BMP-mediated SMAD1/5 phosphorylation (IC50 = 1.14 µM for inhibition of BMP4-stimulated SMAD1/5 phosphorylation) [1]
PPARγ (Pyrintegrin is not a direct agonist) [1]

In a dose-dependent manner (IC50 of 1.14 μM), pyrintegrin (0-10 μM; 1 hour; hASC) administration suppresses BMP4-mediated phosphorylation of BMP-responsive SMAD1/5 [1]. Pyrintegrin induces human adipose stem/progenitor cells (hASC) to differentiate into lipid adipocytes in vitro by upregulating CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPARγ). Additionally, the secretion of adiponectin, leptin, glycerol, and total triglycerides increases in differentiated cells. By phosphorylating SMAD1/5 through BMP, pyrintegrin reduces Runx2 and Osx [1]. In cultured cells, pyrintegrin administration reduces injury-induced declines in focal adhesions, F-actin stress fibers, and active β1-integrin levels [2]. Western blot examination

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Pyrintegrin at 2 µM significantly upregulated the mRNA expression of adipogenic transcription factors PPARγ and C/EBPα in human adipose stem/progenitor cells (hASCs) cultured in adipogenesis induction medium (AIM) compared to AIM alone or control medium (DMEM). [1]
Time-course analysis showed that in hASCs treated with AIM supplemented with 2 µM Pyrintegrin, PPARγ expression began to increase by day 4, peaked between days 7-14, and declined by day 21, while C/EBPα expression increased from day 4 and continued to escalate up to day 21. [1]
hASCs cultured for 2 or 4 weeks in AIM supplemented with 2 µM Pyrintegrin secreted significantly higher levels of adiponectin, leptin, total triglycerides, and glycerol into the supernatant compared to cells in DMEM or AIM alone. [1]
Pyrintegrin (2 µM) robustly augmented lipid droplet accumulation in hASCs cultured in AIM for 4 weeks, as visualized by lipid staining. Pyrintegrin alone (without adipogenic supplements) failed to induce lipid accumulation or upregulate PPARγ/C/EBPα. [1]
In hASCs treated with osteogenesis induction medium (OIM), the addition of 2 µM Pyrintegrin drastically attenuated mineralization (as shown by reduced Alizarin Red staining) and significantly downregulated the expression of osteogenic markers Runx2 and Osterix over a 21-day culture period. [1]
Concurrently, in the same OIM, 2 µM Pyrintegrin upregulated PPARγ and C/EBPα expression and stimulated lipid droplet formation at days 7, 14, and 21. Pyrintegrin alone in DMEM did not induce lipid accumulation. [1]
Pyrintegrin induced lipid accumulation in hASCs in the presence of dexamethasone alone (0.1-10 µM) and in the absence of other adipogenic supplements like insulin and cAMP activators. [1]
A human PPARγ luciferase receptor assay showed that Pyrintegrin at concentrations of 0.02, 0.2, or 2 µM did not activate PPARγ, unlike the positive control Rosiglitazone. [1]
Western blot analysis demonstrated that Pyrintegrin downregulated the phosphorylation of SMAD1/5 (a downstream target of BMP signaling) but did not inhibit the phosphorylation of p42/44 MAPK. [1]
Pyrintegrin inhibited BMP4-mediated phosphorylation of SMAD1/5 in a dose-dependent manner in hASCs, with a half maximal inhibitory concentration (IC50) of 1.14 µM. It did not attenuate TGFβ1-mediated SMAD2 phosphorylation. [1]
At higher doses (5 and 10 µM) in the presence of BMP4, Pyrintegrin activated phosphorylation of p38 MAPK. [1]

Pyrintegrin (10 mg/kg; i.p.; once; C57BL/6J mice) treatment protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of mouse glomeruli showed that LPS administration reduced active β1 integrin levels in podocytes, and co-treatment with Pyrintegrin prevented this [2]. ? In rats, Pyrintegrin reduces peak proteinuria caused by puromycin aminonucleoside-induced nephropathy [2]. ? Pyrintegrin induces transplanted adipose stem/progenitor cells (ASCs) and recruits endogenous cells to form postnatal adipose tissue in vivo. In vivo, Pyrintegrin-treated human adipose stem/progenitor cells (ASCs) in 3D bioprinted scaffolds generated ectopically formed adipose tissue expressing human PPARγ when transplanted into the back of athymic mice. Notably, Pyrintegrin-adsorbed collagen gel implanted in the inguinal fat pad promoted adipogenesis formed by host endogenous cells, demonstrating its ability to induce in situ adipogenesis without the need for cell transplantation [1].

