Bcl-2 (IC50 = 2 nM); Bcl-xL (IC50 = 5.9 nM)
Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor (IC₅₀ = 1-100 nM for BCL-2). It shows >500-fold selectivity over BCL-xL (IC₅₀ >1 μM) and minimal activity against MCL-1. [1][2]

Lisaftoclax (10–1000 nM) induced dose-dependent apoptosis in venetoclax-resistant AML cell lines (MOLM-13/Ven-R, IC₅₀ = 42 nM), with 80% Annexin V⁺ cells at 100 nM after 48h. Western blot showed cleavage of caspase-9 and PARP. [2]

In co-culture systems, Lisaftoclax (200 nM) reprogrammed M2 macrophages to M1 phenotype, increasing IL-1β secretion by 8-fold (p<0.001) and TNF-α by 6-fold (p<0.01) via NLRP3 inflammasome activation. [3]

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Lisaftoclax (compound 6) IC50 values in Bcl-2-dependent RS4;11 cells and Bcl-xl-dependent Molm13 cells are 5.5 nM and 6.4 nM, respectively [1].

The combination of lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115) exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein.[2]

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In venetoclax-resistant AML PDX models, Lisaftoclax (100 mg/kg p.o. QD) reduced tumor burden by 78% (p<0.001) after 28 days. Combination with alrizomadlin achieved complete regression in 5/8 mice. [2]

In MC38 syngeneic models, Lisaftoclax (150 mg/kg p.o.) increased tumor-infiltrating CD8⁺ T cells by 3.2-fold (p<0.01) and synergized with anti-PD-1 (tumor volume reduction: 92% vs. 45% monotherapy). [3]

Apoptosis assay: Cells treated with Lisaftoclax (0.1–10 μM) for 24–72h were stained with Annexin V/PI. Flow cytometry quantified apoptosis rates. [2]

Immunoblotting: Treated cells lysed for BCL-2 family protein analysis (Bax, Bak, cleaved caspase-9). β-actin used as loading control. [2]

Cytokine profiling: ELISA measured IL-1β, TNF-α, and IL-10 in macrophage supernatants after 24h treatment. [3]

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The main challenges in the use of immune checkpoint inhibitors (ICIs) are ascribed to the immunosuppressive tumor microenvironment and the lack of sufficient infiltration of activated CD8+ T cells. Transforming the tumor microenvironment (TME) from "cold" to "hot" and thus more likely to potentiate the effects of ICIs is a promising strategy for cancer treatment. We found that the selective BCL-2 inhibitor APG-2575 can enhance the antitumor efficacy of anti-PD-1 therapy in syngeneic and humanized CD34+ mouse models. Using single-cell RNA sequencing, we found that APG-2575 polarized M2-like immunosuppressive macrophages toward the M1-like immunostimulatory phenotype with increased CCL5 and CXCL10 secretion, restoring T-cell function and promoting a favorable immunotherapy response. Mechanistically, we demonstrated that APG-2575 directly binds to NF-κB p65 to activate NLRP3 signaling, thereby mediating macrophage repolarization and the activation of proinflammatory caspases and subsequently increasing CCL5 and CXCL10 chemokine production. As a result, APG-2575-induced macrophage repolarization could remodel the tumor immune microenvironment, thus improving tumor immunosuppression and further enhancing antitumor T-cell immunity. Multiplex immunohistochemistry confirmed that patients with better immunotherapeutic efficacy had higher CD86, p-NF-κB p65 and NLRP3 levels, accompanied by lower CD206 expression on macrophages. Collectively, these data provide evidence that further study on APG-2575 in combination with immunotherapy for tumor treatment is required.[3]

Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115).[2]

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For PDX studies: NSG mice received Lisaftoclax (100 mg/kg) or vehicle (30% PEG400/5% solutol) orally daily. Alrizomadlin (10 mg/kg) was administered IV twice weekly. [2]

For immunotherapy studies: C57BL/6 mice bearing MC38 tumors received Lisaftoclax (150 mg/kg in 0.5% methylcellulose) QD + anti-PD-1 (200 μg IP) every 3 days. [3]

Oral bioavailability in mice: 65%. Plasma half-life (t_1/2) = 4.3 hours. Steady-state concentration (C_min) > 200 nM at a once-daily dose of 100 mg/kg. [2] Plasma protein binding: 93% across species. [2]

No significant weight loss or hematologic toxicity was observed at the therapeutic dose. Platelet count remained >150×10⁹/L. [2]

[1]. N-(PHENYLSULFONYL)BENZAMIDES AND RELATED COMPOUNDS AS BCL-2 INHIBITORS. Patent. WO2018027097A1.

[2]. Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies. Clin Cancer Res. 2023 Jan 4;29(1):183-196.

[3]. The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation. Cell Mol Immunol. 2024 Jan;21(1):60-79.

