Structure-Activity Relationships of Pyrimidine Nucleotides Containing a 5′-α,β-Methylene Diphosphonate at the P2Y₆ Receptor

Abstract

The G_q-coupled P2Y₆ receptor (P2Y₆R) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2Y₆R agonist and P2Y₁₄R partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,β-methylene bridge as P2Y₆R agonists and identified compatible affinity-enhancing pyrimidine modifications. A distal binding region on the receptor was explored with 4-benzyloxyimino cytidine 5′-diphosphate analogues and their potency determined in a calcium mobilization assay. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest human P2Y₆R potency (MRS4554, 0.57 μM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over human P2Y₁₄R, respectively. However, 3-alkyl (31 – 33, 37, 38), β-D-arabinofuranose (39) and 6-aza (40) substitution prevented P2Y₆R activation. Thus, we have identified new α,β-methylene bridged N⁴-extended CDP analogues as P2Y₆R agonists that are highly selective over the P2Y₁₄R.

Graphical Abstract

Among the eight metabotropic P2Y receptors (P2YRs) are four subtypes that respond to uracil mono- and dinucleotides.¹ The human (h) G_q protein-coupled P2Y₂ receptor (P2Y₂R) and P2Y₄R are potently activated by UTP, but UDP is ~900- and ~160-fold weaker, respectively.² However, UDP acts as an agonist and partial agonist at the P2Y₆R (G_q-coupled) and P2Y₁₄R (G_i-coupled), respectively.¹ The P2Y₁₄R is potently activated by endogenous UDP-sugars, principally UDP-glucose,¹ and the P2Y₆R is also activated by certain synthetic nucleoside 5′-triphosphate and dinucleoside triphosphate analogues.²,³ UTP is inactive at the hP2Y₆R (EC₅₀ >10 μM) and P2Y₁₄R, while UMP does not activate any hP2YR.^1-6 Thus, it is logical to compare and contrast the P2Y₆R and P2Y₁₄R due to their common endogenous agonist UDP, even though they are linked to different second messenger pathways.^7–9

We focus here on the structure-activity relationship (SAR) of pyrimidine mononucleotides as P2Y₆R agonists (e.g., 1 – 10, Chart 1) and demonstrate the ability to distinguish the P2Y₆R and P2Y₁₄R subtypes with analogues of UDP 1 or CDP that contain a 5′-α,β-methylene diphosphonate. Among known P2Y₆R selective UDP analogues, 5-iodo-UDP 2, 5-methoxy-UDP 3, 3-phenylacetyl-UDP 5 and MRS4383 6 are 5′-diphosphates that tend to be hydrolyzed in biological tissue by ectonucleotidases.^2,3,10 These enzymes include nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), 5′-nucleotidase (CD73, hydrolyzes 5′-monophosphates) and other ectonucleotidases.^10,11 A 5′-α,β-methylene diphosphonate group in nucleotide analogues, e.g. 9 and 10, lends greater, but not absolute, stability toward enzymatic hydrolysis compared to the native 5′-diphosphate and is compatible with P2Y₆R activation (Table 1) ^3-5,10,12 We have also reported 5′-α,β-methylene diphosphonates that potently activate the P2Y₁₄R, such as MRS2905 14b (2-thiouridine-5′-O-(α,β-methylene)diphosphate, the structure shown in Table 2 footnote).⁹ In addition to phosphonate bridges, another means of increasing the resistance to hydrolytic degradation is to include an α-borano-diphosphate moiety as in potent P2Y₆R agonist 4.¹³ Dinucleotide P2Y₆R agonists are also more stable toward hydrolysis by CD39 and CD73 in tissues than 5′-diphosphate agonists such as UDP.¹⁰ However, dinucleotides are cleaved by ectonucleotide pyrophosphatase/phosphodiesterases (ENPPs).¹⁰

Chart 1.

