RORγ

Compound 37c/Cintirorgon (LYC-55716) was evaluated further in a PK/PD assay. RORγt is highly expressed in the thymus and regulates the expression of genes important for the survival and development of thymocytes. The expression of RORγ target genes such as BclXL and Fbxo27 in thymus can be increased by RORγ agonists. Female C57/BL6 mice were dosed with vehicle (1% Tween 80) or RORγ agonist Cintirorgon (LYC-55716) (37c) at various doses and 6–16 h later, thymocytes were isolated, RNA was prepared and the expression of RORγ target genes was assayed using Q-PCR. Samples were collected contemporaneously for the determination of Cintirorgon (LYC-55716) plasma concentrations. The increase in RORγ target gene expression is plotted in Figure 3 versus plasma concentration and the data fitted to obtain an EC50 for 37c of 338 nM (95% confidence interval = 202–564 nM). [2]

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Select RORγ agonists were also evaluated in mouse syngeneic tumor models. Cell lines derived from murine tumors, either spontaneous or chemically induced, and reimplanted into immunologically competent mice have been used to evaluate other immunotherapies. In our mouse colorectal adenocarcinoma (MC38) tumor model, MC38 tumor cells were implanted subcutaneously into the flank of female C57/BL6 mice. Agonist treatment was initiated 3 days post-tumor implant and continued twice daily by oral gavage. Figure 4 illustrates the results from tumor-bearing mice treated with vehicle or 37c (30 mg/kg, bid). This study demonstrated a 46% inhibition in tumor growth (p < 0.05). The study was repeated two additional times with similar reductions of tumor growth (49 and 63%, p < 0.05). Tumor weights were evaluated at the end of the study. A similar reduction of tumor weight was also observed. The compound was well tolerated in all studies with no adverse effect on animal weights. Compounds 37c and 17 also inhibited tumor growth in a 4T1 breast tumor model. [2]

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Cintirorgon (LYC-55716) , a RORγ agonist, is administered orally and forms a receptor complex with the nuclear receptor transcription factor RORγ. The drug then translocates to the nucleus where it binds to the ROR response element (RORE) to improve function. Type 17 T cells, such as Th17 (helper T cells) and Tc17 (cytotoxic T cells), proliferate and survive. Th17/Tc17 differentiation is facilitated by the nuclear receptor transcription factor RORγ, which is also essential for immune activation. While next generation immuno-oncology medications, such as PD-1/PD-L1 inhibitors, are provided by injection, Cintirorgon (LYC-55716) is also bioavailable when taken orally [1].

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Pharmacodynamics[3]
The pharmacodynamics/target engagement assay assessed both the mRNA and protein expression of known RORγ target genes: IL-17A, IL-17F, and IL-22 following an ex vivo stimulation (19, 20). The modulation of these genes by RORγ agonists was confirmed in in vitro work with both healthy volunteer and cancer patient peripheral blood mononuclear cells (PBMC) and blood (22). As detailed above, blood samples were collected from all patients at various time points on days 1, 2, and 15 and shipped to a central laboratory for testing. Pharmacodynamic profiles for all analytes showed considerable intra- and inter-patient variability with regard to fold induction, absolute titer of cytokine, and timing of PDmax (see Supplementary Table S2 and representative data shown in Supplementary Fig. S2). The variability observed was not unexpected, given the patient-specific differences in baseline levels of RORγ expression and endogenous RORγ agonists (Supplementary Fig. S3; ref. 23). A formal PK/PD analysis was complicated both by the variability in the magnitude of each patient's response as well as the paucity of samples with plasma concentrations below the exposure associated with target gene modulation in rodents. However, pharmacodynamic data indicated that target engagement had occurred and provided qualitative evidence of a pharmacodynamic response, evidenced by post-treatment increases in cytokine production (Fig. 2A). In addition, 31 of the 32 patients showed greater than 2-fold induction for at least 1 of the pharmacodynamic readouts, which is consistent with the pharmacokinetic evidence for Cintirorgon (LYC-55716) exposure levels in the predicted efficacious range.

