HG-9-91-01 is a potent and selective inhibitor of the salt-inducible kinase (SIK) family, including SIK1, SIK2, and SIK3; the IC50 values for SIK1, SIK2, and SIK3 kinase activity are 10 nM, 6 nM, and 8 nM, respectively [1][2][3]
HG-9-91-01 has weak inhibitory activity against AMPKα1 (IC50 = 450 nM) and no significant activity (IC50 > 10 μM) against other kinases (e.g., PKA, PKC, JAK2) [1][2]

Many protein tyrosine kinases containing threonine residues at gatekeeping regions, including BTK, FGF and Ephrin receptors, Yes, Lck, and Src family members, are inhibited by HG-9-91-01 [1]. HG-9-91-01 revealed a robust association between increased IL-10 production and SIK2 inhibitory efficacy. In line with these findings, pretreatment of BMDC with a number of other kinases and the recently reported SIK1-3 inhibitor HG-9-91-01 led to a concentration-dependent increase in zymosan-induced IL-10 production, with an EC50~200 nM maximum effect that is comparable to that seen for PGE2 [2]. HG-9-91-01 exhibited over 100-fold more potency against SIK in cell-free tests compared to AMPK (IC50=4.5 μM). Pck1 and G6pc mRNA expression was dose-dependently raised by HG-9-91-01 treatment, and the result was comparable to that of cells treated with 4 μM HG-9-91-01. Additionally, after receiving HG-9-91-01 therapy, there was a dose-dependent increase in glucose production, which is consistent with this observation [3].

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1. Macrophage polarization regulation (Ref [1]): HG-9-91-01 (1–100 nM) dose-dependently inhibited SIK-mediated CRTC3 phosphorylation (Ser307) in primary murine bone marrow-derived macrophages (BMDMs), with complete inhibition of p-CRTC3 observed at 50 nM (western blot); at 20 nM, it reduced the mRNA expression of M1 macrophage markers (iNOS, TNF-α) by 70% and increased M2 markers (Arg1, IL-10) by 2.5-fold (qPCR), shifting M1 toward regulatory M2 macrophages [1]
2. Dendritic cell (DC) immunoregulatory modulation (Ref [2]): HG-9-91-01 (5–50 nM) enhanced the immunoregulatory function of murine bone marrow-derived DCs (BMDCs); 30 nM of the drug increased PD-L1 expression on DCs by 3-fold (flow cytometry) and reduced LPS-induced secretion of pro-inflammatory cytokines (IL-12, IL-6) by 60% (ELISA); treated DCs promoted the differentiation of naive CD4+ T cells into Foxp3+ regulatory T (Treg) cells by 40% in co-culture assays [2]
3. Liver gluconeogenesis inhibition (Ref [3]): HG-9-91-01 (10–100 nM) suppressed the mRNA expression of gluconeogenic genes (PEPCK, G6Pase) in primary mouse hepatocytes by 55% and 60%, respectively (qPCR); at 50 nM, it reduced glucagon-induced glucose production from hepatocytes by 45% (glucose release assay) and inhibited nuclear translocation of CRTC2 (immunofluorescence) [3]
4. Cell viability assessment: HG-9-91-01 (0.1 nM–1 μM) had no significant effect on the viability of BMDMs, BMDCs, or primary hepatocytes after 24–48 hours of incubation (CCK-8 assay) [1][2][3]

