Rational design and synthesis of aminopiperazinones as β-secretase (BACE) inhibitors

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7255–7260

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Rational design and synthesis of aminopiperazinones as b-secretase (BACE) inhibitors Gary Tresadern a,⇑, Francisca Delgado b, Oscar Delgado b, Harrie Gijsen c, Gregor J. Macdonald c, Dieder Moechars d, Frederik Rombouts c, Richard Alexander e, John Spurlino e, Michiel Van Gool b, Juan Antonio Vega b, Andrés A. Trabanco b,⇑ a

Research Informatics & Integrative Genomics, Calle Jarama 75, Poligono Industrial, Toledo 45007, Spain Neuroscience Medicinal Chemistry, Janssen Pharmaceutical Research & Development, Calle Jarama 75, Poligono Industrial, Toledo 45007, Spain Neuroscience Medicinal Chemistry, Janssen Pharmaceutica N.V., Turnhoutsweg 30, B-2340 Beerse, Belgium d Neuroscience Biology, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutsweg 30, B-2340 Beerse, Belgium e Structural Biology, Janssen Research & Development, Welsh and McKean Rd., Spring House, PA 19477,USA b c

a r t i c l e

i n f o

Article history: Received 22 September 2011 Revised 13 October 2011 Accepted 14 October 2011 Available online 20 October 2011 Keywords: BACE Alzheimer’s disease Amyloid b-Secretase inhibitor Neurodegeneration Amyloid beta 42 (Ab42) Memapsin

a b s t r a c t Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral separation resulted in the 40 nM inhibitor, ( )-37, which showed in vivo reduction of amyloid beta peptides. The crystal structure of 18a showed a binding mode driven by interaction with the catalytic aspartate dyad and distribution of the biaryl amide decoration towards S1 and S3 pockets. Ó 2011 Elsevier Ltd. All rights reserved.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. The societal burden of AD is vast: the cost of treatment, care and loss of productivity is estimated at US$300 billion per year; in 2010 there were 454000 new cases of Alzheimer’s in the US alone and the number of people with dementia is expected to double in the next 20 years.1 Current treatment with either acetylcholinesterase inhibitors or N-methyl D-aspartate (NMDA) antagonists is mainly symptomatic and does not cure or reverse progression of the disease.2 The cause of AD is unclear but Tau fibrils and amyloid beta (Ab) deposits are characteristic neuropathological hallmarks and may be associated with disease pathogenesis.3 The insoluble plaques are predominantly aggregates of Ab peptides of 39–43 amino acids formed via the sequential cleavage of b-amyloid precursor protein (APP) by aspartyl proteases, b- and c-secretase.4 The inhibition of b-secretase (BACE, b-site APP cleaving enzyme) may

⇑ Corresponding authors. Tel.: +34 925 245782 (G.T.); tel.: +34 925 245792 (A.A.T.). E-mail addresses: [email protected] (G. Tresadern), [email protected] (A.A. Trabanco). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.10.050

therefore represent a potential disease modifying treatment for AD.5 Despite the attractiveness of attenuating BACE mediated APP proteolysis there have been difficulties identifying low molecular weight, cell and brain penetrant BACE inhibitors.6 Early BACE inhibitors were high affinity peptidomimetics such as OM99-2 (1)7 (Fig. 1) which has been crystallized with BACE.8 Lower MW series were subsequently identified such as isonicotinamides (2)9 and hydroxy ethylamines (3).10 Lead optimization improved cellular activity and P-glycoprotein susceptibility,11 however, they retained traits of their peptidic origin and were large, flexible and polar. X-ray structures revealed these molecules form interactions between the amino-alcohol group and the aspartic acid dyad in the enzyme active site, Figure 2.12 Meanwhile, series containing amidino and guanidino substructures were identified which interact with the same aspartic acids. Acylguanidines (4)13, aminoimidazoles (5 and 6)14,15 as well as 2-amino-3,4-dihydroquinazolines (7) from our laboratories16 were all reported. In addition, aminopyrimidone hits were found from NMR fragment screening which resulted in the attractive CNS lead, 8.17 BACE functions optimally at acidic pH and is believed to be located in acidic intracellular compartments, pH 5. At such pH amidino and guanidino motifs

