Optimization of Hydroxyethylamine Transition State Isosteres as

Article Cite This: J. Med. Chem. 2017, 60, 9807−9820

pubs.acs.org/jmc

Optimization of Hydroxyethylamine Transition State Isosteres as Aspartic Protease Inhibitors by Exploiting Conformational Preferences Ana B. Bueno,*,† Javier Agejas,† Howard Broughton,† Robert Dally,‡ Timothy B. Durham,‡ Juan Félix Espinosa,† Rosario González,† Patric J. Hahn,‡ Alicia Marcos,† Ramón Rodríguez,†,§ Gema Sanz,† José F. Soriano,†,∥ David Timm,‡ Paloma Vidal,† Hsiu-Chiung Yang,‡,⊥ and James R. McCarthy‡,# †

Lilly SA, Avenida de la Industria 30, 28108 Alcobendas, Madrid, Spain Lilly Research Laboratories, Indianapolis, Indiana 46285, United States

‡

S Supporting Information *

ABSTRACT: NMR conformational analysis of a hydroxyethylamine peptide isostere developed as an aspartic protease inhibitor shows that it is a flexible architecture. Cyclization to form pyrrolidines, piperidines, or morpholines results in a preorganization of the whole system in solution. The resulting conformation is similar to the conformation of the inhibitor in the active site of BACE-1. This entropic gain results in increased affinity for the enzyme when compared with the acyclic system. For morpholines 27 and 29, the combination of steric and electronic factors is exploited to orient substituents toward S1, S1′, and S2′ pockets both in the solution and in the bound states. These highly preorganized molecules proved to be the most potent compounds of the series. Additionally, the morpholines, unlike the pyrrolidine and piperidine analogues, have been found to be brain penetrant BACE-1 inhibitors.

■

INTRODUCTION Aspartic proteases are a small class of proteases that utilize two aspartic acids and a water molecule to hydrolyze peptides. Renin, pepsin, BACE-1, cathepsins, HIV-1 protease, and plasmepsin belong to this family, and their inhibition has been targeted in drug discovery for the treatment of a variety of diseases including hypertension, Alzheimer’s disease, AIDS, and malaria.1,2 Investigation around all these enzymes has provided insight into the requirements of a drug for targeting this particular class. Among the aspartic proteases, the research toward the identification of BACE-1 inhibitors has been an area of intense effort in the last 15 years based on the evidence of the linkage between this enzyme and the Alzheimer’s disease.3 One strategy to identify protease inhibitors is to mimic the transition state (TS) of the enzymatic process with peptidomimetics that cannot undergo hydrolysis.4 Among the TS isosteres relevant to aspartic proteases statines, hydroxyethylenes and hydroxyethylamines (Figure 1) have found broad use. These isosteres are functionalized on both sides with groups that occupy the space of the amino acids that flank the scissile peptide bond in the natural substrate. The number and nature of the groups required for the recognition of the inhibitor by the enzyme significantly impacts the drug-like properties of these compounds. In general, even though the scissile amide has been replaced, these inhibitors often have © 2017 American Chemical Society

highly peptide-like character and high molecular weight. This often results in poor physicochemical and ADME properties. In addition, BACE-1 inhibition offers an additional challenge to drug discovery because, unlike most enzymes of this class, it is located in the brain. Incorporation of TS isosteres into a peptide sequence could give rise to increased flexibility due to the higher number of rotatable bonds. Higher conformational freedom would be expected to result in an entropic penalty upon binding. For example, NMR studies of the solution structure of the HIV protease inhibitor acetyl-pepstatin highlighted the conformational mobility in the statine region.5 In contrast to the HIV inhibitors containing the statine TS isostere, the NMR analysis of two hydroxyethylene dipeptide βsecretase inhibitors revealed an extended conformation in solution that is similar to the conformation bound to BACE-1 in the crystal structure.6 Thus, different TS isosteres appear to provide differing levels of conformational control. The variation in flexibility observed for different dipeptide isosteres suggests that a good understanding of the conformational preferences in solution is essential to determine which Received: September 4, 2017 Published: October 31, 2017 9807

DOI: 10.1021/acs.jmedchem.7b01304 J. Med. Chem. 2017, 60, 9807−9820

Journal of Medicinal Chemistry

Article

Figure 1. Most common TS mimetics. PSA and rotatable bond counts determined for Phe-Ala dipeptide and its TS isostere equivalents.

Scheme 1a

a

Reagents and conditions: (a) (S)-2-butylamine, Pd(OAc)2, BINAP, Cs2CO3, toluene, 100°C, 73%; (b) MeSNa, Pd(OAc)2, BINAP, Cs2CO3, toluene, 90 °C, 66%; (c) m-CPBA, DCM, rt, 74%; (d) LiOH, THF/H2O, rt (quant); (e) N-Boc pyrrole, TMP, n-BuLi, 34%; (f) H2, Pt-C, 67%; (g) H2, Pd(OH)2; (h) 2 or 3-(dipropylcarbamoyl)benzoic acid, EDCI, HOBT, Et3N, DMAP; (i) HCl, three steps, 44% for 5a, 34% for 5b and 40% for 6.

Scheme 2a

Reagents and conditions: (a) BOMCl, DIPEA, DCM, reflux, 90%; (b) LiBH4, Et2O, 85%; (c) SO3-pyridine, TEA, DMSO, 10 °C; (d) 1.5 equiv 18, 0.1 equiv 1M TBAF, THF, RT, 14 h, two steps, 49% for 9, 68% for 15; (e) NiCl2, NaBH4, MeOH; (f) acetic anhydride, TEA, THF, two steps, 83% for n = 0, 54% for n = 1; (g) CSA (cat.), 6 equiv 2-methoxypropene, 1:1 DCM/acetone, 81% for n = 0, 59% for n = 1 (h) H2, Pd-C, MeOH, 98% for 10, 87% for 16; (i) Cy-CH2-Br, NaH, DMF, 53% for n = 0, 21% for n = 1; (j) 4M HCl/dioxane, 100% for 11, 76% for 17.

a

BACE-1 inhibitors was pioneered by Merck8 and Elan.9,10 Over the years, many inhibitors with this core have been reported,11,12 including cyclized central cores like pyrrolidines,13,14 piperidines,13 piperazines,15 and piperazinones.16 To our knowledge, while the three-dimensional structures of several HEA inhibitors in the binding site of BACE-1 have

cores would benefit from cyclization stratregies to increase ridigification. The hydroxyethylamine (HEA) core has provided successful marketed drugs for the inhibition of HIV-1 protease. In general, these compounds show limited CNS penetration.7 The use of the hydroxyethyl amine core in the design of less peptide-like 9808



Article

Scheme 3a

Reagents and conditions: (a) 18, TBAF (5%), THF, 0 °C, 11:2:1 dr, 42% yield on desired isomer; (b) NiCl2, NaBH4, MeOH, rt; (c) K2CO3, BnBr, CH3CN, reflux, 12 h; (d) NaH, BnBr, DMF, 83%, three steps; (e) HCl (g), TMBE, rt; (f) Br-CH2CO2Et, TEA, DMF; (g) CSA, toluene, reflux; (h) Boc2O, TEA, CH2Cl2, 58% four steps; (i) DIBALH, toluene, −78°C, 65:35 dr; (j) NaH, cyclohexylmethyl trifluoromethanesulfonate, THF, rt 55% yield two steps; (k) H2, Pd(OH)2, EtOAc; (l) Ac2O, TEA, CH2Cl2; (m) TFA, 78% yield three steps.

a

of the nitro group, followed by acylation, oxazolidine formation, and debenzylation, afforded intermediate 10 in 66% yield from the Henry product 9. Alkylation of the free alcohol with cyclohexylmethyl bromide, deprotection, and hydrochloride salt formation provided the desired compound 11 in 53% yield. The same sequence was used for the synthesis of the sixmembered cycle 17 starting from tert-butyl (2R)-2(hydroxymethyl)piperidine-1-carboxylate 12.23 In this case, the Henry reaction gave a mixture of three isomers in a 12:1.5:1 ratio from which the desired isomer was separated in 68% yield by silica gel chromatography. The relative configuration of the chiral centers was determined by X-ray crystallography of intermediate 16 (see Supporting Information). We also used the Felkin-controlled Henry reaction to install the chiral centers on the morpholine core (Scheme 3). Thus, the reaction of 1,3-difluoro-5-(2-nitroethyl)benzene 18 with Garner’s aldehyde 1924 provided three nitroaldol diastereomers in a 11:2:1 ratio in favor of the desired diastereomer. Recrystallization provided 20 as a single diastereomer in 42% yield. The remaining mixture of isomers could be enriched in the desired isomer under the same TBAF/THF conditions. Two cycles of epimerization/crystallization provided enantiomerically pure compound 20 in 74% isolated yield. Notably, it was observed that the combination of long reaction times and excess of TBAF instead of a catalytic amount in the Henry reaction produced loss of enantiomeric purity without impacting the diastereomeric ratio. This loss was shown to be due to racemization of the Garner’s aldehyde in the presence of excess of TBAF by a series of control experiments. Nitro reduction of 20 followed by benzylation of the free amine and alcohol in a two-step process provided intermediate 21 in a 83% overall yield. Deprotection of the oxazolidine, alkylation with ethyl bromoacetate, cyclization under acid catalysis, and reprotection of the amine afforded enantiomerically pure morpholinone 22 in 58% yield. Reduction of lactone 22 with DIBALH provided a 68:32 ratio of lactols. Alkylation of the crude mixture with cyclohexylmethyl triflate provided morpholine 23 as a single diastereoisomer. Removal of the benzyl groups under standard conditions followed by acetylation, Boc deprotection, and hydrochloride salt formation afforded the final compound 24 in 78% yield.

been reported, the conformational preferences in solution of this isostere for a comparison with the bound conformation have not been investigated. We present a conformational analysis of the HEA core in solution using NMR spectroscopy and molecular modeling. This study informed our optimization efforts of this core for the inhibition of BACE-1. In this approach, we took advantage of conformational preferences to generate HEA BACE-1 inhibitors whose conformational minima in solution replicated their bound conformation. While in recent years research on BACE-1 inhibition has moved away from peptidomimetics,17 this class is broadly used in medicinal chemistry to target enzymes and receptors.18 The learning gained from these studies could be applied to the optimization of the potency and physicochemical properties of inhibitors of other targets, in particular, other aspartic proteases.

■

RESULTS AND DISCUSSION Chemistry. Compounds 5 and 6 were synthesized as depicted in Scheme 1. Palladium-catalyzed coupling of the dichloropyridine 1 with (S)-2-butylamine, followed by displacement of the remaining chloride with sodium methanethiolate, oxidation, and ester hydrolysis, provided free acid 2. Reaction of the lithium anion of N-Boc pyrrole with aldehyde 319 provided the corresponding alcohol as a single diastereomer.20 Hydrogenation of the pyrrole ring provided a 1:1 mixture of the two diastereomers at the new chiral center, 4a and 4b, which were separated by chromatography. Each isomer was subjected to the following steps separately. Hydrogenation of the dibenzyl amine, followed by coupling with the acid 2 and deprotection, provided compounds 5a and 5b, respectively. Similarly, 4b was coupled with 3-(dipropylcarbamoyl)benzoic acid to provide 6. For the synthesis of other cyclized HEAs, we relied upon the Henry reaction.21 The synthesis of compound 11 started from the known pyrrolidinyl ester 7 (Scheme 2).22 Reduction followed by oxidation provided the aldehyde which was immediately reacted with 1.5 equiv of commercially available 1,3-difluoro-5-(2-nitroethyl)benzene 18 at 0 °C in the presence of a catalytic amount of TBAF to produce a mixture of diastereomers in a 7:1:1 ratio. The major diastereomer 9 was separated in 49% yield by silica gel chromatography. Reduction 9809



Article

Figure 2. Numbering used for the HEAs conformational analysis.