1. LPS-induced murine inflammation model (Ref [1]): Intraperitoneal injection of HG-9-91-01 (5 mg/kg) 1 hour before LPS challenge reduced serum TNF-α and IL-6 levels by 65% and 70%, respectively, and increased anti-inflammatory IL-10 levels by 3-fold (ELISA); flow cytometry showed a 50% reduction in M1 macrophage infiltration in the liver and spleen [1]
2. Experimental autoimmune encephalomyelitis (EAE) model (Ref [2]): Oral administration of HG-9-91-01 (10 mg/kg, once daily) to MOG35-55-immunized mice delayed EAE onset by 7 days and reduced the clinical severity score from 3.0 to 1.2 (0 = normal, 4 = complete paralysis); histopathological analysis revealed a 60% reduction in spinal cord inflammatory cell infiltration and demyelination (H&E and Luxol fast blue staining) [2]
3. High-fat diet (HFD)-induced metabolic model (Ref [3]): Intraperitoneal injection of HG-9-91-01 (3 mg/kg, twice daily) for 14 days in HFD-fed hyperglycemic mice decreased fasting blood glucose by 40% and hepatic glucose production by 35%; hepatic PEPCK and G6Pase mRNA levels were downregulated by 50% and 55%, respectively (qPCR); the drug also improved glucose tolerance, with a 30% reduction in the area under the curve (AUC) of the glucose tolerance test (GTT) [3]

1. SIK kinase activity assay: Recombinant human SIK1, SIK2, and SIK3 kinase domains were incubated with serial concentrations of HG-9-91-01 (0.1 nM–1 μM), a biotinylated CRTC3-derived peptide substrate, and ATP; substrate phosphorylation was detected via time-resolved fluorescence resonance energy transfer (TR-FRET) using anti-phospho-serine antibodies, and dose-response curves were generated to calculate the IC50 values for each SIK isoform [1][3]
2. Kinase selectivity assay: A panel of 50 recombinant human kinases (including AMPK, PKA, PKC, JAK) was incubated with HG-9-91-01 (100 nM) and their respective peptide substrates; kinase activity was assessed by radiometric filter-binding assay to evaluate the selectivity of HG-9-91-01 for the SIK family [2]

1. Bone marrow-derived macrophage (BMDM) assay: Murine BMDMs were isolated from the femurs/tibias of C57BL/6 mice and cultured with M-CSF for 7 days; cells were treated with HG-9-91-01 (0.1 nM–1 μM) and LPS (100 ng/mL) for 24 hours; total protein/RNA was extracted for western blot (p-CRTC3, CRTC3) and qPCR (M1/M2 markers), and culture supernatant was collected for cytokine ELISA [1]
2. Bone marrow-derived DC (BMDC) assay: Murine BMDCs were generated from bone marrow cells with GM-CSF and IL-4 for 6 days; cells were treated with HG-9-91-01 (5–50 nM) and LPS (1 μg/mL) for 48 hours; PD-L1 expression was detected by flow cytometry, IL-12/IL-6 levels were measured by ELISA, and DCs were co-cultured with naive CD4+ T cells for 5 days to assess Treg differentiation (CD4+Foxp3+) via flow cytometry [2]
3. Primary hepatocyte assay: Primary mouse hepatocytes were isolated by collagenase perfusion, plated on collagen-coated plates, and treated with HG-9-91-01 (10–100 nM) and glucagon (10 nM) for 16 hours; qPCR was used to quantify PEPCK/G6Pase mRNA, glucose release was measured by a glucose assay kit, and CRTC2 subcellular localization was detected by immunofluorescence [3]
4. Cell viability assay: BMDMs, BMDCs, and primary hepatocytes were seeded in 96-well plates and treated with HG-9-91-01 (0.1 nM–1 μM) for 24–48 hours; CCK-8 reagent was added, and absorbance at 450 nm was measured to assess cell viability [1][2][3]

1. LPS-challenged inflammation model: 8–10-week-old C57BL/6 mice were randomized into vehicle and HG-9-91-01 groups (n=8 per group); HG-9-91-01 was dissolved in 10% DMSO, 40% PEG400, and 50% normal saline, and administered intraperitoneally at 5 mg/kg 1 hour before LPS (5 mg/kg, i.p.); serum was collected 6 hours post-LPS for cytokine ELISA, and liver/spleen tissues were harvested for flow cytometry analysis of macrophage subsets [1]
2. EAE autoimmune model: C57BL/6 mice were immunized with MOG35-55 peptide in complete Freund's adjuvant (CFA) to induce EAE; HG-9-91-01 was dissolved in 0.5% CMC-Na solution and administered orally at 10 mg/kg once daily for 21 days starting from immunization; clinical severity was scored daily (0–4 scale), and spinal cord tissues were collected for H&E (inflammation) and Luxol fast blue (demyelination) staining [2]
3. HFD-induced metabolic model: C57BL/6 mice were fed a 60% kcal fat diet for 12 weeks to induce hyperglycemia; mice were randomized into vehicle and HG-9-91-01 groups (n=10 per group); HG-9-91-01 was administered intraperitoneally at 3 mg/kg twice daily for 14 days; fasting blood glucose was measured weekly, GTT was performed at study end, and liver tissues were collected for qPCR and glucose production assays [3]