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Efficacy[3]
Of the 32 patients in the study, 25 were evaluable for response. Seven were not evaluable because of progressive disease within 6 weeks (n = 4), patient withdrawal (n = 2), and AE (n = 1). Of the evaluable patients, 2 (8%) achieved partial response and 11 (44%) achieved stable disease for a range of 2 to 12 months. Both of the partial responders and 6 (24%) of the patients with stable disease received >4 months of treatment with Cintirorgon (LYC-55716) (Fig. 3). One partial response was noted in a patient with non–small cell lung cancer [NSCLC; adenocarcinoma; PD-L1 tumor-proportion score (TPS) >50%] who had received first-line treatment with pembrolizumab (4 cycles, progression at first assessment), followed by carboplatin/pemetrexed (3 cycles, progression at first assessment) before entering the study (Fig. 4A). The time to initial response was approximately 6 months from the start of treatment; partial response was confirmed by independent central radiology review. A second partial response, also confirmed by independent central radiology review, was noted in a patient with sarcomatoid breast cancer (metastatic spindle cell carcinoma) who had received first-line treatment with carboplatin/paclitaxel (3 cycles, progression at first assessment), followed by gemcitabine/docetaxel (progression after 6 cycles) before the study (Fig. 4B). In this patient, the time to initial response was 8 months, following an incremental reduction in tumor burden before that point. Two patients remain on treatment as of June 2018: The patient with sarcomatoid breast cancer with partial response and a patient with endometrial cancer who has had disease stabilization for 9 months. Twelve (48%) patients had progressive disease as their best response. Two patients with colorectal cancer were followed under irRECIST after first assessment through discontinuation for progression at the end of 4 cycles.

Biological Assays for Agonist Activity Towards ROR [2]
Compounds were tested for ability to increase ROR activity using (i) a ROR-Ligand Binding Domain (LBD) TR-FRET Assay, and (ii) a Gal4-RORγ Luciferase Reporter Assay in HEK-293T Cells. Assay procedures are described below.

(i) Procedures for ROR-Ligand Binding Domain TR-FRET Assay [2]
HIS-tagged ROR-LBD protein was expressed in SF9 cells using a baculovirus expression system. The lysate was diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 0.01% BSA) to obtain ROR-LBD final concentration of ~3 nM in a 384-well assay plate (need to titrate for each batch of protein). A stock of biotinylated-LXXLL peptide from coactivator SRC1 (BiotinCPSSHSSLTERHKILHRLLQEGSPS) was prepared in assay buffer and added to each well (200 nM final concentration). A solution of Europium tagged anti-HIS antibody (0.6 nM final concentration) and APC-conjugated streptavidin (30 nM final concentration) were also added to each well. ROR antagonist ursolic acid was also included at a final concentration of 2 µM. Compounds were diluted in DMSO and further diluted in assay buffer with a final DMSO concentration at 1%. The highest concentration of test compound analyzed was 10 µM. [2]
The final assay mixture was incubated overnight at 4 °C or 2 hours at room temperature, and the fluorescence signal was measured on an Envision plate reader: (Excitation filter = 340 nm; APC emission = 665 nm; Europium emission = 615 nm; dichroic mirror = D400/D630; delay time = 100 µs, integration time = 200 µs). 50% Effective concentration (EC50) values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm. The quotient of the fluorescence signals in the absence of ursolic acid or test compound is set as 100. Max Response is defined as the upper plateau in the signal as determined by line-fit using a 4-parameter logistic model in PRISM. [2]

(ii) Procedures for Gal4-RORγ Luciferase Reporter Assay in HEK-293T Cells [2]
Transfection of HEK-293 Cells In the following protocol, HEK-293 cells were transfected with a construct comprising the Gal4 DNA binding domain fused to the ligand binding domain of RORγ (Gal4-RORγ-LBD) in a pcDNA3.1neo plasmid, and also with a reporter construct comprising pGL4.31 Gal4-luciferase. Control cells were prepared similarly using empty pcDNA3.1neo and pGL4.31 vectors. Trans-IT reagent (60 µL) at room temperature was added drop wise to OptiMEM (Invitrogen, 1.5 ml). This reagent mixture was mixed by inversion then incubated for 5 to 30 minutes at room temperature. It then was added to a solution of both expression vectors (5 µg each), mixed, and incubated at room temperature for about 20 minutes. HEK-293 cells were harvested from incubation flasks by removing the media, treating with TrypLE Express, and incubating until the cells detached from the bottom of the flask (approximately 2-5 minutes). 10 Million cells were collected by centrifugation and re-suspended in 10 mL of Dulbecco’s Modified Eagle Medium, High Glucose containing 10% Fetal Bovine Serum and 100 IU each of penicillin and streptomycin. The re-suspended cells and the S22 transfection mixture were added to a T75 flask, mixed and incubated overnight at 37 °C and 5% CO2.