Structures of known P2Y₆R agonists that are UDP analogues.^3,4 The reported potency in activation of PLC (EC₅₀), is shown in italics after each compound, unless noted (“a” indicates EC₅₀ in nM in the calcium mobilization assay).

Table 1.

Potencies of various uridine 5’-diphosphate (UDP) and α,β-methylene-UDP analogues in comparison to known UDP derivatives for activation of the human P2Y₆R, as determined in an assay of calcium mobilization, unless noted.^a

Compound	x =	Y =	Z =	P2Y6R, EC50, μM (Ca2+, unless noted)
5′-Diphosphates
1 UDP	H	H	O	0.0416±0.007
2	I	H	O	0.0491±0.0195, 0.015±0.002b
11	H	H	NOCH3	0.493±0.128
12	H	H	NNHCOOCH3	1.54f
13	H	H	NOCH2C6H5	0.793±0.149
5′-α,β-Methylene diphosphonates
14ac	H	H	O	0.70±0.11b
15c	I	H	O	0.0491±0.0195,c 0.13±0.02b
16c	F	H	O	0.203 ± 0.030c
17c	Cl	H	O	0.097 ± 0.011
18c	Br	H	O	0.104 ± 0.010
9	H	H	NOCH3	0.42±0.08d
10	H	H	NOCH2C6H5	2.36±0.73d
19	H	H	NOCH2C6H5-3-F	2.70 ± 0.78
20	H	H	NOCH2C6H5-3-Cl	1.45 ± 0.22
21	H	H	NOCH2C6H5-3-CF3	1.91 ± 0.47
22	H	H	NOCH2C6H5-3-CO2CH3	0.902 ± 0.05
23	H	H	NOCH2C6H5-4-Cl	1.32 ± 0.08
24	H	H	NOCH2C6H5-4-CH3	5.12 ± 0.28
25c	H	H	NOCH2C6H5-4-CF3	0.571 ± 0.196
26	H	H	NOCH2C6H5-4-CO2CH3	1.67 ± 0.34
27	H	H	NOCH2C6H5-4-SF5	1.59 ± 0.09
28c	F	H	NOCH2C6H5	0.549 ± 0.070
29c	CH3	H	NOCH2C6H5	1.09 ± 0.32
30c	H	H	N4-CO-Ph	1.39 ± 0.223c
Inactive (% activation at 3 μM of <10%)
31c	H	CH2CH3	O	3.5%
32c	H	(CH2)2CH3	O	2.7%
33c	H	CH2C6H5	O	5.3%
34c	F	-	-	5.7%
35c	I	-	-	3.4%
36c	CH3	-	-	3.0%
37c	H	CH3	NOCH2C6H5	3.6%c
38c	H	CH2CH3	NOCH2C6H5	5.7%
Other inactives (% activation at 3 μM of <10%)
39c	Structure shown in footnotee			2.4%
40c	Structure shown in footnotee			5.2%

^aAgonist potencies reflect P2Y₆R-dependent calcium mobilization in 1321N1 human astrocytoma cells stably expressing the human P2Y₆R. Potencies are presented in the form of EC₅₀ values, which represent the concentration of agonist at which 50% of the maximal effect on calcium mobilization 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, unless noted, with each molecule.

^bEC₅₀ values from Maruoka et al. (PLC assay).⁴

^cAll syntheses for indicated compounds were reported in Junker et al., as well as the P2Y₆R Ca²⁺ assay results for compound 30.³³

^dReported in Toti et al. (Ca²⁺ assay).³

^eStructures of 39: 40:

^fn = 1 (tested once).

Table 2.