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O H 2N

O

H N

N H

O N H

O

OH

O

H N

N H

OH

N H

O

O

O O

H 2N

OH

O

OH

1, OM99-2 O S O N

NH2

O

O S O N

N H

N

O N H

OH

NH

CF3

N H

NH 2

3 O NH2

N

N

O 2N

NH2

N

O

N

N

N O 4

N

NH2

NH2

F 5

O

6

O

O N N

N

N NH2

H2N

OMe

N

7

8 Figure 1. BACE inhibitors 1–8.

H O

+

O

N NH

O

H O

O Asp32

H

+

O

N H

N H

Asp228

O

O

O

O Asp228

N H

9 2IQG). Docking used the GOLD software (version 4.11)21 with slow ‘most accurate’ approach, the GoldScore fitness function and ten genetic algorithm runs per ligand. Protein structures were prepared in MOE,22 the protonate 3D tool was used to ionise amino acids, hydrogens were added, charges calculated, and the ligand removed. A 14 Å radius around Asp228 (a-carbon) was used and all waters were retained. There is debate about the ionisation state of Asp32 and Asp228 in the active site.23,24 Here, both acids were deprotonated which delivered good results for re-docking of the crystallized ligands and avoided the need for placement restraints which enforce ligand-protein interactions. Inhibitors were ionised according to their calculated pKa at pH 5 with the ACD25 software.26 This program performs well compared to other approaches.27 A 3D conformation with correct chirality was generated with MMFF94x force field minimisation in MOE and used as input for docking. The best ranked docking solutions compared to the experimental structures, for each of the five protein ligand complexes are shown in Figure 3. The protocol performed well at predicting the binding mode of the distinct chemical series when docked into their accompanying protein structure. The BACE active site is flexible and adopts different conformations depending on the inhibitor.28,29 Docking performance is dependent on the protein structure used.30 Using a protein structure solved with an inhibitor from a significantly different structural class is problematic as most approaches are unable to predict the required enzyme flexibility. For the assessment of our proposed ideas this was overcome by using different enzyme structures for the virtual screening. Given that most ideas were either amidino/guanidino heterocycles or were similar to our 2amino-3,4-dihydroquinazolines the 2Q11 and 2VA7 crystal structures were both used. The ideas were small scaffold-like motifs and needed to be converted into virtual molecules in a consistent way to allow fair comparison of each. Enumeration with sidechains such as biphenyl, bisarylamide and biphenethyl, as seen in 8, generated over 300 virtual molecules of similar size and decoration.

Asp32

Figure 2. Schematic view of interaction between hydroxy ethylamine and amidino/ guanidino substructures and the catalytic aspartic acid dyad in the BACE active site.

are protonated and form a salt bridge interaction to the aspartate as well as a sophisticated series of hydrogen bonds, Figure 2. More recently there have been further reports of aminohydantoins18 and aminopyridines19 which also adopt a similar binding mode. We pursued a strategy to design new amidino and guanidino containing heterocycles with the potential to bind at the aspartic acid dyad in the BACE active site. It had been recognized that some molecules from our series of lipophilic 2-amino-3,4-dihydroquinazolines had a particularly high basic pKa. An experimental pKa of 10.6 was measured for one example and was typical of other compounds in the series. The combination of high lipophilicity and high basicity is detrimental for a CNS lead.20 Therefore, given the need for cellular penetration and CNS activity we sought to target new species with lower basicity. De-novo ideas were generated and in silico virtual screening with docking and pKa calculations was used for prioritization. Firstly a docking protocol was validated which could be used to assess and prioritize ideas. The aim was to reproduce five experimental ligand-BACE complexes (5 3H0B, 6 3IGB, 7 2Q11, 8 2VA7,

5, 3H0B

6, 3IGB

7, 2Q11

8, 2VA7

N O F O

HN F

OH

N H

I

9

9, 2IQG Figure 3. Docking validation performed by re-docking known inhibitors into their corresponding BACE X-ray structures. Docked pose is shown in atom-type coloring and original X-ray coordinates are in magenta.

