Structure-based design, synthesis, and memapsin 2 (BACE) inhibitory activity of carbocyclic and heterocyclic peptidomimetics

J. Med. Chem. 2005, 48, 5175-5190

5175

Structure-Based Design, Synthesis, and Memapsin 2 (BACE) Inhibitory Activity

of Carbocyclic and Heterocyclic Peptidomimetics

Stephen Hanessian,*^,†Hongying Yun,^†Yihua Hou,^†Gaoqiang Yang,^†Malken Bayrakdarian,^†Eric Therrien,^†

Nicolas Moitessier,^§Silvio Roggo,^‡Siem Veenstra,^‡Marina Tintelnot-Blomley,^‡Jean-Michel Rondeau,^‡

Christian Ostermeier,^‡Andre´ Strauss,^‡Paul Ramage,^‡Paolo Paganetti,^‡Ulf Neumann,^‡and Claudia Betschart^‡

Department of Chemistry, Universite´ de Montre´al, C. P. 6128, Succursale Centre-Ville, Montre´al, P. Q., Canada, H3C 3J7, and

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Postfach, CH-4002 Basel, Switzerland

Received February 14, 2005

Molecular modeling based on the X-ray crystal structure of the Tang-Ghosh heptapeptide

inhibitor 1 (OM99-2) of BACE led to the design and synthesis of a series of constrained P₁′

analogues. A cyclopentane ring was incorporated in 1 spanning the P₁′ Ala methyl group and

the adjacent methylene carbon atom of the chain. Progressive truncation at the P₂′-P₄′ sites

led to a potent truncated analogue 5 with good selectivity over Cathepsin D. Using the same

backbone replacement concept, a series of cyclopentane, cyclopentanone, tetrahydrofuran,

pyrrolidine, and pyrrolidinone analogues were synthesized with considerable variation at the

P and P′ sites. The cyclopentanone and 2-pyrrolidinone analogues 45 and 57 showed low nM

BACE inhibition. X-ray cocrystal structures of two analogues 5 and 45 revealed excellent

convergence with the original inhibitor 1 structure while providing new insights into other

interactions which could be exploited for future modifications.

Introduction

have subsequently reported the synthesis of low nM

inhibitors with related structures and reduced molec-

ular weights by appropriate truncation and functional

modification of the original lead structure. Reports from

other laboratories²⁷on inhibitors incorporating the

hydroxyethylene^28-31transition state mimetic group

followed. Further peptidomimetic isosteres that have

successfully been implemented for inhibitors of BACE

are statine,^32-34bis-statine,³⁵phenylnorstatine,^36,37hy-

droxymethylcarbonyl,³⁸and hydroxyethylamine.^39-40

Clearly, the inhibition of BACE relying on conceptu-

ally novel approaches to structure-based design presents

a major intellectual and operational challenge. Herein,

we report our results on the design and synthesis of

novel nM inhibitors of BACE. Molecular modeling based

on the X-ray structure of 1 with BACE¹⁷led us to a

subtle modification at the P₁′ Ala position with the

introduction of a fused cyclopentane ring (Figure 1).

Indeed, an energy minimized CP (cyclopentano) ana-

logue of 1, in which the methyl group of the P₁′ Ala and

the adjacent methylene carbon atom were joined as a

constrained entity, was well matched with the X-ray

conformation of 1. Our first objective was to ascertain

the viability of the CP as a backbone replacement for a

segment of the hydroxyethylene isostere. As a proof of

concept, we prepared three analogues, 3-5, of 1 incor-

porating a CP ring, with progressive truncation at the

P₃′-P₄′ C-terminus (Figure 1). We also prepared a

truncated acyclic analogue 2 as a control for compound

5.

A major histopathological hallmark of Alzheimer’s

disease is extracellular plaques of deposited â-amyloid

(Aâ) in the brain. There is compelling evidence that

â-amyloid (Aâ) plays a prominent role in the pathogen-

esis of Alzheimer’s disease, either as extracellular or

intracellular deposits, or as small soluble aggregates.¹

Reducing the production of Aâ or increasing its clear-

ance therefore are attractive strategies for the treatment

of Alzheimer’s disease.^2-6The generation of Aâ depends

on two consecutive enzymatic steps. The endoproteolytic

cleavage of â-amyloid precursor protein (APP) is initi-

ated by the membrane-bound aspartic protease BACE

(Asp-2, memapsin 2), which exhibits all the character-

istics assigned to the â-secretase activity.^7-11BACE

releases the large ectodomain of APP (APPsâ) and

produces a membrane-bound C99 fragment. This latter

is then the substrate of γ-secretase for the release of

Aâ.¹²

BACE is considered as an ideal target for small

molecule inhibitors, with the ultimate aim of developing

an effective therapeutic agent.^13-16The first crystal

structure of the extracellular domain of BACE com-

plexed with a synthetic heptapeptide inhibitor (1)

harboring an unnatural hydroxyethylene spacer as a

transition state mimic was reported by Hong, Ghosh,

and Tang.¹⁷This disclosure, followed by additional

crystal structures of BACE with^18-22and without¹⁹

complexed inhibitor, as well as studies on the substrate

specificity^22-24provide valuable insights for structure-

based design of inhibitors. Indeed, Ghosh and Tang^21,25,26

Results and Discussion

Chemistry. CP/1 and P′ Truncation. N-Boc L-

leucinal was converted to the acetylenic ester 6 in

analogy to a known procedure⁴¹albeit in low yields (25-

30%). However, using the less basic and more oxophilic

cerium methyl propiolate⁴²resulted in a significant

* To whom correspondence should be addressed. Phone: 514-343-

6738. Fax: 514-343-5728. E-mail: stephen.hanessian@umontreal.ca.

^†Universite´ de Montre´al.

^§Present address: McGill University, 801 Sherbrooke St. W.,

Montre´al, Que´bec H3A 2K6, Canada.

^‡Novartis Pharma AG.

Published on Web 07/15/2005

5176 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

systematically. This protocol of stepwise addition of

protected amino acids gave more consistent results and

better yields compared to a block coupling with di- or

tripeptides. Thus, 12 was hydrolyzed to the carboxylic

acid, which was coupled with the respective peptidic

precursors to give 13-15 as benzyl esters in good to

excellent yields. Removal of the N-Boc and acetal groups

by acid hydrolysis provided the corresponding amino

alcohols which were individually coupled with Boc-

Asn(NTr)-OH to give 16-18, respectively. Cleavage of

the N-Boc group and coupling with Boc-Val-OH led to

the respective products 19-21, which were further

elaborated to the protected full length CP oligopeptides

22-24. Treatment with TFA, followed by catalytic

hydrogenation afforded the desired constrained P₁′-P₄′

analogue 3 of 1 as well as its P₃′-P₄′ truncated

congeners 4 and 5 (Figure 1, Scheme 1). For comparison

of biological data we also prepared the P₃′-P₄′ truncated

peptide analogue 2.⁴⁵

P₂-P₄Modification and P′ Truncation of CP/1.

We pursued a new series of truncated analogues main-

taining the CP constraint and the P₂′ Ala-OH terminus,

while changing the P₂Asn residue to Met. Tang and

Ghosh²⁶have shown that this replacement in a short-

ened analogue of 1 had a small beneficial effect, possibly

also diminishing the hydrophilic nature of the original

inhibitor.

Thus, the common CP intermediate 10 was trans-

formed to the corresponding Ala-OCH₂CCl₃ester 25,

followed by liberation of the amine and coupling with

Boc-Met-OH to give 28 (Scheme 2). Further chain

elongation by coupling with Ac-Leu-OH afforded 31 and

eventually the free acid 34. The keto and deoxy CP

analogues 35 and 36 were prepared following the same

protocol starting with 11 and 12 respectively as shown

in Scheme 2. Sequential single amino acid coupling was

found to minimize racemization compared to alternative

dipeptide coupling strategies which required longer

reaction times at room temperature. To assess the

importance of the P₂′ amino acid residue, we also

synthesized the CP Val-OH analogue 40 (Scheme 2).

Our intention was also to diminish the peptidic

character of the CP series. Toward this end, we replaced

the P₃′-P₄′ Glu-Phe residues by a simple butyramide

moiety, while maintaining the new prototypical CP

structures represented by 45, 46, and 47 (Scheme 3).

The intermediate 11 was transformed to the corre-

sponding Val-butyramide derivative 41. Cleavage of the

N-Boc and acetal groups and coupling with Boc-Met-

OH gave 43 which was cleaved to the amine and further

extended with Ac-LeuOH to give the intended oxo-CP

target 45. Following a similar procedure, the CP inter-

mediate 12 was elaborated via 42 and 44 to give the

P₂′ Ala butyramide analogue 46. The CP Val butyramide

47 was prepared from precursor 40. To assess the

importance of the P₂′ amino acid residues in this series,

and the implication of a constrained CP subunit, we also

prepared the truncated n-butyramide analogue 50, as

well as the acyclic analogue 51⁴⁵(Scheme 3).

Figure 1. A. Structure of inhibitors 1 and 1a (OM99-2,

OM00-3) and proposed constrained P₁′ region; B. P₃′, P₄′

truncated analogue of 1; C. Constrained CP (cyclopentano)

backbone modification of 1; D, E. P₃′ and P₄′ truncated

constrained CP modifications.

improvement, affording 6 in 72% yield as a 5:1 mixture

of isomers (Scheme 1). Conversion to the mixed acetal

7 and Lindlar reduction afforded the cis-ester 8 which

was subjected to Pd-catalyzed cycloannulation with

2-(acetoxymethyl)-3-allyltrimethylsilane 9, according to

Trost’s method⁴³to give the corresponding exo-methyl-

ene CP adduct as a mixture of epimers. Treatment with

sodium methoxide in MeOH resulted in complete epimer-

ization to the enantiopure trans-isomer 10 in excellent

overall yield. Oxidative cleavage with sodium meta-

periodate and catalytic osmium tetroxide led to the

cyclopentanone analogue 11 which was deoxygenated

by transformation to the tosylhydrazone and treatment

with catecholborane⁴⁴to give the core CP intermediate

12 in excellent overall yield. The structural and con-

figurational confirmation of 12 was ascertained from a

single-crystal X-ray analysis.

In an effort to probe the carbocyclic CP space in the

P′ and P₂-P₃truncated analogues shown in Scheme 3,

we also prepared four heterocyclic variants.⁴⁵Thus, the

pyrrolidine 53 and lactam analogues 55 and 57 as well

as tetrahydrofuran analogue 59 were prepared by

We found it more practical to introduce the P₂′-P₄′

subunits first, then to elongate the chain on the P sites

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5177

Scheme 1^a

(a) i: LDA, methyl propiolate, THF, -78 °C; ii: CeCl₃, THF, -78 °C; iii: N-Boc-L-leucinal, -78 °C, 72%; (b) dimethoxypropane, TsOH,

Me₂CO, 64% (6, major isomer); (c) H₂, Lindlar’s catalyst, benzene, 99%; (d) toluene, Pd(OAc)₂, P(OiPr)₃, 85 °C, 90%; (e) NaOMe, MeOH,

99%; (f) NaIO₄, OsO₄cat., THF/H₂O, 90%; (g) H₂N-NHTs, Na₂SO₄, MeOH, 99%; (h) catecholborane, CHCl₃, then NaOAc‚H₂O, 68%; (i) i:

KOH, THF/H₂O; ii: PyBOP, DIPEA, CH₂Cl₂, H-Ala-Glu(OBn)-Phe-OBn, 57% (13, two steps), H-Ala-Glu(OBn)-OBn, 76% (14, two steps),

H-Ala-OBn, 99% (15, two steps); (j) i: TFA, CH₂Cl₂, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Asn(NTr)-OH, 74% (16, two

steps), 66% (17, two steps), 67% (18, two steps); (k) i: HCl, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Val-OH, 59% (19, two

steps), 85% (20, two steps), 78% (21, two steps); (l) i: HCl, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Glu(OBn)-OH, 77% (22,

two steps), 56% (23, two steps), 78% (24, two steps); (m) TFA, CH₂Cl₂; (n) H₂, Pd/C, MeOH, 30% (3, two steps), 54% (4, two steps), 60%

(5, two steps).

standard coupling with the appropriate amino acid

partners as described for the CP analogues (Scheme 4).

Biological Data and Structure-Activity Rela-

tionships. Truncation of the P′ Side. The suitability

of the CP moiety as a rigid dipeptide isostere of the

known inhibitor 1 and its P′-truncated variants was

validated since analogues 3-5 maintained substantial

inhibitory activity as shown in Table 1. The full length

analogue 3 has an IC₅₀of 16 nM, about an order of

magnitude weaker than 1, but, remarkably, the P₄′-

P₃′ truncated CP compound 5 is only 3-fold less potent

than its counterpart 2. While the activity on BACE

hardly changed with stepwise elimination of amino

acids from the P′ side, the Cathepsin D (CathD) activity

decreased sharply. Hence, a substantial increase in

selectivity toward BACE is afforded by the combination

of the P′ truncation with the CP modification, while

maintaining decent potency. The binding of the P₃′ Glu

and P₄′ Phe groups to BACE has been shown to be

affected by the nature of the P₂′ residue. In the OM00-3

(P₂′ ) Val) crystal structure¹⁸the P₃′ Glu and P₄′ Phe

groups make clearly defined interactions with the

enzyme. In comparison, these residues are not well

defined in the OM99-2 (P₂′ ) Ala) complex, suggesting

that they are relatively mobile. This observation can be

interpreted as being the result of a stabilization of the

position of the P₃′ and P₄′ residues due to the more

tightly bound P₂′ Val. Compounds 3-5 have an alanine

in P₂′, and the contribution of the P₃′-P₄′ groups to

binding is most probably minimal. Moreover, with the

P₃′ AlaGlu-OH analogue 4, the loss of the P₄′ Phe

interactions is likely compensated by a favorable elec-

trostatic interaction between the terminal carboxylate

group and the guanidinium moiety of Arg128. The

observed 60-fold decrease of the CathD activity in going

from 3 to 4 indicates a stronger contribution of the P₄′

groups to CathD binding, which is not compensated by

new interactions. Since Arg128 is substituted by a

valine in Cath D,⁴⁶the electrostatic interaction proposed

hereabove for BACE cannot take place.

5178 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

Scheme 2^a

(a) i: NaOH, THF/H₂O; ii: PyBOP, DIPEA, CH₂Cl₂, H-Ala-OCH₂CCl₃, 75% (25, two steps), 75% (26, two steps), 71% (27, two steps);

(b) i: HCl, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Met-OH, 33% (28, two steps), 75% (29, two steps), 75% (30, two steps);

(c) i: HCl, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Ac-Leu-OH, 44% (31, two steps), 76% (32, two steps), 52% (33, two steps); (d)

Zn, KH₂PO₄, 71% (34), 77% (35), 91% (36); (e) i: KOH, THF/H₂O; ii: 1 N HCl; iii: PyBOP, DIPEA, CH₂Cl₂, H-Val-OBn, 98% (two steps);

(f) i: TFA, CH₂Cl₂, then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Met-OH, 68% (two steps); g) i: HCl, then satd NaHCO₃; ii: EDC,

HOBt, CH₂Cl₂/H₂O, Ac-Leu-OH, 58% (two steps); h) 10% HCO₂H, MeOH, Pd Black, 74%.

Scheme 3^a

(a) i: NaOH, THF/H₂O; ii: PyBOP, DIPEA, CH₂Cl₂, H-Val-NHBu, 95%, (41, two steps), H-Ala-NHBu 75%, (42, two steps); (b) i: HCl,

then satd NaHCO₃; ii: PyBOP, DIPEA, CH₂Cl₂, Boc-Met-OH, 72% (43, two steps), 75% (44, two steps); (c) i: HCl, then satd NaHCO₃; ii:

EDC, HOBt, CH₂Cl₂/H₂O, Ac-Leu-OH, 78% (45, two steps), 78% (46, two steps); (d) PyBOP, NMM, DMF, BuNH₂, 20%; (e) i: NaOH,

MeOH/H₂O, then satd NH₄Cl; ii: EDC, HOBt, DMAP, BuNH₂, CH₂Cl₂, 89% (two steps); (f) i: TFA, CH₂Cl₂, then satd NaHCO₃; ii: PyBOP,

DIPEA, CH₂Cl₂, Boc-Met-OH, 74% (two steps); (g) i: HCl, dioxane, then satd NaHCO₃; ii: EDC, HOBt, CH₂Cl₂/H₂O, Ac-Leu-OH, 54%

(50, two steps).

