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. 2019 Nov 3;11(3):346-352.
doi: 10.1021/acsmedchemlett.9b00402. eCollection 2020 Mar 12.

Structure-Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Theresa D Manz  1   2   3 , Sindhu C Sivakumaren  1   2 , Adam Yasgar  4 , Matthew D Hall  4 , Mindy I Davis  4 , Hyuk-Soo Seo  1   2 , Joseph D Card  5   6   7 , Scott B Ficarro  5   6   7 , Hyeseok Shim  8 , Jarrod A Marto  5   6   7 , Sirano Dhe-Paganon  1   2 , Atsuo T Sasaki  9 , Matthew B Boxer  4 , Anton Simeonov  4 , Lewis C Cantley  8 , Min Shen  4 , Tinghu Zhang  1   2 , Fleur M Ferguson  1   2 , Nathanael S Gray  1   2
Affiliations

Structure-Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Theresa D Manz et al. ACS Med Chem Lett. .

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure-activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

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Conflict of interest statement

The authors declare the following competing financial interest(s): N.S.G. is a founder, SAB member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. N.S.G. and T.Z. are inventors on a patent application covering chemical matter in this publication, owned by Dana-Farber Cancer Institute. L.C.C. is a founder and member of the Board of Directors (BOD) of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals. These companies are developing novel therapies for cancer. J.A.M. serves on the SAB of 908 Devices.

