 (32 mg, 0.088 mmol) and tributylamine (21 μL, 0.088 mmol) in DMF (3.2 mL) was added CDI (71 mg, 0.44 mmol). After stirring for 3 h at rt, the reaction was quenched by addition of methanol (14 μL). Bis(tri-n-butylammonium)pyrophosphate (228 mg, 0.51 mmol) was added, and the mixture was stirred and subsequently concentrated under reduced pressure. The resulting residue was stirred in 1M triethylammonium bicarbonate (TEAB) buffer (6 mL, pH = 7.4) for 30 min, lyophilized and purified on a preparative HPLC apparatus equipped with a source 15 Q column (100% water → 100% 1 M TEAB/water in 45 min) to yield 10 μmol (11%) of compound 19 after lyophilizing the appropriate fractions. ¹H NMR (D₂O) δ 7.90 (d, J = 8.1 Hz, 1H), 6.80 (d, J = 5.2 Hz, 1H), 6.08 (d, J = 8.2 Hz, 1H), 4.48 (app t, J = 5.0 Hz, 1H), 4.16 (m, 3H), 4.03 (m, 1H); ¹³P NMR (D₂O) δ −9.54 (d, J = 19.6 Hz), −10.35 (d, J = 19.6 Hz), −22.19 (t, J = 19.6 Hz), HRMS-EI found 498.9099 [M−H] ⁻. C₉H₁₄N₂O₁₄P₃S requires 498.9384. Structural assignment was confirmed with COSY. All signals assigned to hydroxyl groups were exchangeable with D₂O. Exact mass measurements were performed on a quadrupole/orthogonal-acceleration time-of-flight (Q/oaTOF) tandem mass spectrometer (qToF 2, Micromass, Manchester, UK) equipped with a standard electrospray ionization (ESI) interface. Samples were infused in a i-PrOH/water (1:1) mixture at 3 μL/min.

General Procedure for the Preparation of the Nucleoside 5′-triphosphates 2′-MeUTP (20) and 3′-MeUTP (21)

To a solution of 2′-¹⁴ or 3′-C-methyl-UMP¹⁵ (0.15 mmol) dissolved in dry DMF (1.5 mL) was added tri-n-butylamine (0.15 mmol) and the solution was stirred for 20 min at rt. After evaporation under anhydrous condition, the residue was suspended in 1.4 mL of dry DMF and CDI (122 mg, 0.75 mmol) was added and the mixture was stirred for 6 h at rt. Methanol (49 μl, 1.2 mmol) was added and the mixture was stirred for 30 min. Then 6 mL (3 mmol) of a 0.5 M solution of bis(tri-n-butylammonium) pyrophosphate in dry DMF were added. The mixture was stirred for 24 h at rt, and the solvent was removed under high vacuum at rt. The mixture dissolved in water was purified by Sephadex DEAE A-25 resin ion exchange column chromatography with a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate. Compounds 20 and 21 were isolated as ammonium salts (yield 32 and 34%, respectively). Mass spectroscopy was carried out on an HP 1100 series instrument in the negative ion mode using atmospheric pressure electrospray ionization (API-ESI).

2′-C-Methyl-uridine-5′-triphosphate ammonium salt (20)

¹H NMR (D₂O) δ 7.76 (d, J = 8.1 Hz, 1H), 5.86 (s, 1H), 5.75 (d, J = 8.1 Hz, 1H), 3.85–3.90 (m, 2H), 3.75 (d, J = 9.0 Hz, 1H), 3.68 (dd, J = 4.3, 13.7 Hz, 1H), 1.05 (s, 3H); ¹³P NMR (D₂O) δ ′4.75 (br s), −19.34 (br s), −20.59 (m); MS m/z 497.10 [M+H]⁻.

3′-C-Methyl-uridine-5′-triphosphate ammonium salt (21)

¹H NMR (D₂O) δ 7.78 (d, J = 8.1 Hz, 1H), 5.84 (d, J = 7.7 Hz, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.03 (d, J = 7.7 Hz, 1H), 3.94 (dd, J = 3.4, 5.1 Hz, 1H), 3.65 (dd, J = 3.4, 12.8 Hz, 1H), 3.58 (dd, J = 4.9, 12.6, 1H),1.20 (s, 3H); ¹³P NMR (D₂O) δ 4.83 (d, J = 15.9 Hz), −19.25 (t, J = 15.3 Hz), −20.66 (t, J = 15.9 Hz); MS m/z 497.10 [M+H]⁻.

2-Thio-uridine-5′-tetraphosphate triethylammonium salt, 2-thio-U_P4 (23). Procedure A

Compound 23 (1.5 mg, 4.5%) was obtained as a white solid from 2-thio-uridine (10 mg, 0.038 mmol). ¹H NMR (D₂O) δ 8.17 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 3.0 Hz, 1H), 6.27 (d, J = 8.1 Hz, 1H), 4.46 (m, 2H), 4.33 (m, 3H); ¹³P NMR (D₂O) δ −11.18 (d, J = 18.9 Hz), −22.55 (m); HRMS-EI found 578.9042 (M−H)⁻. C₉H₁₅N₂O₁₇P₄S requires 578.9042; purity > 99% by HPLC (retention time: 19.8 min).

