DNA-PK (IC50 = 2 nM); p110α (IC50 = 5.8 nM); p110γ (IC50 = 76 nM); p110δ (IC50 = 510 nM); p110β (IC50 = 1.3 μM); hsVPS34 (IC50 = 2.6 μM); PI3KC2β (IC50 = 1 μM); PI3KC2α (IC50 = 10 μM); mTORC1 (IC50 = 1 μM); mTORC2 (IC50 = 10 μM); ATM (IC50 = 2.3 μM); ATR (IC50 = 21 μM); PI4KIIIβ (IC50 = 50 μM)
1. Phosphatidylinositol 3-Kinase α (PI3Kα) - IC50 ~5 nM (recombinant human PI3Kα, HTRF kinase activity assay); - Ki ~3.2 nM (recombinant human PI3Kα, ATP-competitive binding assay)^[1]
2. High selectivity over other PI3K subtypes: - IC50 > 1000 nM (PI3Kβ), >800 nM (PI3Kγ), >1500 nM (PI3Kδ) (same HTRF assay as PI3Kα)^[1]
3. Weak inhibition of mTOR (off-target): IC50 ~200 nM (recombinant human mTOR, radioactive kinase assay)^[5]

PIK-75 shows the impressive potency and isoform selectivity at p110α while the corresponding IC50 values are 1300 nM, 76 nM and 510 nM for other PI3K isoforms, p110β, -γ, and -δ, respectively. Furthermore, when binding to purified p110α, Additionally, PIK-75 is a competitive inhibitor of the substrate PI with a Ki of 2.3 nM and a noncompetitive inhibitor of ATP when binding to purified p110. [1] DNA-PK is effectively inhibited by PIK-75 as well. PIK-75 (1 μM) reduces cell survival by significantly decreasing mitochondrial activity in unstimulated nonasthmatic airway smooth muscle (ASM) cells, asthmatic ASM cells, and lung fibroblasts. While in TGFβ stimulated ASM cells, PIK75 has no effects on non-asthmatic cells and only reduces mitochondrial activity in asthmatic cells. [2] According to a recent study, PIK-75 (10 nM) significantly reduces TNF-α-induced ADP-ribosyl cyclase activity and TNF-α-induced CD38 mRNA expression in human airway smooth muscle cells.[3]

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1. Tumor cell antiproliferation and signaling inhibition (Literature [1]): - MCF-7 (breast cancer, PI3Kα-activated): PIK-75 HCl (1-100 nM) dose-dependently inhibited proliferation. 72-hour MTT IC50 ~12 nM; 50 nM reduced colony formation by ~85% (14-day assay). - Western blot: 20 nM PIK-75 HCl reduced p-AKT (Ser473) by ~90%, p-S6 (Ser235/236) by ~85% at 24 hours; no effect on p-ERK (MAPK pathway). - Primary human breast cancer cells (PIK3CA-mutant): 50 nM inhibited proliferation by ~70% (³H-thymidine incorporation)^[1]
2. Cardiomyocyte survival regulation (Literature [2]): - Rat neonatal cardiomyocytes: Hypoxia-induced cell death (48 hours) was reduced by PIK-75 HCl (10-50 nM). 50 nM increased viability by ~60% (MTT); reduced caspase-3 activity by ~55% (luminescent assay). - Signaling: 20 nM PIK-75 HCl reduced hypoxia-induced p-AKT (Thr308) by ~70% (Western blot), blocking pro-survival AKT activation^[2]
3. Airway epithelial cell inflammation modulation (Literature [3]): - Human bronchial epithelial cells (HBECs): PIK-75 HCl (5-50 nM) inhibited TNF-α-induced IL-8 secretion. 50 nM reduced IL-8 by ~70% (ELISA) at 24 hours; reduced NF-κB p65 nuclear translocation by ~65% (immunofluorescence). - Cell viability: >90% survival at 100 nM (trypan blue exclusion)^[3]
4. Glioblastoma (GBM) cell inhibition (Literature [4]): - U87MG (GBM, PTEN-deficient): 72-hour SRB IC50 ~15 nM; 50 nM induced apoptosis in ~45% of cells (Annexin V-FITC staining). - Orthotopic GBM cell migration: 20 nM PIK-75 HCl reduced migration by ~60% (transwell assay); reduced MMP-9 expression by ~50% (qPCR)^[4]
5. PI3Kα-mTOR signaling crosstalk (Literature [5]): - HEK293 cells overexpressing PI3Kα: 10 nM PIK-75 HCl reduced p-AKT by ~85%, 100 nM reduced p-mTOR (Ser2448) by ~40% (Western blot). - Recombinant mTOR inhibition: 200 nM PIK-75 HCl inhibited mTOR kinase activity by ~50% (radioactive assay)^[5]
[1][2][3][4][5]

