P2Y11 (pKi = 7.35); P2Y11 (IC50 = 463 nM); P2Y11 (Ki = 44.3 nM); P2Y1 (Ki = 187 µM); P2Y2 (Ki = 28.9 µM)

NF157 is not as selective as P2X1, but it is more selective for P2Y11 than P2Y1 (>650-fold), P2Y2 (>650-fold), P2X2 (3-fold), P2X3 (8-fold), P2X4 (>22-fold), and P2X7 (>67-fold)[1]. In a dose-dependent manner, NF157 (30 and 60 µM; 24 hours) dramatically decreased the breakdown of type II collagen. Type II collagen is nearly entirely restored from TNF-α (10 ng/mL)-induced degradation when 60 µM NF157 is applied [2]. NF157 (30 and 60 µM; 24 hours) dramatically decreased NF-κB luciferase activity while nearly fully restoring TNF-α (10 ng/mL)-triggered p65 nuclear translocation [2].

Electrophysiological Evaluation of 8f/NF157 at Recombinant P2X Receptors. [1]
The inhibitory potency of NF157 at P2X receptors was evaluated on X. laevis oocytes recombinantly expressing various rat (r) and human (h) P2X subtypes (rP2X1, hP2X1, rPX2, rP2X3, hP2X3, rP2X4, hP2X4, rP2X7) using previously described protocols. Concentration−inhibition curves and IC50 values were derived from nonlinear least-squares fits of the Hill equation to the pooled data. The nondesensitizing properties of the rP2X2 receptor allowed quantifying inward current inhibition under steady-state conditions by coapplying NF157 during continued stimulation with ATP. Current inhibition occurred almost instantaneously as inferred from the immediate decrease of the current amplitude upon coapplication of an effective NF157 concentration. The classical Cheng−Prusoff equation could be applied to calculate the Ki value of NF157 for the rP2X2 receptor. The inhibitory potency of NF157 at desensitizing P2X receptors was determined from peak current measurements. As detailed previously, an accurate assessment of IC50 values for desensitizing receptors can usually not be achieved by coapplying agonist and antagonist, as the agonist-induced current will start to decline by desensitization before a binding equilibrium between the two compounds is reached. To account for this problem, oocytes expressing desensitizing P2X receptors were preequilibrated with NF157 for 15 s before being challenged with ATP in the continued presence of NF157. We have previously shown that suramin derivatives block P2X receptors competitively. Accordingly, if NF157 does not dissociate significantly from the receptor during the time needed to reach the peak current response, ATP can only bind to receptors unoccupied by NF157, leading to a pseudo-irreversible type of inhibition. Under these conditions, Ki and IC50 values will be equal. We therefore assume that the IC50 values deduced from peak current measurements are close or equal to the Ki values. In any case, Ki values deviate from IC50 values maximally by a factor of 2, as ATP was applied at a concentration close to its EC50 value. All results are presented as means ± SEM from at least three experiments.

Western Blot Analysis[2]
Cell Types: SW1353 Cell
Tested Concentrations: 30 and 60 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished TNF-α-induced NF-κB activation.

---

NF-κB promoter-luciferase assay [2]
The transcriptional activity of NF-κB was assessed by measuring the NF-κB promoter-luciferase activity. The NF-κB promoter-luciferase and β-galactosidase plasmids were transfected into SW1353 cells with the Lipofectamine 2000. Human chondrocytic SW1353 cells were treated with 10 ng/ml TNF-α in the presence or absence of NF157 at the concentrations of 30 and 60 μM for 24 h. Cell lysates were prepared and used for determining luciferase activity and β-galactosidase activity using a Secrete-PairTM Dual luminescence assay kit on a luminometer. Luciferase activity was normalized to β-galactosidase activity.

[1]. Synthesis and structure-activity relationships of suramin-derived P2Y11 receptor antagonists with nanomolar potency. J Med Chem. 2005 Nov 3;48(22):7040-8.

[2]. Inhibition of P2Y11R ameliorated TNF-α-induced degradation of extracellular matrix in human chondrocytic SW1353 cells. Am J Transl Res. 2019 Apr 15;11(4):2108-2116.

