4-IPP (4-iodo-6-phenylpyrimidine) is a specific, irreversible suicide substrate inhibitor of macrophage migration inhibitory factor (MIF). [1]

4-IPP is a drift suicide substrate of MIF that inhibits MIF's biological action by binding to it covalently and irreversibly [1]. 4-IPP blocks MIF's interaction with the thioredoxin-responsive protein-p65 complex, hence inhibiting RANKL-induced p65 phosphorylation and nuclear translocation [1]. 4-IPP promotes osteocyte-mediated osteoclastogenesis and suppresses NF-κB receptor activator ligand (RANKL)-induced osteoclastogenesis. In a dose-dependent manner, 4-IPP (0.5 - 200 μM; 24-72 hours) suppresses osteoclastogenesis. generation of osteoclasts[1]. 4-IPP (5 – 20 μM); 5[1] mineralizes and forms bones.

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4-IPP inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner in bone marrow macrophages (BMMs). At concentrations of 5, 10, and 20 μM, it significantly reduced the number and mean area of TRAP-positive multinucleated osteoclasts and decreased the number of podosomal actin belts. The distribution of osteoclasts with 5-10 or >10 nuclei was also decreased. [1]
4-IPP inhibited the bone resorption activity of mature osteoclasts. BMM-derived osteoclasts reseeded on bone discs and treated with 5, 10, or 20 μM of 4-IPP for 72 hours showed a dose-dependent reduction in the resorbed pit area. [1]
4-IPP promoted osteoblast differentiation and mineralization. Primary calvarial osteoblasts treated with 2.5, 5, or 10 μM of 4-IPP for 7 days showed a dose-dependent increase in ALP-positive cells. After 21 days of osteogenic differentiation, a dose-dependent increase in mineralized bone nodule formation (Alizarin Red S staining) and calcium deposition was observed. [1]
Mechanistically, 4-IPP inhibited RANKL-induced early NF-κB signaling. In BMMs, 20 μM of 4-IPP reduced the phosphorylation of IκBα and p65, and prevented the nuclear translocation of NF-κB p65. This led to the suppression of late osteoclast marker genes such as NFATc1, CTSK, TRAP, c-Fos, TCIRG1, and DC-STAMP at both the mRNA and protein levels. [1]
In osteoblasts, 4-IPP (2.5, 5, 10 μM) potentiated the dephosphorylation of p65 induced by osteogenic media, thereby alleviating the suppressive effects of NF-κB on bone formation. This was accompanied by a dose-dependent increase in the protein expression of Runx2 and increased mRNA expression of osteoblast marker genes Runx2, ALP, OCN, and OPN after 21 days. [1]
Using co-immunoprecipitation assays in RAW264.7 cells and BMMs, it was found that upon RANKL stimulation, MIF interacts with TXNIP and p65 in a complex. Treatment with 4-IPP (20 μM) inhibited this interaction. [1]
4-IPP showed no significant cytotoxicity in BMMs at concentrations up to 20 μM for 24 and 72 hours. The calculated IC50 at 72 hours was 104.3 μM. In primary calvarial osteoblasts, no adverse effects were observed up to 10 μM for 24 and 72 hours. The calculated IC50 at 72 hours was 85.0 μM. [1]

4-IPP (1 mg/kg, 5 mg/kg; every 2 days; for 8 weeks) increases osteoblastic bone formation and decreases osteoclast activity to improve bone loss related to female food deprivation.[1]

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In a murine model of Ti particle-induced calvarial osteolysis, subcutaneous injection of 4-IPP at 1 mg/kg or 5 mg/kg every 2 days for 2 weeks dose-dependently protected against bone destruction. μCT analysis showed that 4-IPP treatment resulted in higher bone volume (BV/TV) and lower bone porosity compared to the vehicle control. Histological assessment confirmed reduced osteolytic bone destruction, fewer TRAP-positive osteoclasts, and decreased osteoclast surface per bone surface (OcS/BS). Immunofluorescence staining showed higher expression of the osteoblastic marker Runx2 on the bone surface in 4-IPP-treated mice. [1]
In a murine model of ovariectomy (OVX)-induced osteoporosis, intraperitoneal injection of 4-IPP at 1 mg/kg or 5 mg/kg every 2 days for 8 weeks partially prevented the decline in bone mass and deterioration of trabecular microarchitecture. μCT analysis revealed that 4-IPP treatment preserved BV/TV, Tb.N, and Tb.Th, and reduced Tb.Sp compared to OVX vehicle controls. Histomorphometry showed fewer TRAP-positive osteoclasts and reduced OcS/BS. Serum levels of the bone resorption marker CTX-1 were reduced, while serum levels of the bone formation marker P1NP were elevated in 4-IPP-treated mice. Immunofluorescence staining showed higher expression of Runx2 and OCN on the bone surface. qPCR analysis of bone tissue showed decreased expression of osteoclastic genes (NFATc1, CTSK, TRAP) and increased expression of osteoblastic genes (Runx2, ALP, OCN, OPN) following 4-IPP treatment. [1]