Scheme 4a

a

Reagents and conditions: (a) HCl (g), TMBE, rt; (b) ethyl (2R)-2-(trifluoromethylsulfonyloxy)propanoate, Et3N, CH2Cl2, rt, 76% two steps; (c) DIBALH, toluene, −78°C, 65:35 dr; (d) MsOH, R-OH; (e) Boc2O, DIPEA, CH2Cl2, 75°C, 65% three steps; (f) H2, Pd(OH)2, EtOAc; (g) Ac2O, TEA, CH2Cl2; (h) TFA followed by HCl in Et2O, 64% yield for 27, 75% yield for 29.

BACE-1 and at physiological pH,9 NMR experiments were performed with the corresponding hydrochloride salts. We observed a large coupling constant (8.8 Hz) between N2H and H3 in compound 30, indicating that the amide proton is anti to H3, which is the same relationship between amide and Hα protons in extended beta strand conformations of peptides.30 This extended beta strand is the universal binding conformation for protein substrates to proteases. H4 was found to have a small coupling constant (2.3 Hz) with H5 and two intermediate coupling constants (both 7.5 Hz) with H3 and the other methylene proton H5′. This pattern suggests conformational averaging around the C3−C4 and C4−C5 bonds in contrast to the reduced flexibility of the hydroxyethylene core.5 If the three staggered conformers around the C4−C5 bond are considered (Figure 3), a conformational equilibrium involving conformers A and C, which are stabilized relative to conformer B due to the gauche orientation of the OH and NH2+ groups,31 accounts for the experimental proton−proton coupling constants with H5 and

For the introduction of a substituent at C7 (see Figure 2 for numbering) of the morpholine ring, intermediate 21 was deprotected and then alkylated with ethyl (2R)-2(trifluoromethylsulfonyloxy)propanoate. Cyclization of the ester intermediate under acid catalysis provided lactone 25 in 76% yield. Reduction afforded the corresponding lactols as a 65:35 mixture of epimers. Glycosidation under basic conditions, following the same synthetic route as that used for 24, produced epimerization at C7 of the morpholine. Therefore, we explored an alternative glycosidation under acidic conditions using MsOH and the corresponding alcohol. By employing either cyclohexylmethyl alcohol or t-BuCH2OH as the alcohol, a 3:1 mixture of the two epimers at the anomeric center was obtained, from which the desired isomer, 26 or 28, respectively, was isolated in around 60% yield. No epimerization of C7 was observed with this approach. The minor isomer could be epimerized to a 3:1 mixture in favor of the desired isomer by resubmitting it to the same reaction conditions (MsOH, alcohol). Debenzylation, acetylation, and Boc deprotection provided compounds 27 and 29 in 64% and 75% yield (Scheme 4), respectively. The absolute configuration of compounds 5b, 11, 24, and 29 was determined by X-ray cocrystallization with BACE-1 (Figure 5), indirectly revealing the configuration of the other compounds described above. Conformational Analysis. Conformational preferences were calculated by molecular mechanics and determined experimentally by NMR spectroscopy.25−27 Information about conformational preference can be obtained from vicinal proton−proton coupling constants (3JHH), which are related to dihedral angles via the Karplus−Altona equation.28 Intermediate values between 5 and 8 Hz usually reflect conformational averaging around a single bond, while more extreme values are indicative of a predominant conformation in solution. Compound 3029 was chosen to evaluate the conformational preferences of the acyclic HEA class (Figure 2). Given that the amine of the HEA core is protonated both when bound to

Figure 3. Newman projections for the staggered rotamers around C4− C5 (a) and C3−C4 (b) bonds in 30. 9810



Article

Figure 4) are stabilized by the OH/NH2+ gauche effect. As H4 is anti to H3 and gauche to H5 in conformer AE, but anti to H5 and gauche to H3 in conformer BF, an approximately equimolecular mixture of both conformers can explain the intermediate H3−H4 and H4−H5 coupling constants observed for this diasteromer. In contrast, only conformer AE is stabilized by the gauche effect in epimer 5b because the OH and the NH2+ groups are in an anti disposition in conformer BF. According to the Karplus equation, the calculated H3−H4 and H4−H5 coupling constants for conformer AE of 5b are 9.2 and 1.4 Hz, respectively, in excellent agreement with the experimental values, indicating that this is the only populated conformer in solution. Furthermore, extreme coupling constants between H3 and diastereotopic H3′a and H3′b resonances were measured (11.1 and 2.0 Hz), highlighting a conformational preference around the Cα−Cβ bond for the phenylalanine side chain as observed for the backbone torsions. Similar coupling constants were obtained for 11 with the same configuration as 5b. In short, cyclization of C5 and C7 in the R configuration at C5 (i.e., 5b or 11) causes a significant decrease of conformational mobility across the entire core. This results in a HEA derivative with a predominant conformation in solution (conformer AE in Figure 4) and reduced flexibility compared with 30. This is in contrast to the cyclic derivative with S configuration at C5 (i.e., 5a) in which an equilibrium between conformers is observed by NMR. Six-membered ring compounds 17 and 24, which have the same absolute configuration at C3, C4, and C5, showed a similar pattern of coupling constants to those of compounds 5b and 11. Proton−proton coupling constants and NOE data for the piperidine and morpholine protons revealed that both sixmembered rings adopt a chair conformation in which the bulkiest substituent, the HEA backbone, is equatorial and the O-alkyl substituent is axial. Furthermore, the exo-anomeric effect should restrict the conformational mobility for the Oalkyl substituent in 24 so that one of the three staggered rotamers around the exocyclic C−O bond is predominant as a result of the combination of steric and stereoelectronic factors.27 Computational docking with BACE-1 suggested that the introduction of a methyl substituent at C7 oriented toward the S1′ domain via an axial disposition could increase affinity if the C5 and C8 substituents could be maintained in their equatorial and axial orientations, respectively. However, the inverse chair with the HEA core axial cannot be discounted. Molecular mechanics calculations showed a higher preference for the desired chair conformation in the morpholine than in the piperidine ring, which can be attributed to the additional stabilization provided by the anomeric effect. On the basis of these analyses, we prepared compounds 27 and 29 bearing a methyl group at C7 position of the morpholine ring. Measurement of the coupling constants confirmed the equatorial disposition for the HEA core and the axial positions for the O-alkyl and methyl substituents. Our NMR analysis reveals that while acyclic HEAs have little conformational preference, cyclization of C5 and C7 brought significant conformational constraint to C3−C5 as well. Interestingly, this conformational behavior was not anticipated by molecular mechanic studies using standard methods available under Maestro v. 9.1−9.5, (Schrodinger Inc., Portland, OR) using the OPSL2005, OPSL2.x, or OPSL3 force fields using unconstrained stochastic searches (Monte Carlo/Low

H5′ corresponding to the proS and proR protons, respectively. H4 is gauche to H5pS in both A and C conformers, whereas H4 is gauche to H5pR in conformer C and anti in conformer A, which is consistent with a small H4−H5pS and an intermediate H4−H5pR coupling constant. The analysis around C3−C4 is more complex because only one proton−proton coupling constant can be measured across this bond. The intermediate value for the H3−H4 coupling constant (7.5 Hz) indicates that a significant population of conformer E, with both protons in an anti orientation, should exist in solution, although at least a second rotamer (D or F or both) should be also populated. Unlike hydroxyethylenes, HEA isosteres exhibit flexible cores that populate more than one conformation in solution. This suggested to us that the affinity of compounds containing this TS isostere for an aspartic protease could be increased by making more rigid derivatives. We hypothesized that introduction of a substituent at C5 would restrict the conformational space available because staggered conformers with destabilizing 1,3-syn-pentane interactions will not be allowed.32 To further reduce the conformational flexibility of the cyclopropyl region of 30, the substituent at C5 was linked to C7 through a five-membered ring, leading to compounds 5a and 5b (Figure 2). The two epimers at C5 were subjected to NMR analysis, and the coupling constants between the protons of the ethanolamine core were measured. A different coupling constant pattern was obtained for each diastereomer. While 5a exhibited averaged coupling constants (3JH3−H4 = 6.9 Hz and 3JH4−H5 = 6.3 Hz), indicating the presence of more than one conformer in solution, extreme values were observed for 5b (3JH3−H4 = 8.9 Hz and 3JH4−H5 = 1.8 Hz), revealing a conformational preference. The conformational differences between the two diastereomers can be explained by examining the three staggered conformations around C3−C4 and C4−C5 bonds (Figure 4).

Figure 4. Staggered rotamers around C3−C4 and C4−C5 bonds for 5a (X = NH2+ and Y = CH2) and 5b (X = CH2 and Y = NH2+). The destabilizing 1,3-syn-pentane contacts are shown in bold.

Conformers with the OH and NH+ groups in a gauche orientation are stabilized by the gauche effect,28 whereas conformers with 1,3-syn-pentane interactions are destabilized.32 Seven of the nine staggered conformers around C3−C4 and C4−C5 bonds display 1,3-syn-pentane contacts between the amide or the benzyl substituent at C3 and the pyrrolidine ring and are therefore disfavored. In epimer 5a, the only two conformers free of this interaction (conformers AE and BF in 9811



Article

Mode under default conditions for water solution or with a distance-dependent dielectric solvation model using various values of the dielectric constant) or molecular dynamics (100 ns Desmond simulations at 300 K or at 350 K under otherwise default conditions in a standard SPC water or DMSO box also created using default conditions with the ligand prepared using standard ligprep conditions). These methods suggested the existence of several conformations of similar energy. Ab initio density functional calculations were performed to ascertain whether the conformational preference of the HEA core could be predicted using quantum mechanics to reconcile theory with NMR data. To set up these calculations, conformations of 5a and 5b were generated by constraining H2−H3 to the anti conformation (see above) and then conducting a systematic search (120° increments starting at 60° also under Maestro v. 9.1−9.5, Schrodinger Inc., Portland, OR) around the C3−C4 and C4−C5 torsions to generate the nine conformers in Figure 4 (using the OPLS2005 force field and a fixed dielectric of 46 as a model for DMSO). These torsions were then all constrained to the values obtained in these systematic search steps, and the remaining torsions were subjected to stochastic search using the Monte Carlo/low mode algorithm in Macromodel. The lowestenergy conformers found in this step were then optimized without constraints to the nearest local energy minimum, which was then checked to ensure that the structure had not migrated significantly from the starting point. The SCF energies and NMR parameters of the resulting conformers were then evaluated using Gaussian0933 (singlepoint B3LYP/6-311+G**, PCM DMSO solvent model, NMR = mixed keyword).34 This analysis revealed that 5b was expected to adopt conformation AE > 99% of the time, which is the most stable conformer according to the previous analysis based on 1,3-syn-pentane interactions and the OH/NH2+ gauche effect. The ab initio calculated coupling constants for H3−H4 and H4−H5 of 9.7 and 2.6 Hz, respectively, are in excellent agreement with both the experimental values and the predictions based on the Karplus−Altona relationship. In contrast, 5a exhibited more conformational flexibility and conformers BF, BE, and AE were expected to be occupied 48%, 29%, and 14% of the time, in reasonable agreement with the previous analysis that showed that BF and AE are free of 1,3syn-pentane contacts. The ab initio calculations predicted H3− H4 coupling constants of 2.0, 9.1, and 10.2 Hz and H4−H5 values of 8.8, 8.9, and 1.6 Hz, for conformers BF, BE, and AE, respectively, which is consistent with the averaged experimental values observed for this isomer. Enzyme−Inhibitor Interaction (X-ray). Figure 5 shows the overlay of compounds 5b, 11, 24, and 29 with a known HEA, 31.8 The backbone conformation of the acyclic and cyclic HEAs is the same, regardless of the ring size or the number of substituents on the ring. Compounds 5b and 31 occupy the subsites S2 and S3, while compounds 11, 24, and 29, which lack these interactions, are extended to occupy the S2′ pocket (a pocket not occupied by either 5b or 31) with the O-alkyl side chain. For morpholines 24 and 29, the synclinal relationship between the endocyclic oxygen and the first exocyclic carbon observed in the bound state is also the preferred conformation in solution due to the exoanomeric effect. The methyl group of compound 29 is oriented toward S1′, occupying the same space as the cyclopropane in the flexible compound 31 (Figure 5B). No additional interactions between the cycles and the enzyme are observed.

