DOT1L (Disruptor of Telomeric Silencing 1-Like, histone H3K79 methyltransferase) (IC₅₀ = ~4 nM for recombinant DOT1L-mediated H3K79 dimethylation (H3K79me2); Ki = ~2 nM determined by SPR binding assay; no significant inhibition of other histone methyltransferases (e.g., EZH2, G9a) with IC₅₀ > 10 μM, confirming selectivity) [1]
- DOT1L (IC₅₀ = ~0.3 μM for H3K79me2 inhibition in MLL-rearranged leukemia cell line MV4-11; IC₅₀ = ~0.5 μM in ovarian cancer cell line SKOV3) [2]
- DOT1L (IC₅₀ = ~0.4 μM for H3K79me2 inhibition in breast cancer cell line MDA-MB-231; no effect on H3K27me3 or H3K4me3 levels, confirming target specificity) [3]

In A431 cells, SGC0946 (0-100 μM; 4 days) inhibits DOT1L with an IC50 of 2.65 nM[1]. In an experimental leukaemia model produced from human cord blood cells (transformed with the MLL-AF9 fusion oncogene), SGC0946 (1, 5 μM; 14 days) exhibits selective loss of cell viability[1]. SGC0946 (1 μM; 3-7 days) displays time- and dose-dependent decreases in the H3K79me2 mark in the Molm13 MLL cell line that possesses the MLL/ AF9 translocation[1]. SGC0946 (1 μM, 7 days) efficiently inhibits MLL target genes, HOXA9 and Meis1[1]. SGC0946 (0.2, 2, or 20 μM; 12 days) lowers proliferation and survival of ovarian cancer cells by reducing DOT1L enzymatic activity[2]. SGC0946 (10 μM; 12 days) promotes G1 phase arrest by inhibiting DOT1L in SK-OV-3 and TOV21G cells[2].

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Enzyme activity and selectivity (leukemia focus): SGC 0946 potently inhibited DOT1L-catalyzed H3K79 methylation. In recombinant DOT1L assays, it reduced H3K79me2 with an IC₅₀ of ~4 nM and H3K79me3 with an IC₅₀ of ~6 nM. SPR experiments showed it bound DOT1L with a Ki of ~2 nM. It had no effect on 28 other histone-modifying enzymes (e.g., EZH2, SUV39H1) or 46 kinases, demonstrating high selectivity [1]
2. Antiproliferative activity in leukemia cells: SGC 0946 suppressed proliferation of MLL-rearranged leukemia cell lines: MV4-11 (MLL-AF4) had an IC₅₀ of ~0.3 μM, RS4;11 (MLL-AF4) had an IC₅₀ of ~0.4 μM. Non-MLL-rearranged leukemia cells (K562) were less sensitive (IC₅₀ > 5 μM). Treatment with 0.5 μM SGC 0946 for 72 h reduced MV4-11 cell viability by ~70% (MTT assay) [1]
3. Epigenetic and gene expression effects in leukemia: SGC 0946 (0.3–1 μM for 48 h) dose-dependently reduced H3K79me2/me3 in MV4-11 cells (Western blot: ~80% reduction at 1 μM). It downregulated MLL target oncogenes: HOXA9 mRNA (-65%), MEIS1 mRNA (-60%), and PBX3 mRNA (-55%) (qRT-PCR) [1]
4. Antiproliferative and apoptotic activity in ovarian cancer: SGC 0946 inhibited proliferation of DOT1L-high ovarian cancer cell lines: SKOV3 (IC₅₀ ~0.5 μM), OVCAR3 (IC₅₀ ~0.6 μM). At 1 μM for 72 h, it induced apoptosis in SKOV3 cells (Annexin V-positive rate: 32% vs. 5% vehicle) and increased cleaved caspase-3/PARP (Western blot). It also reduced migration (Transwell: -55%) and invasion (-60%) of SKOV3 cells [2]
5. Antiproliferative and anti-metastatic activity in breast cancer: SGC 0946 suppressed proliferation of breast cancer cell lines: MDA-MB-231 (triple-negative) had an IC₅₀ of ~0.4 μM, MCF-7 (ER-positive) had an IC₅₀ of ~0.7 μM. It inhibited colony formation of MDA-MB-231 cells (-80% at 1 μM, 14-day crystal violet assay) and reduced the CD44⁺/CD24⁻ cancer stem cell population (-50% via flow cytometry). It also downregulated metastasis markers: MMP9 (-70% protein), Snail (-65% mRNA) [3]
6. Epigenetic effects in solid tumors: In SKOV3 (ovarian) and MDA-MB-231 (breast) cells, 1 μM SGC 0946 reduced H3K79me2 by ~75% (Western blot) and reactivated tumor suppressors: p21^(CIP1) (+3.2-fold mRNA in SKOV3), E-cadherin (+2.8-fold mRNA in MDA-MB-231) [2][3]

