A series of optimized Menin-Mixed Lineage Leukemia (MLL) protein-protein interaction inhibitors are reported leading to second generation probe ML399. HTS the Molecular Libraries Probe Production Centers Network (MLPCN) screening led to several chemotypes, including a piperidine class which successfully led to first generation probe ML227. Metabolic instability, potency, and ancillary pharmacology activity of ML227 were identified as limiting features. In order to enhance the utility of a menin-MLL inhibitor probe for in vivo proof-of-mechanism studies, a medicinal chemistry effort was reinitiated with a structure-based design approach incorporated. Novel menin-MLL inhibitors with improved potency and DMPK properties were identified leading to declared probe ML399. SAR and characterization of compounds within the series is described.

Probe Structure & Characteristics

ML399

1. Recommendations for scientific use of the probe

This probe (ML399, CID 71777738, SID 164849617) represents a second generation menin-MLL small molecule protein-protein interaction inhibitor with nanomolar inhibitory activity.1 This probe will be used by the research community to further elucidate the importance of the menin-MLL interaction in MLL-mediated leukemogenesis and further support efforts to understand if therapeutic intervention of the menin-MLL pathway with small molecule inhibitors is viable as a novel approach to treat acute lymphoid and myeloid leukemias with MLL rearrangements. Translocations of MLL result in acute leukemias with poor prognosis and development of novel therapeutic strategies is highly desired. Leukemogenic activity of MLL fusion proteins is dependent of their interactions with menin, validating the importance of this interaction as a potential drug target for leukemia.2, 3

Currently, there are no systemically available menin-MLL inhibitor tool compounds for advanced animal model studies. Based upon the profile available to date ML399 represents the first in vivo probe for further understanding the potential of menin inhibitors as a novel approach for treatment of treat acute lymphoid and myeloid leukemias with MLL rearrangements, as well as other cancers where menin is known to have critical interaction with wild-type MLL1 and MLL2 histone methyltransferases.4 The potential for expanded disease areas involving menin-MLL inhibitors is a new area of intense and promising research and thus there is a high demand for suitable in vivo tool compounds for the field since it remains uncertain if effective menin-MLL inhibition will demonstrate efficacy in relevant preclinical animal models. ML399 is a potent and cell penetrant menin-MLL inhibitor that can be used for further studying the biology of MLL and MLL fusion protein mediated leukemogenesis, as well as additional menin mediated pathways of interest in cancer research.

2. Materials and Methods

2.2. Probe Chemical Characterization

Solubility. Solubility in PBS at pH 7.4 was excellent at 86.9 ± 8.2 μM or 39 μg/mL. ML399 is readily soluble up to 40 mM DMSO which is currently used for original stocks of novel compounds for testing.

Stability. Stability was determined for ML399 in PBS buffer at room temperature. After 24 hour (T = 1441 min.), the percent of parent compound remaining was 98.6%, indicating excellent stability after exposure to PBS. After 48h (T=2.884 min.) 96.1% remained as determined by LC-MS.

Compounds added to the SMR collection (MLS#s): MLS005886373 (ML399, CID 71777738, 22.7 mg); MLS005886374 (CID 46926614, 14.1 mg); MLS005886375 (CID 71777749, 14.3 mg); MLS005886376 (CID 6926630, 7.0 mg); MLS005886377 (CID 46926612, 5.6 mg); MLS005886378 (CID 73297282, 12.9 mg).

2.3. Probe Preparation

Synthetic procedure and spectral data for ML399 (CID 71777738, SID 164849617)

Scheme 1Preparation of ML399 (CID 71777738)