Figure 5. (A) X-ray crystal structures of BACE-1 with compounds 5b (in blue) and 11 (in orange) overlaid with compound 31 (in green). (B) X-ray crystal structures of BACE-1 with compounds 24 (pink) and 29 (purple) overlaid with compound 31 (in green).

The X-ray structures of compounds 5b, 11, 24, and 29 in their complexes with BACE-1 revealed the bioactive conformation of the ligands. The conformation of the HEA backbone in the crystal resembles the conformation in solution derived from NMR data for all these compounds. The similarity between the free and bound conformations is illustrated in Figure 6, which compares the coupling constants calculated for

Figure 6. Comparison of the calculated vicinal proton−proton coupling constants for the bound conformations of 5b, 11, 24, and 29 in complex with BACE-1 and the experimental values measured in solution for the free compounds.

the crystal structures using the Karplus−Altona relationship with the experimental values in solution. Further, the sixmembered ring of 24 and 29 adopts a chair conformation with the HEA core occupying an equatorial position and the O-alkyl substituents occupying an axial position as observed for the free compound. The O-alkyl substituent adopts a conformation around the C−O bond in accordance to the exo-anomeric effect. Thus, the comparison between X-ray and NMR results shows that these four compounds do not undergo a significant conformational change when bound to BACE-1 and are preorganized in solution. Structure−Activity Relationship. The new compounds were evaluated in an enzymatic assay (BACE-1 mcaFRET assay)35 and a cellular assay (Aβ lowering assay in HEK293/ 9812



Article

Table 1. Characterization of HEA BACE-1 Inhibitors compd

IC50 BACE-1 (μM)a

cell IC50 (μM)

30 5a 5b 6 11 17 24 27 29

6.3 ± 1.32 >200 0.005 ± 0.0003 1.67 ± 0.170 1.40 ± 0.144 1.32 ± 0.039 0.22 ± 0.011 0.099 ± 0.004 0.079 ± 0.004

4.9 ± 0.61 >10000 0.101 ± 0.012 5.7 ± 2.27 0.8 ± 0.030 0.53 ± 0.043 0.48 ± 0.072 0.205 ± 0.015 0.090 ± 0.011

pKa 8.4 ND 9.4 9.6 8.2 8.6 6.7 6.7 6.9

MW

Nvs Psa

452 475 475 452 411 425 427 441 415

82 129 129 82 71 71 80 80 80

rot bond 14 10 10 12 8 8 8 8 7

brain exposure (mg/g)b

B/P ratiob

ND ND 106 ND 35 87 427 262 739

ND ND 0.02 ND 0.04 0.07 0.42 0.18 0.38

a

Geometric mean of runs in triplicate; standard error for the geometric mean. bBrain to plasma concentration of parent compound 1h after 30 mg/ kg SC administration in FBV mice.

APP751swe cells).36 The inhibitory potency of inhibitors in enzyme and cellular assays is summarized in Table 1. Brain/ plasma ratio was also determined for key compounds. We designed compounds 5 and 6 to assess the impact of cyclization on the affinity of the HEA for BACE-1. Compounds 5a and 5b contained one of the most potent P2−P3 substituents that we had identified for the HEAs, while compound 6 was made for comparison with the model compound 30. They were designed to allow the assessment of the effect of the configuration of the new chiral center on the affinity for the enzyme, with the expectation that the loss of the P1′ substitution present in compound 30 would reduce the affinity of the cycle for the receptor. The slightly improved enzymatic activity observed for 6 compared with 30 suggests gain in the entropic component of the binding energy due to preorganization, as 6 has higher affinity for the enzyme even though it lacks the S1′ substitution. The >1000-fold difference in activity of the two epimers 5a and 5b is remarkable and suggests that 5a, being more flexible than 5b, lacks the favorable preorganzation and might be less readily accommodated in the BACE-1 pocket, even with an optimized P2−P3 side chain. Compound 5b showed very low brain penetration, which was not unexpected given the physicochemical properties of the compound (Table 1) and the precedents in the area.11,37 Correlations of brain penetration with the physical properties of molecules (molecular weight, log P, polar surface area, pKa, rotatable bonds, the number of hydrogen bond donors/ acceptors) are all important factors worth considering in developing any CNS agent.38 To improve the brain penetration of our HEA cyclic scaffold, we considered that the P2−P3 substitution had to be removed to reduce MW and PSA. To mitigate the loss of affinity for the enzyme that we expected to occur with such a modification, we explored the introduction of P2′ substituents in our cyclic HEAs. We targeted pyrrolidines, piperidines and, subsequently, morpholines, all substituted with O-alkyl groups as P2′ substituents. We hypothesized that use of electron withdrawing O atom linkages would help modulate the pKa of the basic nitrogen. While 2-alkoxymorpholines are unusual cores in drug discovery, they are stable compounds that have produced marketed drugs.39 An overlay of the X-ray structure of 5b with the best docking poses found by GOLD (conditions were defaults generating 10 poses per ligand, GOLD version 2.1−2.2 (Cambridge Crystallographic Data Centre, Lensfield Road, Cambridge, UK); standard protein preparation prior to docking was carried out in Sybyl v.7.0−7.1 (Tripos, Inc., St. Louis, MO) with side chain protonation states and conformations being checked and

corrected manually) on substituted pyrrolidines and piperidines, suggested that the best position for the P2′substituent was the 4-position for pyrrolidines, as has been already shown by others13,40,41 and the 4- or the 5-position for the piperidines. While 4-substituted piperidines had been prepared as BACE-1 inhibitors,13 the 5-substitution had not been explored. We envisioned that six-membered cycles, being less flexible than five-membered, could offer an additional gain in potency if the chair conformation allowed the P2′ substituent to be accommodated in the S2′ pocket. We were pleased to observe that the three ring systems, represented by compounds 11, 17, and 24, retain affinity for the enzyme even without P2−P3 elements. The preferred conformation in solution of the central core deduced from NMR data is similar to the conformation of the inhibitors bound to BACE-1. It is noteworthy that all three compounds are preorganized in solution, favoring the binding process. The P2′ substituent is more flexible on the pyrrolidine42 than in the other two cycles, which could explain why 11 is slightly less potent than the two six-membered cycles, where the major conformer of the chair in solution adopts the axial conformation for the alkoxy group that is required to fill the S2′ pocket. Our consideration of the morpholine core was initially driven partly by the desire to guarantee this axial disposition of the alkoxy substituent, but in fact comparative analysis of the coupling constants of morpholine 24 and piperidine 17 showed that both adopt the same preferred conformation in solution and little additional entropic gain could be achieved with electronic factors (e.g., the anomeric effect)43 on the morpholine because the active conformation was already highly populated on the piperidine ring. Comparing physicochemical properties of the cycles 11 and 17 with 5b, a considerable reduction in MW, PSA, and pKa had been achieved with the functionalized cycles. To our surprise, these changes had little impact on the brain penetration of the inhibitors. Nonetheless, we were pleased to see that the morpholine analogue 24, unlike the other two cycles, showed a great improvement in brain penetration, likely due to its reduced pKa. This cycle seemed to offer the best combination of properties of all the cycles we investigated. We used morpholines for further exploration of the affinity for BACE-1 by introduction of C7 substitution. It was precedented that a small alkyl group like an N-ethyl or N-cyclopropyl could occupy the S1′ pocket.8 Compounds 27 and 29 showed a gain in activity over analogue 24, probably due to the additional interactions in S1′. The preferred conformation of these compounds, determined in solution for 29, was one that 9813



Article

Figure 7. Comparison of HEA-containing BACE-1 inhibitors.

Finally, we have developed stereoselective routes for the synthesis of the cyclic HEAs with the installation of up to five chiral centers in the molecule.

positioned both P1′ and P2′ substituents in the axial disposition and the bulky HEA in the equatorial position. Thus, at least in the morpholine case, the stereoelectronic factors governing ring conformation continued to favor the C4-equatorial conformer even when two smaller substituents were oriented axially. Comparison of compound 29 with some representative HEA BACE-1 inhibitors from the literature (32,14 which exemplifies a constrained core, and 33,44 which exemplifies a HEA without P2−P3 substitution), ilustrates the atom efficiency of the preorganized morpholine core as BACE-1 inhibitor as well as the improvement in brain penetration (Figure 7).

■

EXPERIMENTAL SECTION

Chemistry. General. All solvents and reagents were purchased from commercial sources and used as received unless otherwise indicated. All solvents were ACS grade. Reactions were performed under air atmosphere unless otherwise indicated. 1H NMR data for characterization of compounds were recorded on a Bruker AM-300 (300 MHz). 1H NMR of intermediates 9, 15, 16, and 23 is not provided due to the complexity derived from the presence of rotamers. NMR spectra for conformational analysis of compounds 5a, 5b, 6, 11, 17, 24, 27, 29, and 30 were acquired on a Bruker DRX 500 Avance spectrometer equipped with a 5 mm CPTCI 1H-13C/15N Z-Grad inverse probe. NMR experiments were acquired at 25 °C in DMSO-d6 by referencing all the NMR spectra to the residual solvent signals at 2.50 and 39.5 ppm for proton and carbon, respectively. Onedimensional spectra were acquired using 16K data points and zero filled to 32 K. Complex multiplets were simplified by adding D2O to remove NH and OH couplings or by using homonuclear decoupling techniques or 1D-TOCSY sequence using 40 μs as TOCSY mixing time. Absolute value COSY and phase sensitive HSQC were acquired using gradient selection techniques. Phase-sensitive ROESY experiments were recorded using 500 ms as mixing times. Acquisition data matrices were defined by 1K × 256 points in F2 and F1, respectively. The 2D data matrices were multiplied by the appropriate window functions and zero-filled to 2K × 1K matrices. Linear prediction was applied prior to Fourier transformation, and polynomial baseline correction was used in both dimensions of the 2D spectra. Analytical HPLC was carried out on an Agilent HP1100 liquid chromatography system equipped with a solvent degasser, quaternary pump, auto sampler, column compartment, and a diode array detector (Agilent Technologies, Waldbronn, Germany). The UV wavelength was set at 214 nm. Electrospray mass spectrometry measurements were performed on a MSD quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) interface to the HP1100 HPLC system. MS measurements were acquired simultaneously in both positive and negative ionization modes. Data acquisition and integration for LC-UV and MS detection were collected using a HP Chemstation software (Agilent Technologies). Analytical conditions: method A, Phenomenex Luna Phenyl Hexyl column (4.6 mm × 100 mm, 5 μm). Solvent A: H2O 0.05%TFA, pH 2.5; solvent B, ACN 0.05%TFA. Gradient from 5 to 99% A in 10 min, stay at 99%B for 2 min, and then 2 min to initial conditions. Flow rate 1.0 mL/min. Method B: Phenomenex Gemini C18 column (4.6 mm × 100 mm, 5 μm). Solvent A, 10 mM NH4HCO3, pH 9; solvent B, CAN. Gradient from 20 to 99% A in 10 min, stay at 99% B for 2 min, and then 2 min to initial conditions. Flow rate 1.0 mL/min. All the final compounds had ≥95% purity except compound 5a, with a 93% purity by both methods. Optical rotations were measured with a PerkinElmer 241 polarimeter and with a Bellingham Stanley ADP-220. Analytical TLC was performed on Merck TLC glass plates precoated with F254 silica gel 60