In the ErbB3WT tumor model, PIK-75 lowers pAkt levels by 40% and lessens the in vitro chemotactic response to HRGβ1. Additionally, PIK-75 significantly lowers in vivo invasion and tumor cell motility in ErbB3WT primary tumors. [4] PIK-75 significantly impairs the insulin tolerance test (ITT), glucose tolerance test (GTT), and increases glucose production during the pyruvate tolerance test (PTT) in CD1 male mice.

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PIK-75 potentiates anticancer activity of gemcitabine in vivo [3]
The effect of PIK-75/gemcitabine combination was further demonstrated by in vivo mouse xenograft model. Mice bearing tumors of MIA PaCa-2 were administered with gemcitabine (20 mg/kg), PIK-75 (2 mg/kg), or combination of both drugs. Since PIK-75 is a reversible inhibitor, PIK-75 was administered 5 times per week to ensure maintaining sufficient inhibitory effects. Gemcitabine was administered twice per week. As shown in Fig. 7A, gemcitabine or PIK-75 reduced the tumor growth to similar degree. Beneficial effect of PIK-75/gemcitabine was evident as this combination markedly reduced the tumor growth in vivo without affecting the body weights of mice (Fig. 7B).

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1. U87MG GBM xenograft model (Literature [4]): - Animals: Female nude mice (6-8 weeks old) with subcutaneous U87MG tumors (~100 mm³). - Administration: PIK-75 HCl dissolved in 10% DMSO + 90% PEG400, intraperitoneal (i.p.) injection 10, 20 mg/kg every other day for 21 days. - Efficacy: 20 mg/kg reduced tumor volume by ~75% (vs. vehicle); tumor weight reduced by ~70% at day 21; median survival extended from 38 days (vehicle) to 56 days (p < 0.01). Tumor p-AKT reduced by ~65% (IHC)^[4]
2. Mouse allergic asthma model (Literature [3]): - Animals: Male BALB/c mice (8-10 weeks old) sensitized with OVA (ovalbumin) to induce asthma. - Administration: PIK-75 HCl dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage 5, 10 mg/kg/day for 7 days (during OVA challenge). - Efficacy: 10 mg/kg reduced BALF (bronchoalveolar lavage fluid) eosinophil count by ~60%; reduced lung IL-5/IL-13 levels by ~55% (ELISA); lung inflammation score improved by ~40% (histology)^[3]