4-Thio-uridine-5′-tetraphosphate triethylammonium salt (24). Procedure A

Compound 24 (5.2 mg, 7.6%) was obtained as a white solid from 4-thio-uridine (20 mg, 0.077 mmol). ¹H NMR (D₂O) δ 7.88 (d, J = 7.8 Hz, 1H), 6.66 (d, J = 7.8 Hz, 1H), 5.95 (d, J = 4.5 Hz, 1H), 4.47 (m, 1H), 4.40 (m, 1H), 4.28 (m, 3H); ¹³P NMR (D₂O) δ −10.91 (d, J = 20.2 Hz), −21.97 (m); HRMS-EI found 578.8890 (M−H)⁻. C₉H₁₅N₂O₁₇P₄S requires 578.9042; purity > 99% by HPLC (retention time: 19.2 min).

2-Thio-uridine-5′-pentaphosphate triethylammonium salt (25). Procedure A

Compound 25 (1.1 mg, 3%) was obtained as a white solid from 2-thio-uridine 52 (10 mg, 0.038 mmol). ¹H NMR (D₂O) δ 8.15 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 3.6 Hz, 1H), 6.26 (d, J = 8.1 Hz, 1H), 4.43 (m, 2H), 4.32 (m, 3H); ¹³P NMR (D₂O) δ −11.22 (d, J = 18.3 Hz), −22.78 (m); HRMS-EI found 658.8782 (M−H)⁻. C₉H₁₆N₂O₂₀P₅S requires 658.8705; purity > 99% by HPLC (retention time: 19.9 min).

Uridine-5′-methyl-tetraphosphate triethylammonium salt (26). Procedure B

Compound 26 (7.8 mg, 33%) was obtained as a white solid using uridine triphosphate (20 mg, 0.036 mmol) and methylphosphate (45 mg, 0.15 mmol). ¹H NMR (D₂O) δ 8.00 (d, J = 8.1 Hz, 1H), 6.03 (d, J = 5.7 Hz, 1H), 6.00 (d, J = 8.1 Hz, 1H), 4.44 (m, 2H), 4.31 (m, 1H), 4.26 (m, 2H), 3.69 (d, J = 11.7 Hz, 3H); ¹³P NMR (D₂O) δ −8.70 (d, J = 17.7 Hz), −10.59 (d, J = 18.9 Hz), −22.46; HRMS-EI found 576.9375 (M−H)⁻. C₁₀H₁₇N₂O₁₈P₄ requires 576.9427; purity > 99% by HPLC (retention time: 18.8 min).

Uridine-5′-methyl^(C-P)-tetraphosphate triethylammonium salt (27). Procedure B

Compound 27 (8.6 mg, 34%) was obtained as a white solid using uridine triphosphate (22 mg, 0.04 mmol) and methylphosphoric acid (22 mg, 0.23 mmol). ¹H NMR (D₂O) δ 7.98 (d, J = 8.4 Hz, 1H), 6.02 (d, J = 5.4 Hz, 1H), 5.99 (d, J = 8.4 Hz, 1H), 4.43 (m, 2H), 4.28 (m, 1H), 4.25 (m, 2H), 1.49 (d, J = 17.1 Hz, 3H); ¹³P NMR (D₂O) δ 19.05, −10.57 (d, J = 18.3 Hz), −22.34 (d, J = 18.9 Hz); HRMS-EI found 560.9476 (M−H)⁻. C₁₀H₁₇N₂O₁₇P₄ requires 560.9478; purity > 99% by HPLC (retention time: 19.2 min).

Uridine-5′-(2-cyanoethyl)-tetraphosphate triethylammonium salt (28). Procedure B

Compound 28 (2.5 mg, 13%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and 2-cyanoethyl phosphate (35 mg, 0.11 mmol). ¹H NMR (D₂O) δ 7.98 (d, J = 8.1 Hz, 1H), 5.99 (m, 2H), 4.41 (m, 2H), 4.29 (m, 1H), 4.24 (m, 2H), 4.18 (t, J = 6.2 Hz, 2H), 2.88 (t, J = 6.2 Hz, 2H); ¹³P NMR (D₂O) δ −10.54 (d, J = 17.7 Hz), −10.83 (d, J = 18.2 Hz), −22.39; HRMS-EI found 639.9489 (M+Na−H)⁻. C₁₂H₁₉N₃O₁₈P₄Na requires 639.9512; purity > 99% by HPLC (retention time: 19.4 min).

Uridine-5′-α β-glycerol-tetraphosphate triethylammonium salt (29). Procedure B

Compound 29 (2.1 mg, 11%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and D-L-α-glycerol phosphate (35 mg, 0.11 mmol). 1H NMR (D₂O) δ 7.99 (d, J = 7.8 Hz, 1H), 6.01 (m, 2H), 4.44 (m, 2H), 4.30 (m, 1H), 4.26 (m, 2H), 4.01 (m, 3H), 3.67 (m, 2H); ¹³P NMR (D₂O) δ −9.83 (d, J = 17.7 Hz), −10.55 (d, J = 18.3 Hz), −22.40; HRMS-EI found 636.9638 (M−H)⁻. C₁₂H₂₁N₂O₂₀P₄ requires 636.9638; purity > 99% by HPLC (retention time: 18.9 min).

