HDAC; SFN-NAC acts as an orally active histone deacetylase (HDAC) inhibitor . It activates the Nrf2 (Nuclear factor erythroid 2-related factor 2) pathway by disrupting the Keap1-Nrf2 complex, leading to the transcriptional activation of antioxidant response element (ARE)-driven genes . Additionally, SFN-NAC modulates ERK1/2 signaling (specifically activating phosphorylation at Thr202/Tyr204), leading to the downregulation of α-tubulin and stathmin-1, as well as the upregulation of Hsp70 . The compound also induces caspase activation and PARP inactivation .

SFN-NAC (24 h) reduces cell viability with IC50 values of 60.08 μM, 35.20 μM, 39.11 μM, and 36.20 μM for HA cells, U87MG cells, U373MG cells, and U87/TR cells, respectively[1]. Cell Cycle Analysis[1] Cell Line: U87MG and U373MG cells Concentration: 0, 30 μM Incubation Time: 24 h Result: Induced cell-cycle arrest in the G2/M phase and triggered apoptosis at the same time.

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SFN-NAC induced cell-cycle arrest in the G2/M phase and dose-dependently induced intracellular ERK activation, autophagy, and α-tubulin downregulation. These SFN-NAC-induced effects were reversed by inhibiting the ERK pathway with its inhibitor PD98059. U87MG and U373MG cells were transfected with LC3 small interfering RNA, and the subsequent inhibition of autophagy reversed the downregulation of α-tubulin by SFN-NAC. Furthermore, co-immunoprecipitation experiments and confocal microscopy confirmed that SFN-NAC promotes the binding of LC3 with α-tubulin in the cytoplasm. Cell viability experiments demonstrate that SFN-NAC inhibits the growth of U87MG and U373MG cell colonies. [1]

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SFN-NAC exhibits potent anti-proliferative activity across multiple cancer cell lines in a dose-dependent manner. Following 24 hours of treatment, the IC₅₀ values are: 60.08 μM for HA glioblastoma cells, 35.20 μM for U87MG glioblastoma cells, 39.11 μM for U373MG glioblastoma cells, and 36.20 μM for temozolomide-resistant U87/TR glioblastoma cells . In U87MG and U373MG cells, treatment with 30 μM SFN-NAC for 24 hours induces significant G2/M phase cell cycle arrest and triggers apoptosis . Western blot analysis reveals that within a concentration range of 10 to 50 μM, SFN-NAC activates ERK1/2 phosphorylation, downregulates α-tubulin expression, and induces autophagy in a dose-dependent manner . In HepG2-C8 cells, SFN-NAC at 75 μM increases ARE expression, though its potency on ARE-related gene expression is roughly 8-fold less than sulforaphane . In MRC-5 fibroblast cells, SFN-NAC decreases the levels of TGF-β1-induced fibronectin, alpha-smooth muscle actin (α-SMA), and collagen, which are major mediators of fibrosis .

SFN-NAC (10 μmol; 6 h; oral gavage; single dose) significantly inhibited HDAC activity in the colonic mucosa of mice[2].
In mice given a single oral dose of 10 μmol SFN, or 10 μmol of the metabolite SFN–N-acetylcysteine (SFN–NAC), HDAC activity was inhibited significantly in the colonic mucosa at 6 h (Fig. 3). In a longer-term study [19], Apcmin mice ingested ∼6 μmol SFN/day for 70 days, and this resulted in significant inhibition of spontaneous intestinal polyps, compared with controls fed AIN93 diet alone (Fig. 3 center). There was a concomitant increase in global histone H3 and H4 acetylation, and chromatin immunoprecipitation assays performed on mouse colon and intestinal tissues revealed an increase in acetylated histones associated with the promoter region of the P21 gene (Fig. 3, right), as well as bax [19]. Collectively, these findings supported a role for SFN as an HDAC inhibitor in vivo, with evidence for decreased HDAC activity in various tissues and increased global as well as local histone acetylation.[2]

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In an HDAC inhibition model, oral administration of SFN-NAC (10 μmol, single dose by oral gavage) for 6 hours significantly inhibited histone deacetylase (HDAC) activity in the colon mucosa of mice . The compound is orally active and demonstrates better blood-brain barrier permeability compared to its parent compound sulforaphane . Both SFN and SFN-NAC have shown anti-pulmonary fibrotic effects by decreasing key fibrosis mediators, though specific in vivo fibrosis model details are described in the literature .