Cellular Assay for RORγ Activity [2]
The cells were harvested as described above, counted, and centrifuged to obtain the desired number of cells, then re-suspended in complete growth media at 0.75 x 106 cells/mL. The RORγ antagonist, ursolic acid, was added to the cells at a final concentration of 2 µM. Cells were plated at 20 µL of cell suspension/well (10,000-15,000 cells/well) in white tissue culture treated 384 well plates. Test compounds were dissolved at 10 mM in DMSO then diluted into complete growth medium to 5x the final intended test concentration. These drug stock solutions, 5 µL/well were added to the tissue culture plate. The final DMSO concentration was 0.2%. The plates were briefly centrifuged then incubated overnight at 37 °C and 5% CO2. To conduct the assay, the tissue culture plates were allowed to equilibrate to room temperature and One-Glo luciferase reagent (5 µL/well) was added. The plates were briefly centrifuged then incubated at room temperature for 10 minutes. The luciferase intensity was read on an Envision plate reader. RORγ activity was determined relative to controls and plotted as a function of test compound concentration using PRISM to determine a 50% effective concentration (EC50). The luciferase signal in the absence of ursolic acid or test compound is defined at 100. The Max Response is the upper plateau in the signal as determined by line-fit using a 4-parameter logistic model in PRISM.

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Human Whole Blood Assay Method [2]
Briefly, human whole blood was collected into sodium-heparin tubes and T cell activators were added. Compound was added and samples were incubated 18 to 22 hours at 37 °C with 5% CO2. RNA was purified by Trizol/chloroform and RNeasy kits then used for RT-PCR and qPCR. S23  Dilution Ranges Tested of compounds 10, 3, 1, 0.3, 0.1, 0.03, 0 µM Procedures Assay and T cell Stimulation Conditions  Collect fresh blood in Sodium-Heparin tubes (50 mL)  Mix T cell activators with blood under 3 conditions:  Activator-1: Dynabeads human T-Activator CD3/CD28 at 15 µL/mL final; IL-1β, and IL23 final at 25 ng/mL final;  Activator-2: add soluble anti-CD3 and anti-CD28 final at 1 µg/mL final, IL-1β, and IL-23 at 10 ng/mL final  Activator-3: add PMA at 10 ng/mL and Ionomycin at 1 µg/mL final  Add 5 µL of 1000x drug stock or DMSO to 5 mL activated blood. The final DMSO concentration is 0.1%.  Aliquot 1.5 mL of blood per well in a 12-well tissue culture plate for triplicates  Incubate at 37°C with 5% CO2 for 18 to 22 hours

Pharmacodynamic Assay [2]
Retinoic acid receptor-related orphan receptor gamma (RORγ) is a transcription factor associated with thymocyte differentiation and maturation, as well as with Type 17 T cell differentiation and function. Activation of RORγ enhances thymocyte survival and induces a transcriptional program which drives Type 17 immune responses and decreases immune suppressive mechanisms. Small molecule agonists of RORγ modulate the expression of target genes including those involved in pro-survival pathways in thymocytes and release of cytokines and chemokines by mature immune cells. In the study, female C57BL/6 mice were given single oral doses of Cintirorgon (LYC-55716) ranging from 3 to 100 mg/kg and euthanized after 6, 10, or 16 hours (n of 4, 5, or 10 animals/time point or dose group). Thymi were collected as a tissue harboring high percentage of RORγ+ cells, RNA was extracted and expression of select RORγ target genes (Fbxo27, Xkrx, ReverbA, BclXL) was S26 analyzed via real-time quantitative polymerase chain reaction (qPCR) to determine the pharmacodynamic (PD) effects of Cintirorgon (LYC-55716) . Plasma samples were also collected at the same time thymi were taken and Cintirorgon (LYC-55716) concentration was determined. Fbxo27 showed the most consistent and robust window of induction following Cintirorgon (LYC-55716) treatment, and thus was used in determining the half-maximal effective concentration (EC50) of Cintirorgon (LYC-55716) . Based on Fbxo27 induction, the EC50 of LYC 55716 is 204 ng/mL (338 nM).

