HDAC2 ( IC50 = 119 pM ); HDAC6 ( IC50 = 434 nM )
CAY10683 (Santacruzamate A) targets HDAC1 (IC50 = 0.015 μM), HDAC2 (IC50 = 0.022 μM), HDAC3 (IC50 = 0.089 μM), HDAC4 (IC50 = 1.2 μM), HDAC6 (IC50 = 0.34 μM), HDAC8 (IC50 = 0.11 μM), and HDAC10 (IC50 = 0.27 μM) [1]
CAY10683 (Santacruzamate A) targets class I HDACs (HDAC1/2/3/8) and class II HDACs (HDAC4/6/10) [2]

Santacruzamate A (also known as CAY10683) is a potent and specific inhibitor of histone deacetylase (HDAC), showing >3600-fold selectivity over other HDACs with an IC50 of 119 pM for HDAC2. A clinically approved histone deacetylase (HDAC) inhibitor used to treat refractory cutaneous T-cell lymphoma, suberoylanilide hydroxamic acid [SAHA, trade name Vorinostat], and Santacruzamate A share several structural features. Santacruzamate A is isolated from cyanobacterium. Santacruzamate A selectively inhibits HDAC2, a Class I HDAC, at the picomolar level while only slightly inhibiting HDAC4 or HDAC6, two Class II HDACs. To further explore the role of PRMT5 and HDAC2 in CRC invasion and metastasis, researchers treated SW620 cells with inPRMT5 and inHDAC2 (CAY10683 (Santacruzamate A)). Both treatments reduced liver metastasis of CRC cells and completely inhibited the distant metastasis of SW620 cells in nude mice (Figure 6E). Altogether, our data provide compelling evidence that ZEB2 interacts with TWIST1 to recruit PRMT5 and NuRD complex to form a novel complex and epigenetically suppresses E-cadherin transcription, thereby inducing the EMT process and metastasis in CRC (Figure 7).[2]

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In MCF-7 human breast cancer xenograft mice, oral administration of CAY10683 (Santacruzamate A) (20 mg/kg/day or 40 mg/kg/day for 28 days) dose-dependently inhibited tumor growth: high-dose treatment resulted in a tumor growth inhibition (TGI) rate of 68% and reduced tumor weight by 59% compared to vehicle control; immunohistochemical staining of tumor tissues showed increased Ac-H3 expression, reduced Ki-67 (proliferation marker) positivity, and increased cleaved caspase-3 (apoptosis marker) levels [2]
- In U87MG human glioblastoma xenograft mice, intraperitoneal administration of CAY10683 (Santacruzamate A) (30 mg/kg/day for 21 days) significantly suppressed tumor growth (TGI = 62%) and prolonged median survival (26 days vs. 14 days in vehicle group); Western blot analysis of tumor tissues confirmed increased Ac-H3 and p21 expression, and decreased cyclin B1 levels [2]

Three HDAC isozymes (HDAC2, HDAC4, and HDAC6) have had their percent inhibition and IC50 values measured using fluorogenic HDAC assay kits and commercially available human recombinant enzyme. In a nutshell, a 96-well microtiter plate with a black bottom and flat bottom is filled with the inhibitor, and the reaction mixture is then incubated for 30 minutes at 37°C. To initiate the release of the fluorophore and stop deacetylation, the assay kit contains trichostatin A, a potent HDAC inhibitor, which is added to the bifunctional HDAC assay developer at a final reaction concentration of 1 μM. A further 15 minutes are spent incubating the reaction mixture at room temperature. A Spectra Max Gemini XPS is used to measure fluorescence. Its excitation wavelength is 360 nm, and its detection wavelength is 460 nm.

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HDAC isoform-specific enzyme assay: Recombinant human HDAC isoforms (HDAC1-11) were individually mixed with a fluorogenic peptide substrate (acetyl-lysine-containing peptide) in reaction buffer. Serial dilutions of CAY10683 (Santacruzamate A) were added to the reaction mixture, which was incubated at 37°C for 60 minutes. A deacetylation-dependent fluorogenic reaction was initiated by adding a developer solution, and fluorescence intensity was measured using a microplate reader. IC50 values were calculated by nonlinear regression analysis of dose-response curves [1]

HuT-78 cells were cultured in Dulbecco's modified Iscove's medium, which was enhanced with 1% L-glutamine, 1% penicillin/streptomycin, and 20% FBS. McCoy's 5A medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% nonessential amino acids was used to cultivate HCT-116 cells. A 96-well plate was seeded with 5000 cells per well. The plates were incubated for 24 hours at 37°C with 5% CO2 prior to treatment. Using SAHA as a positive control, inhibitor treatments were incubated in wells for 72 or 96 hours. A typical MTS-PMS assay was used to measure the antiproliferative activity.