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5179

Scheme 4^a

(a) i: NaOH, MeOH-H-H₂O, 65 °C, and then 1 N HCl; ii: H-Val-NHBu, PyBOP, DIPEA, CH₂Cl₂; (b) i: TFA, DCM, and then NaHCO₃;

ii: Boc-Met-OH, PyBOP, DIPEA, CH₂Cl₂; (c) i: TMSI, CH₂Cl₂; and then Na₂S₂O₃, NaHCO₃; ii: Ac-Leu-OH, EDC, HOBt, DCM/H₂O; (d)

i: Pd black, HCOOH/MeOH; ii: Boc₂O, NaHCO₃, MeOH; iii: TBAF, THF.

Table 1. Truncation of the P′ Side

compd

R )

IC₅₀BACE, µM IC₅₀CathD, µM

compd

R )

IC₅₀BACE, µM

IC₅₀CathD, µM

1

2

AlaGluPheOH

AlaOH

0.0006

0.012

0.006

0.098

3

4

5

AlaGluPheOH

AlaGluOH

AlaOH

0.016

0.040

0.039

<0.01 (90% @ 0.01 µM)

0.060

6.0

backbone encompassed by the P₂′-P₂residues, confirm-

ing that the rigid CP moiety does not alter substantially

the positioning of the P₂′ alanine and of the modified

transition state mimic. Small positional shifts of 0.6-

0.8 Å are observed for P₁-P₂′ atoms of analogue 5

relative to the corresponding 1 atoms. The distance

between the catalytic aspartic acid residues and the

hydroxyethylene isostere are similar in the OM99-2

complex (2.4 Å to Asp32 Oδ1, 2.8 Å to Asp228 Oδ2) and

in the complex with analogue 5 (2.6 Å to Asp32 Oδ1,

2.6 Å to Asp228 Oδ2). A remarkable feature of the X-ray

of 5 are the two specific interactions of the Ala carbox-

ylate with Tyr198 and Arg128. In the OM99-2 complex,

the carbonyl oxygen atom of the P₃′-P₄′ amide bond is

hydrogen-bonded to the hydroxyl of Tyr198. In the

complex with analogue 5, one of the carboxylate oxygens

is mimicking this interaction, while in addition, the

second oxygen forms a tight interaction with Arg128.

This is possible due to a more than 3 Å move of the Arg

Figure 2. Overlay of cocrystal structures of 5 (yellow) and

1¹⁷(orange) with interactions discussed in text (distances in

Å).

The P₂′ Ala-OH analogue 5 afforded suitable co-

crystals with the enzyme that were amenable to an

X-ray analysis at 2.25 Å resolution (Figure 2). The

superposition of the cocrystal structures of 5 and 1 with

the enzyme showed good agreement along the peptidic

5180 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Table 2. SAR in P₂′: Further Truncation on P- and P′-Side

Hanessian et al.

Table 3. SAR of Ring Element: Val-NHBu Derivatives

compd

R )

IC₅₀BACE, µM

IC₅₀CathD, µM

36

40

46

47

50

AlaOH

0.600

0.190

0.185

0.025

1.82

0.090

ValOH

0.025

AlaNHBu

ValNHBu

NHBu

<0.01 (95% @ 0.01 µM)

<0.01 (100% @ 0.01 µM)

0.060

side-chain when compared to its position in the struc-

ture of 1 (Figure 2).

Thus, the X-ray data show that the loss of the P₃′

group in the Ala-OH analogue 5 can be compensated

by new polar and electrostatic interactions between

Tyr198, Arg128 and the free carboxylate of the inhibitor,

explaining the relatively small effect of the truncation

on the inhibitory potency. As already pointed out

previously, the loss of activity of compound 5 against

CathD can, at least in part, be ascribed to the Arg128

to Val substitution in CathD.⁴⁶

SAR in P₂′. On the basis of the subsite specificity

published by Turner²³and in accordance with published

SAR data,²⁶one can assume that it is beneficial to

replace P₃Val by Leu, and that the P₂Asn can be

replaced by the less hydrophilic Met without loss of

activity. The mentioned published data also revealed

that replacement of P₄Glu in 1 by a hydrogen resulted

in only about 20-fold loss of activity, suggesting that the

P4 residue can be truncated without too much penalty.

Indeed, exchanging the P₄-P₂residues in 5 from

GluValAsn to AcLeuMet as in 36, led to only about 15-

times lower inhibitory activity than 5. However, as

expected due to the more hydrophobic S₂pocket in

CathD,⁴⁶the inhibitory activity for CathD was in-

creased. Further studies were pursued using 36 as

reference compound. Thus, in accordance with the

results of Turner²³replacement of the P₂′-Ala by Val

led to improved activity (compare 36 versus 40, 46

versus 47). A Val residue was shown to fill the S₂′ pocket

better than Ala.¹⁸Capping the carboxylate as a butyl-

amide further improved activity on BACE (compare 36

versus 46, and 40 versus 47). Deletion of P₂′ as in 50

resulted in only weak activity, while additional shorten-

ing of 50 on the N-terminal side going from Ac-Leu to

4-methylpentoyl (as a mimic of Leu) led to a compound

without activity. Therefore, 47 was chosen as a suitable

reference for an SAR study on the cyclopentyl peptido-

mimetic moiety.

(Table 3), which might be explained by two facts. First,

the S₁′ pocket in CathD is more lipophilic than in BACE

(Arg235 in BACE corresponds to Val238 in CathD),

hence less favorable to accommodate a polar carbonyl

group. Second, the Val332 in BACE corresponds to

Ile320 in CathD, resulting in a smaller pocket possibly

leading to unfavorable steric interactions.

The cyclopentanone derivative 45 was cocrystallized

in BACE and an X-ray structure was solved to 2.05 Å

resolution. Overall, the binding conformation of 45

corresponded well with the binding conformation of 5

(Figure 3a). In particular, the positioning of the P₁group

and the modified transition state isostere in 45 is,

within experimental error, identical in the two com-

plexes. As mentioned above, the P₂′ residue has been

shown to influence the binding of the P₃′ and P₄′ side

chains. A structural comparison with the OM00-3¹⁸(P₂′

) Val) complex is therefore interesting (Figure 3b). It

shows that the P₂′ Val binding is not affected by the

rigid CP modification, and that the butylamide cap

takes the place of the P₃′ Glu of OM00-3. The hydrogen-

bonded interaction to Tyr198 is maintained in analogue

45. Furthermore, the nitrogen atom of the butylamide

is H-bonded to the carbonyl oxygen of Pro70 and its

alkyl chain is in van der Waals contact to Pro70, Tyr71

and Thr72 of the enzyme flap, thereby contributing to

the stabilization of the closed conformation of the

enzyme.

SAR of the Cyclopentyl Peptidomimetic Moiety.

The water-filled S1′ pocket¹⁷suggested an extension of

the CP concept toward more polar analogues. An obvi-

ous example is the cyclopentanone 45 (Table 3). From

a study of a cocrystal X-ray structure, the ring carbonyl

of 45 forms a network of hydrogen bonds via water

molecules to several residues lining the hydrophilic S₁′

pocket (see discussion below). This modified pattern of

water-mediated interactions contributes to only a small

improvement of the activity on BACE compared to 5.

On the other hand, the activity on CathD was decreased

An analysis of the solvent structure in the S₁′ pocket,

as observed in the published complexes with OM99-2

and OM00-3¹⁸and in this study with compounds 5 and

45, shows that it is affected by the nature of the groups

in position P₂and P₁′ of the inhibitors, and by Arg235,

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5181

Figure 3. (a) Overlay of cocrystal structures of 5 (yellow) and 45 (green). (b) Overlay of 45 (green) and 1a (blue) (distances in Å).

Figure 4. Binding interactions in the water-filled S₁′ pocket (distances in Å). (a) comparison of 5 (yellow) and 1 (orange). (b)

comparison of 5 (yellow) and 45 (green).

Table 4. SAR of Ring Element: Ala-OH Derivatives

whose orientation varies in response to the bulkiness

and chemical nature of the P₂substituent. Nevertheless,

two waters in the S₁′ pocket are conserved in all four

complexes, and two additional waters are found in three

complexes. As depicted in Figure 4a, the introduction

of the cyclopentyl moiety in P₁′ (compound 5 compared

to 1) hardly effects the pattern of conserved water

molecules. In contrast, introduction of the ring carbonyl

group and replacement of P₂Asn by Met (compound 45)

result in major changes in the interaction pattern

(Figure 4b). The carbonyl oxygen forms hydrogen bonds

with two conserved waters. The movement of Arg235

(now forming a hydrogen bond with one of the waters)

together with the spatially more demanding carbonyl

displaces two water molecules.

With the observed binding pattern of the carbonyl

group in the cocrystal complex of 45 and BACE (Figure

4), we were not surprised that the lactam 57 which

deployed its carbonyl group in the same direction

exhibited almost equal inhibition compared to 45 and

significantly better than the isomeric lactam 55. The

the water-mediated interaction pattern in S₁′ resulting

in less favorable interactions.

A comparable effect of the ring carbonyl was found

in the P₂′-Ala-OH series (Table 4): the keto derivative

slight additional gain in activity for 57 might result from

favorable conformational changes due to steric con-

straints imposed by the rigid lactam motif.

The substantial loss of activity of the oxa-analogue

59 and the isomeric lactam 55 might be explained by

different conformational preferences due to the intro-

duction of electronegative groups adjacent to the amide.

In addition, the loss of activity for BACE of compound

55 is consistent with the negative effect of a hydrophobic

group in the hydrophilic S₁′ environment. The inhibition

of CathD, which comprises a hydrophobic S₁′ pocket, is

much less affected by these changes. The loss of activity

of the tertiary amine 53 is more difficult to explain. Most

likely the positive charge is substantially influencing

35 showed similar activity on BACE as the unsubsti-

tuted compound 36, while the activity for CathD was

decreased. In agreement with the above-mentioned

results for 55, the lipophilic methylene derivative 34

exhibited an almost complete loss of activity for BACE,

while it was still active on CathD.

Conclusion

We have demonstrated the validity of introducing a

carbocyclic constraint in the hydroxyethylene subunit

of the original Tang-Ghosh BACE inhibitor 1. A cocrys-

5182 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

layer was washed with saturated NaHCO₃(3 × 50 mL) and

brine (50 mL), dried (Na₂SO₄), filtered, and concentrated in

vacuo. The residue was purified by column chromatography

(30% EtOAc in hexanes) to afford 6 as a 5:1 mixture of

tal structure of the CP AlaOH analogue 5 with the

enzyme showed a good match with 1. Introduction of a

keto group in the CP ring of P′ truncated analogues as

in 45 results in excellent BACE activity (IC₅₀10nM) as

well as a 5-fold activity loss against CathD. A cocrystal

structure of 45 with BACE shows excellent superposi-

tion with 1a. Heterocyclic replacements of the CP unit

resulted in the single digit nanomolar lactam inhibitor

57. This compound exhibits an at least 7-fold selectivity

toward BACE over CathD. Disappointingly, despite the

high potency in the enzymatic assay (IC₅₀< 10 nM),

the compound exhibited an only weak activity (IC₅₀0.9

µM) in the cellular assay (Aâ-production in CHO cells

transfected with APP_wt). All other inhibitors showed

even lower, mostly nonrelevant inhibition at 10 µM

concentration.⁴⁵The character of the compounds is still

peptidic and is limiting penetration across cell mem-

branes. Although much remains to be done, the results

obtained so far demonstrate that the combination of

truncation and functional manipulation in hydroxy-

ethylene transition state mimetics can produce potent

inhibitors of BACE with excellent potential for selectiv-

ity over CathD.

1

diastereomers (3.05 g, 72%) as a yellow oil; H NMR (CDCl₃)

δ 4.74 (m, 1H), 4.50 (m, 1H), 3.84-3.70 (m, 1H), 3.76 (s, 3H),

1.67 (m, 1H), 1.58-1.20 (m, 2H), 1.43 (s, 9H), 0.93 (m, 6H);

¹³C NMR (CDCl₃) δ 156.6, 153.0, 86.2, 80.0, 65.1, 52.9, 52.7,

38.6, 28.1, 24.6, 23.2, 21.7, 21.5.

(4S,5S)-Isobutyl-5-methoxycarbonylethynyl-2,2-dimeth-

yl-oxazolidine-3-carboxylic Acid tert-Butyl Ester (7). A

solution of 6 (1.27 g, 3.74 mmol), 2,2-dimethoxypropane (15

mL, 0.123 mol), and dry acetone (70 mL) was heated to reflux

for 5 min under argon atmosphere. Then TsOH was added

until a permanent dark red solution was observed. The

reaction mixture was refluxed for an additional 2 h, cooled to

room temperature, and concentrated in vacuo. Saturated

NaHCO₃(20 mL) was added to the residue and extracted with

ethyl acetate (3 × 60 mL). The combined organic layer was

dried (Na₂SO₄), filtered, and concentrated to give a crude

product as a yellow oil, which was purified by column chro-

matography (3-4% EtOAc in hexanes) to afford 7 as a single

isomer (0.92 g, 64%) as a yellow oil; [R]²⁰_D-25.6 (c 1.5, CHCl₃);

IR (neat) 2960, 2874, 2238, 1723, 1703, 1457, 1436, 1381, 1254,

1175, 1128, 1089, 1068, 1027, 851, 752 cm^-1; ¹H NMR (CDCl₃)

δ 4.58 (s, 1H), 4.28-3.90 (m, 1H), 3.77 (s, 3H), 1.73 (s, 3H),

1.62-1.30 (m, 6H), 1.48 (s, 9H), 0.95 (t, J ) 6.2 Hz, 6H); ¹³C

NMR (CDCl₃) δ 153.9, 151.6, 118.1, 96.4, 91.0, 86.6, 68.8, 62.5,

53.1, 43.4, 42.5, 28.8, 28.0, 26.3, 24.0, 21.9; MS (FAB): m/z

340 [M + 1]⁺; HRMS calcd for C₁₈H₃₀NO₅[M + 1]⁺340.2124;

found 340.2108.

Experimental Section

Chemistry. Solvents were distilled under positive pressure

of dry argon before use and dried by standard methods; THF

and ether, from Na/benzophenone; CH₂Cl₂and toluene, from

CaH₂. All commercially available reagents were used without

further purification. All reactions were performed under argon

atmosphere. NMR (¹H, ¹³C) spectra were recorded on Bruker

AMX-300 and ARX-400 spectrometers in CDCl₃or CD₃OD or

D₂O with solvent resonance as the internal standard. Low-

and high-resolution mass spectra were recorded on VG Mi-

cromass, AEIMS 902, or Kratos MS-50 spectrometers using

fast atom bombardment (FAB). The purity of the target

compounds was determined to be >95% by LC/MS obtained

on a Finnigan Surveyor MSQ spectrometer. Purity of the

compounds was determined by method [A] Alltech Prevail C18

column (250 × 4.6 mm) at 0.5 mL/min flow rate using a

gradient of 20-90% acetonitrile-water (0.1% trifluoroacetic

acid) and method [B] Alltech Prevail C18 column (250 × 4.6

mm) at 0.5 mL/min flow rate using 65% methanol-water (0.1%

trifluoroacetic acid). Optical rotations were recorded on a

Perkin-Elmer 241 polarimeter in a 1 dm cell at ambient

temperature. Analytical thin-layer chromatography was per-

formed on Merck 60F 254 precoated silica gel plates. Flash

column chromatography was performed using (40-60 µm)

silica gel at increased pressure. All melting points are uncor-

rected.

(5S)-tert-Butoxycarbonylamino-(4S,R)-hydroxy-7-

methyl-oct-2-ynoic Acid Methyl Ester (6). To a solution

of i-Pr₂NEt (2.548 g, 25.2 mmol) in 20 mL of THF at -78 °C

was added butyllithium (2.5 M in hexanes, 8.4 mL, 21 mmol).

The solution was warmed to 0 °C for 0.5 h and then cooled to

-78 °C. To the LDA solution was added methyl propiolate

(2.548 g, 25.2 mmol) dropwise, and the reaction mixture was

stirred at -78 °C for 0.5 h (solution A).