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Human adipose stem/progenitor cells (hASCs) primed with 2 µM Pyrintegrin in AIM for 2 weeks were seeded in 3D-bioprinted polycaprolactone scaffolds and implanted subcutaneously in the dorsum of athymic mice. After 4 weeks, the retrieved scaffolds showed adipose tissue formation positive for lipid staining (Oil Red O) and expressed significantly higher levels of human PPARγ mRNA compared to scaffolds with AIM-primed cells alone, unprimed cells, or cell-free scaffolds. [1]
3D-printed polycaprolactone scaffolds adsorbed with Pyrintegrin (10 µg/mL in collagen gel) and implanted into the inguinal fat pad of C57BL/6 mice (without any cell transplantation) promoted the formation of adipose tissue by host endogenous cells after 4 weeks, as evidenced by positive Oil Red O staining and significantly higher mouse PPARγ gene expression compared to control scaffolds without Pyrintegrin. [1]

Western blot analysis
Cell Types: Human adipose stem/progenitor cells (hASC)[2]
Tested Concentrations: 0 µM, 0.2 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, 10 µM
Incubation Duration: 1 hour
Experimental Results: BMP4- Inhibition mediates phosphorylation of BMP-responsive SMAD1/5 in a dose-dependent manner.

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For adipogenic differentiation, human adipose stem/progenitor cells (hASCs) were plated at a density of 5 x 10^3 cells per cm^2 and culture-expanded until confluence. Cells were then treated with adipogenic induction medium (AIM) containing 1 µM dexamethasone, 10 µg/ml insulin, 0.5 µM isobutylmethylxanthine, and 60 µM indomethacin. Pyrintegrin was dissolved in DMSO and supplemented into AIM at specified concentrations. Control groups included cells treated with growth medium (DMEM) and vehicle (DMSO). Medium was changed twice a week. [1]
For lipid staining, cell cultures were washed with PBS, fixed with 4% paraformaldehyde, and then stained with a neutral lipid stain for 10-15 minutes. [1]
For gene expression analysis (qRT-PCR), total RNA was isolated using a commercial kit. RNA was reverse-transcribed into cDNA. qPCR reactions were performed using TaqMan Universal PCR Master Mix and gene-specific primers for PPARγ, C/EBPα, Runx2, and Osterix, with GAPDH as a housekeeping gene. [1]
For analysis of secreted factors, culture medium and cell lysate supernatant were collected at specific time points. Secreted human adiponectin and leptin were measured using ELISA. Total triglycerides and glycerol content were measured using a commercial glycerol determination kit. DNA content was measured using a fluorescent dsDNA assay kit. [1]
For osteogenic differentiation, hASCs were treated with osteogenic induction medium (OIM) containing 100 nM dexamethasone, 0.05 mM L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate, with or without 2 µM Pyrintegrin. Mineralization was assessed by Alizarin Red staining after 3 weeks. [1]
For signaling studies, hASCs were serum-starved overnight and then treated with varying doses of Pyrintegrin (0-10 µM) in the presence or absence of BMP4 (30 ng/mL) or TGFβ1 (0.5 ng/mL) for 1 hour. Cell extracts were prepared, and protein phosphorylation (SMAD1/5, SMAD2, p38 MAPK, p42/44 MAPK) was analyzed by western blot. [1]
For the PPARγ reporter assay, reporter cells engineered to express human PPARγ and a luciferase gene under a PPARγ-responsive promoter were treated with Pyrintegrin or Rosiglitazone (positive control). After overnight incubation, luciferase activity was measured using a luminometer. [1]

Animal/Disease Models: Female wild-type C57BL/6J mice (10 weeks old) were injected with LPS [2]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection;
Experimental Results: These animals were provided with significant protection against Effects of LPS-induced proteinuria and foot process (FP) effacement.

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For the transplantation experiment, human adipose stem/progenitor cells (hASCs) were culture-expanded in DMEM, AIM, or AIM supplemented with 2 µM **Pyrintegrin** for 2 weeks. Cells (10 x 10^6 cells/mL) were homogenously suspended in 3 mg/mL type I collagen solution. The cell-collagen mixture was infused into the microchannels of 3D-printed polycaprolactone scaffolds (5 mm diameter x 3 mm height). The scaffolds were incubated at 37°C for 1 hour to allow gelation. [1]
Subcutaneous pockets were surgically created on the dorsum of 8-9 week-old athymic nude male mice under anesthesia. The cell-seeded or cell-free scaffolds were implanted into these pockets. The skin was closed with sutures. Mice received post-surgical analgesic injections. Scaffolds were retrieved 4 weeks after implantation following euthanasia by CO2 asphyxiation. [1]
For the endogenous cell recruitment experiment, **Pyrintegrin** was adsorbed into neutralized type I collagen solution at a concentration of 10 µg/mL. The **Pyrintegrin**-adsorbed or **Pyrintegrin**-free collagen gel was infused into the microchannels of similar polycaprolactone scaffolds and allowed to crosslink at 37°C for 1 hour. [1]
These scaffolds were then surgically implanted into the inguinal fat pad of C57BL/6 male mice (10-11 weeks old). A small pocket was created within the fat pad for scaffold implantation. The fat pad and skin were closed with sutures. Mice received post-surgical analgesic injections. Scaffolds were retrieved 4 weeks after implantation following euthanasia by CO2 asphyxiation. [1]