Cytotoxicity assay[1]
Cell Types: BMMs
Tested Concentrations: 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM , 50 μM, day) inhibits RANKL-induced osteoclast resorption and bone resorption [1]. 100 μM, 200 μM
Incubation Duration: 24 hrs (hours), 72 hrs (hours)
Experimental Results: Inhibition of osteoclastogenesis in a dose-dependent manner.

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Western Blot Analysis [1]
Cell Types: BMMs
Tested Concentrations: 5 μM, 10 μM, 20 μM
Incubation Duration: 1 day, 3 days, 5 days
Experimental Results: Inhibition of RANKL-induced osteoclast differentiation and bone resorption.

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Bone marrow macrophages (BMMs) were isolated from the femora and tibiae of 6-week-old mice. Cells were seeded in 24-well plates at 5×10⁴ cells/well in α-MEM containing 40 ng/ml M-CSF and 50 ng/ml RANKL, without or with various concentrations of 4-IPP (5, 10, 20 μM) for 5-7 days. Media were replenished every other day. At the end of the culture, cells were fixed and stained for TRAP activity. TRAP-positive multinucleated osteoclasts (≥5 nuclei) were scored and quantified. [1]
For the bone resorption assay, BMMs were stimulated with RANKL for 3 days, then dissociated and reseeded onto sterilized bone discs. After 6 hours, cells were treated with 4-IPP (5, 10, 20 μM) for a further 72 hours. Cells were then removed by sonication, and resorption pits were imaged via scanning electron microscopy. The resorbed area was measured using ImageJ software. [1]
For podosomal actin belt staining, BMM-derived osteoclasts were fixed, permeabilized, and incubated with rhodamine-conjugated phalloidin. Nuclei were counterstained with DAPI. Fluorescence images were captured using confocal microscopy, and the number of actin belts was quantified. [1]
Primary calvarial osteoblasts were isolated from 2-day-old mice. For osteogenesis, confluent cells were cultured in osteogenic induction medium (containing dexamethasone, β-glycerophosphate, and ascorbic acid) without or with 4-IPP (2.5, 5, 10 μM). For ALP staining, cells were cultured for 7 days. For mineralization (Alizarin Red S) staining, cells were cultured for 21 days. Calcium deposits were quantified by cetylpyridinium chloride extraction and measuring OD at 550 nm. [1]
For cytotoxicity assays, BMMs or calvarial osteoblasts were seeded in 96-well plates and treated with increasing concentrations of 4-IPP (0.5 to 200 μM) for 24 or 72 hours. Cell viability was assessed using the CCK-8 reagent, and absorbance was read at 450 nm. [1]
For Western blot analysis, total cellular proteins were extracted with RIPA lysis buffer. Proteins were resolved on SDS-PAGE gels, transferred to PVDF membranes, probed with specific primary antibodies (against NF-κB p65, p-p65, IκBα, p-IκBα, c-Fos, NFATc1, TRAP, Runx2, etc.), and then with HRP-conjugated secondary antibodies. Reactivity was detected via chemiluminescence. [1]
For immunofluorescence staining of NF-κB p65 nuclear translocation, wild-type or MIF-KO BMMs were pretreated without or with 20 μM 4-IPP for 2 hours, then stimulated with RANKL for 20 minutes. Cells were fixed, permeabilized, stained with anti-p65 antibody, followed by an Alexa Fluor 546-conjugated secondary antibody. Nuclei were counterstained with DAPI. The percentage of nuclear p65-positive cells was counted. [1]
For co-immunoprecipitation (Co-IP), total cellular proteins from RAW264.7 cells or BMMs were extracted with RIPA lysis buffer without SDS. Lysates were incubated with specific antibodies (against TXNIP or p65) and protein G-sepharose beads overnight. The bound protein complexes were washed, boiled, and then visualized by Western blot with corresponding antibodies (against MIF, TXNIP, and p65). [1]
For real-time quantitative PCR (qPCR), total RNA was extracted using an RNA extraction kit. First-strand cDNA was synthesized from 1 μg of total RNA. qPCR was performed using SYBR Green master mix with gene-specific primers for NFATc1, CTSK, TRAP, c-Fos, TCIRG1, DC-STAMP, Runx2, ALP, OCN, OPN, and GAPDH (as an internal control). Data were analyzed using the 2⁻ΔΔCt method. [1]

