The target of C188-9 is Signal Transducer and Activator of Transcription 3 (STAT3), and it also has potent activity against STAT1.
- For STAT3: In SPR assays, the IC₅₀ for inhibiting recombinant STAT3 binding to phosphododecapeptide ligand is not explicitly given in numerical value, but it binds to STAT3 with high affinity (K_{D} was calculated from sigmoidal binding curve with concentrations of C188-9 ranging from 0.305 to 10,000 nM) [3]
- For G-CSF-induced pSTAT3 in AML cell lines: IC₅₀ values are between 4 and 8 μM [2,4]
- For constitutive pSTAT3 in UM-SCC-17B (HNSCC cell line): IC₅₀ was calculated from dose-response curves with C188-9 concentrations ranging from 0 to 100 μM [3]
- For constitutive pSTAT1 in UM-SCC-17B (HNSCC cell line): IC₅₀ was calculated from dose-response curves with C188-9 concentrations ranging from 0 to 100 μM [3]

C188-9 (Kd = 4.7 nM) is a Stat3 inhibitor [1]. The range of 4-7 μM and 8-18 μM is where C188-9's IC50s for inhibiting Stat3 activation in AML cell lines and primary AML samples are found. In order to measure apoptotic cells for annexin V-labeled cells in apoptosis investigations, C188-9 is applied to AML cell lines and primary samples for a duration of 24 hours. From 6 μM to over 50 μM, the EC50s for inducing apoptosis exhibit significant variability [2].

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1. In cancer cachexia-related cell models: C188-9 blocked the activation of p-Stat3 in C2C12 myotubes induced by conditioned medium from C26 colon carcinoma cells (15 min treatment). It reduced the average size loss of C2C12 myotubes after 72-h incubation in C26-conditioned medium, suppressed the expression and activation of caspase-3 (pro-caspase-3 and cleaved caspase-3), and inhibited the upregulation of C/EBPδ and myostatin in C2C12 myotubes treated with C26-conditioned medium. Additionally, it decreased the loss of myosin heavy chain protein in C2C12 myotubes induced by C26-conditioned medium (72 h treatment), and reduced the mRNA levels of MAFbx/Atrogin-1 and MuRF-1 in C2C12 myotubes (24 h treatment) [1]
2. In AML cell lines: C188-9 inhibited G-CSF-induced Stat3 phosphorylation in a dose-dependent manner (1 h pretreatment with C188-9 followed by 15 min stimulation with 100 ng/mL G-CSF), with IC₅₀ values between 4 and 8 μM for 6 AML cell lines, but did not alter total Stat3 protein levels. It did not affect the tyrosine phosphorylation levels of most tyrosine kinases on a tyrosine kinase protein array, nor inhibit the activity of the MAPK/ERK pathway or the Akt pathway (assessed by pERK1/2 and pAkt immunoreactivities). It inhibited G-CSF-induced expression of Stat3 target genes (SOCS3 and PIM1 mRNA) in 3 AML cell lines (10 μM C188-9 pretreatment for 1 h followed by 1 h stimulation with 100 ng/mL G-CSF), and induced apoptosis in AML cell lines after 24 h treatment (quantified by annexin V–PE labeling, EC₅₀ values estimated from dose-response curves) [2,4]
3. In primary pediatric AML samples: C188-9 inhibited G-CSF-induced pStat3 in a dose-dependent manner (assessed by FACS), induced apoptosis in CD34⁺ primary AML cells after 48 h incubation (quantified by annexin V staining), and reduced colony formation in primary AML samples grown in methylcellulose medium with growth factors (colonies ≥ 30 cells, normalized to untreated control) [2,4]
4. In HNSCC cell lines: C188-9 inhibited constitutive pSTAT3 and pSTAT1 in UM-SCC-17B cells in a dose-dependent manner after 24 h treatment (0 to 100 μM). It inhibited anchorage-dependent growth of UM-SCC-17B cells after 48 h treatment (0 to 100 μM, viable cells quantitated by MTT, IC₅₀ calculated from dose-response curves), and inhibited anchorage-independent growth of multiple HNSCC cell lines (SCC-35, SCC-61, UM-SCC-17B, HN30) after 72 h treatment (IC₅₀ values calculated from dose-response curves) [3]