Lisaftoclax is a novel, orally effective, selective, and potent BCL-2 inhibitor. Lisaftoclax is an orally bioavailable and selective anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) inhibitor with potential pro-apoptotic and anti-tumor activity. After oral administration, Lisaftoclax targets and binds to Bcl-2 activity, thereby restoring the apoptotic process in tumor cells. Bcl-2 is overexpressed in various cancers and plays an important role in the negative regulation of apoptosis; its expression is associated with increased drug resistance and improved tumor cell survival. Objective: This global Phase I clinical trial aims to investigate the safety, efficacy, pharmacokinetics, and pharmacodynamics of Lisaftoclax (APG-2575). Lisaftoclax is a novel, orally active, potent, and selective B-cell lymphoma 2 (BCL-2) inhibitor for the treatment of patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs). Patients and Methods: The maximum tolerated dose (MTD) and the recommended dose for Phase II were evaluated. The primary efficacy endpoint was safety and tolerability, and the secondary efficacy endpoints were pharmacokinetic variables and antitumor effects. The pharmacodynamics of ritaroxate in patient tumor cells was also investigated. Results: The maximum tolerated dose was not reached in 52 patients treated with ritaroxate. Treatment-exclusive adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (both 28.8%), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (both 17.3%), and arthralgia (15.4%). Grade ≥3 hematologic treatment-emergent adverse events (TEAEs), including neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), did not lead to treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax has limited plasma residence time and systemic exposure, and rapidly clears malignant cells. The median number of treatment cycles was 15 (range 6–43). Of the 22 evaluable patients with relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (R/R CLL/SLL), 14 achieved partial remission, resulting in an objective response rate of 63.6%. The median time to response was 2 (range 2–8) cycles. Conclusion: Lisaftoclax was well tolerated, and no evidence of tumor lysis syndrome was observed. The highest dose level did not reach dose-limiting toxicities. Lisaftoclax has unique pharmacokinetic characteristics, making it suitable for a potentially more convenient daily (rather than weekly) escalation regimen and for inducing rapid clinical remission in CLL/SLL patients, warranting further clinical investigation. Clin Cancer Res. 2023 Jul 5;29(13):2385-2393. https://pubmed.ncbi.nlm.nih.gov/37074726/

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Lisaftoclax overcomes veneclax resistance by disrupting BCL-2/MCL-1 heterodimerization and activating Bax-dependent apoptosis. [2]
Phase Ib/II clinical trials for hematologic malignancies are underway (NCT03537482, NCT04771546). [3]

C45H48CLN7O8S

882.422728538513

881.297

C, 61.25; H, 5.48; Cl, 4.02; N, 11.11; O, 14.50; S, 3.63

2180923-05-9

137355972

Light yellow to yellow solid powder

7.5

3

12

12

62

1690

1

ClC1C=CC(=CC=1)C1=C(CN2CCN(C3C=CC(C(NS(C4C=CC(=C(C=4)[N+](=O)[O-])NC[C@H]4COCCO4)(=O)=O)=O)=C(C=3)OC3C=NC4=C(C=CN4)C=3)CC2)CCC2(C1)CCC2

FNBXDBIYRAPDPI-BHVANESWSA-N

InChI=1S/C45H48ClN7O8S/c46-33-4-2-30(3-5-33)39-25-45(12-1-13-45)14-10-32(39)28-51-16-18-52(19-17-51)34-6-8-38(42(23-34)61-35-22-31-11-15-47-43(31)49-26-35)44(54)50-62(57,58)37-7-9-40(41(24-37)53(55)56)48-27-36-29-59-20-21-60-36/h2-9,11,15,22-24,26,36,48H,1,10,12-14,16-21,25,27-29H2,(H,47,49)(H,50,54)/t36-/m0/s1

4-[4-[[8-(4-chlorophenyl)spiro[3.5]non-7-en-7-yl]methyl]piperazin-1-yl]-N-[4-[[(2S)-1,4-dioxan-2-yl]methylamino]-3-nitrophenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide

Bcl2/Bclxl inhibitor 1; APG2575; APG-2575; OSL3FEZ1IF; UNII-OSL3FEZ1IF; LISAFTOCLAX [INN]; LISAFTOCLAX [WHO-DD]; 4-(4-((6-(4-CHLOROPHENYL)SPIRO(3.5)NON-6-EN-7-YL)METHYL)-PIPERAZIN-1-YL)-N-((3-NITRO-4-(((2S)-1,4-DIOXAN-2-YLMETHYL)AMINO)PHENYL)SULFONYL)-2-(1HPYRROLO(2,3-B)PYRIDIN-5-YLOXY)BENZAMIDE; ...; 2180923-05-9; Bcl-2/Bcl-xl inhibitor 1; OSL3FEZ1IF; APG 2575; UNII-OSL3FEZ1IF; LISAFTOCLAX [INN]; Lisaftoclax; APG-2575

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)

DMSO : ~100 mg/mL (~113.32 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)