Potency of α,β-methylene analogues of UDP and CDP at the human P2Y₆R and P2Y₁₄R.^a

Compound	hP2Y6R EC50 ± SEM (μM) or %	hP2Y14R IC50 ± SEM (μM)
14a	0.339b	0.011b
14bc	1.99b	0.00092b
16	0.203 ± 0.030	0.362 ± 0.090
17	0.097 ± 0.011	16.6 ± 10.3
20	1.45 ± 0.22	>100
23	1.32 ± 0.08	>100
25	0.571 ± 0.196	>100
28	0.549 ± 0.070	21.5 ± 11.1
30	1.39 ± 0.223	6.66 ± 4.74

^aFLIPR (Ca²⁺) assay in hP2Y₆R-1231N1 astrocytoma cells, expressed as EC₅₀ or % activation at 3 µM, unless noted. Fluorescent antagonist was used as a tracer for flow cytometry of hP2Y₁₄R-CHO cells, unless noted.

^bPotency in a phospholipase C assay of hP2Y₆R activation (Maruoka et al., 2010)⁴ or in a cAMP assay of hP2Y₁₄R activation (Das et al., 2010).⁹

^cStructure of 14b:

P2Y₆R antagonists have broader envisioned therapeutic use than P2Y₆R agonists, for treating cancer, inflammation, neurodegeneration, and diabetes.^14-22 However, P2Y₆R agonists are also proposed for neurodegeneration, glaucoma, asthma, and other conditions.^13,23-27 A nucleoside precursor prodrug of a P2Y₆R agonist entered a Phase 1b clinical trial for Alzheimer’s disease to target microglial cells (ClinicalTrials.gov: NCT02254369, NCT02386306).^26,27 Unlike other G_q-coupled P2YRs, P2Y₆R counteracts apoptosis in various cell lines and tissue,³ and it also has vascular effects, including both vasoconstriction and vasodilation,²⁸ and synergizes with the hypertensive effects of angiotensin II.^21,22 P2Y₆R agonists that might be relatively stable and suitable for use as in vivo pharmacological probes are also needed. Previously, we reported that UDP-related mononucleotides and Up3U-related dinucleotide analogues that contained a rigidified, carbocyclic ribose moiety, i.e. a South (S)-bicyclo[3.1.0]hexane ((S)-methanocarba) ring system as in methoxyimino derivative MRS4383 6, retained P2Y₆R agonist potency and efficacy with high selectivity.^3,17 The ability of MRS4383 to stimulate PLC activity in mouse adipocytes disappeared in adipocytes derived from P2Y₆R-knockout mice, demonstrating its selectivity.¹⁷ However, the (S)-methanocarba modification was not compatible with a stability-enhancing α,β-methylene 5′-diphosphonate.³

Here, we have modified the pyrimidine ring 4-position for extension in the form of (aryl)alkoxyimino moieties, in the ribose series of α,β-methylene 5′-diphosphonates (Table 1). A chemically stable N⁴-methoxyimino or N⁴-benzyloxyimino modification of the pyrimidine ring in UDP/CDP and α,β-methylene-UDP/CDP analogues was shown to be permissive for P2Y₆R activation.^3,4,29 Thus, an (aryl)alkoxyimino moiety binds in a P2Y₆R region that is not highly sterically constrained, likely facing the extracellular side based on the freedom of chain extension at that position.²⁹ Here, we have empirically explored effects on P2Y₆R of ring substitutions of the N⁴-benzyloxyimino moiety and other modifications. Since the G_q protein pathway activates phospholipase C (PLC) β and subsequent calcium mobilization, we have used for ligand characterization a Ca²⁺ mobilization assay in an astrocytoma cell line stably expressing the hP2Y₆R^4,30,31 To demonstrate selectivity against the hP2Y₁₄R, we have used a fluorescent binding assay in Chinese hamster ovary (CHO) cells stably expressing the receptor.³⁴

The pyrimidine nucleotides (Table 1) were synthesized as shown in Schemes 1 and 2. Synthesis of compounds 10 and 19 – 27 required the preparation of various aryl-substituted benzyloxyamines (42 – 51). These benzyloxyamines were produced following the procedure already reported,³⁰ starting from the corresponding substituted benzyl bromides. The synthesis of compounds 14a, 14b, 15 – 18, 25, and 28 – 40 was reported.^9,33

Scheme 1.

