Search

J. Neurochem. (2011) 118, 1016–1031. Abstract The EphA4 receptor and its ephrin ligands are involved in astrocytic gliosis following CNS injury. Therefore, a strategy aimed at the blockade of EphA4 signaling could have broad therapeutic interest in brain disorders. We have identified novel small molecule inhibitors of EphA4 kinase in specific enzymatic and cell-based assays. In addition, we have demonstrated in two in vitro models of scratch injury that EphA4 receptor kinase is activated through phosphorylation and is involved in the repopulation of the wound after the scratch. A potent EphA4 kinase inhibitor significantly inhibited wound closure and reduced the accumulation of the reactive astrocytes inside the scratch. We have also shown that after the transient focal cerebral ischemia in rats, a large glial scar is formed by the accumulation of astrocytes and chondroitin sulfate proteoglycan surrounding the infarcted tissue at 7 days and 14 days of reperfusion. EphA4 protein expression is highly up-regulated in the same areas at these time points, supporting its potential role in the glial scar formation and maintenance. Taken together, these results suggest that EphA4 kinase inhibitors might interfere with the astrogliosis reaction and thereby lead to improved neurological outcome after ischemic injury.

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK(a) of these inhibitors and the pH of the BACE-1 enzyme binding assay.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.

BACE-1 is a flexible enzyme with experimentally determined motion in the flap region, the catalytic aspartates, and the 10s loop. Four in-house crystallographically determined complexes of tertiary carbinamine inhibitors revealed 10s loop motion in the S 3 pocket. These X-ray structures were used to correlate K i values, which span over five orders of magnitude, with the calculated interaction energy, using the Merck Molecular Force Field for a series of 19 tertiary carbinamine inhibitors.

A convenient one pot method for the synthesis of 2,5-disubstituted 1,3,4-oxadiazoles from acids and acyl hydrazides is reported. Acid activation with CDI, followed by coupling with the desired acylhydrazide and dehydration in the same pot with Ph 3 P and CBr 4 affords the corresponding 1,3,4-oxadiazoles in good yield. The scope of the acid and acylhydrazide components is presented.

An asymmetric route to dihydroquinazolinones via the addition of ortho metalated substrates to tert-butanesulfinyl imines is reported. The scope of the nucleophile and electrophile components and the absolute stereochemical outcome are presented.

In the preceding communication,[1] the proposed synthesis plan identified the two principal pectenotoxin-4 subunits II and III (Figure 1). It was our intention to couple these fragments through the alkylation of the metalloenamine derived from hydrazone III, readily available from the coupling of advanced intermediates IV and V (transform T2), by epoxide II. However, this investigation revealed that the above bond construction was not feasible due to the decomposition of metalloeneamine III under the reaction conditions.[2] Accordingly, the objective in the present communication is the synthesis of the subunits IV and V, and the completion of the syntheses of pectenotoxin-4 (1) and pectenotoxin-8 by a revised fragment coupling strategy, where epoxide alkylation (transform T1) precedes diene formation (transform T2). The plan for the construction of the F-ring tetrahydrofuran IV was to involve a C37 hydroxy-directed epoxidation of olefin VI with a subsequent ring closure by the C32 hydroxy moiety (transform T3). Finally, the stereoselective formation of the E-ring tetrahydrofuran V from its acyclic precursor VII was based on an iodoetherification precedent provided by Bartlett and Rychnovsky (transform T4). The synthesis of the ring-E synthon V began with the known aldol adduct adduct 2 (Scheme 1).[4] Reduction of 2 (LiBH4, THF, 0 8C), and selective protection of the primary alcohol (TBSCl, Im, CH2Cl2, 100% over two steps) afforded allylic alcohol 3.[5] Acylation of 3 with the PMB-protected lactic acid 4[6] (DCC, DMAP, CH2Cl2, 52%), followed by carbonyl olefination of 5a with Tebbe reagent[7] afforded the 1,5-diene 5b. Claisen rearrangement of 5b in refluxing toluene gave the desired rearrangement product 6 in 82% yield for the two steps. Chelate-controlled reduction of the resulting ketone (Zn(BH4)2, Et2O, 78 8C, 86%, d.r. 86:14) provided the precursor for the key iodoetherification reaction. In spite of the modest selectivity that was observed for the formation of the desired tetrahydrofuran 7 (NIS, CH3CN, 40 8C, 89%, d.r. 72:28), this outcome proved sufficient to pursue the planned route. Successive radical dehalogenation of 7 (Bu3SnH, AIBN, toluene, 100%) and deprotection of the primary TBS ether (TBAF, THF, 95%) afforded alcohol 8. Oxidation with Dess± Martin reagent[8] (py, CH2Cl2, 99%), Wittig homologation (EtOC(O)CC(CH3)PPh3, THF, 65 8C; 100% E :Z> 95:5), and ester reduction (LiAlH4, Et2O, 0 8C, 92%) completed the carbon assembly of the E-ring fragment. Benzyl protection (NaH, BnBr, TBAI, THF/DMF, 94%) followed by PMB deprotection (DDQ, CH2Cl2/pH 7 buffer, 95%) gave alcohol 10. Oxidation to the methyl ketone[8] (Dess±Martin periodinane, py, CH2Cl2, 93%), and hydrazone formation (TMSCl, CH2Cl2/Me2NNH2, 100%) completed the synthesis of hydrazone 11. As summarized in Figure 1, the first stage of the synthesis of the ring-F fragment IV will be simplified to the construction of the C31±C35 phosphonium salt, the C36±C40 aldehyde, and their union through a Wittig coupling to afford the Zolefin VI. The synthesis of the C31±C35 phosphonium salt began with the known triol derivative 12 (Scheme 2).[9] Protection of the hydroxy group at C33 of 12 as a PMB ether (PMBBr, NaH, THF/DMF, 95%) followed by reductive ozonolysis (O3, EtOH, then DMS, then NaBH4, 95%) afforded alcohol 13. Transformation of 13 to the corresponding iodide (I2, Im, Ph3P, CH2Cl2, 0 8C, 89%) proceeded smoothly, but careful control of the temperature was required to access phosphonium salt 14 (Ph3P, CH3CN, 55 8C, 89%).[10] The synthesis of the aldehyde partner 17 began with protection of the hydroxy group at C37 of aldol adduct 15[11] as a base-sensitive triphenylsilyl ether (TPSCl, Im, DMAP, DMF, 0 8C, 98%; Scheme 2). Half reduction of the S-phenyl thioester[12] (Pd/C, Et3SiH, acetone, 95%), and olefination under modified Lombardo conditions[13] ([Cp2ZrCl2], Zn dust, CH2I2, THF, 0 8C, 84%) afforded olefin 16. RhodiumCOMMUNICATIONS