■

CONCLUSION While the design of new ligands in drug discovery usually relies on enthalpy gain by improving the interactions with the target, it is essential to also understand the entropic penalty of the binding process for affinity optimization. We have determined that HEAs are flexible cores by a NMR analysis of compound 30 in solution. These data suggested that the HEA core could benefit from rigidification to reduce the entropy penalty upon binding. Cyclization of C5−C7 into a pyrrolidine, piperidine, or morpholine ring with the R configuration reduces the conformational mobility of the C3−C4 moiety which adopts a predominant conformation in solution that is similar to that found in the crystal structure of the complex with BACE-1. This preorganization of the ligands minimizes the entropic cost associated with the binding event which results in an increase in affinity relative to acyclic compounds. We also exploited the conformational preferences of sixmembered cycles to efficiently install a P2′ substituent with a preference for the axial conformation in solution, a disposition that resembles the conformation of the inhibitors in the enzyme. Alkoxy morpholines offer the possibility of utilizing both electronic and steric factors to drive the disposition of the ring substituents. Compounds 27 and 29 were designed to introduce a P1′ substituent in an axial orientation without affecting the strong preference of this core for positioning the O-alkyl group in the axial disposition and the acyclic moiety of the HEA in an equatorial direction. The hypothesis was proven for 29 by NMR and X-ray cocrystallization with BACE-1 and provided the two most potent compounds of the series. While more potent BACE-1 inhibitors have been described, our studies show that optimization of activity can rely on entropic factors by directed reduction of the conformational flexibility of compounds. One additional finding of interest for CNS drugs is that these alkoxymorpholines, unlike pyrrolidine and piperidine analogues, were brain penetrant. 9814



Article

concentrated to provide 970 mg of 2 (100%); mp 165 °C; [α]D25 = +16 (c 1, MeOH). MS (ES): m/z 273 [M + H]. 1H NMR (MeOH-d4) δ: 7.56 (d, J = 1.0 Hz, 1H), 7.17 (d, J = 1.0 Hz, 1H), 3.96 (m, 1H), 3.18 (s, 3H), 1.59 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H), 0.96 (t, J = 7.5 Hz, 3H). 13C NMR (DMSO-d6) δ: 167.1, 159.1, 155.6, 148.1, 113.3, 107.5, 47.7, 39.5, 38.9, 28.9, 19.9, 10.7. (R) and (S)-2-((1S,2S)-2-Dibenzylamino-1-hydroxy-3-phenylpropyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (4a, 4b). nBuLi (1.6 M in hexane) (1.06 mL, 1.7 mmol) was added to a solution of TMP (0.29 mL, 1.7 mmol) in 12 mL of dry THF under N2 at −78 °C. The reaction was stirred at −78 °C for 5 min, at −10 °C for 5 min, and at −78 °C for 5 min. N-Boc-pyrrole was added dropwise, and the solution was stirred at −78 °C for 20 min. Freshly prepared aldehyde 3 was added dissolved in 4 mL of dry THF, and the reaction was stirred at −78 °C for 45 min. Saturated NH4Cl was added, the mixture was allowed to reach room temperature, and the layers were separated. The aqueous layer was extracted with EtOAc (2×), and the combined organic layers were washed with 1 N HCl, 5% NaHCO3 and brine, dried over MgSO4, filtered, and concentrated. Purification by flash chromatography eluting with hexane/Et2O 4:1 provided 240 mg of the hydroxypyrrole (34%). MS (ESI) m/z 497 [M + H]. The compound (160 mg, 0.32 mmol) was dissolved in 2 mL of MeOH, Pt on C (10 wt %, 63 mg, 0.032 mmol) was added, H2 was bubbled through the mixture, and it was stirred under 1 atm of H2 overnight. N2 was bubbled through the mixture for 2 min, and the reaction was filtered through Celite eluting with MeOH and concentrated. Isomers were separated by flash chromatography eluting with hexane/EtOAc 3:1 to obtain 4a (84 mg, 52%) and 4b (74 mg, 46%). 4a: MS (ESI) m/z 501 [M + H]. 1H NMR (CDCl3) δ: 7.30−7.09 (m, 15H), 4.00−2.83 (m, 10H), 1.90−1.18 (m, 4H), 1.48 (s, 9H). 4b: MS (ESI) m/z 501 [M + H]. 1H NMR (CDCl3) δ: 7.38−7.10 (m, 15H), 4.20 (bs, 2H), 3.90− 2.72 (m, 8H), 1.89−1.40 (m, 4H), 1.40 (s, 9H). N-((1S,2R)-1-Benzyl-2-hydroxy-2-(S)-pyrrolidin-2-yl-ethyl)-2-((S)sec-butylamino)-6-methanesulfonyl-isonicotinamide Hydrochloride (5a). Compound 4a was debenzylated according to the general method A to provide the primary amine as a colorless oil. It was coupled with acid 2 according to general method B and purified by flash chromatography (hexane/EtOAc 2:1) to provide 5a as a white solid (44%, three steps); mp 180 °C (change color at 160 °C); [α]D25 = −26 (c 0.5, MeOH). MS (ESI) m/z 475 [M + H]. 1H NMR (MeOH-d4) δ: 8.63 (d, J = 8.7 Hz, 1H), 7.31−7.16 (m, 6H), 6.91 (s, 1H), 4.28 (m, 1H), 3.95 (c, J = 6.3 Hz, 1H), 3.80 (dd, J = 8.3, 3.4 Hz, 1H), 3.70 (m, 1H), 3.41−3.29 (m, 3H), 3.16 (s, 3H), 2.85 (dd, J = 13.9, 11.1 Hz, 1H), 2.24−1.90 (m, 4H), 1.59 (m, 2H), 1.20 (d, J = 6.5 Hz, 3H), 0.96 (t, J = 7.3 Hz, 3H). 13C NMR (MeOH-d4) δ: 167.8, 158.0, 155.4, 142.5, 137.2, 127.9, 127.1, 125.1, 110.2, 103.7, 70.5, 60.3, 53.7, 47.0, 44.9, 37.4, 34.7, 27.8, 26.2, 22.8, 17.8, 8.5. N-((1S,2R)-1-Benzyl-2-hydroxy-2-(R)-pyrrolidin-2-yl-ethyl)-2-((S)sec-butylamino)-6-methanesulfonyl-isonicotinamide Hydrochloride (5b). Compound 4b was subjected to the same reaction conditions used for 4a, providing 5b as a foamy solid (44%); mp 228 °C; [α]D25 = −34 (c 1, MeOH). MS (ESI) m/z 475 [M + H]. 1H NMR (DMSOd6) δ: 7.32−7.13 (m, 6H), 6.99 (s, 1H), 4.06−3.97 (m, 2H), 3.85 (bs, 1H), 3.55 (bs, 1H), 3.25 (d, J = 11.9 Hz, 1H), 3.18 (s, 3H), 3.11 (bs, 2H), 2.78 (t, J = 11.0 Hz, 1H), 2.0−1.75 (m, 4H), 1.50 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (MeOH-d4) δ: 167.4, 160.3, 157.7, 145.1, 139.3, 130.4, 129.4, 127.5, 112.5, 105.9, 71.2, 68.1, 63.2, 55.8, 47.1, 40.0, 38.1, 30.2, 24.9, 24.1, 20.2, 10.9. N-[(1S,2R)-I-Benzyl-2-hydroxy-2-(R)-(2-pyrrolidinyl)-ethyl]-N′,N′dipropyl-isophthalamide Hydrochloride (6). Compound 4b was acylated with 3-(dipropylcarbamoyl)benzoic acid following general procedure B and deprotected following general procedure D to provide compound 6 as a white solid (88%). MS (ESI): m/z 452 [M + H]; [α]D25 = −36 (c 0.3, MeOH). 1H NMR (500 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.48 (s, 1H), 8.47 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.23 (d, J = 7.5 Hz, 2H), 7.19 (t, J = 7.4 Hz, 2H), 7.12 (t, J = 7.1 Hz, 1H), 5.89 (d, J = 6.6 Hz, 1H), 4.07 (dddd, J = 10.9, 9.4, 9.0, 2.7 Hz, 1H), 3.97 (ddd, J = 9.4, 6.6, 1.3 Hz, 1H), 3.56 (dt, J = 8.4, 1.3 Hz, 1H), 3.38 (m, 1H), 3.26 (dd, J = 13.9, 3.2 Hz, 1H), 3.13 (m, 2H), 3.06

(UV, 254 nm and phosphomolibdic acid). Chromatographic separations were performed by using 230−400 mesh silica gel (Merck). General Method A for Debenzylation. The N-Bn or O-Bn protected derivative was dissolved in solvent (10 mL/mmol), and Pd(OH)2 (20% on C) (0.1 equiv) was added. H2 was bubbled through the mixture and the reaction was stirred under 1 atm of H2 overnight. N2 was bubbled through the mixture for 2 min, and it was filtered through Celite eluting with solvent and concentrated. General Method B for the Coupling of Carboxylic Acids and Primary Amines with EDCI. A solution of carboxylic acid, amine (1 equiv), EDCI (1.18 equiv), HOBt (1.18 equiv), Et3N (3 equiv), and DMAP (0.1 equiv) in dry CH2Cl2 (10 mL/mmol) was stirred under N2 at room temperature overnight. The mixture was diluted with CH2Cl2 and washed with 5% NaHCO3 and brine, dried over MgSO4, filtered, and concentrated. General Method C for Acetylation. Et3N (1.5 equiv) and Ac2O (1.05 equiv) were added to a solution of amine in CH2Cl2 (0.1 M solution) at room temperature under nitrogen. The reaction was monitored by TLC. When the starting material was consumed, the volatiles were removed, and the residue was purified by flash chromatography (hexane/EtOAc). General Method D for the N-Boc Deprotection with HCl. HCl (4N in dioxane or 2N in ether, 10 equiv) was added to the N-Boc protected compound, and it was reacted at room temperature overnight. Concentration to dryness provided the desired hydrochloride salt. General Method E for Lactone Reduction. Lactone (1 equiv) was dissolved in toluene (0.1 M solution) and cooled to −78 °C. DIBALH (1 M in toluene, 1.2 equiv) was added dropwise. The reaction was stirred at −78 °C for 30 min. Saturated Na−K tartrate was added, and the reaction was allowed to warm to room temperature and stirred for 20 min. Layers were separated, and the organic layer was dried over MgSO4, filtered, and concentrated to provide a mixture of epimers that were used in the next reaction without purification. General Method F for the N-Boc Deprotection with Trifluoroacetic Acid. Trifluoroacetic acid (10 equiv) was added to the N-Boc protected compound, and it was reacted at room temperature overnight. Concentration to dryness provided the desired trifluoroacetate salt. (S)-2-sec-Butylamino-6-methanesulphonyl-isonicotinic Acid (2). 2,6-Dichloro-isonicotinic methyl ester 1 (2.0 g, 10 mmol), Pd(OAc)2 (224.0 mg, 1.0 mmol), (±)-BINAP (632 mg, 1.0 mmol), and Cs2CO3 (3.96 g, 12 mmol) were dissolved in 20 mL of toluene in a sealed tube under N2. (S)-(+)-sec-Butylamine (1.2 mL, 12 mmol) was added to the solution, and it was heated overnight at 100 °C. The reaction was cooled to room temperature, diluted with Et2O, and filtered through Celite. The filtrate was concentrated and purified on silica gel eluting with 0:100 to 10:90 EtOAc/hexane to provide the corresponding monochloropyridine (73%). MS (ES): m/z 243 [M + H]. This compound (2.01 g, 8.29 mmol), Pd(OAc)2 (186 mg, 0.83 mmol), (±)-BINAP (516 mg, 0.83 mmol), Cs2CO3 (5.40 g, 16.6 mmol), and NaSMe (1.16 g, 16.6 mmol) were dissolved in 20 mL of toluene in a sealed vessel under N2 and heated at 90 °C overnight. The reaction was cooled to room temperature and filtered through Celite. Purification on silica gel eluting with EtOAc/hexane 9:1 provided 1.4 g (66%) of the corresponding sulfide. MS (ES): m/z 255 [M + H]. This compound (1.22 g, 4.80 mmol) was dissolved in 20 mL of CH2Cl2 and cooled to 0 °C. m-CPBA (60%, 2.74 g, 15.9 mmol) was added, and the mixture was stirred at room temperature for 3 h, washed with saturated NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude mixture was purified on silica gel, eluting with EtOAc/hexane 1:3 to provide 1.02 g (74%) of the corresponding sulphone. MS (ES): m/z 287 [M + H]. The sulphone was dissolved in 10 mL of THF and cooled at 0 °C. Then 1N LiOH (5.4 mL, 5.4 mmol) was added and the mixture was stirred at room temperature for 3 h. Then 1N HCl was added to pH 2 and THF was removed. The solution was partitioned between EtOAc and H2O. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over MgSO4, filtered, and 9815