To a 250 mL flame-dried flask were added anhydrous CeCl₃

(5.16 g, 21 mmol) and THF (40 mL). The reaction mixture was

vigorously stirred at room temperature for 2 h and then cooled

to -78 °C. To this suspension was added solution A (-78 °C)

in one portion. The reaction mixture was stirred at the same

temperature for 1 h. A solution of Boc-leucinal (3.012 g, 14

mmol) in THF (12 mL) was added at -78 °C, and the resulting

reaction mixture was stirred at -78 °C for 3 h, quenched by

addition of a solution of acetic acid (4 mL) in THF (16 mL) at

-78 °C, and allowed to warm to room temperature. The

mixture was extracted with diethyl ether (100 mL) and washed

with 10% citric acid (2 × 40 mL), and the separated organic

(4S,5S)-Isobutyl-5-(2-methoxycarbonyl-vinyl)-2,2-di-

methyl-oxazolidine-3-carboxylic Acid tert-Butyl Ester

(8). Benzene (30 mL), Lindlar catalyst (60 mg, 5 wt %), and

quinoline (0.24 mL) were placed in a 100 mL round-bottom

flask and stirred for 20 min at room temperature. Compound

7 (1.2 g, 3.54 mmol) in benzene (20 mL) was added to the flask,

and the mixture was stirred for an additional 20 min at room

temperature. The flask was then partially charged with H₂

(not fully inflated balloon), and the suspension was stirred

vigorously at room temperature overnight. The catalyst was

removed by filtration through a pad of Celite and thoroughly

washed with ethyl acetate. The filtrate was concentrated, and

the residual yellow oil was purified by column chromatography

(5% Et₂O in CH₂Cl₂) to give 8 (1.09 g, 90%) as a yellow oil;

[R]²⁰+24.0 (c 1.2, CHCl₃); IR (neat) 2959, 2873, 1743, 1700,

D

1388, 1367, 1256, 1175, 1092, 922, 870, 770 cm^-1

;

¹H NMR

(CDCl₃) δ 6.28 (m, 1H), 5.84 (m, 1H), 5.47 (m, 1H), 3.80-3.62

(m, 1H), 3.68 (s, 3H), 1.70-1.47 (m, 9H), 1.44 (s, 9H), 0.89 (d,

J ) 2.9 Hz, 3H), 0.85 (d, J ) 6.2 Hz, 3H); ¹³C NMR (CDCl₃) δ

166.2, 152.1, 147.6, 121.5, 80.3, 75.6, 61.9, 53.3, 51.9, 43.6, 28.9,

28.0, 26.3, 25.4, 24.3, 21.9. MS (FAB): m/z 342 [M + 1]⁺;

HRMS calcd for C₁₈H₃₂NO₅[M + 1]⁺342.2281; found 342.2274.

(4S)-Isobutyl-(5S)-((2R)-methoxycarbonyl-4-methylene-

cyclopentyl)-2,2-dimethyl-oxazolidine-3-carboxylic Acid

tert-Butyl Ester (10). To a solution of 8 (1.0 g, 2.93 mmol) in

toluene (5 mL) was added 2-trimethylsilanyl- methylallyl

acetate 9 (0.63 g, 3.52 mmol) followed by triisopropyl phosphite

(0.43 mL, 0.37 g, 1.76 mmol) and palladium acetate (46 mg,

0.2 mmol) at room temperature. The reaction mixture was

heated to 100 °C under an argon atmosphere for 16 h and

cooled, and toluene was evaporated under reduced pressure.

The resulting residue was purified by column chromatography

(5% EtOAc in hexanes) to afford an inseparable ∼2:1 epimeric

mixture as a colorless oil (1.04 g, 90%).

To a solution of the epimeric mixture (0.85 g, 2.15 mmol) in

methanol (10 mL) was added a 0.5 M solution of NaOMe (8.6

mL, 4.3 mmol) in methanol at room temperature, and the

reaction mixture was refluxed for 6 h. Methanol was removed

under reduced pressure, H₂O (5 mL) was added, and the

aqueous layer was extracted with EtOAc (3 × 10 mL). The

combined organic layers were dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was purified by column

chromatography (5% EtOAc in hexanes) to afford exclusively

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5183

the trans isomer 10 as a colorless oil (0.85 g, 99%); [R]²⁰_D-29.5

was separated and washed with saturated NaHCO₃(10 mL)

and brine (10 mL), dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was purified by column chromatography

(50% EtOAc in hexanes) to afford 13 (100 mg, 57%) as a

1

(c 2.9, CHCl₃); H NMR (CDCl₃) δ 4.88 (d, J ) 6.7 Hz, 2H),

3.71 (m, 1H), 3.70 (s, 3H), 2.60-2.30 (m, 6H), 1.60-1.45 (m,

10H), 1.47 (s, 9H), 0.93 (d, J ) 4.8 Hz, 6H); ¹³C NMR (CDCl₃)

δ 175.7, 152.0, 148.8, 106.9, 95.1, 80.8, 60.9, 59.2, 52.1, 46.6,

37.3, 34.2, 28.8, 27.7, 25.6, 24.6, 21.7, 14.5. MS (FAB): m/z

396 [M + 1]⁺; HRMS calcd for C₂₂H₃₇NO₅[M]⁺395.2672; found

395.2682.

colorless oil; [R]²⁰-40.9 (c 2.0, CHCl₃); ¹H NMR (CDCl₃) δ

D

7.50-7.00 (m, 15H), 5.11 (m, 6H), 4.86 (q, J ) 6.2 Hz, 1H),

4.53 (q, J ) 7.0 Hz, 2H), 3.75 (s, 1H), 3.60 (s, 2H), 3.11 (dd, J

) 13.9 Hz, J ) 6.0 Hz, 1H), 3.04 (dd, J ) 13.9 Hz, J ) 6.5 Hz,

1H), 2.49 (m, 1H), 2.41 (m, 3H), 2.06 (m, 1H), 1.96-1.34 (m,

17H), 1.46 (s, 9H), 1.29 (d, J ) 5.2 Hz, 3H), 0.92 (m, 6H); ¹³C

NMR (CDCl₃) δ 175.1, 173.0, 172.3, 170.8, 170.4, 151.6, 135.5,

134.9, 129.1, 128.4, 128.3, 128.2, 128.1, 126.9, 79.5, 67.1, 66.4,

58.8, 53.3, 52.1, 48.6, 48.3, 46.2, 37.6, 31.4, 30.0, 28.4, 27.6,

25.0, 24.2, 21.4, 18.3; MS (FAB): m/z 897.6 [M + 1]⁺; HRMS

calcd for C₅₁H₆₉N₄O₁₀[M + 1]⁺897.5014; found 897.5043.

Compound 14. Prepared from 12 according to the general

procedure for preparation of 13 (124 mg, 76%); [R]²⁰_D-35.1 (c

0.4, CHCl₃); ¹H NMR (CDCl₃) δ 7.40-7.22 (m, 10H), 7.20 (d, J

) 7.6 Hz, 1H), 6.44 (d, J ) 7.4 Hz, 1H), 5.13 (m, 2H), 5.05 (s,

2H), 4.63 (m, 2H), 3.72 (br, 2H), 2.56-2.12 (m, 5H), 1.98 (m,

1H), 1.96-1.10 (m, 23H), 1.45 (s, 9H), 1.33 (d, J ) 6.9 Hz,

3H), 0.90 (d, J ) 6.9 Hz, 6H); ¹³C NMR(CDCl₃) δ 175.6, 172.9,

172.8, 171.6, 152.1, 136.0, 135.6, 129.0, 128.9, 128.8, 128.7,

128.3, 94.1, 82.9, 81.6, 80.0, 78.4, 67.7, 66.9, 60.8, 59.4, 52.1,

49.0, 48.9, 46.9, 44.4, 43.0, 32.0, 30.5, 28.9, 27.5, 27.4, 25.6,

24.7, 22.0, 21.5, 18.9, 14.6; MS (FAB): m/z 750.3 [M + 1]⁺;

HRMS calcd for C₄₂H₆₀N₃O₉[M + 1]⁺750.4285; found 750.4324.

(4S)-Isobutyl-(5S)-((2R)-methoxycarbonyl-4-oxo-cyclo-

pentyl)-2,2-dimethyl-oxazolidine-3-carboxylic Acid tert-

Butyl Ester (11). To a solution of 10 (700 mg, 1.77 mmol) in

THF and H₂O (27 mL, 2:1) was added NaIO₄(1.52 g, 7.08

mmol), followed by addition of OsO₄(44.3 mg, 0.177 mmol) at

room temperature. The reaction mixture was stirred for 14 h

at room temperature. A saturated solution of Na₂S₂O₃(10 mL)

was added and stirred for 10min. The partitioned aqueous

layer was extracted with EtOAc (5 × 20 mL), and the combined

organic layers were dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was purified by column chromatography

(20% EtOAc in hexanes) to afford 11 (0.633 g, 90%) as a

colorless oil; [R]²⁰-38.8 (c 0.7 CHCl₃); ¹H NMR (CDCl₃) δ

D

3.95 (br, 1H), 3.75 (s, 3H), 3.61 (br, 1H), 3.03 (br, 1H), 2.72

(m, 1H), 2.56 (d, J ) 33.5 Hz, J ) 8.4 Hz, 1H), 2.44 (dd, J )

16.0 Hz, J ) 8.2 Hz, 1H), 2.40 (m, 2H), 1.65-1.30 (m, 9H),

1.47 (s, 9H), 0.95 (d, J ) 6.2 Hz, 3H), 0.93 (d, J ) 5.7 Hz, 3H);

¹³C NMR (CDCl₃) δ 215.0, 174.8, 151.9, 94.8, 89.1, 82.0, 80.4,

59.2, 52.7, 43.7, 43.2, 42.0, 38.8, 28.9, 27.3, 25.6, 24.7, 21.8;

MS (FAB): m/z 398 [M + 1]⁺; HRMS calcd for C₂₁H₃₆NO₆[M

+ 1]⁺398.2543; found 398.2550.

Compound 15. Prepared from 12 according to the general

procedure for preparation of 13 (210 mg, 99%); [R]²⁰_D-27.2 (c

0.5, CHCl₃); ¹H NMR (CD₃OD) δ 7.40-7.30 (m, 5H), 6.14 (d, J

) 7.2 Hz, 1H), 5.19 (m, 2H), 4.66 (m, 1H), 3.75 (m, 1H), 3.64

(m, 1H), 2.52 (m, 1H), 2.37 (m, 1H), 1.93-1.39 (m, 15H), 1.45

(4S)-Isobutyl-(5S)-((2R)-methoxycarbonyl-cyclopentyl)-

2,2-dimethyl-oxazolidine-3-carboxylic Acid tert-Butyl

Ester (12). To a solution of 11 (0.4 g, 1.01 mmol) in MeOH

(20 mL) were added NH₂NHTs (205 mg, 1.11 mmol) and

Na₂SO₄(2 g). The reaction mixture was refluxed for 2 h, cooled

to room temperature, and stirred overnight. Methanol was

removed under reduced pressure, and the residue was purified

by column chromatography (33% EtOAc in hexanes) to afford

the hydrazone as a white solid (0.58 g, 99%).

(s, 9H), 1.42 (d, J ) 5.2 Hz, 3H), 0.92 (d, J ) 6.4 Hz, 6H); ¹³

C

NMR (CDCl₃) δ 175.2, 173.4, 152.1, 135.7, 129.0, 128.9, 128.6,

93.9, 82.9, 80.0, 67.6, 59.4, 48.8, 48.5, 46.8, 44.3, 32.6, 32.0,

30.0, 28.9, 27.9, 27.6, 25.6, 24.7, 21.9, 18.9; MS (FAB): m/z

531.2 [M + 1]⁺; HRMS calcd for C₃₀H₄₇N₂O₆[M + 1]⁺531.3434;

found 531.3459.

To a solution of the hydrazone (0.58 g, 1.01 mmol) in CHCl₃

(6 mL) was added catecholborane (1 M in THF, 2.16 mL, 2.16

mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2

h and overnight at room temperature. NaOAc‚3H₂O (0.412 g)

was added, and the reaction mixture was refluxed for 3 h,

cooled to room temperature, diluted with CH₂Cl₂(30 mL), and

washed with saturated Na₂S₂O₃(10 mL). The aqueous layer

was extracted with CH₂Cl₂(3 × 10 mL), and the combined

organic layers were dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was purified by column chromatography

Compound 16. To a solution of 13 (80 mg, 0.089 mmol) in

CH₂Cl₂(0.5 mL) was added TFA (0.2 mL). The reaction

mixture was stirred at room temperature for 1 h, then

quenched by addition of saturated NaHCO₃(5 mL) and

extracted with EtOAc (4 × 10 mL). The combined organic

layers were dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was dissolved in CH₂Cl₂(4 mL), followed by

addition of Boc-Asn(NTr)-OH (63.7 mg, 0.134 mmol), PyBOP

(70 mg, 0.134 mmol) and i-Pr₂NEt (60 µL, 0.268 mmol) at 0

°C. The reaction mixture was stirred at room-temperature

overnight, EtOAc (15 mL) was added, and the separated

organic layer was washed with 1 N HCl (2 × 10 mL), saturated

NaHCO₃(2 × 10 mL) and brine (10 mL). The organic layer

was dried (Na₂SO₄), filtered, and concentrated in vacuo. The

residue was purified by column chromatography (66% EtOAc

(10% EtOAc in hexanes) to afford 12 (283 mg, 68%) as colorless

1

crystals; mp: 75-77 °C; [R]²⁰-35.2 (c 0.8, CHCl₃); H NMR

D

(CDCl₃) δ 3.75-3.50 (m, 2H), 3.68 (s, 3H), 2.43 (m, 2H), 2.00-

1.35 (m, 15H), 1.45 (s, 9H), 0.92 (d, J ) 6.7 Hz, 6H); ¹³C NMR

(CDCl₃) δ 176.9, 152.1, 84.0, 79.9, 59.6, 52.1, 47.2, 31.6, 28.9,

28.0, 27.7, 27.2, 25.8, 25.6, 25.1, 24.7, 21.6; MS (FAB): m/z

383 [M]⁺; HRMS calcd for C₂₁H₃₇NO₅[M]⁺383.2672; found

383.2658.

in hexanes) to afford 16 (80 mg, 74%) as a colorless oil; [R]²⁰

D

-30.8 (c 1.2, CHCl₃); ¹H NMR (CDCl₃) δ 7.50-7.00 (m, 30H),

6.92 (br, 1H), 6.31 (br, 1H), 5.08 (d, J ) 2.6 Hz, 4H), 4.80 (m,

1H), 4.38 (m, 1H), 4.25 (m, 1H), 3.90 (m, 1H), 3.56 (br, 1H),

3.33 (m, 1H), 3.06 (m, 2H), 2.72 (m, 2H), 2.58 (m, 1H), 2.34

(m, 2H), 2.05 (m, 1H), 1.86 (m, 1H), 1.77-1.20 (m, 14H), 1.44

Compound 13. To a solution of KOH (28 mg, 0.5 mmol) in

MeOH (4 mL) and H₂O (2 mL) was added 12 (75 mg, 0.195

mmol). The reaction mixture was stirred at 65 °C for 3 h and

at room-temperature overnight. 1 N HCl (10 mL) was added,

and the aqueous phase was extracted with CH₂Cl₂(4 × 15 mL).

The combined organic layers were dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was used without further

purification. To a solution of Boc-Ala-Glu(OBn)-Phe-OBn (192

mg, 0.3 mmol) in CH₂Cl₂was added TFA (233 µL). The reaction

mixture was stirred at room temperature for 3 h, quenched

by addition of saturated NaHCO₃(1.5 mL), and extracted with

EtOAc (5 × 10 mL). The combined organic layers were dried

(Na₂SO₄), filtered, and concentrated in vacuo. The residue was

dissolved in CH₂Cl₂(3 mL) and followed by addition of NH₂-

Ala-Glu(OBn)-Phe-OBn (192 mg, 0.3 mmol), PyBOP (156 mg,

0.3 mmol), and i-Pr₂NEt (134 µL, 0.6 mmol) at 0 °C. The

reaction mixture was stirred for 4 h at 0 °C, then partitioned

in EtOAc (20 mL) and 1 N HCl (10 mL). The organic layer

(s, 9H), 1.28 (d, J ) 5.3 Hz, 3H), 0.90 (d, J ) 4.9 Hz, 6H); ¹³

C

NMR (CDCl₃) δ 177.0, 174.7, 173.8, 173.6, 172.6, 171.8, 171.3,

170.6, 156.6, 144.7, 136.4, 136.1, 135.5, 129.6, 129.1, 129.0,

128.9, 128.8, 128.7, 128.6, 128.3, 127.4, 80.8, 74.8, 71.1, 67.6,

66.9, 60.8, 53.9, 53.0, 52.7, 50.9, 50.5, 48.2, 46.9, 41.6, 38.1,

31.0, 30.9, 28.7, 27.5, 27.0, 25.1, 24.6, 23.7, 22.3, 21.5, 19.6,

17.8, 14.6, 14.2; MS (FAB): m/z 1213.3 [M + 1]⁺.

