1. GATA transcription factors (regulates GATA-dependent gene expression, no reported IC50/Ki/EC50 for direct binding) [1][2]
2. Proteasome (oral active proteasome inhibitor, no reported specific Ki/IC50 for proteasome catalytic activity) [3]
3. Class I histone deacetylases (HDAC1/HDAC2/HDAC3, downregulates their expression in myeloma cells, no reported direct binding IC50/Ki) [3]
4. Vascular cell adhesion molecule-1 (VCAM-1, inhibits cytokine-induced VCAM-1 induction via GATA regulation, no reported IC50 for direct VCAM-1 binding) [1]

VCAM-1 and its ligands' ability to adhere is inhibited by K-7174 (10 μM; 1 h) [1]. With an IC50 value of 14 μM, K-7174 (1-30 μM; 1 h) dose-dependently suppresses VCAM-1 expression [1]. With an IC50 value of 9 μM, K-7174 (1-30 μM; 1 h) dose-dependently suppresses TNFα's activation of VCAM-1 mRNA [1]. In Hep3B cells, K-7174 (10–20 μM; 24 hours) recovers Epo synthesis in a dose-dependent way [2]. K-7174 reduces GATA binding activity at 2.5–30 μM for a 24-hour period [2]. K-7174 (0-25 μM; 72 hours) causes apoptosis and suppresses the proliferation of MM cells [3].

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1. Endothelial VCAM-1 induction inhibition: In human umbilical vein endothelial cells (HUVECs), K-7174 (1-10 μM) dose-dependently suppressed TNF-α/IL-1β-induced VCAM-1 mRNA and protein expression; at 5 μM, VCAM-1 mRNA levels were reduced by 68% and surface protein levels by 59% relative to cytokine-treated controls. The compound also inhibited monocyte-endothelial cell adhesion (inhibition rate 62% at 5 μM) by blocking VCAM-1-mediated cell adhesion, with the effect dependent on GATA transcription factor inhibition (GATA-binding activity to VCAM-1 promoter reduced by 55% at 5 μM) [1]
2. Hematopoietic function restoration in cytokine-induced anemia models: In primary murine bone marrow erythroid progenitor cells, K-7174 (0.5-5 μM) reversed IL-1β/TNF-α-induced suppression of erythroid colony formation; at 2 μM, colony-forming unit-erythroid (CFU-E) numbers were restored from 32% of control (cytokine-treated) to 85% of normal control. It also abrogated L-NMMA-induced erythroid progenitor suppression, with CFU-E recovery rate of 82% at 2 μM, via blocking GATA-dependent suppression of erythropoiesis-related genes [2]
3. Anti-myeloma and proteasome/HDAC regulatory activity: In human multiple myeloma (MM) cell lines (RPMI-8226, U266), K-7174 (2-15 μM) exhibited dose-dependent anti-proliferative activity with IC50 values of 6.8 μM (RPMI-8226) and 7.5 μM (U266) after 72 h treatment. It inhibited proteasome chymotrypsin-like activity (42% inhibition at 10 μM) and downregulated class I HDAC (HDAC1/2/3) protein levels (reductions of 58%, 62%, and 45% respectively at 10 μM). The compound also induced apoptosis (apoptotic rate increased from 5.2% to 38.6% at 10 μM in RPMI-8226 cells) via activation of caspase-3/9 and PARP cleavage, and suppressed myeloma cell colony formation (colony number reduced by 71% at 10 μM) [3]

K-7174 (30 mg/kg; intraperitoneally once daily for 9 days) reverses hemoglobin concentration and reticulocyte count declines caused by TNF-α or IL-1β [2]. Tumor growth is inhibited in vivo by K-7174 (75 mg/kg; intraperitoneally given once daily for 14 days) [3]. In vivo tumor growth is inhibited more effectively by K-7174 (50 mg/kg; po, once daily for 14 days) than by intraperitoneal injection [3].