1. Acute toxicity: HG-9-91-01 was well tolerated in mice at intraperitoneal injection doses up to 50 mg/kg and oral doses up to 100 mg/kg, with no deaths or serious clinical symptoms (weight loss, lethargy) observed [1][2][3] 2. Subchronic toxicity: In a 14-day mouse study, intraperitoneal injection of HG-9-91-01 (3, 5, 10 mg/kg/day) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine) [3] 3. Plasma protein binding: HG-9-91-01 has a plasma protein binding rate of approximately 90% in human plasma and 88% in mouse plasma (ultrafiltration method) [2] 4. Organ toxicity: Histological analysis of liver, kidney and spleen tissues from mice treated with HG-9-91-01 showed no signs of inflammation, necrosis or fibrosis [1][3]

[1]. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated andregulatory macrophages. Proc Natl Acad Sci U S A. 2012 Oct 16;109(42):16986-91.

[2]. Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells. Proc Natl Acad Sci U S A. 2014 Aug 26;111(34):12468-73.

[3]. The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver. Nat Commun. 2014 Aug 4;5:4535.

HG-9-91-01 belongs to the phenylurea class of compounds and is a potent inhibitor of salt-induced kinase 2 (Skin-induced kinase 2), a potential target protein for ovarian cancer treatment. It can be used both as an anti-tumor drug and as a Skin-induced kinase 2 inhibitor. It is a dimethoxybenzene, aminopyrimidine, N-arylpiperazine, N-alkylpiperazine, secondary amino compound belonging to the phenylurea class of compounds.

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1. HG-9-91-01 is a first-generation small molecule SIK inhibitor used as a research tool compound to study the biological function of the SIK-CRTC signaling pathway[1][2][3]
2. HG-9-91-01's mechanism of action involves competitive binding to the ATP-binding pocket of SIK, inhibiting its kinase activity and subsequent phosphorylation of CRTC2/CRTC3, thereby regulating gene expression in macrophage polarization, DC immune regulation, and hepatic gluconeogenesis[1][2][3]
3. HG-9-91-01 is a preclinical tool compound and has not yet been developed for clinical use; there are no clinical trial reports or FDA warning information reports[1][2][3]
4. HG-9-91-01 has potential therapeutic implications for inflammatory/autoimmune diseases (e.g., multiple sclerosis) and metabolic diseases (e.g., type 2 diabetes) by targeting the SIK-CRTC pathway [2][3]

C32H37N7O3

567.6813

567.295

1456858-58-4

78357808

White to yellow solid powder

1.2±0.1 g/cm3

779.7±70.0 °C at 760 mmHg

425.3±35.7 °C

0.0±2.8 mmHg at 25°C

1.630

4.57

2

8

8

42

825

0

UYUHRKLITDJEHB-UHFFFAOYSA-N

InChI=1S/C32H37N7O3/c1-22-7-6-8-23(2)31(22)36-32(40)39(27-14-13-26(41-4)19-28(27)42-5)30-20-29(33-21-34-30)35-24-9-11-25(12-10-24)38-17-15-37(3)16-18-38/h6-14,19-21H,15-18H2,1-5H3,(H,36,40)(H,33,34,35)

1-(2,4-dimethoxyphenyl)-3-(2,6-dimethylphenyl)-1-[6-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]urea

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 : ≥ 150 mg/mL (~264.23 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.40 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 2: ≥ 2.08 mg/mL (3.66 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 3: ≥ 2.08 mg/mL (3.66 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.)