Step 1. (1-(3-chloropropyl)piperidin-4-yl)(pyridin-2-yl)methanone

To a solution of piperidin-4-yl(pyridin-2-yl)methanone (791 mg, 2.72 mmol) in dry DMF (9.1 mL) was added potassium carbonate (1.13 g, 8.16 mmol) followed by 1-bromo-3-chloropropane (514 mg, 3.26 mmol). Mixture was warmed to 50 °C and stirred for 4 h. The reaction was quenched with H₂O and extracted three times with EtOAc. The organic layers were combined and washed with sat. aqueous NaCl, then dried over Na₂SO₄ and filtered. Concentration in vacuo provided the crude product which was purified by flash chromatography (9:1 CH₂Cl₂/MeOH) to yield the desired product in 790 mg (83%) as a light yellow oil: ¹H NMR (400MHz,CDCl₃) δ (ppm) 8.66 (1H, d, J = 4.4 Hz), 8.01 (1H, d, J = 8.3 Hz), 7.82 (1H, t, J = 7.8 Hz), 7.44 (1H, m), 3.83 (1H, m), 3.59 (2H, t, J = 6.5 Hz), 2.95 (2H, d, J = 11.2 Hz), 2.50 (2H, t, J = 7.2 Hz), 2.16 (2H, m), 1.94 (4H, m), 1.77 (2H, m); ¹³C NMR (100.6 MHz, CDCl₃) δ (ppm) 203.7, 152.7, 148.8, 136.9, 126.9, 122.4, 55.7, 53.2, 43.3, 42.2, 30.0, 28.1; HRMS (ES+, M+H) calcd for C₁₄H₂₀N₂OCl: 267.1264, found: 267.1263

Step 2. 4-(3-(4-((3-fluorophenyl)(hydroxy)(pyridin-2-yl)methyl)piperidin-1-yl)propoxy)benzonitrile

A solution of (1-(3-chloropropyl)piperidin-4-yl)(pyridin-2-yl)methanone (100 mg, 0.35 mmol.) in THF (1.41 mL) was cooled to 0 °C. To this was added 3-Fluorophenylmagnesium bromide (1.0 M in THF) dropwise with stirring. The solution was then slowly warmed to ambient temperature, and stirring continued for 2 h, at which point starting material was consumed by TLC analysis. The reaction was quenched with saturated aqueous NH₄Cl and extracted with EtOAc. The organic layers were combined and dried over Na₂SO_4. Solution was then transferred to a round-bottom flask, concentrated in vacuo to remove solvent, and then dissolved in DMF (1.0 mL). K₂CO₃ (76 mg, 0.55 mmol) was added, followed by 4-hydroxybenzonitrile (131 mg, 1.10 mmol). Mixture was warmed to 50 °C and stirred for 6 h. The reaction mixture was quenched with H₂O and extracted with EtOAc. The organic layers were combined and washed with saturated aqueous NaCl and dried over Na₂SO₄. Concentration in vacuo provided the crude product, which was purified by reverse-phase HPLC chromatography to provide the desired product as an off-white powder in 106 mg (65%). Chiral Separation: Semi-preparative purifications were carried out via stacked injections on a Waters Investigator SFC using a 10 × 250 mm Chiral Technologies CHIRALPAK ID column heated to 40 °C. The eluent was 50% IPA (0.1% DEA) in CO₂ at a flow rate of 15 mL/minute. Backpressure was maintained at 100 bar. The first eluting peak (ML399), retention time = 3.09 min. The second eluting peak (CID 71777745), retention time = 3.79 min. ¹H NMR (500 MHz,CDCl₃) δ (ppm) 8.48 (1H, d, J = 4.8 Hz), 7.69 (1H, dt, J = 7.8, 2.0 Hz), 7.56 (2H, d, J = 9.0 Hz), 7.46 (1H, d, J = 7.9 Hz), 7.37 (2H, t, J = 9.0 Hz), 7.27 (1H, m), 7.18 (1H, dd, J = 5.2, 2.1 Hz) 6.96 (3H, m), 6.02 (1H, br s) 4.06 (2H, t, J = 6.8), 3.06 (2H, m), 2.62 (2H, s), 2.45 (1H, s), 2.22-1.90 (4H, m), 1.77 (2H, m), 1.56 (1H, m), 1.11 (1H, m); ¹³C NMR (125.8 MHz, CDCl₃) δ (ppm) 164.1, 162.4, 162.1 (d, J_CF = 12 Hz), 148.4 (d, J_CF = 12 Hz), 147.4, 137.4, 134.1, 129.8 (d, J_CF = 12 Hz), 122.4, 121.5, 120.4, 119.4, 115.3, 113.8 (d, J_CF = 18 Hz), 113.4 (d, J_CF = 18 Hz), 104.0, 78.6, 66.7, 54.9, 54.0, 44.5, 26.5, 25.6; HRMS (ES+, M+H) calcd for C₂₇H₂₉N₃O₂F: 446.2244, found: 446.2248.

