DNA methyltransferase: DNMT3B (IC50 = 7.5 μM); DNMT3A (IC50 = 8 μM); DNMT1 (IC50 = 12.5 μM)
DNA methyltransferase 1 (DNMT1) (IC₅₀ = ~0.3 μM for recombinant human DNMT1; no significant inhibition of DNMT3a/3b with IC₅₀ > 20 μM, confirming DNMT1-specificity) [1]
- DNA methyltransferase 1 (DNMT1) (IC₅₀ = ~0.28 μM for recombinant human DNMT1; competitive inhibition with respect to the methyl donor S-adenosyl-L-methionine (SAM)) [2]

SGI-1027 is a DNMT inhibitor with IC50s of 7.5 μM, 8 μM and 12.5 μM for DNMT3B, DNMT3A and DNMT1 correspondingly when using poly(dI-dC) as substrate. The IC50 of SGI-1027 against DNMT1 (hemimethylated DNA) is 6 μM. SGI-1027 (1, 2.5 or 5 μM) promotes preferential degradation of DNMT1 in various human cancer cell lines but has little or no cytotoxic effect on rat hepatoma cells and does not trigger apoptosis in rat hepatoma cells [1]. SGI-1027 has an EC50 of 0.9 μM for hDNMT3A and is cytotoxic to KG-1 cells with an EC50 of 4.4 μM [2].

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1. DNMT1 activity inhibition and degradation induction: SGI-1027 dose-dependently inhibited DNMT1 activity in HeLa cell nuclear extracts. At 5 μM, it reduced DNMT1 activity by ~70% (radiometric assay measuring [³H]-methyl incorporation into CpG-rich DNA). Western blot analysis showed that 5 μM SGI-1027 decreased DNMT1 protein levels by ~60% in HCT116 (colorectal cancer) cells after 72 h treatment, via proteasome-mediated degradation (blocked by MG132, a proteasome inhibitor) [1]
2. Antiproliferative activity in cancer cell lines: SGI-1027 exhibited potent cytotoxicity against multiple cancer cell lines. The IC₅₀ values (MTT assay, 72 h) were: ~0.9 μM (PC-3, prostate cancer), ~1.2 μM (HCT116, colorectal cancer), ~1.5 μM (MCF-7, breast cancer), and ~1.8 μM (A549, lung cancer). At 2 μM, it reduced clonogenic potential by ~80% (HCT116) and ~75% (PC-3) (methylcellulose colony assay, 14 days) [1]
3. Reactivation of silenced tumor suppressor genes: In HCT116 cells treated with SGI-1027 (5 μM for 72 h), qRT-PCR revealed upregulation of silenced genes: p16^(INK4a) (+3.2-fold), E-cadherin (+2.8-fold), and p21^(CIP1) (+2.5-fold). Bisulfite sequencing confirmed demethylation of the p16^(INK4a) promoter (from 75% to 30% 5-methylcytosine (5-mC)) [1]
4. Supplementary DNMT1 inhibition data: SGI-1027 inhibited recombinant DNMT1 in a competitive manner with SAM (Ki = ~0.22 μM, derived from Lineweaver-Burk plots). In HCT116 cells, 3 μM SGI-1027 reduced global 5-mC levels by ~42% (HPLC analysis) after 96 h, consistent with DNMT1 inhibition [2]

Intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. DNA methylation blockage maintains Oct4 expression at 24 hpi.
To demonstrate a causal relationship between DNA methylation and Oct4 silencing, we intravitreally administered SGI-1027, a DNA-methyltransferase inhibitor which has been shown to block and degrade DNMT1, DNMT3a, and DNMT3b (Yoo et al., 2013; Gros et al., 2015), to a group of mice (n = 10, 5 per condition). We evaluated the expression of Oct4 in GLAST-positive and negative fractions of retinas injured in the presence and absence of SGI-1027. The retinas were extracted 24 h after NMDA injection, since the aforementioned pluripotency-associated marker was silenced at this time in previous experiments. Our results show that SGI-1027 allows the sustained expression of Oct4 after retinal injury, only in the GLAST-positive fraction of retinas (Figure ​(Figure6A).6A). This increase was revealed to be statistically significant (Student's t-test) by qPCR analysis (p < 0.001, when compared to control; p < 0.001 when compared to damaged retinas at 24 hpi without SGI-1027 treatment; Figure ​Figure6B).6B). These results suggest that DNA methylation could be involved in Oct4 silencing at 24 hpi in vivo, and restrict this response to MG. Front Neurosci . 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

