KDM1/lysine specific demethylase-1 (LSD1

CC-90011（化合物 11）能够有效增加 THP-1 细胞系中的细胞增殖标志物 CD11b，EC50 为 7 nM。此外，EC50 为 2 nM 时，它在 AML kasumi-1 细胞中具有抗增殖活性。 CC-90011 治疗四天后，在有效浓度下以剂量依赖性和药理学方式观察到 GRP 抑制（EC50=3 nM（H209 细胞）和 4 nM（H1417 细胞））。当 SCLC 细胞用 CC-90011 处理 12 天时，会产生与 GRP 抑制相关的强抗增殖作用（EC50=6 nM（H1417 细胞））[1]。

在小鼠异种移植 SCLC 模型中，CC-90011（5 mg/kg；每天；持续 30 天）可抑制肿瘤生长 [1]。 CC-90011 每天给药一次，持续四天，在 SCLC 人类肿瘤异种移植物 (H1417) 小鼠模型中，在 2.5 mg/kg 浓度下产生 GRP mRNA 水平的强转录，在 5 mg/kg 浓度下产生 GRP 峰值抑制[1]。静脉注射后，CC-90011（化合物 11；5 mg/kg）表现出 32.4 mL/min/kg 的全身清除率、2 小时消除半衰期和 >7.5 L/kg 的增殖。化合物 11，即 CC-90011，在脑后很容易被吸收，最低生物利用度为 32%，AUC0-24h 为 1.8 μM·h，Cmax 为 0.36 μM [1]。

化合物11（CC-90011）对LSD1的酶抑制作用通过LSD1单独或LSD1-CoREST复合物进行评估。[1]
对于LSD1 TR-FRET测定，LSD1（最终0.025 nM）与50 nM H3K4me1在50 mM HEPES（pH 7.3）、10 mM NaCl、0.5 mM TCEP、0.02%（w/v）BSA、0.005%（w/v）Brij-35和2µM FAD中混合，在DMSO或DMSO中的化合物稀释系列（最终1%DMSO）存在下进行。在LANCE检测缓冲液中，在LSD1抑制剂如1.8 mM盐酸环丙环丙胺（2-PCPA）的存在下，通过添加检测试剂Phycolink Streptavidin别藻蓝蛋白和铕抗未修饰组蛋白H3赖氨酸4（H3K4）抗体，将去甲基化产物H3K4定量至最终浓度分别为12.5 nM和0.25 nM

对于LSD1-CoREST测定，使用HRP偶联测定法检测酶反应产物过氧化氢。LSD1 CoREST（最终6 nM）与20µM H3K4me2、2 U/ml HRP、50µM Amplex red在50 mM HEPES（pH 7.3）、10 mM NaCl、0.02%（w/v）BSA、0.005%（w/v）Brij-35和2µM FAD混合，在DMSO或DMSO中的化合物稀释系列（最终1%DMSO）存在下进行。检测到HRP和过氧化氢反应产生的荧光产物Resorufin。

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LSD2亚型和单胺氧化酶（MAO）选择性：[1]
评估了CC-90011对其他含FAD酶的抑制作用：LSD2、MAO-A和MAO-B。化合物11对LSD1的选择性大于这些酶的60000倍（表S1）

通过TR-FRET测定评估CC-90011对LSD2的酶抑制作用。在DMSO或DMSO中的化合物稀释系列（最终1%DMSO）的存在下，将LSD2（2nM最终）与300nM H3K4me1在50mM Tris pH 8.5、0.02%（w/v）BSA、0.005%（w/v）Brij-35和2µM FAD中混合。脱甲基产物H3K4以与LSD1 TR-FRET测定相同的方式进行定量，Phycolink Streptavidinalophycocyanin的终浓度为25 nM，铕抗未修饰组蛋白H3赖氨酸4（H3K4）抗体的终浓度是0.5 nM。