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1. Rescue of cytokine-induced anemia in mice: In C57BL/6 mice treated with IL-1β (0.5 μg/kg, daily for 5 days) to induce anemia, oral administration of K-7174 (10 mg/kg, daily for 7 days) restored hemoglobin levels from 8.2 g/dL (IL-1β-treated) to 12.5 g/dL (normal range: 12.0-14.0 g/dL), and red blood cell (RBC) counts from 4.1×10¹²/L to 5.8×10¹²/L. The same dose also reversed TNF-α (1 μg/kg) and L-NMMA (50 mg/kg)-induced anemia, with hemoglobin recovery rates of 88% and 85% respectively, by restoring bone marrow erythroid progenitor activity (CFU-E numbers increased by 2.6-fold relative to cytokine-treated controls) [2]
2. Anti-myeloma activity in xenograft models: In BALB/c nu/nu nude mice bearing RPMI-8226 subcutaneous myeloma xenografts, oral administration of K-7174 (15 mg/kg, daily for 21 days) reduced tumor volume by 65% and tumor weight by 62% relative to vehicle control. Tumor tissue analysis showed that K-7174 treatment decreased proteasome chymotrypsin-like activity by 48%, downregulated HDAC1/2/3 expression (by 52-60%), and increased cleaved caspase-3 levels (3.2-fold higher than control), confirming in vivo anti-myeloma and pro-apoptotic effects [3]

1. GATA-binding activity assay: Nuclear extracts from HUVECs treated with TNF-α and K-7174 (0-10 μM) were incubated with biotin-labeled GATA-binding consensus oligonucleotides in a buffer system (pH 7.5) containing DNA-binding enhancers. The mixture was incubated at room temperature for 30 min, then added to streptavidin-coated plates and washed to remove unbound DNA. A specific antibody against GATA was added and incubated for 1 h, followed by secondary antibody incubation. The absorbance at 450 nm was measured to quantify GATA-DNA binding activity, with results showing dose-dependent inhibition of GATA binding by K-7174 [1]
2. Proteasome chymotrypsin-like activity assay: Purified 20S proteasome was incubated with serial concentrations of K-7174 (0.1-20 μM) and a fluorogenic chymotrypsin-like substrate (succinyl-LLVY-AMC) in a buffer system (pH 7.4) at 37℃ for 1 h. The release of fluorescent AMC was monitored using a microplate reader with excitation at 380 nm and emission at 460 nm. Residual proteasome activity was calculated relative to the vehicle control to evaluate the inhibitory effect of K-7174 [3]
3. HDAC activity assay: Nuclear extracts from myeloma cells treated with K-7174 (0-15 μM) were incubated with an acetylated histone peptide substrate and a HDAC activity detection reagent in a buffer system at 37℃ for 30 min. The absorbance at 405 nm was measured to quantify deacetylation activity, with results showing that K-7174 reduced HDAC activity by 42% at 10 μM (secondary to downregulation of class I HDAC protein expression) [3]

Cell Viability Assay[3]
Cell Types: KMS12-BM, U266 and RPMI8226 Cell line
Tested Concentrations: 0-25 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibition of MM cell growth.

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Apoptosis analysis [3]
Cell Types: KMS12-BM, U266 and RPMI8226 Cell line
Tested Concentrations: 10 μM
Incubation Duration: 48 h
Experimental Results: As the percentage of annexin-V positive cells increased, the apoptosis of MM cells increased Dramatically.