Out of the roughly 13,528 distinguishable genes, C188 modifies the levels of 37 gene transcripts (17 down-regulated and 20 up-regulated, fdr <0.01, fold change≥1.5), 7 of which are identified as STAT3 gene targets. 76 genes that were previously identified as being regulated by STAT3 (38 down-regulated and 38 up-regulated) are among the much larger number of oncogenesis-related genes that C188-9 affects (384 total, 95 down- and 289 up-regulated). Naturally, C188-9 treatment results in the downregulation of 24 (63%) of the 38 genes that have been previously reported to be upregulated by STAT3. Furthermore, C188-9 downregulated 10 additional genes (fdr <0.01, fold change ≥1.5) that had previously been demonstrated to be upregulated by STAT1. Thus, STAT1 has been shown to positively regulate 40 of 48 (83.3%) genes that were previously downregulated by C188-9, including sixteen genes that have been demonstrated to be co-regulated by STAT3 and STAT1. This analysis suggests that C188-9 may affect both STAT3 and STAT1 in order to mediate its effect on gene transcript levels in HNSCC tumors[3].

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1. In cancer cachexia animal models: CD2F1 mice bearing C26 tumors (5 days post-tumor implantation) were treated with C188-9 or D5W twice daily for 14 days. C188-9 suppressed the loss of body weight and muscle mass (gastrocnemius, tibialis anterior), improved the distribution of myofiber sizes in tibialis anterior muscles, increased muscle grip strength, enhanced muscle protein synthesis, and reduced protein degradation in soleus and extensor digitorum longus (EDL) muscles. It also decreased the expression of C/EBPδ, myostatin, MAFbx/Atrogin-1, and the 14-kDa actin fragment (indicator of caspase-3 activity) in gastrocnemius muscles, and inhibited proteasome activity in muscle tissues (measured by fluorogenic peptide LLVY-AMC as substrate) [1]
2. In HNSCC xenograft models: Athymic nude mice (8–10 week old male) were injected with 1.5 × 10⁶ UM-SCC-17B cells into the tongues. Once tumors were established (average volume ~15-20 mm³), mice were treated with intraperitoneal injections of C188-9 (100 mg/Kg) 5 times a week. C188-9 prevented tumor xenograft growth (tumor volumes measured twice weekly, normalized to volume at first day of treatment, p<0.05 by t test), and reduced pSTAT3 and pSTAT1 levels in tumor lysates (assessed by Western blot, normalized to β-actin) [3]

1. SPR assay for STAT3-ligand binding inhibition: Recombinant STAT3 (200 nM) was incubated with different concentrations of C188-9 (0.1 to 1000 μM), then added to a Biacore sensor-chip immobilized with phosphododecapeptide ligand (12 amino acids surrounding and including pY1068 within EGFR). The equilibrium binding levels were measured, normalized (resonance with compound ÷ resonance without compound × 100), and plotted against Log [nM] C188-9 to calculate IC₅₀ [3]
2. Fluorescence-based binding assay for STAT3 affinity: A constant concentration (80 nM) of fluorescently labeled STAT3 (aa residues 127-722) was incubated with increasing concentrations of C188-9 (0.305 to 10,000 nM). Fluorescence was measured continuously before and after application of an infrared laser. The change in fluorescence (F_{norm}) was calculated (ratio of fluorescence immediately before heating and 30 seconds after heating), plotted against the logarithm of C188-9 concentrations, and the dissociation constant K_{D} was calculated from the sigmoidal binding curve [3]
3. Luminex assay for pSTAT3/pSTAT1 quantification: Cell lysates (from AML or HNSCC cell lines treated with C188-9) were assayed for pSTAT3, pSTAT1, and GAPDH levels by Luminex. GAPDH-normalized pSTAT3 or pSTAT1 values were divided by the ratio for untreated cells and expressed as percentage, then plotted against Log [M] C188-9 to calculate IC₅₀ [3,4]

