Interaction of papain-like cysteine proteases with dipeptide-derived nitriles

7688

J. Med. Chem. 2005, 48, 7688-7707

Interaction of Papain-like Cysteine Proteases with Dipeptide-Derived Nitriles^†

Reik Lo¨ser,^‡Klaus Schilling,^§Elke Dimmig,^§and Michael Gu¨tschow*^,‡

Pharmazeutisches Institut, Rheinische Friedrich-Wilhelms-Universita¨t Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany, and

Institut fu¨r Biochemie I, Klinikum, Friedrich-Schiller-Universita¨t Jena, Nonnenplan 2, D-07743 Jena, Germany

Received July 19, 2005

A series of 44 dipeptide nitriles with various amino acids at the P²position and glycine nitrile

at position P¹were prepared and evaluated as inhibitors of cysteine proteinases. With respect

to the important contribution of the P²-S²interaction to the formation of enzyme-inhibitor

complexes, it was focused to introduce structural diversity into the P²side chain. Nonprotei-

nogenic amino acids were introduced, and systematic fluorine, bromine, and phenyl scans for

phenylalanine in the P²position were performed. Moreover, the N-terminal protection was

varied. Kinetic investigations were carried out with cathepsin L, S, and K as well as papain.

Changes in the backbone structure of the parent N-(tert-butoxycarbonyl)-phenylalanyl-glycine-

nitrile (16), such as the introduction of an R-configured amino acid or an azaamino acid into

P²as well as methylation of the P¹nitrogen, resulted in a drastic loss of affinity. Exemplarily,

the cyano group of 16 was replaced by an aldehyde or methyl ketone function. Structure-

activity relationships were discussed with respect to the substrate specificity of the target

enzymes.

Introduction

determines the primary substrate specificity.¹⁰Besides

housekeeping tasks in the turnover of cellular proteins,

thiol-dependent cathepsins also execute specific and

individual functions in physiological processes.^11,12Their

activity is controlled by limited proteolysis of latent

precursors as well as by endogenous inhibitors.^8,13To

an ever increasing extent it becomes obvious that

cysteine proteases are implicated in pathological condi-

tions. For example, the cathepsins B, C, L, and X have

been shown to be key players in the proliferation,

vascularization, and invasion of multiple pancreatic islet

tumors and cervical cancer.¹⁴

Cathepsin L (EC 3.4.22.15) shows the highest pro-

teolytic activity of all lysosomal proteases and is widely

expressed in various tissues. Increased activities of

cathepsin L were observed in several tumors. Very

recently, this protease has been demonstrated to act as

central mediator of the invasive capacity of S-adenosyl-

methionine decarboxylase-transformed malignant fi-

broblasts, probably by proteolytic degradation of extra-

cellular matrix constituents.¹⁵Its involvement in the

metastasis of human melanoma cells has also been

reported.¹⁶

Cathepsin S (EC 3.4.22.27) can be found in lymphatic

organs such as lymph nodes and spleen. It is crucial for

MHC class II mediated antigen presentation in B-cells,

dendritic cells, and nonprofessional antigen presenting

cells such as intestinal epithelial cells. In detail, cathe-

psin S cleaves off the CLIP peptide from the invariant

chain Ii, which is associated with MHC class II Râ

heterodimers in order to prevent premature antigen

binding.^17,18The role of this protease in the pathogenesis

of the autoimmune disease myasthenia gravis has been

elaborated recently.¹⁹

The huge class of cysteine proteinases comprises

enzymes participating in a multitude of biological

processes and can be subdivided into several clans,

including the clans CA (papain-like enzymes), CD

(caspases and related proteases), and PA (picornaviral

proteases). A more detailed classification can be found

in the MEROPS database.^1-3To the first clan belong

proteases from parasites as well as from mammals, and

papain-like cysteine proteases are promising targets for

therapeutic invention in parasitic infections and sys-

temic human diseases, respectively.^4,5At present, 11

human papain-like cysteine proteases have been de-

scribed: the cathepsins B, C, H, F, K, L, O, S, V, W,

and X. They show a high degree of homology and are

mainly located in lysosomes but can also be secreted as

inactive precursors from various cells, such as macroph-

ages, fibroblasts, osteoclasts, and malignant cells.⁶The

mature enzymes share a common fold consisting of two

domains, the N-terminal L (left) and the C-terminal R

(right) domain. The V-shaped active site cleft is located

between the two domains binding the substrate in an

extended conformation (for review, see refs 7 and 8). The

catalytic center is constituted of the residues Cys 25,

His 159, and Asn 175 (papain numbering). The latter

residue does not directly contribute to catalysis, but acts

as hydrogen bond acceptor toward the imidazole entity

of His 159 and thereby enables the formation of a

permanent thiolate-imidazolium ion pair. Substrates

are mainly recognized by the S², S¹, and S^1′binding sites

(nomenclature according to Schechter and Berger).⁹The

S²pocket represents the best defined binding site and

* To whom correspondence should be addressed. Phone: +49 228

732317. Fax: +49 228 732567. E-mail: guetschow@uni-bonn.de.

^†Dedicated to Prof. Dr. Bernd Wiederanders, Jena, on the occasion

of his 65th birthday.

Cathepsin K (EC 3.4.22.38) is predominantly ex-

pressed in osteoclasts.²⁰Its collagenolytic capability is

even higher than that of the matrix metalloproteinases,

because it is able to cleave native collagen at multiple

^‡Universita¨t Bonn.

^§Universita¨t Jena.

Published on Web 11/03/2005

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7689

Scheme 1^a

sites.²¹It has been shown that the collagenase activity

of cathepsin K requires the formation of supramolecular

complexes between the protease and chondroitin 4-sul-

fate.²²Bone remodeling is an essential physiological

process balanced between degradation and reconstruc-

tion. Bone resorption starts with acid-induced deminer-

alization, followed by proteolytic degradation of the

organic matrixsmainly consisting of type I collagens

preponderantly mediated by cathepsin K secreted from

osteoclasts. Enhanced degradation causes diseases such

as osteoporosis. Hence, cathepsin K is a promising drug

target for the treatment of osteoporosis, and strong

efforts have been focused on the development of inhibi-

tors for this enzyme.

a

Reagents and conditions: (a) (1) N-methylmorpholine,

Due to the vital role of papain-like cysteine proteases

in human physiology and pathology, the development

of potent and selective inhibitors for these enzymes

represents a great challenge. Several classes of com-

pounds with electrophilic sites able to interact with the

active thiol have been reported to inhibit cysteine

cathepsins.^1,23,24Most of them are peptide derived

inhibitors such as peptidyl aldehydes, ketones, halo-

methyl ketones, diazoketones, O-acylhydroxamates, ep-

oxides, vinylsulfones, and nitriles. The latter class has

gained much attention in recent years.^25,26Peptide

nitriles bind to the active site thiol via reversible

formation of a covalent thioimidate linkage (Chart

1).^27,28Nitriles are class-selective protease inhibitors

which strongly inhibit cysteine proteases while serine

proteases are not or only weakly affected.²⁹This selec-

tivity can be explained from two different perspectives.

According to the HSAB principle, the addition of the

soft, polarizable sulfur nucleophile of the cysteine pro-

teases to the soft carbon electrophile of the C-N triple

bond is favored over that of the hard oxygen nucleophile

of the serine proteases. From an enzymological point of

view, there is a difference in stabilization of the tetra-

hedral transition state of amide bond hydrolysis. Cys-

teine protease-catalyzed reactions pass through a less

constrained transition state as compared to serine

proteases; the hydrogen bonding to the oxyanion hole

is not as crucial as in serine proteases.^30,31Hence, and

in contrast to serine proteases, cysteine proteases should

be more prone to accept the planar thioimidate formed

from a nitrile inhibitor instead of the tetrahedral

transition state formed upon the competing hydrolysis

of a substrate molecule.

ClCO₂CH₂CH(CH₃)₂, THF; (2) H₂NCH₂CN‚0.5H₂SO₄or CH₃-

NHCH₂CN‚HCl, H₂O, 1 N NaOH, -25 °C to room temperature.

Scheme 2^a

a

Reagents and conditions: (a) phenyl boronic acid, Na₂CO₃,

Pd(PPh₃)₄, DME/H₂O, microwave.

ments and limitations of the P²side chain of the peptidic

inhibitors. Further variations were introduced at the

N-terminal group and the backbone structure, whereas

glycine was kept constant at P¹. In addition, the

bioactivity of a selected nitrile was compared to that of

the analogous aldehyde and methyl ketone inhibitors,

as well as the corresponding propargyl- and allylamide.

Results and Discussion

Synthesis. Wieland’s mixed anhydride method using

isobutyl chloroformate as activating agent, N-methyl-

morpholine as tertiary base, and THF as solvent pro-

vides a powerful and reliable synthetic tool for the

formation of amide bonds in solution.³²This method was

used to couple proteinogenic and nonproteinogenic N-

protected amino acids carrying different aliphatic and

aromatic side chains, activated in situ, with aminoac-

etonitrile to obtain dipeptide nitriles 1-36 and 38

mostly in high yields and purity (Scheme 1). The

N-methylated compound 37 was prepared by using

N-methyl aminoacetonitrile instead of aminoacetoni-

trile. For the preparation of the biphenyl derivatives 39

and 40 a microwave-assisted, palladium-catalyzed Su-

zuki cross coupling of the phenyl bromides 29 and 30

with phenylboronic acid was applied (Scheme 2).^33-35

The acyl derivatives 42-44 were synthesized by depro-

tection of 16 using formic acid and subsequent acylation

of the free dipeptide nitrile 41 under Schotten-Bau-

mann conditions to yield 42 and 43 (Scheme 3). To

prepare 44, biphenyl-4-carbonic acid was activated as

mixed anhydride and reacted with 41. The synthetic

route to the aza analogue 48 includes the multistep

reductive alkylation of tert-butyl carbazate with benz-

aldehyde using sodium cyanoborohydride as reductant

(Scheme 4). The intermediate cyanoborane complex 46

was isolated and subsequently destroyed with sodium

hydroxide solution.³⁶The resulting compound 47 was

Chart 1. Reversible Formation of Thioimidates from

Peptide Nitriles and Cysteine Proteases

Herein we report a detailed study on the structure-

activity relationships of dipeptide derived nitriles as

inhibitors of cysteine proteases. A series of 48 com-

pounds including 44 nitriles were prepared and evalu-

ated toward the canonical papain and the therapeuti-

cally relevant cathepsins L, S, and K on the basis of

the inhibition kinetics. With respect to the known

importance of the P²-S²interaction for specificity and

affinity, the design was aimed to explore the require-

7690 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

Scheme 3^a

Scheme 6^a

a

Reagents and conditions: (a) (1) N-methylmorpholine,

ClCO₂CH₂CH(CH₃)₂, THF; (2) propargylamine or allylamine, -25

°C to room temperature.

preparation of the methyl ketone 53 was accomplished

by reacting Weinreb amide 51 with methyl Grignard

reagent.³⁹The phenylalanine derived propargyl- and

allylamides 54 and 55, respectively, were prepared from

the corresponding amines by the mixed anhydride

method (Scheme 6).

a

Reagents and conditions: (a) (1) HCOOH, room temperature;

(2) 70% HClO₄/iPrOH; (b) Ac₂O or BzCl, sat. NaHCO₃, 4 °C; (c)

(1) biphenyl-4-carbonic acid, N-methylmorpholine, ClCO₂CH₂-

CH(CH₃)₂in THF; (2) 41 in H₂O/1 N NaOH, -25 °C to room

temperature.

Inhibition Kinetics. The dipeptide nitriles 1-40,

42-44, and 48, the aldehyde 52, the methyl ketone 53,

and the unsaturated amides 54 and 55 were investi-

gated toward four cysteine proteases, the plant enzyme

papain, and the mammalian cathepsins L, S, and K.