Reagents and conditions: Synthesis of the modified uridine/cytidine nucleotides. Reagents and conditions: a. (Boc)₂NOH, DBU, DMF, 50 °C, 2 h; b. HCl, 4 N in dioxane, rt, 12 h; c. desired benzyl-hydroxylamine (53 – 62), pyridine, 80 °C, 12 h; d. Methylenebis(phosphonic dichloride), (CH₃O)₃PO, 0 °C, 3 h.

Scheme 2.

Synthesis of the cytidine nucleotide 12. Reagents and conditions: a. (i) dioxane, DMAP, TEA, 2,4,6-triisopropylbenzenesulfonyl chloride, rt, 18 h; (ii) NH₂NHCOOMe, rt, 18 h; (iii) 1 N NH₃ in methanol, rt, 18 h, 6%; b. (i) PO(OMe)₃, Proton Sponge®, POCl₃, 0 °C, 2 h; (ii) DMF, 2[Bu₃NH]⁺HPO₄²⁻, 0 °C → rt, 15 min, 5.3%.

The reaction of cytidine (52) with the desired substituted benzyloxyamine (42 – 51) in pyridine at 80 °C, gave the desired intermediates (53 – 62) in good yields. 5′-O-Phosphonylation of the latter was performed using methylenebis(phosphonic dichloride) in trimethyl phosphate to obtain the desired final compounds 10, 19-27 in acceptable yields (Scheme 1).³⁵

Compound 12 was designed to probe if replacement of the O of the hydroxylamine moiety with N was possible. To prepare compound 12, the oxygen at the 4-position of pyrimidine base of protected uridine (63) was activated using 2,4,6-triisopropylbenzenesulfonyl chloride and then reacted with methyl hydrazinecarboxylate (Scheme 2). This intermediate was unstable to purification by silica-gel chromatography; hence the acetates were deprotected without purification by treating with dilute ammoniacal methanol to give 64 in low isolated yields. Compound 64 was converted to corresponding diphosphate 12 under standard phosphorylation conditions by employing phosphoryl oxychloride and tributylammonium pyrophosphate in low yields.

The P2Y₆R potency of the nucleotide derivatives was determined in concentration-response assays of calcium mobilization, using fluorescence-based calcium mobilization in 1321N1 human astrocytoma cells expressing the human P2Y₆R, as was previously reported.³ This functional data is only indirectly comparable to data previously reported by us and others using a PLC assay.^2,4 It is to be noted that EC₅₀ values of UDP analogues in the calcium assay often underestimate their potency when compared to P2Y₆R-induced PLC stimulation.^3,36 Representative concentration-response curves are shown in Figure 1.

Figure 1.

Representative concentration-response curves for P2Y6R activation in a calcium mobilization assay, using 1321N1 cells expressing the human P2Y₆R (EC₅₀ values (µM): 24, 5.47; 26, 1.65; 27, 1.69).

All of the compounds synthesized here contained a α,β-methylene bridge, while 14 – 30 and 37, 38 additionally contained an alkyloxyimino-pyrimidine nucleobase. Several of the compounds were previously reported by us as intermediates and as P2Y₆R agonists in the synthesis of CD73 inhibitors.³³ Selected analogues were also measured in a fluorescent P2Y₁₄R binding assay by flow cytometry of whole CHO cells expressing the hP2Y₁₄R to establish selectivity (Table 2).