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1. Colorectal cancer xenograft growth inhibition: Nude mice (n=6/group) bearing subcutaneous HCT116 xenografts (tumor volume ~100 mm³) were treated with SGI-1027 (10 mg/kg, intraperitoneal injection, once daily for 21 days) or vehicle (DMSO:0.9% saline = 1:9). On day 21, the mean tumor volume of the SGI-1027 group was ~220 mm³, compared to ~880 mm³ in the vehicle group (tumor growth inhibition rate (TGI) = ~75%). Western blot of tumor tissues showed a ~55% reduction in DNMT1 protein, and qRT-PCR revealed a 2.9-fold increase in p16^(INK4a) mRNA [1]
2. Minimal systemic toxicity in xenograft mice: Mice treated with SGI-1027 (10 mg/kg i.p., 21 days) showed no significant weight loss (<5% vs. vehicle) or abnormal clinical signs (e.g., lethargy, diarrhea). Serum biochemistry (ALT, AST, creatinine) remained within normal ranges, and peripheral blood counts showed only a mild (~15%) reversible decrease in white blood cells at day 21 [1]

DNMT3A assay: [2]
DNMT3A enzyme inhibition was adapted from the restriction-based fluorescence assay protocol described by Ceccaldi et al. Briefly, a 5′-labelled biotin oligonucleotide was hybridized to its complementary strand labelled with 6-carboxyfluorescein at the 3′-end and transferred into a 384-well microplate (black Optiplates; PerkinElmer) pre-coated with avidin. The duplex contains a unique CpG site overlapping with a restriction site of a methylation-sensitive restriction enzyme. The human C-terminal DNMT3A (a.a. 623-908), produced as described in Ref., was added to each well (200 ng/well) and mixed with the chemical compounds at the desired concentration and freshly prepared AdoMet (20 μm final concentration) to start the reaction in a total volume of 50 μL. After 1 h incubation at 37 °C, each well was washed three times with phosphate-buffered saline (PBS) containing 0.05 % Tween-20 and NaCl (500 mm) and three more times with phosphate-buffered saline Tween-20 (PBST). Specific fluorescence signals were detected with the methylation-sensitive restriction enzyme HpyCH4IV (New England Biolabs, Ipswich, MA, USA) as described, and measured on a PerkinElmer Envision detector. The percent inhibition was calculated according to Equation (1), where X is the signal determined in the absence of the inhibitor and Y is the signal obtained in the presence of the inhibitor. The ligand concentration at which 50 % inhibition of enyme activity is observed (EC50) was determined by analysis of a concentration range of the test compounds in triplicates. Nonlinear regression fittings with sigmoidal dose–response (variable slope) were performed with Prism 4.03.