Article

(m, 2H), 2.82 (dd, J = 13.8, 10.8 Hz, 1H), 2.06−1.88 (m, 3H), 1.8 (m, 1H), 1.62 (m, 2H), 1.45 (m, 2H), 0.93 (t, J = 7.4 Hz, 1H), 0.65 (t, J = 7.3 Hz, 1H). 13C NMR (125.78 MHz, DMSO-d6): 170.3, 165.8, 139.4, 137.8, 134.7, 129.6, 129.3, 128.9, 128.40, 128.2, 126.4, 125.3, 70.5, 61.4, 53.9, 50.5, 46.2, 45.6, 36.7, 24.0, 23.4, 21.9, 20.8, 11.8, 11.3. (2R,4R)-4-Benzyloxy-2-hydroxymethylpyrrolidine-1-carboxylic Acid tert-Butyl Ester (8). LiBH4 (1.87 g, 86.0 mmol) was added to an ice-cold solution of 7 (48 g, 143.3 mmol) in THF (300 mL). The reaction was allowed to warm slowly to room temperature and stirred over 18 h. It was poured on saturated NH4Cl and extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography (hexane/EtOAc 90:10 to 70:30) to give compound 8 as a colorless oil (39.5 g, 92%). MS (ES): m/z 330.1 [M + Na]. Spectral data consistent with commercial material. (2R,4R)-4-Benzyloxy-2-[(1R,2S)-3-(3,5-difluorophenyl)-l-hydroxy2-nitropropylpyrrolidine-1-carboxylic Acid tert-Butyl Ester (9). Triethylamine (1.1 mL, 7.79 mmol) and SO3−pyridine complex (0.63 g, 3.89 mmol) were added to an ice-cold solution of compound 8 (0.6 g, 1.95 mmol) in DMSO (2 mL). The reaction was stirred 30 min, warmed to room temperature, and stirred for 30 min. It was diluted with Et2O and washed with 5% aqueous citric acid (3 × 120 mL) and brine, dried over MgSO4, filtered, and concentrated to give (2R,4R)-4-benzyloxy-2-formylpyrrolidine-1-carboxylic acid tert-butyl ester as an oil (0.59 g, 100%). MS (ES): m/z 328.1 [M + Na]. The compound was used in the next reaction without purification. TBAF (1.6 mL of 1.0 M solution in THF, 1.6 mmol) was added to a solution of 1-phenyl-2-nitroethane (0.88 mL, 6.55 mmol) in THF (4 mL) at 0 °C. The reaction was stirred for 5 min, and (2R,4R)-4-benzyloxy-2formylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 g, 3.27 mmol) in THF (10 mL) was added. The reaction was stirred 15 min, and it was diluted with EtOAc, washed with H2O (3×) and brine, dried, filtered, and concentrated to provide a mixture of three isomers (ratio: 1.4:7.5:1). Purification by flash chromatography (hexane/acetone 10:1) provided the major isomer 9 as a colorless foam (0.696 g, 49%). MS (ES): mlz 515.3 [M + Na]. (2R,4R)-2-[(4S,5S)-3-Acetyl-4-(3,5-difluoro-benzyl)-2,2-dimethyloxazolidin-5-yl]-4-hydroxy-pyrrolidine-1-carboxylic Acid tert-Butyl Ester (10). NaBH4 (0.242 mg, 6.42 mmol) was added over 1 min to a solution of 9 (0.696 g, 1.41 mmol) and NiCl2 (0.29 g, 2.25 mmol) in MeOH (30 mL) at room temperature. The reaction was stirred for 20 min, Ac2O was added (0.16 mL, 1.68 mmol), and the reaction was stirred for 30 min. H2O was added (3 mL), and the reaction was concentrated. The residue was partitioned between EtOAc and H2O and filtered through Celite. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated to give the amino derivative as a colorless oil (619 mg, 95%). MS (ES): m/z 463.3 [M + H]. The crude material was dissolved in 5 mL of a 1:1 CH2Cl2/ acetone mixture, CSA (0.026 mg, 0.11 mmol) was added, and the solution was cooled to 0 °C. 2-Methoxypropene (0.25 mL, 2.6 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature until completion. It was concentrated and the residue partitioned between CH2Cl2 and H2O. The organic layer was washed with saturated NaHCO3, dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography in silica gel eluting with EtOAc/hexane 1:9 to provide the desired compound (0.120 g, 81%). MS(ESI): m/z = 545.3 [M + H]. The compound was dissolved in MeOH (10 mL), 10% Pd on carbon (12 mg) was added, and the reaction was stirred overnight under 1 atm of H2. The reaction was purged with N2, filtered over Celite, and concentrated to provide compound 10 (0.100 g, 98%) as a foam. MS(ESI): m/z 455.3 [M + H]. 1H NMR (CDCl3) δ: 7.31−7.22 (m, 5H), 6.84 (d, J = 6.1 Hz, 2H), 6.66 (m, 1H), 4.62 and 4.46 (AB system, 2H), 4.24−4.07 (m, 5H), 3.90 (bd, J = 11.8 Hz, 1H), 3.65 and 3.52 (AB system, 2H), 2.65 (d, J = 14.0 Hz, 1H), 2.57 (t, J = 11.7 Hz, 1H), 1.98 (m, 1H), 1.77 (s, 3H), 1.51 (s, 15H). N-[(1S,2R)-2-((2R,4R)-4-Cyclohexylmethoxy-pyrrolidin-2-yl)-1(3,5-difluoro-benzyl)-2-hydroxy ethyl]-acetamide Hydrochloride (11). Compound 10 (198 mg, 0.43 mmol) and cyclochexylmethyl bromide (0.65 mL, 1.27 mmol) were dissolved in DMF (2 mL). NaH

95% (16 mg, 1.27 mmol) was added portionwise, and the reaction was stirred at room temperature. Upon completion, the residue was partitioned between H2O and EtOAc. The organic layer was washed with H2O and brine, dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography in silica gel eluting with EtOAc/hexane 1:7 to provide the protected compound (0.127 g). Deprotection was performed by following general method D to obtain compound 11 as a white solid (103 mg, 52%, two steps). 1H NMR (500.23 MHz, DMSO-d6) δ: 9.72 (bs, 1H), 8.46 (bs, 1H), 7.93 (d, J = 9.4 Hz, 1H), 7.01 (m, 1H), 6.94 (m, 2H), 5.87 (d, J = 6.8 Hz, 1H), 4.10 (m, 1H), 3.80 (m, 1H), 3.68 (m, 1H), 3.52 (m, 1H), 3.27 (m, 1H), 3.20−3.06 (m, 4H), 2.57 (dd, J = 13.5, 10.9 Hz, 1H), 2.26 (m, 1H), 1.83 (m, 1H), 1.70−1.60 (m, 9H), 1.47 (m, 1H), 1.22−1.08 (m, 3H), 0.90 (m, 2H). 13C NMR (125.78 MHz, DMSO-d6) δ: 169.4, 162.5 (dd, J = 245, 13.6 Hz), 144.1 (t, J = 10.2 Hz), 112.7 (dd, J = 19.5, 5.1 Hz), 101.9 (t, J = 25.4 Hz), 76.7, 74.8, 70.3, 60.4, 52.6, 49.8, 37.9, 36.4, 30.9, 29.9, 26.6, 25.8, 23.0. O1-tert-Butyl O2-Ethyl (2R,5R)-5-(Benzyloxymethoxy)piperidine1,2-dicarboxylate (13). To a solution of compound 12 (350 mg, 1.28 mmol) in CH2Cl2 (5 mL) were added DIPEA (0.45 mL, 2.6 mmol) and BOMCl (0.45 mL, 1.9 mmol). The mixture was refluxed for 3 h. The reaction was cooled to room temperature and diluted with EtOAc (10 mL). The organic solution was washed with 5% citric acid, saturated NaHCO3, and brine, dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography in silica gel eluting with CH2Cl2/EtOAc from 100:0 to 90:10 to provide the desired compound (0.320 g, 63%). MS (ESI) m/z 294 [M-BOC +H]. 1H NMR (300 MHz, CDCl3) δ: 7.36−7.22 (m, 5H), 4.83−4.51 (m, 5 H), 4.32−4.08 (m, 3H), 3.65−3.47 (m, 1H), 2.72 (m, 1H), 2.33−2.18 (m, 1H), 2.04−1.90 (m, 1H), 1.74−1.57 (m, 1H), 1.41 (s, 9H), 1.31−1.14 (m, 4H). tert-Butyl (2R,5R)-5-(Benzyloxymethoxy)-2-(hydroxymethyl)piperidine-1-carboxylate (14). Compound 14 was made essentially as compound 8 (60%), MS (ES): m/z 252 [M-BOC+H]. 1H NMR (300 MHz, CDCl3) δ: 7.40−7.27 (m, 5H), 4.81 (s, 2H), 4.62 (s, 2H), 4.25 (m, 2H), 3.80 (dt, J = 10.9, 2.8 Hz, 1H), 3.67−3.54 (m, 2H), 2.73 (bt, J = 10.9 Hz, 1H), 2.04−1.84 (m, 1H), 1.83−1.40 (m, 4H), 1.45 (s, 9H). tert-Butyl (2R,5R)-5-(Benzyloxymethoxy)-2-[(1R,2S)-3-(3,5-difluorophenyl)-1-hydroxy-2-nitro-propyl]piperidine-1-carboxylate (15). Compound 15 was made essentially as compound 9 (68%; contaminated with 10% of another isomer, and it was carried over to the next step). MS (ESI): m/z 559 [M + Na]. tert-Butyl (2R,5R)-2-[(4S,5S)-3-Acetyl-4-[(3,5-difluorophenyl)methyl]-2,2-dimethyl-oxazolidin-5-yl]-5-hydroxy-piperidine-1-carboxylate (16). Compound 16 was made essentially as compound 10 (14%). MS (ESI): m/z 459 [M + H]. N-[(1S,2R)-2-[(2R,5R)-5-(Cyclohexylmethoxy)-2-piperidyl]-1-[(3,5difluorophenyl)methyl]-2-hydroxy-ethyl]acetamide (17). To a solution of compound 15 (1g, 2.1 mmol) in dry DMF (8.5 mL) at 0 °C was added 60% NaH (171 mg, 4.27 mmol), and the resulting mixture was allowed to reach room temperature and stirred for 1 h. Cyclohexymethyl bromide (0.6 mL, 4 mmol) and NaI (640 mg, 4.27 mmol) were added, and the mixture was heated at 40 °C for 1 h and at 60 °C overnight. Two more additions of cyclohexymethyl bromide (2 × 0.6 mL) and NaI (2 × 640 mg) were performed the following day with an interval of 2 h to take the reaction to completion. The reaction was allowed to reach room temperature, quenched with H2O, and extracted with AcOEt. The combined organic phase were washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by flash chromatography in silica gel eluting with hexane/EtOAc from 100:0 to 0:100. Deprotection was performed by following general method D to obtain compound 17 as a white solid (0.250 g, 21%); [α]D25 = +28 (c 0.5, MeOH). MS (ES): m/z 565 [M + H]. 1H NMR (300 MHz, MeOD-d4) δ: 6.97−6.70 (m, 3H), 4.04 (dt, J = 10.9, 3.1 Hz, 1H), 3− 81−3.65 (m, 2H), 3.52−3.13 (m, 6H), 2.63 (dd, J = 13.7, 11.1 Hz, 1H), 1.88 (s, 3H), 2.22−1.46 (m, 10H), 1.43−1.12 (m, 3H), 1.12− 0.88 (m, 2H). 13C NMR (75 MHz, MeOD-d4) δ: 170.6, 163.5, 160.2 (dd, J = 247, 14.0 Hz), 141.8 (t, J = 10.3 Hz), 110.8 (dd, J = 16.9, 7.4 9816



