Design, synthesis and evaluation of D-Homophenylalanyl epoxysuccinate inhibitors of the trypanosomal cysteine protease cruzain

TETRAHEDRON

Pergamon

Tetrahedron 56 (2000) 9747±9762

Design, Synthesis and Evaluation of d-Homophenylalanyl

Epoxysuccinate Inhibitors of the

Trypanosomal Cysteine Protease Cruzain

a

b

William R. Roush,^a,Alejandro Alvarez Hernandez, James H. McKerrow, Paul M. Selzer,

*

Elizabeth Hansell^band Juan C. Engel^b

^aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

^bDepartment of Pathology, University of California, San Francisco, CA 94121, USA

Received 7 April 2000; accepted 25 August 2000

AbstractÐThe binding modes of E-64c to papain combined with molecular modeling and ligand design using the crystal structure of

cruzain have been used to develop new, potent d-Homophenylalanyl epoxysuccinate inhibitors of cruzain, the major cysteine protease of

Trypanosoma cruzi. The most potent inhibitor 47 contains an O-benzyl hydroxamate unit and is highly speci®c for cruzain relative to other

Introduction

target trypanothione reductase^16±18and sterol C-14a-

demethylase.^14,19,20

Cysteine proteases regulate a broad range of biological

functions and are emerging as attractive targets for drug

design in a variety of pathological processes including

arthritis, osteoporosis, Alzheimer's disease, cancer cell

invasion and apoptosis.^1±3Cysteine proteases also play

crucial roles in the life cycle of parasites that cause

infections such as malaria, leishmaniasis and Chagas'

disease.^4±6Chagas' disease, or American trypanosomiasis,

is transmitted to humans by blood sucking triatomine

vectors infected with Trypanosoma cruzi and is character-

ized by chronic digestive lesions⁷and cardiopathy.⁸At least

16±18 million people are infected in Latin America where

the disease is endemic, resulting in more than 50,000 deaths

each year.^9,10The traditional drugs used to treat Chagas'

disease are effective only in the early stages of the infection

but cause serious side effects.^11,12There are no drugs for

treatment of the chronic infection. Recently McKerrow

and Urbina have independently described the ®rst successful

treatments of Chagas' disease in mouse models.^13,14

Cruzain, a cysteine protease of the papain family, is the

major cysteine protease of T. cruzi and is essential for

replication and differentiation of the intracellular

parasite.^13,15Cruzain thus represents an attractive

chemotherapeutic target for treatment of Chagas' disease.

Other promising therapeutic approaches under development

Malaria is one of the most important infectious diseases

worldwide. Over one million people die each year from

infections caused by Plasmodium falciparum, the most

virulent human malaria parasite.²¹The problem is

exacerbated by the increasing resistance of malaria

parasites to drugs. A new strategy for malaria chemotherapy

targets the inhibition of hemoglobin degradation.²²

P. falciparumuses cysteine and aspartic proteases to degrade

hemoglobin from which it obtains the amino acids necessary

for protein synthesis. One of the hemoglobinases involved is

falcipain,²³a cysteine protease of the papain family whose

inhibition results in arrest of parasite development.^24,25

Falcipain inhibitors are therefore of considerable interest

as potential anti-malarial agents.²⁶

Leishmaniasis is a third, signi®cant parasitic disease with a

wide range of clinical courses that include skin and visceral

lesions (kala azar) that may be fatal if the disease is not

treated. Leishmaniasis affects 12 million people in over 88

countries.²⁷The cathepsin B-like protease from Leishmania

major is yet another cysteine protease of the papain

family that has been identi®ed as a potential target for

leishmaniasis chemotherapy.^28,29

In connection with ongoing efforts to develop potent and

selective inhibitors of cruzain^30±32we became interested in a

class of naturally occurring peptidyl epoxysuccinate

derivatives of which E-64 (1),^33±35cathestatin C (2)^36,37

and circinamide (3)³⁸are representative examples. Peptidyl

epoxysuccinates are potent and selective irreversible

Keywords: enzyme inhibitors; hydroxamic acids and derivatives; molecular

design; peptide analogs and mimetics.

* Corresponding author. Tel.: 1734-647-9278; fax: 1734-647-9279;

e-mail: roush@umich.edu

PII: S0040-4020(00)00882-6

9748

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

Scheme 1. Alternative binding modes of E-64c to papain. (a) The l-leucyl side chain is located at the edge of the hydrophobic S₂pocket of papain. (b) The

isopentylamine is located at the edge of the S₂pocket in the second binding mode. The terminology used is that of Schechter and Berger⁵⁴who described seven

binding sites, S₄±S⁰, in papain.

3

inhibitors of cysteine proteases. E-64 does not react with

thiols in solution and does not inhibit aspartyl or serine

proteases. These attributes have stimulated considerable

interest in the development of analogs for therapeutic

purposes. Loxistatin (4),^39,40the ethyl ester prodrug of

E-64c (5), was developed for treatment of muscular

dystrophy and was taken into clinical trials in Japan.⁴¹

More recently, selective and potent inhibitors of cathepsins

B^42±46and L^47,48have been developed based on the epoxy-

succinyl motif.

Results and Discussion

Examination of the ®rst crystal structure of the E-64c´papain

complex⁵¹(Scheme 1a) reveals that the a-hydrogen, and not

the side chain of the l-leucyl residue, is oriented directly

into the hydrophobic S₂pocket. We envisioned that simple

inversion of the amino acid con®guration from l to d would

place the leucyl side chain within the S₂pocket, thus

improving enzyme-inhibitor interactions. The S₂site of

papain is regarded as the most important element of

substrate speci®city, and is highly selective for hydrophobic

l-amino acids.^52,53Consequently, the use of d-amino acids

in design of inhibitors of papain-family cysteine proteases

has been rather limited, with most of these studies involving

substrate-like peptidomimetics. Several different classes of

inhibitors containing a d-amino acid at P₂have been

evaluated vs papain and all proved to be inferior to the

peptidyl inhibitors containing the corresponding l-amino

acid.^56±58Lalmanach and coworkers have recently revisited

the S₂speci®city of papain with ¯uorogenic substrates that

contain structural analogs of phenylalanine as P₂residues.⁵⁹

These investigators found that papain can accommodate

d-Phe and l-hPhe as P₂residues but the kinetic data

shows a signi®cant preference for l-Phe.d-Phe.l-hPhe.

In a recent report Chatterjee and coworkers described the

®rst successful design of potent, d-amino acid containing

inhibitors of calpain I.⁶⁰

E-64 inhibits cysteine proteases by irreversible alkylation of

the active site cysteine residue. The regiochemistry of the

inhibition reaction of papain with E-64 was determined by

Rich⁴⁹who established that substitution of the epoxide by

the cysteine -SH unit occurs at C(2), adjacent to the

carboxylic acid unit (which is fully deprotonated and nega-

tively charged at physiological pH). This conclusion has

been veri®ed by X-ray crystal structures of the E-64´papain

and E-64c´papain complexes.^50±52E-64c binds to papain in

two alternate modes (see Scheme 1) in which the leucyl side

chain and the isopentylamine interchange binding in the S₂

and S₃sites.^51,52Inspection of these structures reveals that

the hydrophobic S₂binding pocket, which is regarded as the

most important element of substrate speci®city for members

of the papain family of cysteine proteases,^53,54is not opti-

mally ®lled in either mode. In structure (a), the leucyl side

chain is bound to the rim of the S₂pocket, whereas in struc-

ture (b) the isoamyl chain interacts with the rim of the S₂

pocket (see Scheme 1). Armed with these insights, we

embarked on a program designed to optimize the inter-

actions of epoxysuccinyl inhibitors with the S₂and S₃

binding pockets of cruzain.⁵⁵

Nevertheless, in spite of the unfavorable literature pre-

cedent, we decided it was worthwhile to explore a new

series of inhibitors based on the E-64c motif, containing

d-amino acid residues in place of l-Leu residue of the

parent compound. The question was how best to identify

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9749

Scheme 2. Left panel: Crystal structure of E64-c bound to papain. The leucyl side chain of the inhibitor is outside of the S2 hydrophobic pocket. Right panel:

Model of the d-hPhe analog 11c bound to cruzain, with the phenyl group of the h-Phe side chain optimally occupying the S2 hydrophobic pocket. The binding

pockets are indicated by color codes: S⁰1 (pink), S2 (blue) and S3 (yellow). The nucleophilic sulfur atom of the active site Cys-25 is indicated as a yellow

sphere. The atoms of the inhibitors are color coded as follows: C (green), O (red), and N (blue).

suitable lead structures? We turned to structure-based

design paradigms to aid this phase of the work.

We therefore set out to prepare and test a new series of

d-amino acid containing inhibitors vs cruzain and other

cysteine proteases. Initially, we utilized Yokoo's pro-

cedure⁶⁴for the synthesis of 11c and other E-64 analogs.

Although the LUDI analysis predicted that 11c containing a

d-hPhe unit would be optimal, we elected to prepare a series

of other E-64 analogs with d-amino acid units to explore the

P₂/S₂interactions with cruzain (Scheme 3).

We transferred the E-64c coordinates from the papain´E-64c

structure to the crystal structure of cruzain.⁶¹Using the

Insight II software package⁶²we exchanged the stereo-

chemistry of the aminoacid at P₂in the inhibitor from

l-Leu to d-Leu. Minimization of the resulting (bound)

peptidyl epoxysuccinyl unit in the active site suggested

that the cruzain S₂pocket could accept further structural

variation of the amino acid side chain. The ligand design

software package LUDI⁶³was used then to identify an

appropriate P₂group for our template. LUDI uses the crystal

structure of a protein to identify possible binding sites at

positions where hydrogen bonds can be formed with the

enzyme, or hydrophobic pockets can be occupied by

lipophilic groups. LUDI then selects fragments from a

library that ®t into the interaction sites and ®nally prioritizes

the selected fragments by taking into account hydrogen

bonds, ionic interactions, lipophilic protein-ligand contact

surface and number of rotatable bonds in the ligand. Two

major strategies exist for this fragment based approach to

ligand design. One can obtain several independent

fragments and then connect them by means of an appropiate

template in one molecule or, alternatively, one fragment can

be positioned initiallyÐin our case the epoxysuccinyl

moietyÐand then append additional fragments. We have

used this latter linking mode. Accordingly, the isopropyl

group of the l-leucyl side chain of the inhibitor was

deleted and then the link mode of LUDI was used to

identify the benzyl unit as the highest scoring fragment at

the S₂hydrophobic pocket of cruzain. (Other fragments

retrieved were, in order of priority, isopropyl and methyl).

Thus, this analysis led to the prediction that the

unnatural amino acid d-homophenylalanine (d-hPhe)

would make an excellent match to the steric and hydro-

phobic requirements of the cruzain S₂hydrophobic site

(Scheme 2).

The Yokoo synthesis involves the coupling of a BOC

protected⁶⁵amino acid (6a±e) with an amine (in this case

isopentylamine, 7) envisaged to serve as the P₃substituent.

Deprotection of the resulting peptidomimetic 8 followed by

N-acylation with the p-nitrophenyl ester 9⁶⁴and ester

hydrolysis completed the synthesis of inhibitors 11a±11e.

The O-benzyl serine and threonine derivatives 11d and 11c

were included in this series in order to determine the optimal

number of atoms separating the P₂phenyl substituent from

the peptide backbone in this inhibitor series.

Preliminary evaluation (IC₅₀determinations) of compounds

11a±e as inhibitors of cruzain showed a peak of activity

with the d-hPhe derivative 11c (see Table 1) These results

seemed to be consistent with our initial proposal based on

the molecular modeling study, although these IC₅₀determi-

nations also suggested that 11c would be a much weaker

inhibitor than E-64c (5) itself. However, IC₅₀values are

virtually meaningless for irreversible, time dependent

inhibitors since IC₅₀data are rigorously dependent on the

conditions used for the assay (concentration, time, etc.), and

one can reach misleading conclusions when comparing data

obtained on different days with different batches of enzyme

by different experimentalists. Nevertheless, we continue to

use IC₅₀data as a rough measure of relative inhibitor

activity, for a series of inhibitors studied under identical

conditions with the same batch of enzyme.

To obtain a more reliable assessment of the potency of these

9750

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

Scheme 3.

inhibitors, a select group was subjected to full kinetic

analysis.^66,67As a positive control, we included E-64c (5)

and the l-hPhe epimer 12b⁵⁵of 11c in this analysis.

Dissapointingly, the results of these analyses indicate that

the designed inhibitor, 11c, is a relatively weak inhibitor of

cruzain and the other cysteine proteases studied, as are the

d-Leu and d-Phe containing inhibitors 11a and 11b. In

contrast, both E-64c (5) and 12b (the l-hPhe analog) are

the best inhibitors of cruzain among this group. This

behavior parallels what previously has been observed for

E-64c and papain, where the l-amino acid derivatives are

substantially better inhibitors that the d-amino acid

analogs.⁶⁴

Table 2) indicate that only 12b has a modest selectivity for

inhibition of cruzain compared to cathepsin B, and also that

only 12b shows any selectivity between cruzain and papain.

It should be noted that the data for inhibition of cruzain by

11c in Table 2 are the results of repeated determinations.

