IC50: 0.25 nM (LSD1)[1]

CC-90011 (Compound 11) exhibits strong antiproliferative action in AML kasumi-1 cells with an EC50 of 2 nM and strong activation of on-target cellular differentiation marker CD11b in THP-1 cell line[1]. When CC-90011 is administered for four days, GRP suppression is seen in a dose-dependent manner at pharmacologically relevant concentrations (EC50=3 nM, H209, and 4 nM, H1417). When SCLC cells are treated with CC-90011 for 12 days, they exhibit strong antiproliferative activity (EC50=6 nM, H1417), which is linked with the inhibition of GRP[1].

In patient-derived xenograft SCLC models, CC-90011 (5 mg/kg; oral administration; daily; for 30 days) treatment inhibits tumor growth[1]. In SCLC human tumor xenograft (H1417) mice, CC-90011 (once daily; for 4 days) treatment causes a strong downregulation of GRP mRNA levels at 2.5 mg/kg and maximum suppression of GRP at 5 mg/kg[1]. CC-90011 (Compound 11; 5 mg/kg) has a high volume of distribution of 7.5 L/kg, an elimination half-life of 2 hours, and a systemic clearance of 32.4 mL/min/kg following intravenous administration. Oral administration of CC-90011 (Compound 11; 5 mg/kg) results in a high degree of oral bioavailability (oral bioavailability = 32%, AUC0-24h = 1.8 μM·h, and C/sub>max = 0.36 μM).

LSD1 enzymatic inhibition by compound 11 (CC-90011) is assessed by both LSD1 alone or by LSD1- CoREST complex. [1]
For LSD1 TR-FRET assay, LSD1 (0.025 nM final) is combined with 50 nM H3K4me1 in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.5 mM TCEP, 0.02% (w/v) BSA, 0.005% (w/v) Brij-35, and 2 µM FAD either in the presence of DMSO or compound dilution series in DMSO (final 1% DMSO). Demethylated product H3K4 is quantified by the addition of detection reagent Phycolink Streptavidin-allophycocyanin and Europium-antiunmodified histone H3 lysine 4 (H3K4) antibody in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer to final concentration of 12.5 nM and 0.25 nM respectively.

For LSD1-CoREST assay, enzymatic reaction product hydrogen peroxide is detected using a HRP coupled assay method. LSD1-CoREST (6 nM final) is combined with 20 µM H3K4me2, 2 U/ml HRP, 50 µM Amplex red in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.02% (w/v) BSA, 0.005% (w/v) Brij-35, and 2 µM FAD either in the presence of DMSO or compound dilution series in DMSO (final 1% DMSO). Fluorescent product Resorufin from the reaction of HRP and hydrogen peroxide is detected.

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LSD2 isoform and mono amine oxidases (MAOs) selectivity: [1]
Compound 11 (CC-90011)was evaluated for inhibition of other FAD containing enzymes: LSD2, MAO-A, and MAO-B. The selectivity of Compound 11 for LSD1 over these enzymes is greater than 60,000 fold (Table S1).

LSD2 enzymatic inhibition by compound 11 (CC-90011) is assessed by TR-FRET assay. LSD2 (2 nM final) is combined with 300 nM H3K4me1 in 50 mM Tris pH 8.5, 0.02% (w/v) BSA, 0.005% (w/v) Brij-35, and 2 µM FAD either in the presence of DMSO or compound dilution series in DMSO (final 1% DMSO). Demethylated product H3K4 is quantified in the same way as LSD1 TR-FRET assay with final concentration of Phycolink Streptavidinallophycocyanin at 25 nM and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody at 0.5 nM.

A HRP coupled assay similar to LSD1-CoREST assay is used for MAO-A and MAO-B. In the MAO-A assay, 30 µM of Tyramine and 5 µg/ml MAO-A are combined with compounds or DMSO control (final 1%) and monitored in the presence of 2 U/ml HRP, 50 µM Amplex red in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.02% (w/v) BSA, and 0.005% (w/v) Brij-35.