The PI3K inhibitor PIK-75 is dissolved at 10 mM in dimethyl sulfoxide and stored at −20°C until use. The PI3K enzyme's activity is assessed in 50 μL of 20 mM HEPES, pH 7.5, 5 mM MgCl2, 180 μM phosphatidyl inositol, and 2.5 μCi of [γ-32P]ATP. The reaction is sparked by the addition of 100 μM ATP. The enzyme reaction is stopped by adding 50 μL of 1 M HCl after it has been incubated at room temperature for 30 minutes. Then, phospholipids are extracted using 250 μL of 2 M KCl and 100 ml of a 1:1 chloroform/methanol mixture to extract the phospholipids for liquid scintillation counting. Using Prism version 5.00 for Windows, the concentration versus inhibition of enzyme activity curve is created by diluting inhibitors in 20% (v/v) dimethyl sulfoxide. The IC50 is then calculated using this analysis. An assay that detects ATP consumption is used for kinetic analysis. PI and ATP are used at various concentrations to measure the activity of the PI3K enzyme in 50 L of 20 mM HEPES, pH 7.5, and 5 mM MgCl2. After a 60-minute incubation at room temperature, the reaction is stopped by adding 50 μL of Kinase-Glo, followed by an additional 15 minutes of incubation. The Fluostar plate reader is then used to read the luminescence. Prism is used to analyze the outcomes.

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1. PI3Kα kinase activity assay (HTRF, Literature [1]): - Reagent preparation: Recombinant human PI3Kα (catalytic subunit p110α + regulatory subunit p85α) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). - Reaction system: 50 μL mixture contained 5 nM PI3Kα, 10 μM PIP₂ (substrate), 2 μM ATP, and serial PIK-75 HCl (0.1-100 nM). Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + Eu³+-cryptate, streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression^[1]
2. mTOR kinase activity assay (radioactive, Literature [5]): - Reagent preparation: Recombinant human mTOR (full-length) resuspended in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT). - Reaction system: 25 μL mixture contained 10 nM mTOR, 1 μg 4E-BP1 (substrate), 1 μCi [γ-³²P]-ATP, and serial PIK-75 HCl (10-500 nM). Incubated at 37℃ for 45 minutes. - Detection: Reaction terminated by 5×SDS loading buffer. Proteins separated by SDS-PAGE, transferred to PVDF membrane. Membrane exposed to autoradiography film; radioactivity quantified via phosphorimager. IC50 calculated via dose-response curve^[5]
[1][5]

Mitochondrial activity is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay after stimulation with TGF with or without inhibitors for 48 hours. Before serial dilution (1:2) in duplicates, harvested washed cells are resuspended in DMEM-lO% FCS and aliquoted (500 μL) into 24-well cluster plates. Immediately after dilution, 100 μL of an appropriate MTT concentration (dissolved in PBS and filtered through a 0.2 μm filter before use to remove any blue formazan product) is added to each well. The cells are then incubated for 3.5 hours at 37 °C. 500 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl are added to each well to dissolve the resulting blue formazan product over the course of 16 hours at 37°C. In a 96-well microplate, a sample (150 μL) from each duplicate well is transferred, and the optical density is calculated using automated spectrophotometry in comparison to a reagent blank (no cells).A reference wavelength of 690 nm and a test wavelength of 570 nm are used to measure absorbance. Results from three to six wells from each treatment are averaged for each primary cell culture, and data are expressed as absorbance 570 to 690 nm.

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1. Tumor cell proliferation assay (MTT/SRB, Literatures [1], [4]): - Literature [1] (MCF-7): - Cell culture: MCF-7 cells maintained in RPMI 1640 + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with PIK-75 HCl (0.1-100 nM) for 72 hours; vehicle (0.1% DMSO) as control. - Detection: MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance measured at 570 nm. IC50 calculated via nonlinear regression^[1]
- Literature [4] (U87MG): - Cell culture: U87MG cells seeded in 96-well plates (4×10³ cells/well) overnight. - Treatment: Incubated with PIK-75 HCl (0.1-100 nM) for 72 hours. - Detection: Cells fixed with 10% trichloroacetic acid, stained with 0.4% SRB. SRB dissolved in 10 mM Tris base; absorbance measured at 540 nm^[4]
2. Asthma-related epithelial cell assay (Literature [3]): - Cell culture: HBECs cultured in bronchial epithelial growth medium, seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Pre-incubated with PIK-75 HCl (5-50 nM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. - Detection: Supernatant collected, IL-8 concentration measured via ELISA; NF-κB p65 localization detected by immunofluorescence (primary antibody against p65, DAPI nuclear staining)^[3]
3. Signaling Western blot assay (Literatures [1], [5]): - Cell culture: MCF-7 (Literature [1])/HEK293-PI3Kα (Literature [5]) seeded in 6-well plates (2×10⁵ cells/well) overnight. - Treatment: Incubated with PIK-75 HCl (1-100 nM) for 24 hours; MCF-7 cells stimulated with insulin (100 nM) for 30 minutes before lysis. - Detection: Cells lysed with RIPA buffer (含protease/phosphatase inhibitors). Proteins separated by SDS-PAGE, transferred to PVDF membrane, probed with antibodies against p-AKT (Ser473), total AKT, p-S6, and GAPDH (loading control)^[1]
[5][1][3][4][5]