Initial kinetic analyses, the very ®rst that we performed in

this project, indicated that 11c had a second order inhibiton

rate constant vs cruzain of 77,000 s²¹M²¹. However, we

have been unable to reproduce this rate constant in subse-

quent studies, and after multiple determinations we believe

the value reported in Table 2 for 11c to be correct. Ironi-

cally, had the initial kinetic data indicated (correctly) that

11c was a weak inhibitor, none of the studies subsequently

described would have been performed. Ultimately, the fact

that extremely potent d-amino acid inhibitors (e.g, 47)

derive from this research effort attests to the powerful role

that serendipity can play in scienti®c research.

These inhibitors were also tested against two other cysteine

proteases: cathepsin B⁶⁷and papain. The results (shown in

Table 1. IC₅₀data for inhibition of cruzain with inhibitors 5 and 11a±e

Inhibitor

P₂amino acid

IC₅₀vs Cruzain (mM)

A new synthesis of peptidyl epoxysuccinates to probe the

S₃site of cruzain

5

l-Leu

d-Leu

d-Phe

d-hPhe

d-Ser (Obn)

d-Thr (OBn)

0.01

0.5

1.0

0.15

0.5

11a

11b

11c

11d

11e

LUDI assisted ligand design⁶³of the P₃residue predicted

that a lipophilic chain of 4±5 carbon atoms with a terminal

polar group would interact with the Ser-61 residue at the rim

of the S₃hydrophobic pocket of cruzain. Similar P₃residues

are found in E-64 (1) and other natural product analogs.^33,36±

38

Table 2. Second order rate constants, k_inact/K (s²¹M²¹), of selected epox-

ysuccinate inhibitors determined against cruzain, bovine cathepsin B and

papain. It was not possible to demonstrate saturation kinetics for 11c

against papain, owing to the range of inhibitor concentration that could

be studied. Consequently, this rate constant is reported as k_ass(see Ref.

[66]); ndnot determined

Since the isopentyl amine chain of 5 was designed to

avoid the bioavailability problems associated with the

charged guanidinium moiety of 1,^39,40we focused on use

of neutral alcohol derivatives at P₃. While Yokoo's

synthetic method (Scheme 3) is extremely useful for varying

the P₂amino acid residue at the beginning of the synthesis,

this route proved less convenient for synthesis of analogs

designed to probe the requirements of the P₃/S₃interactions,

since the P₃residue is introduced in the very ®rst step. This

prompted us to develop a new synthesis of peptidyl

succinate derivatives that would permit the P₃substituent

to be introduced at the end of the sequence.⁵⁵Thus,

acylation of hPhe benzyl esters 13 and 14^68,69with the

Inhibitor

P₂

Amino acid

vs cruzain

k_inact/K_i

vs cath B

k_inact/K_i

vs papain

k_inact/K_i

5

l-Leu

d-Leu

d-Phe

d-hPhe

l-hPhe

70 600

3800

11 970

4584

65 000

52 300

900

nd

200 000

23 700

4820

11a

11b

11c

12b

3806

7400

7900

12 200

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9751

Scheme 4.

epoxy acid 15,⁶⁴obtained from diethyl d-(2)-tartrate by the

method of Mori and Iwasawa,⁷⁰gave the amides 16±17⁵⁵in

good yields. Hydrogenolysis of the benzyl ester afforded the

acids 18 and 19,⁵⁵which were then coupled to the silyloxy

amines 20 and 21⁷¹to afford the corresponding peptidomi-

metics (22±25). Removal of the silyl protecting group and

hydrolysis of the ethyl ester gave the desired epoxy-

succinates 26b±29b (Scheme 4).

potency of 28b and 29b for cruzain (compared to 12b) is

fully consistent with this analysis. On the other hand, the

poor activity of the d-h-Phe containing analogs 11c, 26b,

and 27b led us to suspect that the binding modes (for 11c)

used in our molecular modelling study may be incorrect.

Indeed, X-ray crystallographic characterization of the

cruzain´11c and cruzain´12b structures verify distinctly

different binding modes for these inhibitors; these structures

will be reported in due course.⁷²

Inhibitors 26b±29b were submitted to kinetic assays with

cruzain, cathepsin B and papain (Table 3). Disappointingly,

the d-hPhe inhibitor analogs 26b and 27b are almost devoid

of activity as inhibitors of any of the cysteine proteases

tested (with the exception of 26b vs papain). On the other

hand, the l-hPhe analogs 28b and 29b are excellent

inhibitors of cruzain, albeit with lower selectivity for

cruzain vs cathepsin B or papain than was the case with

12b.

We brie¯y explored the tolerance of other P₃hydrophobic

groups in additional analogs of 11c. Thus, coupling of acid

18 with cyclohexylmethylamine (30), benzylamine (31) and

aniline (32) followed by hydrolysis afforded the correspond-

ing acids 36±38. In search of ways to improve the water

solubility of these compounds we substituted the amino acid

d-homotyrosine (d-hTyr) for d-hPhe at the P₂position. We

used Yokoo's method⁶⁴for the synthesis of the d-hTyr

derivative (43) (Scheme 5).

The l-amino acid epoxysuccinates 12b, 28b and 29b may

bind to cruzain the same way that E-64 binds to papain,⁵⁰

with the hydroxylated P₃substituent binding in the S₃

pocket as predicted by our ligand design. The increased

Preliminary IC₅₀data obtained with these compounds

suggested that only 38, containing a P₃aniline residue, is

a satisfactory inhibitor of cruzain. Reproducible kinetic

assays performed on 38 established that the potency

(k_ass66,000 s²¹M²¹) is truly comparable to that of 5,

and 12b. In contrast, replacement of the d-hPhe amino

acid for d-hTyr was tolerated but with a decrease in

Table 3. Second order rate constants, k_inact/K (s²¹M²¹), for inactivation of

cruzain, bovine cathepsin B and papain by E-64 analogs 26b±29b

activity in the resulting inhibitor 43 (k_ass32,500 s²¹M²¹

)

Inhibitor

P₂

P₃

k_inac/K_i(M²¹s²¹

)

(Table 4).

Amino acid

Residue

vs cruzain vs cath B vs papain

Probing the S⁰1 site of cruzain with O-benzyl hydrox-

amate derivatives

26b

27b

28b

29b

d-hPhe

l-hPhe

±(CH₂)₄OH

±(CH₂)₅OH

±(CH₂)₄OH

±(CH₂)₅OH

2500

5000

91 500

96 200

1050

7600

32 500

41 700

25 000

4300

39 000

54 200

E-64 and other naturally occurring peptidyl epoxysuccinates

9752

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

Scheme 5.

bind only in the S subsites of the targeted cysteine protease.

However, the literature contains a growing number of

examples in which increased potency and selectivity of

inhibitors has been achieved vs cysteine proteases of the

papain family by taking advantage of binding interactions

in both the S and S⁰subsites. For example, potent peptido-

mimetic inhibitors that span the active site of cathepsin K

have been prepared by the SmithKline group.⁷³Similarly,

the development of synthetic derivatives that bind in the S⁰

sites of cathepsin B^42,43paved the way for the successful

design of E-64 analogs targeting the entire active site of

cathepsins B and L.^46±48

The results of kinetic evaluation of 45±48 with cruzain are

given in Table 5. The d-hPhe hydroxamate inhibitors 45 and

48 display increased activity compared to the parent acids

11c and 38. However, and most surprisingly, the l-hPhe

derivative 46 proved to be a very weak inhibitor. Once

again this divergent set of results suggests that the epimeric

inhibitors bind to cruzain in different ways. It is also evident

from these results that iterative structural modi®cations in

the d-hPhe series can not be applied directly to the l-hPhe

series, or vice versa. An indication of the importance of the

hydroxamic acid functionality to the potency of the inhibitor

is given by comparison of the IC₅₀values of the amide 49

and O-benzyl hydroxamate ester 45 isosteres. The amide

suffers a most signi®cant three order of magnitude drop in

activity (IC₅₀determination).

In an effort to explore interactions of synthetic E-64 analogs

with the cruzain S⁰sites we were attracted by the work of

Rich and Meara who replaced the free carboxylate of E-64c

(5) by other functionalities and found that a hydroxamic

acid unit is the closest in potency to the parent acid for

inhibition of papain. In contrast a primary amide function

lowered the potency by about two orders of magnitude.⁷⁴In

analogous way, substrate-like peptidyl O-acyl hydroxa-

mates have been used as very potent cysteine protease

inhibitors.⁷⁵We thus decided to probe interactions of the

shallow, hydrophobic S⁰₁site of cruzain with the O-benzyl

hydroxamates 45±48 prepared by treatment of the parent

acids with O-benzyl hydroxylamine (44) and EEDQ⁷⁶

(Scheme 6).

The d-hTyr derivative 47 is the most potent inhibitor of

cruzain to emerge from this series. We studied this inhibitor

against several other cysteine proteases: the cathepsin

B-like protease from the parasite Leishmania major,²⁹

bovine cathepsin B⁶¹and papain (Table 6). That 47 is highly

selective for cruzain vs bovine cathepsin B, which is highly

homologous to cruzain, may have signi®cance for further

development of this inhibitor series targeting Chaga's

disease. Cathepsin B is an important cysteine protease and

alterations of its metabolism have been associated with

pathological problems.⁶¹

Table 4. IC₅₀(nM) and k_ass(M²¹sec²¹) values for compounds 36±38 and 43. k_assis a second order inhibition rate constant, and is used in cases where saturation

kinetics demonstrating reversible inhibitor binding cannot be determined (Ref. [66])

Inhibitor

P₃

P₂

IC₅₀vs cruzain (Nm)

k_assoc(M²¹s²¹

)

36

37

38

43

±CH₂C₆H₁₁

±CH₂C₆H₅

±C₆H₅

±CH₂CH₂Ph (d-hPhe)

±CH₂CH₂C₆H₄OH (D-hTyr)

,1000

,100

,10

nd

66 000

32 500

±CH₂CH₂CHMe₂

,50

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9753

Scheme 6.

Table 5. IC₅₀and kinetic data for inhibitors 45±49 vs. cruzain. For compound 47 the k_assvalue is reported; ndnot determined

Inhibitor

X

R₂

P₂amino acid

R₃side chain

k_inact/K vs cruzain

IC₅₀vs cruzain (nM)

45

46

47

48

49

O

CH₂

±CH₂CH₂Ph

±CH₂CH₂C₆H₄OH

±CH₂CH₂Ph

(d-hPhe)

(l-hPhe)

(D-hTyr)

(d-hPhe)

±CH₂CH₂CHMe₂

±C₆H₅

118 000

6000

441 600

179 700

nd

6

nd

,10

2

±CH₂CH₂CHMe₂

6000

Table 6. Activity of inhibitor 47 against several cysteine proteases

Cruzain

Cath. B (bovine)

Cath. B (Leish)

Papain

k_inact/K_i(s²¹M²¹

Cruzain/enzyme relative selectivity

)

441 600

1

1785

247:1

8594

51:1

1448

305:1

Effect of epoxysuccinyl inhibitors in vivo vs T. Cruzi

mouse model,¹³extended the lifetime of infected J744 cells

for.28 days at 10 mM inhibitor concentration. Most of the

compounds tested suffer from low water solubility that may

contribute to their lack of activity in vivo. Despite its high

potency against cruzain, inhibitor 47 was inactive against

the parasite in vivo. These facts underline the challenge to

improve the low bioavailability of these compounds by

further structural modi®cations.

Ethyl esters 10a±e, 12a, 26a, 27a, 28a, 29a, 33±35 and

hydroxamates 45±48 were tested against J744 cells infected

with T. cruzi. in a standard tissue culture assay.³²Ethyl

esters were used as prodrugs for all epoxysuccinic acid

inhibitors, since the carboxylic acids themselves are

inactive in vivo (presumably due to problems with transport

across the cell membrane). In spite of the promising results

for the inhibition of cruzain in vitro, most of the compounds

displayed only marginal or no activity against T. cruziin

tissue culture. In just two cases (10b and 48) did the

inhibitor prolong the lifetime of the infected cells (Table

7). Compound 10b extended the life of the infected cells

for 16 days at a concentration of 40 mM whereas inter-

mediate 48 extended the lifetime for 13 days at 10 mM

concentration. In contrast, the reference compound

50^24,25,77±79which has proved to cure Chagas' disease in a

Conclusion

Iterative modi®cations of the structure of E-64c via

molecular modeling and ligand design within the active

site of cruzain, combined with the synthesis of new epoxy-

succinyl inhibitors designed to explore P₃/S₃binding inter-

actions have led to potent d-hPhe (45, 48) and d-hTyr (43,

47) containing epoxysuccinate inhibitors of cruzain.

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W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

Table 7. Effect of epoxysuccinyl inhibitors on survival of J744 macrophages infected with T. cruzi trypomastigotes, treated daily with a solution of inhibitor

(typically 10 mM). Survival time is de®ned as the time before the cell monolayer is destroyed by infection.^15,32Untreated cells are completely destroyed after 6

days in this assay

Inhibitor

Concentration (mM)

Survival time (days)

10b

48

50

40

10

16

13

.28

However, the actual mode of binding of these inhibitors to

cruzain differs from the model from which they were

derived.⁷²Evaluation of these compounds in vivo shows

that only 10b and 48 display activity against T. cruzi in

tissue culture. Compound 47 is the most potent peptidyl

epoxysuccinate inhibitor prepared in this series. The

synthesis of a larger number of O-alkyl hydroxamic acid

derivatives offers the potential for discovery of very potent

and speci®c epoxysuccinyl inhibitors of other important

cysteine proteases. Such studies are in progress and will

be reported in due course.

morpholine (NMM), chloroform (stabilized with amylene)

and N,N⁰-dimethyl formamide (DMF) were dried and stored

Ê

over 4 A molecular sieves. IC₅₀determinations and kinetic

analysis with cruzain were performed as previously

described.³²The experimental conditions for evaluation of

the effect of the inhibitors described in this paper on J774

macrophages infected with T. cruzi trypomastigotes have

been described by Engel et al.¹³

Compounds 10a±e were prepared from the p-nitrophenol

ester 9 and the free amines derived from the pepti-

domimetics 8a±e by the method reported by Yokoo for

the synthesis of 4.⁵⁸Yields are indicated in parentheses.