In the MAO-B assay, 300 µM Benzylamine and 5 µg/ml MAO-B are combined with compounds or DMSO control (final 1%) and monitored in the presence of 2 U/ml HRP, 50 µM Amplex red in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.02% (w/v) BSA, and 0.005% (w/v) Brij-35.

In vitro cell biology: Compound 11 (CC-90011) demonstrated potent anti-proliferative activity in a Kasumi-1 cell line model of AML with an IC50 of 0.0024 M (Figure S1A) and showed no effect (IC50 of >10 M) in a normal human fibroblast cell model (Figure S1B).[1]

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Cell-MTS colorimetric plate based assay was used to quantify the amount of newly generated NADH in the presence or absence of compound 11. NADH levels were used as a proxy for the quantification of cellular proliferation. Cells were incubated in the presence of an 11-point dilution series of compound 11 (CC-90011) for 168 hours at 37 °C, 5% CO2. At the end of this compound incubation, CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay solution (Promega) was added and the OD490 was determined. IC50 values were calculated using the IDBS Xlfit software package and include background subtracted OD490values and normalization to DMSO controls. Cell Titer Glo Luminescence Cell Viability Assay (Promega) measured the number of viable cells based on quantitation of the ATP present. Cells were incubated in the presence of an 8-point dilution series of compound 11 and incubated at 37°C, 5% CO2 for 7 days for AML cell lines and 12 days for H1417 cell line. At the end of the incubation, Cell Titer Glo reagent was added to the wells and the plate was read for luminescence. IC50 values were calculated using the IDBS Xlfit software package.[1]

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Modulation of CD11b expression in THP1 by compound 11 (CC-90011): CD11b was shown to be upregulated with LSD1 inhibition. In order to assess compound 11 (CC-90011) ability to inhibit LSD1 in AML, a quantitative FACS assay was utilized to measure CD11b protein expression in the AML cell line, THP-1. In this assay, compound 11 (CC-90011) increased CD11b protein expression with an EC50 value of 7 nM (Figure S2).[1]

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THP-1 CD11b: Cells were incubated for 96 hours with compound 11 (CC-90011). Cells were analyzed by FACS for CD11b expression. The percent of CD11b positive cells relative to compound 11 concentration was plotted in Xlfit to generate an EC50 value.

Animal/Disease Models: BALB/c nude mice bearing small cell lung carcinoma (SCLC)[1]
Doses: 5 mg/kg
Route of Administration: Oral administration; daily; for 30 days
Experimental Results: demonstrated a tumor growth inhibition (TGI) of 78% at 5 mg /kg with no body weight loss.

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In vivo PK/PD analysis of GRP expression in SCLC: [1]
Nude mice implanted with the SCLC cell line H1417 tumor were dosed orally daily with 2.5, 5, and 10 mg/kg compound 11 (CC-90011) for 4 consecutive days (3 mice for each group). Tumors, which were approximately 100 mm3 at the initiation of the study, were harvested 24h following the last dose. Total RNA was used to make cDNA for qPCR assessment. Total levels of human GRP transcript were normalized to human RPL19 levels. Quantification of target inhibition was generated by calculating Ct values of compound 11 treated vs vehicle GRP transcript.

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In vivo efficacy of compound 11 (CC-90011) in H1417 SCLC xenograft: [1]
The efficacy and tolerability of compound 11, dosed orally at 2.5 and 5 mg/kg, was evaluated in the NCIH1417 small cell lung cancer (SCLC) xenograft model in athymic nude mice. Mean tumor growth in the control progressed over the course of the study exhibited a 2.5 fold increase in mean tumor volume from Day 0 to Day 65. Tumors in the compound 11 (CC-90011) treated groups regressed after Day 14, resulting in a net loss in mean tumor volume at study end. The differences in the distribution of tumor volumes on Day 65 for compound 11 treated versus control animals were significant with a calculated probability (p) ≤ 0.001 and ≤ 0.0001 for the 2.5 and 5 mg/kg dose levels, respectively. Compound 11 appeared well tolerated, and animals receiving the 2.5 or 5 mg/kg doses exhibited respective mean body weight gains of 1% and 7.5% at study end (Figure S4). All animals survived the duration of the study.