Papain was assayed with the chromogenic substrate

Z-Phe-Arg-NHNp (Np ) 4-nitrophenyl). To reduce en-

zyme consumption, more sensitive fluorimetric assays

were chosen to characterize the inhibitory activity of

these compounds toward the cathepsins. The specific

substrates Z-Phe-Arg-NHMec, Z-Val-Val-Arg-NHMec,

and Z-Leu-Arg-NHMec (Mec ) 4-methylcoumarin-7-yl),

containing the fluorescent C-terminal leaving group

7-amino-4-methylcoumarin (AMC), were employed to

monitor the activities of cathepsin L, cathepsin S, and

cathepsin K, respectively. The K_mvalues of the sub-

strates were required for the kinetic analyses and had

to be determined under the same experimental condi-

tions used in the inhibition experiments. The determi-

nation of the K_mvalue of the papain substrate Z-Phe-

Arg-NHNp was difficult due to low affinity (K_m> 500

µM) and poor solubility even in the presence of 12%

DMSO (<500 µM). As described in the Experimental

Section, K_mwas determined by monitoring the complete

papain-catalyzed hydrolysis at a preferably high initial

substrate concentration. The data were analyzed with

differential equations 3 and 4 describing both the

catalytic activity and the slow, but significant, inactiva-

tion of the enzyme over a comparably long period of time

to obtain K_m) 965 ( 244 µM. The K_mvalues of the

cathepsins were determined as usual in separate mea-

surements in the presence of different substrate con-

centrations and were calculated from the initial veloci-

ties.

Scheme 4^a

a

Reagents and conditions: (a) benzaldehyde, THF, room

temperature; (b) NaBH₃CN, AcOH, THF; (c) 1 N NaOH, MeOH;

(d) CDI, triethylamine, H₂NCH₂CN‚0.5H₂SO₄, DMF, 4 °C to room

temperature.

Scheme 5^a

a

Reagents and conditions: (a) H₃CNHOCH₃‚HCl, 1 N NaOH,

dioxane, room temperature; (b) (1) 4 N HCl/dioxane, room tem-

perature; (2) 70% HClO₄/iPrOH; (c) (1) Boc-Phe-OH, N-methyl-

morpholine, ClCO₂CH₂CH(CH₃)₂, THF; (2) 50 in H₂O/1 N NaOH,

-25 °C to room temperature; (d) (1) LiAlH₄, Et₂O; (2) sat. NH₄Cl;

(e) (1) Mg, CH₃I, Et₂O; (2) sat. NH₄Cl.

All nitrile inhibitors showed time-independent inhibi-

tion. It could be concluded from the inhibitor structures

and literature data that these dipeptide nitriles act as

reversible, active site directed inhibitors and exhibit

competitive behavior. Thus, the slopes of the progress

curves could directly be used to determine K_i′ values

from which K_ivalues were calculated. A representative

kinetic analysis is illustrated in Figure 1, showing the

inhibition of cathepsin S by the naphthylalanine-derived

compound 34. The velocities of the enzyme-catalyzed

reaction were influenced by the inhibitor concentration

already at the beginning of the measurement and kept

constant until its end. It could be deduced from the

reacted with the azolide formed in situ from N,N′-

carbonyldiimidazole and aminoacetonitrile sulfate,³⁷to

yield the azadipeptide nitrile 48. Attempts to prepare

the aldehyde 52 (Scheme 5) by oxidation of the corre-

sponding primary alcohol with the hypervalent iodine

reagent iodoxybenzoic acid (IBX) resulted in a complex

mixture of products. Therefore, the Weinreb amide 51

was prepared in three steps and subsequently reduced

with lithium aluminum hydride³⁸to furnish 52. The

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7691

to an isopropyl moiety. The carba-analogue 13 was

accepted by the four proteases to a similar extent as

the parent methionine derivative 12. These data dem-

onstrate that hydrophobic interactions between the P²

aliphatic residues and the S²binding sites account for

the affinity of the dipeptide nitriles toward the target

enzymes.

The proline derived compound 14 did not exhibit

inhibitory activity. A successive extension of the spacer

between the R-carbon and the phenyl ring was realized

to provide inhibitors 15, 16, 21, and 22. The phenylg-

lycine derivative 15 showed weak activities with a K_i

value toward papain being 3 orders of magnitude higher

than that of the phenylalanine inhibitor 16. Nitrile 16

was a potent inhibitor of papain, and both cathepsins

L and S. Further extension diminished enzyme inhibi-

tion, however, even the introduction of a three-atom-

linker in the O-benzylserine derivate 22 resulted in K_i

values of 11 and 2.2 µM toward papain and cathepsin

S, respectively.

Further structural alterations were made on the basis

of inhibitor 16. Bioisosteric replacement of the benzene

ring by five-membered hetarenes yielded 18-20. These

structures were well tolerated by the target proteases.

The thiophene derivatives were significantly more

potent that the 2-furyl compound 18. The attachment

of the thiophene ring at position 2′, compared to position

3′, was somewhat preferred by the four enzymes (19

versus 20).

For the benzene ring of 16, systematic fluorine and

bromine scans were performed. The fluorine scan was

done to evaluate the fluorophilic/fluorophobic potential

of the S²pockets. Monofluorination (24-26) was well

tolerated, but no enhancement of the affinities was

observed. Perfluorination, as realized in 27, significantly

decreased the affinity, when compared to inhibitor 16.

This could be explained by electrostatic repulsions due

to the directly opposed quadrupole moments of perfluo-

robenzenes⁴⁰rather than by a raised sterical demand

of the pentafluorophenyl group. From these results, it

can be concluded that the S²pockets of the four

proteases are slightly fluorophobic. Studies in which the

systematic fluorination of aromatic residues in thrombin

inhibitors led to improved affinities were recently

published.^41,42The systematic bromine substitution of

the aromatic ring in 16 was also accepted by the

proteases and resulted in mostly equipotent inhibitors

(28, 29, 30 versus 16).

Figure 1. (A) Monitoring of the human cathepsin S-catalyzed

hydrolysis of Z-Val-Val-Arg-NHMec (40 µM) in the presence

of increasing concentrations of compound 34 (50 mM potas-

sium phosphate pH 6.5, 50 mM NaCl, 2 mM EDTA, 0.01%

Triton X-100, 25 µM DTT, and 1% DMSO, 37 °C). The reaction

was initiated by addition of the enzyme. Data were obtained

by measuring the fluorescence emission at 440 nm after

excitation at 360 nm. Ordinate values were corrected for

background fluorescence. Color scheme: red, [I] ) 0; yellow,

[I] ) 0; dark green, [I] ) 0.05 µM; dark blue, [I] ) 0.1 µM;

magenta, [I] ) 0.2 µM; light blue, [I] ) 0.3 µM; light green, [I]

) 0.4 µM; gray, [I] ) 0.6 µM; violet, [I] ) 1 µM; turquoise, [I]

) 5 µM; brown, [I] ) 10 µM. (B) Plot of the rates of hydrolysis

of Z-Val-Val-Arg-NHMec versus concentrations of 34. The

linear dependence shown in the Dixon plot (inset) indicates

competitive inhibition. Nonlinear regression gave an apparent

inhibition constant K_i′ ) (1 + [S]/K_m)K_i) 0.25 ( 0.02 µM. A

Michaelis constant K_m) 19.2 ( 0.8 µM was determined

separately.

The usefulness of bicyclic aromatic cores was inves-

tigated with the inhibitors 31-34. The tryptophan and

3′-thionaphthenylalanine derived compounds 31 and 32

behaved almost equally toward each of the four enzymes

and inhibited cathepsin L and cathepsin S nearly to the

same extent. The replacement of the indole and thion-

aphthene moieties by the bioisosteric naphthalene

resulted in the case of the R-substitution (33) in a loss

of inhibitory activity. In contrast, introduction of â-naph-

thalene (34) furnished very potent inhibitors of cathe-

psin S, cathepsin L, and papain.

To investigate substituents, in which the two aromatic

rings are connected by a single bond, the three possible

biphenyl derivatives 35, 39, and 40 were synthesized

and evaluated. While the para and meta biphenylala-

nine-derived compounds showed remarkable inhibitory

instantaneously attained equilibrium that the enzyme-

inhibitor interaction involves the rapid formation of the

covalent thioimidate complex.

General Trends in Cysteine Protease Inhibition.

The inhibition constants of 37 dipeptide nitriles toward

papain and the cathepsins L, S, and K are summarized

in Table 1. An aliphatic R²residue attached at the P²

position required at least two carbon atoms to achieve

inhibition. Introduction of a further methyl group at the

R²chain mostly enhanced the inhibitory potency (5

versus 4; 7 versus 6). Rigidization of the geminal methyl

groups in 7 led to the cyclopropyl derivative 10 and

resulted in a loss of inhibition, particularly toward

cathepsins L and K. This might be due to the reduced

sterical demand of a cyclopropyl group in comparison

7692 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

Table 1. Inhibition of Papain, Cathepsin L, Cathepsin S, and Cathepsin K by Dipeptide Nitriles 1-35, 39, and 40

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7693

Table 1 (Continued)

7694 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Table 1 (Continued)

Lo¨ser et al.

Table 2. Inhibition of Papain, Cathepsin L, Cathepsin S, and

Cathepsin K by Glycine Derivatives 1, 2, and 38

The intervention in the peptidic backbone had a

dramatic influence on the bioactivity. N-Methylation

(37) decreased the affinity by 3 orders of magnitude. The

calculated changes in the free enthalpies of binding are

in the range ∆∆G_obs> 2 kcal/mol, suggesting the amide

N-H to be a strong H-bond donor. The exchange of the

phenylalanine of 16 by azaphenylalanine as realized in

48 was not tolerated. The change of the configuration

from S to R (36) also led to a drastic reduction in the

binding affinity. Our observations with the azadipeptide

nitrile are in accordance with those reported by Cala-

bretta et al. on azapeptidyl methylketones as substrate

analogue inhibitors of papain and cathepsin B.³⁶The

poor affinity resulting from the C/N-replacement was

explained by the geometry changed from tetrahedral to

nearly trigonal-planar. Furthermore, the capacity of the

amide N-H bond to act as an H-bond donor is reduced.

Quantum chemical calculations have shown that aza-

peptides cannot adopt â-strand conformations, which is

crucial for protease-peptide interactions, because the

dihedral angle φ is restricted.^43,44However, cystatin

derived azapeptides with azaglycine in the P¹position

have been shown to be very potent inhibitors of papain-

like cysteine proteases.⁴⁵Although the P²benzyl group

of the R-configured compound 36 could be expected to

fit worse than that of 48, the latter compound appeared

to be even less potent than 36. Therefore, the inhibitor

geometry might be more distorted in the aza analogue

48 than in the R-configured 36.

K_i(µM)

bovine

human

cathepsin cathepsin cathepsin

compd

R

papain

>3000

L

S

K

1^a

Boc

Z

>1000 >1000

>1000

2^a

1599 ( 109 >1000 >1000

38 2-naphthyl- 10 ( 0.2

25 ( 5 2.5 ( 0.2 >100

sulfonyl

^aCompounds are also included in Table 1.

potency toward papain, cathepsin L, and cathepsin S,

the ortho linkage led to a dramatic decrease in inhibi-

tory activity.

The data given in Table 1 underscore the importance

of the P²residue. Accordingly, the glycylglycine deriva-

tives 1 and 2 are not active. Nevertheless, one compound

lacking a P²residue showed significant inhibitory

properties obviously mediated by the 2-naphthylsulfonyl

group of 38 (Table 2).

Further structural variations, outlined in Table 3,

were carried out starting from inhibitor 16 and main-

taining its phenylalanine and cyanomethyl substruc-

tures. Compounds 16, 17, and 42-44 (Table 3) differ

in the nature of N-terminal protecting group and share

the remaining molecular structure. Without exception,

these phenylalanine derivatives exhibited affinities in

the lower micromolar or nanomolar range. However, the

small acetyl moiety was less favorable and all bulkier

substituents including carbamate and acyl protecting

groups enhanced the interaction (42 versus 16, 17, and

43, 44, respectively).

Furthermore, the nitrile warhead was exchanged by

the electrophilic carbonyl group as realized in the

aldehyde 52 and the methyl ketone 53 (Table 4). The

dipeptidyl aldehyde 52 showed significantly increased

affinity to all enzymes. This can be understood by the

stronger electrophilicity of the aldehyde carbonyl carbon

when compared to the cyano carbon. The strong elec-

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7695

Table 3. Inhibition of Papain, Cathepsin L, Cathepsin S, and Cathepsin K by Phenylalanine Derivatives

K_i(µM)

bovine

cathepsin L

human

cathepsin S

human

cathepsin K

compd

R¹

R²

X

conf

papain

16^a

17^a

42

43

44

37

36

48

tBuO

H

Me

H

CH

N

S

R

0.26 ( 0.01

0.36 ( 0.01

0.38 ( 0.02

0.062 ( 0.002

0.016 ( 0.001

>100

0.52 ( 0.02

0.18 ( 0.02

5.8 ( 0.5

0.23 ( 0.02

0.55 ( 0.07

>100

0.62 ( 0.05

0.51 ( 0.05

1.4 ( 0.1

0.077 ( 0.004

0.24 ( 0.03

>100

4.1 ( 0.2

0.26 ( 0.01

7.5 ( 0.5

1.5 ( 0.1

0.30 ( 0.03

>100

BnO

Me

Ph

4-biphenyl

tBuO

41 ( 2

>100

46 ( 13

>100

725 ( 21

>100

>50

>100

^aCompounds are also included in Table 1.