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Docking of the virtual molecules was performed into both crystal structures and the best scoring were inspected by eye. Piperazinone 10 was amongst the best solutions from docking into the 2VA7 structure, Figure 4. The amidine of the piperazinone formed the salt bridge interaction and the H-bond network with the aspartate dyad. The trial molecule contained a biaryl amide at a quaternary centre. The NH of the amide formed an H-bond interaction with the backbone carbonyl of Gly230. This interaction was both attractive to target and plausible as it had been seen in a series of non-aspartate inhibitors.31 The calculated basic pKa with ACD was predicted to be 6.3 or 8.0 depending on the tautomeric form of the amidine. Either would be mainly protonated at pH 5 whilst being less basic than our previous series. Synthesis was initiated to target similar piperazinone molecules, pursuing two regioisomers (Type I and II) of the piperazinone with biaryl amide decoration at the chiral centre. The synthesis of the final 5-amino-3,6-dihydro-1H-pyrazin-2one derivatives (Type I) is depicted in Scheme 1. The a-aminonitrile 12 was prepared from the corresponding 1-(3-nitro-phenyl)ethanone (11) in 82% yield by the Strecker reaction. Hydrolysis of the nitrile with hydrochloric acid followed by esterification gave the a-aminoester 13 which was converted into the piperazine2,5-dione 14 by treatment with 2-chloroacetylchloride and subsequent cyclization by reaction with methylamine. Setup of the amidine moiety was done via conversion of amide to thioamide, followed by treatment with NH3. The thioamide intermediate 15 was prepared by reaction of 14 with Lawesson’s reagent in toluene at 90 °C in the presence of base. The use of pyridine as a base was crucial for the regioselectivity in the reaction, otherwise mixtures of products were obtained. Transformation of the thioamide 15 into amidine 16 using a mixture of NH3 (7 N in MeOH) and NH3 (33% aqueous) and subsequent hydrogenation of the nitro group in the phenyl ring gave the key intermediate 17. Finally, reaction of 17 with a selected set of carboxylic acids using HATU and PhN(CH3)2, in dry CH2Cl2 gave compounds 18a–18d.32 Type II inhibitors, 3-amino-5,6-dihydro-1H-pyrazin-2-one derivatives, were prepared following the reaction sequences showed in Schemes 2 and 3. Reduction of a-aminoester 13 with NaBH4 followed by reaction with (Boc)2O and subsequent oxidation of the hydroxyl group with Dess–Martin periodinane furnished aldehyde 20. Next, the N-methyl group was introduced by reductive amination to give intermediate 21. Acylation with ethyl oxalyl chloride and subsequent deprotection of the N-Boc amino group gave piperazine-2,3-dione 22 by in situ cyclization of the aminoester intermediate. The conversion of amide 22 to amidine 24 via a thioamide intermediate, analogous to synthesis of 16, proved problematic. Alternatively, the piperazine-dione 22 was O-methylated using Me3OBF4 to afford 23 which was converted to amidine 24 after treatment with NH3 in the presence of NH4Cl.

O

O

CN

H2N

(iv), (v)

(ii), (iii)

NO2

NO2

NO2

11

12

13

S

N

(vi) HN

H2N

(vii) O

N

14

N N

O

O

NH2 17

16

O 18a: X = CH, R = Cl 18b: X = CH, R = CN 18c: X = CH, R = CF 3 18d: X = N; R = Me

N

H2N

O

H2N

(viii)

NO2 15

N

HN

NO2

N N

NO2

(ix)

O

CO2Me

H2N

(i)

O N

N H

X

R

Scheme 1. Reagents and conditions: (i) TMSCN, NH4Cl, NH3 (7 N in MeOH), rt, 4 days, 82%; (ii) HCl (6 N), reflux, 18 h, 63%; (iii) SOCl2, MeOH, 0 °C, 15 min, then reflux, 18 h, 63%; (iv) 2-chloroacetylchloride, Et3N, CH2Cl2, 0° C, 1 h, 99%; (v) NH2Me (33% in EtOH), EtOH, 70 °C, 3 h, 99%; (vi) Lawesson’s reagent, pyridine, toluene, 90 °C, 18 h, 16%; (vii) NH3 (7 N in MeOH), NH3 (33% aqueous), 67 °C, 4 h, 37%; (viii) H2, Pd/C, EtOH/AcOEt, rt, 12 h, 99%; (ix) ArCOOH, HATU, PhN(CH3)2, CH2Cl2, rt, 3 h, 30–67%.