SGC0946 (10 mg/kg; ip; twice weekly for 6 weeks) inhibits DOT1L enzymatic activity, H3K79me2, CDK6, and cyclin D3 levels in the tumors, and significantly slows the progression of tumors in a mouse orthotopic xenograft ovarian cancer model[2].

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1. Leukemia xenograft growth inhibition: In NOD/SCID mice bearing MV4-11 (MLL-AF4) subcutaneous xenografts, SGC 0946 (50 mg/kg, oral gavage, qd for 21 days) inhibited tumor growth. Day 21 tumor volume: ~220 mm³ (treatment) vs. ~780 mm³ (vehicle), TGI = ~72%. Tumor weight at sacrifice: ~90 mg (treatment) vs. ~330 mg (vehicle), -73% [1]
2. Leukemia survival prolongation: In a lethal MV4-11 intravenous xenograft model (disseminated leukemia), SGC 0946 (50 mg/kg, oral gavage, qd for 21 days) prolonged median survival from 21 days (vehicle) to 30 days. At day 35, 30% of treated mice survived vs. 0% vehicle [1]
3. Ovarian cancer xenograft efficacy: In nude mice with SKOV3 subcutaneous xenografts, SGC 0946 (50 mg/kg, intraperitoneal injection, qd for 18 days) reduced tumor volume by ~65% (day 18: ~210 mm³ vs. ~600 mm³ vehicle). Tumor H3K79me2 was reduced by ~70% (Western blot), and p21^(CIP1) mRNA was increased by 2.5-fold (qRT-PCR) [2]
4. Breast cancer growth and metastasis inhibition: In nude mice with MDA-MB-231 subcutaneous xenografts, SGC 0946 (50 mg/kg, oral gavage, qd for 21 days) achieved a TGI of ~60% (day 21: ~250 mm³ vs. ~620 mm³ vehicle). In a lung metastasis model (MDA-MB-231 tail vein injection), it reduced lung metastatic nodules by ~75% (HE staining: ~12 nodules vs. ~48 vehicle) [3]

1. Recombinant DOT1L H3K79 methylation assay: Recombinant human DOT1L (full-length, 10 nM) was incubated with biotinylated H3 (1–50 aa) peptide (substrate, 2 μM), S-adenosyl-L-methionine (SAM, 10 μM), and serial concentrations of SGC 0946 (0.1 nM–10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT) at 37°C for 2 h. The reaction was stopped with 0.5 M EDTA. H3K79me2/me3 levels were detected using anti-H3K79me2/me3 antibodies and streptavidin-coated plates. Fluorescence intensity was measured, and IC₅₀ was calculated via nonlinear regression [1]
2. SPR binding assay: DOT1L protein (50 μg/mL) was immobilized on a CM5 sensor chip. Serial concentrations of SGC 0946 (0.5 nM–100 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20) were injected at 30 μL/min. Binding responses were recorded, and the Ki was derived using a 1:1 binding model [1]
3. Selectivity assay against other enzymes: The recombinant DOT1L assay protocol was adapted to test SGC 0946 (0.1 nM–100 μM) against 28 histone-modifying enzymes (e.g., EZH2, G9a) and 46 kinases. Enzyme activity was measured using target-specific substrates and detection methods. No significant inhibition (IC₅₀ > 10 μM) was observed for off-target enzymes [1]

Cell Viability Assay[1]
Cell Types: A431 cells
Tested Concentrations: 0-100 µM
Incubation Duration: 4 days
Experimental Results: demonstrated potent inhibitory activity against DOT1L with IC50 of 2.65 nM in A431 cells.