1. C2C12 myotube assay (cancer cachexia): C2C12 myotubes were treated with conditioned medium from C26 colon carcinoma cells with or without C188-9 for different durations (15 min, 24 h, 72 h). Western blot was performed to detect p-Stat3, Stat3, caspase-3, cleaved caspase-3, pro-caspase-3, C/EBPδ, myostatin, and myosin heavy chain. The average size of C2C12 myotubes was measured after 72 h incubation. RT-PCR was used to detect mRNA levels of MAFbx/Atrogin-1 and MuRF-1 after 24 h treatment [1]
2. AML cell line assay: AML cell lines (Kasumi-1, THP-1, etc.) were serum-starved for 1 hour, pretreated with C188-9 (0 to 10 μM) for 1 hour, then stimulated with 100 ng/mL G-CSF for 15 minutes. FACS was used to analyze pStat3 levels (gated by forward/side scatter to exclude debris, pStat3⁻/pStat3⁺ gates based on isotype control). Western blot was performed to detect pStat3, total Stat3, pERK1/2, and pAkt. Quantitative RT-PCR was used to measure SOCS3 and PIM1 mRNA levels (ΔΔC_{t} method). Apoptosis was quantified by annexin V–PE labeling after 24 h treatment with C188-9 [2,4]
3. Primary AML cell assay: Primary pediatric AML samples were treated with C188-9 for 48 h, and apoptosis was quantified by annexin V staining. For colony formation assay, primary AML samples were plated in methylcellulose medium with growth factors and increasing doses of C188-9, colonies (≥30 cells) were counted after 10–14 days, and normalized to untreated control [2,4]
4. HNSCC cell assay: UM-SCC-17B, SCC-35, SCC-61, and HN30 cells were treated with C188-9 (0 to 100 μM) for 24–72 h. For pSTAT3/pSTAT1 detection, cell lysates were analyzed by Luminex. For anchorage-dependent growth assay, cells were cultured in 96-well plates for 48 h, viable cells were quantitated by MTT, relative % viability was calculated (viability after treatment ÷ viability of untreated cells × 100), and IC₅₀ was calculated from dose-response curves. For anchorage-independent growth assay, cells were treated for 72 h, and IC₅₀ was calculated similarly [3]

D5W (5g of dextrose in 100 ml of water); 12.5 mg/kg; i.p.
CD2F1 female mice received isogenic C26 tumor cell via s.c injection

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1. Cancer cachexia model (CD2F1 mice): C26 colon carcinoma cells were implanted into CD2F1 mice to induce cachexia. After 5 days of tumor implantation, mice were treated with C188-9 or D5W twice daily for 14 days (administration route not specified). At the end of treatment, body weight, muscle mass (gastrocnemius, tibialis anterior), myofiber size distribution, muscle grip strength, muscle protein synthesis/degradation rates, and protein expression (caspase-3, C/EBPδ, myostatin, MAFbx/Atrogin-1) in gastrocnemius muscles were measured. Proteasome activity in muscle tissues was assessed using fluorogenic peptide LLVY-AMC as substrate [1]
2. HNSCC xenograft model (nude mice): 1.5 × 10⁶ UM-SCC-17B cells were injected into the tongues of athymic, 8–10 week old, male nude mice. Once tumors reached an average volume of ~15-20 mm³, mice were randomized into groups (5 mice/group) and received intraperitoneal injections of DMSO, C188 (50 mg/Kg), or C188-9 (100 mg/Kg) 5 times a week. Tumor volumes were measured twice weekly (calculated as 0.5 × (long dimension) × (short dimension)²), normalized to the volume at the first day of treatment. After treatment, mice were euthanized, tumor lysates were prepared, and Western blot was performed to detect pSTAT3, total STAT3, β-actin, pSTAT1, and total STAT1 [3]

[1]. Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia. J Biol Chem. 2015 Apr 24;290(17):11177-87.