Table 4. Inhibition of Papain, Cathepsin L, Cathepsin S, and Cathepsin K by Dipeptide Analogues 16, 52, and 53

papain

bovine cathepsin L

human cathepsin S

human cathepsin K

K_i

(µM)

K_i

(µM)

k_on

k_off

(10^-3s^-1

K_i

(µM)

k_on

k_off

(10^-4s^-1

K_i

(µM)

k_on

k_off

compd

R

(M^-1s^-1

)

(M^-1s^-1

)

(M^-1s^-1

)

(10^-4s^-1

)

16^aCN

0.32 ( 0.01

0.52 ( 0.02

0.62 ( 0.05

4.1 ( 0.2

52

53

CHO 0.015 ( 0.0005 0.022 ( 0.001

0.025 ( 0.001

0.074 ( 0.004

COMe 187 ( 24

2.3 ( 0.3

515 ( 32 1.2 ( 0.25 2.1 ( 0.1

336 ( 19 7.1 ( 0.7 2.1 ( 0.3

228 ( 21 4.8 ( 1.2

^aCompound is also included in Table 1.

trophilic aldehyde is thought to be the determining

factor for the affinity. The thiohemiacetal formed by the

nucleophilic attack of the cysteine sulfur also accounts

for the pronounced activity of 52. The thiohemiacetal

has a tetrahedral geometry different from the planar

thioimidate that results from the corresponding nitrile

inhibitors. A crystal structure of cathepsin S complexed

with a dipeptidyl aldehyde revealed that the formation

of the tetrahedral adduct is not stereospecific, because

complexes with S- and R-configurations could be ob-

served.⁴⁶Thus, the aldehyde carbonyl can be attacked

at both the si and the re face, forming adducts in which

either the hydrogen atom or the hydroxy group points

toward the oxyanion hole, respectively, which may

further account for the enhanced affinity. It was found

for inhibitor 52 that the relative impact of the specific-

ity-mediating P²-benzyl group was reduced resulting in

lower differences in inhibitory potency toward the four

enzymes. For comparison, the nitrile 16 revealed a

discriminating affinity for cathepsins L and S over

cathepsin K. In contrast, the dipeptidyl methyl ketone

53 exhibited K_ivalues which were 1 order of magnitude

higher toward cathepsins L and S than those of com-

pound 16, while inhibition of cathepsin K was not

affected by this structural replacement. Interestingly,

inhibitor 53 showed time-dependent inhibition against

the cathepsins (Figure 2). The reactions were analyzed

by the formalism of slow-binding kinetics, i.e., eq 5 and

the corresponding data analysis as noted in the Experi-

mental Section. A one-step mechanism could be con-

cluded from the linear dependence of the pseudo-first-

order rate constant, k_obs, on the inhibitor concentration,

[I], as well as from the initial velocities, v_i, being

independent of the inhibitor concentration.⁴⁷The drop

in the activity caused by the attachment of the methyl

group (53 versus 52) might be due to the enhanced steric

hindrance in the ketone-derived tetrahedral complex,

rather than the reduced electrophilicity of the ketone

carbonyl group. The methyl group might not fit into the

oxyanion hole and thus the carbonyl would only be

attacked at the re face. The methyl ketone 53 completely

lacked selectivity for the cathepsins with K_ivalues

between 2.1 and 2.3 µM. The second-order rate con-

stants for the formation of the enzyme-inhibitor com-

plex, k_on, were very similar.

To investigate the contributions of the covalent and

noncovalent interactions to enzyme-ligand recognition,

the cyano group of 16 was replaced by nonelectrophilic

entities such as the ethynyl group in 54 and the vinyl

group in 55 (Table 5). The propargylamide 54 resembles

16 in the unbound state, and the allylamide 55 mimics

the geometry of the enzyme-bound thioimidate. The poor

affinities of 54 and 55 demonstrate the importance of

the covalent interaction. This result, combined with the

fact that the glycine derivative 1 is by 4 orders of

magnitude weaker than 16, indicates the high cooper-

ativity between the covalent and noncovalent interac-

tion. The finding that the non-nitriles 54 and 55 are

somewhat active against papain might result from ion-

7696 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

Table 5. Inhibition of Papain, Cathepsin L, Cathepsin S, and

Cathepsin K by Dipeptide Analogues 1, 16, 54, and 55

K_i(µM)

bovine

human

compd R¹

1^a

R²

papain cathepsin L cathepsin S cathepsin K

H

CtN

>3000 >1000 >1000 >1000

0.26 ( 0.01 0.52 ( 0.02 0.62 ( 0.05 4.1 ( 0.2

16^aBn CtN

54 Bn CtCH 497 ( 14

55 Bn CdCH₂497 ( 52

>1000

>100

>1000

^aCompounds are also included in Table 1.

induced dipole interaction between the active site thi-

olate and the C-C multiple bonds.

Inhibition of Papain. Papain was scarcely affected

by compounds with aliphatic residues in P²(Table 1).

Strong inhibition could be achieved by introduction of

various aromatic structures into P². As most potent

nitriles, 35 (Table 1) and 44 (Table 3) were identified,

both bearing a 4-biphenyl moiety, either in P²(35), or

as the N-terminal acyl residue (44), respectively. The

biphenyl structural element is considered as a “privi-

leged structure” in drug discovery, since the two aro-

matic units are combined in a well-balanced degree of

flexibility and rigidity.⁴⁸However, the bromine scan

showed that a bromine atom attached in the para

position improved the affinity to nearly the same extent

(28 versus 35, Table 1). Thus, the enhancement of the

activity of the parent compound 16 by para-phenyl

substitution could be attributed to the hydrophobic

character of the para-substituent (phenyl or bromo)

rather than to a specific aromatic interaction within the

S²pocket. An impact of a para-substituent to lower K_m

has been found within a series of substrates containing

phenylalanine analogues.⁴⁹The most dramatic struc-

tural impact was observed upon switching the linkage

of the naphthalene ring to the dipeptide scaffold from

R to â position, lowering K_iby 3 orders of magnitude

(33 versus 34).

With regard to the substrate specificity, it is known

that papain mainly accepts substrates containing a

bulky nonpolar amino acid in P², e.g. phenylalanine. The

S²binding pocket is defined by the hydrophobic, non-

aromatic side chains of Pro 68, Val 133, Val 157, Asp

158, and Ala 166. Crystal structures of the enzyme

complexed with corresponding chloromethyl ketones

revealed van der Waals contacts of the phenyl group

with the valine side chains⁵⁰as well as with Asp 158

and Pro 168.⁵¹However, the structural basis for the

preference of aromatic over aliphatic residues in P²is

not completely understood. The present study confirmed

the strong preference of papain for aromatic side chains

in the P²position. However, phenylalanine was not the

best accepted amino acid in P². Certain, well-defined

modifications of the benzene ring enhanced the activity

of the resulting structures, others drastically reduced

it. Noteworthy, the differences in the binding affinity

within the group of dipeptide nitriles bearing an aro-

matic structure in P²were more obvious for papain than

for the cathepsins.

Figure 2. (A) Monitoring of the human cathepsin S-catalyzed

hydrolysis of Z-Val-Val-Arg-NHMec (40 µM) in the presence

of increasing concentrations of compound 53 (50 mM potas-

sium phosphate pH 6.5, 50 mM NaCl, 2 mM EDTA, 0.01%

Triton X-100, 25 µM DTT, and 1% DMSO, 37 °C). The reaction

was initiated by addition of the enzyme. Data were obtained

by measuring the fluorescence emission at 440 nm after

excitation at 360 nm. Ordinate values were corrected for

background fluorescence. Color scheme: red, [I] ) 0; yellow,

[I] ) 0; dark green, [I] ) 5 µM; dark blue [I] ) 10 µM; magenta,

[I] ) 15 µM; light blue [I] ) 30 µM; light green, [I] ) 40 µM;

gray, [I] ) 50 µM; violet, [I] ) 60 µM; turquoise, [I] ) 75 µM;

brown, [I] ) 100 µM. (B) Plot of the steady state rates of the

hydrolysis of Z-Val-Val-Arg-NHMec versus concentrations of

53. The linear dependence shown in the Dixon plot (inset)

indicates competitive inhibition. Nonlinear regression gave an

apparent inhibition constant K_i′ ) (1 + [S]/K_m)K_i) 6.6 ( 0.5

µM. A Michaelis constant K_m) 19.2 ( 0.8 µM was determined

separately. (C) Plot of the k_obsvalues versus concentrations of

54. The linear dependence indicates a one-step mechanism for

the enzyme-inhibitor interaction. Linear regression gave an

apparent second order rate constant k_on′ ) k_on/(1 + [S]/K_m) )

109 ( 6 M^-1s^-1. From the corresponding k_onvalue, a first order

rate constant k_off) k_onK_i) 7.1 × 10^-4( 0.7 × 10^-4s^-1was

calculated.

Inhibition of Cathepsin L. The trend observed with

papain, that less efficient inhibition was achieved by

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7697

Table 6. Comparison of the Inhibition of Bovine and Human Cathepsin L by Selected Compounds

compounds with aliphatic residues in P²(Table 1), is

also valid for cathepsin L. Monocyclic hetarenes in P²

side chains were less well tolerated by cathepsin L than

by the other enzymes. Bicyclic aromatic structures (31,

32, 34, and to a lower extent 33) were explicitly accepted

in the S²pocket. The â-naphthylalanine derivative 34

is the best inhibitor against cathepsin L (K_i) 0.12 µM)

of all compounds investigated in this study. With respect

to the binding of bicyclic aromatic structures, the S²

pocket of cathepsin L is more flexible than in the case

of papain. When the tert-butoxycarbonyl group was

replaced by benzyloxycarbonyl, inhibition became more

pronounced (7 versus 8; 16 versus 17). The Z-protected

compound 17 was the second-best inhibitor of the

dipeptide nitrile series and exhibited a slight preference

toward cathepsin L over cathepsin S. In addition, the

differences in the inhibitory behavior toward the bovine

and the human cathepsin L were investigated by

evaluating selected compounds at the human enzyme.

The results are shown in Table 6. The, at best, marginal

differences indicated that the human enzyme can be

substituted by bovine cathepsin L, at least for inhibition

studies with derivatives addressing the nonprimed

binding sites.

ing either valine or leucine in P²; the substrate specific-

ity is less restricted.⁵⁴In accordance, potent inhibitors

of the present series bear aliphatic as well as aromatic

residues in the P²position, a feature which distin-

guished cathepsin S from the other examined enzymes

(Table 1). This observation reflects the plasticity of the

S²site, which can be attributed to a conformational

switch of Phe 205, a phenomenon discovered upon

comparison of enzyme-inhibitor complexes containing

either P²-Leu or P²-Phe derived inhibitors.⁴⁶The leu-

cine-derived inhibitors 7 and 8 possessed high potency.

Prolongation and cyclization of the leucine side chain

by three methylene groups led to the cyclohexylalanine

derivative 11, which was the most potent inhibitor (K_i

) 0.044 µM) among all considered nitriles. It inhibited

cathepsin S with high selectivity over the cathepsins K

and L. This observation accords with that made by Ward

et al.²⁶A further particularly active compound was the

â-naphthylalanine derivative 34, which also inhibited

cathepsin L in a comparable manner. Replacement of

the phenyl group in 16 by a 2-thienyl moiety in 19

intensified the inhibition of cathepsin S. The thienyla-

lanine derivative 19 showed a weak preference for

cathepsin S, while the biphenyl compounds 35 and 39

did not differentiate well between cathepsin L and S.