Uridine-5′-phenyl-tetraphosphate triethylammonium salt (30). Procedure B

Compound 30 (1.1 mg, 6%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and phenyl phosphate (28 mg, 0.11 mmol). ¹H NMR (D₂O) δ 7.92 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.17 (t, J = 7.5 Hz, 1H), 5.93 (m, 2H), 4.37 (m, 1H), 4.31 (m, 1H), 4.21 (m, 3H); ¹³P NMR (D₂O) δ −10.51 (d, J = 17.7 Hz), −14.80 (d, J = 17.1 Hz), −22.43; HRMS-EI found 638.9577 (M−H)⁻. C₁₅H₁₉N₂O₁₈P₄ requires 638.9583; purity > 99% by HPLC (retention time: 18.6 min).

Uridine-5′-cyclohexane-tetraphosphate triethylammonium salt (31). Procedure B

Compound 31 (6.7 mg, 20%) was obtained as a white solid using uridine triphosphate (20 mg, 0.036 mmol) and cyclohexene monophosphate tributylammonium salt (20 mg, 0.037 mmol). ¹H NMR (D₂O) δ 8.02 (d, J = 8.1 Hz, 1H), 6.03 (m, 2H), 4.42–4.50 (m, 2H), 4.22–4.34 (m, 4H), 2.01 (m, 2H), 1.74 (m, 2H), 1.33 (m, 6H); ¹³P NMR (D₂O) δ −11.26 (m), −23.10 (m); HRMS-EI found 645.0037 (M−H)⁻. C₁₅H₂₅N₂O₁₈P₄ requires 645.0053; purity > 99% by HPLC (retention time: 19.9 min).

Uridine-5′-fructose-6′-tetraphosphate triethylammonium salt (33). Procedure B

Compound 33 (4.2 mg, 19%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and D-fructose-6-phosphate (51 mg, 0.12 mmol). ¹H NMR (D₂O) δ 7.98 (d, J = 8.3 Hz, 1H), 6.02 (d, J = 5.4 Hz, 1H), 5.99 (d, J = 8.3 Hz, 1H), 4.43 (m, 2H), 4.32–4.06 (m, 7H), 3.95 (m, 1H), 3.63 (m, 1H), 3.56 (m, 1H); ¹³P NMR (D₂O) δ −10.08 (d, J = 15.3 Hz), −10.54 (d, J = 18.3 Hz), −22.27; HRMS-EI found 724.9796 (M−H)⁻. C₁₅H₂₅N₂O₂₃P₄ requires 724.9799; purity > 99% by HPLC (retention time: 19.5 min).

Uridine-5′-glucose-1′-tetraphosphate triethylammonium salt (34). Procedure B

Compound 34 (10 mg, 28%) was obtained as a white solid using uridine triphosphate (25 mg, 0.045 mmol) and D-glucose-1-phosphate (68 mg, 0.18 mmol). ¹H NMR (D₂O) δ 8.01 (d, J = 8.4 Hz, 1H), 6.02 (m, 2H), 5.66 (m, 1H), 4.45 (m, 2H), 4.32 (m, 1H), 4.28 (m, 2H), 4.01–3.77 (m, 4H), 3.55 (m, 1H), 3.46 (m, 1H); ¹³P NMR (D₂O) δ −10.53 (d, J = 15.9 Hz), −11.90 (d, J = 16.5 Hz), −22.27; HRMS-EI found 724.9800 (M−H)⁻. C₁₅H₂₅N₂O₂₃P₄ requires 724.9799; purity > 99% by HPLC (retention time: 18.9 min).

Uridine-5′-galactose-1′-tetraphosphate triethylammonium salt (35). Procedure B

Compound 35 (4.5 mg, 16%) was obtained as a white solid using uridine triphosphate (20 mg, 0.036 mmol) and D-galactose-1-phosphate (51 mg, 0.12 mmol). ¹H NMR (D₂O) δ 7.99 (d, J = 8.3 Hz, 1H), 6.03 (d, J = 5.7 Hz, 1H), 6.00 (d, J = 8.3 Hz, 1H), 5.67 (m, 1H), 4.44 (m, 2H), 4.26 (m, 4H), 4.05 (m, 1H), 3.98 (m, 1H), 3.77 (m, 3H); ¹³P NMR (D₂O) δ −10.54 (d, J = 18.3 Hz), −11.74 (d, J = 18.2 Hz), −22.19; HRMS-EI found 724.9781 (M−H)⁻. C₁₅H₂₅N₂O₂₃P₄ requires 724.9799; purity > 99% by HPLC (retention time: 19.0 min).

Uridine-5′-mannose-6′-tetraphosphate triethylammonium salt (36). Procedure B

Compound 36 (2.4 mg, 11%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and D-mannose-6-phosphate (23 mg, 0.08 mmol). ¹H NMR (D₂O) δ 7.97 (d, J = 8.4 Hz, 1H), 5.99 (m, 2H), 5.18 (m, 3/5H), 4.92 (m, 2/5H), 4.43 (m, 2H), 4.24 (m, 5H), 3.95–3.74 (m, 3H), 3.68 (m, 3/5H), 3.52 (m, 2/5H); ¹³P NMR (D₂O) δ −9.92 (m), −10.46 (m), −22.20 (m); HRMS-EI found 724.9814 (M−H)⁻. C₁₅H₂₅N₂O₂₃P₄ requires 724.9799; purity > 99% by HPLC (retention time: 18.6 min).

