Nurr1
Nurr1 (NR4A2, orphan nuclear receptor). C-DIM12 activates Nurr1 and enhances its transcriptional activity. No IC50, Ki, EC50, or DC50 values are provided in these manuscripts [1,2,3].

C-DIM12 induces Nurr1 and DA gene expression in cell lines and primary neurons[3]. In vitro, C-DIM12 inhibits astrocyte inflammatory signaling. Nitric oxide synthase (NOS2), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) are NF-B-regulated genes that are inhibited by lipopolysaccharide (LPS) in BV-2 microglia. Nurr1-RNA interference attenuated these effects. Additionally, C-DIM12 increased nuclear translocation of Nurr1 primary microglia while decreasing NF-B activation in NF-B-GFP reporter cells. C-DIM12 increases Nurr1 binding to the p65-binding site while decreasing lipopolysaccharide-induced p65 binding to the NOS2 promoter. Additionally, CoREST (Corepressor for Repressor Element 1 Silencing Transcription Factor) and NCOR2 (Nuclear Receptor Corepressor 2) binding was stabilized by C-DIM12[2].

---

In BV-2 microglial cells, C-DIM12 (10 μM) suppressed LPS-induced mRNA expression of NF-κB-regulated inflammatory genes including NOS2, TNFα, IL-6, CCL5, and CCL2 in a dose-dependent manner (0.1, 1, 10 μM). Knockdown of Nurr1 by RNAi abolished this suppression, indicating a Nurr1-dependent mechanism [2].
In NF-κB/GFP HEK293 reporter cells, C-DIM12 (100 μM) significantly reduced TNFα (30 ng/ml)-induced NF-κB/GFP expression, comparable to the positive control Bay-11 (50 μM) [2].
C-DIM12 did not prevent nuclear translocation of p65 in BV-2 microglia treated with LPS; rather, it enhanced nuclear levels of p65. However, it decreased LPS-induced phosphorylation of p65 at serine (trend) [2].
ChIP assays in BV-2 cells showed that C-DIM12 (10 μM) reduced p65 binding to the NOS2 promoter, increased Nurr1 recruitment to the p65 binding site, and enhanced binding of corepressors NCOR2 and CoREST to the NOS2 promoter [2].
In primary mouse microglia, C-DIM12 (10 μM) with LPS (1 μg/ml) for 24 hours increased nuclear translocation of Nurr1 [2].
In N2A and N27 dopaminergic neuronal cell lines, C-DIM12 (5-10 μM) induced time- and dose-dependent increases in mRNA expression of Nurr1, tyrosine hydroxylase (TH), and vesicular monoamine transporter 2 (VMAT2). Knockdown of Nurr1 by RNAi abolished these effects, indicating a Nurr1-dependent mechanism [3].
In N2A cells transfected with FLAG-tagged human Nurr1, C-DIM12 (10 μM, 24 h) increased nuclear fluorescence of FLAG and Nurr1 [3].
In primary mouse dopaminergic neurons, C-DIM12 (10 μM, 24 h) increased expression of Nurr1 and TH in TH-positive neurons [3].
In differentiated MN9D and undifferentiated N2A cells, C-DIM12 (10 μM) co-treatment with 6-OHDA (100 μM for MN9D, 10 μM for N2A) significantly increased cell viability compared to 6-OHDA alone (P<0.05 for N2A, P<0.01 for MN9D) [3].

C-DIM12 has the neuroprotective activity in MPTPp-treated mice[2].
In a mouse model of Parkinson's disease using MPTP and probenecid (MPTPp), C-DIM12 (50 mg/kg, oral gavage daily for 7 days, administered after 7 days of MPTPp) significantly protected against loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra pars compacta (SNpc) and prevented further loss of TH fiber density in the striatum compared to MPTPp14d vehicle controls. Stereological counts showed significant preservation of TH+ neurons [1].
C-DIM12 treatment attenuated MPTPp-induced activation of microglia (IBA-1+) and astrocytes (GFAP+) in the SN and striatum, reducing the number of activated glial cells [1].
C-DIM12 decreased MPTPp-induced expression of NF-κB/EGFP reporter in the SN of transgenic NF-κB/EGFP mice, indicating suppression of NF-κB activity. Co-localization analysis showed reduced EGFP in both astrocytes and microglia [1].
C-DIM12 prevented MPTPp-induced neuronal death as measured by MAP2+ neuron counts and reduced nitrotyrosine adduct formation and γH2AX (DNA damage marker) in TH+ neurons [1].
C-DIM12 preserved striatal TH, DAT, and VMAT2 protein levels in MPTPp-treated mice and improved hind-limb stride length (a measure of motor function) [1].
C-DIM12 increased Nurr1 expression and promoted nuclear localization of Nurr1 in TH+ neurons of the SNpc, even at 21 days after MPTPp treatment [1].
Quantitative PCR array analysis of SN tissue showed that C-DIM12 reduced MPTPp-induced expression of NF-κB-regulated inflammatory genes including TNFα, IL-1α, CCL2, Caspase-1, and TLR4 [1].

For up to 24 hours, NF-B-GFP HEK cells are exposed to 30 ng/ml of TNF in the presence of 100 μM C-DIM12. C-DIM12 effectively inhibits NF-B-GFP expression in the NF-B-GFP HEK cells after TNF treatment, showing a statistically significant decrease in total GFP fluorescence per cell.