[2]. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood. 2011 May 26;117(21):5701-9.

[3]. Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma. Oncotarget. 2016 May 3;7(18):26307-30.

[4]. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood. 2011;117(21):5701-5709.

C-188-9, a STAT3 inhibitor, is an orally bioavailable binatol sulfonamide inhibitor of signal transduction and transcription activator 3 (STAT3) with potential antitumor activity. After oral administration, C-188-9 specifically targets and binds to the phosphotyrosine peptide binding site within the Src homology 2 (SH2) domain of STAT3. This inhibits Janus kinase (JAK)-mediated STAT3 tyrosine phosphorylation and activation. This prevents STAT3 nuclear translocation, blocks STAT3 binding to response gene promoters, and disrupts STAT3-mediated gene expression regulation. STAT3 regulates gene transcription involved in various cellular functions. STAT3 is persistently activated in various human cancers and plays a crucial role in tumor transformation, uncontrolled tumor cell proliferation, anti-apoptosis, metastasis, immune escape, tumor angiogenesis, epithelial-mesenchymal transition (EMT), and the Warburg effect.

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1. C188-9 is a novel small molecule Stat3 inhibitor derived from C188 (a previously discovered Stat3 inhibitor) through a lead compound discovery program. It binds to STAT3 with high affinity and is significantly improved over C188 in inhibiting the binding of STAT3 to its pY peptide ligand, cytokine-stimulated pSTAT3, and constitutive pSTAT3/STAT1 activity in cancer cells [3]
2. Mechanism of action in cancer cachexia: C188-9 inhibits Stat3 activation, thereby inhibiting caspase-3 (by reducing its transcription and activity) and the ubiquitin-proteasome system (by inhibiting the C/EBPδ → myostatin → MAFbx/Atrogin-1/MuRF-1 pathway), ultimately maintaining muscle mass by reducing protein loss in muscle [1]
3. Mechanism of action in cancer cells: C188-9 selectively inhibits the phosphorylation and activity of Stat3 (and STAT1), blocks the expression of Stat3-dependent genes (SOCS3, PIM1), induces cancer cell apoptosis, and inhibits cancer cell proliferation (anchor-dependent and non-anchor-dependent). In head and neck squamous cell carcinoma (HNSCC), it regulates STAT3-regulated genes involved in tumorigenesis and radioresistance, as well as STAT1-regulated radioresistance genes [2,3,4]. 4. Clinical application potential: C188-9 has the potential to treat cancer cachexia, acute myeloid leukemia (AML), and radioresistant head and neck squamous cell carcinoma (HNSCC), and can be used alone or in combination with radiotherapy. Baylor College of Medicine (DJT as the principal inventor) has filed patents for C188 and C188-9, and StemMed Ltd. holds an exclusive license to these compounds [3].

C27H21NO5S

471.52

471.114

C, 68.78; H, 4.49; N, 2.97; O, 16.97; S, 6.80

432001-19-9

1324494

Pinky beige solid powder

1.4±0.1 g/cm3

680.9±65.0 °C at 760 mmHg

365.6±34.3 °C

0.0±2.2 mmHg at 25°C

1.732

5.24

3

6

5

34

776

0

S(C1C=CC(=CC=1)OC)(NC1=CC(=C(C2=CC=CC=C12)O)C1=C(C=CC2=CC=CC=C12)O)(=O)=O

QDCJDYWGYVPBDO-UHFFFAOYSA-N

InChI=1S/C27H21NO5S/c1-33-18-11-13-19(14-12-18)34(31,32)28-24-16-23(27(30)22-9-5-4-8-21(22)24)26-20-7-3-2-6-17(20)10-15-25(26)29/h2-16,28-30H,1H3

N-[4-hydroxy-3-(2-hydroxynaphthalen-1-yl)naphthalen-1-yl]-4-methoxybenzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (4.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)