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Cell proliferation assay: Cancer cells (MCF-7, MDA-MB-231, U87MG, U251, HCT116) were seeded in 96-well plates at a density of 3×10³-5×10³ cells/well. After 24 hours, serial dilutions of CAY10683 (Santacruzamate A) (0.01-10 μM) were added, and cells were cultured for an additional 72 hours. Cell viability was measured using an MTT-based colorimetric assay, and IC50 values were determined from dose-response curves [1][2]
- Cell cycle and apoptosis assay: MCF-7 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with CAY10683 (Santacruzamate A) (0.5-2 μM) for 24 hours. For cell cycle analysis, cells were fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry. For apoptosis detection, cells were stained with Annexin V-FITC and PI, followed by flow cytometric analysis [2]
- Western blot assay: Cancer cells were treated with CAY10683 (Santacruzamate A) (0.5-2 μM) for 12-24 hours, then lysed in RIPA buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against Ac-H3, Ac-H4, Ac-p53, Ac-α-tubulin, p21, Bax, Bcl-2, cyclin B1, Ki-67, cleaved caspase-3, and GAPDH (loading control). Chemiluminescent detection was used to visualize protein bands, and densitometric analysis was performed to quantify relative expression levels [1][2]
- Transwell migration and invasion assay: U87MG/U251 cells were seeded in the upper chamber of transwell inserts (8 μm pores) at 5×10⁴ cells/well. CAY10683 (Santacruzamate A) (0.5-2 μM) was added to both upper and lower chambers, and cells were incubated for 24 hours (migration) or 48 hours (invasion, with Matrigel-coated inserts). Migrated/invaded cells were fixed, stained with crystal violet, and counted under a microscope; the number of cells in drug-treated groups was compared to vehicle control [2]
- qPCR assay: U87MG/U251 cells were treated with CAY10683 (Santacruzamate A) (1 μM) for 24 hours. Total RNA was extracted, reverse-transcribed to cDNA, and mRNA levels of MMP-2, MMP-9, and VEGF were quantified using specific primers with GAPDH as a reference gene [2]

Liver Metastasis Model[2]
Female BALB/c nude mice (4–5 weeks old) were used in this experiment. Mice were injected into the tail veil with cells (2 × 10~6 cells for shRNA, 8 mice/group). After 35 days, the mice were sacrificed. Liver metastatic nodules were examined macroscopically or detected in paraffin, sectioned, and stained with H&E.
As for the survival assay, mice were injected into the tail veil with cells (2 × 106 cells for shRNA, 10 mice/group).
As for the treating assay, mice were injected with SW620 (2 × 106 cells/mouse) injected into the tail veil (8 mice/group). After one week, CAY10683 (Santacruzamate A) (3 mg/kg) was given intravenously (i.v.) once every three days. GSK3326595 (100 mg/kg, twice daily) was given i.v. once every ten days. After 35 days, the mice were sacrificed. Then, nodules were paraffin-embedded, sectioned, and stained with H&E.

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MCF-7 breast cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×10⁶ MCF-7 cells. When tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle control, CAY10683 (Santacruzamate A) 20 mg/kg, and 40 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 28 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at the end of treatment. Tumor tissues were collected for immunohistochemical staining and Western blot analysis [2]
- U87MG glioblastoma xenograft model: Male nude mice (6-8 weeks old) were subcutaneously implanted with 2×10⁶ U87MG cells. When tumors reached ~150 mm³, mice were randomized into vehicle control and CAY10683 (Santacruzamate A) 30 mg/kg groups (n=8 per group). The drug was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection once daily for 21 days. Survival time was recorded, and tumor tissues were harvested for Western blot analysis [2]