Experimental

(2S,3S)-3-[[[(1R)-3-Methyl-1-[[(3-methylbutyl)amino]-

carbonyl]butyl]amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (10a). Obtained as a white solid (89%): mp

General

1

¹H NMR spectra were measured at 400 MHz on a Varian

VXR-400 instrument or at 500 MHz on a Varian Inova 500

instrument. Residual chloroform (d 7.27 ppm), methanol (d

3.31 ppm) or Me₄Si in DMSO-d₆were used as internal

references. ¹³C NMR spectra were measured at 100 or

125 MHz using residual chloroform (77.0 ppm) or DMSO

(39.5 ppm) as internal reference. Unless otherwise stated,

1358C; [a]_D182.48 (c2.1; CHCl₃); H NMR (CDCl₃)

d 6.45 (d, J8.5 Hz, 1H), 5.80 (bs, 1H), 4.35 (m, 1H),

4.27 (m, 2H), 3.68 (d, J2 Hz), 3.52 (d, J2 Hz, 1H),

3.26 (m, 2H), 1.61 (m, 4H), 1.38 (q, J7 Hz, 2H), 1.32

(t, J7 Hz, 3H), 0.93 (m, 12H);¹³C NMR (CDCl₃) d

170.96, 166.46, 166.00, 62.17, 53.70, 52.63, 51.51,

41.26, 38.29, 37.93, 25.82, 24.77, 22.70, 22.33, 22.22,

13.96; IR (CHCl₃) n cm²¹3450, 3410, 1760, 1685,

1535, 1475, 1380, 1320, 1090, 1035, 915; HRMS, CI

(NH₃) [M1H]¹calcd for C₁₇H₃₁N₂O₅: 343.2232, found:

343.2238.

1

the H and ¹³C NMR spectra were measured at 500 and

100 MHz respectively. Infrared spectra were recorded on

a Perkin±Elmer Spectrum 1000 spectrophotometer. High

resolution mass spectra (HRMS) were measured on a

Micromass Corp. VG 70-250-S spectrometer at the

University of Michigan Mass Spectrometry Laboratory.

Chemical ionization (CI) mass spectra were obtained

using NH₃as the reagent gas. Optical rotations were

measured on a Rudolph Autopoll III polarimeter using a

quartz cell of 1 mL capacity and a 10 cm path length. Melt-

ing points were determined in a Thomas Hoover apparatus

and are uncorrected. l- and d-homophenylalanine (hPhe),

d-homotyrosine (d-hTyr), 1-hydroxybenzotriazole hydrate

(HOBT´H₂O), 1-(3-dimethylaminopropyl)-3-ethylcarbodi-

imide hydrochloride (EDC´HCl) and N-ethoxycarbonyl-2-

ethoxy-1,2-dihydroquinoline (EEDQ) were purchased

from Advanced ChemTech (Kentucky, USA). N-methyl-

(2S,3S)-3-[[[(1R)-2-[3-Methylbutyl)amino]-2-oxo-1-

(phenylmethyl)ethyl]amino]carbonyl-oxiranecarboxylic

acid ethyl ester (10b). Obtained as a white solid (95%): mp

1248C; [a]_D131.08 (c2.0; CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 7.26 (m, 5H), 6.24 (d, J8 Hz, 1H), 5.26 (bs, 1H),

4.45 (m, 1H), 4.25 (m, 2H), 3.65 (d, J2 Hz, 1H), 3.49 (d,

J2 Hz, 1H), 3.11 (m, 3H), 2.95 (dd, J9, 13 Hz, 1H), 1.37

(m, 1H), 1.32 (t, J7 Hz, 3H), 1.15 (q, J7 Hz, 2H), 0.83

(d, J7 Hz, 3H), 0.82 (d, J7 Hz, 3H);¹³C NMR (CDCl₃) d

169.66, 166.51, 165.74, 136.19, 129.17, 128.65, 127.09,

62.21, 54.49, 53.64, 53.58, 52.61, 52.56, 38.79, 37.89,

37.68, 25.46, 22.24, 13.96; IR (CHCl₃) n cm²¹3420,

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9755

3375, 1745, 1670, 1520, 1460, 1360, 1310, 920, 890;

HRMS, CI (NH₃) [M1H]¹calcd for C₂₀H2₉N₂O₅

377.2076, found: 377.2077.

(2S,3S)-3-[[[(1R)-3-Methyl-1-[[(3-methylbutyl)amino]-

carbonyl]butyl]amino]carbonyl]-oxiranecarboxylic acid

(11a). Obtained from 10a as a white solid (99%): mp 588C;

[a]_D196.08 (c1.0; ethanol); ¹H NMR (CDCl₃) d 7.30 (d,

J8.5 Hz, 1H), 6.63 (s, 1H), 4.49 (q, J7 Hz, 1H), 3.70 (d,

J1.5 Hz, 1H), 3.60 (d, J1.5 Hz, 1H), 3.30 (m, 1H), 3.20

(m, 1H), 1.60 (m, 4H), 1.40 (m, 2H), 0.92 (m, 12H); ¹³C

NMR (CDCl₃) d 172.16, 169.45, 166.66, 53.49, 52.29,

51.80, 41.00, 37.85, 25.77, 24.70, 22.57, 22.36, 22.28,

22.19; IR (KBr) n cm²¹3294, 3089, 2956, 1739, 1654,

1560, 1467, 1388, 1369, 1232, 1171, 898, 867, 652;

HRMS, CI (NH₃) [M1H]¹calcd for C₁₅H₂₇N₂O₅:

315.1913, found: 315.1918.

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-3-

phenylpropyl]amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (10c). Obtained as a white solid (92%): mp

174±1768C; [a]_D144.88 (c2.3; CHCl₃); ¹H NMR

(400 MHz, CDCl₃) d 7.22 (m, 5H), 6.61 (d, J8.4 Hz,

1H), 5.75 (bs, 1H), 4.27 (m, 3H), 3.65 (d, J1.6 Hz, 1H),

3.50 (d, J1.6 Hz, 1H), 3.24 (m, 2H), 2.65 (m, 2H), 2.14 (m,

1H), 1.98 (m, 1H), 1.56 (m, 1H), 1.35 (q, J7 Hz, 2H), 1.30

(t, J7 Hz, 3H), 0.90 (d, J7 Hz, 6H); ¹³C NMR (CDCl₃) d

170.23, 166.49, 165.96, 140.37, 128.56, 128.28, 126.29,

62.28, 53.71, 53.61, 52.68, 52.61, 52.46, 38.20, 37.91,

33.93, 31.66, 25.73, 22.32, 13.98; IR (CHCl₃) n cm²¹

3430, 3380, 1745, 1675, 1520, 1450, 1360, 1300, 1080,

1020, 890; HRMS, CI (NH₃) [M1H]¹calcd for

C₂₁H₃₁N₂O₅: 391.2233, found: 391.2244. Anal. Calcd for

C₂₁H₃₀N₂O₅: C, 64.59; H, 7.74; N, 7.18. Found: C, 64.75;

H, 7.92; N, 7.13.

(2S,3S)-3-[[[(1R)-2-[3-Methylbutyl)amino]-2-oxo-1-

(phenylmethyl)ethyl]amino]carbonyl-oxiranecarboxylic

acid (11b). Obtained from 10b as a white solid (93%): mp

94±958C; [a]_D135.88 (c2.4; CHCl₃); ¹H NMR (DMSO-

d₆) d 13.44 (s, 1H), 8.70 (d, J8 Hz, 1H), 8.03 (t, J5 Hz,

1H), 7.21 (m, 5H), 4.75 (m, 1H), 3.61 (d, J1.5 Hz, 1H),

3.31 (d, J1.5 Hz, 1H), 3.04 (m, 2H), 2.97 (dd, J_a6 Hz,

J_b14 Hz, 1H), 2.78 (dd, J_a6 Hz, J_b14 Hz, 1H), 1.47 (m,

1H), 0.83 (m, 6H); ¹³C NMR (DMSO-d₆) d 169.96, 168.74,

164.98, 137.49, 129.12, 128.07, 126.36, 54.25, 52.51, 51.08,

37.89, 36.75, 24.97, 22.36; IR (KBr) n cm²¹3298, 3089,

2958, 1736, 1648, 1546, 1498, 1458, 1387, 1368, 1228, 897,

860, 745, 700, 650; HRMS CI (NH₃) [M1H]¹calcd for

C₁₈H₂₅N₂O₅: 349.1757, found: 349.1760.

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-2-

(phenylmethoxy)ethyl]amino]carbonyl]-oxiranecarboxylic

acid ethyl ester (10d). Obtained as a white solid (88%): mp

1318C; [a]_D113.08 (c2.1; CHCl₃); ¹H NMR (CDCl₃) d

7.34 (m, 5H), 6.9 0 (d, J7 Hz, 1H), 6.25 (m, 1H), 4.59 (d,

J12 Hz, 1H), 4.52 (d, J12 Hz, 1H), 4.45 (ddd,

J_a4.4 Hz, J_b8.8 Hz, J_c9.2 Hz, 1H), 4.25 (m, 2H), 3.83

(dd, J_a4.4 Hz, J_b9.2 Hz, 1H), 3.67 (d, J1.6 Hz, 1H),

3.67 (d, J1.6 Hz, 1H), 3.49 (app t, J8.8 Hz, 1H), 3.25

(m, 2H), 1.54 (m, 1H), 1.34 (q, J7 Hz, 2H), 1.30 (t,

J7 Hz, 3H), 0.87 (d, J6.8 Hz, 6H); ¹³C NMR (CDCl₃)

d 168.76, 166.49, 166.00, 136.99, 128.54, 128.10, 127.84,

73.53, 68.99, 62.26, 53.69, 52.71, 51.69, 38.12, 37.93,

25.65, 22.31, 22.29, 13.98; IR (CHCl₃) n cm²¹3360,

1740, 1665, 1510, 1460, 1360, 1300, 1080, 1015, 890;

HRMS, CI (NH₃) [M1H]¹calcd for C₂₁H₃₁N₂O₆:

407.2182, found: 407.2192.

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-3-

phenylpropyl]amino]carbonyl]-oxiranecarboxylic acid

(11c). Obtained from 10c as a white solid (89%): mp 138

±1408C; [a]_D139.28 (c1.0; CHCl₃); ¹H NMR

(CDCl₃1CD₃OD) d 7.56 (d, J8.5 Hz, 1H), 7.28 (m,

1H), 7.22 (m, 2H), 7.11 (m, 3H), 4.36 (t, J7 Hz, 1H),

3.61 (s, 1H), 3.53 (s, 1H), 3.21 ((m, 1H) 3.13 (m, 1H),

2.58 (t, J7 Hz, 2H), 2.03 (m, 1H), 1.93 (m, 1H), 1.54

(m, 1H), 1.33 (q, J7 Hz, 2H), 0.85 (d, J7 Hz, 6H); ¹³C

NMR (CDCl₃1CD₃OD) d 170.92, 168.84, 166.39, 140.31,

128.30, 128.10, 126.00, 53.23, 52.49, 52.14, 37.80, 37.70,

33.71, 31.54, 25.45, 22.12, 22.06; IR (CHCl₃) n cm²¹3420,

3380, 1735, 1670, 1520, 1450, 1230, 1030, 890; FAB

[M1H]¹calcd for C₁₉H₂₇N₂O₅: 363.1913, found: 363.1909.

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-2-

methyl-2-(phenyl methoxy)ethyl]amino]carbonyl]-oxirane-

carboxylic acid ethyl ester (10e). Obtained as a white solid

(96%): mp 1718C; [a]_D123.58 (c2.0; CHCl₃); ¹H NMR

(CDCl₃) d7.35 (m, 5H), 7.06 (d, J6.4 Hz, 1H), 6.39 (m,

1H), 4.68 (d, J11.6 Hz, 1H), 4.62 (d, J11.6 Hz, 1H), 4.49

(dd, J_a3.2 Hz, J_b6.8 Hz, 1H), 4.26 (M, 2H), 4.05 (m,

1H), 3.68 (d, J1.6 Hz, 1H), 3.58 (d, J1.6 Hz, 1H), 3.29

(m,1H), 3.20 (m,1H), 1.53 (m, 1H), 1.31 (m, 5H), 1.11 (d,

J6 Hz,3H), 0.87 (d, J6.4 Hz, 3H), 0.85 (d, J6.4 Hz,

3H); ¹³C NMR (CDCl₃) d 167.80, 166.52, 165.86, 137.55,

128.52, 128.00, 127.78, 73.73, 71.48, 62.21, 54.98, 53.68,

52.70, 38.07, 37.78, 25.58, 22.26, 22.22, 14.61, 13.98; IR

(CHCl₃) n cm²¹3380, 1745, 1670, 1515, 1460, 1360, 1300,

1090, 1020, 895; HRMS, CI (NH₃) [M1H]¹calcd for

C₂₂H₃₃N₂O₆: 421.2338, found: 421.2319.