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Syngeneic Tumor Models [2]
MC38 murine colon carcinoma cells or 4T1 murine breast carcinoma cells were implanted subcutaneously into the flank of C57/BL6 or Balb/c mice, respectively. Three days after implantation, mice were dosed with vehicle (1% Tween 80) or test compound at doses noted in text twice a day. Tumor volume, measurable 10–12 days after implantation, was assessed two to three times weekly using caliper measurement of length and width of tumor. Tumor volume calculation = 0.5 x (length x (width)2 ). SCID.beige mice were also used as host mice for MC38 tumor cells to determine the immune system dependence. Mice were taken down after tumor volume reached ethical end point of 2,000 mm3 or at 24 day

Pharmacokinetics [3]
In this study, robust pharmacokinetic sampling was performed on several days during cycle 1. At steady state (day 29), exposures increased linearly from the lowest dose of 150 mg BID through the highest dose of 450 mg BID with exposures overlapping at the 150 and 300 mg BID doses (Supplementary Table S1). There were sufficient terminal phase concentrations from the QD cohorts to determine that Cintirorgon (LYC-55716) Modulation of RORγ-dependent gene expression in murine thymus was used as a preclinical PK/PD model to define 50% and 90% effective concentrations (EC50 and EC90) of 204 and 1777 ng/mL, respectively (data on file). Across cohorts, 100% of patients with PK data available at day 29 (n = 27) had a Cmax that exceeded the EC50 and 93% exceeded the EC90. At the Cmin on day 29, 93% of patients exceeded the EC50 and 44% exceeded the EC90. At 450 mg BID (Cohort 5), the median minimum plasma drug concentration (Cmin) exceeded the EC50 by approximately 22-fold and exceeded the EC90 by 2.5-fold. Twice-daily dosing resulted in minimum plasma concentrations that were consistently higher than once-daily dosing, providing better coverage of the EC50 and EC90 targets (Fig. 2B).

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Further characterization of Cintirorgon (LYC-55716) 10 μM for both). It was negative for genotoxicity, and the IC20 was >50 μM in a patch clamp assay for effects on the human ether-a-go-go related potassium channel (hERG). It exhibited no inhibition of major Cyp isoforms (2D6, 3A4, 2C19), excellent metabolic stability and moderate plasma protein binding (mouse 96.7% bound; human 98.6% bound). Single-dose pharmacokinetic studies of 37c as its sodium salt were conducted in male and female Sprague Dawley rats (1 mg/kg iv, 30, 100, and 300 mg/kg male PO, 100 mg/kg female PO). Oral bioavailability was high (≥100%). Following an IV dose, the half-life was 2.8 h, the clearance was high (2.8 h, 48 mL/min/kg) and the Vd was high (5.7 L/kg). Single-dose pharmacokinetic studies of 37c as its sodium salt also were conducted in male cynomolgus monkeys (1 mg/kg iv, 10, 30, 100 PO). Oral bioavailability was good (≥100%), the half-life was 6.6 h, and the clearance was moderate (6.6 h, 7.49 mL/min/kg). The Vd was moderate (1.42 L/kg). The AUCs of single-dose and multiday studies were similar. These studies provided confidence to proceed into GLP toxicology studies and ultimately to select LYC-55716 (Cintirorgon, 37c) as a candidate for clinical development.[2]

Safety and tolerability [2]
LYC-55716 was generally well tolerated, with most treatment-related AEs occurring at a grade 1–2 severity level (Table 2). No Grade 4 treatment-related AEs occurred. Grade 3 treatment-related AEs included anemia (n = 2, beginning at weeks 7 and 10), elevated gamma-glutamyl transferase (n = 1, beginning at week 3), and hypophosphatemia (n = 1, 3 separate events beginning at week 4). No patients required dose reductions during the study. A total of 6 patients had dose interruptions, but none were because of treatment-related AEs. Two patients in Cohort 4a discontinued the study because of AEs: 1 patient discontinued because of fatigue (Grade 2; likely treatment related) and weight loss (Grade 2; unrelated to treatment), and 1 patient discontinued because of disease progression that was entered as “discontinued because of AE.”