Compound 17. Prepared from 14 according to the general

procedure for preparation of 16 (110 mg, 66%); [R]²⁰_D-44.8 (c

1

0.6, MeOH); H NMR (CDCl₃) δ 7.50-7.00 (m, 25H), 6.94 (d,

J ) 6.4 Hz, 1H), 6.86 (d, J ) 4.8 Hz, 1H), 6.26 (s, 1H), 5.13 (d,

J ) 12.3 Hz, 1H), 5.08 (d, J ) 12.3 Hz, 1H), 5.03 (s, 2H), 4.51

(q, J ) 7.7 Hz, 1H), 4.30(m, 2H), 3.85 (m, 1H), 3.38 (m, 1H),

2.80 (m, 1H), 2.66 (m, 1H), 2.51 (m, 1H), 2.41-2.06 (m, 5H),

2.00 (m, 1H), 1.90-1.01 (m, 13H), 1.40 (s, 9H), 0.86 (m, 6H);

5184 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

¹³C NMR (CDCl₃) δ 176.3, 173.0, 172.6, 172.0, 171.3, 170.0,

144.1, 135.5, 135.0, 128.5, 128.4, 128.3, 128.1, 127.8, 126.9,

80.3, 74.4, 70.6, 67.2, 66.4, 60.3, 51.9, 51.5, 50.7, 49.5, 47.7,

46.4, 40.8, 37.5, 30.4, 30.0, 28.1, 26.8, 26.5, 24.5, 23.9, 23.1,

21.7, 20.9, 17.2, 14.0, 13.6; MS (FAB): m/z 1067.0 [M + 1]⁺.

Compound 18. Prepared from 15 according to the general

procedure for preparation of 16 (120 mg, 67%); [R]²⁰_D-51.0 (c

0.8, CHCl₃); ¹H NMR (CDCl₃) δ 7.60-7.00 (m, 20H), 6.89 (d, J

) 8.0 Hz, 1H), 6.68 (d, J ) 6.4 Hz, 1H), 6.22 (d, J ) 6.0 Hz,

1H), 5.14 (q, J ) 12.3 Hz, 2H), 5.08 (q, J ) 12.3 Hz, 2H), 4.53

(m, 1H), 4.30 (m, 1H), 3.85 (m, 1H), 3.68 (d, J ) 5.0 Hz, 1H),

3.37 (m, 1H), 2.83 (m, 1H), 2.61 (m, 2H), 2.30 (m, 1H), 1.90-

1.48 (m, 8H), 1.41 (s, 9H), 1.47-1.10 (m, 3H), 0.87 (m, 6H);

¹³C NMR (CDCl₃) δ 175.6, 173.2, 172.1, 170.0, 156.0, 144.2,

135.3, 129.1, 129.0, 128.7, 128.5, 128.4, 127.4, 80.7, 74.9, 71.1,

67.4, 60.8, 52.1, 51.9, 48.7, 48.2, 47.1, 41.2, 38.2, 31.0, 30.8,

28.7, 26.4, 25.8, 24.5, 24.0, 23.8, 22.2, 21.5, 19.5, 18.2, 14.6,

14.1; MS (FAB): m/z 848.0 [M + 1]⁺.

aqueous layer was extracted with EtOAc (4 × 10 mL). The

combined organic layers were dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was dissolved in N,N-

dimethylacetamide (1 mL), and followed by addition of N-Boc-

Glu(OBn)-OH (42 mg, 0.125 mmol), PyBOP (66 mg, 0.125

mmol), and i-Pr₂NEt (36 µL, 0.21 mmol). The reaction mixture

was stirred at room temperature for 24 h, EtOAc (15 mL) was

added, and the separated organic layer was washed with 1 N

HCl (2 × 10 mL), saturated NaHCO₃(2 × 10 mL), and brine

(10 mL), dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was purified by column chromatography (80%

EtOAc in hexanes) to afford 22 (50 mg, 77%) as a white solid;

[R]²⁰_D-39.3 (c 0.1, MeOH); ¹H NMR (CD₃OD) δ 7.39-7.02 (m,

35H), 5.08 (m, 6H), 4.65 (m, 2H), 4.36 (m, 1H), 4.23 (m, 2H),

3.95 (m, 1H), 3.04 (m, 2H), 2.80 (m, 2H), 2.60-2.22 (m, 7H),

2.07 (m, 4H), 1.89 (m, 4H), 1.63 (m, 5H), 1.46-1.10 (m, 14H),

1.40 (s, 9H), 0.90 (m, 12H); ¹³C NMR (CD₃OD) δ 177.5, 174.3,

173.6, 173.3, 172.2, 172.1, 172.0, 171.6, 170.9, 156.9, 144.9,

136.9, 136.5, 135.9, 129.3, 129.1, 128.6, 128.5, 128.4, 128.2,

127.8, 127.7, 126.9, 126.8, 126.5, 79.8, 74.3, 70.9, 67.1, 66.4,

60.6, 58.9, 54.4, 52.5, 49.8, 48.7, 48.5, 48.3, 48.0, 47.8, 47.6,

47.4, 37.3, 31.4, 30.5, 30.4, 30.1, 27.8, 27.7, 27.6, 24.9, 24.7,

22.9, 21.6, 17.4, 16.7, 13.5; MS (FAB): m/z 1533.1 [M + 1]⁺.

Compound 19. A solution of 16 (68 mg, 0.056 mmol) in

HCl (4 M in dioxane, 1 mL, 4 mmol) was stirred at 0 °C for 2

h, then saturated NaHCO₃(10 mL) was added and the aqueous

layer was extracted by EtOAc (4 × 10 mL). The combined

organic layers were dried (Na₂SO₄), filtered, and concentrated

in vacuo. To a solution of the residue in CH₂Cl₂(2 mL) were

added Boc-Valine (36.4 mg, 0.168 mmol), PyBOP (88 mg, 0.168

mmol), and i-Pr₂NEt (126 µL, 0.56 mmol). The reaction

mixture was stirred at room temperature for 24 h, EtOAc (15

mL) was added, and the separated organic layer was washed

with 1 N HCl (2 × 10 mL), saturated NaHCO₃(2 × 10 mL),

and brine (10 mL), dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was purified by column chromatography

Compound 23. Prepared from 20 according to the general

procedure for the preparation of 22 (57 mg, 56%); [R]²⁰_D-35.4

1

(c 2.3, MeOH); H NMR (CD₃OD) δ 7.46-7.00 (m, 30H), 5.08

(m, 6H), 4.62 (m, 1H), 4.48 (m, 1H), 4.36-4.20 (m, 2H), 3.88

(m, 1H), 3.64 (m, 1H), 3.36 (m, 1H), 2.81 (m, 1H), 2.44 (m,

8H), 2.23-1.20 (m, 28H), 1.42 (s, 9H), 1.28 (d, J ) 6.0 Hz,

2H), 0.96 (m, 16H); ¹³C NMR (CD₃OD) δ 177.3, 174.9, 174.6,

173.3, 173.0, 172.1, 171.8, 156.9, 144.9, 136.6, 129.1, 128.6,

128.4, 128.2, 127.8, 126.8, 79.8, 74.1, 67.1, 66.4, 58.5, 54.3, 51.9,

51.5, 51.2, 49.5, 31.4, 31.1, 30.5, 30.1, 27.7, 27.1, 26.7, 24.8,

22.9, 21.6, 18.8, 17.3, 16.9; MS (FAB): m/z 1384.9 [M + 1]⁺.

(66% EtOAc in hexanes) to afford 19 (43 mg, 59%) as a white

1

foam; [R]²⁰-32.8 (c 2.0, MeOH); H NMR (CD₃OD) δ 7.41-

D

7.05 (m, 30H), 5.08 (d, J ) 3.4 Hz, 4H), 4.67 (m, 2H), 4.38 (m,

1H), 4.26 (m, 1H), 4.05-3.70 (m, 2H), 3.08 (m, 2H), 2.80 (m,

2H), 2.55 (m, 1H), 2.36 (m, 2H), 2.06 (m, 2H), 1.86 (m, 1H),

1.78-1.18 (m, 25H), 1.28 (d, J ) 7.2 Hz, 3H), 0.91 (m, 12H);

¹³C NMR (CD₃OD) δ 178.5, 175.2, 174.3, 172.5, 171.8, 145.8,

137.8, 136.9, 130.3, 130.0, 129.5, 129.2, 128.7, 127.8, 75.2, 68.1,

67.4, 62.1, 61.3, 55.4, 53.5, 52.3, 52.0, 51.3, 50.8, 47.3, 38.7,

38.2, 32.4, 31.9, 31.1, 28.7, 28.1, 25.8, 25.7, 23.8, 22.5, 19.8,

19.7, 18.3, 18.2, 17.6; MS (FAB): m/z 1315 [M + 2]⁺.

Compound 24. Prepared from 21 according to the general

procedure for the preparation of 22 (63 mg, 78%); [R]²⁰_D-48.4

1

(c 1.0, MeOH); H NMR (CD₃OD) δ 7.33-7.16 (m, 25H), 5.10

(m, 4H), 4.37 (m, 1H), 4.20 (m, 1H), 4.08 (m, 1H), 3.90 (m,

1H), 3.70 (m, 1H), 3.36 (m, 1H), 2.86 (m, 1H), 2.70 (m, 1H),

2.58-2.26 (m, 4H), 2.06 (m, 3H), 1.95-1.52 (m, 8H), 1.51-

1.22 (m, 8H), 1.42 (d, J ) 3.3 Hz, 9H), 0.92 (m, 12H); ¹³C NMR

(CD₃OD) δ 178.5, 174.3, 173.1, 172.5, 171.8, 171.2, 157.9, 145.9,

137.5, 137.3, 130.0, 129.5, 129.2, 129.1, 128.7, 128.6, 127.8,

80.8, 75.2, 71.8, 71.7, 67.8, 67.4, 59.8, 55.2, 52.5, 52.0, 47.0,

42.1, 39.2, 33.1, 32.4, 31.5, 28.7, 28.4, 27.9, 26.5, 25.9, 25.7,

23.9, 23.4, 22.9, 22.5, 19.8, 18.3, 17.2; MS (FAB): m/z 1165.4

[M + 1]⁺.

Compound 20. Prepared from 17 according to the general

procedure for preparation of 19 (87 mg, 85%); ¹H NMR

(CD₃OD) δ 7.29 (m, 25H), 5.14 (d, J ) 7.1 Hz, 2H), 5.09 (d, J

) 3.5 Hz, 2H), 4.63 (t, J ) 7.0 Hz, 1H), 4.51 (m, 1H), 4.30 (q,

J ) 7.0 Hz, 1H), 3.95 (d, J ) 5.6 Hz, 1H), 3.87 (m, 1H), 2.79

(m, 2H), 2.55 (m, 1H), 2.43 (m, 2H), 2.31 (m, 1H), 2.18 (m,

1H), 1.99 (m, 3H), 1.82 (m, 1H), 1.64 (m, 7H), 1.42 (s, 9H),

1.45-1.20 (m, 6H), 1.27 (d, J ) 7.1 Hz, 3H), 0.94 (d, J ) 6.6

Hz, 6H), 0.88 (d, J ) 6.8 Hz, 6H); ¹³C NMR (CD₃OD) δ 177.3,

174.5, 173.1, 173.0, 171.1, 170.8, 167.5, 144.9, 136.5, 136.1,

129.1, 128.6, 128.5, 128.4, 128.2, 128.1, 127.7, 126.8, 79.7, 74.0,

70.8, 67.1, 66.4, 60.3, 51.9, 51.5, 51.1, 31.1, 30.0, 27.8, 26.7,

24.8, 24.7, 22.8, 21.5, 18.8, 17.3, 16.8; MS (FAB): m/z 1168.8

[M + 1]⁺.

Compound 3. A solution 22 (40 mg, 0.026 mmol) in TFA

(1 mL) was stirred at room temperature for 1 h. MeOH (2 mL)

was added to quench the reaction, and the solvents were

removed under reduced pressure at room temperature. The

residue was purified by column chromatography (16% MeOH

in CH₂Cl₂) to afford a white solid (13 mg, 42%); [R]²⁰_D-47.5 (c

0.1, MeOH). The white solid was dissolved in MeOH (1 mL),

10% Pd on carbon (3 mg) was added, and the mixture was

hydrogenated for 2 h at 1 atm. Filtration and evaporation

afforded 3 as a white solid (7.1 mg, 71%); ¹H NMR (D₂O) δ

7.35-7.05 (m, 5H), 5.00-4.78 (m, 4H), 4.33 (m, 1H), 4.10 (m,

3H), 3.90 (m, 1H), 3.86 (m, 1H), 3.10 (m, 1H), 2.87 (m, 1H),

2.72 (dd, J ) 15.7 Hz, J ) 5.8 Hz, 1H), 2.72 (dd, J ) 15.7 Hz,

J ) 5.8 Hz, 1H), 2.61 (dd, J ) 15.1 Hz, J ) 8.1 Hz, 1H), 2.54

(m, 1H), 2.40-1.78 (m, 9H), 1.79-1.41 (m, 6H), 1.38-1.20 (m,

2H), 1.25 (d, J ) 6.6 Hz, 3H), 0.86 (d, J ) 6.0 Hz, 6H), 0.77

(m, 6H); MS (FAB): m/z 919.2 [M + 1]⁺; HRMS calcd for

C₄₃H₆₇N₈O₁₄[M + 1]⁺919.4732; found 919.4772; LC/MS

retention time: [A] 11.41 min; [B] 10.45 min.

Compound 4. Prepared from 23 according to the general

procedure for the preparation of 3 (15.5 mg, 54%). [R]²⁰_D-42.0

(c 0.6, H₂O); ¹H NMR (D₂O) δ 4.30 (m, 1H), 4.20 (m, 1H), 4.05

(m, 1H), 3.90 (m, 1H), 2.65 (m, 1H), 2.44 (m, 1H), 2.40-2.10

(m, 8H), 2.06-1.95 (m, 9H), 1.86 (m, 2H), 1.67-1.38 (m, 5H),

1.28 (m, 4H), 0.90 (m, 22H); ¹³C NMR (D₂O) δ 179.7, 178.8,

176.0, 175.0, 172.7, 171.5, 170.1, 169.9, 74.2, 61.8, 60.1, 60.0,

52.9, 51.7, 50.2, 48.1, 46.1, 40.9, 37.1, 32.0, 31.6, 30.6, 27.8,

Compound 21. Prepared from 18 according to the general

procedure for preparation of 19 (91 mg, 78%); [R]²⁰-23.9 (c

D

3.2, CHCl₃); ¹H NMR (CDCl₃) δ 7.77 (d, J ) 6.0 Hz, 1H), 7.47

(s, 1H), 7.37-7.15 (m, 20H), 6.79 (d, J ) 9.1 Hz, 1H), 6.62 (br,

1H), 5.18-5.02 (m, 4H), 4.59-4.49 (m, 2H), 3.99-3.85 (m, 1H),

3.82 (t, J ) 5.9 Hz, 1H), 3.53 (br, 1H), 3.39 (br, 1H), 2.76-

2.60 (m, 3H), 2.29 (m, 1H), 2.08-1.86 (m, 1H), 1.75 (m, 2H),

1.47 (m, 4H), 1.47-1.34 (m, 5H), 1.40 (s, 9H), 0.92 (m, 12H);

¹³C NMR (CDCl₃) δ 176.1, 174.6, 173.7, 172.8, 171.3, 170.6,

156.6, 156.3, 144.7, 135.9, 129.1, 129.0, 128.7, 128.5, 128.4,

127.4, 80.7, 80.3, 74.1, 71.1, 67.3, 60.7, 59.8, 51.7, 51.4, 48.5,

47.9, 47.1, 41.1, 38.2, 31.2, 31.0, 30.3, 28.7, 28.6, 26.1, 25.1,

24.5, 23.8, 23.2, 22.2, 19.8, 19.7, 18.3, 18.0; MS (FAB): m/z

946.5 [M + 1]⁺; HRMS calcd for C₅₅H₇₂N₅O₉[M + 1]⁺946.5330;

found 946.5332.