The first members of the pectenotoxin family of marine natural products were isolated off the northeastern coast of Japan in 1985. Subsequently, ten members of this group have been identified.[1] The structural diversity within the pectenotoxins stems from variations in the oxidation state at C43, as well as the differing configurations of the AB spiroketal portion of the structures. Pectenotoxin-2 (C431⁄4Me) is cytotoxic towards human lung, colon, and breast cancer cell lines with LC50 values in the nanomolar range.[1c] PectenotoxCOMMUNICATIONS

During our ongoing efforts to develop a small molecule inhibitor targeting the β-amyloid cleaving enzyme (BACE-1), we discovered a class of compounds bearing an aminoimidazole motif. Initial optimization led to potent compounds that have high Pgp efflux ratios. Crystal structure-aided design furnished conformationally constrained compounds that are both potent and have relatively low Pgp efflux ratios. Computational studies performed after these optimizations suggest that the introduction of the constraint enhances potency via additional hydrophobic interactions rather than conformational restriction.

The first total synthesis of a spongipyran macrolide, altohyrtin C, is described. The convergent synthesis strategy relies on a regioselective macrolactonization, a stereoselective Wittig coupling of the two major synthetic fragments, a complex anti aldol reaction to join the C1–C15 and C16–C28 spiroketal regions, and an anomeric sulfone acylation to join the C29–C37 and C38–C43 pyran regions. The incorporation of the C44–C51 sidechain in the final stages of the synthesis establishes a viable route for the construction of variants in this pharmacologically important region. Methodological developments en route to the total synthesis include a 1,5 anti-selective methyl ketone aldol reaction and a diastereoselective approach to Lewis acid mediated β-C-glycosidation. Completion of the synthesis has confirmed the stereochemical assignments proposed in the altohyrtin series and has established the identity of the altohyrtin and spongistatin marine macrolides.

A short, efficient synthesis of elenic acid, a marine natural product with interesting biological activity, has been completed. Critical features of the synthesis are the development of methodology for the one-pot elaboration of an alkyne to an (E)-β,γ-unsaturated ester with a stereocenter at the α-position and the use of the zipper reaction of a 1-arylalkyne. This reaction has not been previously reported with aromatic substrates. The synthesis strategy provides considerable flexibility for the preparation of structural analogues.

Alkenyl-substituted ketals 6 readily afford Claisen rearrangement substrates 7 upon treatment with TESOTf/DIPEA. Thermal rearrangement completes this efficient and general process for generation of functionally rich ϵ-oxy-γ,δ-enones 8.