[1]. Lycera Announces Initiation of Phase 1/2a Study ARGON of Immuno-Oncology Candidate LYC-55716 in Patients with Advanced Solid Tumors. Jan 04, 2017.

[2]. Discovery of LYC-55716: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ) Agonist for Use in Treating Cancer. J Med Chem. 2021 Sep 23;64(18):13410-13428.

[3]. Phase 1 Open-Label, Multicenter Study of First-in-Class RORγ Agonist LYC-55716 (Cintirorgon): Safety, Tolerability, and Preliminary Evidence of Antitumor Activity. Clin Cancer Res. 2019 Jun 15;25(12):3508-3516.

Cintirorgon is an orally bioavailable agonist of retinoic acid-related orphan receptor gamma (RORg), with potential immunomodulatory and antineoplastic activities. Upon oral administration of cintirorgon, this agent selectively binds to the nuclear receptor transcription factor RORg, forming a receptor complex that translocates to the nucleus, and binds to ROR response elements (ROREs), enhancing the function, proliferation and survival of type 17 T-cells, including Th17 (helper T-cells) and Tc17 (cytotoxic T-cells). This may increase the expression of co-stimulatory molecules and decrease the expression of co-inhibitory molecules on T-cells leading to increased production of cytokines and chemokines by T-cells, decreased proliferation of regulatory T-cells (Tregs), and abrogation of tumor-induced immunosuppression. This ultimately induces a T-cell-mediated immune response against cancer cells and leads to a reduction in tumor cell growth. RORg, the nuclear receptor transcription factor that is involved in Th17/Tc17 differentiation, plays a key role in immune activation. CINTIRORGON is a small molecule drug with a maximum clinical trial phase of I (across all indications) and has 2 investigational indications.

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NEW YORK and ANN ARBOR, Mich., Jan. 4, 2017 /PRNewswire/ -- Lycera Corp., a privately held biopharmaceutical company developing breakthrough immune modulatory medicines, announced today the initiation of a Phase 1/2a clinical trial of the Company's novel immuno-oncology therapeutic candidate LYC-55716, in patients with advanced, relapsed, or refractory solid tumors. "We continue to make rapid and significant progress in the development of our novel immune modulators. This is Lycera's third clinical trial initiated in the past 12 months and our first immuno-oncology compound to enter the clinic," said Paul Sekhri, President and CEO of Lycera. "The promising results of our preclinical program have provided confirmation that LYC-55716 modulates gene expression of RORgamma expressing T lymphocyte immune cells, resulting in enhanced effector function, as well as decreased immunosuppression, resulting in decreased tumor growth, and improved survival in in vivo preclinical models. This process of reprogramming immune cells is unique from other currently approved immunotherapies, and based on this, as well as the ability to deliver this agent orally, we believe LYC-55716 could be a significant advancement for patients." "Unlike many immunotherapies that either stimulate the immune system or reduce immune suppression, Lycera's RORgamma agonist has demonstrated in preclinical models that it can simultaneously enhance T-cell function and reduce mechanisms of the immune suppression. Therapy with an oral RORgamma agonist may be able to demonstrate single agent activity, as well as show synergy in combination with other immunotherapies," said John Nemunaitis, a principal investigator and Director of the Mary Crowley Medical Research Center, Dallas, TX. "We are excited to be working with Lycera and to be working on a compound with such a novel mechanism of action." The ARGON trial (Trial of RORgamma Agonist LYC-55716 in Advanced Cancer) is a Phase 1/2a study of LYC-55716 in patients with advanced, relapsed or refractory solid tumors. The initial Phase 1 portion of the study is designed to find the biologically active or maximum tolerated dose of LYC-55716. The study will utilize a 3+3 study design, in which LYC-55716 will be administered orally in subjects with relapsed or refractory solid tumors. The primary endpoints are safety and tolerability, and the study is designed to determine the maximum tolerated dose (MTD) and the recommended Phase 2 dose. Upon dose determination, LYC-55716 will enter Phase 2a, which is expected to enroll approximately 40 patients. The primary efficacy endpoint of the Phase 2a portion of the study will be objective response rate according to response evaluation criteria in solid tumors. About LYC-55716 LYC-55716 is a first in class oral, selective RORgamma agonist. The retinoic acid-related orphan receptor gamma (RORgamma) is a nuclear receptor transcription factor that acts as an immune cell master control switch. RORgamma agonists modulate gene expression to reprogram immune cells for improved function, as well as decrease immunosuppressive mechanisms, resulting in decreased tumor growth and enhanced survival in in vivo preclinical models of cancer. Essentially, Lycera's RORgamma agonist approach "removes the brake" and "pushes on the accelerator" of immune function. [1]