Compound 22. A solution of 19 (54 mg, 0.042 mmol) in

HCl (4 M in dioxane, 3 mL, 12 mmol) was stirred at 0 °C for

1 h. Saturated NaHCO₃(5 mL) was added, and the separated

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5185

27.5, 27.0, 26.6, 25.5, 25.3, 24.5, 22.9, 21.6, 18.7, 18.1, 16.9;

MS (FAB): m/z 772.2 [M + 1]⁺; HRMS calcd for C₃₄H₅₈N₇O₁₃

[M + 1]⁺772.4048; found 772.4092; LC/MS retention time: [A]

9.32 min; [B] 8.54 min.

over Na₂SO₄, filtered, and concentrated in vacuo. The residue

was purified by column chromatography (50% EtOAc in

hexanes) to afford 28 (24 mg, 33%) as a white solid; [R]²⁰

D

1

-34.5 (c 0.4, CHCl₃); H NMR (CDCl₃): δ 7.57 (m, 1H), 7.27

(m, 1H), 5.11 (d, J ) 7.6 Hz, 1H), 5.03 (d, J ) 12.1 Hz, 1H),

4.85 (d, J ) 10.4 Hz, 1H), 4.62 (d, J ) 7.8 Hz, 1H), 4.61 (m,

1H), 4.56 (m, 1H); 4.09 (m, 1H), 3.58 (m, 1H), 3.32 (m, 1H),

2.68 (m, 2H), 2.56 (m, 4H), 2.39 (m, 2H), 2.10 (s, 3H), 2.02 (m,

1H), 1.88 (m, 1H), 1.73 (m, 1H), 1.60 (m, 1H), 1.54 (d, J ) 7.4

Hz, 3H), 1.42 (s, 9H), 1.37 (m, 1H), 0.91 (d, J ) 6.5 Hz, 3H),

0.89 (d, J ) 6.5 Hz, 3H); ¹³C NMR (CDCl₃): δ 175.6, 173.9,

173.7, 156.4, 148.6, 106.8, 95.0, 81.2, 76.8, 74.7, 54.7, 53.2, 49.7,

49.1, 46.9, 40.3, 38.5, 36.0, 30.9, 30.7, 28.7, 25.3, 23.7, 22.1,

17.4, 15.8; MS (FAB): m/z 674.2 [M + 1]⁺; HRMS calcd for

C₂₈H₄₇Cl₃N₃O₇S [M + 1]⁺674.2200; found 674.2218.

Compound 5. Prepared from 24 according to the general

procedure for the preparation of 3 (10.9 mg, 60%) except that

the hydrogenation was carried out in MeOH/H₂O (1:1) instead

of MeOH for 12 h. [R]²⁰_D-37.8 (c 0.2, MeOH); ¹H NMR (D₂O)

δ 4.10 (m, 3H), 3.82 (m, 1H), 3.45 (m, 3H), 2.73 (m, 12H), 2.60-

2.31 (m, 4H), 2.22 (m, 1H), 2.17-1.78 (m, 6H), 1.76-1.41 (m,

6H), 1.34 (m, 4H), 0.90 (m, 2H); ¹³C NMR (D₂O) δ 180.5, 180.0,

178.7, 175.2, 173.1, 172.9, 172.0, 170.3, 74.3, 74.1, 60.4, 52.4,

51.7, 51.0, 49.1, 48.1, 46.1, 40.9, 37.0, 31.4, 30.6, 29.3, 26.9,

26.3, 25.2, 24.5, 23.8, 22.8, 21.6, 18.7, 18.0, 16.4; MS (FAB):

m/z 645.5 [M + 1]⁺; HRMS calcd for C₂₉H₅₁N₆O₁₀[M + 1]⁺

643.3622; found 643.3662; LC/MS retention time: [A] 8.38 min;

[B] 7.67 min.

Compound 29. A solution of 26 (76 mg, 0.13 mmol) in HCl

(4 M in dioxane, 2 mL) was stirred at room temperature for 1

h. The solvent was removed under reduced pressure, then the

residue was dissolved in CH₂Cl₂(3 mL) and cooled to 0 °C.

Boc-Met-OH (48.6 mg, 0.19 mmol) was added followed by

PyBOP (74.3 mg, 0.14 mmol) and iPr₂NEt (90 µL, 0.52 mmol).

The reaction mixture was allowed to warm to room temper-

ature over a period of 3 h. The solution was diluted with EtOAc

(15 mL), washed with 1 N HCl (1 mL) and saturated NaHCO₃

(1 mL). The organic phase was dried over Na₂SO₄, filtered,

and concentrated in vacuo. The residue was purified by column

chromatography (70% EtOAc in hexanes) to afford 29 (65 mg,

Compound 25. Ac-Leu-OCH₂CCl₃(110 mg, 0.34 mmol) was

stirred with HCl (4 M in dioxane, 1 mL) at room temperature

for 1 h. The solvent was removed under reduced pressure, then

the residue was dissolved in CH₂Cl₂(3 mL) and cooled to 0

°C. The acid from 10 (78 mg, 0.20 mmol) was added followed

by PyBOP (120 mg, 0.20 mmol) and i-Pr₂NEt (155 µL, 0.80

mmol). The reaction mixture was allowed to warm to room

temperature over a period of 3 h. The reaction mixture was

diluted with EtOAc (15 mL) and washed with 1 N HCl (2 mL)

and saturated NaHCO₃(2 mL). The organic phase was dried

over Na₂SO₄and concentrated in vacuo. The residue was

purified by column chromatography (50% EtOAc in hexanes)

to afford 25 (77 mg, 75%) as a colorless oil; [R]²⁰_D-22.5 (c 1.5,

75%) as a white solid; [R]²⁰-24.4 (c 0.5, CHCl₃); ¹H NMR

D

(CDCl₃): δ 7.33 (d, J ) 9.1 Hz, 1H), 7.26 (m, 1H), 5.21 (d, J )

8.6 Hz, 1H), 4.62 (m, 1H), 4.61 (d, J ) 16.0 Hz, 1H), 4.13 (m,

1H), 3.97 (m, 1H), 3.38 (m, 1H), 3.02 (m, 1H), 2.54 (m, 4H),

2.40 (m, 3H), 2.10 (s, 3H), 2.04 (m, 1H), 1.93 (m, 1H), 1.62 (m,

2H), 1.54 (d, J ) 9.8 Hz, 3H), 1.41 (s, 9H), 1.33 (m, 2H), 0.91

(d, J ) 8.9 Hz, 3H), 0.89 (d, J ) 8.9 Hz, 3H); ¹³C NMR (CDCl₃):

δ 216.9, 174.0, 173.6, 172.4, 156.3, 95.0, 81.2, 74.8, 74.7, 54.9,

51.1, 48.9, 44.6, 43.9, 41.3, 41.0, 39.6, 31.1, 30.8, 28.6, 25.2,

23.7, 22.1, 17.7, 15.8; MS (FAB): m/z 676.1 [M + 1]⁺; HRMS

calcd for C₂₇H₄₅Cl₃N₃O₈S [M + 1]⁺676.1993; found 676.2004.

1

CHCl₃); H NMR (CDCl₃): δ 6.09 (d, J ) 7.4 Hz, 1H), 4.93 (d,

J ) 11.9 Hz, 1H), 4.88 (m, 2H), 4.76 (m, 1H), 4.64 (d, J ) 11.9

Hz, 1H), 3.81 (m, 1H), 3.64 (m, 1H), 2.62 (m, 3H), 2.52 (m,

2H), 2.42 (m, 1H), 1.75 (m, 1H), 1.58 (s, 3H), 1.50 (d, J ) 7.3

Hz, 3H), 1.46 (s, 9H), 1.44 (s, 3H), 1.40 (m, 2H), 0.91 (d, J )

6.4 Hz, 6H); ¹³C NMR (CDCl₃): δ 174.1, 171.9, 152.1, 148.9,

107.2, 94.9, 80.2, 78.5, 77.7, 74.6, 58.9, 48.4, 48.0, 46.5, 37.8,

33.1, 28.9, 27.2, 25.5, 24.8, 21.9, 18.6; MS (FAB): m/z 583, [M

+ 1]⁺; HRMS calcd for C₂₆H₄₂Cl₃N₂O₆[M + 1]⁺583.2108; found

583.2123.

Compound 30. Prepared from 27 according to the general

procedure for the preparation of 28 (75%); [R]²⁰-40.4 (c )

D

1

Compound 26. Prepared from 11 according to the general

procedure for the preparation of 25 (75%); [R]²⁰_D-65.6 (c 0.4,

CHCl₃); ¹H NMR (CDCl₃): δ 7.26 (m, 1H), 6.22 (d, J ) 7.4 Hz,

1H), 4.94 (d, J ) 11.9 Hz, 1H), 4.76 (m, 1H), 4.64 (d, J ) 11.9

Hz, 1H), 3.57 (m, 1H), 2.93 (q, J ) 9.1 Hz, 1H), 2.73 (dd, J )

8.2 Hz, J ) 7.4 Hz, 1H), 2.56 (d, J ) 9.4 Hz, 2H), 2.39 (m,

2H), 1.84 (m, 1H), 1.56 (s, 3H), 1.53 (d, J ) 7.2 Hz, 3H), 1.45

(s, 12H), 1.38 (m, 2H), 0.91 (m, 6H), ¹³C NMR (CDCl₃): δ 214.8,

172.8, 171.6, 152.0, 94.8, 80.5, 74.7, 58.9, 48.6, 45.2, 43.6, 42.9,

38.4, 28.9, 27.0, 25.4, 24.7, 21.9, 18.6; MS (FAB): m/z 585.1

[M + 1]⁺; HRMS calcd for C₂₅H₃₉Cl₃N₂O₇[M + 1]⁺585.1901;

found 585.1889.

1.9, CHCl₃); H NMR (CDCl₃): δ 7.26 (br, 1H), 6.96 (br, 1H),

5.04 (d, J ) 6.0 Hz, 1H), 5.00 (d, J ) 12.1 Hz, 1H), 4.66 (m,

1H), 4.63 (d, J ) 12.1 Hz, 1H), 4.11 (m, 1H), 3.58 (b, 1H), 3.32

(b, 1H), 2.55 (d, J ) 11.9 Hz, 1H), 2.46 (m, 1H), 2.28 (m, 1H),

2.09 (s, 3H). 2.01 (m, 2H), 1.85 (m, 3H), 1.65 (m, 5H), 1.52 (t,

J ) 7.0 Hz, 2H), 1.43 (s, 9H), 0.90 (d, J ) 6.6 Hz, 3H), 0.88 (d,

J ) 6.6 Hz, 3H); ¹³C NMR (CDCl₃): δ 176.5, 173.8, 156.4, 95.0,

81.0, 77.7, 74.7, 54.6, 53.5, 50.2, 49.0, 47.2, 40.4, 32.4, 31.0,

30.7, 28.8, 28.6, 25.4, 24.4, 23.6, 22.2, 17.4, 15.8; MS (FAB):

m/z 662.3 [M + 1]⁺; HRMS calcd for C₂₇H₄₇N₃O₆Cl₃[M + 1]⁺

662.2200; found 662.2182.

Compound 31. A solution of 28 (20.0 mg, 0.030 mmol) in

a mixture of formic acid (98%, 1 mL) and H₂O (20 µL) was

stirred at room temperature for 2 h. The mixture was

concentrated under reduced pressure, the residue was dis-

solved in EtOAc (5 mL) and washed with NaHCO₃(1 N, 0.5

mL). The solvent was evaporated, and the residue was dis-

solved in a mixture of CH₂Cl₂(1.0 mL) and water (1.0 mL)

and cooled to 0 °C. Ac-L-Leu-OH (10.4 mg, 0.060 mmol) was

added followed by HOBt (8.1 mg, 0.060 mmol) and EDC (12.7

mg, 0.060 mmol). The mixture was stirred at 0 °C for 24 h,

then diluted with EtOAc (15 mL) and washed with HCl (1 N,

0.2 mL) and NaHCO₃(1 N, 0.2 mL). The organic layer was

dried over Na₂SO₄, filtered, and concentrated in vacuo. The

residue was purified by column chromatography (EtOAc then

10% MeOH in CH₂Cl₂) to afford 31 (9.5 mg, 44%) as a white

solid; [R]²⁰_D-50.3 (c 0.4, CHCl₃); ¹H NMR (CDCl₃): δ 7.39 (m,

1H), 7.26 (m, 2H), 6.59 (m, 1H), 5.00 (d, J ) 12.0 Hz, 1H),

4.87 (m, 2H), 4.77 (t, J ) 7.2 Hz, 1H), 4.62 (d, J ) 12.0 Hz,

1H), 4.57 (m, 1H), 4.39 (m, 1H), 3.99 (m, 1H), 3.49 (m, 1H),

2.81 (m, 1H), 2.70 (m, 1H), 2.55 (m, 4H), 2.49 (m, 1H), 2.31

(m, 1H), 2.09 (m, 1H), 2.08 (s, 3H), 2.03 (s, 3H), 1.62 (m, 4H),

1.53 (d, J ) 7.2 Hz, 3H), 1.49 (m, 2H), 1.34 (m, 2H), 0.90 (m,

12H); ¹³C NMR (CDCl₃): δ 175.0, 173.1, 173.0, 171.6, 171.4,

Compound 27. Prepared from 12 according to the general

procedure for the preparation of 25 (71%); [R]²⁰_D-27.8 (c 0.5,

1

CHCl₃); H NMR (CDCl₃): δ 6.01 (d, J ) 7.3 Hz, 1H), 4.93 (d,

J ) 11.9 Hz, 1H), 4.73 (m, 1H), 4.62 (d, J ) 11.9 Hz, 1H), 3.76

(m, 1H), 3.65 (m, 1H), 2.51 (m, 1H), 2.41 (m, 1H), 1.93 (m,

1H), 1.80 (m, 2H), 1.58 (s, 3H), 1.49 (d, J ) 7.2 Hz, 3H), 1.45

(s, 12H), 1.80-1.40 (m, 6H), 0.91 (d, J ) 6.2 Hz, 6H); ¹³C NMR

(CDCl₃): δ 175.3, 172.0, 152.1, 94.9, 94.0, 80.1, 77.7, 74.6, 74.5,

59.3, 48.7, 48.4, 46.7, 32.0, 30.8, 28.9, 27.5, 26.2, 25.7, 25.6,

24.7, 21.9, 18.6; MS (FAB): m/z 571 [M + 1]⁺; HRMS calcd

for C₂₅H₄₂N₂O₆Cl₃[M + 1]⁺571.2108; found 571.2097.

Compound 28. A solution of (67.2 mg, 0.115 mmol) in a

mixture of formic acid (98%, 2.0 mL), CH₂Cl₂(1.0 mL), and a

drop of water was stirred at room temperature for 3 h. The

solvents were removed at room temperature under reduced

pressure, and the residue was dissolved in CH₂Cl₂(3 mL) and

cooled to 0 °C. Boc-Met-OH (43.0 mg, 0.172 mmol) was added

followed by PyBOP (58.0 mg, 0.115 mmol) and i-Pr₂NEt (89

µL, 0.46 mmol). The reaction mixture was allowed to warm to

room temperature over a period of 3 h. The solution was

diluted with EtOAc (10 mL) and washed with 1 N HCl (1 mL)

and saturated NaHCO₃(1 mL). The organic layer was dried

5186 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

107.4, 95.0, 74.6, 73.9, 53.5, 52.9, 51.6, 48.4, 48.0, 41.8, 41.0,

36.3, 33.5, 30.6, 30.4, 30.1, 25.3, 23.7, 23.4, 23.1, 22.7, 22.3,

18.3, 15.7; MS (FAB): m/z 729.2 [M + 1]⁺; HRMS calcd for

C₃₁H₅₂Cl₃N₄O₇S [M + 1]⁺729.2622; found 729.2621.

22.9, 22.4, 21.5, 21.2, 21.1, 17.9, 14.3; MS (FAB): m/z 601.1

[M + 1]⁺; HRMS calcd for C₂₈H₄₉N₄O₈S [M + 1]⁺601.3226;

found 601.3267; LC/MS retention time: [A] 12.31 min; [B]

11.27 min.