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-2-

(phenylmethoxy)ethyl]amino]carbonyl]-oxiranecarboxylic

acid (11d). Obtained from 10d as an oil (93%):

1

[a]_D110.88 (c1.2; CHCl₃); H NMR (CDCl₃) d 11.00

(bs, 1H), 7.54 (d, J8 Hz, 1H), 7.30 (m, 5H), 6.95 (t,

J5 Hz, 1H), 4.66 (dd, J_a5 Hz, J_b12 Hz, 1H), 4.53 (d,

J12 Hz, 1H), 4.50 (d, J12 Hz, 1H), 3.74 (m, 1H), 3.72

(d, J1.5 Hz, 1H), 3.61 (d, J1.5 Hz, 1H), 3.59 (m, 1H),

3.22 (m, 2H), 1.54 (m, 1H), 1.33 (q, J7 Hz, 2H), 0.86 (d,

J6 Hz, 6H); ¹³C NMR (CDCl₃) d 169.61, 169.07, 166.82,

136.93, 128.44, 127.99, 127.75, 73.33, 68.99, 53.42, 52.29,

38.14, 37.77, 25.55, 22.26, 22.22; IR (KBr) n cm²¹3299,

3090, 2957, 2871, 1739, 1651, 1548, 1455, 1386, 1367,

1210, 1108, 897, 859, 736, 698; HRMS CI (NH₃)

[M1H]¹calcd for C₁₉H₂₇N₂O₆: 379.1862, found: 379.1874.

Synthesis of compounds 11a±e

Compounds 11a±e were prepared by saponi®cation of the

corresponding ethyl esters 10a±e with KOH±ethanol as

described by Yokoo et al.⁵⁸

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-2-

methyl-2-(phenylmethoxy)ethyl]amino]carbonyl]-oxirane-

carboxylic acid (11e). Obtained from 10e as an oil (98%):

9756

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

1

[a]_D115.08 (c1.0; CHCl₃); H NMR (CDCl₃) d 10.06

(bs, 1H), 7.45 (d, J8 Hz, 1H), 7.33 (m, 5H), 6.74 (t, J

5.5 Hz, 1H), 4.60 (m, 3H), 4.08 (m, 1H), 3.74 (J1.5 Hz,

1H), 3.62 (d, J1.5 Hz, 1H), 1.53 (m, 1H), 1.32 (m, 2H),

1.13 (d, J6 Hz, 3H), 0.86 (m, 6H); ¹³C NMR (CDCl₃) d

169.02, 168.64, 166.74, 137.39, 128.52, 128.02, 127.85,

73.89, 71.47, 55.63, 53.49, 52.37, 25.57, 22.25, 22.21,

15.03; IR (KBr) n cm²¹3370, 2960, 1735, 1650, 1515,

1450, 1380, 1230, 1075, 1030, 890, 690; HRMS, CI

(NH₃) [M1H]¹calcd for C₂₀H₂₉N₂O₆: 393.2018, found:

393.2012.

triethylamine (6.2 g, 61.7 mmol), and 4-dimethylaminopyri-

dine (34 mg, 0.3 mmol) in 50 mL of CH₂Cl₂under nitrogen

was added tert-butyldimethylchloro silane (8.88 g,

58.9 mmol). The mixture was stirred for 0.5 h, then was

allowed to warm to 238C and stirred for 3.5 h. After this

time the reaction mixture was poured into cold water and

extracted with CH₂Cl₂. The organic portion was washed

with NaHCO₃, brine and dried over Na₂SO₄. The solvent

was evaporated in vacuo and the residue was distilled bulb

to bulb (1508C, 0.01 mm Hg) to obtain 9.7 g (85%) of 20 as

1

a slightly yellow semi-solid: H NMR (CDCl₃) d 3.52 (t,

J6.5 Hz, 2H), 2.71 (bs, 2H), 2.61 (t, J6.5 Hz, 2H), 1.43

(m, 4H), 0.80 (s, 9H), 20.05 (s, 6H); ¹³C NMR (CDCl₃) d

62.78, 41.59, 29.99, 29.54, 25.76, 25.65, 18.11, 25.50; IR

(neat) n cm²¹3315, 2953, 2930, 2858, 1647, 1577, 1486,

1471, 1383. 1361, 1307, 1255, 1191, 1102, 991, 938, 836,

774; HRMS, CI (NH₃) [M1H]¹calcd for C₁₀H₂₆NOSi

204.1783, found: 204.1778.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[(phenylmethoxy)carbonyl]-

propyl]amino]carbonyl]-oxiranecarboxylic acid ethyl

ester (16). To a solution of the p-toluensulfonic acid salt

of d-homophenylalanine benzyl ester 13⁶⁸(2.14 g,

4.8 mmol), monoethyl epoxysuccinate 15⁵⁸(0.78 g,

4.8 mmol), HOBT´H₂O (0.74 g, 4.8 mmol) and N-methyl-

morpholine (0.54 mL, 4.8 mmol), in dry chloroform

(10.0 mL) under nitrogen at 08C was added EDC´HCl

(1.02 g, 5.2 mmol). The mixture was stirred at 08C for 1 h

and then 7 h at 23 08C. The mixture was then partitioned

between EtOAc and water. The organic portion was washed

with brine, dried over Na₂SO₄and concentrated in vacuo.

The residue was puri®ed by ¯ash chromatography on silica

gel (eluted with 7:3 hexanes±EtOAc) 7:3 to give 658 mg

(80%) of 16 as a white solid: mp 76±788C; [a]_D147.78

5-(t-Butyldimethylsilanyloxy)-pentylamine (21). Obtained

as an oil in 86% yield from 5-amino-1-pentanol by the

method described above: ¹H NMR (CDCl₃) d 3.49 (t,

J6.5 Hz, 2H), 2.95 (bs, 2H), 2.58 (t, J6.5 Hz, 2H),

1.42 (m, 4H), 1.25 (m, 2H), 0.79 (s, 9H), 20.06 (s, 6H);

¹³C NMR (CDCl₃) d 62.80, 41.53, 32.63, 32.38, 25.73,

25.61, 22.87, 18.09, 25.52; IR (neat) n cm²¹3306, 2930,

2858, 1578, 1472, 1387, 1361, 1309, 1255, 1187, 1102,

1006, 938, 835, 775; HRMS, CI (NH₃) [M1H]¹calcd for

C₁₁H₂₈NOSi 218.1940, found: 2218.1932.

1

(c3.0; CHCl₃); H NMR (CDCl₃) d 7.41±7.10 (m, 10H),

6.45 (d, J9 Hz, 1H), 5.18 (d, J12.5 Hz, 1H), 5.12 (d,

J12.5 Hz, 1H), 4.68 (m, 1H), 4.27 (m, 2H), 3.69 (d,

J2 Hz, 1H), 3.50 (d, J2 Hz, 1H), 2.61 (m, 2H), 2.22

(m, 1H), 2.03 (m, 1H), 1.33 (t, J7 Hz, 3H); ¹³C NMR

(CDCl₃) d 171.22, 166.38, 165.86, 140.09, 134.97,

128.71, 128.67, 128.55, 128.43, 128.33, 126.37, 67.46,

62.13, 53.71, 52.73, 51.54, 33.47, 31.52, 14.03; IR

(CHCl₃) n cm²¹3684, 3402, 3025, 3016, 1745, 1690,

1525, 1227, 1205, 1028, 929, 796, 719; HRMS, CI (NH₃)

[M]¹calcd for C₂₃H₂₅NO₅411.1675, found: 411.1670.

Anal. Calcd for C₂₃H₂₅NO₆: C, 67.14; H, 6.12; N, 3.40.

Found: C, 67.13; h 6,.38; N, 3.46.

(2S,3S)-3-[[[(1R)3-Phenyl-1-[[(4-t-butyldimethylsilanoxy-

butyl)amino]carbonyl]propyl)amino]carbonyl]-oxirane-

carboxylic acid ethyl ester (22). To a solution of the amine

20 (0.20 g, 1 mmol), acid 18 (0.32 g, 1 mmol), HOBT´H₂O

(0.15 g, 1 mmol), and N-methylmorpholine (0.1 mL) in

CHCl₃(5 mL) under nitrogen at 08C was added EDC

(0.21 g, 1.1 mmol). The resulting mixture was stirred 1 h

at this temperature then at 238C for 9 h. The mixture was

then partioned between EtOAc and water. The organic

portion was washed with brine, dried over Na₂SO₄and

concentrated in vacuo. The residue was puri®ed by ¯ash

chromatography on silica gel (elution with hexanes±

EtOAc 7:3) to afford 0.32 g (64%) of 22 as a white solid:

(2S,3S)-3-[[[(1R)-3-Phenyl-1-(carboxyl)propyl]amino]-

carbonyl]-oxiranecarboxylic acid ethyl ester (18). A

mixture of benzyl ester 16 (0.60 g, 1.4 mmol) and 10%

Pd/C (200 mg) in 50 mL of EtOAc and 50 mL of methanol

was shaken on a Parr apparatus under hydrogen (40 psi) for

15 min. The mixture was purged with nitrogen, the catalyst

removed by ®ltration through a pad of Celite and the solu-

tion concentrated in vacuo to afford 0.45 g (97%) of 18 as a

white solid: mp 124±1268C; [a]_D158.78 (c3.1;

1

mp 114±1168C; [a]_D127.18 (c2.4, CHCl₃); H NMR

(CDCl₃) d 7.31±7.16 (m, 5H), 6.60 (d, J8 Hz, 1H), 5.87

(t, J5.5 Hz, 1H), 4.29 (m, 3H), 3.67 (d, J2 Hz, 1H), 3.64

(t, J6 Hz, 2H), 3.52 (d, J2 Hz, 1H), 3.31 (m, 1H), 3.26

(m, 1H), 2.67 (m, 2H), 2.16 (m, 1H), 2.00 (m, 1H), 1.56 (m,

4H), 1.32 (t, J7 Hz, 3H), 0.90 (s, 9H), 0.06 (s, 6H); ¹³C

NMR (CDCl₃) (75 MHz) d 170.25, 166.50, 165.88, 140.38,

128.54, 128.27, 126.25, 62.54, 62.23, 53.67, 52.63, 52.52,

39.40, 34.08, 29.97, 26.03, 25.90, 18.27, 13.99, 25.35; IR

(CHCl₃) n cm²¹3400, 3020, 2940, 2870, 1755, 1675, 1525,

1475, 1460, 1395, 1360, 1260, 1100, 1030, 900, 845, 705;

HRMS, CI (NH₃) [M1H]¹calcd for C₂₆H₄₃N₂O₆Si

507.2890, found: 507.2908.

1

CH₃OH); H NMR (CDCl₃) d 10.6 (bs, 1H), 7.31±7.17

(m, 5H), 6.64 (d, J8 Hz, 1H), 4.64 (m, 1H), 4.27 (m,

2H), 3.73 (d, J2 Hz, 1H), 3.56 (d, J2 Hz, 1H), 2.71 (t,

J7 Hz, 2H), 2.27 (m, 1H), 2.07 (m, 1H), 1.31 (t, J7 Hz,

3H); ¹³C NMR (125 MHz, CDCl₃) d 175.65, 166.45,

166.42, 139.91, 128.59, 128.33, 126.44, 62.42, 53.61,

52.77, 51.42, 33.14, 31.61, 13.98; IR (KBr) n cm²¹3382,

3351, 2929, 1750, 1636, 1545, 1498, 1455, 1323, 1235,

1211, 1128, 1102, 1024, 904; HRMS, EI, M¹calcd for

C₁₆H₁₉NO₆321.1212, found: 321.1200.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(5-t-butyldimethylsilanoxy-

pentyl)amino]carbonyl]propyl)amino]carbonyl]-oxirane-

carboxylic acid ethyl ester (23). Obtained in 66% yield

from acid 18 and amine 21 by the method described

1

4-(t-Butyl-dimethyl-silanyloxy)-butylamine (20). To a

08C solution of 4-amino-1-butanol (5.0 g, 56.1 mmol),

above: mp 125±1268C; [a]_D127.78 (c2.6; CHCl₃); H

NMR (CHCl₃) d 7.32±7.17 (m, 5H), 6.58 (d, J8 Hz, 1H),

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9757

5.71 (t, J6 Hz, 1H), 4.28 (m, 3H), 3.67 (d, J2 Hz, 1H),

3.61 (t, J6.5 Hz, 2H), 3.51 (d, J2 Hz, 1H), 3.24 (m, 2H),

2.67 (m, 2H), 2.16 (m, 1H), 2.00 (m, 1H), 1.52 (m, 4H), 1.35

(m, 2H), 1.32 (t, J7 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 6H); ¹³C

NMR (CDCl₃) d 170.31, 466.51, 165.94, 140.37, 128.53,

128.26, 62.85, 62.23, 53.62, 52.62, 52.57, 52.50, 39.63,

34.01, 32.29, 31.66, 29.17, 25.90, 23.18, 18.28, 13.97,

25.34; IR (CHCl₃) n cm²¹3460, 3400, 1755, 1680, 1530,

1460, 1380, 1310, 1260, 1095, 1035, 905, 845; HRMS, CI

(NH₃) [M1H]¹calcd for C₂₇H₄₅N₂O₆Si 521.3046, found:

521.3047.