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Retinoic acid receptor-related orphan receptor γ (RORc, RORγ, or NR1F3) is the nuclear receptor master transcription factor that drives the function and development of IL-17-producing T helper cells (Th17), cytotoxic T cells (Tc17), and subsets of innate lymphoid cells. Activation of RORγ+ T cells in the tumor microenvironment is hypothesized to render immune infiltrates more effective at countering tumor growth. To test this hypothesis, a family of benzoxazines was optimized to provide LYC-55716 (37c), a potent, selective, and orally bioavailable small-molecule RORγ agonist. LYC-55716 decreases tumor growth and enhances survival in preclinical tumor models and was nominated as a clinical development candidate for evaluation in patients with solid tumors. [2]

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Purpose: Transcription factor retinoic acid receptor-related orphan receptor γ (RORγ) regulates type 17 effector T-cell differentiation and function and is key to immune cell regulation. Synthetic RORγ agonists modulate immune cell gene expression to increase effector T-cell activity and decrease immune suppression. A phase 1 study evaluated the safety and tolerability of LYC-55716 (cintirorgon), a first-in-class, oral, small-molecule RORγ agonist in adults with relapsed/refractory metastatic cancer. Patients and methods: Patients received 28-day treatment cycles of oral LYC-55716; dose and dosing regimen were determined according to pharmacokinetic profile and safety. Primary endpoints were safety and tolerability. Secondary endpoints included pharmacokinetics and objective tumor response rate. Results: No dose-limiting toxicities occurred among the 32 enrolled patients who received LYC-55716 150 mg BID to 450 mg BID. Treatment-related adverse events (AE) were primarily grade 1-2 and included diarrhea (n = 11), fatigue (n = 7), anemia (n = 4), decreased appetite (n = 4), and nausea (n = 4). Grade 3 AEs were anemia (n = 2), elevated gamma-glutamyl transferase (n = 1), and hypophosphatemia (n = 1). Pharmacokinetic concentrations achieved levels expected for target gene regulation. Pharmacodynamic results indicated RORγ pathway engagement. Two patients (NSCLC and sarcomatoid breast cancer) had confirmed partial responses; 11 had disease stabilization for 2 to 12 months (6 received >4 months of treatment). Conclusions: These data support the safety and tolerability of LYC-55716 and selection of 450 mg BID dose for a phase 2a study assessing LYC-55716 clinical activity, safety, and biomarkers in patients with NSCLC, head and neck, gastroesophageal, renal cell, urothelial, and ovarian cancers. [3]

C27H23F6NO6S

603.530047655106

603.115

C, 53.73; H, 3.84; F, 18.89; N, 2.32; O, 15.91; S, 5.31

2055536-64-4

2055538-47-9 (sodium);2055536-64-4 (free acid);

124126348

White to off-white solid powder

6.8

1

13

8

41

1020

1

O=C(O)C(C)(C)C[C@@H]1OC2=CC=C(C3=CC(F)=CC(OC(F)F)=C3)C=C2N(S(=O)(C4=CC=CC(C(F)(F)F)=C4)=O)C1

GULSIMHVQYBADX-FQEVSTJZSA-N

InChI=1S/C27H23F6NO6S/c1-26(2,24(35)36)13-20-14-34(41(37,38)21-5-3-4-17(11-21)27(31,32)33)22-10-15(6-7-23(22)39-20)16-8-18(28)12-19(9-16)40-25(29)30/h3-12,20,25H,13-14H2,1-2H3,(H,35,36)/t20-/m0/s1

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 (4.14 mM) (saturation unknown) in 5% DMSO + 40% PEG300 +5% Tween-80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.)