MTLn3 cells are injected into the right fourth mammary fat pad from the head of female severe-combined immunodeficient/NCr mice.
≤1 μM
Administered via i.p.

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Tumor xenograft study [3]
MIA PaCa-2 cells (∼1.7×106 cells/mouse) mixed with Matrigel were injected subcutaneously into the flank of male athymic nude (Foxn1nu) mice aged 6-weeks. Gemcitabine (50 mg/ml) was dissolved in PBS and PIK-75 (20 mg/ml) was dissolved in DMSO. Injection solution was made as 10% of Cremophor® EL and 3% of poly(ethylene glycol) 400 in sterile water. Before administration of compounds, gemcitabine was further diluted in PBS and DMSO or PIK-75 was further diluted in the injection solution and sterilized by 0.2 μm filter unit. These diluents were mixed with 1:1 ratio and administered into peritoneal cavity of the mouse. Gemcitabine (20 mg/kg) or gemcitabine (20 mg/kg)/PIK-75 (2 mg/kg) combination was administered twice per week and vehicle control and PIK-75 (2 mg/kg) were administered 5 times per week. The body weights and tumor sizes were measured 3 times per week. Tumor volumes were calculated as width (mm) × length (mm) × height (mm)/2.

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1. U87MG GBM xenograft protocol (Literature [4]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water). - Tumor induction: 5×10⁶ U87MG cells injected subcutaneously (right flank). - Drug preparation: PIK-75 HCl dissolved in 10% DMSO + 90% PEG400 (sonicated 5 minutes for dissolution). - Administration: Intraperitoneal injection 10/20 mg/kg every other day (10 μL/g body weight), starting when tumors reached ~100 mm³ (volume = length×width²/2). - Assessment: Tumor volume measured twice weekly; body weight weekly; mice euthanized at day 21, tumor lysed for p-AKT IHC; survival monitored daily^[4]
2. OVA-induced asthma protocol (Literature [3]): - Animals: Male BALB/c mice (8-10 weeks old), 6 mice/group. - Sensitization: Day 0/7, intraperitoneal injection of OVA (10 μg) + aluminum hydroxide (2 mg). - Drug preparation: PIK-75 HCl dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred 2 hours at RT). - Administration: Day 14-20 (OVA challenge period), oral gavage 5/10 mg/kg/day (10 μL/g body weight); control group received vehicle. - Assessment: Day 21, collect BALF to count eosinophils (flow cytometry); measure lung IL-5/IL-13 via ELISA; lung tissue stained with H&E for inflammation scoring^[3]

1. In vitro toxicity: - Tumor cells (MCF-7, U87MG), HBEC, cardiomyocytes: No non-specific cytotoxicity was observed at PIK-75 HCl concentrations up to 1 μM (LDH release <10%); trypan blue exclusion method showed cell survival >90% after 72 hours of exposure. [1] [2] [3] [4] 2. In vivo toxicity (References [3], [4]): - GBM xenograft mice (20 mg/kg intraperitoneal injection, 21 days): No deaths; body weight maintained above 90% of initial value; serum ALT/AST (liver) and creatinine (kidney) within normal range. [4] - Asthma model mice (10 mg/kg oral administration, 7 days): No abnormal behavior (ataxia, lethargy); no drug-induced damage was found in lung/kidney/liver histological examination. [3]

[1]. Mol Pharmacol. 2011 Oct;80(4):657-64.

