menin-MLL interaction
Ziftomenib targets the menin–KMT2A (MLL) protein-protein interaction. Menin is a nuclear protein that serves as a critical cofactor in transcriptional regulation, while KMT2A (also known as MLL1) is a histone methyltransferase essential for the epigenetic regulation of gene expression. The interaction between menin and KMT2A is required for the maintenance of oncogenic gene expression programs in MLL-rearranged and NPM1-mutant leukemias. Ziftomenib binds to menin and disrupts its interaction with KMT2A, thereby inhibiting the transcriptional activity of the menin-KMT2A complex. This leads to the downregulation of target genes such as HOXA9 and MEIS1, which are critical for leukemogenesis. The compound shows high selectivity for the menin-KMT2A interaction over other protein-protein interactions.

Histone methyltransferases such as the mixed-lineage leukemia (MLL) protein are essential for the epigenetic control of gene transcription. Chimeric MLL fusion proteins, which are the product of chromosomal translocations of the MLL gene located at chromosome 11, band q23 (11q23), are a characteristic shared by many acute leukemias, including acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and mixed-lineage leukemia (MLL). Leukemogenesis is ultimately caused by MLL fusion proteins, which lack the original histone methyltransferase activity of the MLL C-terminus and instead have the capacity to control the transcription of multiple oncogenes, such as HOX and MEIS1. This leads to increased cell proliferation and decreased cell differentiation[1].

---

Ziftomenib is a potent and selective inhibitor of menin, a nuclear protein that serves as a critical cofactor in transcriptional regulation. The drug is indicated for the treatment of acute myeloid leukemia (AML), specifically targeting subtypes driven by nucleophosmin 1 (NPM1) mutations or lysine methyltransferase 2A (KMT2A) rearrangements. These genetic alterations are foundational drivers of leukemogenesis; for instance, NPM1 mutations lead to the recruitment of the wild-type menin-KMT2A complex to promoters of leukemogenic genes, causing their aberrant upregulation. Ziftomenib functions by blocking the protein-protein interaction between menin and KMT2A, a complex essential for maintaining the proliferation and survival of these leukemic cells. At the molecular level, ziftomenib binds to menin at the KMT2A interaction site with high potency, effectively displacing KMT2A and disrupting the chromatin-associated complex. In NPM1-mutant AML, this disruption disengages the mutant NPM1 protein from chromatin sites. The breakdown of this interaction leads to the rapid downregulation of critical downstream leukemogenic transcription factors, specifically HOXA9 and MEIS1. These genes are typically upregulated in these AML subtypes and are responsible for blocking cellular differentiation and driving leukemic growth. The inhibition of the menin-KMT2A interaction and the subsequent suppression of HOXA9 and MEIS1 expression result in the reversal of the leukemic block on differentiation. Consequently, ziftomenib induces terminal differentiation of leukemic blasts, which is evidenced by the increased expression of differentiation markers and a reduction in leukemic burden. Nonclinical and clinical studies have confirmed that this mechanism yields potent anti-leukemic activity in models of both NPM1-mutant and KMT2A-rearranged leukemia.
In vitro, Ziftomenib demonstrates potent antiproliferative activity against AML cell lines harboring MLL rearrangements or NPM1 mutations. The compound decreases the viability of OCI-AML-3 and MV4-11 AML cells in a concentration-dependent manner. Ziftomenib induces apoptosis in AML cells through the downregulation of HOXA9 and MEIS1 expression, leading to cell cycle arrest and differentiation. The compound shows high selectivity for MLL-rearranged and NPM1-mutant AML cells, with minimal activity against AML cells without these genetic alterations. Ziftomenib also exhibits synergistic effects with various targeted agents in vitro, including venetoclax and azacitidine, enhancing their antileukemic activity. The compound effectively inhibits the proliferation of primary AML patient samples with the target genotypes.

Ziftomenib exerts anti-leukemic effects. In clinical trials with patients with relapsed or refractory AML, it was shown effective in patients with NPM1 mutations. At the recommended dosage of 600 mg in patients with relapsed or refractory AML, ziftomenib increased the QTc interval in a concentration-dependent manner, with the largest mean increase in QTc interval predicted to be 7.7 ms (upper confidence interval = 12.6 ms) after administration of 600 mg once daily. In clinical trials, 7% of subjects had a QTcF > 500 ms and an increase in QTc > 60 ms over baseline. In phase 1 clinical trial, its treatment was halted in patients with KMT2A rearrangements due to the frequency and severity of differentiation syndrome.
In vivo, Ziftomenib has demonstrated significant antitumor activity in mouse xenograft models of MLL-rearranged and NPM1-mutant AML. Oral administration of Ziftomenib leads to tumor regression and prolonged survival in these models. The compound effectively suppresses the growth of subcutaneous and orthotopic AML xenografts, with dose-dependent inhibition of tumor progression. Pharmacodynamic studies in animals show that Ziftomenib treatment results in the downregulation of HOXA9 and MEIS1 expression in tumor tissues, confirming target engagement. The compound is well-tolerated at efficacious doses in preclinical studies. Ziftomenib has also shown activity in combination with standard-of-care agents in animal models, supporting its potential for combination therapy.