The EphA4 receptor and its ephrin ligands are involved in astrocytic gliosis following CNS injury. Therefore, a strategy aimed at the blockade of EphA4 signaling could have broad therapeutic interest in brain disorders. We have identified novel small molecule inhibitors of EphA4 kinase in specific enzymatic and cell-based assays. In addition, we have demonstrated in two in vitro models of scratch injury that EphA4 receptor kinase is activated through phosphorylation and is involved in the repopulation of the wound after the scratch. A potent EphA4 kinase inhibitor significantly inhibited wound closure and reduced the accumulation of the reactive astrocytes inside the scratch. We have also shown that after the transient focal cerebral ischemia in rats, a large glial scar is formed by the accumulation of astrocytes and chondroitin sulfate proteoglycan surrounding the infarcted tissue at 7 days and 14 days of reperfusion. EphA4 protein expression is highly up-regulated in the same areas at these time points, supporting its potential role in the glial scar formation and maintenance. Taken together, these results suggest that EphA4 kinase inhibitors might interfere with the astrogliosis reaction and thereby lead to improved neurological outcome after ischemic injury.

Alkenyl-substituted ketals 6 readily afford Claisen rearrangement substrates 7 upon treatment with TESOTf/DIPEA. Thermal rearrangement completes this efficient and general process for generation of functionally rich ϵ-oxy-γ,δ-enones 8.

A short, efficient synthesis of elenic acid, a marine natural product with interesting biological activity, has been completed. Critical features of the synthesis are the development of methodology for the one-pot elaboration of an alkyne to an (E)-β,γ-unsaturated ester with a stereocenter at the α-position and the use of the zipper reaction of a 1-arylalkyne. This reaction has not been previously reported with aromatic substrates. The synthesis strategy provides considerable flexibility for the preparation of structural analogues.

The first total synthesis of a spongipyran macrolide, altohyrtin C, is described. The convergent synthesis strategy relies on a regioselective macrolactonization, a stereoselective Wittig coupling of the two major synthetic fragments, a complex anti aldol reaction to join the C1–C15 and C16–C28 spiroketal regions, and an anomeric sulfone acylation to join the C29–C37 and C38–C43 pyran regions. The incorporation of the C44–C51 sidechain in the final stages of the synthesis establishes a viable route for the construction of variants in this pharmacologically important region. Methodological developments en route to the total synthesis include a 1,5 anti-selective methyl ketone aldol reaction and a diastereoselective approach to Lewis acid mediated β-C-glycosidation. Completion of the synthesis has confirmed the stereochemical assignments proposed in the altohyrtin series and has established the identity of the altohyrtin and spongistatin marine macrolides.

The optimization of tertiary carbinamine derived inhibitors of BACE1 from its discovery as an unstable lead to low nanomolar cell active compounds is described. Five-membered heterocycles are reported as stable and potency enhancing linkers. In the course of this work, we have discovered a clear trend where the activity of inhibitors at a given assay pH is dependent on pKa of the amino group that interacts directly with the catalytic aspartates. The potency of compounds as inhibitors of Αβ production in a cell culture assay correlated much better with BACE1 enzyme potency measured at pH 7.5 than at pH 4.5.

This Letter describes the one pot synthesis of tertiary carbinamine 3 and related analogs of brain penetrant BACE-1 inhibitors via the alkylation of the Schiff base intermediate 2. The methodology developed for this study provided a convenient and rapid means to explore the P1 region of these types of inhibitors, where the P1 group is installed in the final step using a one-pot two-step protocol. Further SAR studies led to the identification of 10 which is twofold more potent in vitro as compared to the lead compound. This inhibitor was characterized in a cisterna magna ported rhesus monkey model, where significant lowering of CSF Abeta40 was observed.

This letter describes replacements for the P3 amide moiety present in previously reported tertiary carbinamine macrolactones. Although P-gp efflux issues associated with these amide-macrolactones were solved and full brain penetration was measured in one case, potency was compromised in the process.

This Letter describes the design and synthesis of tertiary carbinamine macrocyclic inhibitors of the β-secretase (BACE-1) enzyme. These macrocyclic inhibitors, some of which incorporate novel P2 substituents, display a 2- to 100-fold increase in potency relative to the previously described acyclic analogs while affording greater stability.

We describe the discovery and optimization of tertiary carbinamine derived inhibitors of the enzyme beta-secretase (BACE-1). These novel non-transition-state-derived ligands incorporate a single primary amine to interact with the catalytic aspartates of the target enzyme. Optimization of this series provided inhibitors with intrinsic and functional potency comparable to evolved transition state isostere derived inhibitors of BACE-1.

A convenient one pot method for the synthesis of 2,5-disubstituted 1,3,4-oxadiazoles from acids and acyl hydrazides is reported. Acid activation with CDI, followed by coupling with the desired acylhydrazide and dehydration in the same pot with Ph 3 P and CBr 4 affords the corresponding 1,3,4-oxadiazoles in good yield. The scope of the acid and acylhydrazide components is presented.

An asymmetric route to dihydroquinazolinones via the addition of ortho metalated substrates to tert-butanesulfinyl imines is reported. The scope of the nucleophile and electrophile components and the absolute stereochemical outcome are presented.

We describe the development of cell-permeable β-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell-based assay (IC50 < 100 nM), these inhibitors display impressive selectivity against other biologically relevant aspartyl proteases.