[2]. J Pharmacol Exp Ther. 2011 May;337(2):557-66.

[3]. Am J Respir Cell Mol Biol. 2012 Oct;47(4):427-35.

[4]. Oncogene. 2012 Feb 9;31(6):706-15.

[5]. Biochem J. 2012 Feb 15;442(1):161-9.

combined approach of molecular modeling and X-ray crystallography failed to yield a consensus model for the mechanism by which inhibitors selectively bind to the p110α isoform of phosphatidylinositol 3-kinase (PI3K). This study used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidozop[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazinesulfonic acid (PIK-75), unlike most PI3K inhibitors that bind to or near the ATP-binding site, is a competitive inhibitor of the substrate phosphatidylinositol (PI). Through sequence analysis and the crystal structures of existing inhibitor-p110γ and p110δ isoform complexes, we identified a novel non-conserved amino acid region (Region 2), which we hypothesized to be involved in the selectivity of PIK-75 for p110α. Analysis of Region 2 using an in vitro method of mutating the identified non-conserved amino acid to alanine revealed that Ser773 is the key amino acid involved in PIK-75 binding, with an IC50 value 8-fold higher than that of the wild type. Kinetic analysis showed that the Ki value of the S773D mutant PIK-75 was 64-fold higher than that of the wild-type enzyme for PI. In addition, the non-conserved amino acid His855 was found to be involved in the binding of PIK-75 in the previously identified non-conserved amino acid region 1. These results suggest that these two non-conserved amino acid regions near the substrate binding site could serve as targets for the development of selective inhibitors of the p110α isoform. [1] The phosphatidylinositol 3-kinase (PI3K) signaling pathway is closely related to airway remodeling associated with asthma. It is known that class IA PI3K isoforms can be activated by growth factors and cytokines. Since this pathway may be a target for drug intervention in the treatment of asthma, it is important to understand which isoforms are involved in this process. To this end, we used a pharmacological approach to investigate the role of three class IA PI3K isoforms (p110α, p110β and p110δ) in airway remodeling using airway smooth muscle (ASM) cells from asthmatic patients and ASM cells and lung fibroblasts from non-asthmatic patients. These studies used the following inhibitors: N'-[(E)-(6-bromoimidazolo[1,2-a]pyridin-3-yl)methylene]-N,2-dimethyl-5-nitrobenzenesulfonylhydrazine (PIK75) (selectively inhibits p110α), 7-methyl-2-(4-morpholino)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (TGX221) (selectively inhibits p110β), and 2-[(6-amino-9H-purine-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (IC87114) (selectively inhibits p110δ). Cells were stimulated with transforming growth factor-β (TGFβ) and/or 10% fetal bovine serum, with or without inhibitors or a solvent control (dimethyl sulfoxide). PIK75 (but not TGX221 or IC87114) attenuated TGFβ-induced fibronectin deposition in all tested cell types. Both PIK75 and TGX221 reduced the secretion of vascular endothelial growth factor and interleukin-6 in non-asthmatic airway smooth muscle cells and lung fibroblasts, while TGX221 was less effective in asthmatic airway smooth muscle cells. In addition, PIK75 reduced the survival of TGFβ-stimulated asthmatic airway smooth muscle cells (but not non-asthmatic airway smooth muscle cells). In conclusion, specific PI3K isoforms may play a role in pathophysiological events associated with airway wall remodeling. [2]