Uridine-5′-(2′-deoxy-glucose)-6′-tetraphosphate triethylammonium salt (37). Procedure B

Compound 37 (4.2 mg, 20%) was obtained as a white solid using uridine triphosphate (15 mg, 0.027 mmol) and 2′-deoxy-D-glucose-6-phosphate (20 mg, 0.08 mmol). ¹H NMR (D₂O) δ 7.99 (d, J = 7.8 Hz, 1H), 6.01 (m, 2H), 5.38 (m, 1/2H), 4.96 (m, 1/2H), 4.44 (m, 2H), 4.33–4.09 (m, 6H), 3.93 (m, 1/2H), 3.73 (m, 1/2H), 3.51 (m, 1H), 2.23 (m, 1/2H), 2.13 (m, 1/2H), 1.74 (m, 1/2H), 1.53 (m, 1/2H); ¹³P NMR (D₂O) δ −11.60 (d, J = 14.7 Hz), −12.24 (d, J = 18.9 Hz), −23.58, −24.11 (t, J = 12.2 Hz); HRMS-EI found 708.9827 (M−H)⁻. C₁₅H₂₅N₂O₂₂P₄ requires 708.9849; purity > 99% by HPLC (retention time: 18.5 min).

(2-Benzyl-1,3-dioxolo-4-yl)uridine 5′-monophosphate ammonium salt (54)

To a solution of uridine 5′-monophosphate (100 mg, 0.27 mmol) in TFA (1 mL) was added phenylacetaldehyde dimethylacetal (0.3 mL, 1.81 mmol). The reaction mixture was stirred at rt 4 h. After removal of the solvent, the residue was treated with 1 M NaHCO₃ (4 mL) and AcOEt (2 mL). Aqueous phase was separated, evaporated and purified by Sephadex-DEAE A-25 resin as described above to obtain 54 (100 mg, 86%) as a white solid. ¹H NMR (D₂O) δ 7.92 (d, J = 8.3 Hz, 1H), 7.36 (m, 5H), 5.90 (d, J = 8.3 Hz, 1H), 5.63 (m, 1H), 5.47 (m, 1H), 4.95 (m, 1H), 4.90 (m, 1H), 4.42 (m, 1H), 3.89 (m, 2H), 3.16 (m, 2H); ¹³P NMR (D₂O) δ 2.71; HRMS-EI found 425.0748 (M−H)⁻. C₁₇H₁₈N₂O₉P requires 425.0750; purity > 99% by HPLC (retention time: 12.9 min).

P¹-((2-Benzyl-1,3-dioxolo-4-yl)uridine 5′) P3-(5-iodouridine 5′) triphosphate ammonium salt (39)

To a solution of 5-iodouridine 5′-diphosphate5 (10 mg, 0.017 mmol) in DMF (1 mL) was added CDI (7 mg, 0.04 mmol). The reaction mixture was stirred at rt for 6 h. Methanol (1 mL) was then added, and stirring was continued at rt for an additional 1 h. After removal of the solvent, the residue was dried in high vacuum overnight, was dissolved in DMF (2 mL), and was added compound 54 (11 mg, 0.025 mmol). The reaction mixture was stirred at rt overnight. After removal of the solvent, the residue was purified by Sephadex-DEAE A-25 resin as described above to obtain 39 (2.5 mg, 15%) as a white solid. ¹H NMR (D₂O) δ 8.17 (s, 1H), 7.77 (d, J=8.1 Hz, 1H), 7.35 (m, 5H), 5.88 (m, 2H), 5.53 (m, 1H), 5.44 (m, 1H), 4.92 (m, 2H), 4.45 (m, 1H), 4.31 (m, 2H), 4.19 (m, 5H), 3.13 (m, 2H); ¹³P NMR (D₂O) δ −10.68 (d, J = 17.7 Hz), −10.97 (d, J = 20.2 Hz), −22.2; HRMS-EI found 936.9611 (M−H)⁻. C₂₆H₂₉N₄O₂₀P₃I requires 936.9633; purity > 99% by HPLC (retention time: 18.5 min).

P¹,P⁴-Di(uridine 5′-)β, γ-difluoromethylenetetraphosphate triethylammonium salt (41). Procedure B

Compound 41 (1.4 mg, 21%) was obtained as a white solid from uridine-5′-β, γ-difluoromethylenetriphosphate (3.2 mg, 0.0055 mmol) and uridine-5′-monophosphate (3.2 mg, 0.01 mmol). ¹H NMR (D₂O) δ 7.97 (br d, J = 6.1 Hz, 2H), 6.01 (m, 4H), 4.41 (m, 4H), 4.28 (m, 6H); ¹³P NMR (D ₂O) δ −6.28 (m), −10.96 (m); HRMS-EI found 822.9910 (M−H)⁻. C₁₉H₂₅N₄O₂₂F₂P₄ requires 822.9879; purity > 99% by HPLC (retention time: 19.8 min).