Figures

Figure 1
Figure 1
Reported PI5P4K inhibitors.
Figure 2
Figure 2
Cocrystal structure of PI5P4Kα and THZ-P1-2 (dark gray). The pocket bound by the inhibitor is highlighted in pale-green. H-bonds are shown as dashed bonds. For clarity, only Asn198, Val199, Phe134, and Phe200 are shown as capped sticks. PDB-ID: 6OSP.
Scheme 1
Scheme 1. Synthesis of THZ-P1-2 and Compounds 6–11
Reagents and conditions: (a) (HO)2B-R or respective pinacol boronic acid esters, NaHCO3, Pd(PPh3)2Cl2, ACN/H2O, 90–100 °C, 2.5–19 h, 53–83% yield; (b) 4-methylaniline, TEA, BuOH, 140 °C, 1 h or 3/4-phenylaniline, TEA, EtOH, 120 °C, 3 h, 99%-quant. yield; (c) m-phenylenediamine, DIEA, NMP, 140–160 °C, 1.5 h to 2 d, 37–95% yield; (d) 4-nitrobenzoyl chloride, pyridine or TEA/DCM, rt, 5 h or 4-((tert-butoxycarbonyl)amino)benzoic acid, HATU, Hunig’s Base, DMF, rt, 1 h, then TFA/DCM, rt, 2 h, 37% yield; (e) SnCl2·2H2O, EtOAc/MeOH, 80 °C, 4.5 h to 2 d, or H2, 10% Pd/C, EtOAc/MeOH or MeOH, rt, 16 h, 4–87% yield over 2 steps; (f) 4-bromocrotonyl chloride, DIEA, DCM or ACN, 0 °C, then dimethylamine, THF, rt, 1 h, 24–71% yield; (g) 4-dimethylaminocrotonic acid, DIEA, HATU, DCM, or oxalylic chloride, DMF rt, 1–12 h, 6–30% yield; (h) 1.0 M NaOH/1,4-dioxane, rt, 4–6 h, 8–60% yield.
Scheme 2
Scheme 2. Synthesis of Compounds 14–19
Reagents and conditions: (a) anilines, DIEA, NMP, 150 °C, 6–8 h, then TFA/DCM, rt, 1 h, 67–84% yield; (b) anilines, DIEA, NMP, 150 °C, 8 h to overnight, 46–60% yield; (c) nitroaniline, Cs2CO3, X-Phos, Pd2(dba)3, 1,4-dioxane, 104 °C, 3 h, then SnCl2, MeOH, 80 °C, 8 h, 61% yield; (d) carboxylic acid chlorides, pyridine, rt, overnight, then SnCl2·2H2O, EtOAc/MeOH, 80 °C, 1.5–2 d, 23–39% yield over 2 steps; (e) carboxylic acid chlorides, DIEA or TEA, DCM, 0 °C, 5 min or rt, 1.5 h, then H2, 10% Pd/C, MeOH or EtOAC/MeOH, rt, 12 h to overnight, 36–51% over 2 steps; (f) carboxylic acids, HATU, DIEA, DCM, rt, 0.5–3 h, then HCl, MeOH or EtOAc, rt, 0.5 h to overnight, 54–85% over 2 steps; (g) 4-bromocrotonyl chloride, DIEA, DCM or ACN, 0 °C, 5 min to 0.5 h, then dimethylamine (2.0 M in THF), rt, 0.5–2 h, 30–91% yield; (h) 4-dimethylaminocrotonic acid hydrochloride, DIEA, HATU, DCM, rt, 12 h, 30% yield; (i) 1.0 M NaOH/1,4-dioxane, 10 °C to rt, 3–6 h, 10–46% yield.
Scheme 3
Scheme 3. Synthesis of Compound 22
Reagents and conditions: (a) tert-butyl 3-aminopiperidine-1-carboxylate, DIEA, NMP, 110 °C, overnight, 50% yield; (b) TFA/DCM, rt, overnight, quant. yield; (c) 3-nitroaniline, triphosgene, TEA, DCM, 0 °C, 2 h, then rt, overnight, 83% yield; (d) SnCl2, EtOAc/MeOH, reflux, overnight, 74% yield; (e) 4-bromocrotonyl chloride, DIEA, DCM, 0 °C, 5 min, then dimethylamine (2.0 M in THF), rt, 1 h, 68% yield; (f) 1.0 M NaOH/1,4-dioxane, 10 °C to rt, 4 h, 30% yield.
Scheme 4
Scheme 4. Synthesis of Compound 25
Reagents and conditions: (a) 2-(3-aminophenyl)acetic acid, DIEA, NMP, 140 °C, 12 h, 6% yield; (b) tert-butyl piperidin-4-ylcarbamate, HATU, DIEA, DMF, rt, 0.5 h, 51% yield; (c) HCl, EtOAc, rt, 0.5 h, 90% yield; (d) 4-dimethylaminocrotonic acid hydrochloride, DIEA, HATU, DCM, rt, 0.5 h, 50% yield; (e) 1.0 M NaOH/1,4-dioxane, rt, 4 h, 56% yield.
Scheme 5
Scheme 5. Synthesis of Compound 30
Reagents and conditions: (a) 4-iodotoluidine, trans-1,2-cyclohexanediamine, K3PO4, CuI, toluene, 110 °C, 1 d, 48% yield; (b) m-phenylenediamine, DIEA, NMP, 150 °C, 4 d, quant. yield; (c) 4-nitrobenzoyl chloride, pyridine, rt, overnight; (d) SnCl2·2H2O, EtOAc/MeOH, 80 °C, 1 d, 40% yield over 2 steps; (e) 4-bromocrotonyl chloride, DIEA, ACN, 0 °C; (f) dimethylamine (2.0 M in THF), rt, 1 h, 4% yield over 2 steps.
Figure 3
Figure 3
(A) KINOMEscan results for THZ-P1-2 and compound 30. Selectivity profile was determined for 469 kinases as percent DMSO control at 1 μM inhibitor concentration (excluding mutant, atypical, and pathogen). (B) Table comparing percent DMSO control for top five off-targets of THZ-P1-2 to compound 30.
Figure 4
Figure 4
(A) Melting-curves (Tm) of PI5P4Kα and PI5P4Kβ. Treatment with THZ-P1-2 at 10 μM for 1 h, in comparison to DMSO. (B) Representative Western blot of isothermal CETSA at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors at 1 or 10 μM. (C) Quantified relative band intensity (%) of isothermal CETSA experiment (Figure 4B). Mean of three independent experiments. (D) Dose–response curve of isothermal CETSA for PI5P4Kα at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors. Mean of three independent experiments (representative Western blot, see Supporting Figure 5A). (E) Dose–response curve of isothermal CETSA for PI5P4Kβ at 52 °C (HEK 293T cells) with selected PI5P4Kα/β inhibitors. Mean of three independent experiments (representative Western blot, see Supporting Figure 5B).
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