Structure-activity relationships of imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human insulin-degrading enzyme

Abstract

Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.

Keywords: Amyloid-beta peptides; Docking; Enzymes; Inhibitors; Structure–activity relationships; X-ray diffraction.

Figure 1

Structures of hit 1 discovered by screening, binding to hIDE (PDB code 4DTT) and hit-to-lead optimization strategy.

Figure 2

Binding of 1 and analogues 22, 33 and 44 at the exosite of hIDE-CF-E111Q. O (red); N (blue); C (blue for IDE N-terminal domain, orange for IDE C-terminal domain, gray for inhibitors); hydrogen contacts in dotted lines. A) 1: X-Ray structure PDB code: 2YPU; B) 33 X-Ray structure PDB code: 4QIA; C) 22 X-Ray structure PDB code: 4GSF; D) 44 X-Ray structure PDB code: 4GS8.

Figure 3

Binding of 1 and analogues 44 and 46 at the catalytic site of hIDE-CF. Zn (magenta sphere); O (red); N (blue); C (blue for IDE N-terminal domain, orange for IDE C-terminal domain, gray for inhibitors); hydrogen contacts in dotted lines. A) 1: X-Ray structure PDB code: 4DTT; B) Docking of 44 in PDB code: 4DTT; C) Docking of 46 in PDB code: 4DTT; D) Structures of 1, 44 and 46.

Figure 4

Summary of Structure-Activity Relationships.

Scheme 1a

Synthesis of analogues 1-4. ^a Reagents and conditions: (a) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h 2) L-aminoacid methyl esters, anhydrous DIEA, anhydrous DMF, Argon, room temp., overnight.

Scheme 2a

Synthesis of analogues 5-14, 16-23. ^a Reagents and conditions: (a) R-Br, MeOH, DIEA , room temp., 2-12 h, 27-76%; (b) SOCl₂, MeOH, 0°c then room temp., 18 h ; (c) RCOCl, DIEA, DCM or RCOOH, EDCI, HOBt, DIEA, DCM, room temp., 18 h; (d) NaOH, MeOH, H₂O, 1-6 h, room temp.; (e) benzylchloroformate, NaOH 2N, 0 °C then room temp., 2 h, 64%; (f) Boc₂O, dioxane, NaOH 2 N, 0 °C then room temp., 18 h, 67%. (g) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h 2) L-Histidine methyl ester dihydrochloride, anhydrous DIEA, anhydrous DMF, Argon, room temp., overnight. (h) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h 2) H-His(1-Trt)-OMe HCl, anhydrous DIEA, anhydrous DMF, Argon, room temp., overnight. (i) TFA, TIS, DCM, room temp., 5min-4 h. (j) 1) DCC, THF, overnight, room temp. 2) L-Histidine methyl ester dihydrochloride, DIEA, THF, 5 h, room temp. (k) HCl(g), DCM, 2 h, room temp.

Scheme 3a

Synthesis of analogues 15 and 24. ^a Reactants and conditions a) chloroacetylchloride, NaHCO₃, DCM, room temp., 10 min; b) tert-butylbromoacetate, DIEA, THF, 0 °C then room temp., overnight; c) NaHCO₃, DMF, reflux, 24 h; d) TFA, TIS, DCM, room temp. 2 h; e) L-Histidine methyl ester dihydrochloride, EDCI, HOBt, DIEA, DCM, room temp., 18 h.

Scheme 4a

Synthesis of analogue 25. ^a Reagents and conditions: (a) LAH, THF, 0 °C then reflux, 18 h, 70%; (b) TsCl, TEA, DCM, room temp., 18 h, 44%; (c) KCN, DMSO, 80 °C, 3.5 h; (d) H₂SO₄, H₂O, reflux, 3 days, 40% (e) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h or DCC, THF, 5 h, room temp. 2) L-H-Histidine-methylamide, anhydrous DIEA, anhydrous DMF, Argon, room temp., overnight.

Scheme 5a

Synthesis of analogues 26-31. ^a Reagents and conditions: (a) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h (b) ammoniac, DMF 100%; (c) TritylO-NH₂, DIEA, DMF, room temp., 18 h, 93%; (d) 20% SOCl₂ / MeOH, room temp., 18 h, 100%; (e) Br(CH₂)₂COOtBu, K₂CO₃, KI, acetone, reflux, 36 h; (f) NaOH, MeOH, 5 days, 40 °C; (g) Boc₂O, NaOH 2N, dioxane, room temp.overnight; (h) potassium tert-butylate 1 M in THF, diethyl squarate, THF, 4 °C, 15 min, 55%; (i) N Benzylglycine hydrochloride, TEA, MeOH, room temp., 18 h, 70%. (j) EDCI, HOBt, DIEA, DCM, L-His-OMe.2HCl, room temp., 18 h, or i. chlorure d’oxalyle, DCM, DMF cat., 0 °C, 30 min; ii. L-(1-Trt)-His-OMe.HCl, DIEA, DCM, room temp., 18 h, 38%; (k) TFA, TIS, DCM, room temp., 5 min-4 h. (l) SOCl₂, MeOH, room temp., 2 h.

Scheme 6a

Synthesis of 32. ^a Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, L-H-Histidine-methylamide, room temp., 18 h, 80%; (b) TFAA/pyridine, THF, 0 °C; (c) NaN₃, ammonium chloride, DMF, 90 °C, 60 h.

Scheme 7a

Synthesis of compounds 33-47. ^a Reactants and conditions: (a) i. MeNH₂, EtOH, MeOH, reflux; (b) SOCl₂, iBuOH, 60 °C, 48 h; (c) EDCI, DMAP, isopropanol, DCM, reflux, 24h, 56 % (d) HClg, DCM, 1h, 100%; (e) amine, EDCI, HOBt, TEA, DMF, room temp., 16 h; (f) i. TBTU, DIEA, DMF, 10 min, room temp.; ii. amidoxime, DMF, room temp. 4.5 h then reflux 1.5 h. (g) i) N-benzyl-iminodiacetic acid, TFAA 2% in acetic anhydride, 50-70 °C, 5 h, ii) amine, anhydrous DIEA, anhydrous DMF, argon, room temp., overnight ; (h) i) diacid 13a, TFAA 2% in acetic anhydride, 50-70 °C, 5 h, ii) amine, anhydrous DIEA, anhydrous DMF, argon, room temp., overnight, (i) TFA/DCM 50/50, TIS; (j) NH₃g, dioxane/MeOH (5/2), room temp. 10 h, (k) NaOH, H₂O, MeOH, room temp., 4 h.

Scheme 8a

Synthesis of 48-49. ^a Reagents and conditions: (a) trifluoroacetic anhydride 2% in acetic anhydride, 50-70 °C, 5 h; (c) MeOH, DIEA, reflux, overnight; (c) amine, DMF, HOBt, EDCI , NMM, room temp. 22 h; (d) TFA/DCM TIS, room temp. 4h.