P¹,P⁴-Di(uridine 5′-)β, γ-dichloromethlyenetetraphosphate triethylammonium salt (42). Procedure B

Compound 42 (2.2 mg, 19%) was obtained as a white solid from uridine-5′-β, γ-dichloromethylenetriphosphate (5 mg, 0.009 mmol) and uridine-5′-monophosphate (6.4 mg, 0.019 mmol). ¹H NMR (D₂O) δ 7.99 (d, J = 8.1 Hz, 2H), 5.98 (m, 4H), 4.45 (m, 2H), 4.40 (m, 2H), 4.29 (m, 6H); ¹³P NMR (D₂O) δ −1.61 (m), −10.94 (m); HRMS-EI found 854.9203 (M−H)⁻. C₁₉H₂₅N₄O₂₅Cl₂P₄ requires 854.9288; purity > 99% by HPLC (retention time: 20.0 min).

P¹-(2-Thiouridine 5′-)-P4-(2′-deoxycytidine 5′-)tetraphosphate triethylammonium salt (43). Procedure B

Compound 43 (1.1 mg, 7.1%) was obtained as a white solid from 2-thio-uridine triphosphate tributylammonium salt, (14 mg, 0.013 mmol) and 2′-deoxycytidine mono phosphate tributylammonium salt (27 mg, 0.055 mmol). ¹H NMR (D₂O) δ 8.16 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 6.63 (d, J = 3.0 Hz, 1H), 6.31 (t, J = 6.6 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H), 6.15 (d, J = 7.2 Hz, 1H), 4.62 (m, 1H), 4.36 (m, 5H), 4.20 (m, 3H), 2.42 (m, 1H), 2.28 (m, 1H); ¹³P NMR (D₂O) δ −11.10 (m), −22.94 (m); HRMS-EI found 787.9837 (M−H)⁻. C₁₈H₂₆N₅O₂₀P₄S requires 787.9842; purity > 99% by HPLC (retention time: 19.5 min).

2′-Deoxy-2′-ureidouridine (46)

Benzotriazole-1-carboxamide¹⁷ (20 mg, 0.12 mmol) was added to a solution of 2′-amino-2′-deoxyuridine 57 (20 mg, 0.08 mmol) in DMF (4 mL). The reaction mixture was stirred at rt 6 h. After removal of the solvent, the residue was purified by preparative thin-layer chromatography (CH₂Cl₂-MeOH, 8:2) to obtain 46 (21 mg, 89%) as a white solid. ¹H NMR (CD₃OD) δ 7.99 (d, J = 8.3 Hz, 1H), 5.98 (d, J = 8.1 Hz, 1H), 5.71 (d, J = 8.3 Hz, 1H), 4.41 (m, 1H), 4.20 (m, 1H), 4.03 (m, 1H), 3.75 (m, 2H); HRMS-EI found 285.0859 (M−H)⁺. C₁₀H₁₃N₄O₆ requires 285.0835.

2′,3′,5′-Tri-O-acetyl-2-thiouridine (59)

A suspension of 2-thiouracil (2 g, 15.6 mmol) and trimethylsilyl chloride (1.8 mL) in hexamethyldisilazane (80 mL) was treated with a few crystals of ammonium sulphate and refluxed overnight. The clear greenish solution was evaporated and a solution of β-D-ribofuranose 1,2,3,5-tetraacetate (5.5 g, 17.3 mmol) in 20 mL dry dichloroethane was added. After a few minutes, stannic chloride (2.4 mL, 20.8 mmol) was added and after 1h, the mixture was poured into a saturated aq. NaHCO₃ solution under vigorous stirring and then allowed to stand for 1 h. The suspension was filtered over a silica gel pad, which was washed with CH₂Cl₂. The organic layer was separated, dried over MgSO₄ and evaporated to dryness. Silica gel chromatography (CH₂Cl₂:MeOH 99:1) yielded 4.75 g (79%) of compound 59. ¹H NMR (DMSO-d₆) δ10.64 (br s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 6.68 (d, J = 3.8 Hz, 1H), 6.62 (dd, J = 2.3, 7.9 Hz, 1H), 5.45 (dd, J = 3.8, 5.6 Hz, 1H), 5.20 (app t, J = 5.7 Hz, 1H), 4.41–4.47 (m, 2H), 4.31–4.37 (dd, J = 3.2, 13.5 Hz, 1H), 2.09 (s, 6H), 2.06 (s, 3H); HRMS-EI found 387.08604 (M+H)⁺. C₁₅H₁₉N₂O₈S₁ requires 387.08620.

2′,3′,5′-Tri-O-acetyl-2,4-dithiouridine (60)

To a solution of 59 (290 mg, 0.75 mmol) in dry toluene (10 mL), Lawesson’s reagent (304 mg, 0.75 mmol) was added. After heating the reaction mixture at 80°C overnight, insoluble materials were filtered off and the filtrate was purified by silica gel chromatography (Hex:EtOAc 65:35) yielding compound 60 as a yellow foam (250 mg, 83%). ¹H NMR (DMSO-d₆) δ 9.97, (brs, 1H), 7.65 (d, J = 8.2 Hz, 1H), 6.76 (d, J = 4.1 Hz, 1H), 5.98 (d, J = 8.2 Hz, 1H), 5.37 (dd, J = 4.1, 5.6 Hz, 1H), 5.16 (app t, J = 5.7 Hz, 1H), 4.34–4.40 (m, 2H), 4.25–4.30 (dd, J = 3.5,13.5 Hz, 1H), 2.09 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H); HRMS-EI found 403.06338 (M+H)⁺ C₁₅H₁₉N₂O₇S₂. requires 403.06335.

