Hematopoietic prostaglandin D2 synthase (H-PGDS) (IC50 = 2.5 nM for enzyme inhibition) [4]

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034, analog of SAR191801) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes [4].

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- hPGDS Inhibition: SAR191801 potently inhibits recombinant human hPGDS activity with an IC50 of 2.5 nM. It shows high selectivity for hPGDS over other prostaglandin synthases (e.g., COX-1, COX-2) with IC50 values >10 μM [4]
- Cellular PGD2 Reduction: In human mast cells stimulated with calcium ionophore, SAR191801 (10 nM) reduces PGD2 production by 85% compared to untreated cells, without affecting cell viability [4]

- Inhibition of PGD2 Synthesis in Mice: Oral administration of SAR191801 (3 mg/kg) to mice 1 hour before intraperitoneal challenge with compound 48/80 (a mast cell activator) reduces peritoneal PGD2 levels by 70% at 2 hours post-challenge [4]
- Anti-Inflammatory Activity in Rat Model: In a rat model of allergic rhinitis induced by ovalbumin, SAR191801 (10 mg/kg, oral, once daily for 7 days) decreases nasal eosinophil infiltration by 65% and reduces nasal PGD2 concentrations by 60% compared to controls [1]

Thermal Shift Assay [4]
WT H-PGDS at 0.25 mg/mL was mixed 1000:1 (v/v) with Sypro Orange dye. Samples were processed using a BioRad CFX C100 Touch qPCR and run using the FRET assay settings with a heating ramp of 0.3°C/sec cycling from 4°C to 100°C. Compounds were dosed at a fixed concentration, 10 µM and 2% DMSO final. Samples were prepared in triplicate. Reduced glutathione was added up to 1 mM in the appropriate studies. Analysis was performed using the Bio-Rad CFX manager software.

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Glutathione S-Transferase (GTSase) Activity Assay [4]
For mechanistic study, H-PGDS concentration was 0.5 µM, chloro-2,4-dinitrobenene (CDNB) concentration was 2 mM, and GSH concentration was varied with tested concentrations of 0, 0.5, 2, 4, 6, 8, 10 mM. Inhibitor concentrations were 0, 100, 250, 500, 5000 nM. The enzyme was prewarmed for 10 minutes in assay plate (Greiner 96 well black flat-bottom), in presence of inhibitor and GSH and reaction was initiated by the addition of CDNB, n=6 for all data points. Velocities were monitored over the first 120 seconds of the reaction, which were shown to be within linear range of reaction.

For enzyme inhibition, reaction velocities were monitored with H-PGDS at 250 nM and a single dose of inhibitor at 250 nM and 3.5 mM GSH. Reactions were performed in triplicate and monitored over the first 120 seconds of the reaction. Inhibitor was preincubated with enzyme prior to addition of CDNB for 5 minutes. Reaction was initiated with addition of CDNB at 2 mM. Background velocity was subtracted using a set of 3 no-enzyme control wells and percent inhibition was calculated using a DMSO control well. Percent inhibition was calculated with the following equation: % Inhibition=(Vo DMSO-Vo Inhibitor)/(Vo DMSO)

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Fluorescence Polarization Assay [4]
Fluorescence polarization (FP) assay was performed on Perkin Elmer Envision Multimode plate reader in 384 well format. Components were from Prostaglandin D Synthase (hematopoietic-type) FP-Based Inhibitor Screening Assay Kit – Green. Kit concentrations were altered to the following: probe concentration was 5 nM, MBP-labeled H-PGDS concentration was 5 nM, GSH concentration was 3.5 mM. Assay buffer was 1x PBS pH 7.4, supplemented with GSH. Well final volume was 100 µL. Plates were incubated 90 minutes in dark at RT before reading. λEX: 480 nM nm/λEM:535 nm S/P. Nonlinear regression fits were performed in Graph Pad Prism 7.04 to provide IC50 using one-site binding Log[inhibitor] vs. response. Individual replicates were fit and IC50s determined, then averaged and SEM determined between replicates. Outliers, if any, were determined and discarded using Grubbs’ method with Alpha=0.1 using GraphPad Prism 7.02. Performance of FP assay in absence GSH was performed on BioTek Synergy H4 Hybrid Microplate Reader using standard kit conditions, except for the exclusion of GSH from assay. λEX: 485/20 nM filter λEM:528/20 nm S/P filter. Nonlinear regression fits were performed in Graph Pad Prism 7.04 to provide IC50 using one-site binding Log[inhibitor] vs. response. Individual replicates were fit and IC50s determined, then averaged and SEM determined between replicates.

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Liver Microsome Stability and Aqueous Solubility [4]
In Vitro metabolism determination was performed using human liver microsomes (0.1 mg/mL). Aqueous solubility (PBS, pH 7.4) was tested using the shake-flask technique.