With respect to Z or Boc as the N-protecting group,

cathepsin S behaved similarly. Surprisingly, when the

Z-derivative 17 was shortened by the CH₂O unit, as

realized in the benzoyl-protected 43, the potency in-

creased by nearly 1 order of magnitude (Table 3).

Inhibition of Cathepsin K. The substrate specificity

of cathepsin K is similar to that of cathepsin S; leucine

is favored over phenylalanine in P².²⁰Its S²subsite is

defined by Tyr 67, Met 68, Ala 133, Leu 157, Ala 160,

and Leu 205 (papain numbering).⁵⁵Cathepsin K showed

the highest affinities toward dipeptide nitriles with a

leucine side chain (7, 8, Table 1). Every structural

The P²substrate specificity of cathepsin L resembles

that of papain. The S²site is constituted by the side

chains of Asp 158, Gly 160, Met 157, Ala 205, Met 68,

and Leu 67 (papain numbering).⁵²The pronounced

preference for aromatic binding partners, as confirmed

in this study, might be explained by sulfur-arene

interactions mediated by one of the two methionine side

chains, as it has been observed in several protein-

ligand complexes.⁴⁰

Inhibition of Cathepsin S. The S²pocket of cathe-

psin S is formed by the side chains of Phe 67, Met 68,

Gly 133, Val 157, Gly 160, and Phe 205 (papain

numbering).⁵³This protease prefers substrates contain-

7698 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

aluminum sheets. Preparative column chromatography was

performed on silica gel 60 (Fluka) 70-230 mesh. Preparative

HPLC was performed on a Eurospher 100 C₁₈column (250 ×

20 mm) with a mixture of MeOH and H₂O at a flow rate of 20

mL/min. Microwave assisted syntheses were done in a CEM

discover system. ¹H NMR spectra (500 MHz) and ¹³C NMR

spectra (125 MHz) were recorded on a Bruker Avance 500.

Mass spectra were obtained on a API 2000 spectrometer from

Applied Biosystems (ESI, sprayed from a 10^-5M solution in 2

mM NH₄OAc/MeOH 1:1; volumetric flow rate 10 µL/min).

Optical rotations were determined on a Perkin-Elmer 241

Polarimeter. Enzymatic activity of papain and cathepsins was

measured with chromogenic substrates spectrophotometrically

at a Varian Cary Bio 50 UV/vis spectrophotometer or fluoro-

metrically at a Perkin-Elmer luminescence spectrometer LS

50 B, respectively. Papain was purchased from Sigma, Ger-

many. Bovine liver cathepsin L and human cathepsin L, His

Tag were purchased from Calbiochem, Germany. Recombinant

human cathepsin S was either expressed in insect cells⁵⁶or

purchased from Calbiochem; the two preparations showed a

similar K_mtoward Z-Val-Val-Arg-NHMec. Recombinant hu-

man Cathepsin K (expressed in yeast) was a gift of D.

Bro¨mme.⁵⁷Human cathepsin L, His Tag was purchased from

Calbiochem.

change of this moiety increased the inhibition constant,

which then mostly escaped from the nanomolar range.

Thus, cathepsin K was unique in that it possessed minor

promiscuity with respect to the acceptance of a variety

of P²residues, in particular those from nonproteinogenic

amino acids. Especially certain space-filling aromatic

substituents at P²were not at all tolerated, e.g. the

O-benzylserine (22), tyrosine (23), para-bromophenyl-

alanine (28), and â-naphthylalanine (34) derivatives, all

of which were found to be substantially less potent,

compared to the other proteases. Enhanced inhibitory

activity was observed when the N-terminal group was

changed from tert-butoxycarbonyl to benzyloxycarbonyl

(7 versus 8; 16 versus 17). The replacement of the tert-

butoxycarbonyl group by benzoyl (16 versus 43, Table

3) had no remarkable effect, whereas a 4-phenylbenzoyl

group shifted the inhibition constant into the nanomolar

range (44 versus 43). These observations implicated that

an aromatic entity needs to have an exact distance to

the P²-nitrogen for favorable interactions.

The substrates, Z-Phe-Arg-NHNp, Z-Phe-Arg-NHMec,

Z-Val-Val-Arg-NHMec, Z-Leu-Arg-NHMec, were from Bachem,

Bubendorf, Switzerland. The amino acid derivatives were

purchased from Novabiochem, La¨ufelfingen, Switzerland,

Bachem, Bubendorf, Switzerland, Acros, Geel, Belgium, Ald-

rich, Steinheim, Germany, and Synthetech Inc., Albany, NY.

N-(2-Naphthylsulfonyl)-glycine was prepared as described

in the literature.⁵⁸DTT () (()-threo-2,3-dihydroxy-1,4-

butanedithiol), Brij 35 P, and Triton X-100 were obtained from

Fluka; CHAPS () 3-[(3-cholamidopropyl)dimethylammonio]-

1-propanesulfonate) was from Sigma. Mathematical data

analyses were done with the programs Grafit 4 (Erithacus

Software), GraphPad Prism 4 (GraphPad Software) and EASY-

FIT.⁵⁹

N-(tert-Butoxycarbonyl)-glycyl-glycine-nitrile (1). Gen-

eral Procedure for Dipeptide Nitriles 1-38. Boc-Gly-OH

(1 g, 5.7 mmol) was dissolved in THF (15 mL) and cooled to

-25 °C. Subsequently, N-methylmorpholine (1.73 mL, 5.7

mmol) and isobutyl chloroformate (0.75 mL, 5.7 mmol) were

sequentially added under vigorous stirring. Immediately after

the formation of a white precipitate (N-methylmorpholine

hydrochloride), a solution of aminoacetonitrile sulfate (0.66 g,

6.3 mmol) in H₂O (0.5 mL), precooled on ice, and 1 N NaOH

(6.3 mL) were added. The resulting mixture was allowed to

warm to room temperature. After 2 h, THF was removed under

reduced pressure and the residual aqueous mixture was

diluted with a small volume of H₂O, adjusted to pH 1 (10%

NaHSO₄) and extracted with ethyl acetate (3 × 20 mL). The

combined organic layers were washed with H₂O (10 mL), sat.

NaHCO₃(2 × 10 mL), and brine (10 mL), dried over Na₂SO₄,

and evaporated to dryness. The solid residue was recrystallized

from cyclohexane/ethyl acetate to obtain 1 (0.83 g, 68%): mp

110-112 °C (lit.⁶⁰mp 108-110 °C); ¹H NMR (400 MHz, CDCl₃)

δ 1.39 (s, 9H), 3.78 (d, J ) 5.6 Hz, 2H), 4.11 (d, J ) 5.8 Hz,

2H), 5.41 (br s, 1H), 7.27 (t, J ) 5.2 Hz, 1H); ¹³C NMR (125

MHz, CDCl₃) δ 27.41, 28.31, 44.18, 80.83, 116.06, 156.44,

170.29; MS (ESI) m/z (rel intensity) (pos.) 236 (35, [M + Na]⁺),

231 (100, [M + NH₄]⁺), 214 (66, [M + H]⁺), 158 (62, [M -

(CH₃)₂CCH₂+ H]⁺), 114 (5, [M - (CH₃)₂CCH₂- CO₂+ H]⁺)

(neg.) 272 (57, [M + CH₃COO]^-), 212 (86, [M - H]^-), 138 (100,

[M - tBuOH - H]^-). Anal. (C₉H₁₅N₃O₃) C, H, N.

Conclusions

Potent inhibitors of papain-like cysteine proteases

were designed due to the introduction of structural

diversity into the P²side chain of dipeptidyl nitriles with

an aminoacetonitrile portion at the P¹position. This

approach is based on the major contribution of the P²-

S²interaction to facilitate strong binding of the inhibi-

tors to the enzyme. Although the formation of a covalent

thioimidate complex is well established for the reaction

of cysteine proteinases with nitrile inhibitors, the

cooperative effect of both the covalent attachment and

the P²-S²interaction are crucial factors for inhibition.

Among the four enzymes used in this study, cathepsin

K showed a rather distinct profile with respect to

inhibitor structures. Papain, cathepsin L, and cathepsin

S share the P²substrate specificity and tended to exhibit

similar affinities to a number of inhibitors. As papain

represents the best characterized and cheapest cysteine

protease, it might be used as model enzyme in the

preliminary search for inhibitors of the cathepsins L and

S. It can be concluded from the data of this study that

selectivity for one of the target enzymes over the other

closely related proteases is difficult to achieve with

dipeptide-derived compounds. The cysteine proteases

did not show promiscuity with respect to changes within

the backbone structure or methylation at the P¹nitro-

gen as found by systematic structural changes of a

parent compound 16. All enzymes behaved sensitively

to subtle changes in the P²entity. It was demonstrated

that various nonproteinogenic amino acids could be

introduced into the P²position to result in an enhanced

affinity. The present series was mainly focused on

variations within the P²side chain. However, the

N-protecting group was found to be a suitable unit to

accentuate the enzyme-inhibitor interaction. Thus, a

combination of the P²side chain, as established in this

study, with diverse groups at the P²nitrogen might be

a promising strategy for future investigations.

N-(Benzyloxycarbonyl)-glycyl-glycine-nitrile (2). The

crude product was recrystallized from cyclohexane/ethyl ac-

1

etate, yield 90%: mp 144-146 °C (lit.⁶¹mp 148-150 °C); H

NMR (500 MHz, CDCl₃) δ 3.65 (d, J ) 6.3 Hz, 2H), 4.12 (d, J

) 5.7 Hz, 2H), 5.03 (s, 2H), 7.25-7.40 (m, 5H), 7.52 (t, J ) 6.2

Hz, 1H), 8.58 (t, J ) 5.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃)

δ 27.14, 43.49, 65.74, 117.68, 127.87, 127.95, 128.48, 137.07,

156.63, 170.11; MS (ESI) m/z (rel intensity) (pos.) 270 (32,

[M + Na]⁺), 265 (100, [M + NH₄]⁺), 248 (52, [M + H]⁺), 204

Experimental Section

General Methods and Materials. Melting points were

determined on a Bu¨chi 510 oil bath apparatus and are not

corrected. Thin-layer chromatography was performed on Merck

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7699

(38, [M - CO₂+ H]⁺) (neg.) 306 (32, [M + CH₃COO]^-), 246

(45, [M - H]^-), 138 (100, [M - BnOH - H]^-). Anal.

(C₁₂H₁₃N₃O₃) C, H, N.

N-(Benzyloxycarbonyl)-leucyl-glycine-nitrile (8). In

addition to the General Procedure, the free carboxylic acid

Z-Leu-OH was released from its commercially available

dicyclohexylammonium salt by the following method: Z-Leu-

OH × dicyclohexylamine (1 g, 2.2 mmol) was suspended

in ethyl acetate (20 mL) and shaken with ice-cold 2 N H₂SO₄

(6 mL). The organic phase was separated, the aqueous phase

was diluted with H₂O (10 mL) and extracted with ethyl

acetate (2 × 20 mL). The combined organic layers were

washed with H₂O (10 mL) and brine (10 mL), dried over

Na₂SO₄, and concentrated to dryness under reduced pres-

sure. The oily residue obtained thereby was subjected to

the standard mixed anhydride coupling to obtain a pure

N-(tert-Butoxycarbonyl)-alanyl-glycine-nitrile (3). The

oily residue obtained after removal of ethyl acetate solidified

in the refrigerator, and the solid was suspended in petrol ether

and collected by filtration, yield 72%: mp 92-95 °C (lit.⁶⁰90-

1

92 °C); [R]²⁰) -26.0 (c ) 1.13, MeOH); H NMR (500 MHz,

D

DMSO-d₆) δ 1.16 (d, J ) 7.3 Hz, 3H), 1.37 (s, 9H), 3.95 (m,

1H), 4.10 (d, J ) 5.7 Hz, 2H), 7.00 (d, J ) 7.0 Hz, 1H), 8.46 (t,

J ) 5.4 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 17.87, 27.23,

28.32, 49.68, 78.27, 117.71, 155.24, 173.58; MS (ESI) m/z (rel

intensity) (pos.) 266 (5, [M + K]⁺), 250 (9, [M + Na]⁺), 245

(36, [M + NH₄]⁺), 228 (35, [M + H]⁺), 172 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 128 (68, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.)