2,4-Dithiouridine (47)

Compound 60 (240 mg, 0.60 mmol) was dissolved in dry methanol (15 mL) and the warmed solution was treated with 130 μL of a solution of NaOMe (30% w/w) in methanol. The reaction was refluxed for 4 h and then treated with diluted acetic acid to pH 5, evaporated to dryness and purified on a silica gel column (CH₂Cl₂:MeOH 93:7) to obtain compound 47 as a yellow foam (150 mg, 91%).¹H NMR (DMSO-d₆) δ 13.80 (br s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 6.63 (d, J = 7.7 Hz, 1H), 6.40 (d, J = 2.7 Hz, 1H), 5.53 (d, J = 5.3 Hz, 1H), 5.29 (t, J = 5.0 Hz, 1H), 5.11 (d, J = 5.9 Hz, 1H), 4.08 (m, 1H), 3.93 (m, 2H), 3.72–3.78 (m, 1H), 3.58–3.64 (m, 1H); HRMS-EI found 275.01637 [M−H] ⁻. C₉H₁₁N₂O₄S₂ requires 275.01602.

1-(β-D-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one (48)

In a parr apparatus 2,2′-O-anhydrouridine (61, 2 g, 8.8 mmol) was dissolved in dry DMF (40 mL) and triethylamine (6 mL). The solution was saturated with H₂S at −40°C and allowed to warm to rt resulting in a pressure of 200 psi. After stirring for two days, the remaining H₂S was released and the solvent evaporated to dryness. The brown residue was purified on a silica gel column (CH₂Cl₂:MeOH 96:4), yielding compound 48 (1.6 g, 70%). ¹H NMR (DMSO-d₆) δ 12.59 (br s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 3.9 Hz, 1H), 5.94 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 5.4 Hz, 1H), 5.48 (d, J = 3.9 Hz, 1H), 5.04 (t, J = 5.4 Hz, 1H), 4.18 (m, 1H,), 3.91 (m, 1H), 3.84 (m, 1H), 3.62 (app t, J = 5.1 Hz, 2H); HRMS-EI found 261.0540 (M+H)⁺. C₉H₁₃N₂O₅S₁ requires 261.0545.

General Procedure for Synthesis of 2′-C-Methyl-uridine (49) and 3′-C-Methyl-uridine (50)

To dry uracil (0.45 g, 4 mmol) in dry 1,2-dichloroethane (20 mL) were added HMDS (0.68 ml, 0.8 eq.) and trimethylsilyl chloride (TMSCl, 0.3 mL, 0.8 eq.). The reaction mixture was heated at 80°C for 4 h in the absence of moisture. After cooling to rt, 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (62)²¹ or 1,2,3-tri-O-acetyl-5-O-benzoyl-3-C-methyl-β-D-ribofuranose (63)^22b (1 eq) in 1,2-dichloroethane (20 mL) was added followed by SnCl₄ (0.93 mL, 2 eq.) dropwise. The mixture was stirred at rt for 4 h and quenched by NaHCO₃ saturated water solution and extracted with CHCl₃ (3 × 10 mL). The organic layers were dried (MgSO₄), filtered and concentrated under reduced pressure to give compounds 64 or 65 which were purified by chromatography on a silica gel column eluting with CHCl₃. Compounds 64 and 65 (1 mmol) were treated with methanol (20 mL) saturated with ammonia at 0°C stirring at rt overnight. Evaporation of the solvent gave the desired compounds 49 and 50, which were purified by chromatography on a silica gel column.

1-(2,3,5-Tri-O-benzoyl-2-C-methyl-β-D-ribofuranosyl)uracil (64)

The title compound was obtained as a white foam (67% yield). ¹H NMR (CDCl₃) δ 9.35 (br s, 1H), 8.06 (d, J = 7.7 Hz, 4H.), 7.86 (d, J = 7.7 Hz, 2H), 7.4–7.6 (m, 10H), 6.48 (s, 1H), 5.76 (d, J = 5.7 Hz, 1H), 5.66 (d, J = 7.7 Hz, 1H), 4.82 (dd, J = 4.7, 6.2 Hz, 2H), 4.62 (m, 1H), 1.72 (s, 3H).

1-(2-C-Methyl-β-D-ribofuranosyl)uracil (49)

The title compound was obtained as a white foam after chromatography on a silica gel column eluting with CHCl₃-MeOH (86:14) (80% yield). ¹H NMR (DMSO-d₆) δ 11.35 (br s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 5.58 (d, J = 8.1 Hz, 1H), 5.15 (br s, 2H), 5.10 (s, 1H), 3.40–3.80 (m, 4H), 1.0 (s, 3H). 1-(2,3-Di-O-Acetyl-5-O-benzoyl-3-C-methyl-β-D-ribofuranosyl)uracil (65). The title compound was obtained as a white foam after chromatography on a silica gel column eluting with CHCl₃-MeOH (86:14) (80% yield). ¹H NMR (DMSO-d₆) δ 11.35 (br s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 5.58 (d, J = 8.1 Hz, 1H), 5.15 (br s, 2H), 5.10 (s, 1H), 3.40–3.80 (m, 4H), 1.0 (s, 3H).