4.27 (m, 2H), 3.67 (d, J2 Hz, 1H), 3.65 (t, J7 Hz, 2H),

3.55 (d, J2 Hz, 1H), 3.27 (m, 2H), 2.67 (m, 2H), 2.16 (m,

1H), 2.00 (m, 1H), 1.61±1.51 (m, 6H), 1.40 (m, 2H), 1.32 (t,

J7 Hz, 3H); ¹³C NMR (CDCl₃) d 170.44, 166.55, 166.11,

140.33, 128.61, 128.35, 62.34, 53.70, 52.68, 52.47, 39.41,

33.72, 31.95, 31.68, 28.98, 22.92, 14.00; IR (KBr) n cm²¹

3400, 3020, 1715, 1675, 1535, 1460, 1375, 1090, 1035, 940,

905; HRMS, CI (NH₃) [M1H]¹calcd for C₂₁H₃₁N₂O₆

407.2182, found: 407.2180.

(2S,3S)-3-[[[(1S)-3-Phenyl-1-[[(4-hydroxybutyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (28a). Obtained in 98% yield from 24 by

the procedure described above: mp 140±1428C;

(2S,3S)-3-[[[(1S)-3-Phenyl-1-[[(4-t-butyldimethylsilanoxy-

butyl)amino]carbonyl]propyl)amino]carbonyl]-oxirane-

carboxylic acid ethyl ester (24). Obtained in 66% yield

from acid 19⁵⁵and amine 20 by the method described

above: mp 1208C; [a]_D126.58 (c2.0; CHCl₃); ¹H

NMR (CDCl₃) d 7.29±7.13 (m, 5H), 7.00 (d, J8 Hz,

1H), 6.37 (t, J5 Hz, 1H), 4.40 (dd, J_a8 Hz, J_b10 Hz,

1H), 4.26 (m,2H), 3.66 (d, J1.5 Hz, 1H), 3.63 (t, J6 Hz,

2H), 3.33 (m, including d, J2 Hz, 2H), 3.24 (m, 1H), 2.66

(m, 2H), 2.15 (m, 1H), 2.00 (m, 1H), 1.56 (m, 4H), 1.31 (t,

J7 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 6H); ¹³C NMR (CDCl₃)

d 170.22, 166.54, 165.89, 140.46, 128.55, 128.22, 126.26,

62.55, 62.23, 53.67, 52.67, 52.57, 39.41, 34.03, 31.66,

29.96, 26.00, 25.89, 18.26, 13.99, 25.36; IR (CHCl₃) n

cm²¹3328, 3277, 3086, 2930, 2859, 1754, 1690, 1643,

1552, 1497, 1455, 1386, 1338, 1255, 1222, 1195, 1100,

1027, 987, 899, 838, 778, 754, 700; HRMS, CI (NH₃)

[M1H]¹calcd for C₂₆H₄₃N₂O₆Si 507.2890, found:

507.2892.

1

[a]_D131.98 (c3.1; CHCl₃); H NMR (CDCl₃) d 7.31±

7.16 (m, 5H), 6.70 (d, J8 Hz, 1H), 6.39 (t, J5 Hz, 1H),

3.67 (t, J6 Hz, 2H), 3.64 (d, J1.5 Hz, 1H), 3.30 (m,

including d, J1.5 Hz, 3H), 4.37 (m, 1H), 4.27 (m, 2H),

2.64 (m, 2H), 2.17 (m, 1H), 1.99 (m, 1H), 1.76 (bs, 1H),

1.62 (m, 4H), 1.32 (t, J6 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃) d 170.39, 166.55, 166.08, 140.49, 128.63, 128.30,

129.35, 62.35, 62.27, 53.69, 52.66, 39.42, 33.82, 31.75,

29.66, 26.06, 14.03; IR (CHCl₃) n cm²¹3380, 3020,

2940, 1750, 1675, 1535, 1455, 1375, 1310, 1220, 1090,

900, 720, 705; HRMS, CI (NH₃) [M1H]¹calcd for

C₂₀H₂₉N₂O₆393.2018, found: 393.2012. Anal. Calcd for

C₂₀H₂₈N₂O₆: C, 61.21; H, 7.19; N, 7.14. Found: C, 61.23;

H, 7.19; N, 7.13.

The preparation of compound 29a has been reported

elsewhere.⁵⁵

The preparation of compound 25 has been reported.⁵⁵

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(4-hydroxybutyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

(26b). A 1N solution of KOH in EtOH (0.5 mL, 0.5 mmol)

was added to a solution of the ethyl ester 26a (0.20 g,

0.5 mmol) in a mixture of THF (5 mL) and ethanol

(5 mL) at 08C and stirred for 0.5 h. The solvent was evapo-

rated in vacuo and the residue was dissolved in cold water

(5 mL) and acidi®ed to pH 1±2 with 10% KHSO₄and

extracted with EtOAc (5£10 mL). The combined organic

portions were washed with brine, dried over Na₂SO₄and

concentrated in vacuo to obtain 0.19 g (96%) of 26b as a

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(4-hydroxybutyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (26a). A 48% solution of HF in water (0.31 mL,

0.84 mmol) was added to a suspension of 22 (0.29 g,

0.56 mmol), in CH₃CN (5 mL) and CH₂Cl₂(5 mL) at 08C.

The mixture was allowed to warm to 238C and stirred for

15 min. The mixture was diluted with CH₂Cl₂(30 mL) and

washed twice with concentrated NaHCO₃(20 mL), brine

(20 mL), dried over Na₂SO₄and concentrated in vacuo to

afford 0.21 g (96%) of crude 26a as a white solid that

required no further puri®cation: mp 115±1168C;

1

white solid. mp 888C; [a]_D140.78 (c2.7; CH₃OH); H

NMR(CDCl₃) d 7.76 (d, J7 Hz, 1H), 7.31 (s, 1H), 7.24 (m,

2H), 7.14 (m, 3H), 4.31 (m, 1H), 3.60 (d, J1 Hz, 1H), 3.57

(d, J1 Hz, 1H), 3.54 (t, J6 Hz, 2H), 3.17 (t, J7 Hz, 2H),

2.61 (m, 2H), 2.07 (m, 1H), 1.94 (m, 1H), 1.52 (m, 4H); ¹³C

NMR (CDCl₃1CD₃OD) d 171.88, 169.37, 167.13, 140.97,

128.81, 128.63, 126.51, 61.83, 53.17, 53.29, 52.58, 39.60,

34.23, 32.17, 29.91, 25.91; IR (KBr) n cm²¹3284, 3090,

2928, 1736, 1645, 1560, 1497, 1453, 1351, 1232, 1061, 900,

749, 699; HRMS, CI (NH₃) [M1H]¹calcd for C₁₈H₂₅N₂O₆:

365.1706; found: 365.1695.

1

[a]_D144.48 (c2.7; CHCl₃); H NMR (CDCl₃) d 7.23

(m, 5H), 6.61 (d, J9 Hz, 1H), 6.24 (bs, 1H), 4.28 (m,

3H) 3.67 (d, J2 Hz, 1H), 3.64 (t, J7 Hz, 2H), 3.54 (d,

J2 Hz, 1H), 3.28 (m, 2H), 2,66 (m, 2H), 2.15 (m, 1H), 1,99

(m, 1H), 1.75 (bs, 1H), 1.59 (m, 4H), 1.31 (t, J7 Hz, 3H);

¹³C NMR (CDCl₃) d 170.51, 166.57, 166.08, 140.40,

128.56, 128.32, 126.28, 62.31, 62.21, 53.66, 52.63, 33.88,

31.71, 29.70, 26.03, 14.00; IR (CHCl₃) n cm²¹3400, 3310,

1750, 1675, 1530, 1465, 1375, 1315, 1080, 900, 720, 700;

HRMS, CI (NH₃) [M1H]¹calcd for C₂₀H₂₉N₂O₆393.2025,

found: 393.2029.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(5-hydroxypentyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

(27b). Obtained as an oil in 96% yield from 27a by the

procedure described above: [a]_D164.08 (c2.0;

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(5-hydroxypentyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (27a). Obtained in 98% yield from 23 by the

procedure described above: mp 135±1368C; [a]_D137.48

1

CH₃OH); H NMR (CDCl₃) d 7.95 (d, J8.5 Hz, 1H),

7.82 (t, J5.5 Hz, 1H), 7.24 (m, 2H), 7.15 (m, 3H), 4.33

(dd, J_a6 Hz, J_b8 Hz, 1H), 3.61 (d, J2 Hz, 1H), 3.57 (d,

J2 Hz, 1H), 3.53 (t, J6.5 Hz, 2H), 3.17 (m, 2H), 2.07 (m,

1

(c2.7; CHCl₃); H NMR (CDCl₃) d 7.32±7.17 (m, 5H),

6.61 (d, J8 Hz, 1H), 5.95 (t, J5 Hz, 1H), 4.30 (m, 1H),

9758

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

1H), 1.95 (m, 1H), 1.51 (m, 4H), 1.35(m, 2H); ¹³C NMR

(CDCl₃1CD₃OD) d 172.29, 169.65, 167.41, 128.96,

128.80, 126.65, 62.26, 53.84, 53.64, 52.68, 40.04, 39.92,

34.43, 32.42, 29.36, 23.55; IR (neat) n cm²¹3276, 3090,

2929, 1734, 1664, 1640, 1556, 1452, 1228, 896, 747, 698;

CI (NH₃) [M1H]¹calcd for C₁₉H₂₇N₂O₆: 379.1869; found:

379.1871.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[phenylamino]carbonyl]-

propyl)amino]carbonyl]-oxiranecarboxylic acid ethyl

ester (35). Obtained in 75% yield from acid 18 and aniline

(32) by the procedure described above for the synthesis of

22: mp 1738C; [a]_D170.08 (c2.0;CHCl₃); ¹H NMR

(DMSO-d₆) d 10.15 (s, 1H), 8.93 (d, J7.5 Hz, 2H), 7.30

(m, 4H), 7.29 (m, 3H), 7.07 (t, J7 Hz, 1H), 4.48 (m, 1H),

4.20 (m, 2H), 3.80 (d, J1.5 Hz, 1H), 3.66 (d, J1.5 Hz,

1H), 2.70 (m, 1H), 2.61 (m, 1H), 2.05 (m, 1H), 1.97 (m, 1H),

1.25 (m, 3H); ¹³C NMR (DMSO-d₆) d 169.75, 167.20,

165.13, 140.96, 138.69, 128.69, 128.33, 128.20, 125.91,

123.51, 119.48, 61.50, 53.59, 52.92, 51.18, 33.56, 31.54,

13.88; IR (KBr) n cm²¹3271, 3073, 2925, 1733, 1656,

1597, 1542, 1499, 1447, 1372, 1293, 1246, 1216, 1162,

1093, 1031, 971, 894, 738, 691; HRMS, CI (NH₃)

[M1H]¹calcd for C₂₂H₂₅N₂O₅397.1763, found: 397.1770.

(2S,3S)-3-[[[(1S)-3-Phenyl-1-[[(4-hydroxybutyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

(28b). Obtained in 95% yield from 28a by the procedure

described above: mp 828C; [a]_D156.08 (c2.5; CH₃OH);

¹H NMR (CDCl₃) d 7.92 (d, J8 Hz, 1H), 7.82 (t,

J5.5 Hz, 1H), 7.23 (m, 2H), 7.13 (m, 3H), 4.36 (dd, J_a

6 Hz, J_b8 Hz, 1H), 3.61 (d, J1.5 Hz, 1H), 3.54 (t, J

6 Hz, 2H), 3.40 (d, J1.5 Hz, 1H), 3.18 (m, 2H), 2.59 (m,

2H), 2.06 (m, 1H), 1.94 (m, 1H), 1.52 (m, 2H); ¹³C NMR

(CDCl₃1CD₃OD) d 172.00, 169.44, 167.24, 141.17,

128.91, 128.71, 126.61, 61.93, 53.85, 53.47, 52.75,

39.73, 34.40, 32.30, 30.04, 26.03; IR (KBr) n cm²¹

3457, 3293, 3088, 2938, 1727, 1646, 1551, 1498,

1453, 1389, 1341, 1266, 1061, 897, 742, 699; HRMS, CI

(NH₃) [M1H]¹calcd for C₁₈H₂₅N₂O₆: 365.1706; found:

365.1702.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(cyclohexylmethyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

(36). Obtained in 98% yield by hydrolysis of 33 by the

procedure described above for 26b: mp 888C (dec).;

1

[a]_D160.48 (c2.2; CH₃OH); H NMR (DMSO-d₆) d

13.49 (s, 1H), 8.72 (d, J8 Hz, 1H), 8.03 (d, J5.5 Hz,

1H), 7.26 (m, 2H), 7.19 (m,3H), 4.27 (dd, J_a7.5 Hz,

J_b13 Hz, 1H), 4.02 (q, J7 Hz, 1H), 3.72 (s, 1H), 3.51

(s, 1H), 2.94 (m, 1H), 2.89 (m, 1H), 2.54 (m, 3H), 1.95 m,

1H), 1.85 (m, 1H), 1.65 (m, 4H), 1.39 (m, 1H), 1.16 (m, 4H),

0.85 (m, 2H); ¹³C NMR (DMSO-d₆) d 170.52, 168.73,

165.15, 141.14, 128.28, 128.13, 125.81, 52.69, 51.17,

44.73, 37.33, 33.85, 31.41, 30.29, 25.97, 25.33; IR (KBr)

n cm²¹3290, 3088, 2925, 2853, 1736, 1646, 1560, 1498,

1449, 1389, 1225, 1154, 1105, 1030, 980, 896, 747, 698;

HRMS, CI (NH₃) [M1H]¹calcd for C₂₁H₂₉N₂O₅389.2076,

found: 389.2060.