Reference compounds 1, 2, 9, 10, and 15 are included in Table 1.³,⁴,³⁶ The P2Y₆R potency of UDP 1 (EC₅₀ 0.0416 μM) was reduced 12- to 19-fold with 4-imino-oxyalkyl modifications in 11 and 13. Nevertheless, we sought to explore the interaction of bound to a nucleotide agonist with this distal region of the receptor, especially in combination with a stabilizing methylene phosphonate. Previously, considerable P2Y₆R agonist potency was observed upon 4-imino-oxyalkylaryl substitution of UDP.⁴

Although the N⁴-methoxy α,β-methylene analogue 9 (0.42 μM) was more potent than the corresponding N⁴-benzyloxy analogue 10 (2.36 μM), we examined functional group substitution of the phenyl ring of 10. 3-Substituted benzyloxo groups (19 – 22) displayed EC₅₀ values in the range of 0.9–2.7 μM, while 4-substituted benzyloxo groups (23 – 27) displayed EC₅₀ values in the range of 0.57–2.3 μM. Notably, the IC₅₀ values for hP2Y₁₄R binding of compounds 20, 23 and 25 were >100 μM, indicating a high degree of selectivity. A 4-trifluoromethyl-benzyl substituent in 25 provided the highest potency (0.57 μM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over P2Y₁₄R, respectively. N⁴-Benzoyl derivative 30 was a potent P2Y₆R agonist (1.39 μM), but nonselective. A 4-pentafluorosulfanyl group (SF₅) group³⁷ in 27 maintained P2Y₆R potency. 5-Methyl substitution of the cytosine ring in 29 led to an EC₅₀ of 1.09 μM, i.e. slightly more potent than 10. 3-Alkyl or aryl alkyl (31 – 33, 37, 38), β-D-arabinofuranose 39, 6-aza 40 substitution, as previously synthesized,³³ was shown here to completely prevent P2Y₆R activation. The 5-substituted cytosine derivatives 34–36 in the α,β-methylene series were inactive at P2Y₆R, in contrast to closely related, potent uracil derivatives (15, 16). Curiously, alkylation of N³-position of N⁴-benzyloxy analogues (37, 38) led to a complete loss of P2Y₆R activity. 5-Fluoro-5′-αβ-methylene-UDP 16 was nonselective between P2Y₆R and P2Y₁₄R (affinity 0.2–0.3 μM).

P2Y₆ agonist 26 and 27 and P2Y₁₄ agonist 14b were chosen as representative UDP analogues containing a stabilizing α,β-methylene bridge for the determination of off-target activities at 46 receptors, channels and transporters (Supporting information) by the Psychoactive Drug Screening Program (PDSP).³⁸ 14b showed no significant (i.e. >50% binding inhibition at 10 μM) off-target activities. At 10 μM, 26 inhibited binding at the D₃ dopamine receptor (85%), and 27 inhibited binding at the σ₁ receptor (59%). Nevertheless these nucleotides are non-promiscuous in binding.

N⁴-Benzyloxy-CDP analogues containing a stabilizing α,β-methylene bridge were prepared and evaluated as P2Y₆R agonists. The reactions between cytidine and alkoxy amines afforded single condensation products, which we represent with a Z-exo C-N double bond, consistent with previous reports, including X-ray crystallographic structures obtained for model compounds.³⁹,⁴⁰ Considerable freedom of substitution of the benzyl ring is present, while substitution of the pyrimidine C5 position (halo, methyl) but not the N³ position (alkyl) is compatible with P2Y₆R activation. SAR studies of several other P2YRs have revealed that substitution of the phosphate moiety of agonists with an α,β-methylene or β,γ-methylene bridge greatly reduces affinity. For example, at the P2Y₁R, agonist activity is lost in α,β-methylene-ATP.⁴¹ However, β,γ-methylene-or β,γ-dihalomethylene analogues of ATP are potent P2Y₁₂R antagonists.⁴² A previous study comparing α,β-methylene bridged UDP analogues showed a tendency toward P2Y₁₄R selectivity in comparison to P2Y₆R.⁹ Here, we have demonstrated that a wide range of α,β-methylene-CDP derivatives are suitable as selective P2Y₆R agonists, but that is dependent on the presence of a N⁴-benzyloxyimino group. The potency may not be as high as α,β-methylene-UDP derivatives, but these CDP derivatives have high P2Y₆R selectivity compared to the P2Y₁₄R. Additional P2Y₆R agonists are needed to explore P2Y₆R physiological effects and interactions of the P2Y₆R with other proteins, such as the angiotensin 1 (AT1) receptor. This significance of increased P2Y₆R mRNA expression as a signature of human tumors needs to be explored.⁴³ We have not tested these P2Y₆R agonists in vivo. Still, because of the phosphonate group, they might display a longer duration of action than various UDP analogues that have been used in vivo and isolated in tissue studies.^17,28