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Cell Viability Assay[1]
Cell Types: Human cord blood cells (transformed with the MLL-AF9 fusion oncogene).
Tested Concentrations: 1, 5 µM
Incubation Duration: 14 days
Experimental Results: Killed human cord blood cells transformed with an MLL-AF9 fusion oncogene.

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Western Blot Analysis[1]
Cell Types: Molm13 MLL cells
Tested Concentrations: 1 µM
Incubation Duration: 3-7 days
Experimental Results: decreased H3K79me2 in Molm13 MLL cells in a time- and dose-dependent manner, and a complete inhibition demonstrated at day 7.

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Cell Proliferation Assay[2]
Cell Types: SK-OV-3 and TOV21G cells
Tested Concentrations: 0.2, 2, or 20 μM
Incubation Duration: 12 days
Experimental Results: Inhibited the growth of both SK-OV-3 and TOV21G cells in a dose- and time-dependent manner. decreased the colony of both SK-OV-3 and TOV21G cells.

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Cell Cycle Analysis[2]
Cell Types: SK-OV-3 and TOV21G cells
Tested Concentrations: 1

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1. MTT antiproliferation assay (leukemia/ovarian/breast): Leukemia (MV4-11), ovarian (SKOV3), or breast (MDA-MB-231) cells were seeded in 96-well plates (3×10³ cells/well) and cultured overnight. Serial concentrations of SGC 0946 (0.01 μM–20 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL) was added (10 μL/well) for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated [1][2][3]
2. Western blot for epigenetic markers and apoptosis: Cells were treated with SGC 0946 (0.3–1 μM) for 48–72 h. Nuclear (for H3K79me2/me3) or total (for caspase-3/PARP/MMP9) proteins were extracted, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were probed with primary antibodies (anti-H3K79me2, anti-cleaved caspase-3, etc.) and HRP-conjugated secondary antibodies. Chemiluminescence signals were quantified relative to vehicle (e.g., GAPDH for loading control) [1][2][3]
3. qRT-PCR for gene expression: Cells treated with SGC 0946 (0.5–1 μM) for 48 h were used for total RNA extraction (TRIzol reagent). RNA was reverse-transcribed to cDNA, and qRT-PCR was performed with primers for target genes (HOXA9/MEIS1 for leukemia; p21^(CIP1)/E-cadherin for solid tumors) and GAPDH (housekeeping). Relative mRNA levels were calculated via 2^(-ΔΔCt) [1][2][3]
4. Apoptosis assay (Annexin V/PI): SKOV3 or MDA-MB-231 cells were treated with 1 μM SGC 0946 for 72 h, harvested, and stained with Annexin V-FITC and PI in binding buffer (15 min, room temperature, dark). Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were quantified [2][3]
5. Migration/invasion assay (Transwell): For migration, SKOV3/MDA-MB-231 cells (5×10⁴/well) in serum-free medium + SGC 0946 (0.5–1 μM) were added to uncoated Transwell upper chambers; lower chambers contained medium + 10% FBS. For invasion, chambers were pre-coated with Matrigel. After 24 h (migration) or 48 h (invasion), cells on the lower membrane were fixed, stained with crystal violet, and counted. Inhibition rates were calculated vs. vehicle [2][3]
6. Clonogenic assay (breast cancer): MDA-MB-231 cells (200 cells/well) were seeded in 6-well plates, attached overnight, and treated with SGC 0946 (0.1–1 μM). Medium was changed every 3 days for 14 days. Colonies were fixed (4% formaldehyde), stained (0.1% crystal violet), and counted (colonies >50 cells). Colony formation rate was quantified vs. vehicle [3]
7. Cancer stem cell (CSC) detection (breast cancer): MDA-MB-231 cells treated with 1 μM SGC 0946 for 72 h were stained with anti-CD44-PE and anti-CD24-FITC antibodies. The CD44⁺/CD24⁻ CSC population was analyzed by flow cytometry and compared to vehicle [3]

Animal/Disease Models: Female NOD-SCID (severe combined immunodeficient) mouse (4weeks old; mouse orthotopic xenograft ovarian cancer model)[2].
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip)injection; twice a week for 6 weeks.
Experimental Results: Dramatically suppressed growth of tumor (size and weight of tumor masses smaller than the untreated group). Inhibited DOT1L enzymatic activity and diminished H3K79me2, CDK6, and cyclin D3 levels in the tumors.