3. Results

3.1. Dose Response Curves for Probe

Figure 1FP Dose Response Curve for Probe ML399

4. Discussion

4.1. Comparison to existing art and how the new probe is an improvement

Peptide fragments were first reported which disrupt the menin-MLL interactions in vitro and in cells.2, 5 With respect to peptidomimetics, a series of macrocyclic compounds was reported which were developed using a structure-based design approach.6 The most potent compound described MCP-1 was 18.5 nM (Figure 2). MCP-1 has a MW of 815 and not unexpected for a peptidomimetic of this nature, its ligand efficiency metrics are quite poor (LE < 0.25, LELP >15). Similar to acyclic peptide based inhibitors these tools are typically not viable as starting points for small molecule optimization. The assay provider has independently described a thienopyrimidine class of inhibitors leading to MI2, as shown in Figure 2.4, 7, 8 Similar potency and robust activity has been noted by this class of compounds in cell based assays; however, DMPK and ancillary activity is unknown at this time. Thus, a full comparison of the respective profiles for ML399 versus MI2 cannot be made at this time. In the case of MI2, ligand efficiency and LELP are more closely aligned with ML399. Cellular activity is reported to be above 5 μM in the case of MI2 and interestingly, this class of inhibitors does not occupy the tail group Trp341 pocket, nor engage in a hydrogen bond with Trp341 as occupied by the ML399 class. This differential binding feature of the disclosed piperidine class allows for an additional interaction with menin that may lead to inhibitors with enhanced potency. Beyond the thienopyrimidine class, no other reports of small molecule PPIs of the menin-MLL interaction have been reported at this time.

Figure 2

Reported classes of peptidomimetic and small molecule menin binders.

5. References

1.

He S, Senter TJ, Pollock J, Han C, Upadhyay SK, Purohit T, Gogliotti RD, Lindsley CW, Cierpicki T, Stauffer SR, Grembecka J. High-affinity small-molecule inhibitors of the menin-mixed lineage leukemia (MLL) interaction closely mimic a natural protein-protein interaction. J Med Chem. 2014;57:1543–56. [PMC free article: PMC3983337] [PubMed: 24472025]

2.

Caslini C, Yang Z, El-Osta M, Milne TA, Slany RK, Hess JL. Interaction of MLL amino terminal sequences with menin is required for transformation. Cancer Res. 2007;67:7275–83. [PMC free article: PMC7566887] [PubMed: 17671196]

3.

Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I, Herr W, Cleary ML. Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol Cell Biol. 2004;24:5639–49. [PMC free article: PMC480881] [PubMed: 15199122]

4.

Cierpicki T, Grembecka J. Challenges and opportunities in targeting the menin-MLL interaction. Future Med Chem. 2014;6:447–62. [PMC free article: PMC4138051] [PubMed: 24635524]

5.

Grembecka J, Belcher AM, Hartley T, Cierpicki T. Molecular basis of the mixed lineage leukemia-menin interaction: implications for targeting mixed lineage leukemias. J Biol Chem. 2010;285:40690–8. [PMC free article: PMC3003368] [PubMed: 20961854]

6.

Zhou H, Liu L, Huang J, Bernard D, Karatas H, Navarro A, Lei M, Wang S. Structure-based design of high-affinity macrocyclic peptidomimetics to block the menin-mixed lineage leukemia 1 (MLL1) protein-protein interaction. J Med Chem. 2013;56:1113–23. [PubMed: 23244744]

7.

Hess JL, Grembecka J, Cierpicki T. Compositions and Methods for Treatment of Leukemia. 2011

8.

Shi A, Murai MJ, He S, Lund G, Hartley T, Purohit T, Reddy G, Chruszcz M, Grembecka J, Cierpicki T. Structural insights into inhibition of the bivalent menin-MLL interaction by small molecules in leukemia. Blood. 2012;120:4461–9. [PMC free article: PMC3512226] [PubMed: 22936661]