286 (20, [M + CH₃COO]^-), 226 (100, [M - H]^-), 152 (79, [M -

tBuOH - H]^-). Anal. (C₁₀H₁₇N₃O₃) C, H, N.

crude product, yield 94%: mp 112-113 °C; [R]²⁰) -22.3

D

(c ) 2.15, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.89 (d,

J ) 6.0 Hz, 3H), 0.91 (d, J ) 6.3 Hz, 3H), 1.49-1.56 (m, 1H),

1.60-1.68 (m, 2H), 4.06 (d, J ) 5.7 Hz, 2H), 4.17-4.27

(m, 1H), 5.06 (d, J ) 12.3 Hz, 1H), 5.10 (d, J ) 12.3 Hz, 1H),

5.38 (d, J ) 8.2 Hz, 2H), 7.21 (br s, 1H), 7.29-7.35 (m, 5H);

¹³C NMR (125 MHz, CDCl₃) δ 21.79, 22.82, 24.62, 27.38,

40.68, 53.20, 67.43, 115.90, 127.96, 128.37, 128.62, 135.83,

156.61, 172.70; MS (ESI) m/z (rel intensity) (pos.) 326 (6,

[M + Na]⁺), 321 (100, [M + NH₄]⁺), 304 (66, [M + H]⁺), 260

(34, [M - CO₂+ H]⁺) (neg.) 362 (67, [M + CH₃COO]^-), 302

(100, [M - H]^-), 194 (71, [M - BnOH - H]^-). Anal.

(C₁₆H₂₁N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-2-aminobutanoyl-glycine-ni-

trile (4). The oily residue obtained after removal of the solvent

crystallized in the refrigerator, and the solid was suspended

in petrol ether and collected by filtration, yield 80%: mp 80-

1

82 °C; [R]²⁰) -27.8 (c ) 2.79, MeOH); H NMR (500 MHz,

D

CDCl₃) δ 0.94 (t, J ) 7.4 Hz, 3H), 1.43 (s, 9H), 1.59-1.70 (m,

1H), 1.80-1.90 (m, 1H), 4.01-4.09 (m, 1H), 4.14 (d, J ) 5.4

Hz, 2H), 5.14 (d, J ) 7.9 Hz, 1H), 7.31 (br s, 1H); ¹³C NMR

(125 MHz, CDCl₃) δ 9.99, 25.33, 27.29, 28.29, 55.51, 80.67,

115.88, 156.09, 172.58; MS (ESI) m/z (rel intensity) (pos.) 280

(4, [M + K]⁺), 264 (54, [M + Na]⁺), 259 (100, [M + NH₄]⁺),

242 (97, [M + H]⁺), 186 (99, [M - (CH₃)₂CCH₂+ H]⁺), 142

(16, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 300 (57, [M +

CH₃COO]^-), 240 (99, [M - H]^-), 166 (100, [M - tBuOH - H]^-).

Anal. (C₁₁H₁₉N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-isoleucyl-glycine-nitrile (9).

The crude product was recrystallized from petrol ether/ethyl

acetate, yield 48%: mp 130-132 °C; [R]²⁰) 22.5 (c ) 1.23,

D

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.88 (t, J ) 7.4 Hz, 3H),

0.92 (d, J ) 6.6 Hz, 3H), 1.06-1.18 (m, 1H), 1.42 (s, 9H), 1.47-

1.57 (m, 1H), 1.79-1.92 (m, 1H), 3.97 (dd, J ) 8.5, 7.3 Hz,

1H), 4.10 (dd, J ) 17.8, 6.0 Hz, 1H), 4.17 (dd, J ) 17.5, 5.9

Hz, 1H), 5.20 (d, J ) 7.9 Hz), 7.33 (br s, 1H); ¹³C NMR (125

MHz, CDCl₃) δ 11.09, 15.48, 24.81, 27.23, 28.30, 36.92, 58.99,

80.52, 115.80, 156.16, 172.44; MS (ESI) m/z (rel intensity)

(pos.) 308 (4, [M + K]⁺), 292 (9, [M + Na]⁺), 287 (57, [M +

NH₄]⁺), 270 (42, [M + H]⁺), 214 (100, [M - (CH₃)₂CCH₂+ H]⁺),

170 (50, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 328 (26, [M +

CH₃COO]^-), 268 (100, [M - H]^-), 194 (87, [M - tBuOH - H]^-).

Anal. (C₁₃H₂₃N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-valyl-glycine-nitrile (5). The

crude product was recrystallized from petrol ether/ethyl

acetate, yield 54%: mp 110-111 °C; [R]²⁰_D) -28.4 (c ) 1.20,

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.93 (d, J ) 7.0 Hz, 3H),

0.95 (d, J ) 6.9 Hz), 1.43 (s, 9H), 2.03-2.16 (m, 1H), 3.92-

3.99 (m, 1H), 4.09 (dd, J ) 17.3, 5.7 Hz, 1H), 4.18 (dd, J )

17.5, 5.8 Hz, 1H), 5.23 (d, J ) 7.9 Hz, 1H), 7.36 (br s, 1H); ¹³

C

NMR (125 MHz, CDCl₃) δ 18.08, 19.18, 27.23, 28.30, 30.74,

59.82, 80.50, 115.82, 155.21, 172.36; MS (ESI) m/z (rel

intensity) (pos.) 294 (4, [M + K]⁺), 278 (8, [M + Na]⁺), 273

(39, [M + NH₄]⁺), 256 (30, [M + H]⁺), 200 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 156 (47, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.)

314 (25, [M + CH₃COO]^-), 254 (100, [M - H]^-), 180 (67, [M -

tBuOH - H]^-). Anal. (C₁₂H₂₁N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-cyclopropylalanyl-glycine-ni-

trile (10). The corresponding free carboxylic acid was released

from its dicyclohexylammonium salt as described in the

preparation of 8. The crude product was recrystallized from

n-hexane/ethyl acetate, yield 72%: mp 124-125 °C (n-hexane/

ethyl acetate); [R]²⁰_D) -17.3 (c ) 2.28, MeOH); ¹H NMR (500

MHz, DMSO-d₆) δ -0.02-0.04 (m, 1H), 0.05-0.12 (m, 1H),

0.30-0.43 (m, 2H), 0.64-0.77 (m, 1H), 1.31-1.39 (m, 1H), 1.37

(s, 9H), 1.48-1.60 (m, 1H), 3.91-4.00 (m, 1H), 4.05-4.16 (m,

2H), 6.94 (d, J ) 7.9 Hz, 1H), 8.52 (t, J ) 5.7 Hz, 1H); ¹³C

NMR (125 MHz, DMSO-d₆) δ 4.05, 4.60, 7.87, 27.16, 28.37,

36.58, 54.85, 78.21, 117.62, 155.46, 172.94; MS (ESI) m/z (rel

intensity) (pos.) 306 (3, [M + K]⁺), 290 (10, [M + Na]⁺), 285

(41, [M + NH₄]⁺), 268 (38, [M + H]⁺), 212 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 168 (32, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

326 (19, [M + CH₃COO]^-), 266 (100, [M - H]^-), 192 (89, [M -

tBuOH - H]^-). Anal. (C₁₃H₂₁N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-norvalyl-glycine-nitrile (6).

The crude product was recrystallized from petrol ether/ethyl

acetate, yield 44%: mp 120-123 °C; [R]²⁰_D) -23.1 (c ) 1.87,

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.92 (t, J ) 7.4 Hz, 3H),

1.29-1.42 (m, 2H), 1.43 (s, 9H), 1.53-1.62 (m, 1H), 1.74-1.82

(m, 1H), 4.05-4.19 (m, 3H), 5.10 (d, J ) 7.3 Hz, 1H), 7.28 (br

s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 13.64, 18.86, 27.31, 28.28,

33.99, 54.10, 80.69, 115.88, 156.09, 172.77; MS (ESI) m/z (rel

intensity) (pos.) 294 (4, [M + K]⁺), 278 (61, [M + Na]⁺), 273

(93, [M + NH₄]⁺), 256 (100, [M + H]⁺), 200 (94, [M - (CH₃)₂-

CCH₂+ H]⁺), 156 (25, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

314 (62, [M + CH₃COO]^-), 254 (96, [M - H]^-), 180 (100, [M -

tBuOH - H]^-). Anal.(C₁₂H₂₁N₃O₃) C, H; N: calcd 16.46, found

15.94.

N-(tert-Butoxycarbonyl)-leucyl-glycine-nitrile (7). The

oily residue solidified in the refrigerator. It was suspended in

petrol ether and collected by filtration, yield 92%: mp 114-

116 °C; [R]²⁰_D) -23.4 (c ) 2.11, MeOH); ¹H NMR (500 MHz,

CDCl₃) δ 0.90 (d, J ) 6.3 Hz, 3H), 0.93 (d, J ) 6.6 Hz, 3H),

1.43 (s, 9H), 1.46-1.56 (m, 1H), 1.59-1.71 (m, 2H), 4.13 (d, J

) 5.7 Hz, 2H), 4.13-4.21 (m, 1H), 5.08 (d, J ) 4.1 Hz, 1H),

7.41 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 21.77, 22.87,

24.67, 27.33, 28.28, 40.59, 52.73, 80.70, 115.91, 156.17, 173.17;

MS (ESI) m/z (rel intensity) (pos.) 292 (6, [M + Na]⁺), 287 (100,

[M + NH₄]⁺), 270 (60, [M + H]⁺), 214 (100, [M - (CH₃)₂CCH₂

+ H]⁺), 170 (21, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 328

(48, [M + CH₃COO]^-), 268 (100, [M - H]^-), 194 (73, [M -

tBuOH - H]^-). Anal. (C₁₃H₂₃N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-cyclohexylalanyl-glycine-ni-

trile. (11) The corresponding free carboxylic acid was released

from its dicyclohexylammonium salt as described in the

preparation of 8. Recrystallization was done from petrol ether/

ethyl acetate, yield 53%: mp 81-83 °C; [R]²⁰) -12.8 (c )

D

1.08, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.80-1.01 (m, 3H),

1.06-1.27 (m, 3H), 1.43 (s, 9H), 1.53-1.77 (m, 7H), 4.13 (d, J

) 5.4 Hz, 2H), 4.15-4.23 (m, 1H), 5.01 (d, J ) 7.9 Hz, 1H),

7.31 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 25.97, 26.15,

26.31, 27.32, 28.28, 32.47, 33.58, 33.98, 39.18, 52.08, 80.75,

115.88, 156.17, 173.16; MS (ESI) m/z (rel intensity) (pos.) 348

(6, [M + K]⁺), 332 (8, [M + Na]⁺), 327 (46, [M + NH₄]⁺), 310

(42, [M + H]⁺) 254 (100, [M - (CH₃)₂CCH₂+ H]⁺), 210 (65,

[M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 368 (29, [M +

CH₃COO]^-), 308 (100, [M - H]^-), 234 (50, [M - tBuOH - H]^-).

Anal. (C₁₆H₂₇N₃O₃) C, H, N.

7700 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

N-(tert-Butoxycarbonyl)-methionyl-glycine-nitrile (12).

This compound was obtained as a pure crude product, yield

91%: mp 98-99 °C; [R]²⁰_D) -22.2 (c ) 1.50, MeOH); ¹H NMR

(500 MHz, DMSO-d₆) δ 1.37 (s, 9H), 1.72-1.88 (m, 2H), 2.02

(s, 3H), 2.36-2.48 (m, 2H), 3.96-4.06 (m, 1H), 4.10 (d, J )

5.7 Hz, 2H), 7.06 (d, J ) 7.9 Hz, 1H), 8.52 (t, J ) 5.5 Hz, 1H);

¹³C NMR (125 MHz, DMSO-d₆) δ 14.74, 27.28, 28.30, 29.31,

31.39, 53.41, 78.39, 117.66, 155.55, 172.65; MS (ESI) m/z (rel

intensity) (pos.) 326 (6, [M + K]⁺), 310 (13, [M + Na]⁺), 305

(50, [M + NH₄]⁺), 288 (57, [M + H]⁺), 232 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 188 (57, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

346 (14, [M + CH₃COO]^-), 286 (100, [M - H]^-), 212 (82, [M -

tBuOH - H]^-). Anal. (C₁₂H₂₁N₃O₃S) C, H, N.

N-(tert-Butoxycarbonyl)-norleucyl-glycine-nitrile (13).