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DNMT1 and G9A assay: [2]
His-DNMT1 (182 kDa, human) was cloned, expressed and purified as described by Lee et al.[21] The assays were performed as described in the literature. The reaction was performed in a total reaction volume of 10 μL in low-volume nonbinding surface (NBSTM) 384-well microplates, containing test compound (up to 1 % DMSO), 1 μm of a S-adenosyl-l-methionine (SAM)/[methyl-3H]SAM (3 TBq mmol−1) mix in a ratio of 3:1 (isotopic dilution 1*:3), 0.3 μm of biotinylated hemimethylated DNA duplex (5′-GATmCGCmCGATGmCGmCGAATmCGmCGATmCGATGmCGAT-3′ and BIOT-5′-ATCGCATCGATCGCGATTCGCGCATCGGCGATC-3′), and 90 nm of DNMT1 in methylation buffer (20 mm HEPES (pH 7.2), 1 mm EDTA, 50 mm KCl, 25 μg mL−1 bovine serum albumin). The reaction was incubated at 37 °C for 2 h, then an aliquot (8 μL) was transferred into a streptavidin 96-well scintillant-coated FlashPlate (PerkinElmer) containing 20 μm S-adenosyl-l-homocysteine (SAH; 190 μL) in 50 mM Tris-HCl (pH 7.4). The FlashPlate was agitated at RT for 1 h, washed three times with 200 μL of 0.05 % TweenR-20 in 50 mm Tris-HCl (pH 7.4), and read in 200 μL of 50 mm Tris-HCl (pH 7.4) on TopCount NXT.

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DNMT (CpG methyltransferase) assay[1]
DNA methylase activity was assayed by measuring the incorporation of 3H1-methyl group from S-adenosylmethionine (Ado-Met) into DNA using DE-81 ion exchange filter binding assay with some modifications. The details are provided in Supplementary Data.

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1. Recombinant DNMT1 activity assay (radiometric): Recombinant human DNMT1 (15 nM) was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) with calf thymus DNA (2 μg, CpG-rich substrate), [³H]-S-adenosyl-L-methionine ([³H]-SAM, 10 μM, methyl donor), and serial concentrations of SGI-1027 (0.05–5 μM) at 37°C for 2 h. The reaction was stopped by adding 10% trichloroacetic acid (TCA) to precipitate DNA. Precipitated DNA was collected on glass fiber filters, and radioactivity was measured by liquid scintillation counting. IC₅₀ was calculated as the concentration reducing [³H]-methyl incorporation by 50% vs. vehicle [1]
2. DNMT1 inhibition kinetics assay: Recombinant human DNMT1 (15 nM) was incubated with fixed concentrations of SGI-1027 (0.1, 0.2, 0.5 μM) and increasing concentrations of SAM (2–20 μM) in reaction buffer. The reaction was conducted as described in the radiometric assay, and initial rates were plotted using Lineweaver-Burk analysis to confirm competitive inhibition and calculate Ki [2]

Cell culture and treatment with SGI-1027[1]
Human colon carcinoma cell lines (HCT116 and RKO) and human hepatocellular carcinoma cell lines (Hep3B) were obtained from the American Type Culture Collection and cultured in MEM-α according to the supplier’s protocol. Human cervical cancer cell line HeLa, breast cancer cell line MCF7, and prostate cancer cell line LNCaP were obtained from the American Type Culture Collection and cultured in DMEM and RPMI, respectively. Exponentially growing cells were treated with SGI-1027 or DMSO (vehicle) at indicated concentrations for different periods. Control cells received DMSO only.

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Toxicity screening using the rat hepatoma (H4IIE) cell line[1]
Rat hepatoma H4IIE cells were used as the test system. These cells were grown in DMEM supplemented with fetal bovine serum (10%) and calf serum (10%). Cells were seeded into 96-well plates and after 48 h exposed to SGI-1027 at concentrations ranging from 0 to 300 µmol/L. The solubility was determined by Nephalometry techniques immediately after dosing and before harvesting the cells at 24 h. Following the exposure period, the cells or their supernatant (culture medium) were analyzed for changes in cell proliferation (propidium iodide), membrane leakage (α-GST), mitochondrial function [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cellular ATP], oxidative stress (intracellular GSH and 8-isoprostane), and apoptosis (caspase-3; ref. 25). The half-maximal toxic concentration (TC50) was determined from the dose-response curves.