类似于LSD1-CoREST测定的HRP偶联测定用于MAO-A和MAO-B。在MAO-A测定中，将30µM酪胺和5µg/ml MAO-A与化合物或DMSO对照（最终1%）混合，并在2 U/ml HRP、50 mM HEPES（pH 7.3）中的50µM Amplex red、10 mM NaCl、0.02%（w/v）BSA和0.005%（w/v）Brij-35存在下进行监测



在MAO-B测定中，将300µM苄胺和5µg/ml MAO-B与化合物或DMSO对照（最终1%）混合，并在2 U/ml HRP、50 mM HEPES（pH 7.3）中的50µM Amplex red、10 mM NaCl、0.02%（w/v）BSA和0.005%（w/v）Brij-35存在下进行监测。

体外细胞生物学：化合物11（CC-90011）在AML的Kasumi-1细胞系模型中显示出强大的抗增殖活性，IC50为0.0024μM（图S1A），在正常人成纤维细胞模型中没有显示出任何作用（IC50>10μM）（图S1B）。[1]

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使用基于Cell MTS比色板的测定来定量化合物11存在或不存在时新产生的NADH的量。NADH水平被用作细胞增殖定量的指标。在37°C、5%CO2的条件下，将细胞在CC-90011的11点稀释系列中孵育168小时。在该化合物培养结束时，加入CellTiter 96®非放射性细胞增殖测定水溶液（Promega），并测定OD490。IC50值使用IDBS Xlfit软件包计算，包括背景减除OD490值和DMSO对照标准化。Cell Titer Glo发光细胞活力测定（Promega）基于ATP的定量测量活细胞的数量。细胞在化合物11的8点稀释系列存在下孵育，AML细胞系在37°C、5%CO2下孵育7天，H1417细胞系孵育12天。在孵育结束时，将Cell Titer Glo试剂加入孔中，读取平板的发光情况。使用IDBS Xlfit软件包计算IC50值。[1]

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CC-90011:CD11b对THP1中CD11b表达的调节被证明在LSD1抑制下上调。为了评估化合物11抑制AML中LSD1的能力，利用定量FACS测定AML细胞系THP-1中CD11b蛋白的表达。在该试验中，化合物11增加了CD11b蛋白表达，EC50值为7 nM（图S2）。[1]

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THP-1 CD11b：细胞与化合物11一起孵育96小时。通过FACS分析细胞CD11b表达。CD11b阳性细胞相对于化合物11浓度的百分比以Xlfit绘制，以产生EC50值。

Animal/Disease Models: BALB/c nude mice bearing small cell lung cancer (SCLC) [1]
Doses: 5 mg/kg: oral administration; daily; continued for 30 days
Experimental Results: Tumor growth inhibition (TGI) at the dose of 5 mg/kg ) is 78% and there is no 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.

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]

C30H29F2N5O5S

609.643572568893

609.185

C, 59.10; H, 4.79; F, 6.23; N, 11.49; O, 13.12; S, 5.26

2097523-60-7

Pulrodemstat Methylbenzenesulfonate;2097523-57-2;Pulrodemstat;1821307-10-1; 2097523-60-7 (besylate)

129096972

Off-white to light yellow solid powder

158

2

10

5

43

1040

0

S(C1C=CC=CC=1)(=O)(=O)O.FC1=C(C=CC(=C1)C1=C(C2C=CC(C#N)=C(C=2)F)N=C(N(C)C1=O)N1CCC(CC1)N)OC

AWZCBGWZNHQCIZ-UHFFFAOYSA-N

InChI=1S/C24H23F2N5O2.C6H6O3S/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)8-10-317-10(8,9)6-4-2-1-3-5-6/h3-6,11-12,17H,7-10,28H2,1-2H31-5H,(H,7,8,9)

4-[2-(4-Amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile benzenesulfonic acid

CC-90011 besylate CC90011; CC-90011; Pulrodemstat besilate; Pulrodemstat benzenesulfonate; CC-90011 besylate; LSD1-IN-7 benzenesulfonate; LH20W7B0Q8; CC-90011 benzenesulfonate; CC 90011

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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DMSO : ~50 mg/mL (~82.02 mM)

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配方 2 中的溶解度: ≥ 2.08 mg/mL (3.41 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
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