Compound 32. A solution of 29 (42 mg, 0.066 mmol) in

HCl (4 M in dioxane, 2.0 mL) was stirred at room temperature

for 1 h. The solution was concentrated under reduced pressure

at room temperature. The residue was dissolved in EtOAc (5

mL) and washed with NaHCO₃(1 N, 0.5 mL). The organic

layer was concentrated under reduced pressure, and the

residue was dissolved in a mixture of CH₂Cl₂(1 mL) and water

(1 mL). Ac-Leu-OH (11.4 mg, 0.066 mmol) was added at 0 °C

followed by HOBt (8.9 mg, 0.066 mmol) and EDC (14.0 mg,

0.073 mmol). The reaction mixture was stirred at 0 °C for 24

h, diluted with EtOAc (15 mL), and then washed with HCl (1

N, 0.2 mL) and NaHCO₃(1 N, 0.2 mL). The organic phase was

dried over Na₂SO₄, filtered, and concentrated in vacuo. The

residue was purified by column chromatography (EtOAC then

Compound 36. Prepared from 33 according to the general

procedure for the preparation of 34 (17.2 mg, 91%); [R]²⁰_D-53.0

(c 0.4, MeOH); ¹H NMR (CD₃OD): δ 4.44 (m, 1H), 4.35 (t, J )

6.8 Hz, 3H), 4.19 (q, J ) 7.1 Hz, 1H), 3.89 (m, 1H), 3.41 (m,

1H), 2.57 (m, 3H), 2.24 (m, 1H), 2.09 (s, 3H), 2.02 (m, 1H),

2.00 (s, 3H), 1.86 (m, 1H), 1.70 (m, 5H), 1.59 (m, 3H), 1.48 (m,

1H), 1.37 (d, J ) 7.1 Hz, 3H), 1.31 (m, 1H), 0.98 (d, J ) 6.5

Hz, 3H), 0.93 (m, 9H); ¹³C NMR (CD₃OD): δ 180.6, 176.9, 174.3,

172.7, 172.5, 73.7, 53.4, 52.7, 51.4, 51.2, 41.7, 40.7, 31.2, 30.7,

30.3, 26.9, 24.9, 24.8, 24.7, 22.8, 22.4, 21.5, 21.4, 21.0, 17.5,

14.3; MS (FAB): m/z 587.3 [M + 1]⁺; HRMS calcd for

C₂₈H₅₁N₄O₇S [M + 1]⁺587.3434; found 587.3474; LC/MS

retention time: [A] 15.60 min; [B] 14.29 min

Compound 37. To a solution of KOH (280 mg, 5.0 mmol)

in MeOH (20 mL) and H₂O (10 mL) was added 12 (770 mg, 2

mmol). The reaction mixture was stirred at 65 °C for 3 h then

at room-temperature overnight. HCl (1 N, 20 mL) was added,

and the aqueous phase was extracted with CH₂Cl₂(4 × 20 mL).

The combined organic layers were dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was dissolved in CH₂Cl₂

(35 mL), followed by addition of H-Val-OBn‚TsOH salt (1.14

g, 3 mmol), PyBOP (1.59 g, 3 mmol) and i-Pr₂NEt (1.78 mL,

10.2 mmol) at 0 °C. The reaction mixture was stirred for 3.5

h at 0 °C, then was partitioned in EtOAc (80 mL) and 1 N

HCl (20 mL). The separated organic layer was washed with

saturated NaHCO₃(20 mL) and brine (20 mL). The organic

layer was dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was purified by column chromatography (20%

10% MeOH in CH₂Cl₂) to afford 32 (34.2 mg, 76%) as a white

1

solid; [R]²⁰-68.7 (c 0.6, CHCl₃); H NMR (CDCl₃): δ 7.75 (d,

D

J ) 9.3 Hz, 1H), 7.58 (d, J ) 9.8 Hz, 1H), 7.18 (d, J ) 12.3 Hz,

1H), 7.11 (d, J ) 7.6 Hz, 1H), 4.96 (d, J ) 15.9 Hz, 1H), 4.75

(m, 1H), 4.60 (d, J ) 15.9 Hz, 1H), 4.54 (m, 1H), 4.48 (m, 1H),

4.20 (m, 1H), 4.05 (m, 1H), 3.51 (m, 1H), 3.13 (m, 1H), 2.56

(m, 3H), 2.49 (m, 2H), 2.32 (m, 2H), 2.09 (m, 2H), 2.06 (s, 3H),

2.00 (s, 3H), 1.68 (m, 2H), 1.55 (d, J ) 9.7 Hz, 3H), 1.31 (m,

2H), 0.89 (m, 13H); ¹³C NMR (CDCl₃): δ 217.5, 174.0, 173.6,

172.4, 172.3, 171.9, 94.9, 74.6, 73.4, 53.7, 53.6, 50.4, 48.4, 45.3,

44.0, 42.0, 40.92, 40.8, 38.6, 30.7, 30.0, 25.3, 23.7, 23.1, 22.3,

18.3, 15.7; MS (FAB): m/z 731.1 [M + 1]⁺; HRMS calcd for

C₃₀H₅₀Cl₃N₄O₈S [M + 1]⁺731.2415; found 731.2433.

Compound 33. Prepared from 30 according to the general

procedure for the preparation of 32 (52%); [R]²⁰_D-71.5 (c 1.2,

EtOAc in hexanes) to afford 37 (1.1 g, 98%); as a colorless oil;

1

CHCl₃); H NMR (CDCl₃): δ 7.46 (d, J ) 7.0 Hz, 1H), 7.20 (d,

[R]²⁰-21.1 (c 1.0, CHCl₃); H NMR (CDCl₃) δ 7.36-7.19 (m,

D

J ) 8.9 Hz, 1H), 6.96 (d, J ) 7.0 Hz, 1H), 6.60 (m, 1H), 4.97

(d, J ) 11.9 Hz, 1H), 4.71 (m, 1H), 4.62 (d, J ) 11.9 Hz, 1H),

4.54 (m, 1H), 4.44 (m, 1H), 4.06 (m, 1H), 3.92 (m, 1H), 3.45

(m, 1H), 2.62 (m, 1H), 2.53 (m, 1H), 2.26 (m, 1H), 2.08 (s, 3H),

2.04 (m, 2H), 2.02 (s, 3H), 1.71 (m, 3H), 1.61 (m, 6H), 1.51 (d,

J ) 7.3 Hz, 3H), 1.37 (m, 1H), 0.90 (m, 12H); ¹³C NMR (CDCl₃):

δ 176.1, 173.0, 172.9, 171.8, 171.2, 95.0, 77.6, 74.6, 53.5, 52.7,

51.9, 48.6, 48.5, 48.3, 41.7, 41.4, 30.7, 30.6, 30.2, 26.5, 25.2,

24.6, 23.7, 23.4, 23.0, 22.8, 22.4, 18.2, 15.8; MS (FAB): m/z

717.6 [M + 1]⁺; HRMS calcd for C₃₀H₅₂N₄O₇Cl₃[M + 1]⁺

717.2622; found 717.2609.

5H), 6.06 (d, J ) 7.5 Hz, 1H), 5.10 (m, 2H), 4.60 (m, 1H), 3.77

(br, 1H), 3.60 (br, 1H), 2.42 (br, 2H), 2.14 (m, 1H), 1.96-1.20

(m, 15H), 1.40 (d, J ) 5.5 Hz, 9H), 0.92 (m, 12H); ¹³C NMR

(CDCl₃) δ 175.6, 172.3, 152.0, 135.6, 128.9, 128.8, 128.7, 94.1,

82.4, 79.9, 67.4, 60.7, 59.2, 57.2, 49.0, 47.1, 44.5, 43.0, 31.9,

31.7, 30.6, 28.8, 27.9, 27.3, 25.4, 24.7, 21.8, 21.4, 19.5, 19.3,

17.9, 14.6; MS (FAB): m/z 559.4 [M + 1]⁺.

Compound 38. To a solution of 37 (559 mg, 1 mmol) in

CH₂Cl₂(16 mL) was added TFA (4 mL). The reaction mixture

was stirred at room temperature for 1 h, then quenched by

addition of saturated NaHCO₃(15 mL) and extracted with

EtOAc (4 × 20 mL). The combined organic layers were dried

(Na₂SO₄), filtered, and concentrated in vacuo. The residue was

dissolved in CH₂Cl₂(15 mL), followed by addition of Boc-Met-

OH (499 mg, 2 mmol), PyBOP (1.059 g, 2 mmol), and i-Pr₂NEt

(696 µL, 4 mmol) at 0 °C. The reaction mixture was stirred at

0 °C for 3 h. EtOAc (80 mL) was added, and the separated

organic layer was washed by 1 N HCl (2 × 20 mL), saturated

NaHCO₃(2 × 20 mL), and brine (20 mL). The organic layer

was dried (Na₂SO₄), filtered, and concentrated in vacuo. The

residue was purified by column chromatography (66% EtOAc

Compound 34. To a solution of 31 in THF (0.4 mL) were

added KH₂PO₄and zinc powder (100 mg) under Ar atmo-

sphere. The reaction mixture was stirred for 1 h. After the

disappearance of starting material on TLC, the Zn powder was

filtered, and the filtrate was concentrated under reduced

pressure. The residue was purified by column chromatography

(20% MeOH in CH₂Cl₂) to afford 34 (5.0 mg, 71%) as a white

solid; [R]²⁰-51.9 (c 0.2, MeOH); ¹H NMR (CD₃OD): δ 4.84

D

(m, 2H), 4.47 (m, 1H), 4.34 (m, 1H), 4.23 (m, 1H), 3.94 (m,

1H), 3.43 (t, J ) 4.2 Hz, 1H), 2.76 (m, 1H), 2.61 (m, 1H), 2.53

(m, 2H), 2.40 (m, 2H), 2.29 (m, 1H), 2.09 (s, 3H), 2.00 (s, 3H),

1.68 (m, 1H), 1.59 (m, 4H), 1.45 (m, 1H), 1.38 (d, J ) 7.1 Hz,

3H), 1.29 (m, 2H), 0.91 (m, 12H); ¹³C NMR (CD₃OD): δ 175.0,

174.5, 174.0, 172.5, 172.4, 150.3, 105.4, 72.1, 53.1, 52.5, 51.7,

50.8, 49.0, 48.8, 41.4, 40.8, 36.5, 32.4, 31.4, 30.2, 24.9, 24.8,

22.8, 22.3, 21.4, 21.3, 21.0, 18.3, 14.3; MS (FAB): m/z 599.3

[M + 1]⁺; HRMS calcd for C₂₉H₅₁N₄O₇S [M + 1]⁺599.3478;

found 599.3471; LC/MS retention time: [A] 22.33 min; [B]

15.16 min.

in hexanes) to afford 38 (440 mg, 68%) as a colorless oil; [R]²⁰

D

-101.2 (c 0.1, MeOH); ¹H NMR (CDCl₃) δ 7.42-7.19 (m, 5H),

6.91 (d, J ) 6.4 Hz, 1H), 5.84 (d, J ) 8.3 Hz, 1H), 5.63 (d, J )

7.6 Hz, 1H), 5.12 (m, 2H), 4.67 (br, 1H), 4.51 (m, 1H), 4.41 (m,

1H), 4.16 (m, 1H), 3.56 (m, 1H), 3.39 (br, 1H), 2.50 (m, 3H),

2.29 (m, 1H), 2.13 (m, 1H), 2.01 (s, 3H), 1.87 (m, 2H), 1.74 (m,

2H), 1.61 (m, 5H), 1.40-1.21 (m, 1H), 1.36 (d, J ) 8.6 Hz, 9H),

0.84 (m, 12H); ¹³C NMR (CDCl₃) δ 176.7, 174.0, 173.7, 156.4,

135.7, 129.0, 128.8, 128.7, 127.8, 80.7, 76.6, 67.5, 58.7, 54.5,

53.6, 50.3, 47.4, 40.5, 32.1, 31.2, 30.9, 30.6, 28.7, 27.8, 25.3,

24.4, 23.7, 22.1, 19.7, 19.1, 15.7; MS (FAB): m/z 650.4 [M +

1]⁺; HRMS calcd for C₃₄H₅₆N₃O₇S [M + 1]⁺650.3839; found

650.3811.

Compound 35. Prepared from 32 according to the general

procedure for the preparation of 34 (12 mg, 77%); [R]²⁰_D-32.2

(c 0.6, MeOH); ¹H NMR (CD₃OD): δ 4.58 (m,1H), 4.48 (m, 1H),

4.34 (t, J ) 4.7 Hz, 1H), 4.22 (m, 1H), 4.04 (m, 1H), 3.53 (m,

1H), 3.11 (m, 1H), 2.68 (m, 1H), 2.58 (m, 1H), 2.44 (m, 2H),

2.39 (m, 1H), 2.30 (m, 1H), 2.09 (s, 3H), 2.00 (s, 3H), 1.94 (m,

1H), 1.69 (m, 1H), 1.58 (m, 4H), 1.44 (m, 1H), 1.40 (d, J ) 6.9

Hz, 3H), 1.29 (m, 1H), 0.93 (m, 12H); ¹³C NMR (CD₃OD): δ

218.4, 174.5, 174.4, 174.1, 172.7, 72.4, 53.2, 52.7, 51.0, 44.7,

44.6, 44.1, 41.2, 40.7, 38.4, 31.3, 30.3, 27.9, 24.9, 24.8, 24.9,

Compound 39. A solution of 38 (325 mg, 0.5 mmol) in HCl

(4 M in dioxane, 10 mL, 40 mmol) was stirred at room

temperature for 1 h, then the solvent was removed under

reduced pressure. The residue was dissolved in a saturated

solution of NaHCO₃(10 mL) and extracted with EtOAc (4 ×

15 mL). The combined organic layers were dried (Na₂SO₄),

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5187

filtered, and concentrated in vacuo. The residue was dissolved

in CH₂Cl₂(5 mL), followed by addition of water (5 mL), Ac-

Leu-OH (86.5 mg, 0.5 mmol), and HOBt (67.7 mg, 0.5 mmol),

then cooled in an ice bath to 0 °C, and EDC (105 mg, 0.5 mmol)

was added, then stirred at 0 °C for 36 h. Aqueous HCl (1 N, 5

mL) was added, the layers were partitioned, and the organic

layer was washed sequentially with aqueous HCl (0.5 N, 10

mL), brine (10 mL), NaHCO₃(1 N, 2 × 10 mL), and brine (10

mL). The organic layer was dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was purified by column

for 1 h. The solvent was removed under reduced pressure, and

the residue was dissolved in CH₂Cl₂(2.0 mL) and cooled to 0

°C. Boc-Met-OH (25 mg, 0.101 mmol) was added followed by

PyBOP (52.6 mg, 0.101 mmol) and i-Pr₂NEt (48 µL, 2.73

mmol). The mixture was allowed to warm to room temperature

over a period of 2 h, diluted with EtOAc (10 mL), and washed

with HCl (1 N, 1 mL) and saturated NaHCO₃(1 mL)). The

organic phase was dried over Na₂SO₄, filtered, and concen-

trated in vacuo. The residue was purified by column chroma-

tography (EtOAc) to afford 43 (41 mg, 72%) as a white solid;

[R]²⁰_D-41.7 (c 0.6, MeOH); ¹H NMR (CDCl₃): δ 8.19 (br, 1H),

7.56 (br, 1H), 6.29 (br, 1H), 5.22 (br, 1H), 4.28 (m, 1H), 4.28

(m, 1H), 3.87 (m, 1H), 3.61 (m, 2H), 3.31 (m, 1H), 3.20 (m,

1H), 2.86 (m, 1H), 2.56 (m, 4H), 2.36 (m, 1H), 2.11 (s, 3H),

2.09 (m, 1H), 1.92 (m, 1H), 1.50 (m, 2H), 1.46 (m, 2H), 1.41 (s,

9H), 1.33 (m, 3H), 0.85-0.98 (m, 15H); ¹³C NMR (CDCl₃): δ

215.0, 173.8, 173.4, 172.3, 156.1, 80.6, 75.4, 54.2, 52.8, 47.1,

43.6, 43.2, 39.8, 39.2, 31.2, 30.4, 30.1, 29.9, 28.1, 24.7, 23.7,

23.1, 21.5, 19.9, 19.3, 15.5, 15.2, 15.1; MS (FAB): m/z 651.4

[M + 23]⁺; HRMS calcd for C₃₁H₅₇N₄O₇S [M + 1]⁺629.3947;

found 629.3931.

chromatography (EtOAc) to afford 39 (204 mg, 58%) as a white

1

foam; [R]²⁰-23.9 (c 3.2, MeOH); H NMR (CD₃OD) δ 7.40-

D

7.22 (m, 5H), 5.21 (d, J ) 12.3 Hz, 1H), 5.11 (d, J ) 12.3 Hz,

1H), 4.43 (q, J ) 5.0 Hz, 1H), 4.32 (m, 2H), 3.90 (m, 1H), 3.43

(t, J ) 4.6 Hz, 1H), 2.66 (m, 1H), 2.53 (m, 2H), 2.31 (m, 1H),

2.20-1.80 (m, 6H), 2.08 (s, 3H), 1.98 (s, 3H), 1.68 (m, 7H), 1.56

(t, J ) 7.4 Hz, 3H), 1.45 (m, 1H), 1.27 (m, 1H), 0.94 (m, 18H);

¹³C NMR (CD₃OD) δ 178.5, 175.0, 173.3, 173.1, 172.9, 137.5,

129.6, 129.5, 129.4, 74.4, 67.7, 59.7, 54.7, 53.6, 52.7, 42.4, 41.9,

32.0, 31.8, 31.7, 31.5, 26.9, 25.9, 25.8, 25.7, 23.8, 23.2, 22.2,

22.0, 19.2, 18.8, 15.2; MS (FAB): m/z 705.8 [M + 1]⁺.