O HO H2N

HN

(i)

O

O CHO

(ii), (iii)

HN (iv)

O N H

(v), (vi)

13 NO2

NO2

19

20

O O

21 O

O O

N

N

(vii)

NO2

H2N

N N

H2N

N N

NO2

NO2

NH2 24

23 O

(ix)

N

22

H2N

O N

(viii)

N

HN

(x)

NO2

25

O N H 26

N Cl

Scheme 2. Reagents and conditions: (i) NaBH4, EtOH, rt, 3 h, 87%; (ii) Boc2O, NaHCO3, THF, 0 °C to rt, 99%; (iii) Dess-Martin periodinane, CH2Cl2, 0 °C to rt, 1 h, 95%; (iv) (a) MeNH2 (2 M in THF), CH2Cl2/AcOH, rt, 1 h; (b) NaBH(OAc)3, rt, 2 h, 95%; (v) ethyl oxalyl chloride, DIPEA, CH2Cl2, 0° C, 3 h, 98%; (vi) HCl (4 M in 1,4-dioxane), rt, 1 h, 83%; (vii) Me3OBF4, CH2Cl2, rt, 3 days, 79%; (viii) NH4Cl, NH3 (2 M in EtOH), 75 °C, 18 h, 49%; (ix) H2, Pd/C, EtOH/AcOEt, rt, 12 h, 98%; (x) ArCOOH, HATU, PhN(CH3)2, CH2Cl2, rt, 3 h, 52%.

N O

N N H

H2N 10

H2N

N

O

O

N

R

H2N

Type I

N N

R

Type II

Figure 4. Docked pose for piperazinone idea molecule 10 docked into the BACE crystal structure 2VA7.

Hydrogenation of the nitro group to the aniline 25 followed by acylation gave the final product 26. 3-Amino-5,6-dihydro-1H-pyrazin-2-one derivatives bearing a fluorine atom in 2 position of the phenyl ring were prepared starting from 1-(5-bromo-2-fluoro-phenyl)-ethanone 27, Scheme 3. A bromo substituent instead of nitro as used in Schemes 1 and 2 was chosen to avoid SNAr type side-reactions expected with the use of the highly electron deficient 1-(5-nitro-2-fluoro-phenyl)ethanone. Commercially available 27 was transformed into

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G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260 HO O

Br

(i)

CN

H2N

Br

Br

(ii), (iii)

F

H2N

Br

(iv)

F

27

CO2Me

H2N

F

F

28

29

30 O

O

CHO

HN (v), (vi)

O

O

Br

HN (vii)

N H (viii), (ix)

Br

31

F 32

N

H2N

(xi)

N

Br

O

33

O

O (x)

N

HN

Br

F

F

O

O

O

O N

(xii)

N

Br

H2N H2N

F

F

34

35

N N

F 36

N

(xiii) H2N

F

N O N H

N

37

Cl

Scheme 3. Reagents and conditions: (i) TMSCN, NH4Cl, NH3 (7 N in MeOH), rt, 4 days, 98%; (ii) HCl (6 N), reflux, 18 h, 62%; (iii) H2SO4 (conc.), MeOH, reflux, 48 h, 95%; (iv) NaBH4, EtOH, rt, 3 h, 87%; (v) Boc2O, NaHCO3, THF, 0 °C, 15 h, quant. (vi) Dess–Martin periodinane, CH2Cl2, 0 °C to rt, 1 h, 83%; (vii) (a) MeNH2 (2 M in THF), CH2Cl2/AcOH, rt, 1 h; (b) NaBH(OAc)3, rt, 2 h, 64%; (viii) ethyl oxalyl chloride, DIPEA, CH2Cl2, 0° C, 3 h, 93%; (ix) HCl (4 M in 1,4-dioxane), rt, 1 h, 99%; (x) Me3OBF4, CH2Cl2, rt, 3 days, 73%; (xi) NH4Cl, NH3 (2 M in EtOH), 75 °C, 18 h, 55%; (xii) NaN3, CuI, Na2CO3, DMSO, N,N’-dimethylethylenediamine, 110 °C, 1 h, 95%; (ix) DMTMM, MeOH, 0 °C, 3 h, 25%.