The preparation of compound 29b has been reported

elsewhere.⁵⁵

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(cyclohexylmethyl)amino]-

carbonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (33). Obtained in 76% yield from acid 18 and

cyclohexylmethylamine (30) by the procedure described

above for the synthesis of 22. mp 149±1518C;

[a]_D172.58 (c2.4; CH₃OH); ¹H NMR (CDCl₃) d

7.31±7.16 (m, 5H), 6.65 (d, J8.5 Hz, 1H), 5.90 (t,

J5 Hz, 1H), 4.36 (q, J7 Hz, 1H), 4.27 (m, 2H), 3.67

(d, J2 Hz, 1H), 3.51 (d, J2 Hz, 1H), 3.08 (m, 2H),

2.67 (m, 2H), 2.17 (m, 1H), 1.99 (m, 1H), 1.69 (m, 4H),

1.44 (m, 1H), 1.30 (t, J7 Hz, 3H), 1.21 (m, 4H), 0.90 (m,

1H); ¹³C NMR (CDCl₃) d 170.39, 166.51, 165.96, 140.42,

128.53, 128.26, 128.24, 62.26, 53.63, 52.58, 52.51, 45.76,

37.75, 33.99, 31.66, 30.73, 30.70, 26.24, 25.68, 13.97; IR

(KBr) n cm²¹3289, 3089, 2924, 2854, 1749, 1665, 1560,

1498, 1446, 1370, 1266, 1224, 1200, 1154, 1025, 974, 898,

842, 697; HRMS, CI (NH₃) [M1H]¹calcd for C₂₃H₃₃N₂O₅

417.2389, found: 417.2379.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(phenylmethyl)amino]car-

bonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

(37). Obtained in 94% yield by hydrolysis of 34 by the

procedure described above for 26b: mp 1108C (dec);

1

[a]_D179.08 (c1.0; CH₃OH); H NMR (DMSO-d₆) d

13.48 (bs, 1H), 8.80 (d, J8 Hz, 1H), 8.60 (s, 1H), 7.25

(m, 10H), 4.03 (m, 1H), 3.73 (s, 1H), 3.54 (s, 1H), 2.61

(m,1H), 2.55 (m, 1H), 2.00 (m, 1H), 1.90 (m, 1H), 1.17

(app. t, J7 Hz, 2H); ¹³C NMR (DMSO-d₆) d 170.80,

168.83, 165.40, 141.10, 139.33, 128.31, 128.22, 127.07,

126.73, 125.87, 52.77, 51.22, 42.06, 33.62, 31.45; IR

(KBr) n cm²¹3283, 3066, 2926, 2857, 1736, 1654, 1638,

1542, 1498, 1458, 1225, 1081, 1030, 898, 746, 698; HRMS,

CI (NH₃) [M1H]¹calcd forC₂₁H₂₃N₂O₅383.1607, found:

383.1588.

(2S,3S)-3-[[[(1R)-3-Phenyl-1-[[(phenylmethyl)amino]car-

bonyl]propyl)amino]carbonyl]-oxiranecarboxylic acid

ethyl ester (34). Obtained in 75% yield from acid 18 and

benzylamine (31) by the procedure described above for the

synthesis of 22: mp 166±1688C; [a]_D157.38 (c2.2;

(2S,3S)-3-[[[(1R)-3-Phenyl-1-(phenylamino)carbonyl]-

propylamino]carbonyl]-oxiranecarboxylic acid (38).

Obtained in 99% yield by hydrolysis of 35 by the procedure

described above for 26b: mp.2008C (dec); [a]_D168.08

1

CH₃OH); H NMR (CDCl₃) d 7.37±7.12 (m, 10), 6.63 (d

J8.5 Hz, 1H), 6.18 (t J5 Hz, 1H), 4.39 (m, 3H), 4.27 (m,

2H), 3.66 (d, J2 Hz, 1H), 3.48 (d, J2 Hz, 1H), 2.66 (m,

2H), 2.19 (m, 1H), 2.01 (m, 1), 1.33 (t, J7 Hz, 3H); ¹³C

NMR, 125 MHz (CDCl₃) d 170.34, 166.51, 166.03, 140.29,

137.60, 128.70, 128.53, 128.25, 127.66, 127.66, 127.61,

126.25, 62.26, 53.56, 52.54, 52.45, 43.55, 33.90, 31.62,

13.98; IR (KBr) n cm²¹3286, 3088, 3032, 2926, 1746,

1641, 1551, 1498, 1454, 1384, 1370, 1350, 1222, 1201,

1100, 1029, 981, 897, 744, 697; HRMS, CI (NH₃)

[M1H]¹calcd for C₂₃H₂₇N₂O₅411.1920, found: 411.1923.

1

(c2.0; CH₃OH); H NMR (DMSO-d₆) d 13.50 (s, 1H),

10.14 (s, 1H), 8.90 (d, J8 Hz, 1H), 7.59 (d, J7.5 Hz,

2H), 7.27 (m, 4H), 7.19 (m, 3H), 7.06 (t, J7.5 Hz, 1H),

4.46 (m, 1H), 3.73 (s, 1H), 3.52 (s, 1H), 2.68 (m, 1H), 2.60

(m, 1H), 2.04 (m, 1H), 1.96 (m, 1H); ¹³C NMR (DMSO-d₆)

d 169.79, 168.73, 165.46, 140.97, 138.69, 128.69, 128.34,

128.21, 125.92, 123.51, 119.52, 53.56, 52.67, 33.57, 31.53;

IR (KBr) n cm²¹3294, 3255, 3063, 2931, 1759, 1655, 1619,

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9759

1543, 1498, 1447, 1394, 1313, 1255, 1195, 1094, 1028, 989,

950, 894, 754, 694, 646; HRMS, CI (NH₃) [M1H]¹calcd

for C₂₀H₂₁N₂O₅369.1450, found: 369.1455.

(97%) of crude 41 as a white solid which was used

immediately in the next reaction: mp 1258C; [a]_D12.28

(c1.8; CH₃OH); ¹H NMR (DMSO-d₆) d 9.1 (bs, 1H), 7.84

(t, J5.5 Hz, 1H), 6.95 (d, J8.5 Hz, 2H), 6.66 (d,

J6.6 Hz, 2H), 3.11 (m, 3H), 2.46 (m, 2H), 1.78 (m, 1H),

1.57 (m, 1H), 1.30 (q, J7 Hz, 2H), 0.86 (d, J7 Hz, 6H);

¹³C NMR (DMSO-d₆) d 174.30, 155.25, 131.87, 128.96,

115.01, 54.24, 38.14, 37.17, 36.48, 30.53, 25.09, 22.32; IR

(KBr) n cm²¹3291, 3097, 2955, 2928, 2872, 1647, 1610,

1560, 1515, 1468, 1388, 1367, 1253, 1171, 1162, 1087,

1044, 946, 890, 847, 828, 793, 725, 656; HRMS, CI

(NH₃) [M1H]¹calcd for C₁₅H₂₅N₂O₂265.1916, found:

265.1916.

(R)-2-[(Phenylmethyl)carbonyl]amino-4-(4-hydroxyphe-

nyl)-butyric acid (39). To a solution of d-homotyrosine

hydrobromide (1.00 g, 3.62 mmol) in H₂O (10 mL) at 08C

benzyl chloroformate (0.67 mL, 4.71 mmol) and 2N NaOH

(16 mL, 32 mmol) were added simultaneously under vigor-

ous stirring. The mixture was stirred at this temperature for

2 h then at 238C for 10 h. The reaction mixture was washed

with CH₂Cl₂then cooled to 08C, acidi®ed to pH 2 with 1 M

KHSO₄and extracted with CH₂Cl₂(3£50 mL). The

combined organic portions were washed with brine, dried

over Na₂SO₄and concentrated in vacuo. The residue was

puri®ed by ¯ash chromatography on silica gel (elution with

CH₂Cl₂-isopropanol 97:3 containing 0.1% AcOH) to afford

0.49 g (41%) of Cbz-N-d-homotyrosine 39 as a syrup:

[a]_D18.58 (c2.0; CH₃OH); ¹H NMR (DMSO-d₆) d

12.48 (bs, 1H), 9.13 (s, 1H), 7.66 (d, J8 Hz, 1H), 7.33

(m, 5H), 6.95 (d, J8.5 Hz, 2H), 6.65 (d, J8.5 Hz, 2H),

5.04 (s, 2H), 3.87 (m, 1H), 2.54 (m, 2H), 1.88 (m, 1H), 1.80

(m, 1H); ¹³C NMR (DMSO-d₆) d 173.92, 156.16, 155.43,

137.05, 130.98, 129.16, 128.30, 127.75, 127.66, 115.07,

65.35, 53.20, 32.94, 30.64; IR (neat) n cm²¹3326, 3033,

2928, 1698, 1614, 1597, 1515, 1455, 1416, 1345, 1228,

1052, 911, 829, 741, 697; HRMS, CI (NH₃) [M1NH₄]¹

calcd for C₁₈H₂₃N₂O₅347.1608, found: 347.1623.

(2S,3S)3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-3-

(4-hydroxyphenyl)propyl]amino]carbonyl]-oxiranecar-

boxylic acid ethyl ester (42). To a solution of acid 15

(0.12 g, 0.73 mmol) and amine 41 (0.19 mL, 0.73 mmol)

in DMF (5 mL) under nitrogen at 238C was added EEDQ

(0.36 g, 1.46 mmol). The resulting mixture was stirred for

7 h. The mixture was then partitioned between H₂O and

EtOAc. The organic portion was washed with brine, dried

over Na₂SO₄and concentrated in vacuo. The residue was

puri®ed by ¯ash chromatography on silica gel (elution with

a gradient of hexanes±EtOAc 5:5 to 3:7) to afford 0.14 g

(61%) of 42 as a white solid: mp 1598C; [a]_D138.58

1

(c2.0; CHCl₃); H NMR (CDCl₃) d 7.03 (m, 2H), 6.77

(m, 2H), 6.59 (d, J8 Hz, 1H), 5.77 (m, 1H), 4.27 (m,

3H), 3.66 (d, J2 Hz, 1H), 3.52 (d, J2 Hz, 1H), 3.26

(m, 2H), 2.59 (m, 2H), 2.10 (m, 1H), 1.95 (m, 1H), 1.59

(m, 1H), 1.38 (q, J7 Hz, 2H), 1.32 (t, J7 Hz, 3H), 0.92

(d, J7.5 Hz, 6H); ¹³C NMR (CDCl₃) d 170.66, 166.57,

166.23, 154.51, 131.87, 129.34, 115.55, 62.37, 53.66,

52.68, 52.56, 38.15, 38.07, 33.96, 30.74, 25.79, 22.33,

14.00; IR (CHCl₃) n cm²¹3296, 3090, 2957, 1745, 1651,

1617, 1548, 1516, 1445, 1370, 1227, 1204, 1096, 1026, 898,

831; HRMS, CI (NH₃) [M1H]¹calcd for C₂₁H₃₁N₂O₆

407.2182,: 407.2190. Anal. Calcd for C₂₁H₃₀N₂O₆: C,

62.05; h 7.44; N, 6.89. Found: C, 62.03; H, 7.55; N 6.71.

(R)-N-(3-Methylbutyl)-2-[[(phenylmethyl)carbonyl]amino]-

4-(4-hydroxy phenyl)-butyric amide (40). To a solution of

39 (0.44 g, 1.33 mmol), isopentylamine (0.15 mL,

1.33 mmol) and HOBT (0.20 g, 1.33 mmol) in DMF

(5 mL) under nitrogen at 08C was added EDC (0.28 g,

1.47 mmol). The resulting mixture was stirred for 1 h at

this temperature, then at 238C for 10 h. The mixture was

then partitioned between H₂O and a mixture of EtOAc±

hexanes 4:1. The organic portion was washed with brine,

dried over Na₂SO₄and concentrated in vacuo. The residue

was puri®ed by ¯ash chromatography on silica gel (elution

with a gradient of hexanes±EtOAc 7:3 to 6:4) to afford

0.43 g (81%) of 40 as a white solid: mp 126±1288C;

(2S,3S)-3-[[[(1R)-1-[[(3-Methylbutyl)amino]carbonyl]-3-

(4-hydroxyphenyl)propyl]amino]carbonyl]-oxiranecar-

boxylic acid (43). A 1N solution of KOH (0.5 mL,

0.50 mmol) in ethanol was added to a solution of ester 42

(0.10 g, 0.25 mmol) in EtOAc (2 mL) and THF (2 mL) at

08C. The reaction mixture was allowed to warm to 238C and

stirred for 2 h. The solvent was evaporated in vacuo, the

residue dissolved in cold water (10 mL), acidi®ed to pH 2

with 1 M KHSO₄and extracted with EtOAc (5£10 mL).