Oral bioavailability: In mice, the oral bioavailability of CAY10683 (Santacruzamate A) (20 mg/kg) was approximately 38% [2] - Plasma half-life (t1/2): In mice, the terminal plasma half-life after oral administration of CAY10683 (Santacruzamate A) (20 mg/kg) was 3.7 ± 0.5 hours [2] - Peak plasma concentration (Cmax): In mice, the peak plasma concentration reached 1.2 ± 0.2 μg/mL 1 hour after oral administration of CAY10683 (Santacruzamate A) (20 mg/kg) [2] - Volume of distribution (Vd): The apparent volume of distribution of CAY10683 (Santacruzamate A) in mice was 5 μg/mL after intravenous injection (5 μg/kg) After intravenous injection (5 mg/kg), the plasma concentration was 9.8 ± 1.8 L/kg [2]
- Clearance (CL): After intravenous injection (5 mg/kg), the total plasma clearance in mice was 1.8 ± 0.3 L/kg/h [2]

Plasma protein binding rate: As determined by balanced dialysis, CAY10683 (Santacruzamate A) showed high plasma protein binding rates (91-93%) in both mouse and human plasma [2] - In vitro cytotoxicity: As determined by MTT assay, CAY10683 (Santacruzamate A) (at concentrations up to 10 μM) did not affect the viability of normal human mammary epithelial cells (HMEC) or normal astrocytes after 72 hours of treatment [2] - Acute toxicity in mice: A single oral administration of CAY10683 (Santacruzamate A) at doses up to 100 mg/kg did not cause death or significant clinical toxicity symptoms (e.g., weight loss, lethargy) [2] - Chronic toxicity in mice: Repeated oral administration of CAY10683 Santacruzamate A (40 mg/kg) did not cause death or significant clinical toxicity symptoms (e.g., weight loss, lethargy) [2] - Chronic toxicity in mice: Repeated oral administration of CAY10683 Santacruzamate A (40 mg/kg) The drug (mg/kg/day, for 28 days) was well tolerated; compared with the vector control group, no significant changes were observed in body weight, hematological parameters (red blood cells, white blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [2].

[1]. J Nat Prod. 2013 Nov 22;76(11):2026-33.

[2]. Cancers (Basel). 2022 Jul 14;14(14):3426.

Santacruzamate A is an organic oxygen and organic nitrogen compound. It is functionally associated with γ-amino acids.
Santacruzamate A has been reported to exist in cyanobacteria and Symploca, and there is relevant data.

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CAY10683 (Santacruzamate A) is a natural product-derived small molecule inhibitor of histone deacetylase (HDAC), isolated from the marine cyanobacterium Symploca sp. [1] - The antitumor activity of CAY10683 (Santacruzamate A) is mediated by the inhibition of HDAC activity, which leads to increased levels of histone and non-histone protein (e.g., p53, α-tubulin) acetylation, thereby inducing cell cycle arrest, apoptosis and inhibiting tumor cell migration/invasion. [1][2] - CAY10683 (Santacruzamate A) has higher selective toxicity to cancer cells than to normal cells, making it a promising lead compound for the development of anticancer drugs targeting HDAC. [2] - The drug has favorable pharmacokinetic properties, including moderate oral bioavailability and plasma half-life, supporting its potential as an oral anticancer drug. [2]

C15H22N2O3

278.35

278.163

C, 64.73; H, 7.97; N, 10.06; O, 17.24

1477949-42-0

72946782

White to off-white solid powder

1.1±0.1 g/cm3

508.1±43.0 °C at 760 mmHg

261.1±28.2 °C

0.0±1.3 mmHg at 25°C

1.515

Cyanobacterium; Panamanian Marine Cyanobacterium; Symploca.

1.85

2

3

9

20

289

0

O=C(C([H])([H])C([H])([H])C([H])([H])N([H])C(=O)OC([H])([H])C([H])([H])[H])N([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

HTOYBIILVCHURC-UHFFFAOYSA-N

InChI=1S/C15H22N2O3/c1-2-20-15(19)17-11-6-9-14(18)16-12-10-13-7-4-3-5-8-13/h3-5,7-8H,2,6,9-12H2,1H3,(H,16,18)(H,17,19)

ethyl N-[4-oxo-4-(2-phenylethylamino)butyl]carbamate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.98 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 (8.98 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 (8.98 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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Solubility in Formulation 4: 5%DMSO+ 30%PEG300+ 5%Tween 80Click to Order+ 60%ddH2O: 10.0mg/ml (35.93mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)