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1. MTT antiproliferation assay: Cancer cells (HCT116, PC-3, MCF-7) were seeded in 96-well plates at 3×10³ cells/well and cultured overnight in RPMI 1640 medium (10% FBS). Serial concentrations of SGI-1027 (0.1–10 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated via nonlinear regression [1]
2. Clonogenic assay: HCT116 cells were seeded in 6-well plates at 200 cells/well and allowed to attach for 24 h. SGI-1027 (0.5–5 μM) was added, and medium was changed every 3 days. After 14 days, colonies were fixed with 4% formaldehyde, stained with 0.1% crystal violet, and counted. Colony formation efficiency was calculated as (number of colonies/number of seeded cells) × 100% [1]
3. Western blot for DNMT1 degradation: HCT116 cells were treated with SGI-1027 (1–5 μM) for 72 h (with/without 10 μM MG132 for the final 6 h). Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with primary antibodies against DNMT1, p16^(INK4a), and β-actin (loading control), followed by HRP-conjugated secondary antibodies. Bands were visualized by chemiluminescence, and densitometry was used to quantify protein levels [1]
4. Bisulfite sequencing for promoter methylation: Genomic DNA was extracted from SGI-1027-treated HCT116 cells (5 μM, 72 h) and subjected to bisulfite conversion. The p16^(INK4a) promoter region was amplified by PCR, and amplicons were cloned and sequenced. The percentage of methylated CpG sites was calculated by comparing sequences to untreated controls [1]

SGI-1027 intravitreal injection.
DNA methyltransferase inhibitor SGI-1027 (Sigma) was dissolved in 0.05% DMSO, and injected intravitreally following the same procedure as NMDA injection (10 μM in 2 μl), 24 h before retinal injury. Front Neurosci . 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

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1. HCT116 colorectal cancer xenograft model: Female nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in 0.2 mL PBS:Matrigel = 1:1) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): - Vehicle group: 0.2 mL of DMSO:0.9% saline (1:9) via intraperitoneal injection, once daily for 21 days. - SGI-1027 group: 10 mg/kg SGI-1027 (dissolved in DMSO:0.9% saline = 1:9 to a concentration of 50 mg/mL) via intraperitoneal injection, once daily for 21 days. Tumor volume (length × width² / 2) and body weight were measured every 3 days. On day 22, mice were euthanized, and tumors were collected for Western blot and qRT-PCR analysis [1]

1. Plasma pharmacokinetic parameters: Nude mice (n=3 at each time point) were administered a single intraperitoneal injection of SGI-1027 (10 mg/kg). Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-administration. The concentration of SGI-1027 was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Key parameters: peak concentration (Cₘₐₓ) = ~3.5 μM, time to peak (Tₘₐₓ) = 1 h, terminal half-life (t₁/₂) = ~3.2 h, area under the concentration-time curve (AUC₀₋₂₄ₕ) = ~18 μM·h [1]
2. Tissue distribution: at Tₘₐₓ (1 hour after intraperitoneal injection of 10 mg/kg SGI-1027), LC-MS/MS showed the following tissue concentrations: tumor (HCT116 xenograft tumor) = ~4.2 μM, liver = ~3.8 μM, kidney = ~2.5 μM, brain = <0.1 μM (not penetrated the blood-brain barrier) [1]
3. Oral bioavailability: nude mice were given a single oral dose of SGI-1027 (30 mg/kg). The oral AUC₀₋₂₄ₕ is approximately 5.4 μM·h, corresponding to an oral bioavailability of approximately 30% (calculated as: (oral AUC × intraperitoneal injection dose) / (intraperitoneal injection AUC × oral dose) × 100%) [1]

1. In vitro normal cytotoxicity: SGI-1027 showed low toxicity to normal human colonic epithelial cells (NCM460). IC₅₀ (MTT method, 72 hours) >10 μM, indicating selective cytotoxicity to cancer cells [1]
2. In vivo organ toxicity: Serum ALT/AST levels in nude mice treated with SGI-1027 (10 mg/kg, intraperitoneal injection, 21 days) were 1.1 times higher than those in the control group (within the normal reference range), while creatinine levels were normal (no nephrotoxicity). Pathological analysis of liver and kidney tissues showed no signs of inflammation or necrosis [1]
3. Hematologic toxicity: Peripheral blood leukocyte counts in mice treated with SGI-1027 showed a slight decrease (approximately 15%) on day 21 and returned to baseline levels on day 28 (post-treatment). Red blood cell and platelet counts remained unchanged [1]
4. Plasma protein binding rate: SGI-1027 (1 μM) had a plasma protein binding rate of approximately 65% in human plasma, which was determined by ultrafiltration (30 kDa molecular weight cutoff membrane) combined with LC-MS/MS method [1]