Compound 40. To a solution of 39 (84 mg, 0.119 mmol) in

10% formic acid in MeOH was added Pd black (90 mg) under

Ar atmosphere. The mixture was stirred at room temperature

for 30 min. The reaction mixture was filtered through a pad

of Celite and washed with MeOH. The collected filtrate was

evaporated under reduced pressure, and the residue was

purified by column chromatography (20% MeOH in CH₂Cl₂)

Compound 44. Prepared from 42 according to the general

procedure for the preparation of 43 (26.8 mg, 75%); [R]²⁰_D-45.5

(c 1.0, CHCl₃); ¹H NMR (CDCl₃): δ 8.28 (br, 1H), 7.27 (br, 1H),

6.58 (br, 1H), 6.9 (m, 1H), 5.26 (d, J ) 6.9 Hz, 1H), 5.00 (br,

1H), 4.31 (t, J ) 6.4 Hz, 1H), 4.22 (q, J ) 7.0 Hz, 1H), 3.40

(m, 1H), 3.28 (m, 1H), 3.21 (m, 2H), 2.57 (m, 2H), 2.38 (m,

1H), 2.36 (m, 1H), 2.10 (s, 3H), 1.91 (m, 2H), 1.88 (m, 2H),

1.74 (m, 2H), 1.63 (m, 4H), 1.47 (m, 2H), 1.43 (s, 9H), 1.37 (d,

J ) 7.2 Hz, 3H), 1.34 (m, 3H), 0.91 (m, 9H); ¹³C NMR (CDCl₃):

δ 176.8, 174.3, 173.8, 156.4, 80.8, 54.8, 53.6, 50.9, 50.4, 47.3,

40.6, 39.7, 32.9, 31.9, 31.4, 30.7, 29.1, 28.6, 25.4, 24.2, 23.6,

22.2, 20.4, 18.2, 15.7, 14.2; MS (FAB): m/z 587.1 [M + 1]⁺;

609.2 [M + 23]⁺; HRMS calcd for C₂₉H₅₅N₄O₆S [M + 1]⁺

587.3842; found 587.3854.

to afford 40 (54 mg, 74%) as a white solid; [R]²⁰-104.5 (c

D

0.5, MeOH); ¹H NMR (CD₃OD) δ 4.45 (q, J ) 5.0 Hz, 1H), 4.32

(m, 2H), 3.91 (m, 1H), 3.48 (t, J ) 4.3 Hz, 1H), 2.71 (m, 1H),

2.52 (m, 2H), 2.26 (m, 1H), 2.20-1.80 (m, 5H), 2.09 (s, 3H),

1.98 (s, 3H), 1.68 (m, 7H), 1.56 (t, J ) 7.4 Hz, 3H), 1.43 (m,

1H), 1.28 (m, 1H), 0.95 (m, 18H); ¹³C NMR (CD₃OD) δ 177.5,

174.4, 174.1, 172.5, 172.3, 73.2, 58.6, 53.1, 52.6, 51.8, 41.3, 40.8,

31.1, 30.7, 30.5, 30.1, 25.0, 24.9, 24.8, 23.0, 22.4, 21.4, 21.3,

21.1, 18.8, 17.8, 14.3; MS (FAB): m/z 637.3 [M + 23]⁺; HRMS

calcd for C₃₀H₅₅N₄O₇S [M + 1]⁺615.3747; found 615.3789; LC/

MS retention time: [A] 17.89 min; [B] 16.38 min.

Compound 45. A solution of 43 (24.4 mg, 0.038 mmol) in

HCl (4 M in dioxane, 1 mL) was stirred at room temperature

for 1 h. After removal of the solvent under reduced pressure,

the residue was dissolved in EtOAc and washed with NaHCO₃

(1 N, 0.5 mL) and water. The solvent was removed and the

residue dissolved in a mixture of CH₂Cl₂(0.75 mL) and water

(0.75 mL) and cooled to 0 °C. Ac-Leucine (13.1 mg, 0.076 mmol)

was added followed by EDC (16.1 mg, 0.076 mmol) and HOBt

(10.2 mg, 0.076 mmol). The reaction mixture was stirred at 0

°C for 24 h, then diluted with EtOAc (5 mL), washed with HCl

(1 N, 0.2 mL) and NaHCO₃(1 N, 0.2 mL). The organic phase

was dried over Na₂SO₄, filtered, and concentrated in vacuo.

The residue was purified by column chromatography (10%

MeOH in CH₂Cl₂) to give the 45 (20.8 mg, 78%) as a white

Compound 41. Boc-Val-NHBu (43 mg, 0.158 mmol) was

stirred with HCl (4 M in dioxane, 1 mL) at room temperature

for 1 h. The solvent was removed under reduced pressure, and

the compound was dissolved in CH₂Cl₂(2 mL) and cooled to 0

°C. The acid from 11 (48 mg, 0.125 mmol) was added followed

by PyBOP (65 mg, 0.125 mmol) and i-Pr₂NEt (65 µL, 0.375

mmol). The reaction mixture was allowed to warm to room

temperature over a period of 3 h, diluted with EtOAc (10 mL),

and washed with HCl (1 N, 1 mL) and saturated NaHCO₃(1

mL). The organic phase was dried over Na₂SO₄, filtered, and

concentrated in vacuo. The residue was purified by column

chromatography (50% EtOAc in hexanes) to give 41 (64 mg,

solid; [R]²⁰-60.0 (c 0.2, CHCl₃); ¹H NMR (CD₃OD): δ 4.46

D

95%) as a white solid; [R]²⁰-66.6 (c 0.9, CHCl₃); ¹H NMR

(m, 1H), 4.32 (t, J ) 7.0 Hz, 1H), 4.12 (d, J ) 7.8 Hz, 1H),

3.98 (m, 1H), 3.52 (m, 1H), 3.24 (m, 1H), 3.16 (m, 2H), 2.60

(m, 1H), 2.51 (m, 1H), 2.41 (m, 2H), 2.28 (m, 1H), 2.23 (s, 3H),

2.05 (m, 2H), 1.98 (s, 3H), 1.69 (m, 1H), 1.58 (m, 4H), 1.50 (m,

4H), 1.37 (m, 2H), 1.28 (m, 1H), 0.88-0.99 (m, 21H); ¹³C NMR

(CD₃OD): δ 218.0, 174.9, 174.2, 172.6, 172.5, 172.4, 72.3, 59.7,

53.1, 52.7, 51.3, 44.7, 44.1, 41.2, 41.0, 40.8, 39.1, 37.9, 31.5,

31.2, 31.1, 30.3, 24.9, 24.8, 22.9, 22.3, 21.4, 20.1, 18.8, 18.3,

14.3, 13.0; MS (FAB): m/z 684.6 [M + 1]⁺; HRMS calcd for

C₃₄H₆₂N₅O₇S [M + 1]⁺684.4325; found 684.4363; LC/MS

retention time: [A] 20.33 min; [B] 18.28 min.

D

(CDCl₃): δ 6.59 (br, 1H), 6.08 (br, 1H), 4.27 (t, J ) 7.0 Hz, 1H),

4.02 (m, 1H), 3.56 (br, 2H), 3.30 (m, 1H), 3.20 (m, 1H), 4.27

(q, J ) 8.8 Hz, 1H), 2.62 (m, 1H), 2.56 (m, 1H), 2.49 (m, 1H),

2.38 (m, 2H), 2.08 (m, 1H), 1.88 (br, 2H), 1.47 (s, 3H), 1.40 (m,

5H), 1.46 (s, 9H), 1.34 (m, 3H), 0.95 (m, 15H); ¹³C NMR

(CDCl₃): δ 214.6, 172.6, 170.5, 156.0, 79.9, 58.5, 58.1, 45.2, 42.2,

39.2, 37.4, 31.4, 28.3, 26.30, 24.8, 24.2, 21.2, 19.8, 19.1, 18.0,

13.5; MS (FAB): m/z 538.2 [M + 1]⁺; HRMS calcd for

C₂₉H₅₂N₃O₆[M + 1]⁺538.3856; found 538.3834.

Compound 42. Prepared from 10 according to the general

procedure for the preparation of 41 (26.8 mg, 75%); [R]²⁰_D-51.1

Compound 46. Prepared from 44 according to the general

1

procedure for the preparation of 45 (17 mg, 78%); [R]²⁰_D-55.1

(c 1.3, CHCl₃); H NMR (CDCl₃): δ 6.63 (br, 1H); 6.40 (t, J )

1

6.8 Hz, 1H), 4.52 (t, J ) 7.0 Hz, 1H), 3.74 (m, 1H), 3.63 (m,

1H), 3.24 (q, J ) 6.7 Hz, 2H), 2.47 (m, 1H), 2.37 (m, 1H), 1.88

(m, 1H), 1.72 (m, 1H), 1.67 (m, 2H), 1.64 (m, 4H), 1.58 (s, 3H),

1.48 (m, 2H), 1.45 (s, 12H), 1.36 (d, J ) 6.9 Hz, 3H), 1.33 (m,

3H); 0.92 (m, 9H); ¹³C NMR (CDCl₃): δ 175.6, 172.6, 152.1,

80.0, 77.6, 59.4, 49.2, 48.0, 46.9, 39.6, 32.0, 28.9, 27.5, 25.6,

24.7, 21.9, 20.4, 19.2, 14.1; MS (FAB): m/z 496.2 [M + 1]⁺;

HRMS calcd for C₂₇H₅₀N₃O₅[M + 1]⁺496.3750; found 496.3768.

(c 0.2, CHCl₃and MeOH (1:1)); H NMR (CD₃OD): δ 4.64 (m,

1H), 4.42 (t, J ) 6.4 Hz, 1H), 4.31 (q, J ) 7.0 Hz, 1H), 3.92

(m, 1H), 3.36 (m, 1H), 3.19 (m, 2H), 2.59 (m, 2H), 2.50 (m,

1H), 2.27 (m, 1H), 2.16 (s, 3H), 2.09 (m, 3H), 1.98 (s, 3H), 1.90

(m, 2H), 1.70 (m, 6H), 1.56 (m, 3H), 1.46 (m, 3H), 1.35 (d, J )

7.3 Hz, 3H), 1.30 (m, 2H), 0.96 (m, 15H); ¹³C NMR (CD₃OD):

δ 177.1, 174.5, 174.3, 172.5, 172.3, 73.8, 53.1, 52.5, 51.2, 49.7,

47.1, 41.5, 40.8, 39.1, 31.5, 31.1, 30.7, 30.2, 26.7, 24.9, 24.8,

24.7, 22.9, 22.3, 21.4, 21.3, 21.1, 20.0, 17.4, 14.3, 13.1; MS

(FAB): m/z 642.3 [M + 1]⁺; 664.4 [M + 23]⁺; HRMS calcd for

Compound 43. A solution of 41 (49 mg, 0.091 mmol) in

HCl (4 M in dioxane, 1 mL) was stirred at room temperature

5188 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16

Hanessian et al.

C₃₂H₆₀N₅O₆S [M + 1]⁺642.4264; found 642.4242; LC/MS

Compound 50. A solution of 49 (103.2 mg, 0.2 mmol) in

HCl (4 M in dioxanes, 5 mL, 20 mmol was stirred at room

temperature for 1 h. The solvent was removed under reduced

pressure, and the residue was dissolved in saturated NaHCO₃

(10 mL) and extracted with EtOAc (4 × 10 mL). The combined

organic layers were dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was dissolved in CH₂Cl₂(2 mL) followed

by addition of water (2 mL), Boc-Leucine (38.1 mg, 0.22 mmol),

and HOBt (26.9 mg, 0.22 mmol). The reaction mixture was

cooled in an ice bath to 0 °C, and EDC (42 mg, 0.22 mmol)

was added. The resulting reaction mixture was stirred at 0

°C for 36 h. Aqueous HCl (1 N, 5 mL) was added, and the

layers were partitioned. The organic layer was washed se-

quentially with aqueous HCl (0.5 N, 10 mL), brine (10 mL),

NaHCO₃(1 N, 2 × 10 mL), and water (10 mL). The organic

layer was dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was purified by column chromatography (10%

retention time: [A] 20.21 min; [B] 18.51 min.

Compound 47. To a solution of 40 (30 mg, 0.049 mmol) in

N,N-dimethylacetamide (0.5 mL) were sequentially added

PyBOP (27.2 mg, 0.051 mmol), N-methylmorpholine (5.6 µL,

0.051 mmol), and BuNH₂(5.8 µL, 0.058 mmol). The reaction

mixture was stirred at 0 °C for 3 h. The reaction mixture was

diluted with EtOAc (40 mL) and washed with 1 N HCl (2 × 8

mL), NaHCO₃(2 × 8 mL) and brine (8 mL). The organic layer

was dried (NaSO₄), filtered, and concentrated in vacuo. The

residue was purified by column chromatography (4% of MeOH

in CH₂Cl₂) to afford 47 (6.5 mg, 20%) as a white solid; [R]²⁰

D

-78.4 (c 0.3, MeOH); ¹H NMR (CD₃OD) δ 4.45 (q, J ) 5.0 Hz,

1H), 4.35 (m, 1H), 4.09 (m, 1H), 3.90 (m, 1H), 3.46 (m, 2H),

3.19 (m, 2H), 2.68 (m, 1H), 2.60 (m, 1H), 2.52 (m, 1H), 2.28

(m, 1H), 2.09 (s, 3H), 1.99 (s, 3H), 1.87 (m, 3H), 1.80-1.08 (m,

22H), 0.95 (m, 18H); ¹³C NMR (CD₃OD) δ 177.3, 174.1, 172.7,

172.5, 172.4, 73.2, 59.7, 53.8, 52.6, 40.8, 31.5, 30.9, 30.3, 30.2,

24.9, 24.8, 22.4, 21.4, 21.3, 21.1, 20.1, 18.8, 18.3; MS (FAB):

m/z 670.5 [M + 1]⁺; HRMS calcd for C₃₄H₆₄N₅O₆S [M + 1]⁺

670.4533; found 670.4571; LC/MS retention time: [A] 26.98

min; [B] 24.71 min.

MeOH in EtOAc) to afford 50 (62.0 mg, 54%) as a white foam;

1

[R]²⁰-94.1 (c 1.0, MeOD); H NMR (CD₃OD) δ 4.46 (q, J )

D

5.1 Hz, 1H), 4.35 (t, J ) 7.6 Hz, 1H), 3.95 (m, 1H), 3.43 (t, J

) 4.6 Hz, 1H), 3.20 (m, 2H), 2.54 (m, 3H), 2.28 (m, 1H), 2.12

(m, 1H), 2.00 (s, 3H), 1.99 (m, 1H), 1.84 (m, 1H), 1.70 (m, 6H),

1.60-1.23 (m, 10H), 0.95 (m, 15H); ¹³C NMR (CD₃OD) δ 177.2,

174.0, 172.5, 172.2, 73.4, 59.7, 53.8, 53.1, 52.6, 51.6, 48.8, 41.5,

40.8, 39.3, 31.7, 31.2, 30.9, 30.2, 25.5, 24.9, 24.8, 22.3, 21.4,

21.1, 20.2, 14.3, 13.1; MS (FAB): m/z 571.39 [M + 1]⁺; HRMS

calcd for C₂₉H₅₅N₄O₅S [M + 1]⁺571.3893; found 571.3900; LC/

MS retention time: [A] 21.72 min; [B] 19.89 min.