---

BV-2 microglia: Cells were pretreated with C-DIM12 (0.1-10 μM) for 1 hour in serum-free medium, then stimulated with LPS (1 μg/ml) for 24 hours. RNA was isolated and qPCR performed for inflammatory genes (NOS2, TNFα, IL-6, IL-1β, CCL2, CCL5). For RNAi, Nurr1 siRNA or DsiRNA was transfected 24 hours before treatment. For ChIP, cells were fixed with formaldehyde, chromatin sheared, immunoprecipitated with anti-p65, anti-Nurr1, anti-NCOR2, or anti-CoREST antibodies, and qPCR performed on NOS2 promoter region [2].
NF-κB/GFP HEK293 reporter cells: Cells were treated with TNFα (30 ng/ml) ± C-DIM12 (25-100 μM) or Bay-11 (30-50 μM) for 24 hours. GFP fluorescence was quantified and normalized to DAPI [2].
Immunofluorescence: BV-2 or primary microglia fixed with paraformaldehyde or methanol, stained with anti-p65, anti-Nurr1, anti-CD11b, DAPI, imaged by fluorescence or confocal microscopy. Nuclear fluorescence intensity quantified [2].
N2A and N27 cells: Treated with C-DIM12 (5-10 μM) for 4-24 hours, qPCR for Nurr1, TH, VMAT2. For RNAi, Nurr1 DsiRNA transfected 48 hours before treatment. For transfection, FLAG-Nurr1 plasmid transfected 24 hours before C-DIM12 treatment, then immunofluorescence for FLAG and Nurr1 [3].
Primary dopaminergic neurons: Isolated from E18 mouse ventral midbrain, cultured 1 week, treated with C-DIM12 (10 μM) or DMSO for 24 hours, then immunostained for TH and Nurr1, fluorescence intensity quantified [3].
Cell viability: MN9D (differentiated with sodium butyrate) or N2A cells treated with 6-OHDA ± C-DIM12 (10 μM) for 24 hours, viability measured by PrestoBlue reagent [3].

Transgenic NF-κB/EGFP reporter mice (C57Bl/6 background)
50 mg/kg
oral administration
IV, Oral gavage

---

MPTPp mouse model: Adult NF-κB/EGFP reporter mice (C57Bl/6) received probenecid (250 mg/kg, i.p.) followed 4 h later by MPTP (20 mg/kg, s.c.) every other day for 7 days (total MPTP 80 mg/kg). After day 7, C-DIM12 (50 mg/kg) dissolved in corn oil was administered once daily by oral gavage for 7 days (MPTPp14d group). Control groups received saline or corn oil vehicle. Mice were terminated at days 7 and 14. Tissues were perfused with 4% paraformaldehyde, brains sectioned at 40 μm for immunohistochemistry. Behavioral assessment: hind-limb stride length measured at day 0, 7, 14 using a plexiglass trackway and video recording [1].
For Western blot, striatal tissue was homogenized in RIPA buffer, proteins separated by SDS-PAGE, immunoblotted for TH, DAT, VMAT2, β-actin [1].
For qPCR array, RNA isolated from SN, cDNA synthesized, and RT-qPCR performed using NF-κB pathway-specific 384-well plate (PAMM-025Z) [1].

[1] Toxicol Sci . 2015 Feb;143(2):360-73.

[2] Mol Pharmacol . 2015 Jun;87(6):1021-34.

[3] Neurosci Lett . 2015 Oct 21;607:83-89.

C-DIM12 is a para-chlorophenyl substituted diindolylmethane (C-DIM) that activates the orphan nuclear receptor Nurr1 (NR4A2). It was selected for in vivo efficacy based on structure-activity relationship studies showing that smaller, more polar para substituents on the phenyl group enhance anti-inflammatory activity. C-DIM12 has a chlorine substituent at the para position of the phenyl ring. It is hypothesized to work by enhancing Nurr1-mediated transrepression of NF-κB-regulated inflammatory genes in glial cells, thereby protecting dopaminergic neurons from neuroinflammatory injury [1,2,3]. The compound also directly supports dopaminergic neuronal phenotype by inducing expression of TH and VMAT2 via Nurr1 [3].

C23H17CLN2

356.85

356.108

C, 77.41; H, 4.80; Cl, 9.93; N, 7.85

178946-89-9

5302583

Light Brown solid powder

1.3±0.1 g/cm3

585.6±45.0 °C at 760 mmHg

336.1±14.3 °C

0.0±1.6 mmHg at 25°C

1.747

6.13

2

0

3

26

443

0

ClC1C=CC(C(C2=CNC3=CC=CC=C23)C2=CNC3=CC=CC=C23)=CC=1

FIDOCHXHMJHKRW-VHQVDBNASA-N

InChI=1S/C33H45NO10/c1-17(35)44-33-21-19(14-31(38,28(42-6)26(33)36)27(21)43-29(37)18-10-8-7-9-11-18)32-20(40-4)12-13-30(16-39-3)15-34(2)25(32)22(33)23(41-5)24(30)32/h7-11,19-28,36,38H,12-16H2,1-6H3/t19-,20+,21-,22+,23+,24-,25?,26+,27-,28+,30+,31-,32+,33-/m1/s1

[(1S,2R,3R,4R,5R,6S,7S,8R,9R,13S,16S,17R,18R)-8-acetyloxy-5,7-dihydroxy-6,16,18-trimethoxy-13-(methoxymethyl)-11-methyl-11-azahexacyclo[7.7.2.12,5.01,10.03,8.013,17]nonadecan-4-yl] benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

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.)