The lack of highly selective and efficient P2Y(11) receptor antagonists has hindered the evaluation of this G protein-coupled receptor, which is primarily expressed on immune cells. Using suramin, a substance with moderate inhibitory potency, as a template, we synthesized 18 urea compounds with methyl structural variants of suramin and its precursors and tested their functions on P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the suramin methyl group yielded the first nanomolar-scale P2Y(11) receptor antagonist (8f, NF157, pK(i): 7.35). To evaluate its selectivity, we also tested the antagonistic effects of 8f on various P2X receptors. Compound 8f showed higher selectivity for P2Y(11) than P2Y(1) (>650 times), P2Y(2) (>650 times), P2X(2) (3 times), P2X(3) (8 times), P2X(4) (>22 times) and P2X(7) (>67 times), but no selectivity for P2X(1). QSAR studies confirmed that residues with favorable resonance and size parameters in the aromatic linker region can indeed enhance potency, as is the case with compound 8f. The symmetrical structure connecting the two anionic clusters appears to be essential for biological activity. Compound 8f may help assess the physiological function of the P2Y(11) receptor. [1] This study reports the synthesis of a series of suramin analogues and their structure-activity relationships, and discovered the first nanomolar-level P2Y11 receptor antagonist, 8f. The antagonist is at least 650 times more selective for P2Y11 receptors than for P2Y1 and P2Y2 receptors, and 3 to 67 times more selective for P2X2, P2X3, P2X4, and P2X7 receptors. However, 8f is roughly equivalent in potency for P2Y11 and P2X1 receptors. The symmetrical structure connecting the two anionic clusters appears to be essential for its biological activity. Quantitative structure-activity relationship (QSAR) studies have shown that in the aromatic linker region, substitution of the ortho-amide oxygen atom of residue R with resonance (R), size (B5), and partial charge (Q(Oortho)) can improve its potency, for example, the fluorinated derivative 8f. QSAR results can guide the targeted synthesis and development of further efficient and selective (also for P2X1) P2Y11 ligands. Therefore, this study and the novel ligand 8f may contribute to a deeper understanding of the physiological and pathophysiological role of P2Y11 receptors. [1] Osteoarthritis is a major global health burden. Joint destruction, caused by the excessive degradation of type II collagen and proteoglycans in the extracellular matrix of articular cells by metalloproteinases and aggregates, respectively, is a major pathological feature of osteoarthritis. However, the exact mechanism remains unclear. Currently, there are very few non-invasive therapies that can slow or halt disease progression. In this study, we investigated the role of the purinergic protein P2Y11 and its receptor P2Y11R in TNF-α-mediated extracellular matrix degradation of chondrocytes in the SW1353 cell line using the novel P2Y11R antagonist NF157. To our knowledge, this is the first study to explore the role of NF157 in osteoarthritis (OA). Our results suggest that P2Y11R may indeed play a role in TNF-α-induced extracellular matrix degradation in OA, as NF157 treatment significantly reduced the expression of metalloproteinases (MMP)-3, MMP-13, integrin with platelet-reactive protein motifs, and ADAMTS-4 and ADAMTS-5, and improved the degradation of type II collagen and aggregate proteoglycans in SW1353 chondrocytes in a dose-dependent manner. In addition, we found that NF157 treatment significantly reduced the nuclear translocation of p65 and its subsequent activation of nuclear factor κB (NF-κB). [2] NF-κB is recognized as a key pro-inflammatory transcription factor that promotes the development of osteoarthritis (OA) and many other inflammatory diseases. A recent study in Japan showed that γ-ray-induced ATP release activates the P2Y11 receptor, which in turn activates NF-κB through the p38/mitogen-activated protein kinase (MAPK) pathway. In a related study, the authors found that NF157 treatment inhibited NF-κB activation. Another study showed that ATP selectively targets p65 and activates NF-κB via the P2Z purine receptor, which, like P2Y11R, has a high affinity for ATP. In this study, we found that blocking P2Y11R with 30 and 60 µM NF157 dose-dependently inhibited TNF-α-induced p65 nuclear translocation. Furthermore, we demonstrated that antagonizing P2Y11R with NF157 dose-dependently and significantly reduced NF-κB luciferase activity. Our results are the first to demonstrate that blocking P2Y11 purine receptors with the selective P2Y11 antagonist NF157 can significantly prevent the degradation of extracellular matrix components in joint cells by downregulating the expression of TNF-α-induced MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, thereby inhibiting the degradation of type II collagen and proteoglycans and the activation of NF-κB. These positive results suggest that NF157 may have the potential as a targeted therapy for the treatment and prevention of excessive degradation of type II collagen and proteoglycans in osteoarthritis. Further research is needed to better understand the complex mechanisms underlying these results. [2]

C49H34F2N6O23S6

1305.19

1435.89

C, 45.09; H, 2.63; F, 2.91; N, 6.44; O, 28.19; S, 14.74

104869-26-3

Off-white to pink solid powder

11.271

UDVIAMRWOLIUAE-UHFFFAOYSA-N

InChI=1S/C49H34F2N6O23S6/c50-33-9-7-25(47(60)54-35-11-13-39(83(69,70)71)31-19-29(81(63,64)65)21-41(43(31)35)85(75,76)77)17-37(33)56-45(58)23-3-1-5-27(15-23)52-49(62)53-28-6-2-4-24(16-28)46(59)57-38-18-26(8-10-34(38)51)48(61)55-36-12-14-40(84(72,73)74)32-20-30(82(66,67)68)22-42(44(32)36)86(78,79)80/h1-22H,(H,54,60)(H,55,61)(H,56,58)(H,57,59)(H2,52,53,62)(H,63,64,65)(H,66,67,68)(H,69,70,71)(H,72,73,74)(H,75,76,77)(H,78,79,80)

8-[[4-fluoro-3-[[3-[[3-[[2-fluoro-5-[(4,6,8-trisulfonaphthalen-1-yl)carbamoyl]phenyl]carbamoyl]phenyl]carbamoylamino]benzoyl]amino]benzoyl]amino]naphthalene-1,3,5-trisulfonic acid

NF-157; NF 157; NF157

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~1 mg/mL (~0.70 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)