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For the transplantation experiment, human adipose stem/progenitor cells (hASCs) were culture-expanded in DMEM, AIM, or AIM supplemented with 2 µM Pyrintegrin for 2 weeks. Cells (10 x 10^6 cells/mL) were homogenously suspended in 3 mg/mL type I collagen solution. The cell-collagen mixture was infused into the microchannels of 3D-printed polycaprolactone scaffolds (5 mm diameter x 3 mm height). The scaffolds were incubated at 37°C for 1 hour to allow gelation. [1]
Subcutaneous pockets were surgically created on the dorsum of 8-9 week-old athymic nude male mice under anesthesia. The cell-seeded or cell-free scaffolds were implanted into these pockets. The skin was closed with sutures. Mice received post-surgical analgesic injections. Scaffolds were retrieved 4 weeks after implantation following euthanasia by CO2 asphyxiation. [1]
For the endogenous cell recruitment experiment, Pyrintegrin was adsorbed into neutralized type I collagen solution at a concentration of 10 µg/mL. The Pyrintegrin-adsorbed or Pyrintegrin-free collagen gel was infused into the microchannels of similar polycaprolactone scaffolds and allowed to crosslink at 37°C for 1 hour. [1]
These scaffolds were then surgically implanted into the inguinal fat pad of C57BL/6 male mice (10-11 weeks old). A small pocket was created within the fat pad for scaffold implantation. The fat pad and skin were closed with sutures. Mice received post-surgical analgesic injections. Scaffolds were retrieved 4 weeks after implantation following euthanasia by CO2 asphyxiation. [1]

[1]. Pyrintegrin Induces Soft Tissue Formation by Transplanted or Endogenous Cells. Sci Rep. 2017 Jan 27;7:36402.

[2]. A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules. J Am Soc Nephrol. 2015 Nov;26(11):2741-52.

[3]. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci U S A. 2010 May 4;107(18):8129-34.

Pyrintegrin is a 2,4-disubstituted pyrimidine small molecule initially identified for its ability to promote human embryonic stem cell survival. [1]
This study reports that Pyrintegrin promotes adipogenic differentiation of human adipose stem/progenitor cells and induces adipose tissue formation in vivo, both from transplanted cells and recruited host endogenous cells. [1]
The proposed mechanism involves Pyrintegrin attenuating BMP-mediated SMAD1/5 phosphorylation, which is associated with osteogenesis, thereby shifting the balance towards adipogenesis. It upregulates key adipogenic transcription factors PPARγ and C/EBPα, but not through direct agonism of PPARγ. Its adipogenic activity requires the presence of glucocorticoids like dexamethasone. [1]
The ability of Pyrintegrin to promote endogenous adipogenesis without cell transplantation suggests potential therapeutic applications in soft tissue reconstruction and augmentation for conditions like lipoatrophy, lumpectomy, or facial trauma. [1]
Pyrintegrin's attenuation of osteogenesis may have potential usage in promoting soft tissue healing and avoiding ectopic mineralization. [1]

451.167

1228445-38-2

1228445-38-2;

46223724

Off-white to light yellow solid powder

1.4±0.1 g/cm3

729.3±70.0 °C at 760 mmHg

394.9±35.7 °C

0.0±2.5 mmHg at 25°C

1.679

3.26

3

8

7

32

720

0

S(C1C=CC(=CC=1)NC1=NC=CC(=N1)N1C2C=CC(=CC=2CCC1)O)(NCC1CC1)(=O)=O

QRJTZIJWDLJKQO-UHFFFAOYSA-N

InChI=1S/C23H25N5O3S/c29-19-7-10-21-17(14-19)2-1-13-28(21)22-11-12-24-23(27-22)26-18-5-8-20(9-6-18)32(30,31)25-15-16-3-4-16/h5-12,14,16,25,29H,1-4,13,15H2,(H,24,26,27)

N-Cyclopropylmethyl-4-[4-(6-hydroxy-3,4-dihydro-2H-quinolin-1-yl)-pyrimidin-2-ylamino]-benzenesulfonamide

Pyrintegrin PTN

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 : ~250 mg/mL (~553.66 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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