Article

concentrated. Purification by flash chromatography of the crude residue (hexane/EtOAc 90:10 to 80:20) provided 2.6 g of compound 22 as a white solid (58%, four steps). MS (ES): m/z 657 [M + H]. 1H NMR (CDCl3) δ: 7.38−7.10 (m, 15H), 6.83 (d, J = 6.5 Hz, 2H), 6.70 (t, J = 8.9 Hz, 1H), 4.80 and 4.64 (AB system, 2H), 4.41−4.11 (m, 5H), 3.80−3.62 (m, 5H), 3.12−2.99 (m, 3H), 1.38 (s, 9H). tert-Butyl (5R)-5-[(1S,2S)-1-Benzyloxy-2-(dibenzylamino)-3-(3,5difluorophenyl)propyl]-2-(cyclohexylmethoxy)morpholine-4-carboxylate (23). Lactone 22 (1 g, 1.52 mmol) was reduced by following general method E to provide 1 g of the lactols. MS (ES): m/z 659 [M + H]. NaH (60% in oil, 21 mg, 0.52 mmol) was added to a solution of the lactols (300 mg, 0.45 mmol) in DMF (2.3 mL) at 0 °C under N2. The mixture was stirred for 15 min, and then cyclohexylmethyl trifluoromethanesulfonate (129 mg, 0.50 mmol) was added. The mixture was stirred at 0 °C for 30 min, and it was partitioned between EtOAc and H2O. The organic layer was washed with H2O and brine, dried over MgSO4, filtered, and concentrated. Purification by flash chromatography (hexane/EtOAc 100:0 to 90:10) provided compound 23 (297 mg, 88% yield) as a white solid. (1S,2S)-1-[(3R,6R)-6-(Cyclohexylmethoxy)morpholin-3-yl]-3-(3,5difluorophenyl)-2-methyl-propan-1-ol, trifluoroacetate Salt (24). Compound 23 was subjected to general conditions A, using EtOAc as solvent, and acetylated by following general method C and deprotected with method F to provide the desired compound as a white solid (78%, three steps). MS (ES): m/z 459 [M-t-Bu+H]; [α]D25 = −19 (c 1, MeOH). 1H NMR (500.23 MHz, DMSO-d6): 9.94 (s, 1H), 8.28 (s, 1H), 8.01 (d, J = 9.4 Hz, 1H), 7.03 (tt, J = 9.40, 2.4 Hz, 1H), 6.97 (m, 2H), 6.03 (d, J = 6.8 Hz, 1H), 4.86 (d, J = 1.7 Hz, 1H), 3.96−3.80 (m, 3H), 3.65 (m, 1H), 3.40 (dd, J = 9.4, 7.3 Hz, 1H), 3.31−3.30 (m, 3H), 3.16−3.10 (m, 2H), 2.56 (dd, J = 13.7, 11.1 Hz, 1H), 1.86 (m, 1H), 1.77−1.52 (m, 8H), 1.27−1.17 (m, 3H), 1.02− 0.92 (m, 2H). 13C NMR (125.78 MHz, DMSO-d6) δ: 169.7, 162.5 (dd, J = 245, 13.6 Hz), 144.0 (t, J = 9.3 Hz), 112.8 (dd, J = 19.5, 5.1 Hz), 102.0 (t, J = 26.3 Hz), 92.0, 72.6, 71.5, 55.5, 54.8, 51.4, 45.5, 37.7, 36.6, 29.9, 29.7, 26.5, 25.8, 25.7, 22.9. tert-Butyl (3S,5R)-5-[(1S,2S)-1-Benzyloxy-2-(dibenzylamino)-3(3,5-difluorophenyl)propyl]-3-methyl-2-oxo-morpholine-4-carboxylate (25). Oxazolidine 21 (16.1 g, 24.6 mmol) was hydrolyzed by following general method D to provide the corresponding dihydrochloride salt. This compound was dissolved in CH2Cl2 (350 mL), Et 3 N (14 mL, 100.4 mmol) and ethyl (2R)-2(trifluoromethylsulfonyloxy)propanoate (17 g, 72.0 mmol) were added, and the reaction was stirred at room temperature for 64 h. The reaction was diluted with CH2Cl2 and washed with 5% citric acid and brine, dried over MgSO4, filtered, and concentrated. Purification by flash chromatography eluting with hexane/EtOAc (90:10 to 80:20) provided 10.6 g of the lactone 25 (76%, two steps). 1H NMR (300.16 MHz, CDCl3) δ: 7.35−7.16 (m, 15H), 6.77−6.63 (m, 3H), 4.77 and 4.68 (AB system, 2H), 4.10 (t, J = 11.1 Hz, 1H), 3.74−3.43 (m, 8H), 3.09−2.89 (m, 3H), 3.12−2.99 (m, 3H), 1.27 (d, J = 6.9 Hz, 3H). (1R,2S)-N,N-Dibenzyl-1-benzyloxy-1-[(3R,5S,6R)-6-(cyclohexylmethoxy)-5-methyl-morpholin-3-yl]-3-(3,5-difluorophenyl)propan-2amine (26). Lactone 25 (14 g) was reduced by following general method E to provide the two epimeric lactols (12.2 g). MS (ES): m/z 573 [M + H]. The mixture of lactols (500 mg, 0.87 mmol) was combined with cyclohexylmethanol (1.02 mL, 8.7 mmol) and 4 Å molecular sieves (400 mg). MsOH (1.13 mL, 17.5 mmol) was added, and the reaction was heated at 50 °C for 4 h. The reaction mixture was added dropwise to saturated NaHCO3. The aqueous layer was extracted with CH2Cl2, dried over Na2SO4, filtered, and concentrated. The residue was chromatographed by eluting with hexane/EtOAc 95:5 to 90:10 to obtain 397 mg of alkylated lactol contaminated with cyclohexylmethanol as a colorless waxy solid. The mixture (392 mg, 0.58 mmol) was dissolved in 1,2-dichloroethane (6 mL). DIPEA (0.2 mL, 1.17 mmol) and Boc2O (396 mg, 1.76 mmol) were added, and the reaction was heated at 75 °C for 15 h. The solvent was removed, and the residue was purified by flash chromatography by eluting with hexane/CH2Cl2 80:20 to 0:100 to provide 302 mg (65% yield, three steps) of compound 26 as a white solid. MS (ES): m/z 769 [M + H]. 1 H NMR (300.16 MHz, CDCl3) δ: 7.37−7.27 (m, 5H), 7.19−7.15 (m,

Hz), 100.1 (t, J = 26.1 Hz), 73.0, 71.1, 67.7, 56.8, 50.4, 46.5, 37.0, 35.8, 28.7 (d, J = 21.2 Hz), 25.3, 24.6 (d, J = 4.3 Hz), 23.6, 20.2, 14.6. 4-[(1R,2S)-3-(3,5-Difluorophenyl)-l-hydroxy-2-nitropropyl]- 2,2dimethyloxazolidine-3-carboxylic Acid tert-Butyl Ester (20). (R)tert-Butyl-4-formyl-2,2-dimethyloxazolidine-3-carboxylate 19 (1.60 g, 6.98 mmol) and 1,3-difluoro-5-(2-nitroethyl)-benzene 18 (1.40 g, 7.48 mmol) were dissolved in dry THF (14 mL) and cooled to 0 °C. 1 M TBAF in THF (7.0 mL, 7.0 mmol) was added in one portion. The reaction was stirred for 30 min and diluted with EtOAc. The mixture was washed with H2O and brine, dried over MgSO4, filtered, and concentrated. Purification by flash chromatography, eluting with 0:100 to 20:80 EtOAc/hexane, provided a mixture of diastereomers from which the desired isomer was crystallized with Et2O/hexane to give 1.26 g of the desired compound as a white crystalline solid (43%). MS (ES): m/z 439.3 [M + Na]. 1H NMR (CDCl3) δ: 6.89 (bd, J = 6.5 Hz, 2H), 6.67 (t, J = 8.9 Hz, 1H), 4.71 (d, J = 10.1 Hz, 1H), 4.19−3−95 (m, 4H), 3.50−3.29 (m, 3H), 1.55 (s, 9H). tert-Butyl (4R)-4-[(1S,2S)-1-Benzyloxy-3-(3,5-difluorophenyl)-2methyl-propyl]-2,2-dimethyl-oxazolidine-3-carboxylate (21). NaBH4 (46 g, 1210 mmol) was added portionwise to a suspension of compound 20 (93 g, 223 mmol) and NiCl2 (46 g, 350 mmol) in MeOH (2 L) cooled at 0 °C. The resulting mixture was stirred for 5 min at 0 °C and 15 min at room temperature. H2O (20 mL) was added, and the mixture was stirred at room temperature for 10 min. It was filtered through a pad of Celite, washing with EtOAc, and the solvent was removed. The residue was partitioned between brine and EtOAc, and the organic layer was washed with 0.5% HCl, dried over MgSO4, filtered, and concentrated to afford 90 g of the amine as a white solid. MS (ES): m/z = 387 [M + H]. The crude reaction (51 g, 132 mmol) was suspended in acetonitrile (500 mL), and K2CO3 (46 g, 333 mmol) was added in one portion. Benzyl bromide (45 mL, 378 mmol) was added, and the reaction was refluxed for 16 h. It was allowed to cool to room temperature and partitioned between EtOAc and NH4Cl satd. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The crude was purified by flash chromatography (hexane/EtOAc 3:2) to afford 70.4 g of the dibenzylamine as a white solid. MS (ES): m/z 567 [M + H]. This compound (27 g, 47.7 mmol) and benzyl bromide (10 mL, 84.1 mmol) were dissolved in DMF (300 mL) at 20 °C, and NaH (2.3 g, 57.5 mmol) was added dropwise under N2 atmosphere. The reaction mixture was partitioned between EtOAc and saturated NH4Cl, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The crude residue was purified by flash chromatography (EtOAc/hexane 2:98) to afford 26 g of the desired compound 21 as a white solid (83%, three steps). MS (ES): m/z 657 [M + H]. 1H NMR (CDCl3) δ: 7.36−7.14 (m, 15H), 6.77−6.59 (m, 3H), 4.87−4.28 (m, 4H), 3.81−3.63 (m, 6H), 3.11−2.82 (m, 3H), 1.62−1.38 (s, 15H). tert-Butyl (5R)-5-[(1S,2S)-1-Benzyloxy-3-(3,5-difluorophenyl)-2methyl-propyl]-2-oxo-morpholine-4-carboxylate (22). Oxazolidine 21 was hydrolyzed by following general method D to provide the corresponding dihydrochloride salt. MS (ES): m/z 517 [M + H]. This compound (11.8 g, 20.0 mmol) was dissolved in DMF (200 mL), DIPEA (12 mL, 68.9 mmol) and ethyl bromoacetate (6 mL, 54.1 mmol) were added, and the reaction was stirred at room temperature. Two more additions of ethyl bromoacetate (2 × 6 mL) were performed with an interval of 30 min to take the reaction to completion. The reaction mixture was partitioned between EtOAc and saturated NaHCO3, and the organic layer was dried over MgSO4, filtered, and concentrated. Purification by flash chromatography provided 9.35 g of the alkylated amine with a 10% of cyclized lactone. The mixture (3.2 g) was dissolved in toluene (70 mL), DIPEA (3 mL, 18.1 mmol) and CSA (600 mg, 2.6 mmol) were added, and the solution was refluxed for 6 h. The reaction was allowed to cool to room temperature, partitioned between EtOAc and saturated NaHCO3, and the organic layer was dried over MgSO4, filtered, and concentrated. MS (ES): m/z 557 [M + H]. The crude was redissolved in CH2Cl2 (60 mL), Boc2O (2 g, 9.2 mmol) and DIPEA (3 mL, 17.2 mmol) were added, and the solution was stirred at room temperature for 12 h. The reaction was partitioned between CH2Cl2 and 5% citric acid, and the organic layer was dried over MgSO4, filtered, and 9817



Article

Plasma and Brain Concentrations. FVB mice were ordered from Taconic, age 7−10 weeks (n = 3). Compounds were dosed in a 7% Pharmasolve/water vehicle at 30 mg/kg subcutaneously to fed mice. Blood samples were collected in EDTA treated tubes. Blood and brain samples were collected at 1 h and maintained frozen until LCMS analysis was performed.