The corresponding free carboxylic acid was released from its

dicyclohexylammonium salt as described in the preparation

of 8. The product was obtained as colorless oil, which solidified

in the refrigerator; it was suspended in petrol ether and

collected by filtration, yield 82%: mp 67-75 °C; [R]²⁰_D) -17.1

(c ) 2.11, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 0.85-0.90

(m, 3H), 1.25-1.36 (m, 4H), 1.42 (s, 9H), 1.53-1.64 (m, 1H),

1.74-1.85 (m, 1H), 4.05-4.20 (m, 3H), 5.17 (d, J ) 7.9 Hz,

1H), 7.39 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 13.81, 22.28,

27.30, 27.67, 28.28, 31.78, 54.29, 80.63, 115.90, 156.09, 172.87;

MS (ESI) m/z (rel intensity) (pos.) 308 (4, [M + K]⁺), 292 (10,

[M + Na]⁺), 287 (43, [M + NH₄]⁺), 270 (35, [M + H]⁺), 214

(100, [M - (CH₃)₂CCH₂+ H]⁺), 170 (52, [M - (CH₃)₂CCH₂-

CO₂+ H]⁺) (neg.) 328 (24, [M + CH₃COO]^-), 268 (100, [M -

H]^-), 194 (56, [M - tBuOH - H]^-). Anal. (C₁₃H₂₃N₃O₃) C, H,

N: calcd 15.60; found 15.06.

N-(tert-Butoxycarbonyl)-prolyl-glycine-nitrile (14). The

crude product was recrystallized from petrol ether/ethyl

acetate, yield 39%: mp 139-140 °C; [R]²⁰_D) -49.2 (c ) 1.39,

MeOH); ¹H NMR (500 MHz, DMSO-d₆) δ 1.33 & 1.39 (s, 9H),

1.68-1.85 (m, 3H), 2.02-2.19 (m, 1H), 3.23-3.33 (m, 1H),

3.35-3.41 (m, 1H), 4.00-4.05 (m, 1H), 4.05-4.16 (m, 2H),

8.49-8.54 & 8.54-8.61 (m, 1H); ¹³C NMR (125 MHz, DMSO-

d₆) δ 23.35 (i) & 24.03 (w), 27.13, 28.06 (i) & 28.26 (w), 30.79

(i) & 30.98 (w), 46.54 (i) & 46.78 (w), 59.52 (w) & 59.77 (i),

78.86 (i) & 78.90 (w), 117.61 (i) & 117.67 (w), 153.27 (i) &

153.80 (w), 172.98 (w) & 173.39 (i); MS (ESI) m/z (rel intensity)

(pos.) 292 (6, [M + K]⁺), 276 (8, [M + Na]⁺), 271 (29, [M +

NH₄]⁺), 254 (48, [M + H]⁺), 198 (100, [M - (CH₃)₂CCH₂+ H]⁺),

154 (87, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 312 (28, [M +

CH₃COO]^-), 252 (100, [M - H]^-), 178 (47, [M - tBuOH - H]^-).

Anal. (C₁₂H₁₉N₃O₃) C, H, N.

- CO₂+ H]⁺) (neg.) 362 (33, [M + CH₃COO]^-), 302 (100, [M

- H]^-), 228 (83, [M - tBuOH - H]^-). Anal. (C₁₆H₂₁N₃O₃) C,

H, N.

N-(Benzyloxycarbonyl)-phenylalanyl-glycine-nitrile

(17). The crude product was recrystallized from petrol ether/

ethyl acetate, yield 72%: mp 139-141 °C; [R]²⁰_D) -12.9 (c )

2.07, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 3.01-3.09 (m, 2H),

3.99 (d, J ) 5.7 Hz, 2H), 4.46 (m, 1H), 5.04 (s, 2H), 5.61 (d, J

) 6.7 Hz, 1H) 7.12-7.18 (m, 3H), 7.21-7.37 (m, 8H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.24, 38.29, 55.96, 67.21, 115.64, 127.17,

127.91, 128.26, 128.55, 128.77, 129.22, 135.85, 135.88, 156.13,

171.57; MS (ESI) m/z (rel intensity) (pos.) 360 (12, [M + Na]⁺),

355 (100, [M + NH₄]⁺), 338 (50, [M + H]⁺), 294 (62, [M - CO₂

+ H]⁺), (neg.) 396 (25, [M + CH₃COO]^-), 336 (100, [M - H]^-),

228 (51, [M - BnOH - H]^-). Anal. (C₁₉H₁₉N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-2′-furylalanyl-glycine-ni-

trile (18). The commercially available tert-butylammonium

salt of the corresponding protected amino acid was employed

directly in the mixed anhydride procedure. Therefore, this

reaction was performed without N-methylmorpholine. Frac-

tionation of the crude material on silica gel using petrol ether/

ethyl acetate (2:1) as eluent led to a colorless oil, which

crystallized in the refrigerator overnight, yield 31%: mp 112-

113 °C; [R]²⁰_D) -10.3 (c ) 1.07, MeOH); ¹H NMR (500 MHz,

CDCl₃) δ 1.41 (s, 9H), 3.05-3.17 (m, 2H), 4.07 (dd, J ) 17.2,

5.2 Hz, 1H), 4.14 (dd, J ) 17.4, 6.0 Hz, 1H), 4.42 (br s, 1H),

5.17 (d, J ) 7.6 Hz, 1H), 6.12 (d, J ) 3.2 Hz, 1H), 6.28 (dd, J

) 3.0, 2.1 Hz, 1H), 6.93 (br s, 1H), 7.31-7.34 (m, 1H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.36, 28.24, 30.44, 53.62, 80.98, 108.21,

110.57, 115.57, 142.23, 150.18, 155.68, 171.43; MS (ESI) m/z

(rel intensity) (pos.) 332 (6, [M + K]⁺), 316 (10, [M + Na]⁺),

311 (36, [M + NH₄]⁺), 294 (42, [M + H]⁺), 238 (100, [M -

(CH₃)₂CCH₂+ H]⁺), 194 (90, [M - (CH₃)₂CCH₂- CO₂+ H]⁺)

(neg.) 352 (13, [M + CH₃COO]^-), 292 (100, [M - H]^-), 218 (76,

[M - tBuOH - H]^-). Anal. (C₁₄H₁₉N₃O₄) C: calcd 57.33, found

58.18; H: calcd 6.53, found 6.94; N: calcd 14.33, found 13.14.

N-(tert-Butoxycarbonyl)-2′-thienylalanyl-glycine-ni-

trile (19). Pure product was obtained by recrystallization from

n-hexane/ethyl acetate, yield 61%: mp 117-119 °C; [R]²⁰

)

D

1

-5.0 (c ) 1.21, MeOH); H NMR (500 MHz, CDCl₃) δ 1.42 (s,

9H), 3.27 (dd, J ) 15.0, 6.5 Hz, 1H), 3.32 (dd, J ) 15.0, 6.5

Hz, 1H), 4.07 (dd, J ) 17.5, 5.2 Hz, 1H), 4.15 (dd, J ) 17.4,

6.0 Hz, 1H), 4.37 (m, 1H), 5.05 (d, J ) 5.4 Hz, 1H), 6.82 (br s,

1H), 6.85 (dd, J ) 3.5, 0.95 Hz, 1H), 6.93 (dd, J ) 5.1, 3.5 Hz,

1H), 7.17 (dd, J ) 5.2, 1.1 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃)

δ 27.39, 28.25, 31.91, 55.38, 81.11, 115.49, 125.08, 127.02,

127.25, 137.60, 155.68, 171.33; MS (ESI) m/z (rel intensity)

(pos.) 348 (6, [M + K]⁺), 332 (15, [M + Na]⁺), 327 (52, [M +

NH₄]⁺), 310 (40, [M + H]⁺), 254 (100, [M - (CH₃)₂CCH₂+ H]⁺),

210 (82, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 368 (17, [M +

CH₃COO]^-), 308 (100, [M - H]^-), 234 (70, [M - tBuOH - H]^-).

Anal. (C₁₄H₁₉N₃O₃S) C, H, N.

N-(tert-Butoxycarbonyl)-phenylglycyl-glycine-ni-

trile (15). Recrystallization of the crude product was done from

petrol ether/ethyl acetate, yield 66%: mp 176-177 °C; [R]²⁰

D

) 96.5 (c ) 2.01, MeOH); ¹H NMR (500 MHz, DMSO-d₆) δ

1.37 (s, 9H), 4.13 (d, J ) 5.7 Hz, 2H), 5.17 (d, J ) 8.2 Hz, 1H),

7.26-7.30 (m, 1H), 7.31-7.35 (m, 2H), 7.35-7.39 (m, 2H), 7.42

(d, J ) 6.6 Hz, 1H), 8.80 (t, J ) 5.5 Hz, 1H); ¹³C NMR (125

MHz, DMSO-d₆) δ 27.30, 28.27, 57.81, 78.65, 117.48, 127.53,

127.89, 128.46, 138.09, 155.11, 171.02; MS (ESI) m/z (rel

intensity) (pos.) 312 (30, [M + Na]⁺), 307 (99, [M + NH₄]⁺),

290 (100, [M + H]⁺), 234 (93, [M - (CH₃)₂CCH₂+ H]⁺), 190

(70, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 348 (22, [M +

CH₃COO]^-), 288 (99, [M - H]^-), 214 (100, [M - tBuOH - H]^-).

Anal. (C₁₅H₁₉N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-3′-thienylalanyl-glycine-ni-

trile (20). Pure product was obtained by recrystallization from

n-hexane/ethyl acetate, yield 42%: mp 137-139 °C; [R]²⁰

)

D

1

-1.3 (c ) 1.06, MeOH); H NMR (500 MHz, CDCl₃) δ 1.40 (s,

9H), 3.04-3.15 (m, 2H), 4.06 (dd, J ) 17.4, 5.3 Hz, 1H), 4.12

(dd, J ) 17.2, 5.9 Hz, 1H), 4.28-4.42 (m, 1H), 5.02 (d, J ) 6.6

Hz, 1H), 6.78 (br s, 1H), 6.92 (dd, J ) 4.9, 1.2 Hz, 1H), 7.02-

7.05 (m, 1H), 7.28 (dd, J ) 5.0, 2.9 Hz, 1H); ¹³C NMR (125

MHz, CDCl₃) δ 27.36, 28.25, 32.37, 54.89, 80.98, 115.54, 123.02,

126.50, 128.22, 136.12, 155.72, 171.78; MS (ESI) m/z (rel

intensity) (pos.) 348 (6, [M + K]⁺), 332 (13, [M + Na]⁺), 327

(47, [M + NH₄]⁺), 310 (41, [M + H]⁺), 254 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 210 (84, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

368 (14, [M + CH₃COO]^-), 308 (100, [M - H]^-), 234 (87, [M -

tBuOH - H]^-). Anal. (C₁₄H₁₉N₃O₃S) C, H, N.

N-(tert-Butoxycarbonyl)-phenylalanyl-glycine-ni-

trile (16). The crude product was recrystallized from cyclo-

hexane/ethyl acetate, yield 59%: mp 136-138 °C (lit.⁶⁰mp

136.5-137.5 °C); [R]²⁰_D) 1.2 (c ) 1.10, MeOH); ¹H NMR (500

MHz, CDCl₃) δ 1.37 (s, 9H), 2.98-3.08 (m, 2H), 4.05 (d, J )

5.8 Hz, 2H), 4.30-4.45 (m, 1H), 5.16 (d, J ) 7.3 Hz, 1H), 6.94

(br s, 1H) 7.13-7.18 (m, 2H), 7.20-7.25 (m, 1H), 7.26-7.31

(m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 27.27, 28.22, 38.20,

55.63, 80.74, 115.57, 127.16, 128.80, 129.21, 136.04, 155.73,

171.92; MS (ESI) m/z (rel intensity) (pos.) 342 (3, [M + K]⁺),

326 (21, [M + Na]⁺), 321 (82, [M + NH₄]⁺), 304 (59, [M + H]⁺),

248 (100, [M - (CH₃)₂CCH₂+ H]⁺), 204 (68, [M - (CH₃)₂CCH₂

N-(tert-Butoxycarbonyl)-homophenylalanyl-glycine-

nitrile (21). The oily crude product crystallized in the

refrigerator; it was suspended in Et₂O and collected by

filtration, yield 47%: mp 95-98 °C; [R]²⁰) -21.0 (c ) 1.12,

D

dioxane); ¹H NMR (500 MHz, CDCl₃) δ 1.44 (s, 9H), 1.87-

1.97 (m, 1H), 2.09-2.19 (m, 1H), 2.61-2.74 (m, 2H), 4.04-

4.16 (m, 3H), 5.13 (br s, 1H), 7.12-7.21 (m, 4H), 7.24-7.28

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7701

(m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 27.33, 28.29, 31.76,

33.23, 53.70, 80.81, 115.80, 126.30, 128.36, 128.58, 140.34,

156.06, 172.42; MS (ESI) m/z (rel intensity) (pos.) 356 (7, [M

+ K]⁺), 340 (37, [M + Na]⁺), 335 (100, [M + NH₄]⁺), 318 (75,

[M + H]⁺), 262 (88, [M - (CH₃)₂CCH₂+ H]⁺), 218 (53, [M -

(CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 376 (41, [M + CH₃COO]^-),

316 (96, [M - H]^-), 242 (100, [M - tBuOH - H]^-); Anal.