The HDAC inhibitory activity of SFN-NAC was evaluated in vivo using a mouse model. Mice were administered a single oral dose of SFN-NAC (10 μmol) by gavage. After 6 hours of treatment, the colon mucosa was harvested and analyzed for histone deacetylase (HDAC) activity, which was found to be significantly inhibited compared to control groups . The Nrf2-activating mechanism involves the electrophilic reaction of SFN-NAC with sulfhydryl groups of specific cysteine residues on Keap1, leading to a conformational change that disrupts the Keap1-Nrf2 complex, allowing Nrf2 to translocate to the nucleus and bind to the Antioxidant Response Element (ARE) .

Western Blot Analysis[1]
Cell Types: U87MG and U373MG
Tested Concentrations: 0, 10, 20, 30, 40, 50μM
Incubation Duration: 24 h
Experimental Results: Activated ERK1/2 (Thr202/Tyr204), downregulated α-tubulin, and induced autophagy in a dose-dependent manner.

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Cell Viability Assay[1]
Cell Types: HA, U87MG, U373MG and U87/TR
Tested Concentrations: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 μM for HA and U87MG cells or 0, 10, 20, 30, 40, 50, 60 μM for U373MG and U87/TR cells
Incubation Duration: 24 h
Experimental Results: Decreased the cell viability of these cell lines in a dose-dependent manner.

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Cell Viability Assay: Cells (HA, U87MG, U373MG, U87/TR) were seeded and treated with varying concentrations of SFN-NAC (0-90 μM) for 24 hours. Cell viability was assessed using standard assays (e.g., MTT or CCK-8). The IC₅₀ values were calculated as 60.08 μM (HA), 35.20 μM (U87MG), 39.11 μM (U373MG), and 36.20 μM (U87/TR) . Cell Cycle Analysis: U87MG and U373MG cells were treated with 30 μM SFN-NAC for 24 hours, then fixed, stained with propidium iodide, and analyzed by flow cytometry, revealing G2/M phase arrest and increased apoptosis . Western Blot Analysis: U87MG and U373MG cells were treated with SFN-NAC (0, 10, 20, 30, 40, 50 μM) for 24 hours. Protein lysates were analyzed using antibodies against ERK1/2 (phosphorylated at Thr202/Tyr204), α-tubulin, Hsp70, and stathmin-1, showing dose-dependent activation of ERK1/2, downregulation of α-tubulin, and induction of autophagy . ARE Reporter Assay: HepG2-C8 cells were treated with 75 μM SFN-NAC, and ARE-driven gene expression was measured, showing increased ARE activity approximately 8-fold less potent than SFN . Anti-Fibrosis Assay: MRC-5 fibroblast cells were stimulated with TGF-β1 and treated with SFN-NAC, followed by measurement of fibronectin, α-SMA, and collagen levels, all of which were decreased .

Animal/Disease Models:mice[2]
Doses: 10 μmol
Route of Administration: Oral gavage; 6 h; single dose
Experimental Results: Significantly inhibited HDAC activity in mouse colon mucosa.

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In the HDAC inhibition study, mice were administered SFN-NAC orally by gavage at a dose of 10 μmol (single dose). After 6 hours of treatment, the animals were sacrificed, and colon mucosa was collected for HDAC activity analysis . For pharmacokinetic studies, Sprague-Dawley rats were used. Intravenous administration was performed at a dose of 0.1 mg/kg, and oral (peroral) administration was performed at a dose of 0.5 mg/kg. Blood samples were collected at various time points post-administration for plasma concentration analysis .

The pharmacokinetic parameters of SFN-NAC have been characterized in rat models . Following intravenous administration (0.1 mg/kg): AUClast = 2.11 ± 0.502 μg·min/mL; AUCinf = 2.16 ± 0.515 μg·min/mL; elimination half-life (t₁/₂) = 33.2 ± 14.2 minutes; mean residence time (MRT) = 15.8 ± 7.7 minutes; volume of distribution at steady-state (Vss) = 711 ± 262 mL/kg; total body clearance (CL) = 48.3 ± 10.5 mL/min/kg . Following oral administration (0.5 mg/kg): Cmax = 0.006 ± 0.003 μg/mL; oral bioavailability (F%) = >24.8% . SFN-NAC is metabolically unstable, particularly in plasma, where it degrades considerably faster than SFN, with SFN being formed from SFN-NAC as a metabolite . The compound demonstrates better blood-brain barrier permeability compared to sulforaphane .