In this study, inhibition of 5′-nucleotidase (CD73) was not measured. If present, this activity could be a caveat during the interpretation of pharmacological data using nucleoside/nucleotide ligands of purinergic receptors. Some of the compounds included here were found to inhibit CD73 by Junker et al.³³ Using a rat CD73 radiometric assay with [³H]AMP as a substrate,⁴⁴ with a substrate concentration of 5 μM AMP, the following IC₅₀ values (μM or % inhibition at 1 μM) were determined: 9, 0.257; 10, 0.112; 14a, 1.83; 14b, 7%; 15, 0.162; 16, >1; 25, 0.030; 28, 0.085; 29, 0.321.

Within the P2YR family, only P2Y₁R and P2Y₁₂R experimental structures are available.¹ Thus, current structural hypotheses for P2Y₆R ligand recognition are necessarily based on homology modeling,³,⁴⁵ which needs refinement. Expanding the range of agonist SAR can be informative for GPCR modeling, and P2Y₆R agonist SAR is already more highly advanced than its antagonist SAR. By analogy, we effectively used molecular dynamics (MD) simulation to recapitulated P2Y₁₄R agonist SAR, based on homology to the P2Y₁₂R, and this homology modeling approach later led to the effective discovery of potent P2Y₁₄R antagonists.⁴⁶,⁴⁷ Although P2Y₁R and P2Y₆R belong to the same G_q-coupled subfamily of P2YRs, their similarity and identity of sequence are only 41% and 28%, respectively ⁴⁵ Thus, a new P2Y₆R homology model would be strengthened and validated by extensively simulating the binding of known ligands, perhaps including the present agonists.

In conclusion, we have synthesized, and evaluated as P2Y₆R agonists in a calcium mobilization assay, various UDP/CDP analogues containing a stabilizing α,β-methylene bridge. A putative distal binding region of the receptor surrounding a 4-benzyloxyimino cytidine group was explored with aryl substitution. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest potency (MRS4554, 0.57 μM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over P2Y₁₄R, respectively. Thus, we have expanded the range of P2Y₆R agonists, and explored the SAR of pyrimidine nucleoside 5′-α,β-methylene diphosphonates that are highly selective over P2Y₁₄R. The distinguishing features are an N⁴-extension in the CDP analogues in the P2Y₆R agonists reported here versus a 2-thio group in uracil-containing P2Y₁₄R-selective agonists, such as MRS2905 14b.

Highlights.

• P2Y₆ receptor (P2Y₆R) agonists proposed for neurodegeneration, glaucoma, asthma, and other conditions.

• We have synthesized and evaluated various UDP/CDP analogues, containing a stabilizing α,β-methylene bridge, in a calcium mobilization assay.

• We identified new P2Y₆R agonists that are highly selective over P2Y₁₄R.

• The distinguishing features are an N⁴-extension in CDP analogues as P2Y₆R agonists versus a 2-thio group in uracil-containing P2Y₁₄R-selective agonists.

Abbreviations:

CDI

carbonyldiimidazole

DCC

dicyclohexylcarbodiimide

DIPEA

diisopropylethylamine

DMF

N,N-dimethylformamide

GPCR

G protein-coupled receptor

PDSP

Psychoactive Drug Screening Program

PLC

phospholipase C

RMSD

root-mean-square deviation

SAR

structure activity relationship

TBAP

tetrabutylammonium dihydrogen phosphate

TEA

triethylamine

TEAB

tetraethylammonium tetrafluoroborate

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TLC

thin layer chromatography

UDP

uridine 5′-diphosphate.