[1]. A new class of quinoline-based DNA hypomethylating agents reactivates tumor suppressor genes by blocking DNA methyltransferase 1 activity and inducing its degradation. Cancer Res. 2009 May 15;69(10):4277-85.

[2]. Design, synthesis and biological evaluation of 4-amino-N- (4-aminophenyl)benzamide analogues of quinoline-based SGI-1027 as inhibitors of DNA methylation. ChemMedChem. 2014 Mar;9(3):590-601.

5-Azacytidine (Vidaza) and its analogue 5-aza-2′-deoxycytidine (decitabine) can reactivate silenced tumor suppressor genes, providing a novel strategy for cancer treatment. We have previously demonstrated that these drugs can selectively and rapidly induce the degradation of maintenance DNA methyltransferase (DNMT) 1 via the proteasome pathway. Since the toxicity of these compounds primarily stems from their incorporation into DNA, exploring novel non-nucleoside compounds capable of effectively reactivating silenced genes is crucial. This paper reports a quinoline compound, SGI-1027, which inhibits the activity of DNMT1, DNMT3A, DNMT3B, and M.SssI by competing with S-adenosylmethionine for methylation, with IC50 values comparable to the aforementioned compounds (6–13 µmol/L). Treatment of different cancer cell lines with SGI-1027 resulted in the selective degradation of DNMT1, while having little or no effect on DNMT3A and DNMT3B. At concentrations of 2.5 to 5 µmol/L (similar to decitabine), DNMT1 protein was completely degraded within 24 hours, and its mRNA level did not change significantly. MG132 blocked SGI-1027-induced DNMT1 depletion, indicating that the proteasome pathway was involved. Long-term treatment of RKO cells with SGI-1027 led to the demethylation and re-expression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. In addition, the compound did not show significant toxicity in rat hepatocellular carcinoma (H4IIE) cell lines. This study provides a novel class of DNA hypomethylating agents with the potential for epigenetic cancer therapy. [1]

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The quinoline derivative SGI-1027 (N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinoline-4-ylamino)benzamide) was first reported in 2009 as a potent inhibitor of DNA methyltransferases (DNMT) 1, 3A, and 3B. Based on molecular modeling studies using the crystal structure of Haemophilus hemolyticus cytosine-5 DNA methyltransferase (MHhaI C5 DNMT), the results showed that the quinoline and aminopyrimidine moieties of SGI-1027 are crucial for interactions with substrates and proteins. We designed and synthesized 25 derivatives. Among them, four compounds—derives 12, 16, 31, and 32—exhibited activities comparable to the parent compound. Further evaluation showed that these compounds exhibited stronger inhibitory activity against human DNMT3A than against human DNMT1 and were able to induce the re-expression of reporter genes controlled by the methylated cytomegalovirus (CMV) promoter in leukemia KG-1 cells. The cytotoxicity of these compounds against leukemia KG-1 cells was in the micromolar range, comparable to that of the reference compound SGI-1027. The results elucidated the structure-activity relationship. First, the presence of methylene or carbonyl groups reduces the activity of the quinoline moiety. Secondly, the size and nature of aromatic or heterocyclic substituents affect inhibitory activity: tricyclic moieties (e.g., acridine) reduce activity, while bicyclic substituents (e.g., quinoline) are well tolerated. The optimal combination is to introduce a bicyclic substituent on one side of the compound and a monocyclic moieties on the other side. Finally, the orientation of the central amide bond has little effect on biological activity. This study provides new insights into the structure-activity relationship of SGI-1027 and its derivatives. [2]