No direct toxicity data (such as LD₅₀, hepatotoxicity, nephrotoxicity) for SFN-NAC were reported in the available literature. However, the compound is classified for research use only and is not intended for human or veterinary use . Cell viability assays indicate that SFN-NAC has a dose-dependent cytotoxic effect on cancer cell lines, with IC₅₀ values ranging from approximately 35 to 60 μM, while normal cell toxicity profiles are not specified in the provided references . For storage, SFN-NAC should be kept at -20°C under desiccated conditions, and stock solutions are recommended to be stored as aliquots in tightly sealed vials at -20°C for up to one month .

[1]. Sulforaphane-N-Acetyl-Cysteine Induces Autophagy Through Activation of ERK1/2 in U87MG and U373MG Cells. Cell Physiol Biochem. 2018;51(2):528-542.

[2]. Dietary histone deacetylase inhibitors: from cells to mice to man[C]//Seminars in cancer biology. Academic Press, 2007, 17(5): 363-369.

Background/Objective: Sulforaphane-N-acetylcysteine (SFN-NAC) is a metabolite of sulforaphane (SFN), with a longer half-life and better blood-brain barrier permeability than SFN. Previous studies have found that SFN-NAC can disrupt microtubules through the ERK pathway and inhibit the growth of lung cancer cells. However, its underlying mechanism is still unclear, and it is unknown whether SFN-NAC can inhibit the growth of glioma. This study is the first to confirm that SFN-NAC can activate autophagy-mediated downregulation of α-tubulin expression through the ERK pathway. Methods: This study used two widely used glioma cell lines—U87MG and U373MG cells. The effects of SFN-NAC on α-tubulin and its interaction with microtubule-associated protein 1 light chain 3 (LC3) were analyzed by apoptosis detection, Western blot analysis, immunoprecipitation, immunostaining and electron microscopy. [1] Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli and broccoli sprouts. This anticancer substance was initially identified as a potent inducer of phase II detoxification enzymes, but mounting evidence suggests that SFN also functions through epigenetic mechanisms. Studies have shown that SFN can inhibit histone deacetylase (HDAC) activity in human colon and prostate cancer cell lines and increase global and local histone acetylation levels, such as acetylation levels in the promoter regions of the P21 and Bax genes. SFN can also inhibit the growth of prostate cancer xenografts and spontaneous intestinal polyps in mouse models, and there is evidence that it alters histone acetylation and HDAC activity in vivo. In human trials, HDAC activity in circulating peripheral blood mononuclear cells was inhibited within 3–6 hours after a single intake of 68 grams of broccoli sprouts, while histone H3 and H4 acetylation levels increased. These findings suggest that one of the cancer chemopreventive mechanisms of SFN is through the inhibition of epigenetic alterations associated with HDAC activity. Other dietary components, such as butyrate, biotin, lipoic acid, organosulfur compounds from garlic, and vitamin E metabolites, also exhibit structural features consistent with HDAC inhibition. Dietary compounds can relieve the suppression of epigenetically silent genes in cancer cells and activate these genes in normal cells, which is of great significance for the prevention and treatment of cancer. In a broader sense, people are increasingly interested in dietary HDAC inhibitors and their effects on the epigenetic mechanisms of other chronic diseases such as cardiovascular disease, neurodegenerative diseases and aging. [2]

C11H20N2O4S3

340.48

340.058

334829-66-2

71772353

White to off-white solid powder

1.4±0.1 g/cm3

106-108ºC

1.608

-0.33

3

7

10

20

377

1

CC(N[C@@H](CSC(NCCCCS(C)=O)=S)C(O)=O)=O

IIHBKTCHILXGOT-KMYGYIBBSA-N

InChI=1S/C11H20N2O4S3/c1-8(14)13-9(10(15)16)7-19-11(18)12-5-3-4-6-20(2)17/h9H,3-7H2,1-2H3,(H,12,18)(H,13,14)(H,15,16)/t9-,20?/m0/s1

(2R)-2-acetamido-3-(4-methylsulfinylbutylcarbamothioylsulfanyl)propanoic acid

SFN-NAC; DL-Sulforaphane N-acetyl-L-cysteine; 334829-66-2; D,L-Sulforaphane N-Acetyl-L-cysteine; CHEMBL4245297; (2R)-2-acetamido-3-(4-methylsulfinylbutylcarbamothioylsulfanyl)propanoic acid;

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 : 10 mg/mL (29.37 mM; with sonication and heat)

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.)