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In vivo efficacy in LXFS 615 SCLC PDX model: [1]
Compound 11 (CC-90011) was shown to be efficacious in LXFS 615 patient derived xenograft model of SCLC with a TGI of 78% at 5 mg/kg (P-value: 0.001). Compound 11 was well tolerated with no body weight loss (Figure S5). [1]
In vivo efficacy of SCLC PDX model (LU2514): Compound 11 (CC-90011) when dosed orally for 28 days was shown to be efficacious in LU-2514 patient derived xenograft model of SCLC with a TGI of 56% at 10 mg/kg (P-value: 0.0024) and 39% at 5 mg/kg (P-value: 0.0109). Compound 11 was well tolerated at all doses examined in this study with mean body weight losses <10%.

[1]. Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1). J Med Chem. 2020 Dec 10;63(23):14522-14529.

Pulrodemstat is an orally available inhibitor of lysine specific demethylase 1 (LSD1), with potential antineoplastic activity. Upon administration, pulrodemstat binds to and inhibits LSD1, a demethylase that suppresses the expression of target genes by converting the di- and mono-methylated forms of lysine at position 4 of histone H3 (H3K4) to mono- and unmethylated H3K4, respectively. LSD1 inhibition enhances H3K4 methylation and increases the expression of tumor (remove hyphen) suppressor genes. This may lead to an inhibition of cell growth in LSD1-overexpressing tumor cells. In addition, LSD1 demethylates mono- or di-methylated H3K9 which increases gene expression of tumor promoting genes; inhibition of LSD1 promotes H3K9 methylation and decreases transcription of these genes. LSD1, an enzyme belonging to the flavin adenine dinucleotide (FAD)-dependent amine oxidase family that is overexpressed in certain tumor cells, plays a key role in tumor cell growth and survival.

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Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).[1]

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Conventional targeted agents for combating cancers have focused on promoting apoptosis in rapidly proliferating cancer cells. These agents have proven effective in reducing tumor bulk, but prolonged treatment has often resulted in tumors developing resistance. An alternative strategy is to suppress proliferation by inducing terminal differentiation of the cancer cells. This strategy is employed less frequently but has been highly effective in subsets of AML. The discovery of CC-90011, a highly potent and reversible inhibitor of LSD1, provides a novel differentiation strategy for the treatment of neuroendocrine tumors and AML. Phase 1 study of CC-90011 in patients with advanced solid tumors has been completed, and the safety, tolerability, and preliminary efficacy have been reported. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier NCT03850067).[1]

C31H31F2N5O5S

623.67

623.201

C, 59.70; H, 5.01; F, 6.09; N, 11.23; O, 12.83; S, 5.14

2097523-57-2

Pulrodemstat benzenesulfonate;2097523-60-7;Pulrodemstat;1821307-10-1

139600313

Typically exists as solid at room temperature

2

10

5

44

1070

0

OZZFOHIBJFKYLY-UHFFFAOYSA-N

InChI=1S/C24H23F2N5O2.C7H8O3S/c1-30-23(32)21(14-5-6-20(33-2)19(26)11-14)22(15-3-4-16(13-27)18(25)12-15)29-24(30)31-9-7-17(28)8-10-31;1-6-2-4-7(5-3-6)11(8,9)10/h3-6,11-12,17H,7-10,28H2,1-2H3;2-5H,1H3,(H,8,9,10)

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxopyrimidin-4-yl]-2-fluorobenzonitrile;4-methylbenzenesulfonic acid

097523-57-2; Pulrodemstat tosylate; UNII-496P6HY485; Pulrodemstat (Methylbenzenesulfonate); 496P6HY485; Benzonitrile, 4-(2-(4-amino-1-piperidinyl)-5-(3-fluoro-4-methoxyphenyl)-1,6-dihydro-1-methyl-6-oxo-4-pyrimidinyl)-2-fluoro-, 4-methylbenzenesulfonate (1:1); Pulrodemstat Methylbenzenesulfonate;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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