Animal/Disease Models: 8weeks old C57BL/6J male mice [1]
Doses: 1 mg/kg, 5 mg/kg
Route of Administration: intraperitoneal (ip) injection; once every 2 days; for 8 weeks
Experimental Results: Alleviation of OVX-induced osteoporosis .

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Ti particle-induced calvarial osteolysis model: Eight-week-old male C57BL/6 mice were used. Ti particles (30 mg in PBS) were implanted under the periosteum at the middle calvaria suture. 4-IPP (1 mg/kg or 5 mg/kg in PBS) or PBS alone (vehicle) was injected subcutaneously into the periosteum every 2 days for 2 weeks. At the end of the experiment, calvariae were removed for μCT and histological analysis. [1]
OVX-induced osteoporosis model: Eight-week-old female C57BL/6 mice underwent bilateral ovariectomy (OVX) or sham surgery. One week after OVX, mice were injected intraperitoneally with 4-IPP (1 mg/kg or 5 mg/kg in PBS) or PBS alone (vehicle) every 2 days for 8 weeks. At the end of the experiment, tibiae and femurs were excised for μCT, histological, and qPCR analysis. Serum was collected for ELISA measurement of CTX-1 and P1NP. [1]

In the in vivo studies, no adverse effects or fatalities were noted in mice treated with 4-IPP at doses of 1 mg/kg or 5 mg/kg (subcutaneous every 2 days for 2 weeks, or intraperitoneal every 2 days for 8 weeks). [1]

[1]. Macrophage migration inhibitory factor (MIF) inhibitor 4-IPP suppresses osteoclast formation and promotes osteoblast differentiation through the inhibition of the NF-κB signaling pathway. FASEB J. 2019 Jun;33(6):7667-7683.

4-Iodo-6-phenylpyrimidine is a pyrimidine compound with an iodine atom at position 4 and a phenyl substituent at position 6. It acts as an inhibitor of macrophage migration inhibitory factors, an antitumor agent, and an inducer of apoptosis. It belongs to the pyrimidine class, biaryl compounds, and organoiodine compounds.

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4-IPP (4-iodo-6-phenylpyrimidine) is a specific, irreversible suicide substrate inhibitor of macrophage migration inhibitory factor (MIF). It covalently and irreversibly binds to MIF to inhibit its biologic activity. [1]
This study demonstrates that pharmacological inhibition of MIF with 4-IPP can be used to regulate bone metabolism by balancing osteoclast-mediated bone resorption and osteoblast-mediated bone formation. It shows therapeutic potential for the treatment of pathologic osteolytic bone disorders (e.g., wear particle-induced osteolysis) and postmenopausal osteoporosis. [1]
The mechanism involves the inhibition of the MIF-TXNIP-p65 axis, which regulates RANKL-induced NF-κB signaling during osteoclast formation. In osteoclasts, 4-IPP prevents the interaction of MIF with TXNIP-p65 complexes, blocking p65 phosphorylation and nuclear translocation, thereby suppressing NFATc1 and downstream osteoclastogenic genes. In osteoblasts, 4-IPP inhibits NF-κB signaling, which is a known suppressor of bone formation, thereby potentiating osteoblast differentiation and mineralization. [1]

C10H7IN2

282.08

281.965

41270-96-6

817368

White to yellow solid powder

1.728±0.06 g/cm3

380.2±30.0 °C

2.748

0

2

1

13

157

0

ZTCJXHNJVLUUMR-UHFFFAOYSA-N

InChI=1S/C10H7IN2/c11-10-6-9(12-7-13-10)8-4-2-1-3-5-8/h1-7H

4-Iodo-6-phenyl-pyrimidine

4 IPP 4IPP 4-IPP

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 : ~100 mg/mL (~354.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.86 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 25.0 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: ≥ 2.5 mg/mL (8.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.86 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 25.0 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.)