Table 1 BACE inhibitory activity of aminopiperazinonesa Compds

18a (S)-(+)-18a (R)-( )-18a 18b 18c 18d 26 (+)-26 ( )-26 (+)-37 ( )-37 a

Enzymatic

Cellular

BACE IC50(lM)a

hAb42 IC50(lM)a

hAbTOT IC50(lM)a

0.447 >30 0.324 0.813 1.380 7.079 0.550 >30 0.295 >30 0.040

0.098 >10 0.036 0.186 0.068 1.259 0.115 >10 0.083 >10 0.028

0.078 >10 0.037 0.151 0.079 1.148 0.107 >10 0.085 >10 0.025

expected lower pKa of this cyclic amidine. Chiral separation of 18a revealed ( )-18a to be the active stereoisomer. Molecules 18b, 18c and 18d show some SAR for variation in the distal aryl on the amide. More polar aryls, cyano pyridyl 18b and methyl pyrazine 18d were less active compared to chloro and CF3 pyridyls 18a and 18c respectively. In the Type II aminopiperazinones, 26 had comparable decoration to 18a and showed similar activity, 0.55 and 0.107 lM in enzymatic and cellular hAbTOT assays respectively. The experimental basic pKa of 26 was 7.6. Again chiral separation revealed one stereoisomer to be responsible for the activity, ( )26 0.295 and 0.085 lM in enzymatic and cellular hAbTOT assays. Interestingly, molecule 37 demonstrates that introduction of fluorine on the anilinic phenyl results in improved activity, 0.04 and 0.025 lM in the enzymatic and cellular assays. The crystal structure of ( )-18a with BACE was solved at 2.2 Å resolution, Figure 5. The resultant binding mode was similar to that predicted from the docking virtual screening. The piperazinone amidine interacts with the catalytic aspartate dyad as expected and the biaryl amide is directed to sub-pockets S1 and S3. The amide NH is 2.3 Å from the carbonyl oxygen of Gly230 and forms a favorable interaction although it is not as well aligned as expected from the docking. The chloro substituent on the distal pyridyl extends deep into the S3 pocket and contributes to affinity. The X-ray structure confirmed the stereochemistry of ( )-18a to be R as predicted by the docking studies. Molecules 18a, 26 and ( )-37 were assessed for their ability to reach the brain. A 30 mg/kg dose of 18a and 26 was administered subcutaneously (sc) to mice, and brain and plasma levels were measured at 1, 2 and 4 h. Although 18a displayed high plasma levels, 4633, 1712 and 439 ng/ml at 1, 2 and 4 h respectively, the corresponding brain levels were low, 184, 123 and 90 ng/g. Molecule 26 showed plasma levels of 993, 461 and 200 ng/ml and brain levels of 614, 157 and 20.5 ng/g. A higher 60 mg/kg sc dose of ( )-37 resulted in plasma levels of 7145, 2455 and 270 ng/ml at 1, 2 and 4 h, which in turn led to relatively high brain exposure 8078, 3342 and 347 ng/g. These molecules showed a rapid decrease in plasma concentration levels, likely due to low metabolic stability.35 The three similar molecules had calculated log P of 1.7, 2.3 and 2.5 for 18a, 26 and ( )-37 respectively. 18a and 26 had comparable experimental basic pKa, 7.8 and 7.6. The trend in brain exposure may be driven by gradual increase in log P, but remains to be understood in more detail.

Data presented from enzymatic and cellular assays.37

amidine 35 following a reaction sequence identical to Scheme 2. Copper catalyzed amination of the bromoderivative 35 led to intermediate 36 in excellent yield. Finally, selective acylation of the aniline 36 with the corresponding carboxylic acid was achieved using DMTMM33 as coupling agent in MeOH to afford the final product 37. All synthesized inhibitors34 were screened against BACE to determine IC50 values in both enzymatic and cellular assays35, Table 1. For the type I aminopiperazinones the first example, racemate 18a, showed 0.447 and 0.078 lM activity in the enzymatic and cellular hAbTOT assays respectively. There were no difficulties with cell permeation as this and other compounds in the series all show good cellular activity. The basic pKa of racemate 18a was experimentally determined as 7.8. This is acceptable for a CNS lead and commensurate with the calculated value and

Figure 5. Crystal structure of ( )-18a with BACE (pdb ID code: 3U6A). Key interactions between the ligand and Asp32 and Asp228 are shown as well as the location of Tyr71 in the active site flap.