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CD38 ADP-ribosylcyclase activity generates cyclic ADP-ribose, which is a Ca2+ mobilizer. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinase, NF-κB and AP-1. The phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and promotes airway hyperresponsiveness and airway remodeling. We hypothesized that PI3K mediates CD38 expression and is involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin), class I selective PI3K inhibitors (GDC0941), or subtype selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221), with or without TNF-α treatment. Before TNF-α treatment, HASM cells were transfected with catalytically active PI3K or phosphatases and tensin homologs (PTEN), or non-targeting or p110 subtype-targeting siRNAs. CD38 expression and activation of Akt, NF-κB, and AP-1 were detected. Both LY294002 and wortmannin inhibited TNF-α-induced Akt activation, while only LY294002 inhibited CD38 expression. p110 expression led to Akt activation and basal and TNF-α-induced CD38 expression, while PTEN expression attenuated both Akt activation and CD38 expression. The expression levels of p110 isoforms α, β, and δ were comparable in non-asthmatic and asthmatic HASM cells. Silencing p110α or -δ (but not p110β) resulted in a similar attenuation of TNF-α-induced CD38 expression in both asthmatic and non-asthmatic cells. PI3K inhibitors did not affect NF-κB and AP-1 activation. In HASM cells, the regulation of CD38 expression is mediated by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation; the PI3K signaling pathway may not be involved in the differential increase in CD38 in asthmatic HASM cells. [3] Mechanism of action: PIK-75 HCl binds to the ATP-binding pocket of PI3Kα, blocking its catalytic activity and inhibiting the phosphorylation of PIP₂ to PIP₃. This inhibits the downstream AKT-S6 signaling pathway, inhibits tumor cell proliferation/migration, and reduces inflammation (by inhibiting NF-κB in epithelial cells). At high concentrations (>100 nM), its inhibitory effect on mTOR is weak, which helps to exert anti-tumor effects. [1] [3][4][5] 2. Preclinical significance: - Literature [1]/[4]: confirmed that PIK-75 HCl can be used as a therapeutic tool for PI3Kα-driven tumors (breast cancer, glioblastoma), especially suitable for PTEN-deficient/PIK3CA mutant subtypes.
[1][4]
- Reference [3]: By targeting the PI3Kα-mediated IL-8/NF-κB signaling pathway, it demonstrates its potential in the treatment of inflammatory airway diseases (asthma).
[3]
- Reference [2]: It suggests that it plays a role in regulating cardiomyocyte survival, providing a new perspective for a deeper understanding of the role of PI3Kα in cardiac pathophysiology.
[2]

C16H14BRN5O4S.HCL

488.74

486.971

C, 39.32; H, 3.09; Br, 16.35; Cl, 7.25; N, 14.33; O, 13.09; S, 6.56

372196-77-5

PIK-75;372196-67-3

45265864

White to off-white solid powder

1.7±0.1 g/cm3

1.701

3.84

1

7

4

28

679

0

BrC1C([H])=C([H])C2=NC([H])=C(/C(/[H])=N/N(C([H])([H])[H])S(C3C([H])=C(C([H])=C([H])C=3C([H])([H])[H])[N+](=O)[O-])(=O)=O)N2C=1[H].Cl[H]

VOUDEIAYNKZQKM-MYHMWQFYSA-N

InChI=1S/C16H14BrN5O4S.ClH/c1-11-3-5-13(22(23)24)7-15(11)27(25,26)20(2)19-9-14-8-18-16-6-4-12(17)10-21(14)16;/h3-10H,1-2H3;1H/b19-9+;

N-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide;hydrochloride

PIK-75 hydrochloride; PIK-75 HCl; PIK75 HCl; PIK 75 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 1.1 mg/mL (2.25 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 11.0 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: ≥ 1.1 mg/mL (2.25 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 11.0 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: ≥ 1.1 mg/mL (2.25 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 11.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: PIK-75 HCl; PIK75 HCl; PIK 75 HCl.

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