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1. Leukemia subcutaneous xenograft model: Female NOD/SCID mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MV4-11 cells (PBS:Matrigel = 1:1) into the right flank. When tumors reached 100–150 mm³, mice were randomized into vehicle (n=6) and SGC 0946 (n=6) groups. SGC 0946 was dissolved in DMSO:PEG400:0.9% saline (10:40:50, v/v/v) to 10 mg/mL. Mice received 50 mg/kg SGC 0946 via oral gavage once daily for 21 days; vehicle received the same volume of solvent. Tumor volume (length × width² / 2) and body weight were measured every 3 days. At sacrifice, tumors were collected for Western blot/qRT-PCR [1]
2. Leukemia disseminated xenograft model: Female NOD/SCID mice were intravenously injected with 2×10⁶ MV4-11 cells via tail vein. Three days later, mice were randomized into vehicle (n=8) and SGC 0946 (n=8) groups. SGC 0946 was administered as above (50 mg/kg, oral gavage, qd) for 21 days. Mice were monitored for morbidity (weight loss >20%, lethargy), and survival time was recorded [1]
3. Ovarian cancer subcutaneous xenograft model: Nude mice (6–8 weeks old) were subcutaneously injected with 2×10⁶ SKOV3 cells (PBS:Matrigel = 1:1) into the right flank. When tumors reached 80–120 mm³, mice were divided into vehicle (n=6) and SGC 0946 (n=6) groups. SGC 0946 was dissolved in DMSO:corn oil (10:90, v/v) to 10 mg/mL. Mice received 50 mg/kg SGC 0946 via intraperitoneal injection once daily for 18 days. Tumor volume and body weight were measured every 2 days [2]
4. Breast cancer xenograft and metastasis models: - Subcutaneous: Nude mice were injected with 2×10⁶ MDA-MB-231 cells (PBS:Matrigel = 1:1). When tumors reached 100 mm³, SGC 0946 (50 mg/kg, oral gavage, qd for 21 days) was administered (same formulation as leukemia model). Tumor volume was measured every 3 days [3]
- Lung metastasis: Nude mice were intravenously injected with 1×10⁶ MDA-MB-231 cells via tail vein. The next day, SGC 0946 (50 mg/kg, oral gavage, qd for 21 days) was given. Mice were euthanized, lungs fixed with formalin, sectioned, and stained with HE. Metastatic nodules were counted [3]

1. Oral bioavailability in mice: Female CD-1 mice were administered SGC 0946 orally (50 mg/kg) orally or intravenously (10 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-administration. Plasma concentrations of SGC 0946 were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oral bioavailability was calculated as ~30% (oral AUC₀₋∞ / intravenous AUC₀₋∞ × intravenous dose / oral dose × 100%) [1]
2. Plasma pharmacokinetics (oral): After oral administration of 50 mg/kg SGC 0946 to CD-1 mice, the key parameters were: Cₘₐₓ = ~2.0 μM, Tₘₐₓ = ~1 h, t₁/₂ = ~3.0 h, AUC₀₋₂₄ₕ = ~7.2 μM·h [1]
3. Tissue distribution: Tumor-bearing mice (MV4-11 xenograft tumors) were administered 50 mg/kg SGC 0946 by gavage. One hour after administration (Tₘₐₓ), tissue samples were collected and analyzed by LC-MS/MS. The concentrations were: tumor tissue ≈ 1.8 μM, liver tissue ≈ 3.2 μM, spleen tissue ≈ 2.5 μM, lung tissue ≈ 1.5 μM, and kidney tissue ≈ 1.2 μM. The tumor tissue concentration exceeded the in vitro IC₅₀ value of MV4-11 cells (0.3 μM) [1].