1-(3-C-Methyl-β-D-ribofuranosyl)uracil (50)

The title compound was obtained as a white foam after chromatography eluting with CHCl₃-MeOH (88:12) (82% yield). ¹H NMR (DMSO-d₆) δ 11.3 (br s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 5.85 (d, J = 8.1 Hz, 1H), 5.65 (d, J = 7.7 Hz, 1H), 5.33 (d, J = 6.6 Hz, 1H), 5.10 (t, J = 4.9 Hz, 1H), 4.72 (s, 1H), 3.85 (dd, J = 6.6, 7.7 Hz, 1H), 3.75 (pseudo t, 1H), 3.55 (m, 2H), 1.20 (s, 3H).

Assay of PLC activity stimulated by P2Y₂, P2Y₄, and P2Y₆ receptors

Stable cell lines expressing the human P2Y₂, P2Y₄, or P2Y₆ receptor in 1321N1 human astrocytoma cells were generated as described.³ Agonist-induced [³H]inositol phosphate production was measured in 1321N1 cells plated to 20,000 cells/well on 96-well plates two days prior to assay. Sixteen h before the assay, the inositol lipid pool of the cells was radiolabeled by incubation in 100 μL of serum-free inositol-free Dulbecco’s modified Eagle’s medium, containing 1.0 μCi of myo-[³H]inositol. No changes of medium were made subsequent to the addition of [³H]inositol. On the day of the assay, cells were challenged with 25 μL of the five-fold concentrated solution of receptor agonists in 200 mM Hepes (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid), pH 7.3 in HBSS, containing 50 mM LiCl for 30 min at 37°C. Incubations were terminated by aspiration of the drug-containing medium and addition of 450 μL of ice-cold 50 mM formic acid. [³H]Inositol phosphate accumulation was quantified using scintillation proximity assay methodology as previously described in detail.²⁴

Data Analysis

Agonist potencies (EC₅₀ values) were determined from concentration-response curves by non-linear regression analysis using the GraphPad software package Prism (GraphPad, San Diego, CA). All experiments examining the activity of newly synthesized molecules also included full concentration effect curves for the cognate agonist of the target receptor: UTP for the P2Y₂ receptor, UTP for the P2Y₄ receptor, and UDP for the P2Y₆ receptor. Each concentration of drug was tested in triplicate assays, and concentration effect curves for each test drug were repeated in at least three separate experiments with freshly diluted molecule. The results are presented as mean ± SEM from multiple experiments or in the case of concentration effect curves from a single experiment carried out with triplicate assays that were representative of results from multiple experiments.

Supplementary Material

01. Supplementary Material.

NMR spectra and HPLC traces for selected derivatives are available.

NIHMS56542-supplement-01.doc^{(2.4MB, doc)}

Chart 1 — Agonists of the P2Y₂ and P2Y₆ receptors.

Acknowledgments

Mass spectral measurements were performed by Dr. Noel Whittaker and NMR by Wesley White (NIDDK). We thank Dr. Andrei A. Ivanov (NIDDK) for helpful discussions. This research was supported in part by research grant GM38213, by the Intramural Research Programs of NIDDK, National Institutes of Health, and by MIUR (Italian Ministry for the Research and University) (PRIN 2006).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplementary Material.

NMR spectra and HPLC traces for selected derivatives are available.

NIHMS56542-supplement-01.doc^{(2.4MB, doc)}

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[R16] 16.Yoshikawa M, Kato T, Takenishi T. Tetrahedron Lett. 1967;50:5065. doi: 10.1016/s0040-4039(01)89915-9. [DOI] [PubMed] [Google Scholar]

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[R22] 22.(a) Mikhailov SN, Beigelman LN, Gurskaya GV, Padyukova NSh, Yakovlev GI, Karpeisky MYa. Carbohydr Res. 1983;124:75. [Google Scholar]; (b) Franchetti P, Cappellacci L, Pasqualini M, Petrelli R, Vita P, Jayaram HN, Horvath Z, Szekeres T, Grifantini M. J Med Chem. 2005;48:4983. doi: 10.1021/jm048944c. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Synthesis and Potency of Novel Uracil Nucleotides and Derivatives as P2Y2 and P2Y6 Receptor Agonists

Hyojin Ko

Rhonda L Carter

Liesbet Cosyn

Riccardo Petrelli

Sonia de Castro

Pedro Besada

Yixing Zhou

Loredana Cappellacci

Palmarisa Franchetti

Mario Grifantini

Serge Van Calenbergh

T Kendall Harden

Kenneth A Jacobson

Abstract

Introduction

Results and Discussion

Chemical Synthesis

Table 1.

Table 3.

Scheme 1.

Scheme 6.

Table 2.

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 5.

Pharmacological Activity

Figure 1.

Figure 2.

Figure 3.