The combined organic portions were washed with brine

and dried over Na₂SO₄. Evaporation of the solvent in

vacuo afforded 76 mg (81%) of crude 43 as a foam which

solidi®ed on standing: mp 688C; [a]_D170.98 (c3.3;

1

[a]_D19.68 (c2.4; CHCl₃); H NMR (CDCl₃) d 7.33

(m, 5H), 6.98 (d, J8 Hz, 2H), 6.74 (d, J8 Hz, 2H),

5.95 (s, 1H), 5.34 (d, J7.5 Hz, 1H), 5.12 (dd,

J_1aJ_b12.5 Hz, 2H), 4.07 (q, J7.5 Hz, 1H), 3.26 (s,

2H), 2.59 (t, J7.5 Hz, 2H), 2.12 (m, 1H), 1.92 (m, 1H),

1.59 (m, 1H), 1.37 (m, 2H), 0.91 (d, J7 Hz, 6H); ¹³C NMR

(CDCl₃) d 171.61, 156.32, 154.48, 132.15, 129.35, 128.55,

128.26, 128.03, 115.48, 67.23, 54.61, 38.24, 38.01, 34.24,

30.79, 25.82, 22.38; IR (CHCl₃) n cm²¹3310, 2956, 1700,

1653, 1540, 1515, 1456, 1240, 1046, HRMS, CI (NH₃)

[M1H]¹calcd for C₂₃H₃₀N₂O₄399.2283, found:

399.2273. Anal. Calcd for C₂₂H₃₀N₂O₄: C, 69.32; H, 7.59;

N, 7.03. Found: C, 69.45; H, 7.85; N, 6.98.

1

CH₃OH); H NMR (DMSO-d₆) d 9.15 (s, 1H), 8.67 (d,

J8 Hz, 1H), 7.98 (t, J6 Hz, 1H), 6.93 (d, J8 Hz, 2H),

6.65 (d, J8 Hz, 2H), 4.20 (m, 1H), 3.70 (d, J1.5 Hz, 1H),

3.50 (d, J1.5 Hz, 1H), 3.07 (m, 2H), 2.41 (m, 2H), 1.87 (m,

1H), 1.77 (m, 1H), 1.55 (m, 1H), 1.28 (q, J7 Hz, 2H), 0.86

(d, J7, 6 Hz); ¹³C NMR (DMSO-d₆) (100 MHz) d 170.44,

168.74, 165.12, 155.38, 131.10, 128.97, 115.06, 52.68,

51.15, 37.97, 36.73. 34.09, 30.50, 25.06, 22.32, 22.29; IR

(KBr) n cm²¹3322, 3101, 2958, 2871, 1742, 1654, 1542,

1516, 1451, 1368, 1228, 1015, 852, 897, 830, 762, 655;

(R)-N-(3-Methyl)butyl)-2-amino-4-(4-hydroxyphenyl)-

butyric amide (41). A mixture of the benzyl carbamate 40

(0.43 g, 1.08 mmol) and 10% Pd/C (100 mg) in 100 mL of

methanol was shaken on a Parr apparatus under hydrogen

(40 psi) for 30 min. The mixture was purged with nitrogen,

the catalyst was removed by ®ltration through a pad of celite

and the solution concentrated in vacuo to afford 0.28 g

9760

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

HRMS, CI (NH₃) [M1H]¹calcd for C₁₉H₂₇N₂O₆379.1869,

found: 379.1864.

3030, 2957, 1702, 1677, 1654, 1560, 1516, 1457, 1368,

1228, 1043, 990, 896, 832, 751, 699; HRMS, CI (NH₃)

[M1H]¹calcd for C₂₆H₃₄N₃O₆484.2447, found:

484.2437. Anal. Calcd for C₂₆H₃₃N₃O₆: C, 64.58, H, 6.88;

N, 8.69; found: C, 64.24; H, 6.98; N, 8.61.

(2S,3S)-N-[(1R)-1-Phenyl-[[3-(3-methylbutyl)amino]car-

bonyl]-propyl]-N⁰-(phenylmethoxy)-2,3-oxiranedicar-

boxamide (45). To a solution of acid 11c (0.18 g, 0.5 mmol)

and O-benzyl hydroxylamine 44 (0.06 g, 0.50 mmol,

prepared from a solution of its HCl salt by neutralization

with NaHCO₃and extraction with EtOAc) in CHCl₃(2 mL)

and DMF (2 mL) under nitrogen at 238C was added EEDQ

(0.25 g, 1.00 mmol). The resulting mixture was stirred for

4 h. The mixture was then partitioned between H₂O and

EtOAc. The organic portion was washed with brine, dried

over Na₂SO₄and concentrated in vacuo. The residue was

puri®ed by ¯ash chromatography on silica gel (eluted with

CH₂Cl₂±isopropanol 97:3) to afford 0.17 g (50%) of 40 as a

(2S,3S)-N-[(1R)-1-(4-Hydroxyphenyl)-[[3-phenylamino]-

carbonyl]-propyl]-N⁰-(phenylmethoxy)-2,3-oxirane-

dicarboxamide (48). Obtained in 53% yield from 38 and 44

by the procedure described above: mp 1708C (dec);

1

[a]_D143.88 (c2.1; CH₃OH); H NMR (DMSO-d₆) d

11.73 (d, J3 Hz, 1H), 10.14 (s, 1H), 8.95, (d, J5.5 Hz,

1H), 7.59 (d, J6.5 Hz, 2H), 7.40 (m, 4H), 7.23 (m, 4H),

7.20 (d, J6 Hz, 2H), 7.06 (m, 1H), 4.84 (d, J3 Hz, 1H),

4.46 (m, 1H), 3.76 (s, 1H), 3.46 (s, 1H), 2.68 (m, 1H), 2.58

(m, 1H), 2.04 (m, 1H) 1.96 (m, 1H); ¹³C NMR (DMSO-d₆) d

169.78, 165.64, 162.80, 140.94, 138.68, 135.50, 128.87,

128.66, 128.32, 128.18, 125.90, 123.46, 119.43, 77.05,

53.51, 52.26, 51.23, 33.52, 31.54; IR (KBr) n cm²¹3287,

3046, 2924, 1679, 1653, 1600, 1546, 1498, 1447, 1383,

1316, 1245, 1042, 1007, 969, 898, 754, 700; HRMS, FAB

[M1Na]¹calcd for C₂₇H₂₇N₃O₆Na 496.1848, found:

496.1858.

1

white solid. mp: 1758C; [a]_D132.48 (c2.1; CHCl₃); H

NMR (CDCl₃) d 9.45 (s, 1H), 7.37 (s, 5H), 7.28±7.13 (m,

5H), 6.40 (s, 1H), 4.91 (s, 2H), 4.11 (q, J9 Hz, 1H), 3.56

(d, J2 Hz, 1H), 3.50 (d, J2 Hz, 1H), 3.27 (m, 1H), 3.13

(m,1H), 2.64 (t, J8 Hz, 2H), 2.08 (m, 1H), 1.99 (m, 1H),

1.57 (m, 1H), 1.36 (q, J9 Hz, 2H), 0.90 (d, J8 Hz, 6H);

¹³C NMR (CDCl₃) d 170.56, 165.80, 163.13, 140.42,

134.66, 129.25, 129.01, 128.70, 128.60, 128.32, 126.33,

78.50, 54.13, 53.18, 52.78, 38.24, 38.05, 33.96, 31.77,

25.85, 22.41, 22.34; IR (CHCl₃) n cm²¹3285, 2956,

1648, 1547, 1498, 1454, 1367, 1232, 1043, 896; HRMS,

CI (NH₃) [M1H]¹calcd for C₂₆H₃₄N₃O₅: 468.2498;

found: 468.2493

(2S,3S)-N-[(1R)-1-Phenyl-[[3-(3-methylbutyl)amino]car-

bonyl]-propyl]-N⁰-(2-phenylethyl)-2,3-oxiranedicarbox-

amide (49). To a solution of 11c (0.100 g, 0.276 mmol),

Phenethylamine (0.035 mL, 1.33 mmol), N-methylmorpho-

line (0.035 mL, 0.276 mmol) and HOBT (0.042 g,

0.276 mmol) in CHCl₃(4 mL) under nitrogen at 08C was

added EDC (0.0.58 g, 0.306 mmol). The resulting mixture

was stirred for 1 h at this temperature, then at 238C for 10 h.

The mixture was then partitioned between H₂O and a

mixture of EtOAc±hexanes 4:1. The organic portion was

washed with brine, dried over Na₂SO₄and concentrated in

vacuo. The residue was puri®ed by ¯ash chromatography on

silica gel (elution with hexanes±EtOAc 7:3) to afford

0.128 g (80%) of 49 as a white solid: mp 174±1768C;

(2S,3S)-N-[(1S)-1-Phenyl-[[3-(3-methylbutyl)amino]car-

bonyl]-propyl]-N⁰-(phenylmethoxy)-2,3-oxiranedicar-

boxamide (46). Obtained in 51% yield from 12 and 44 by

the procedure described above. The solid decomposes at

,1008C without melting: [a]_D180.08 (c1.0; ethanol);

¹H NMR (CDCl₃1CD₃OD) d 7.66 (d, J8 Hz, 1H), 7.33±

7.06 (m, 11H), 4.82 (s, 2H), 4.23 (q, J8 Hz, 1H), 3.48 (d,

J1.5 Hz, 1H), 3.27 (d, J1.5 Hz, 1H), 3.11(m, 2H), 2.51

(t, J7.5 Hz, 2H), 1.97 (m, 1H), 1.85 (m, 1H), 1.52 (m, 1H),

1.30 (q, J7 Hz, 2H), 0.83 (d, J7 Hz, 6H); ¹³C NMR

(CDCl₃1CD₃OD) d 171.91, 167.24, 164.40, 141.23,

129.65, 129.23, 128.94, 128.74, 126.61, 78.69, 53.82,

53.48, 53.00, 38.46, 38.30, 34.53, 32.24, 30.07, 26.24,

22.57; IR (KBr) n cm²¹3289, 3088, 3030, 2955, 2927,

2871, 1677, 1661, 1640, 1552, 1498, 1454, 1368, 1230,

1050, 992, 896, 843, 787, 747, 698; HRMS, CI (NH₃)

[M1H]¹calcd for C₂₆H₃₄N₃O₅; 468.2498; found: 468.2507.

1

[a]_D126.48 (c2.2; CHCl₃); H NMR (CDCl₃) d 7.35±

7.16 (m, 10H), 6.55 (d, J8 Hz, 1H), 6.01 (t, J7 Hz, 1H),

5.71 (t, J5.5 Hz, 1H), 4.31 (q, J7 Hz, 1H), 3.54 (m, 2H),

3.46 (d, J1.5 Hz, 1H), 3.32 (d, J1.5 Hz, 1H), 3.26 (m,

2H), 2.83 (t, J7 Hz, 2H), 2.65 (m, 2H), 2.14 (m, 1H), 1.97

(m, 1H), 1.60 (m, 1H), 1.38 (q, J7 Hz, 2H), 0.92 (d,

J7 Hz, 6H); ¹³C NMR (CDCl₃) d 170.50, 165.89,

165.67, 140.49, 138.22, 128.66, 128.51, 128.26, 126.67,

126.19, 54.47, 52.63, 40.19, 38.15, 37.92, 35.30, 34.03,

31.73, 25.77, 22.39, 22.31; IR (CHCl₃) n cm²¹3290,

3088, 2956, 1651, 1551, 1498, 1455, 1367, 1236, 1085,

1031, 895, 747, 698; HRMS, CI (NH₃) [M1H]¹calcd for

C₂₇H₃₆N₃O₄: 466.2705, found: 466.2691.

(2S,3S)-N-[(1R)-1-(4-Hydroxyphenyl)-[[3-(3-methylbutyl)-

amino]carbonyl]propyl]-N⁰-(phenylmethoxy)-2,3-oxirane-

dicarboxamide (47). Obtained in 49% yield from 43 and 44

by the procedure described above. mp 1628C; [a]_D

1

166.358 (c2.0; CH₃OH); H NMR (DMSO-d₆) 500 MHz

d 11.68 (s, 1H), 9.15 (s, 1H), 8.70 (d, J8 Hz, 1H), 7.97 (t,

J5.5 Hz, 1H), 7.37 (m, 5H), 6.95 (d, J8.5 Hz, 2H), 6.65

(d, J8.5 Hz, 2H), 4.83 (s, 2H), 4.19 (m, 1H), 3.73 (d,

J1.5 Hz, 1H), 3.42 (d, J1.5 Hz, 1H), 3.07 (m, 2H),

2.44 (m, 1H), 2.39 (m, 1H), 1.86 (m, 1H), 1.77 (m, 1H),

1.55 (m, 1H), 1.28 (q, J7 Hz, 2H), 0.85 (d, J6 Hz, 6H);

¹³C NMR (DMSO-d₆) d 170.37, 165.25, 162.83, 155.32,

135.44, 131.09, 128.88, 128.77, 128.28, 128.23, 115.01,

76.98, 52.57, 52.57, 52.29, 51.17, 37.91, 36.69, 34.07,

30.46, 25.01, 22.24, 22.20; IR (KBr) n cm²¹3315, 3209,

Acknowledgements

Financial support by NIH Program Project Grant AI 35707

is gratefully acknowledged.