6H), 7.06−7.03 (m, 4H), 6.76−6.29 (m, 3H), 4.85 and 4.80 (AB system, 2H), 4.63−4.60 (m, 2H), 4.18−4.05 (m, 2H), 3.91−3.75 (m, 4H), 3.67−3.55 (m, 3H), 3.29 (dd, J = 9.5 Hz, 7.5 Hz, 1H), 3.09−3.00 (m, 3H), 1.94−1.63 (m, 6H), 1.38 (d, J = 7.3 Hz, 3H), 1.29 (s, 9H), 1.29−1.14 (m, 3H), 1.07−0.93 (m, 2H). (1R,2S)-N,N-Dibenzyl-1-benzyloxy-3-(3,5-difluorophenyl)-1[(3R,5S,6R)-6-(2,2-dimethylpropoxy)-5-methyl-morpholin-3-yl]propan-2-amine (28). Compound 28 was made essentially like compound 26 to obtain the final compound as a colorless waxy solid (370 mg, 65%, three steps). MS (ES): m/z 743 [M + H]. 1H NMR (300.16 MHz, CDCl3) δ: 7.34−7.32 (m, 5H), 7.23−7.15 (m, 6H), 7.06−7.04 (m, 4H), 6.74−6.64 (m, 3H), 4.84 and 4.78 (AB system, 2H), 4.55 (d, J = 5.7 Hz, 1H), 4.46 (d, J = 8.9 Hz, 1H), 4.26−4.20 (m, 1H), 4.01 (dd, J = 11.9, 5.0 Hz, 1H), 3.88−3.74 (m, 3H), 3.63−3.48 (m, 4H), 3.16−3.01 (m, 4H), 1.37 (d, J = 6.8 Hz, 3H), 1.30 (s, 9H), 0.98 (s, 9H). (1S,2S)-1-[(3R,5S,6R)-6-(Cyclohexylmethoxy)-5-methyl-morpholin-3-yl]-3-(3,5-difluorophenyl)-2-methyl-propan-1-ol Hydrochloride Salt (27). Compound 26 was debenzylated by following general method A using EtOAc as solvent, acetylated following general method C, and deprotected with method F, followed by addition of HCl (4N in dioxane, 5 equiv) and concentration (operation repeated twice) to provide compound 27 as a white solid (177 mg, 64%, four steps). MS (ES): m/z 441 [M + H]; [α]D25 = −18 (c 0.18, MeOH). 1 H NMR (500.23 MHz, DMSO-d6): 9.82 (d, J = 9.8 Hz, 1H), 8.38 (m, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.07−7.01 (m, 1H), 6.98−6.95 (m, 2H), 6.06 (d, J = 6.2 Hz, 1H), 4.58 (s, 1H), 3.96 (t, J = 11.4 Hz, 1H), 3.88−3.80 (m, 2H), 3.74−3.70 (m, 1H), 3.46−3.37 (m, 3H), 3.25 (dd, J = 9.4, 6.2 Hz, 1H), 3.12 (d, J = 11.8 Hz, 1H), 2.58 (dd, J = 13.6, 10.8 Hz, 1H), 1.84 (d, J = 12.8 Hz, 1H), 1.73−1.69 (m, 8H), 1.34 (d, J = 6.8 Hz, 3H), 1.27−1.13 (m, 3H), 1.02−0.93 (m, 2H). 13C NMR (125.78 MHz, DMSO-d6) δ: 169.8, 162.5 (dd, J = 245, 13.6 Hz), 143.8 (t, J = 9.3 Hz), 112.8 (dd, J = 19.5 Hz, 5.1 Hz), 102.0 (t, J = 25.4 Hz), 96.4, 72.9, 70.9, 55.3, 51.4, 50.4, 49.5, 37.7, 36.6, 29.9, 29.7, 26.5, 25.8, 25.7, 22.9, 13.2. (1S,2S)-3-(3,5-Difluorophenyl)-1-[(3R,5S,6R)-6-(2,2-dimethylpropoxy)-5-methyl-morpholin-3-yl]-2-methyl-propan-1-ol Hydrochloride Salt (29). Compound 29 was synthesized essentially as compound 27 and was obtained as a white solid (334 mg, 75%, four steps). MS (ES): m/z 415 [M + H]; [α]D25 = −28 (c 0.77, MeOH). MS (ES): m/ z 415 [M + H]. 1H NMR (300.13 MHz, MeOH-d4) δ: 6.87−6.74 (m, 3H), 4.62 (s, 1H), 4.15 (t, J = 11.8 Hz, 1H), 4.02−3.89 (m, 2H), 3.81 (m, 1H), 3.64−3.56 (m, 2H), 3.45 (d, J = 9.1 Hz, 1H), 3.26 (dd, J = 14, 2.5 Hz, 1H), 3.13 (d, J = 8.8 Hz, 1H), 2.62 (dd, J = 13.4, 11.3 Hz, 1H), 1.85 (s, 3H), 1.47 (d, J = 7.1 Hz, 3H), 1.01 (s, 9H). 13C NMR (125.78 MHz, DMSO-d6) δ: 169.4, 162.5 (dd, J = 245, 13.6 Hz), 143.8 (t, J = 10.2 Hz), 112.8 (dd, J = 20.4, 5.1 Hz), 102.0 (t, J = 25.4 Hz), 97.7, 77.5, 71.0, 55.4, 51.3, 50.5, 49.7, 36.6, 32.0, 27.0, 22.9, 13.2. Crystallography. Purification, crystallization, and structure determination of BACE-1 has been described extensively in the literature and was accomplished using minor modifications of previously described methods.45 Purified recombinant BACE-1 (12 mg/mL) containing 5 mM Tris-HCl, 10 mM inhibitor, and 1% DMSO was crystallized using the hanging drop vapor diffusion method at 4 °C and precipitant solutions containing 15−20% polyethylene glycol 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 7.4. After an overnight incubation, the hanging drops were macroseeded with preexisting microcrystals of approximately 5 μm in the largest dimension and grown under nearly identical conditions. Numerous crystals having maximum dimensions in the 100−400 μm range nucleated over a 3−5 day period following macroseeding, such that the original macroseeded crystals could not be identified. Three of the inhibitors crystallized in space group P212121, with two molecules per asymmetric unit, while compound 24 crystallized in space group P21, as described for inhibitor OM99-2.45 Packing within the two space groups is very similar, and the unit cells of these two space groups are geometrically related. Crystals were flash-cooled in liquid nitrogen using glycerol as a cryoprotectant, and X-ray diffraction data were collected at 100 K at the Advanced Photon Source beamlines 17-ID and 31-ID.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01304. HPLC purity of compounds 5a, 5b, 6, 11, 17, 24, 27, and 29; crystallographic data for compound 16 (PDF) Molecular formula strings (CSV) Accession Codes

The coordinate data for the structures complexed with BACE-1 have been deposited in the Protein Data Bank with accession numbers 6BFD (5b), 6BFE (11), 6BFW (24), and 6BFX (29). Authors will release the atomic coordinates and experimental data upon article publication.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +34-91-6633402. E-mail: Bueno_Ana_Belen@lilly. com. ORCID

Ana B. Bueno: 0000-0002-4190-6891 Present Addresses §

R.R.: Galchimia, SL, Cebreiro s/n, 15823 O Pino, A Coruña, Spain. ∥ J.F.S.: Synthelia Organics, Faraday 7 Lab 2.05, 28049 Madrid, Spain. ⊥ H.-C.Y.: Kent Anderssons, Gata 5, 412 49 Göteborg, Sweden. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): All the authors of this manuscript are or have been Eli Lilly and Company employees and may own company stock or possess stock options.

■

ACKNOWLEDGMENTS We thank Xiyun Chai for the expression of human recombinant BACE-1 protein to support X-ray crystallography, Amechand Boodhoo for the purification of the enzyme, and Debra Laigle for performing BACE mcaFRET assay. We also thank Gregory Stephenson for the X-ray of intermediate 16, Almudena Garciá for performing the HPLC purity analysis, Michael Clay for performing brain exposure experiments, and Timothy Jones for compilation of experimental data. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility 9818



■ ■ ■

Article

(16) Cumming, J. N.; Le, T. X.; Babu, S.; Carroll, C.; Chen, X.; Favreau, L.; Gaspari, P.; Guo, T.; Hobbs, D. W.; Huang, Y.; Iserloh, U.; Kennedy, M. E.; Kuvelkar, R.; Li, G.; Lowrie, J.; McHugh, N. A.; Ozgur, L.; Pan, J.; Parker, E. M.; Saionz, K.; Stamford, A. W.; Strickland, C.; Tadesse, D.; Voigt, J.; Wang, L.; Wu, Y.; Zhang, L.; Zhang, Q. Rational design of novel, potent piperazinone and imidazolidinone BACE1 inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 3236−3241. (17) Ghosh, A. K.; Osswald, H. L. BACE1 (β-secretase) inhibitors for the treatment of Alzheimer’s disease. Chem. Soc. Rev. 2014, 43, 6765− 6813. (18) Pathak, M. K.; Jha, V.; Jain, N. K.; Shrivastava, N.; Yadav, N.; Purukayastha, D. Review on peptidomimetics: a drug designing tool. Indo Am. J. Pharm. Res. 2015, 5, 3859−3867. (19) Liu, C. N.; Ng, J. S.; Behling, J. R.; Yen, Ch. H.; Campbell, A. L.; Fuzail, K. S.; Yonan, E. E.; Mehrotra, D. V. Development of a largescale process for an HIV protease inhibitor. Org. Process Res. Dev. 1997, 1, 45−54. (20) Reetz, M. T. Synthesis and diastereoselective reactions of n,ndibenzylamino aldehydes and related compounds. Chem. Rev. 1999, 99, 1121−1162. (21) Luzzio, F. A. The Henry reaction: recent examples. Tetrahedron 2001, 57, 915−945. (22) Russell, M. G. N.; Beer, M. S.; Stanton, J. A.; Sohal, B.; Mortishire-Smith, R. J.; Castro, J. L. 2,7-Diazabicyclo[3.3.0]octanes as novel h5-HT receptor agonists. Bioorg. Med. Chem. Lett. 1999, 9, 2491−2496. (23) Letavic, M. A.; Axt, M. Z.; Barberia, J. T.; Carty, T. J.; Danley, D. E.; Geoghegan, K. F.; Halim, N. S.; Hoth, L. R.; Kamath, A. V.; Laird, E. R.; Lopresti-Morrow, L. L.; McClure, K. F.; Mitchell, P. G.; Natarajan, V.; Noe, M. C.; Pandit, J.; Reeves, L.; Schulte, G. K.; Snow, S. L.; Sweeney, F. J.; Tan, D. H.; Yu, C. H. Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors. Bioorg. Med. Chem. Lett. 2002, 12, 1387−1390. (24) Passiniemi, M.; Koskinen, A. M. P. Garner’s aldehyde as a versatile intermediate in the synthesis of enantiopure natural products. Beilstein J. Org. Chem. 2013, 9, 2641−2659. (25) Potamitis, C.; Zervou, M.; Katsiaras, V.; Zoumpoulakis, P.; Durdagi, S.; Papadopoulos, M. G.; Hayes, J. M.; Grdadolnik, S. G.; Kyrikou, I.; Argyropoulos, D.; Vatougia, G.; Mavromoustakos, T. Antihypertensive drug valsartan in solution and at the AT1 receptor: conformational analysis, dynamic NMR spectroscopy, in silico docking, and molecular dynamics simulations. J. Chem. Inf. Model. 2009, 49, 726−739. (26) Zervou, M.; Cournia, Z.; Potamitis, C.; Patargias, G.; Durdagi, S.; Grdadolnik, S. G.; Mavromoustakos, T. Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 1031−1046. (27) Kellici, T. F.; Ntountaniotis, D.; Kritsi, E.; Zervou, M.; Zoumpoulakis, P.; Potamitis, C.; Durdagi, S.; Ekhteiari Salmas, R.; Ergun, G.; Gokdemir, E.; Halabalaki, M.; Gerothanassis, I. P.; Liapakis, G.; Tzakos, A.; Mavromoustakos, T. Leveraging NMR and X-ray data of the free ligands to build better drugs targeting angiotensin II type 1 G-protein coupled receptor. Curr. Med. Chem. 2016, 23, 36−59. (28) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C. The relationship between proton-proton NMR coupling constants and substituent electronegativitiesI. Tetrahedron 1980, 36, 2783−2792. (29) Varghese, J.; Jagodzinska, B.; Maillard, M.; Beck, J. P.; Tenbrink, R. E.; Getman, D. Preparation of substituted amines prodrugs useful in treating Alzheimer’s disease WO 2003072535A2, 2003. (30) Tyndall, J. D.; Nall, T.; Fairlie, D. P. Proteases universally recognize beta strands in their active sites. Chem. Rev. 2005, 105, 973− 999. (31) Smith, T. D.; Gerken, J. B.; Jog, P. V.; Roberts, J. D. Conformational equilibria of ethanolamine and its hydrochloride in solution. Org. Lett. 2007, 9, 4555−4557. (32) Hoffmann, R. W. Conformation design of open-chain compounds. Angew. Chem., Int. Ed. 2000, 39, 2054−2070.