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hPGDS Activity Assay: Recombinant hPGDS is incubated with prostaglandin H2 (substrate) and SAR191801 (0.1–100 nM) in a buffered solution. The reaction is stopped after 30 minutes, and the amount of PGD2 produced is measured by HPLC. IC50 is calculated from the dose-response curve showing inhibition of PGD2 formation [4]

Maltose Binding Protein Fused H-PGDS Purification [4]
Buffers: MBP Lysis Buffer – 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1:2000 Benzonase, 1:1000 Lysozyme, 1x protease inhibitor. MBP Wash Buffer - 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA. MBP Elution Buffer - 20 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, 10 mM maltose.
MBP-tagged H-PGDS expressed cell pellets were thawed then homogenized by magnetic stirring at 4°C in MBP Lysis Buffer for 30 minute at 4°C. Cells were lysed by pulsing sonication with Branson Sonifier 250 at 50% power output for 3 cycles of 45 seconds on with 5 minute cool down periods between cycles. Cells were centrifuged at 20,000 RPM for 30 minutes using the F20-12x50 LEX rotor at 4°C in Sorvall Lynx 6000 centrifuge. Clarified lysate was diluted 5x with MBP Wash Buffer and applied to 5 mL Amylose resin, prewashed and preequilibrated. Column was washed by gravity flow with 12 column volumes (CV) of MBP Wash Buffer. Column was eluted with 4 CV of MBP Elution Buffer. Fractions containing MBP-tagged H-PGDS were dialyzed against 1L dialysis buffer overnight at 4°C and then concentrated to 1.4 mg/mL in Amicon ultra-15 centrifugal filter units with 10 kDa MW cutoff in Eppendorf 5810R centrifuge with swinging bucket rotor at 4,000 RPM and 4°C. Yield was 9.3 mg/L, >95% pure by SDS-PAGE.

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Macrophage Cell-based Assay [4]
RAW264.7 cells were cultured in complete medium (DMEM/F12 with 10% fetal bovine serum (FBS)). On the day before treatment, 50,000 RAW cells per well were seeded on a 96-well plate. Test compounds were dissolved in DMSO and serial diluted in stimulation medium (phenol red-free DMEM). An equal volume of stimulation medium containing test compound at 2x concentration or DMSO control was then added to cell containing wells. After incubating overnight with test compound, 0.5 µg/ml of lipopolysaccharide (LPS) from Escherichia coli O55:B5 was added to the cells and incubated for another 6 hours. Then media samples were collected for quantitation of PGD2 production using a Prostaglandin D2 ELISA Kit. Toxicity of test compounds was assessed by WST-1 cell proliferation assay at the end of the 6hr incubation with LPS using a colorimetric assay kit.

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Mast Cell PGD2 Production Assay: Human mast cells are pre-treated with SAR191801 (0.1–100 nM) for 1 hour, then stimulated with calcium ionophore for 3 hours. Supernatants are collected, and PGD2 levels are quantified by ELISA. Cell viability is assessed using a colorimetric assay to confirm non-toxicity [4]

- Mouse Peritoneal PGD2 Model: Male mice are randomized into control and treatment groups. SAR191801 is formulated in 0.5% carboxymethylcellulose and administered orally at 3 mg/kg. One hour later, compound 48/80 is injected intraperitoneally. Peritoneal fluid is collected 2 hours post-challenge, and PGD2 levels are measured by ELISA [4]
- Rat Allergic Rhinitis Model: Rats are sensitized to ovalbumin, then challenged intranasally. SAR191801 (10 mg/kg) or vehicle is administered orally once daily for 7 days starting 1 day before the first challenge. Nasal tissues are harvested, and eosinophils are counted via histology; nasal lavage fluid is analyzed for PGD2 by HPLC [1]

- Oral Bioavailability: In rats, SAR191801 has an oral bioavailability of 45% after a single 10 mg/kg dose [4]
- Plasma Half-Life: In mice, the plasma half-life of SAR191801 is 4.2 hours following oral administration of 3 mg/kg [4]
- Tissue Distribution: In rats, SAR191801 (10 mg/kg, oral) shows highest concentrations in the liver and kidneys at 2 hours post-dose, with brain levels ~20% of plasma concentrations [4]

- Plasma Protein Binding: SAR191801 binds to human plasma proteins at 92% [4]
- No acute toxicity is observed in mice at oral doses up to 300 mg/kg, with no significant changes in body weight or serum chemistry parameters (ALT, AST, creatinine) [4]

[1]. Phenyloxadiazole derivatives as PGD inhibitors and their preparation, pharmaceutical compositions and use in the treatment of allergic and inflammatory disorders. From PCT Int. Appl. (2011), WO 2011044307 A1 20110414.

[2]. Method for treating macular degeneration using syk multikinase inhibitor, an hPGDS inhibitor and a DP antagonist. From PCT Int. Appl. (2010), WO 2010080563 A2 20100715.

[3].W Demonstration on Pilot-Plant Scale of the Utility of 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) as a Catalyst in the Efficient Amidation of an Unactivated Methyl Ester. From Organic Process Research & Development (2012), 16(12), 1967-1969.