(5S)-((2R)-Butylcarbamoyl-cyclopentyl)-(4S)-isobutyl-

2,2-dimethyl-oxazolidine-3-carboxylic Acid tert-Butyl

Ester (48). To a solution of NaOH (40 mg, 1.0 mmol) in MeOH

(4.5 mL) and H₂O (2.3 mL) was added 12 (192.2 mg, 0.5 mmol),

then stirred at 65 °C for 3 h and at room-temperature

overnight. 1 N HCl (10 mL) was added, the aqueous phase

was extracted with CH₂Cl₂(4 × 10 mL), and the combined

organic layers were dried (Na₂SO₄), filtered, and concen-

trated in vacuo. The residue was used without further puri-

fication.

Compound 53. Prepared from intermediate 52⁴⁵according

to the general procedure for the preparation of 46; [R]²⁰

D

-19.56 (c 0.23, MeOH); ¹H NMR (CD₃OD) δ 8.28 (br, 1H); 7.27

(br, 1H); 6.58 (b, 1H); 5.26 (d, J ) 6.9 Hz, 1H); 5.0 (br, 1H);

4.31 (t, J ) 6.4 Hz, 1H); 4.22 (q, J ) 7.0 Hz, 1H); 3.40 (m,

1H); 3.28(m, 1H); 3.21 (m, 2H); 2.57 (m, 2H); 2.38 (m. 1H);

2.36 (m. 1H); 2.10 (s, 3H); 1.91 (m, 2H); 1.88 (m, 2H); 1.74 (m,

2H); 1.63 (m, 4H); 1.47 (m, 2H); 1.43 (s, 9H); 1.37 (d, J ) 7.2

Hz, 3H); 1.34 (m, 3H); 0.91 (m, 9H); ¹³C NMR (CD₃OD) δ 174.7,

174.2, 172.6, 172.3, 172.2, 73.2, 59.8, 59.3, 56.6, 52.7, 48.6, 48.4,

46.3, 40.8, 39.1, 31.5, 30.2, 24.9, 24.8, 22.8, 22.4, 21.4, 21.1,

20.1, 18.8, 18.2, 13.08; MS (FAB): m/z [M + 1]⁺685.4; HRMS

calcd for C₃₄H₆₅N₆O₆[M + 1]⁺685.5686; found 685.4688; LC/

MS retention time: [A] 19.40 min; [B] 17.77 min.

To a solution of the above acid and BuNH₂(50 µL, 0.525

mmol) in CH₂Cl₂(11.5 mL) were added EDC (101 mg, 0.525

mmol) and HOBt (71 mg, 0.525 mmol), followed by DMAP

(64.3 mg, 0.525 mmol) at 0 °C. The reaction mixture was

stirred for 24 h at room temperature and then partitioned

between CH₂Cl₂(20 mL) and 1 N HCl (10 mL). The organic

layer was separated and washed by brine (10 mL), dried

(Na₂SO₄), filtered, and concentrated in vacuo. The residue was

purified by column chromatography (20% EtOAc in hexanes)

to afford 48 (190 mg, 89%) as a white solid; [R]²⁰-23.1 (c

D

Compound 55. Prepared from intermediate 54⁴⁵according

0.4, CHCl₃); ¹H NMR (CDCl₃) δ 5.69 (s, 1H), 3.68 (br, 1H), 3.59

(br, 1H), 3.19 (m, 2H), 2.41 (m, 1H), 2.24 (br, 1H), 1.92-1.10

(m, 19H), 1.39 (s, 9H), 0.83 (m, 9H); ¹³C NMR (CDCl₃) δ 175.3,

152.1, 93.9, 82.9, 81.6, 59.2, 49.2, 46.8, 44.5, 43.0, 39.6, 32.2,

31.0, 28.8, 27.4, 26.2, 25.5, 25.3, 24.7, 21.8, 20.4, 14.1; MS

(FAB): m/z 425.4 [M + 1]⁺; HRMS calcd for C₂₄H₄₅N₂O₄[M +

1]⁺425.3379; found 425.3374.

to the general procedure for the preparation of 46; [R]²⁰_D-49.1

1

(c 0.64, MeOH). H NMR (CD₃OD): δ 0.91 (m, 9H), 1.34 (m,

3H), 1.37 (d, J ) 7.2 Hz, 3H), 1.43 (s, 9H), 1.47 (m, 2H), 1.63

(m, 4H), 1.74 (m, 2H), 1.88 (m, 2H), 1.91 (m, 2H), 2.10 (s, 3H),

2.36 (m, 1H), 2.38 (m, 1H), 2.57 (m, 2H), 3.21 (m, 2H), 3.28

(m, 1H), 3.40(m, 1H), 4.22 (q, J ) 7.0 Hz, 1H), 4.31 (t, J ) 6.4

Hz, 1H), 5.0 (1H, b), 5.26 (d, J ) 6.9 Hz, 1H), 6.58 (b, 1H),

7.27 (b, 1H), 8.28 (b, 1H); ¹³C NMR (CD₃OD): δ 175.26, 175.04,

173.41, 173.37, 173.11, 81.05, 72.73, 70.03, 59.47, 53.92, 53.45,

52.50, 46.89, 42.37, 41.75, 40.03, 32.50, 32.39, 32.20, 31.17,

27.00, 26.50, 25.87, 25.70, 24.94, 23.89, 23.31, 22.63, 22.32,

22.20, 22.06, 21.05, 19.64, 18.69, 15.22, 14.03; IR (film) 3288,

3081, 2958, 2872, 1644, 1538; MS (FAB): m/z 713.5 [M + 1]⁺;

LC/MS retention time: [A] 18.71 min; [B] 17.14 min.

Compound 49. To a solution of 48 (170 mg, 0.4 mmol) in

CH₂Cl₂(6 mL) was added TFA (1.5 mL). The reaction mixture

was stirred at room temperature for 1 h. The reaction was

quenched by addition of saturated NaHCO₃(10 mL) and

extracted by EtOAc (4 × 10 mL). The combined organic layers

were dried (Na₂SO₄), filtered, and concentrated in vacuo. The

residue was dissolved in CH₂Cl₂(7 mL) followed by addition

of N-Boc-Met-OH (299 mg, 1.2 mmol), PyBOP (636 mg, 1.2

mmol) and i-Pr₂NEt (418 µL, 2.4 mmol) at 0 °C. The reaction

mixture was stirred at 0 °C for 3 h, EtOAc (80 mL) was added,

and the separated organic layer was washed with 1 N HCl (2

× 10 mL), saturated NaHCO₃(2 × 10 mL), and brine (10 mL).

The organic layer was dried (Na₂SO₄), filtered, and concen-

trated in vacuo. The residue was purified by column chroma-

tography (66% EtOAc in hexanes) to afford 49 (152 mg, 74%)

as a colorless oil; [R]²⁰_D-58.6 (c 0.5, CHCl₃); ¹H NMR (CDCl₃)

δ 6.63 (d, J ) 8.4 Hz, 1H); 6.30 (br, 1H); 5.40 (br, 1H); 4.19

(m, 1H); 3.94 (m, 2H), 3.40 (s, 1H), 3.19 (m, 2H), 2.53 (m, 3H),

2.17 (m, 1H), 2.00 (m, 1H), 2.06 (d, J ) 3.2 Hz, 3H), 1.86 (m,

2H), 1.79-1.16 (m, 13H), 1.40 (d, J ) 2.8 Hz, 9H), 0.86 (m,

9H); ¹³C NMR (CDCl₃) δ 175.5, 172.1, 155.6, 80.1, 73.8, 53.8,

50.7, 47.7, 47.6, 41.4, 39.2, 31.6, 31.0, 30.1, 29.0, 28.1, 25.5,

24.5, 23.9, 23.2, 21.7, 20.0, 15.2, 13.6; MS (FAB): m/z 516.3

[M + 1]⁺; HRMS calcd for C₂₆H₅₀N₃O₅S [M + 1]⁺516.3471;

found 516.3466.

Compound 57. Prepared from intermediate 56⁴⁵according

to the general procedure for the preparation of 46; [R]²⁰_D-75.0

1

(c 1.0, MeOH); H NMR (MeOD) δ 8.18 (t, J ) 5.2 Hz, 1H),

7.54 (d, J ) 9.3 Hz, 1H), 4.49 (dd, J ) 9.6 Hz, J ) 4.6 Hz, 1H),

4.36 (t, J ) 7.5 Hz, 1H), 4.18 (d, J ) 3.3 Hz, 1H), 4.14 (d, J )

11.7 Hz, 2H), 3.47 (dd, J ) 6.3 Hz, J ) 2.6 Hz, 1H), 3.31 (m,

1H), 3.24 (m, 1H), 2.75 (s, 3H), 2.73-2.50 (m, 4H), 2.38 (m,

1H), 2.26-1.92 (m, 3H), 2.10 (s, 3H), 2.01 (s, 3H), 1.75 (m, 1H),

1.65 (m, 4H), 1.60 (m, 2H), 1.42-1.24 (m, 3H), 0.96 (m, 21H);

¹³C NMR (MeOD) δ176.6, 174.3, 172.6, 172.2, 172.1, 171.5,

72.5, 65.9, 60.0, 52.7, 31.5, 31.0, 30.4, 28.0, 24.9, 24.8, 22.9,

22.4, 21.5, 21.2, 21.1, 20.1, 18.7, 18.5, 14.3, 13.1; MS (FAB):

m/z 699.3 [M + 1]⁺HRMS calcd for C₃₄H₆₃N₆O₇S [M + 1]⁺

;

699.4434; found 699.4474; LC/MS retention time: [A] 18.10

min; [B] 16.58 min.

Compound 59. Prepared from intermediate 58⁴⁵according

to the general procedure for the preparation of 46; [R]²⁰_D-49.1

Carbocyclic and Heterocyclic BACE Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5189

1

(2) Citron, M. Strategies for disease modification in Alzheimer’s

disease. Nature Rev. Neurosci. 2004, 5, 677-685.

(c 0.6, MeOH); H NMR (CDCl₃) δ 8.28 (b, 1H), 7.27 (b, 1H),

7.27 (b, 1H), 6.58 (b, 1H), 5.26 (d, J ) 6.9 Hz, 1H), 5.0(b, 1H),

4.31 (t, J ) 6.4 Hz, 1H), 4.22 (q, J ) 7.0 Hz, 1H), 3.40(m, 1H),

3.28(m, 1H), 3.21 (m, 2H), 2.57(m, 2H), 2.38(m, 1H), 2.36(m,

1H), 2.10(s, 3H), 1.91(m, 2H), 1.88 (m, 2H), 1.74 (m, 2H), 1.63

(m, 4H), 1.47 (m, 2H), 1.43 (s, 9H), 1.37 (d, J ) 7.2 Hz, 3H),

1.34 (m, 3H), 0.91 (m, 9H); ¹³C NMR (CDCl₃): δ 175.26, 175.04,

173.41, 173.41, 173.37, 173.11, 81.05, 72.73, 70.03, 59.47,

53.92, 53.45, 52.50, 46.89, 42.37, 41.75, 40.03, 32.50, 32.39,

32.20, 31.17, 27.00, 26.50, 25.87, 25.70, 24.94, 23.89, 23.31,

22.63, 22.32, 22.20, 22.06, 21.05, 19.64, 18.69, 15.22, 14.03;

IR (film) 3288, 3081, 2958, 2872, 1644, 1538; MS (FAB) m/z

672.4 [M + 1]⁺; HRMS calcd for C₃₃H₆₂N₅O₇S [M + 1]⁺

672.4370; found 672.4371; LC/MS retention time: [A] 22.28

min; [B] 20.79 min.

Modeling of Compounds in BACE. A 10 Å shell around

the inhibitor in the BACE OM99-2 cocrystal structure (PDB

ref 1FKN) was used for calculations. In this binding site model

the Monte Carlo docking/energy minimization protocol of the

MCDOCK routine in the QXP program⁴⁷(within the Flow96

package) was applied. Depending on the size and flexibility of

the ligands 1000 or 2000 search and energy minimization

cycles were performed in order to ensure an in depth confor-

mational search and the exploration of different possible

binding modes.

(3) Golde, T. E. Alzheimer disease therapy: Can the amyloid cascade

be halted? J. Clin. Invest. 2003, 111, 11-18.

(4) Selkoe, D. J.; Schenk, D. Alzheimer’s Disease: Molecular

Understanding Predicts Amyloid-Based Therapeutics. Annu Rev.

Pharmacol. Toxicol. 2003, 43, 545-584.

(5) Conway, K. A.; Baxter, E. W.; Felsenstein, K. M.; Reitz, A. B.

Emerging â-Amyloid Therapies for the Treatment of Alzheimer’s

Disease. Curr. Pharm. Des. 2003, 9, 427-447.

(6) Wolfe, S. M. Therapeutic Strategies for Alzheimer’s Disease.

Nature Rev. Drug Discovery 2002, 1, 859-866.

(7) 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. â-Secretase Cleavage of

Alzheimer’s Amyloid Precursor Protein by the Transmembrane

Aspartic Protease BACE. Science 1999, 286, 735-741.

(8) Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.; Meek, T. D.;

Chapman, C.; Gloger, I. S.; Murphy, K. E.; Southan, C. D.; Ryan,

D. M.; Smith, T. S.; Simmons, D. L.; Walsh, F. S.; Dingwall, C.;

Christie, G. Identification of a Novel Aspartic Protease (Asp 2)

as â-Secretase. Mol. Cell. Neurosci. 1999, 14, 419-427.

(9) Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.;

Pauley, A. M.; Brashler, J. R.; Stratman, N. C.; Mathews, W.

R.; Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.;

Heinrikson, R. L.; Gurney, M. E. Membrane-anchored aspartyl

protease with Alzheimer’s disease â-secretase activity. Nature

1999, 402, 533-537.

Enzyme Inhibition Measurements. Materials: Recom-

binant human BACE (SwissProt accession number P56817,

amino acids 1-453 + a thrombin-cleavable C-kappa tag) was

produced in-house using Sf9 insect cells. The proenzyme was

activated autocatalytically at pH 4.5. Human proCathepsin

D (SwissProt accession number P07339) was expressed in Sf9

cells, purified, and autoactivated by incubation at pH 3.7.

Peptide substrates were obtained from Bachem, Bubendorf,

Switzerland, and all other biochemicals were obtained from

Fluka, Buchs, Switzerland.

Enzyme assays: Human BACE activity was assayed in 100

mM acetate buffer, pH 4.5, containing 0.1% CHAPS, using

Mca-SEVNLDAEFK(DNP)-OH (3 µM), and Cathepsin D activ-

ity was measured in 100 mM formate buffer, pH 3.1, using

Mca-GKPILFFRLK(DNP)-D-R-NH₂(2 µM). Test substances

were serially diluted from 10 µM to 10 nM, inhibition assays

were done in black 96-wellplates (Costar) by adding the

respective enzyme in assay buffer. Plates were incubated at

room temperature for 1 h, and residual enzymatic activity was

measured after addition of substrate in a Spectramax Gemini

fluorescence plate reader (Molecular Devices, Sunnyvale, CA).

IC₅₀values were calculated using the Microsoft Excel extension

XL-fit.

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Acknowledgment. We thank NSERC (Canada) for

financial assistance, Dr. Michel Simard for X-ray analy-

sis, and Dr. Dalbir Sekon (Universite´ de Montre´al) for

LC/MS analyses. S.H. thanks Novartis Pharma AG,

Basel/Canada, for generous financial support. We also

thank Rene´ Hemmig, Sylvie Lehmann, and Sebastien

Rieffel for technical support. X-ray data collection for

the enzyme complexes was performed at the Swiss Light

Source, Paul Scherrer Institut, Villigen, Switzerland.

We are grateful to the machine and beamline groups

whose outstanding efforts have made these experiments

possible.

Supporting Information Available: Additional experi-

mental procedures, copies of selected NMR spectra, LC/MS

data, experimental procedures for crystallization and crystal

structure data, and X-ray crystallographic data for compound

12. This material is available free of charge via the Internet

at http://pubs.acs.org.

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JM050142+

Article Doi

Structure-based design, synthesis, and memapsin 2 (BACE) inhibitory activity of carbocyclic and heterocyclic peptidomimetics

DOI: 10.1021/jm050142+

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Article abstract of DOI:10.1021/jm050142+

Full text of DOI:10.1021/jm050142+

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