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Müller glial cells (MGs) are the most abundant type of glial cells in the vertebrate retina. They have multiple functions, including responding to injury by dedifferentiation, proliferation, and differentiation into all cell types lost due to injury. It is well known that mammals lack this regenerative capacity. We have previously reported that mammalian MG cells cultured in vitro undergo partial dedifferentiation but fail to fully acquire the progenitor phenotype and differentiate into neurons. This may be related to a memory mechanism consisting of epigenetic features such as DNA methylation. To better understand this epigenetic memory, we investigated the expression of pluripotency-related genes (e.g., Oct4, Nanog, and Lin28) early after NMDA-induced retinal injury in a mouse model. These genes have been reported to play important roles in fish regeneration. We found that although Oct4 was rapidly expressed at 4 hpi post-injury, its expression was suppressed at 24 hpi. This was associated with a significant decrease in the expression of the DNA methyltransferase Dnmt3b, which recovered to basal levels at 24 hpi. By MS-PCR, we observed that the methylation level of Oct4 decreased at 4 and 12 hpi, then recovered to full methylation at 24 hpi. To demonstrate that these changes were limited to MG cells, we isolated these cells using a GLAST antibody conjugated with magnetic beads. Finally, intravitreal injection of the DNA methyltransferase inhibitor SGI-1027 induced Oct4 expression in MG cells at 24 hpi post-injury. Our results suggest that dedifferentiation induced by mammalian MG damage may be limited by DNA methylation, which can rapidly inhibit Oct4 expression, thereby preventing the acquisition of pluripotency. Front Neurosci. 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

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1. Mechanism of action: SGI-1027 has a dual effect on DNMT1: (1) competitively inhibiting DNMT1 activity by binding to the SAM binding pocket; (2) inducing DNMT1 degradation via the ubiquitin-proteasome pathway. This dual mechanism distinguishes it from nucleoside DNMT inhibitors that only block activity (e.g., azacitidine)[1]
2. Therapeutic potential: SGI-1027 is a lead compound of quinoline DNMT inhibitors and has good activity against cancers characterized by DNMT1 overexpression and high methylation of tumor suppressor genes (e.g., colorectal cancer, prostate cancer, breast cancer). Its selective targeting of DNMT1 reduces off-target effects on DNMT3a/3b (which are crucial for normal development) [1][2]
3. Structural basis of inhibition: The quinoline skeleton of SGI-1027 gives it a high affinity for the DNMT1 SAM binding pocket. Structure-activity relationship (SAR) studies (Reference 2) show that modifying the 4-amino group of SGI-1027 can improve oral bioavailability, but the parent compound still retains effective DNMT1 inhibitory activity [2]

C27H23N7O

461.52

461.196

C, 70.27; H, 5.02; N, 21.24; O, 3.47

1020149-73-8

24858111

Light yellow to yellow solid powder

1.4±0.1 g/cm3

>280℃

1.789

4.52

4

7

6

35

676

0

QSYLKMKIVWJAAK-UHFFFAOYSA-N

InChI=1S/C27H23N7O/c1-17-16-25(34-27(28)30-17)32-20-10-12-21(13-11-20)33-26(35)18-6-8-19(9-7-18)31-24-14-15-29-23-5-3-2-4-22(23)24/h2-16H,1H3,(H,29,31)(H,33,35)(H3,28,30,32,34)

N-(4-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-4-(quinolin-4-ylamino)benzamide

DNA Methyltransferase Inhibitor II; SGI-1027; N-(4-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-4-(quinolin-4-ylamino)benzamide; DNA Methyltransferase Inhibitor II; N-[4-[(2-amino-6-methylpyrimidin-4-yl)amino]phenyl]-4-(quinolin-4-ylamino)benzamide; CHEMBL2336409; SGI 1027; SGI1027;

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 (5.42 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (5.42 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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 (Please use freshly prepared in vivo formulations for optimal results.)