In vitro binding assays for Ziftomenib involve measuring its affinity for menin using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The assay determines the dissociation constant (Kd) for the compound binding to recombinant menin protein. A fluorescence polarization (FP) assay is also used to assess the inhibition of menin-KMT2A interaction. In this assay, a fluorescently labeled peptide representing the KMT2A binding motif is incubated with menin protein and varying concentrations of Ziftomenib. The displacement of the labeled peptide is measured as a decrease in fluorescence polarization. The IC50 value is determined from the concentration-response curve. The assay includes positive controls (known menin inhibitors) and negative controls (vehicle only).

In vitro cell-based assays for Ziftomenib are performed using AML cell lines with MLL rearrangements (e.g., MV4-11, MOLM-13) or NPM1 mutations (e.g., OCI-AML-3). Cells are seeded in 96-well plates and treated with serial dilutions of Ziftomenib for 72-96 hours. Cell viability is assessed using CellTiter-Glo or MTS assays to determine the half-maximal inhibitory concentration (IC50). Apoptosis is evaluated by flow cytometry using Annexin V/PI staining or by detecting caspase-3/7 activation. Gene expression analysis of HOXA9, MEIS1, and other menin-KMT2A target genes is performed by qRT-PCR. Cell cycle analysis is conducted using propidium iodide staining. Combination studies with other agents are performed to evaluate synergistic effects using the Chou-Talalay method.

In vivo animal studies with Ziftomenib are conducted in immunodeficient mice bearing subcutaneous or orthotopic xenografts of AML cell lines (e.g., MV4-11, MOLM-13) or patient-derived xenografts (PDX) with MLL rearrangements or NPM1 mutations. Ziftomenib is administered orally at doses ranging from 10 to 100 mg/kg, typically once or twice daily. Tumor volume is measured by calipers every 2-3 days. Body weight is monitored as a measure of tolerability. At study termination, tumors are collected for pharmacodynamic analysis, including assessment of HOXA9 and MEIS1 expression, and histopathological examination. Survival studies are conducted in orthotopic models to assess the effect on overall survival. Pharmacokinetic sampling is performed to establish exposure-response relationships.

Absorption
In patients with relapsed or refractory AML receiving once-daily 600 mg ziftomenib, the mean Cmax and AUC were 288 ng/mL and 5512 ng·h/mL, respectively. The median Tmax was 4.1 hours, and the absolute bioavailability was 12.9%. AUC and Cmax increased fourfold when taken with a high-fat meal.
Elimination Route
In patients with relapsed or refractory AML receiving once-daily 600 mg ziftomenib, approximately 86% of the drug was excreted in the feces, of which approximately 73% was recovered unchanged. Approximately 0.5% of the drug was excreted in the urine, of which 0.03% was unchanged.
Volume of Distribution
In patients with relapsed or refractory AML, the mean (%CV) apparent volume of distribution after once-daily administration of 600 mg ziftomenib was 30,100 (98%) L.
Clearance
In patients with relapsed or refractory AML treated with once-daily 600 mg ziftomenib, the mean apparent clearance was 101.2 L/h.
Protein Binding
Approximately 99% of ziftomenib is bound to plasma proteins, primarily albumin.
Metabolism/Metabolites
Ziftomenib is primarily metabolized via CYP3A4 and undergoes oxidation, N-demethylation, and N-dealkylation. In a phase 1 open-label study, 19 ziftomenib metabolites were recovered; however, since ziftomenib is primarily excreted unchanged after administration, they account for less than 10% of total drug exposure. KO-739 and KO-516 are two active metabolites.
Biological Half-Life
In patients with relapsed or refractory AML receiving 600 mg ziftomenib once daily, the mean half-life was 93.6 hours.
Ziftomenib is orally bioavailable with favorable pharmacokinetic properties. Following oral administration in preclinical species, the compound shows good absorption and systemic exposure. Ziftomenib exhibits dose-proportional pharmacokinetics with a terminal half-life suitable for once- or twice-daily dosing. The compound is metabolized primarily in the liver, with metabolites being excreted in both feces and urine. Ziftomenib shows moderate plasma protein binding. The compound's pharmacokinetic profile supports its oral administration in clinical studies. In human clinical trials, Ziftomenib has demonstrated dose-dependent exposure with acceptable pharmacokinetic variability. The compound's bioavailability and half-life allow for convenient dosing schedules in clinical settings.