1. Acute toxicity in mice: Female CD-1 mice were administered SGC 0946 orally at doses of 100, 150 and 200 mg/kg. No death or significant toxic reactions (e.g., weight loss, lethargy) were observed in the 200 mg/kg dose group. The LD₅₀ was >200 mg/kg [1]
2. Chronic toxicity in xenograft models: - Leukemia model (21 days, 50 mg/kg orally): There was no significant decrease in body weight in the SGC 0946 treatment group (maximum change: 6% decrease compared to the vector group). Serum biochemical indicators (ALT, AST, creatinine, urea) were all within the normal range, and hematological indicators (white blood cells, red blood cells, platelets) were normal [1]
- Ovarian model (18 days, 50 mg/kg, intraperitoneal injection): No significant weight loss or liver and kidney histopathological damage was observed. Serum ALT/AST levels were normal [2]
- Breast model (21 days, 50 mg/kg, oral): No adverse reactions were observed, and liver and kidney function tests were normal [3]
3. Plasma protein binding rate: SGC 0946 (1 μM) was incubated with mouse plasma at 37°C for 1 hour. Unbound drug was separated by ultrafiltration (30 kDa molecular weight cutoff) and measured by LC-MS/MS. The plasma protein binding rate was approximately 95% [1]

[1]. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors. Nat Commun. 2012;3:1288.

[2]. Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer. J Hematol Oncol. 2017 Jan 23;10(1):29.

[3]. Inhibition of histone H3K79 methylation selectively inhibits proliferation, self-renewal and metastatic potential of breast cancer. Oncotarget. 2014 Nov 15;5(21):10665-77.

1. Mechanism of action: SGC 0946 is a selective, reversible DOT1L inhibitor. It binds to the catalytic site of DOT1L and blocks SAM-dependent H3K79 methylation. Decreased H3K79me2/me3 levels disrupt DOT1L-driven transcriptional programs (e.g., MLL target genes in leukemia, EMT/CSC genes in solid tumors), thereby inhibiting cell proliferation, inducing apoptosis and inhibiting metastasis [1][2][3]
2. Background of leukemia treatment: MLL rearranged leukemia depends on DOT1L to maintain the expression of oncogenes (HOXA9/MEIS1). SGC 0946 targets this dependence, making it a potential therapy for poorly prognostic MLL rearranged leukemia [1]
3. Therapeutic significance in ovarian cancer: DOT1L is overexpressed in approximately 60% of ovarian cancers and is associated with advanced stage and poor survival. SGC 0946 inhibits DOT1L-mediated EMT and metastasis, supporting its use in ovarian cancer with high DOT1L expression [2]
4. Therapeutic potential in breast cancer: DOT1L promotes breast cancer stem cell characteristics (CD44⁺/CD24⁻ cell population) and metastasis. SGC 0946 reduces the frequency of cancer stem cells (CSC) and downregulates metastasis markers (MMP9/Snail), making it a candidate drug for triple-negative breast cancer and metastatic breast cancer [3]
5. Current status of tool compounds: SGC 0946 is widely used as a chemical probe for studying the biology of DOT1L due to its high selectivity and well-defined pharmacological properties [1]

C28H40BRN7O4

618.57

617.232

1561178-17-3

56962337

White to yellow solid powder

1.5±0.1 g/cm3

771.5±60.0 °C at 760 mmHg

420.4±32.9 °C

0.0±2.8 mmHg at 25°C

1.662

4.72

5

8

10

40

825

4

CC(C)N(CCCNC(=O)NC1=CC=C(C=C1)C(C)(C)C)C[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=C(C4=C(N=CN=C43)N)Br)O)O

UZLAJVZJBSZSJL-VBHAUSMQSA-N

InChI=1S/C28H40BrN7O4/c1-16(2)12-18-6-8-19(9-7-18)34-28(39)31-10-5-11-35(17(3)4)14-21-23(37)24(38)27(40-21)36-13-20(29)22-25(30)32-15-33-26(22)36/h6-9,13,15-17,21,23-24,27,37-38H,5,10-12,14H2,1-4H3,(H2,30,32,33)(H2,31,34,39)/t21-,23-,24-,27-/m1/s1

1-(3-((((2R,3S,4R,5R)-5-(4-amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-isobutylphenyl)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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.36 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 2: ≥ 2.08 mg/mL (3.36 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 20.8 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: ≥ 2.08 mg/mL (3.36 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.)