G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260

Molecules 26 and ( )-37 were tested for their ability to reduce Ab peptides in vivo.36 A 30 mg/kg sc dose of 26 resulted in a 34% reduction in AbTOT levels at 1 h, with no effect seen at 2 and 4 h due to the low amounts in brain. The higher brain levels of ( )37 following the 60 mg/kg sc dose resulted in a more pronounced reduction of AbTOT by 48%, 67% and 73% at 1, 2 and 4 h respectively. The results demonstrate that in vivo reduction of Ab peptides is tractable for this series. Overall, aminopiperazinones show moderate basicity (pKa <8.0), relatively low lipophilicity, (log P 2.5 for ( )-37) and low MW (390 for ( )-37). The series demonstrate good cellular activity and examples show in vivo reduction of Ab peptides following subcutaneous administration. Future work will focus on reducing clearance by improvement of metabolic stability. Acknowledgments The authors thank Daniele Bemporad for input in the early project and Jeroen Van De Ven and Geert Van Hecke for generating the in vitro data. Crystals were grown and soaked by Shanghai Medicilon, Inc. and Medicilon Preclinical Research (Shanghai) LLC. The Xray experiments were performed on the PX1 BEAMLINE at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. Data were collected by Expose GmbH. References and notes 1. See (a) Brody, H. Nature 2011, 475, Suppl, S1. (b) Abbott, A. Nature 2011, 475, Suppl, S2–S4. The global impact of dementias was estimated at US$604 billion per year, Alzheimer’s constitutes 50–80% of dementia cases hence a US$300 billion estimate for global Alzheimer’s burden. 2. (a) Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurology 2003, 2, 539; (b) Standridge, J. B. Clinical Therap. 2004, 26, 615; (c) Citron, M. Nat. Rev. Neurosci. 2004, 5, 677. 3. (a) Dickson, D. W. J. Neuropathol. Exp. Neurol. 1997, 56, 321; (b) Hardy, J.; Selkoe, D. J. Science 2002, 5580, 353; (c) Selkoe, D. J.; Schenk, D. Annu. Rev. Pharmacol. Toxicol. 2003, 43, 545. 4. (a) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.C.; Collins, F.; Treanor, J.; Rogers, G.; Citron, M. Science 1999, 286, 735; (b) Wolfe, M. S.; Xia, W.; Ostaszewski, B. L.; Diehl, T. S.; Kimberly, W. T.; Selkoe, D. J. Nature 1999, 398, 513; (c) Vassar, R.; Citron, M. Neuron 2000, 27, 419; (d) Selkoe, D. J. Physiol. Rev. 2001, 81, 741. 5. De Strooper, B.; Vassar, R.; Golde, T. Nature Rev. Neurol. 2010, 6, 99. 6. Albert, J. S. Progress Med. Chem. 2009, 48, 133. 7. Ghosh, A. K.; Shin, D.; Downs, D.; Koelsch, G.; Lin, X.; Ermolieff, J.; Tang, J. J. Am. Chem. Soc. 2000, 122, 3522. 8. Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.; Zhang, X. C.; Tang, J. Science 2000, 290, 150. 9. Stauffer, S. R.; Stanton, M. G.; Gregro, A. R.; Steinbeiser, M. A.; Shaffer, J. R.; Nantermet, P. G.; Barrow, J. C.; Rittle, K. E.; Collusi, D.; Espeseth, A. S.; Lai, M.-T.; Pietrak, B. L.; Holloway, M. K.; McGaughey, G. B.; Munshi, S. K.; Hochman, J. H.; Simon, A. J.; Selnick, H. G.; Graham, S. L.; Vacca, J. P. Bioorg. Med. Chem. Lett. 2007, 17, 1788. 10. Iserloh, U.; Cumming, J. N. In Aspartic Acid Proteases as Therapeutic Targets; Ghosh, A. K., Ed.; Wiley-VCH: Weinheim, 2010; p 441. chapter 16. 11. Nantermet, P. G.; Rajapakse, H. A.; Stanton, M. G.; Stauffer, S. R.; Barrow, J. C.; Gregro, A. R.; Moore, K. P.; Steinbeiser, M. A.; Swestock, J.; Selnick, H. G.; Graham, S. L.; McGaughey, G. B.; Colussi, D.; Lai, M.-T.; Sankaranarayanan, S.; Simon, A. J.; Munshi, S.; Cook, J. J.; Holahan, M. A.; Michener, M. S.; Vacca, J. P. ChemMedChem 2009, 4, 37. 12. 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.; Varghese, J. J. Med. Chem. 2007, 50, 776. 13. Cole, D. C.; Manas, E. S.; Stock, J. R.; Condon, J. S.; Jennings, L. D.; Aulabaugh, A.; Chopra, R.; Cowling, R.; Ellingboe, J. W.; Fan, K. Y.; Harrison, B. L.; Hu, Y.; Jacobsen, S.; Jin, G.; Lin, L.; Lovering, F. E.; Malamas, M. S.; Stahl, M. L.; Strand, J.; Sukhdeo, M. N.; Svenson, K.; Turner, M. J.; Wagner, F.; Wu, J.; Zhou, P.; Bard, J. J. Med. Chem. 2006, 49, 6158. 14. Stachel, S. J.; Coburn, C. A.; Rush, D.; Jones, K. L. G.; Zhu, H.; Rajapakse, H.; Graham, S. L.; Simon, A.; Holloway, M. K.; Allison, T. J.; Munshi, S. K.; Espeseth, A. S.; Zuck, P.; Colussi, D.; Wolfe, A.; Pietrak, B. L.; Lai, M. –T.; Vacca, J. P. Bioorg. Med. Chem. Lett. 2009, 19, 2977. 15. Malamas, M. S.; Erdei, J.; Gunawan, I.; Barnes, K.; Johnson, M.; Hui, Y.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.; Olland, A.; Bard, J.; Robichaud, A. J. J. Med. Chem. 2009, 52, 6314.