[4].Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors. Bioorg Med Chem Lett . 2021 Feb 15:34:127759. .

The utility of 1,5,7-triazabicyclo[4.4.0]dec-5-ene as a reagent to facilitate efficient amide formation by reaction of an amine with an unactivated ester was demonstrated on pilot-plant scale as a key step in the synthesis of an H-PGDS inhibitor. [3]

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Prostaglandin D2 (PGD2) is released in large quantities during allergic and asthmatic anaphylaxis from activated mast cells, reaching levels 100–1000 times higher than those produced by platelets, macrophages, T-helper and dendritic cells. PGD2 also plays a detrimental pro-inflammatory role in systemic mastocytosis, rheumatoid arthritis, and Duchenne’s muscular dystrophy. PGD2 synthesis from the cyclic endoperoxide arachidonic acid (AA) metabolite, prostaglandin H2 (PGH2), is catalyzed by two enzymes, lipocalin-type PGD2-synthase (l-PGDS) and hematopoietic-type PGD-synthase (H-PGDS) (Scheme 1). l-PGDS expression is largely localized to the brain, whereas H-PGDS expression occurs primarily within the peripheral tissues. PGD2 is the endogenous activating ligand for two prostaglandin receptors, PGD2 receptor 1 (DP1) and PGD2 receptor 2 (DP2 or CRTH2), where these receptors mediate complex downstream effects which can be pro-inflammatory or anti-inflammatory in various instances. H-PGDS is therefore a central protein target for development of selective, potent inhibitors for therapeutic use against PGD2-mediated diseases and conditions.[4]
H-PDGS is a sigma-type glutathione transferase that catalyzes the bi-bi molecular reaction between nucleophilic cofactor glutathione (GSH) and electrophilic substrate PGH2. HQL-79 (Fig. 1) was among the first-generation human H-PGDS inhibitors identified. HQL-79 inhibits H-PGDS competitively with PGH 2 and possesses moderate potency with a Ki of 5 μM. Surface plasmon resonance (SPR) revealed that HQL-79 bound H-PGDS with 12-fold higher affinity in the presence of GSH and Mg2+ than in their absence, supporting that HQL-79 binding is stabilized by enzyme-cofactor-inhibitor interactions, perhaps by a network of hydrogen bonds. [4]

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- Mechanism of Action: SAR191801 is a selective inhibitor of hPGDS, which catalyzes the conversion of prostaglandin H2 to PGD2. By blocking this enzyme, it reduces PGD2-mediated inflammatory responses, including mast cell activation and eosinophil recruitment [1,4]
- Therapeutic Potential: Investigated for treating allergic and inflammatory disorders such as allergic rhinitis, asthma, and atopic dermatitis, as well as age-related macular degeneration in combination with SYK inhibitors and DP antagonists [1,2]

C22H20N6O3

416.433

416.16

C, 63.45; H, 4.84; N, 20.18; O, 11.53

1234708-04-3

1234708-04-3;

46700750

White to off-white solid powder

3.136

2

8

6

31

598

0

OC(C)(C)C1=NC(C2=CC(CNC(C(C=N3)=CN=C3C4=CC=CC=N4)=O)=CC=C2)=NO1

VJCLAPUACUQZOV-UHFFFAOYSA-N

InChI=1S/C22H20N6O3/c1-22(2,30)21-27-18(28-31-21)15-7-5-6-14(10-15)11-26-20(29)16-12-24-19(25-13-16)17-8-3-4-9-23-17/h3-10,12-13,30H,11H2,1-2H3,(H,26,29)

N-[[3-[5-(1-Hydroxy-1-methylethyl)-1,2,4-oxadiazol-3-yl]phenyl]methyl]-2-(2-pyridinyl)-5-pyrimidinecarboxamide

hPGDS-IN-1; hPGDS-IN 1; 1234708-04-3; hPGDS-IN-1; SAR191801; N-(3-(5-(2-hydroxypropan-2-yl)-1,2,4-oxadiazol-3-yl)benzyl)-2-(pyridin-2-yl)pyrimidine-5-carboxamide; N-({3-[5-(2-hydroxypropan-2-yl)-1,2,4-oxadiazol-3-yl]phenyl}methyl)-2-(pyridin-2-yl)pyrimidine-5-carboxamide; N-[[3-[5-(2-hydroxypropan-2-yl)-1,2,4-oxadiazol-3-yl]phenyl]methyl]-2-pyridin-2-ylpyrimidine-5-carboxamide; 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid 3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-benzylamide; N-[[3-[5-(1-Hydroxy-1-methylethyl)-1,2,4-oxadiazol-3-yl]phenyl]methyl]-2-(2-pyridinyl)-5-pyrimidinecarboxamide; hPGDS-IN1; SAR 191801; SAR-191801; SAR191801

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~30 mg/mL (~72.04 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.

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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).

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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)

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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).

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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

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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.

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