(C₁₇H₂₃N₃O₃) C, H, N.

(500 MHz, CDCl₃) δ 1.35 (s, 9H), 2.97-3.08 (m, 1H), 3.16 (dd,

J ) 13.0, 5.1 Hz, 1H), 4.04-4.15 (m, 2H), 4.33-4.45 (m, 1H),

5.12 (d, J ) 7.9 Hz, 1H), 6.97 (br s, 1H), 6.99-7.09 (m, 2H),

7.16-7.25 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 27.32, 28.17,

31.35, 54.70, 80.83, 115.42 (d, J ) 22.1 Hz), 115.54, 123.20 (d,

J ) 15.4 Hz), 124.47 (d, J ) 3.5 Hz), 129.06 (d, J ) 8.2 Hz),

131.69 (d, J ) 4.5 Hz), 155.75, 161.27 (d, J ) 243.4 Hz), 171.57;

MS (ESI) m/z (rel intensity) (pos.) 360 (5, [M + K]⁺), 344 (10,

[M + Na]⁺), 339 (40, [M + NH₄]⁺), 322 (35, [M + H]⁺), 266

(100, [M - (CH₃)₂CCH₂+ H]⁺), 222 (73, [M - (CH₃)₂CCH₂-

CO₂+ H]⁺) (neg.) 380 (15, [M + CH₃COO]^-), 320 (100, [M -

H]^-), 246 (70, [M - tBuOH - H]^-). Anal. (C₁₆H₂₀N₃O₃F) C, H,

N.

N-(tert-Butoxycarbonyl)-pentafluorophenylalanyl-gly-

cine-nitrile (27). The obtained semisolid crude material was

purified by fractionation on silica gel using petrol ether/ethyl

acetate (2:1) as eluent to yield a solid residue, which was

recrystallized from n-hexane/ethyl acetate, yield 12%: mp

139-140 °C (n-hexane/ethyl acetate); [R]²⁰_D) -12.7 (c ) 1.05,

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.36 (s, 9H), 3.03 (dd, J

) 14.5, 9.5 Hz, 1H), 3.27 (dd, J ) 14.4, 4.3 Hz, 1H), 4.11 (dd,

J ) 17.5, 5.5 Hz, 1H), 4.20 (dd, J ) 17.4, 6.0 Hz, 1H), 4.36-

4.48 (m, 1H), 5.16 (d, J ) 8.8 Hz, 1H), 7.15 (br s, 1H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.47, 28.05, 53.32, 81.35, 109.80-110.40

(m), 115.48, 137.46 (dm, J ) 249.8 Hz), 140.44 (dm, J ) 251.5

Hz), 145.46 (dm, J ) 244.1 Hz), 155.76, 170.59; MS (ESI) m/z

(rel intensity) (pos.) 432 (7, [M + K]⁺), 416 (19, [M + Na]⁺),

411 (82, [M + NH₄]⁺), 394 (24, [M + H]⁺), 338 (100, [M -

(CH₃)₂CCH₂+ H]⁺), 294 (64, [M - (CH₃)₂CCH₂- CO₂+ H]⁺)

(neg.) 392 (100, [M - H]^-), 372 (22, [M - HF - H]^-), 318 (25,

[M - tBuOH - H]^-), 298 (32, [M - tBuOH - HF - H]^-). Anal.

(C₁₆H₁₆N₃O₃F₅) C, H; N: calcd 10.68, found 10.26.

N-(tert-Butoxycarbonyl)-O-benzylseryl-glycine-ni-

trile (22). The oily crude product crystallized in the refrigera-

tor; it was suspended in petrol ether and collected by filtration,

1

yield 83%: mp 81-85 °C; [R]²⁰) 3.2 (c ) 1.11, MeOH); H

D

NMR (500 MHz, CDCl₃) δ 1.43 (s, 9H), 3.57 (dd, J ) 9.5, 6.3

Hz, 1H), 3.86-3.91 (m, 1H), 4.06-4.20 (m, 2H), 4.30 (br s, 1H),

4.49 (d, J ) 11.7 Hz, 1H), 4.55 (d, J ) 12.0 Hz, 1H), 5.34 (br

s, 1H), 7.02 (t, J ) 5.7 Hz, 1H), 7.26-7.31 (m, 3H), 7.32-7.37

(m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 27.43, 28.24, 53.92,

69.28, 73.60, 80.78, 115.64, 127.88, 128.14, 128.61, 137.06,

155.45, 170.82; MS (ESI) m/z (rel intensity) (pos.) 372 (6, [M

+ K]⁺), 356 (13, [M + Na]⁺), 351 (43, [M + NH₄]⁺), 334 (35,

[M + H]⁺), 278 (100, [M - (CH₃)₂CCH₂+ H]⁺), 234 (36, [M -

(CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 392 (14, [M + CH₃COO]^-)

332 (100, [M - H]^-), 258 (90, [M - tBuOH - H]^-). Anal.

(C₁₇H₂₃N₃O₄) C, H, N.

N-(tert-Butoxycarbonyl)-tyrosyl-glycine-nitrile (23).

The crude product was recrystallized from cyclohexane/ethyl

acetate, yield 80%: mp 166-168 °C; [R]²⁰) 6.8 (c ) 1.16,

D

MeOH); ¹H NMR (500 MHz, DMSO-d₆) δ 1.30 (s, 9H), 2.63

(dd, J ) 13.7, 9.6 Hz, 1H), 2.82 (dd, J ) 13.7, 4.9 Hz, 1H),

4.03-4.07 (m, 1H), 4.10 (d, J ) 5.7 Hz, 2H), 6.63 (d, J ) 8.2

Hz, 2H), 6.90 (d, J ) 8.5 Hz, 1H), 7.00 (d, J ) 8.2 Hz, 2H),

8.55 (t, J ) 5.35 Hz, 1H), 9.12 (s, 1H); ¹³C NMR (125 MHz,

DMSO-d₆) δ 27.21, 28.28, 36.58, 56.03, 78.24, 115.02, 117.59,

127.94, 130.17, 155.37, 155.94, 172.63; MS (ESI) m/z (rel

intensity) (pos.) 358 (7, [M + K]⁺), 342 (13, [M + Na]⁺), 337

(68, [M + NH₄]⁺), 320 (48, [M + H]⁺), 264 (100, [M - (CH₃)₂-

CCH₂+ H]⁺), 220 (34, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

378 (50, [M + CH₃COO]^-), 318 (100, [M - H]^-), 244 (51, [M -

tBuOH - H]^-). Anal. (C₁₆H₂₁N₃O₄) C, H, N.

N-(tert-Butoxycarbonyl)-4′-bromophenylalanyl-glycine-

nitrile (28). This compound was obtained as a pure crude

product, yield 99%: mp 139-140 °C; [R]²⁰) 3.3 (c ) 2.17,

D

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.38 (s, 9H), 2.95 (dd, J

) 14.1, 7.1 Hz, 1H), 3.04 (dd, J ) 13.9, 6.6 Hz, 1H), 4.05 (dd,

J ) 17.5, 5.8 Hz, 1H), 4.10 (dd, J ) 17.7, 5.7 Hz, 1H), 4.29-

4.37 (m, 1H), 5.06 (d, J ) 8.5 Hz, 1H), 6.89 (br s, 1H), 7.04 (d,

J ) 8.2 Hz, 2H), 7.41 (d, J ) 8.5 Hz, 2H); ¹³C NMR (125 MHz,

CDCl₃) δ 27.35, 28.21, 37.39, 55.36, 81.07, 115.48, 121.21,

130.98, 131.90, 135.04, 155.69, 171.57; MS (ESI) m/z (rel

intensity) (pos.) 406 (45, [M + Na]⁺, ⁸¹Br), 404 (45, [M + Na]⁺,

⁷⁹Br), 401 (99, [M + NH₄]⁺, ⁸¹Br), 399 (100, [M + NH₄]⁺, ⁷⁹Br),

384 (34, [M + H]⁺, ⁸¹Br), 382 (34, [M + H]⁺, ⁷⁹Br), 328 (34, [M

- (CH₃)₂CCH₂+ H]⁺, ⁸¹Br), 326 (35, [M - (CH₃)₂CCH₂+ H]⁺,

⁷⁹Br), 284 (6, [M - (CH₃)₂CCH₂- CO₂+ H]⁺, ⁸¹Br), 282 (6, [M

- (CH₃)₂CCH₂- CO₂+ H]⁺, ⁷⁹Br) (neg.) 382 (95, [M - H]^-,

⁸¹Br), 380 (100, [M - H]^-, ⁷⁹Br), 308 (43, [M - tBuOH - H]^-,

⁸¹Br), 306 (43, [M - tBuOH - H]^-, ⁷⁹Br). Anal. (C₁₆H₂₀N₃O₃-

Br) C, H; N: calcd 10.99, found 10.36.

N-(tert-Butoxycarbonyl)-4′-fluorophenylalanyl-glycine-

nitrile (24). Recrystallization of the crude product was done

from n-hexane/ethyl acetate, yield 38%: mp 140 °C; [R]²⁰

)

D

1

0.1 (c ) 1.15, MeOH); H NMR (500 MHz, CDCl₃) δ 1.38 (s,

9H), 2.97 (dd, J ) 14.0, 7.1 Hz, 1H), 3.06 (dd, J ) 14.0, 6.9,

1H), 4.01-4.13 (m, 2H), 4.27-4.38 (m, 1H), 5.08 (d, J ) 8.2

Hz, 1H), 6.89 (br s, 1H), 6.93-7.01 (m, 2H), 7.09-7.16 (m, 2H);

¹³C NMR (125 MHz, CDCl₃) δ 27.32, 28.21, 37.23, 55.61, 80.97,

115.51, 115.67 (d, J ) 21.1 Hz), 130.78 (d, J ) 8.2 Hz), 131.72

(d, J ) 3.5 Hz), 155.70, 162.01 (d, J ) 244.4 Hz), 171.67; MS

(ESI) m/z (rel intensity) (pos.) 360 (7, [M + K]⁺), 344 (14, [M

+ Na]⁺), 339 (45, [M + NH₄]⁺), 322 (31, [M + H]⁺), 266 (100,

[M - (CH₃)₂CCH₂+ H]⁺), 222 (63, [M - (CH₃)₂CCH₂- CO₂+

H]⁺) (neg.) 380 (10, [M + CH₃COO]^-), 320 (100, [M - H]^-),

246 (88, [M - tBuOH - H]^-). Anal. (C₁₆H₂₀N₃O₃F) C, H, N.