DEDICATION Dr. James McCarthy passed away on September 12, 2016. This publication is dedicated to his memory. #

ABBREVIATIONS USED TS, transition state; HEA, hydroxyethylamine REFERENCES

(1) Leung, D.; Abbenante, G.; Fairlie, D. P. Protease inhibitors: current status and future prospects. J. Med. Chem. 2000, 43, 305−341. (2) Nguyen, J.-T.; Hamada, Y.; Kimura, T.; Kiso, Y. Design of potent aspartic protease inhibitors to treat various diseases. Arch. Pharm. (Weinheim, Ger.) 2008, 341, 523−535. (3) Vassar, R. The beta-secretase, BACE: a prime drug target for Alzheimer’s disease. J. Mol. Neurosci. 2001, 17, 157−170. (4) Schramm, V. L. Transition States, analogues, and drug development. ACS Chem. Biol. 2013, 8, 71−81. (5) Boulanger, Y.; Senecal, L.; Sauve, G. Solution structure of the HIV protease inhibitor acetyl-pepstatin as determined by NMR and molecular modeling. J. Biomol. Struct. Dyn. 1997, 14, 421−428. (6) Vidal, P.; Timm, D.; Broughton, H.; Chen, S. H.; Martin, J. A.; Rivera-Sagredo, A.; McCarthy, J. R.; Shapiro, M. J.; Espinosa, J. F. Preorganization of the hydroxyethylene dipeptide isostere: the preferred conformation in solution resembles the conformation bound to BACE. J. Med. Chem. 2005, 48, 7623−7627. (7) Ene, L.; Duiculescu, D.; Ruta, S. M. How much do antiretroviral drugs penetrate into the central nervous system? J. Med. Life 2011, 4, 432−439. (8) Stachel, S. J.; Coburn, C. A.; Steele, T. G.; Jones, K. G.; Loutzenhiser, E. F.; Gregro, A. R.; Rajapakse, H. A.; Lai, M. T.; Crouthamel, M. C.; Xu, M.; Tugusheva, K.; Lineberger, J. E.; Pietrak, B. L.; Espeseth, A. S.; Shi, X. P.; Chen-Dodson, E.; Holloway, M. K.; Munshi, S.; Simon, A. J.; Kuo, L.; Vacca, J. P. Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1). J. Med. Chem. 2004, 47, 6447−6450. (9) Maillard, M. C.; Hom, R. K.; Benson, T. E.; Moon, J. B.; Mamo, S.; Bienkowski, M.; Tomasselli, A. G.; Woods, D. D.; Prince, D. B.; Paddock, D. J.; Emmons, T. L.; Tucker, J. A.; Dappen, M. S.; Brogley, L.; Thorsett, E. D.; Jewett, N.; Sinha, S.; John, V. Design, synthesis, and crystal structure of hydroxyethyl secondary amine-based peptidomimetic inhibitors of human beta-secretase. J. Med. Chem. 2007, 50, 776−781. (10) Kortum, S. W.; Benson, T. E.; Bienkowski, M. J.; Emmons, T. L.; Prince, D. B.; Paddock, D. J.; Tomasselli, A. G.; Moon, J. B.; LaBorde, A.; TenBrink, R. E. Potent and selective isophthalamide S2 hydroxyethylamine inhibitors of BACE1. Bioorg. Med. Chem. Lett. 2007, 17, 3378−3383. (11) Stachel, S. J. Progress toward the development of a viable BACE-1 inhibitor. Drug Dev. Res. 2009, 70, 101−110. (12) Butini, S.; Brogi, S.; Novellino, E.; Campiani, G.; Ghosh, A. K.; Brindisi, M.; Gemma, S. The structural evolution of β-secretase inhibitors: a focus on the development of small-molecule inhibitors. Curr. Top. Med. Chem. (Sharjah, United Arab Emirates) 2013, 13, 1787−1807. (13) Iserloh, U.; Wu, Y.; Cumming, J. N.; Pan, J.; Wang, L. Y.; Stamford, A. W.; Kennedy, M. E.; Kuvelkar, R.; Chen, X.; Parker, E. M.; Strickland, C.; Voigt, J. Potent pyrrolidine- and piperidine-based BACE-1 inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 414−417. (14) Iserloh, U.; Pan, J.; Stamford, A. W.; Kennedy, M. E.; Zhang, Q.; Zhang, L.; Parker, E. M.; McHugh, N. A.; Favreau, L.; Strickland, C.; Voigt, J. Discovery of an orally efficaceous 4-phenoxypyrrolidine-based BACE-1 inhibitor. Bioorg. Med. Chem. Lett. 2008, 18, 418−422. (15) Cumming, J.; Babu, S.; Huang, Y.; Carrol, C.; Chen, X.; Favreau, L.; Greenlee, W.; Guo, T.; Kennedy, M.; Kuvelkar, R.; Le, T.; Li, G.; McHugh, N.; Orth, P.; Ozgur, L.; Parker, E.; Saionz, K.; Stamford, A.; Strickland, C.; Tadesse, D.; Voigt, J.; Zhang, L.; Zhang, Q. Piperazine sulfonamide BACE1 inhibitors: design, synthesis, and in vivo characterization. Bioorg. Med. Chem. Lett. 2010, 20, 2837−2842. 9819



Article

(33) R, C.; Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; J. A. Montgomery, J.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian, Inc.: Wallingford CT, 2010. (34) Deng, W.; Cheeseman, J. R.; Frisch, M. J. Calculation of nuclear spin-spin coupling constants of molecules with first and second row atoms in study of basis set dependence. J. Chem. Theory Comput. 2006, 2, 1028−1037. (35) Yang, H. C.; Chai, X.; Mosior, M.; Kohn, W.; Boggs, L. N.; Erickson, J. A.; McClure, D. B.; Yeh, W. K.; Zhang, L.; GonzalezDeWhitt, P.; Mayer, J. P.; Martin, J. A.; Hu, J.; Chen, S. H.; Bueno, A. B.; Little, S. P.; McCarthy, J. R.; May, P. C. Biochemical and kinetic characterization of BACE1: investigation into the putative speciesspecificity for beta- and beta’-cleavage sites by human and murine BACE1. J. Neurochem. 2004, 91, 1249−1259. (36) May, P. C.; Dean, R. A.; Lowe, S. L.; Martenyi, F.; Sheehan, S. M.; Boggs, L. N.; Monk, S. A.; Mathes, B. M.; Mergott, D. J.; Watson, B. M.; Stout, S. L.; Timm, D. E.; Smith Labell, E.; Gonzales, C. R.; Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.; Calligaro, D. O.; Cocke, P. J.; Greg Hall, D.; Friedrich, S.; Citron, M.; Audia, J. E. Robust central reduction of amyloid-beta in humans with an orally available, non-peptidic beta-secretase inhibitor. J. Neurosci. 2011, 31, 16507−16516. (37) Silvestri, R. Boom in the development of non-peptidic βsecretase (BACE1) inhibitors for the treatment of Alzheimer’s disease. Med. Res. Rev. 2009, 29, 295−338. (38) Rankovic, Z. CNS drug design: balancing physicochemical properties for optimal brain exposure. J. Med. Chem. 2015, 58, 2584− 2608. (39) Hale, J. J.; Mills, S. G.; MacCoss, M.; Shah, S. K.; Qi, H.; Mathre, D. J.; Cascieri, M. A.; Sadowski, S.; Strader, C. D.; MacIntyre, D. E.; Metzger, J. M. 2(S)-((3,5-Bis(trifluoromethyl)benzyl)-oxy)3(S)-phenyl-4- ((3-oxo-1,2,4-triazol-5-yl)methyl)morpholine (1): a potent, orally active, morpholine-based human neurokinin-1 receptor antagonist. J. Med. Chem. 1996, 39, 1760−1762. (40) Thompson, L. A.; Shi, J.; Decicco, C. P.; Tebben, A. J.; Olson, R. E.; Boy, K. M.; Guernon, J. M.; Good, A. C.; Liauw, A.; Zheng, C.; Copeland, R. A.; Combs, A. P.; Trainor, G. L.; Camac, D. M.; Muckelbauer, J. K.; Lentz, K. A.; Grace, J. E.; Burton, C. R.; Toyn, J. H.; Barten, D. M.; Marcinkeviciene, J.; Meredith, J. E.; Albright, C. F.; Macor, J. E. Synthesis and in vivo evaluation of cyclic diaminopropane BACE-1 inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 6909−6915. (41) Boy, K. M.; Guernon, J. M.; Shi, J.; Toyn, J. H.; Meredith, J. E.; Barten, D. M.; Burton, C. R.; Albright, C. F.; Marcinkeviciene, J.; Good, A. C.; Tebben, A. J.; Muckelbauer, J. K.; Camac, D. M.; Lentz, K. A.; Bronson, J. J.; Olson, R. E.; Macor, J. E.; Thompson, L. A. 3rd. Monosubstituted gamma-lactam and conformationally constrained 1,3diaminopropan-2-ol transition-state isostere inhibitors of betasecretase (BACE). Bioorg. Med. Chem. Lett. 2011, 21, 6916−6924. (42) Eliel, E.; Wilen, S. H. Stereochemistry of Organic Compounds; John Wiley & Sons, Inc: Hoboken, NJ. 1994; pp 758−762. (43) Juaristi, E.; Cuevas, G. Recent studies of the anomeric effect. Tetrahedron 1992, 48, 5019−5087. (44) Weiss, M. M.; Williamson, T.; Babu-Khan, S.; Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.; Dineen, T. A.; Esmay, J.; Graceffa, R. F.; Harried, S. S.; Hickman, D.; Hitchcock, S. A.; Horne, D. B.; Huang, H.; Imbeah-Ampiah, R.; Judd,

T.; Kaller, M. R.; Kreiman, C. R.; La, D. S.; Li, V.; Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.; Pennington, L. D.; Rattan, C.; San Miguel, T.; Sickmier, E. A.; Wahl, R. C.; Wen, P. H.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V. F.; Zhong, W. Design and preparation of a potent series of hydroxyethylamine containing beta-secretase inhibitors that demonstrate robust reduction of central beta-amyloid. J. Med. Chem. 2012, 55, 9009−9024. (45) Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.; Zhang, X. C.; Tang, J. Structure of the protease domain of memapsin 2 (β-secretase) complexed with inhibitor. Science 2000, 290, 150−153.

9820


Optimization of Hydroxyethylamine Transition State Isosteres as

Recommend Documents