Herein we describe the development

Herein, we describe the development of second generation GSM compounds aimed at improving critical physicochemical properties while maintaining the potent activity of the parent AGSM. Ligand design focused on identifying heterocyclic replacements for the hydrophobic D-ring as a means to improve ADME (absorption, distribution, metabolism and excretion) parameters, as well as aqueous solubility. Replacement of the alkyl rich phenyl D-ring by a pyrazole containing scaffold was anticipated to ameliorate the property related shortcomings of the AGSMs. Based on the spatial arrangement of the substituents within AGSM , as well as molecular modeling overlays, tetrahydroindazole, 3--butylpyrazole and cyclopentapyrazole D-ring analogs were selected for preparation. The construction of the three unique series would be accomplished utilizing established Hantzsch chemistry from a common bromoacetophenone precursor enabling the rapid preparation of a diverse set of pyrazole containing scaffolds permitting rigorous evaluation of these novel D-ring substitutions (). A series of 3-aminotetrahydroindazole analogs were synthesized in order to explore the local spatial constraints of the γ-secretase binding cavity with respect to this novel D-ring substitution. The fused ring system of the 2-substituted tetrahydroindazole was envisioned to mirror the alkyl functionalization of AGSM while rigidifying the D-ring and preserving favorable hydrophobic interactions of the parent molecule. 2-Substituted tetrahydroindazoles – displayed modest activity for the reduction of Aβ42 in a SHSY5Y neuroblastoma cell line stably over-expressing human APP and demonstrated a slight gain in potency as the alkyl chain increases in size (compounds –)., However, the alteration in -alkyl connectivity of tetrahydroindazole providing isopropyl derivative , as well as the introduction of a -butyl group in dl 473 led to a slight reduction in activity. Removal of the 2-postion substituent as illustrated by tetrahydroindazole abrogates activity, underscoring the importance of the alkyl functionality with respect to Aβ modulation. Based on the pivotal role the 2-postion substituent plays with respect activity, in addition to the overall tolerance for alkyl substituents of various size, analogs – were prepared to thoroughly probe the chemical space for favorable interactions. The constrained and small 2-cyclobutyl tetrahydroindazole displayed a 2-fold increase in activity when compared to isopropyl analog , suggesting that this region of γ-secretase features a narrow hydrophobic pocket. This assertion is further supported by ligands – which illustrate that as the size of the substituent increases from the 2-cyclopropyl to the larger 2-cyclohexyl derivative ( vs ) the observed activity decreases. Moreover, increasing the polarity of the 2-position substituent through the incorporation of heteroatoms as exemplified by the tetrahydrofuran , tetrahydropyran and 1-methylpiperidine significantly erodes potency. In general, deviation from linear -alkyl substitution is deleterious to suppressing Aβ42 levels, and the introduction of heteroatoms served to exacerbate this trend. Efforts to increase the flexibility of the heterocyclic substituent, as well as extend beyond the constrained hydrophobic region through the insertion of an ethyl or propyl linker failed to restore the loss of activity as evidenced by analogs , and . Consequentially, the incorporation of smaller functionalities within the active 2-postion alkylated analogs was explored. 2-Trifluororethane demonstrated similar activity to parent analog , whereas 2-fluoroethane displayed a slight increase in activity for the reduction of Aβ42. The insertion of oxygen with the alkyl chain was poorly tolerated as demonstrated by 2-methoxyethane , 2-hydroxyethane , and 2-hydroxypropane which are approximately 2-fold less active than the corresponding aliphatic analog. However, increasing hydrophobicity of analog through the addition of two flanking methyl groups resulting in 2-hydroxy-2-methylpropane compound showed an unexpected 3-fold improvement over primary analog thus reinforcing the strong preference for hydrophobic character within this region of the D-ring by the γ-secretase enzyme. Finally, an -methyl derivative of compound was synthesized in order to ascertain the role of the proton with respect to ligand affinity. Unfortunately, -methylated analog loses all activity suggesting either the presence of a hydrogen bond between the ligand and enzyme or an overall reduction in ligand flexibility stemming from the sterical interactions between the D-ring and the added methyl which restrict access to conformations required for target interaction.