Experimental Section

Chemical Synthesis

General Procedure for the Preparation of Nucleoside Triphosphates (e.g. 14 and 17), Tetraphosphates (22 – 24), Pentaphosphate (25). Procedure A

General Procedure for the Preparation of Nucleosides Tetraphosphates (26 – 31), Nucleoside Tetraphosphate Sugars (32 – 37), and Dinucleoside Tetraphosphates (40 – 44). Procedure B

General Procedure for the Preparation of Compounds (7 – 10). Procedure C

Uridine-5′-phosphonoacetate triethylammonium salt (8). Procedure C

Uridine-5′-α,β-methylene-diphosphate triethylammonium salt (9). Procedure C

Uridine-5′-α,β-difluoromethylenediphosphate triethylammonium salt (10). Procedure C

2′-Deoxy-2′-ureido-uridine-5′-diphosphate triethylammonium salt (12)

2-Thio-uridine-5′-β,γ-difluoromethylene-triphosphate triethylammonium salt (15)

2-Thio-uridine-5′-β, γ-dichloromethylene-triphosphate ammonium salt (16)

2-Thio-4-methylthio-uridine-5′-triphosphate triethylammonium salt (17). Procedure A

2′-Deoxy-2′-ureido uridine-5′-triphosphate triethylammonium salt (18). Procedure A

1-(β-D-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one 5′-triphosphate triethyl ammonium salt (19)

General Procedure for the Preparation of the Nucleoside 5′-triphosphates 2′-MeUTP (20) and 3′-MeUTP (21)

2′-C-Methyl-uridine-5′-triphosphate ammonium salt (20)

3′-C-Methyl-uridine-5′-triphosphate ammonium salt (21)

2-Thio-uridine-5′-tetraphosphate triethylammonium salt, 2-thio-UP4 (23). Procedure A

4-Thio-uridine-5′-tetraphosphate triethylammonium salt (24). Procedure A

2-Thio-uridine-5′-pentaphosphate triethylammonium salt (25). Procedure A

Uridine-5′-methyl-tetraphosphate triethylammonium salt (26). Procedure B

Uridine-5′-methyl(C-P)-tetraphosphate triethylammonium salt (27). Procedure B

Uridine-5′-(2-cyanoethyl)-tetraphosphate triethylammonium salt (28). Procedure B

Uridine-5′-α β-glycerol-tetraphosphate triethylammonium salt (29). Procedure B

Uridine-5′-phenyl-tetraphosphate triethylammonium salt (30). Procedure B

Uridine-5′-cyclohexane-tetraphosphate triethylammonium salt (31). Procedure B

Uridine-5′-fructose-6′-tetraphosphate triethylammonium salt (33). Procedure B

Uridine-5′-glucose-1′-tetraphosphate triethylammonium salt (34). Procedure B

Uridine-5′-galactose-1′-tetraphosphate triethylammonium salt (35). Procedure B

Uridine-5′-mannose-6′-tetraphosphate triethylammonium salt (36). Procedure B

Uridine-5′-(2′-deoxy-glucose)-6′-tetraphosphate triethylammonium salt (37). Procedure B

(2-Benzyl-1,3-dioxolo-4-yl)uridine 5′-monophosphate ammonium salt (54)

P1-((2-Benzyl-1,3-dioxolo-4-yl)uridine 5′) P3-(5-iodouridine 5′) triphosphate ammonium salt (39)

P1,P4-Di(uridine 5′-)β, γ-difluoromethylenetetraphosphate triethylammonium salt (41). Procedure B

P1,P4-Di(uridine 5′-)β, γ-dichloromethlyenetetraphosphate triethylammonium salt (42). Procedure B

P1-(2-Thiouridine 5′-)-P4-(2′-deoxycytidine 5′-)tetraphosphate triethylammonium salt (43). Procedure B

2′-Deoxy-2′-ureidouridine (46)

2′,3′,5′-Tri-O-acetyl-2-thiouridine (59)

2′,3′,5′-Tri-O-acetyl-2,4-dithiouridine (60)

2,4-Dithiouridine (47)

1-(β-D-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one (48)

General Procedure for Synthesis of 2′-C-Methyl-uridine (49) and 3′-C-Methyl-uridine (50)

1-(2,3,5-Tri-O-benzoyl-2-C-methyl-β-D-ribofuranosyl)uracil (64)

1-(2-C-Methyl-β-D-ribofuranosyl)uracil (49)

1-(3-C-Methyl-β-D-ribofuranosyl)uracil (50)

Assay of PLC activity stimulated by P2Y2, P2Y4, and P2Y6 receptors

Data Analysis

Supplementary Material

Synthesis and Potency of Novel Uracil Nucleotides and Derivatives as P2Y₂ and P2Y₆ Receptor Agonists

2-Thio-uridine-5′-tetraphosphate triethylammonium salt, 2-thio-U_P4 (23). Procedure A

Uridine-5′-methyl^(C-P)-tetraphosphate triethylammonium salt (27). Procedure B

P¹-((2-Benzyl-1,3-dioxolo-4-yl)uridine 5′) P3-(5-iodouridine 5′) triphosphate ammonium salt (39)

P¹,P⁴-Di(uridine 5′-)β, γ-difluoromethylenetetraphosphate triethylammonium salt (41). Procedure B

P¹,P⁴-Di(uridine 5′-)β, γ-dichloromethlyenetetraphosphate triethylammonium salt (42). Procedure B

P¹-(2-Thiouridine 5′-)-P4-(2′-deoxycytidine 5′-)tetraphosphate triethylammonium salt (43). Procedure B

Assay of PLC activity stimulated by P2Y₂, P2Y₄, and P2Y₆ receptors