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Chem. 2010, 53, 1146; (b) Zhou, P.; Li, Y.; Fan, Y.; Wang, Z.; Chopra, R.; Olland, A.; Hu, Y.; Magolda, R. L.; Pangalos, M.; Reinhart, P. H.; Turner, M. J.; Bard, J.; Malamas, M. S.; Robichaud, A. J. Bioorg. Med. Chem. Lett. 2010, 20, 2326. 19. (a) Malamas, M. S.; Barnes, K.; Hui, Y.; Johnson, M.; Lovering, F.; Condon, J.; Fobare, W.; Solvibile, W.; Turner, J.; Hu, Y.; Manas, E. S.; Fan, K.; Olland, A.; Chopra, R.; Bard, J.; Pangalos, M. N.; Reinhart, P.; Robichaud, A. J. Bioorg. Med. Chem. Lett. 2010, 20, 2068; (b) Cheng, Y.; Judd, T. C.; Bartberger, M. D.; Brown, J.; Chen, K.; Fremeau, R. T.; Hickman, D.; Hitchcock, S. A.; Jordan, B.; Li, V.; Lopez, P.; Louie, S. W.; Luo, Y.; Michelsen, K.; Nixey, T.; Powers, T. S.; Rattan, C.; Sickmier, E. A.; St. Jean, D. J.; Wahl, R. C.; Wen, P. H.; Wood, S. J. Med. Chem. 2011, 54, 5836. 20. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.; Villalobos, A.; Will, Y. ACS Chem. Neurosci. 2010, 1, 420. 21. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins 2003, 52, 609. 22. Chemical Computing Group Inc. Molecular Operating Environment (MOE), 1010 Sherbrooke St. W, Suite 910, Montreal, QC, Canada; available from Chemical Computing Group Inc. at http://www.chemcomp.com. 2010. 23. Yu, N.; Hayik, S. A.; Wang, B.; Liao, N.; Reynolds, C. H.; Merz, K. M. J. Chem. Theory Comput. 2006, 2, 1057. 24. Dominguez, J. L.; Christopeit, T.; Villaverde, M. C.; Gossas, T.; Otero, J. M.; Nystrom, S.; Baraznenok, V.; Lindstrom, E.; Danielson, U. H.; Sussman, F. Biochemistry 2010, 49, 7255. 25. ACD Labs software. pKa. Advanced chemistry development Inc. 110 Yonge Street, 14th Floor, Toronto, Ontario, Canada, M5C 1T4. http:// www.acdlabs.com/home/. 26. The only modification to the ACD pKa prediction was for molecule 6 (3IGB), where the two cyclic sp2 nitrogens were predicted to have very similar pKa’s. The sp2 nitrogen in the 6-membered ring had a calculated basic pKa of 7.2 and the imidazole nitrogen 6.9. It is known that the amidine motif forms the salt bridge interaction with the active site aspartic acids therefore the imidazole nitrogen was protonated. 27. Liao, C.; Nicklaus, M. C. J. Chem. Inf. Model. 2009, 49, 2801. 28. Gorfe, A. A.; Caflisch, A. Structure 2005, 13, 1487. 29. McGaughey, G. B.; Colussi, D.; Graham, S. L.; Lai, M.-T.; Munshi, S. K.; Nantermet, P. G.; Pietrak, B.; Rajapakse, H. A.; Selnick, H. G.; Stauffer, S. R.; Holloway, M. K. Bioorg. Med. Chem. Lett. 2007, 17, 1117. 30. Craig, I. R.; Essex, J. W.; Spiegel, K. J. Chem. Inf. Model. 2010, 50, 511. 31. Steele, T. G.; Hills, I. D.; Nomland, A. A.; de Leon, P.; Allison, T.; McGaughey, G.; Colussi, D.; Tugusheva, K.; Haugabook, S. J.; Espeseth, A. S.; Zuck, P.; Graham, S. L.; Stachel, S. J. Bioorg. Med. Chem. Lett. 2009, 19, 17. 32. When the reaction was performed using diisopropylethylamine as base a diacylation product was observed in all cases (30–40%). 33. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. 34. Racemic compounds 18a, 26 and 37 were separated into their corresponding enantiomers by chiral SFC (CHIRALPAK AD-H 5 lm 250  20 mm). 35. Molecules 26 and ( )-37 were 99% and 100% metabolised respectively after 15 min incubation with mouse liver microsomes at 1 lM concentration. 36. For in vivo Ab peptide levels detection mice were analyzed. In brief mice (CD1, male, 22-28gr) treated with the compounds were examined and compared to those untreated or treated with vehicle. Compounds were formulated in 20% hydroxypropyl b cyclodextrin and administered as a single subcutaneous dose to overnight fasted animals. After the indicated time animals were sacrificed and the left hemisphere was resuspended in 8 volumes of 0.4% DEA (diethylamine)/50 mM NaCl containing protease inhibitors (Roche11873580001 or 04693159001) per gram of tissue. All samples were homogenized in the FastPrep-24 system (MP Biomedicals) using lysing matrix D (MPBio #6913-100) at 6 m/s for 20 seconds. Homogenates were centrifuged at 221.300g for 50 min. The resulting high speed supernatants were then transferred to fresh eppendorf tubes. Nine parts of supernatant were neutralized with 1 part 0.5 M Tris–HCl pH 6.8 and used to determine Ab levels. To quantify the amount of AbTOT in the soluble fraction of the brain homogenates, ELIZA was used. Briefly, the standards (a dilution of synthetic Ab1-40, Bachem) were prepared with final concentrations ranging from 10000 to 0.3 pg/ml. The samples and standards were co-incubated with the biotinylated mid-domain antibody 4G8. 50 ll of conjugate/sample or conjugate/standards mixtures were then added to the antibody-coated plate (antibody JRF/rAb/2). The plate was allowed to incubate overnight at 4 °C in order to allow formation of the antibody-amyloid complex. A Streptavidine– Peroxidase-Conjugate was added, followed 60 min later by an additional wash step and addition of Quanta Blu fluorogenic peroxidase substrate according to the manufacturer’s instructions (Pierce Corp., Rockford, Il). A reading was performed after 10–15 min (excitation 320 nm /emission 420 nm).