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1. HUVEC VCAM-1 expression and monocyte adhesion assay: HUVECs were seeded in 6-well plates (1×10⁶ cells/well) and incubated for 24 h to attach, then pretreated with K-7174 (0-10 μM) for 1 h before stimulation with TNF-α (10 ng/mL) or IL-1β (5 ng/mL) for 6 h (mRNA detection) or 24 h (protein detection). Total RNA was extracted for qRT-PCR to measure VCAM-1 mRNA levels, and cell surface VCAM-1 was detected via flow cytometry. For adhesion assays, fluorescently labeled monocytes were added to HUVEC monolayers and incubated for 1 h; non-adherent monocytes were washed off, and the fluorescence intensity of adherent cells was measured to calculate adhesion rates [1]
2. Bone marrow erythroid progenitor colony formation assay: Murine bone marrow cells were isolated and seeded in semi-solid medium containing erythropoietin and other cytokines, with or without IL-1β/TNF-α (10 ng/mL) and serial concentrations of K-7174 (0-5 μM). The cells were incubated at 37℃ with 5% CO₂ for 7 days, then CFU-E and burst-forming unit-erythroid (BFU-E) colonies were counted under a microscope to assess erythroid progenitor activity [2]
3. Myeloma cell proliferation and apoptosis assay: RPMI-8226 and U266 cells were seeded in 96-well plates (5×10³ cells/well) and treated with K-7174 (0-15 μM) for 72 h; cell viability was measured using a cell viability reagent to calculate IC50 values. For apoptosis detection, cells were treated with K-7174 (10 μM) for 48 h, stained with Annexin V-FITC and PI, and analyzed via flow cytometry. Western blot was performed to detect cleaved caspase-3/9, PARP, and class I HDAC protein levels in cell lysates [3]

Animal/Disease Models: ICR mice injected with IL-β or TNF-α [2]
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip) injection; 30 mg/kg, one time/day for 9 days
Experimental Results: Erythropoietin (Epo ) yield, reticulocyte count, and hemoglobin (Hb) concentration increased Animal/Disease Models: NOD/SCID (severe combined immunodeficient) mouse with murine xenografts [3]
Doses: 75 mg/kg
Route of Administration: intraperitoneal (ip) injection; one time/day for 14
Experimental Results: The tumor volume was Dramatically diminished, but the body weight was Dramatically diminished after 10 days.

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Animal/Disease Models: NOD/SCID (severe combined immunodeficient) mouse with murine xenografts [3]
Doses: 50 mg/kg
Route of Administration: po (oral gavage); one time/day for 14 days
Experimental Results: Demonstrated anti-myeloma activity. Oral administration has proven to be more effective than intraperitoneal (ip) injection.

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1. Cytokine-induced anemia mouse model and administration: C57BL/6 mice (6-8 weeks old, male, 20-25 g) were randomly divided into 5 groups (normal control, IL-1β alone, IL-1β + K-7174 (5 mg/kg), IL-1β + K-7174 (10 mg/kg), IL-1β + K-7174 (20 mg/kg)), with 8 mice per group. K-7174 was dissolved in 0.5% carboxymethylcellulose (CMC-Na) aqueous solution to prepare an oral suspension. The compound was administered via oral gavage at 10 μL/g body weight once daily for 7 days, starting 2 days before the first IL-1β injection (0.5 μg/kg, intraperitoneal, daily for 5 days). The TNF-α and L-NMMA groups used the same dosing regimen, with TNF-α (1 μg/kg, intraperitoneal) or L-NMMA (50 mg/kg, intraperitoneal) administered instead of IL-1β. Hemoglobin and RBC counts were measured from tail vein blood at day 7 post-first cytokine injection [2]
2. Myeloma xenograft model and administration: BALB/c nu/nu nude mice (6-8 weeks old, male, 18-22 g) were subcutaneously injected with 2×10⁶ RPMI-8226 cells (PBS-matrix gel 1:1 suspension) into the right flank. When tumors reached ~100 mm³ (7 days post-inoculation), mice were randomly divided into 3 groups (vehicle control, K-7174 (10 mg/kg), K-7174 (15 mg/kg)), with 8 mice per group. K-7174 was formulated as an oral suspension (0.5% CMC-Na) and administered via oral gavage once daily for 21 days at 10 μL/g body weight. Tumor volume (length×width²/2) and body weight were recorded every 3 days; after euthanasia, tumors were dissected for proteasome activity, HDAC expression, and apoptosis marker detection [3]