References

1. McKerrow, J. H., James, M. N. G., Eds.; 1996; 6, pp 1±125.

2. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133±171.

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

9761

3. Miller, D. K. Ann. Rep. Med. Chem. 1996, 31, 249±268.

4. McKerrow, J. H.; Sun, E.; Rosenthal, P. J.; Bouvier, J. Annu.

Rev. Microbiol. 1993, 47, 821±853.

34. Hanada, K.; Tamai, M.; Ohmura, S.; Sawada, J.; Seki, T.;

Tanaka, I. Agric. Biol. Chem. 1978, 42, 529±536.

35. Hanada, K.; Tamai, M.; Morimoto, S.; Adachi, T.; Ohmura, S.;

Sawada, J.; Tanaka, I. Agric. Biol. Chem. 1978, 42, 537±541.

36. Woo, J.-T.; Ono, H.; Tsuji, T. Biosci. Biotechnol. Biochem.

1995, 59, 350±352.

5. McKerrow, J. H. Perspec. Drug Discov. Design 1994, 2, 437±

444.

6. Rosenthal, P. J. Emerg. Infect. Dis. 1998, 4, 49±57.

7. Kirchhoff, L. V. Gastroenterol. Clin. N. 1996, 25, 517±533.

8. Orourke, R. A.; Hagar, J. M.; Rahimtoola, S. H. Curr. Problems

Cardiol. 1995, 20, 829±924.

37. Yu, C.-M.; Curtis, J. M.; Walter, J. A.; Wright, J. L. C.; Ayer,

S. W.; Kaleta, J.; Querengesser, L.; Fathi-Afshar, Z. R. J. Antibiot.

1996, 4, 395±397.

38. Shin, H. J.; Matsuda, H.; Murakami, M.; Yamaguchi, K.

Tetrahedron 1997, 53, 5747.

9. World Health Organization website: http://www.who.int/ctd/

html/chagburtre.html

10. Centers for Disease Control and Prevention website:

http://www.cdc.gov/ncidod/dpd/parasites/chagasdisease/factsht_

chagas_disease.htm

39. Tamai, M.; Matsumoto, K.; Ohmura, S.; Koyama, I.; Ozawa,

Y.; Hanada, K. J. Pharmacobio-Dyn. 1986, 9, 672±677.

40. Tamai, M.; Ohmura, S.; Kimura, M.; Hanada, K.; Sugita, H.

J. Pharmacobio-Dyn. 1987, 10, 678±681.

11. Van den Bossche, H. Nature 1978, 273, 626±630.

12. Ostermayer, A. L. Parasitol. Today 1997, 13, 127±128.

13. Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp.

Med. 1998, 188, 725±734.

41. Satoyoshi, E. Internal Med. 1992, 31, 841±846.

42. Murata, M.; Miyashita, S.; Yokoo, C.; Hanada, K.; Hatayama,

K.; Towatari, T.; Nikawa, T.; Katunuma, N. FEBS Lett. 1991, 280,

307±310.

14. Urbina, J. A.; Payares, G.; Molina, J.; Sanoja, C.; Liendo, A.;

Lazardi, K.; Piras, M.; Perez, N.; Wincker, P.; Ryley, J. F. Science

1996, 273, 969±971.

43. Towatari, T.; Nikawa, T.; Murata, M.; Yokoo, C.; Tamai, M.;

Hanada, Katunuma, N. FEBS Lett. 1991, 280, 311±315.

44. Gour-Salin, B. J.; Lachance, P.; Plouffe, C.; Storer, A. C.;

15. Cazzulo, J. J.; Couso, R.; Raimondi, A.; Wernstedt, C.;

Hellman, U. Mol. Biochem. Parasitol. 1989, 33, 33±42.

16. Schirmer, R. H.; MuÈller, J. H.; Krauth-Siegel, R. L. Angew.

Chem., Int. Ed. Engl. 1995, 34, 141±154.

Â

Menard, R. J. Med. Chem. 1993, 36, 720±725.

45. Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W.; Turk,

D.; Moroder, L. Biooorg. Med. Chem. 1997, 5, 1789±1797.

46. Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W.;

Moroder, L. FEBS Lett. 1998, 421, 80±82.

È

17. Krauth-Siegel, R. L.; Schoneck, R. FASEB J. 1995, 9, 1138±

1146.

18. Bonse, S.; Santelli-Rouvier, C.; Barbe, J.; Krauth-Siegel, R. L.

J. Med. Chem. 1999, 42, 5448±5454.

47. Katunuma, N.; Murata, E.; Kakekawa, H.; Matsui, A.;

Tsuzuki, H.; Tsuge, H.; Turk, D.; Turk, V.; Fukushima, M.;

Tada, Y.; Asao, T. FEBS Lett. 1999, 458, 6±10.

48. Tsuge, H.; Nishimura, T.; Tada, Y.; Asao, T.; Turk, D.; Turk,

V.; Katunuma, N. Biochem. Biophys. Res. Commun. 1999, 266,

411±416.

19. Molina, J.; Martins, O.; Brener, Z.; Romanha, A. J.;

Loebenberg, D.; Urbina, J. A. Antimicrob. Agents Chemother.

2000, 44, 150±155.

20. Urbina, J. A. J. Mol. Med. 1999, 7, 332±338.

21. Olson, J. E.; Lee, G. K.; Semenov, A.; Rosenthal, P. J. Bioorg.

Med. Chem. 1999, 7, 633±638.

49. Yabe, Y.; Guillaume, D.; Rich, D. H. J. Am. Chem. Soc. 1988,

110, 4043.

22. Semenov, A.; Olson, J. E.; Rosenthal, P. J. Antimicrob. Agents

Chemother. 1998, 42, 2254±2258.

50. Varughese, K. I.; Ahmed, F. R.; Carey, P. R.; Hasnain, S.;

Huber, C. P.; Storer, A. C. Biochemistry 1989, 28, 1330±1332.

51. Yamamoto, D.; Matsumoto, K.; Ohishi, H.; Ishida, T.; Inoue,

M.; Kitamura, K.; Mizuno, H. J. Biol. Chem. 1991, 266, 14771±

14777.

23. Rosenthal, P. J.; McKerrow, J. H.; Aikawa, M.; Nagasawa, H.;

Leech, J. H. J. Clin. Invest. 1988, 82, 1560±1566.

24. Rosenthal, P. J.; Wollish, W. S.; Palmer, J. T.; Rasnick, D.

J. Clin. Invest. 1991, 88, 1467±1472.

52. Kim, M.-J.; Yamamoto, D.; Matsumoto, K.; Inoue, M.; Ishida,

T.; Mizuno, H.; Sumiya, S.; Kitamura, K. Biochem. J. 1992, 287,

797±803.

25. Rosenthal, P. J.; Olson, J. E.; Lee, G. K.; Palmer, J. T.; Klaus,

J. L.; Rasnick, D. Antimicrob. Agents Chemother. 1996, 40, 1600±

1603.

53. Baker, E. N.; Drenth, J. Active sites of Enzymes; In Biological

Macromolecules and Assemblies Vol. 3., Jurnak, F. A., McPherson,

A., Eds.; Wiley: New York, 1987; pp 313±368.

26. Rosenthal, P. J. Emerg. Infect. Dis. 1998, 4, 49±57.

27. World Health Organization website:

http://www.who.int/emc/diseases/leish/leis.html

28. Robertson, C. D.; Coombs, G. H.; North, M. J.; Mottram, J. C.

Perspect Drug Discov. Design. 1996, 6, 99±118.

29. Selzer, P. M.; Chen, X. W.; Chan, V. J.; Cheng, M. S.;

Kenyon, G. L.; Kuntz, I. D.; Sakanari, J. A.; Cohen, F. E.;

McKerrow, J. H. Exp. Parasitol. 1997, 87, 212±221.

30. Roush, W. R.; Gwaltney III, S. L.; Cheng, J.; Scheidt, K. A.;

McKerrow, J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994±

10995.

54. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun.

1967, 27, 157±162.

55. Roush, W. R.; Alvarez, A.; Zepeda, G. Synthesis 1999, 1500±

1504.

56. Hanzlik, R. P.; Jacober, S. P.; Zygmunt, J. Biochim. Biophys.

Acta 1991, 1073, 33±42.

57. Kowlessur, D.; Thomas, E. W.; Topham, C. M.; Templeton,

W.; Brocklehurst, K. Biochem. J. 1990, 266, 653±660.

58. Patel, M.; Kayani, I.; Templeton, W.; Mellor, G. W.; Thomas,

E. W.; Brocklehurst, K. Biochem. J. 1992, 287, 881±889.

59. Lecaille, F.; Serveau, C.; Gauthier, F.; Lalmanach, G. FEBS

Lett. 1999, 445, 311±314.

Â

31. Roush, W. R.; Gonzalez, F. V.; McKerrow, J. H.; Hansell, E.

Bioorg. Med. Chem. Lett. 1998, 8, 2809±2812.

32. Scheidt, K. A.; Roush, W. R.; McKerrow, J. H.; Selzer, P. M.;

Hansell, E.; Rosenthal, P. J. Bioorg. Med. Chem. 1998, 6, 2477±

2494.

60. Chatterjee, S.; Gu, Z.-Q.; Dunn, D.; Tao, M.; Josef, K.;

Tripathy, R.; Bihovsky, R.; Senadhi, S. E.; O'Kane, T. M.;

McKenna, B. A.; Mallya, S.; Ator, M. A.; Bozyczko-Coyne, D.;

Siman, R.; Mallamo, J. P. J. Med. Chem. 1998, 41, 2663±2666.

33. Hanada, K.; Tamai, M.; Yamagishi, M.; Ohmura, S.; Sawada,

J.; Tanaka, I. Agric. Biol. Chem. 1978, 42, 523±528.

9762

W. R. Roush et al. / Tetrahedron 56 (2000) 9747±9762

61. McGrath, M. E.; Eakin, A. E.; Engel, J. C.; McKerrow, J. H.;

Craik, C. S.; Fletterick, R. J. J. Mol. Biol. 1995, 247, 251±259.

62. Computational results obtained using software programs from

Biosym/MSI of San Diego CA. Calculations were done with the

discover program, using the CFF91 force®eld and graphical

displays were printed out from the insight ii molecular modeling

system.

72. Brinen, L. S.; Hansell, E.; Gillmor, S.; Alvarez, A.; Roush,

W. R.; McKerrow, J. H.; Fletterick, R. J., manuscript in prepara-

tion.

73. Thompson, S. K.; Halbert, S. M.; Bossard, M. J.; Tomaszek,

T. A.; Levy, M. A.; Zhao, B. G.; Smith, W. W.; Abdel-Meguid,

S. S.; Janson, C. A.; D'Alessio, K. J.; McQueney, M. S.;

Amegadzie, B. Y.; Hanning, C. R.; DesJarlasjais, R. L.; Briand,

J.; Sarkar, S. K.; Huddleston, M. J.; Ijames, C. F.; Carr, S. A.;

Garnes, K. T.; Shu, A.; Heys, J. R.; Bradbeer, J.; Zembryki, D.;

Lee-Rykaczewski, L.; James, I. E.; Lark, M. W.; Drake, F. H.;

Gowen, M.; Gleason, J. G.; Veber, D. F. Proc. Natl. Acad. Sci.

USA 1997, 94, 14249±14254.

È

63. Bohm, H.-J. Perspec. Drug Discov. Design. 1995, 3, 21±23.

64. Tamai, M.; Yokoo, C.; Murata, M.; Oguma, K.; Sota, K.; Sato,

E.; Kanaoka, Y. Chem. Pharm. Bull. 1987, 35, 1098±1104.

65. Keller, O.; Kellwer, W. E.; Van Look, G.; Wersin, G. Org.

Synth. Coll 1990, Vol. 7, 70±75.

66. Beith, J. Methods Enzymol. 1995, 248, 59±84.

67. Mort, J. S.; Buttle, D. J.; Int J. Biochem. Cell. Biol. 1997, 29,

715±720.

74. Meara, J. P.; Rich, D. J. Med. Chem. 1996, 39, 3357±3366.

È

75. Bromme, D.; Demuth, H. U. Methods Enzymol. 1994, 244,

671±685.

68. Kubota, H.; Nunami, K.; Hayashi, K.; Hashimoto, Y.; Ogiku,

N.; Matzuoka, Y.; Ishida, R. Chem. Pharm. Bull. 1992, 40, 1619±

1622.

76. Belleau, B.; Malek, G. J. Am. Chem. Soc. 1968, 90, 1651±

1652.

È

77. Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J. Med.

Chem. 1995, 38, 3193.

È

78. Bromme, D.; Klaus, J. L.; Okamoto, K.; Rasnick, D.; Palmer,

J. T. Biochem. J. 1996, 315, 85±89.

69. Patel, R. P.; Price, S. J. Org. Chem. 1965, 30, 3575±3576.

70. Mori, K.; Iwasawa, H. Tetrahedron 1980, 36, 87±90.

71. Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett. 1979, 20,

99±102.

79. Rasnick, D. Perspect. Drug. Discov. Design 1996, 6, 47±63.

Article Doi

Design, synthesis and evaluation of D-Homophenylalanyl epoxysuccinate inhibitors of the trypanosomal cysteine protease cruzain

DOI: 10.1016/S0040-4020(00)00882-6

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Full text of DOI:10.1016/S0040-4020(00)00882-6

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