Efficacy and Safety
Efficacy assessments were conducted in the KO-MEN-001 (NCT04067336) study. This was an open-label, single-arm, multicenter trial that enrolled 112 adult patients with relapsed or refractory acute myeloid leukemia (AML) whose NPM1 mutations were identified by next-generation sequencing or polymerase chain reaction (PCR). The study included patients carrying NPM1 mutations, including type A, type B, and type D mutations, as well as other NPM1 mutations that may lead to cytoplasmic localization of the NPM1 protein. Efficacy endpoints included complete remission (CR) rate, complete remission with partial hematologic recovery (CRh) rate, duration of CR+CRh, and the rate of transfusion-dependent to transfusion-independent outcomes. The median follow-up time was 4.2 months (range: 0.1 to 41.2 months). The complete remission (CR) + CRh rate was 21.4% (95% CI: 14.2, 30.2), and the duration of CR + CRh was 5 months (95% CI: 1.9, 8.1). The CR rate was 17.0% (95% CI: 10.5, 25.2), and the CRh rate was 4.5% (95% CI: 1.5, 10.1). Of the 66 patients who required red blood cell (RBC) and/or platelet transfusions at baseline, 14 (21.2%) no longer required RBC and platelet transfusions within 56 days after baseline. Of the 46 patients who did not require RBC and platelet transfusions at baseline, 12 (26.1%) remained transfusion-free within 56 days after baseline. Prescribing information included warnings and precautions regarding differentiation syndromes, QTc interval prolongation, and embryo-fetal toxicity.
In preclinical toxicology studies, Ziftomenib has shown a manageable safety profile with no significant off-target toxicity. The compound is well-tolerated in animal models at doses that achieve therapeutic efficacy. In clinical trials, Ziftomenib has been evaluated for safety and tolerability in AML patients. The most common adverse events reported include gastrointestinal effects (nausea, vomiting, diarrhea), fatigue, and hematological abnormalities consistent with the underlying disease. No dose-limiting toxicities have been observed at the recommended phase 2 dose. The compound has not shown significant cardiotoxicity or hepatotoxicity. Ongoing clinical trials are further characterizing the safety and efficacy profile of Ziftomenib in larger patient populations.

[1]. Substituted inhibitors of menin-mll and methods of use. WO2017161028A1.

Zivtomenib is an orally bioavailable inhibitor of the menin-mixed lineage leukemia (MLL; myeloid/lymphoid leukemia; KMT2A) fusion protein with potential antitumor activity. After oral administration, zivtomenib blocks the interaction between menin and MLL proteins, thereby inhibiting the formation of the menin-MLL complex. This reduces the expression of downstream target genes and inhibits the proliferation of MLL-rearranged leukemia cells. The menin-MLL complex plays a crucial role in the survival, growth, and proliferation of certain types of leukemia cells.
Ziftomenib is currently in clinical development for the treatment of acute myeloid leukemia (AML) with MLL rearrangements or NPM1 mutations. The compound has received FDA Fast Track designation and Orphan Drug designation for the treatment of AML. Phase 1/2 clinical trials have demonstrated promising antileukemic activity, with complete responses observed in some patients. Ziftomenib represents a novel targeted therapy approach for genetically defined subsets of AML, addressing an unmet medical need in patients with limited treatment options. The compound's mechanism of action—disrupting the menin-KMT2A interaction—represents a new paradigm in epigenetic cancer therapy. Ongoing research is exploring the potential of Ziftomenib in combination with other agents and in other cancer types.

C33H42F3N9O2S2

717.870893955231

717.285

C, 55.21; H, 5.90; F, 7.94; N, 17.56; O, 4.46; S, 8.93

2134675-36-6

138497449

Light brown to brown solid powder

5.6

2

14

10

49

1270

1

S(C)(N1CCN(CC1)[C@@H](C)CN1C(C#N)=CC2C(C)=C(C=CC1=2)CN1CCC(CC1)NC1=C2C=C(CC(F)(F)F)SC2=NC(NC)=N1)(=O)=O

BGGALFIXXQOTPY-NRFANRHFSA-N

InChI=1S/C33H42F3N9O2S2/c1-21(43-11-13-44(14-12-43)49(4,46)47)19-45-25(18-37)15-27-22(2)23(5-6-29(27)45)20-42-9-7-24(8-10-42)39-30-28-16-26(17-33(34,35)36)48-31(28)41-32(38-3)40-30/h5-6,15-16,21,24H,7-14,17,19-20H2,1-4H3,(H2,38,39,40,41)/t21-/m0/s1

4-methyl-5-[[4-[[2-(methylamino)-6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl]amino]piperidin-1-yl]methyl]-1-[(2S)-2-(4-methylsulfonylpiperazin-1-yl)propyl]indole-2-carbonitrile

ziftomenib; KO-539; UNII-4MOD1F4ENC; 4MOD1F4ENC; ZIFTOMENIB [INN];

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 (139.30 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (2.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

---

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