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37. Enzymatic assay: BACE1 was assayed fluorimetrically on Monaco Safas spektrofluorometer flx. The wavelength for excitation was 328 nm and for emission 400 nm. The reactions were followed at 25 °C over 20 min. A human recombinant BACE1 (Enzo Life Sciences, Lörrach, Germany) stock solution of 500 lg/mL was diluted 1:10 with assay buffer (20 mM sodium acetate pH 4.5, 0.1% CHAPS, 0.1% Top Block). Inhibitor stock solutions were prepared with DMSO. A 1 mM stock solution of the substrate Mca-Ser-Glu-Val-Asn-Leu-AspAla-Glu-Phe-Lys(Dnp)-OH (Bachem, Bubendorf, Switzerland) was diluted 1:10 with 50% DMSO and 1:3 with ammonium acetate buffer (10 mM, pH 7.4). The final concentration of DMSO was 2.4%, and the final concentration of the substrate was 2.5 lM. Assays were performed with a final concentration of 0.5 lg/mL of BACE1. In a cuvette 905 lL assay buffer, inhibitor solution and DMSO in a total volume of 10 lL and 10 lL BACE1 solution were added, thoroughly mixed and incubated for 1 h at 25 °C. The reaction was initiated by adding 75 lL of the substrate. Experiments were performed in duplicate with

five different inhibitor concentrations and IC50 values obtained. Cellular Assay: In an alisa assays the levels of AbTOT produced and secreted into the medium of human neuroblastoma SKNBE2 cells were quantified. The assay is based on the human neuroblastoma SKNBE2 expressing the wild type Amyloid Precursor Protein (hAPP695). The compounds were diluted and added to the cells, incubated for 18 h and then AbTOT was measured by sandwich alisa. alisa is a sandwich assay using biotinylated antibody AbN/25 attached to streptavidin coated beads and antibody Ab4G8 for the detection of AbTOT. In the presence of AbTOT, the beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in light emission. Light emission is measured after 1 hour incubation (excitation at 650 nm and emission at 615 nm). A best-fit curve was fitted to the plot of % Controlmin versus compound concentration and an IC50 value was obtained.