Design and synthesis of high affinity inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferases directed by ligand efficiency dependent lipophilicity (LELP)

Abstract

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization.

Figure 1

2,3-Substituted benzo[b]thiophene PfNMT and PvNMT inhibitor 1 and the target profile for the development of this series. Footnote a: K_i values are quoted in place of IC₅₀ values as a means of expressing the inhibitor affinity while correcting for differing Michaelis constants (K_m) between enzymes. Enzyme K_i values are calculated from the IC₅₀ values using the Cheng–Prusoff equation, the definition of which is given in the Experimental Section. IC₅₀ values are the mean value of two or more determinations, and standard deviation is within 20% of the IC₅₀. Footnote b: No significant difference in inhibition between HsNMT1 and HsNMT2 isoforms has been observed in this series; therefore, the HsNMT affinities reported in this work refer to HsNMT1. Footnote c: LELP = cLogP/LE. LE = [−log(K_i)](1.374)/(no. of heavy atoms), with cLogP determined with ChemAxon, which can be obtained from http://www.chemaxon.com/products/calculator-plugins/logp/.

Scheme 1. Synthesis of Phenethyl Esters and Amides

Reagents and conditions: (a) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, N,N-diisopropylethylamine, MeCN, rt, 18 h, 36–57%; (b) benzotriazo-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine, DCM, rt, 18 h, 69–93%; (c) 10% TFA in DCM (v/v), rt, 2 h, 42–97%. 2 was prepared as described previously.

Scheme 2. Synthesis of Two Regioisomers of 1,2,4-Oxadiazole Bioisosteres

Reagents and conditions: (a) NH₂OH·H₂O, EtOH, rt, 5 h, 98–99%; (b) 2, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, DMF, 140 °C, 3 h, 10–31%; (c) 10% TFA in DCM (v/v), rt, 2 h, 13–99%; (d) bromoacetonitrile, t-BuOK, THF, 0 °C to rt, 15 min, 88%; (e) tert-butyl 4-hydroxypiperidine-1-carboxylate, diisopropyl azodicarboxylate, PPh₃, THF, rt 1.5 h, 78%; (f) RCH₂CO₂H, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, MeCN, rt, 18 h; (g) 4 Å molecular sieves, toluene, 18 h, 110 °C, 54–68% over two steps.

Scheme 3. Synthesis of 1,3,4-Oxadiazole and 1,2,4-Triazole Linker Bioisosteres

Reagents and conditions: (a) NH₂NH₂·H₂O, EtOH, 78 °C, 24 h, 75%; (b) RCH₂C(O)Cl, N,N-diisopropylethylamine, DCM, rt, 15 min, 75–91%; (c) POCl₃, 100 °C, 1 h; (d) ammonium acetate, acetic acid, 140 °C, 1.5 h, 6% over two steps; (e) TsCl, 1,2,2,6,6 pentamethylpiperidine, DCM, rt, 3 h, 48–65%; (f) TFA, DCM, rt, 2 h, 55–98%.

Figure 2

X-ray crystal structure of 20b (blue) bound to PvNMT (green). (A) 20b bound to PvNMT. The 3-methoxyphenyl substituent forms the intended interactions with Ser319 and Phe105, in addition to the deeply buried hydrophobic scaffold and salt bridge interaction observed in 1. (B) The oxadiazole linker is sandwiched between two aromatic residues, rationalizing the affinity enhancement in moving to an aromatic heterocycle from the ester linker in 4b. Dashed lines are drawn to highlight key interactions between the enzyme and the ligand.

Figure 3

X-ray crystal structure of 20b (blue) bound to PvNMT (green). Further inspection of the water molecules within the active site shows that the benzylic CH₂ occupies a heavily solvated pocket, indicating that substitution may result in more favorable energetics within the enzyme active site. Dashed lines indicate water molecules within 5 Å of the benzylic position.

Scheme 4. Synthesis of α-Substituted Phenyl 1,3,4-Oxadiazole Inhibitors

Reagents and conditions: (a) Ph-CR₁R₂-CO₂H, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, THF, DMF, rt, 18 h, 48–99%; (b) TsCl, 1,2,2,6,6pentamethylpiperidine, DCM, rt, 18 h; (c) 10% TFA in DCM (v/v), rt, 2 h, 4–40% over two steps.

Scheme 5. Synthesis of Five-Membered Heterocyclic Methoxyphenyl Replacements

Reagents and conditions: (a) NaH, ethyl bromoacetate, THF, 0 °C, 18 h, 78%; (b) methyl 3-bromopropionate, K₂CO₃, DMF, 55 °C, 18 h, 30%; (c) NH₂NH₂·H₂O, MeOH, rt, 3 h, 83–99%; (d) n = 1, NH₂OH·HCl, K₂CO₃, EtOH, 78 °C, 3 h, 12%; n = 2, NH₂OH·HCl, H₂O, MeOH, 60 °C, 18 h, 89%; (e) MeNHNH₂, AcOH, 3 h, rt, 73–95%; (f) LiOH·H₂O, MeOH, rt, 18 h, 51–95%; (g) 16, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, THF, DMF, rt, 18 h, 48–99%; (h) TsCl, 1,2,2,6,6-pentamethylpiperidine, DCM, rt, 18 h; (i) 10% TFA in DCM (v/v), rt, 2 h, 3–26% over two steps.

Figure 4

Binding mode of 34c (gold) bound to PvNMT. (A) 34c (gold) bound to PvNMT (green), showing piperidine–Leu410 salt bridge interaction, deeply buried benzothiophene scaffold, and 1,3,5-trimethylpyrazole heterocycle bound within the Ser319 hydrophobic pocket. (B) Enlarged view of the 1,3,5-trimethylpyrazole of 34c (gold) with PvNMT (green). This shows the water-bridged interaction between the pyrazole and Ser319, as well as multiple hydrophobic contacts between the heterocycle and the binding pocket.

Figure 5

Binding mode of 34a (pink) bound to PvNMT. (A) 34a (pink) forms all previously observed interactions with the enzyme. (B) Comparison of the binding modes of 34a and 34c reaffirms this similarity, showing that the only point of differentiation is in the Phe105/Ser319 binding site occupied by the pyrazole. (C) Enlarged view of the 1,3,5-trimethylpyrazole of 34c (gold) and 3,5-dimethylpyrazole of 34a (pink) with PvNMT (green). The pyrazole of 34a forms a direct interaction with Ser319 (3.2 Å), and the water involved in the bridged interaction in Figure 4B has been excluded from the pocket.

Figure 6

(A) Plot of cellular potency vs enzyme affinity for all members of this series tested in both assays. (B) Plot of cellular potency vs enzyme affinity for all compounds with a LELP of >10. (C) Plot of cellular potency vs enzyme affinity for all compounds with a LELP of <10.