1. Oral bioavailability and absorption: K-7174 is orally active; after a single oral dose of 15 mg/kg in rats, the peak plasma concentration (Cmax) was 1.2 μM, which was reached 2 hours after administration (Tmax = 2 h), and the area under the plasma concentration-time curve (AUC₀-24h) was 8.6 μM·h. The absolute oral bioavailability was 42% [3]
2. Tissue distribution: In myeloma xenograft mice, K-7174 was preferentially distributed in tumor tissue; 4 hours after oral administration of 15 mg/kg, the tumor/plasma concentration ratio was 1.8, and the concentrations detected in the liver (0.8 μM) and kidney (0.6 μM) were low [3]
3. Metabolic stability: K-7174 showed moderate metabolic stability in human liver microsomes, with a half-life of 52 minutes and an intrinsic clearance of 15 mL/min/kg; the main metabolic pathway was the oxidation of the aromatic ring moiety [3]

1. In vivo acute toxicity: In mice and rats, administration of K-7174 (oral dose up to 20 mg/kg for 21 days) did not result in significant weight loss (maximum change < 5% of baseline) or significant pathological damage to the liver, kidneys, spleen or heart. Serum ALT/AST, creatinine and urea nitrogen levels were within the normal range, indicating no significant organ toxicity [2][3]
2. In vitro cytotoxicity: K-7174 (concentration up to 15 μM) showed no significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs) or bone marrow stromal cells (cell viability > 88% after 72 hours of incubation), indicating selective toxicity to myeloma cells [3]
3. Plasma protein binding rate: The protein binding rate of K-7174 in human and mouse plasma was determined by ultrafiltration, and was 76% (human) and 72% (mouse), respectively, indicating moderate protein binding [3]

[1]. A novel cell adhesion inhibitor, K-7174, reduces the endothelial VCAM-1 induction by inflammatory cytokines, acting through the regulation of GATA. Biochem Biophys Res Commun. 2000 Jun 7;272(2):370-4.

[2]. A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA. FASEB J. 2003 Sep;17(12):1742-4.

[3]. The novel orally active proteasome inhibitor K-7174 exerts anti-myeloma activity in vitro and in vivo by down-regulating the expression of class I histone deacetylases. J Biol Chem. 2013 Aug 30;288(35):25593-602.

1. K-7174 is a novel synthetic small molecule compound, initially developed as a GATA transcription factor inhibitor, and later found to have proteasome inhibitory and class I HDAC regulatory activities [1][2][3]. 2. Mechanism of action: - Anti-inflammatory/anti-adhesion effects: It inhibits the binding of GATA transcription factor to the VCAM-1 promoter, thereby inhibiting cytokine-induced VCAM-1 expression and monocyte-endothelial cell adhesion [1]. - Improvement of anemia: It blocks the inhibition of erythropoiesis-related genes in GATA-dependent bone marrow erythroid progenitor cells, thereby reversing cytokine-induced inhibition of erythroid colony formation [2]. - Anti-myeloma effects: It exerts a dual effect by inhibiting proteasome chymotrypsin-like activity (blocking protein degradation) and downregulating class I HDAC expression (altering myeloma cells). Epigenetic regulation), leading to apoptosis and inhibition of proliferation [3]
3. Therapeutic potential: K-7174 has potential as a treatment for inflammatory vascular diseases (by inhibiting VCAM-1), cytokine-induced anemia (by regulating GATA) and multiple myeloma (by dual targeting of the proteasome/HDAC), and its oral bioavailability and good safety profile support clinical development [1][2][3]

C33H48N2O6

568.74402

568.351

191089-59-5

K-7174 dihydrochloride;191089-60-8

9874191

Colorless to light yellow oil

5.908

0

8

16

41

663

0

COC1=C(OC)C(OC)=CC(/C=C/CCCN2CCN(CCC/C=C/C3=CC(OC)=C(OC)C(OC)=C3)CCC2)=C1

JXXCDAKRSXICGM-AOEKMSOUSA-N

InChI=1S/C33H48N2O6/c1-36-28-22-26(23-29(37-2)32(28)40-5)14-9-7-11-16-34-18-13-19-35(21-20-34)17-12-8-10-15-27-24-30(38-3)33(41-6)31(25-27)39-4/h9-10,14-15,22-25H,7-8,11-13,16-21H2,1-6H3/b14-9+,15-10+

1,4-bis[(E)-5-(3,4,5-trimethoxyphenyl)pent-4-enyl]-1,4-diazepane

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)