N-(tert-Butoxycarbonyl)-3′-bromophenylalanyl-glycine-

nitrile (29). This compound was obtained as a pure crude

product, yield 99%: mp 139 °C; [R]²⁰_D) 4.7 (c ) 1.71, MeOH);

¹H NMR (500 MHz, CDCl₃) δ 1.39 (s, 9H), 2.95 (dd, J ) 13.8,

7.5 Hz, 1H), 3.08 (dd, J ) 13.7, 6.5 Hz, 1H), 4.07 (dd, J ) 17.8,

6.5 Hz, 1H), 4.12 (dd, J ) 17.5, 5.9 Hz, 1H), 4.30-4.40 (m,

1H), 5.10 (d, J ) 8.2 Hz, 1H), 6.97 (br s, 1H), 7.10 (d, J ) 7.6

Hz, 1H), 7.14-7.19 (m, 1H), 7.33 (s, 1H), 7.37 (d, J ) 7.9 Hz,

1H); ¹³C NMR (125 MHz, CDCl₃) δ 27.36, 28.23, 37.59, 55.43,

81.08, 115.50, 122.72, 127.93, 130.32, 130.35, 132.23, 138.53,

155.73, 171.51; MS (ESI) m/z (rel intensity) (pos.) 406 (38, [M

+ Na]⁺, ⁸¹Br), 404 (38, [M + Na]⁺, ⁷⁹Br), 401 (99, [M + NH₄]⁺,

⁸¹Br), 399 (100, [M + NH₄]⁺, ⁷⁹Br), 384 (28, [M + H]⁺, ⁸¹Br),

382 (29, [M + H]⁺, ⁷⁹Br), 328 (26, [M - (CH₃)₂CCH₂+ H]⁺,

⁸¹Br), 326 (27, [M - (CH₃)₂CCH₂+ H]⁺, ⁷⁹Br), 284 (4, [M -

(CH₃)₂CCH₂- CO₂+ H]⁺, ⁸¹Br), 282 (4, [M - (CH₃)₂CCH₂-

CO₂+ H]⁺, ⁷⁹Br) (neg.) 382 (95, [M - H]^-, ⁸¹Br), 380 (100, [M

- H]^-, ⁷⁹Br), 308 (44, [M - tBuOH - H]^-, ⁸¹Br), 306 (45, [M -

tBuOH - H]^-, ⁷⁹Br). Anal. (C₁₆H₂₀N₃O₃Br) C, H; N: calcd

10.99, found 10.47.

N-(tert-Butoxycarbonyl)-3′-fluorophenylalanyl-glycine-

nitrile (25). Recrystallization of the crude product was done

from n-hexane/ethyl acetate, yield 64%: mp 150 °C; [R]²⁰

)

D

1

-3.0 (c ) 1.25, MeOH); H NMR (500 MHz, CDCl₃) δ 1.38 (s,

9H), 2.99 (dd, J ) 13.9, 7.6 Hz, 1H), 3.09 (dd, J ) 13.7, 6.5

Hz, 1H), 4.02-4.15 (m, 2H), 4.31-4.42 (m, 1H), 5.09 (d, J )

8.2 Hz, 1H), 6.86-6.99 (m, 4H), 7.22-7.29 (m, 1H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.33, 28.20, 37.65, 55.39, 81.07, 114.16

(d, J ) 20.8 Hz), 115.51, 116.14 (d, J ) 21.1 Hz), 124.92 (d, J

) 2.7 Hz), 130.32 (d, J ) 8.2 Hz), 138.61 (d, J ) 7.4 Hz), 155.75,

162.89 (d, J ) 245.1 Hz), 171.58; MS (ESI) m/z (rel intensity)

(pos.) 360 (6, [M + K]⁺), 344 (13, [M + Na]⁺), 339 (48, [M +

NH₄]⁺), 322 (30, [M + H]⁺), 266 (100, [M - (CH₃)₂CCH₂+ H]⁺),

222 (64, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 380 (10, [M +

CH₃COO]^-), 320 (100, [M - H]^-), 246 (84, [M - tBuOH - H]^-).

Anal. (C₁₆H₂₀N₃O₃F) C, H, N.

N-(tert-Butoxycarbonyl)-2′-fluorophenylalanyl-glycine-

nitrile (26). Recrystallization of the crude product was done

from n-hexane/ethyl acetate, yield 42%: mp 137-139 °C (n-

hexane/ethyl acetate); [R]²⁰_D) -4.8 (c ) 1.30, MeOH); ¹H NMR

N-(tert-Butoxycarbonyl)-2′-bromophenylalanyl-glycine-

nitrile (30). The crude product was recrystallized from petrol

ether/ethyl acetate, yield 50%: mp 141-143 °C (petrol ether/

7702 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

1

ethyl acetate); [R]²⁰) -8.8 (c ) 1.17, MeOH); H NMR (500

27.30, 28.19, 38.19, 55.70, 80.95, 115.43, 125.93, 126.34,

127.16, 127.64, 127.66, 128.04, 128.66, 132.50, 133.46, 133.50,

155.69, 171.78; MS (ESI) m/z (rel intensity) 392 (3, [M + K]⁺),

376 (47, [M + Na]⁺), 371 (100, [M + NH₄]⁺), 354 (34, [M +

H]⁺), 298 (47, [M - (CH₃)₂CCH₂+ H]⁺), 254 (5, [M - (CH₃)₂-

CCH₂- CO₂+ H]⁺) (neg.) 412 (5, [M + CH₃COO]^-), 352 (100,

[M - H]^-), 278 (64, [M - tBuOH - H]^-). Anal. (C₂₀H₂₃N₃O₃)

C, H, N.

D

MHz, CDCl₃) δ 1.34 (s, 9H), 3.03-3.18 (m, 1H), 3.22-3.36 (m,

1H), 4.07 (dd, J ) 17.7, 4.8 Hz, 1H), 4.13 (dd, J ) 17.3, 6.0

Hz, 1H), 4.43-4.50 (m, 1H), 5.13 (d, J ) 8.5 Hz, 1H), 6.83 (br

s, 1H), 7.08-7.13 (m, 1H), 7.21-7.26 (m, 2H), 7.53 (d, J ) 8.2

Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 27.38, 28.19, 37.90,

54.51, 80.84, 115.50, 124.79, 127.86, 128.92, 131.64, 133.02,

135.89, 155.69, 171.51; MS (ESI) m/z (rel intensity) (pos.) 406

(31, [M + Na]⁺, ⁸¹Br), 404 (31, [M + Na]⁺, ⁷⁹Br), 401 (99, [M +

NH₄]⁺, ⁸¹Br), 399 (100, [M + NH₄]⁺, ⁷⁹Br), 384 (52, [M + H]⁺,

⁸¹Br), 382 (53, [M + H]⁺, ⁷⁹Br), 328 (39, [M - (CH₃)₂CCH₂+

H]⁺, ⁸¹Br), 326 (40, [M - (CH₃)₂CCH₂+ H]⁺, ⁷⁹Br), 284 (8, [M

- (CH₃)₂CCH₂- CO₂+ H]⁺, ⁸¹Br), 282 (8, [M - (CH₃)₂CCH₂

- CO₂+ H]⁺, ⁷⁹Br) (neg.) 382 (96, [M - H]^-, ⁸¹Br), 380 (100,

[M - H]^-, ⁷⁹Br), 308 (42, [M - tBuOH - H]^-, ⁸¹Br), 306 (42,

[M - tBuOH - H]^-, ⁷⁹Br). Anal. (C₁₆H₂₀N₃O₃Br) C, H, N.

N-(tert-Butoxycarbonyl)-tryptophanyl-glycine-ni-

trile (31). The foamy crude product crystallized upon dissolv-

ing in cyclohexane und subsequent addition of ethyl acetate,

yield 90%: mp 135-138 °C; [R]²⁰_D) 0.7 (c ) 1.20, MeOH); ¹H

NMR (500 MHz, CDCl₃) δ 1.40 (s, 9H), 3.15 (dd, J ) 13.1, 7.4

Hz, 1H), 3.22-3.35 (m, 1H), 3.88 (dd, J ) 17.3, 5.7 Hz, 1H),

3.95 (dd, J ) 17.3, 4.7 Hz, 1H), 4.44 (br s, 1H), 5.16 (br s, 1H),

6.40 (t, J ) 5.8 Hz, 1H), 7.02 (d, J ) 2.2 Hz, 1H), 7.08-7.13

(m, 1H), 7.16-7.21 (m, 1H), 7.34 (d, J ) 7.9 Hz, 1H), 7.58 (d,

J ) 7.9 Hz, 1H), 8.29 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ

27.15, 28.18, 28.25, 55.08, 80.64, 109.76, 111.41, 115.60,

118.53, 119.86, 122.38, 123.57, 127.19, 136.23, 155.59, 172.27;

MS (ESI) m/z (rel intensity) (pos.) 381 (8, [M + K]⁺), 365 (26,

[M + Na]⁺), 360 (100, [M + NH₄]⁺), 343 (71, [M + H]⁺), 287

(96, [M - (CH₃)₂CCH₂+ H]⁺), 243 (15, [M - (CH₃)₂CCH₂-

CO₂+ H]⁺) (neg.) 401 (49, [M + CH₃COO]^-), 341 (100, [M -

H]^-), 267 (37, [M - tBuOH - H]^-). Anal. (C₁₈H₂₂N₄O₃) C, H,

N.

N-(tert-Butoxycarbonyl)-3′-thionaphthenylalanyl-gly-

cine-nitrile (32). Recrystallization of the crude product was

done from n-hexane/ethyl acetate, yield 68%, 144-146 °C;

[R]²⁰_D) -8.8 (c ) 1.15, MeOH); ¹H NMR (500 MHz, CDCl₃) δ

1.37 (s, 9H), 3.10-3.47 (m, 2H), 3.94-4.08 (m, 2H), 4.38-4.60

(m, 1H), 5.15 (d, J ) 8.2 Hz, 1H), 6.70 (br s, 1H), 7.19 (s, 1H),

7.31-7.40 (m, 2H), 7.76 (dd, J ) 7.1, 1.7 Hz, 1H), 7.84 (dd, J

) 6.9, 1.6 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 27.34, 28.21,

31.01, 54.20, 80.94, 115.44, 121.45, 123.03, 124.36, 124.52,

124.60, 130.59, 138.39, 140.48, 155.66, 171.72; MS (ESI) m/z

(rel intensity) (pos.) 398 (6, [M + K]⁺), 382 (15, [M + Na]⁺),

377 (81, [M + NH₄]⁺), 360 (41, [M + H]⁺), 304 (100, [M -

(CH₃)₂CCH₂+ H]⁺), 260 (68, [M - (CH₃)₂CCH₂- CO₂+ H]⁺)

(neg.) 418 (9, [M + CH₃COO]^-), 358 (100, [M - H]^-), 284 (50,

[M - tBuOH - H]^-). Anal. (C₁₈H₂₁N₃O₃S) C, H; N: calcd 11.69,

found 11.18.

N-(tert-Butoxycarbonyl)-4′-biphenylalanyl-glycine-ni-

trile (35). The crude product was recrystallized from petrol

ether/ethyl acetate, yield 68%: mp 164-165 °C; [R]²⁰) 7.3

D

1

(c ) 1.12, MeOH); H NMR (500 MHz, CDCl₃) δ 1.39 (s, 9H),

3.03-3.16 (m, 2H), 4.02-4.15 (m, 2H), 4.33-4.45 (m, 1H), 5.06

(d, J ) 5.4 Hz), 6.73 (br s, 1H), 7.24 (d, J ) 7.9 Hz, 2H), 7.29-

7.34 (m, 1H), 7.38-7.43 (m, 2H), 7.51-7.56 (m, 4H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.31, 28.23, 37.64, 55.59, 80.95, 115.57,

126.97, 127.36, 127.54, 128.80, 129.68, 134.98, 140.14, 140.50,

155.70, 171.76; MS (ESI) m/z (rel intensity) (pos.) 418 (4, [M

+ K]⁺), 402 (12, [M + Na]⁺), 397 (100, [M + NH₄]⁺), 380 (43,

[M + H]⁺), 324 (42, [M - (CH₃)₂CCH₂+ H]⁺), 280 (7, [M -

(CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 378 (100, [M - H]^-), 304

(48, [M - tBuOH - H]^-). Anal. (C₂₂H₂₅N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-^D-phenylalanyl-glycine-ni-

trile (36). The crude product was recrystallized from petrol

ether/ethyl acetate, yield 56%: mp 136-138 °C (petrol ether/

1

ethyl acetate); [R]²⁰) -0.6 (c ) 1.86, MeOH); H NMR (500

D

MHz, CDCl₃) δ 1.38 (s, 9H), 3.00-3.10 (m, 2H), 4.04 (dd, J )

17.4, 5.7 Hz, 1H), 4.09 (dd, J ) 17.4, 6.0 Hz, 1H), 4.30-4.40

(m, 1H), 5.01 (br s, 1H), 6.66 (br s, 1H), 7.14-7.19 (m, 2H),

7.21-7.26 (m, 1H), 7.27-7.33 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃) δ 27.29, 28.23, 38.01, 55.66, 80.88, 115.46, 127.24,

128.88, 129.22, 136.00, 155.66, 171.75; MS (ESI) m/z (rel

intensity) (pos.) 342 (3, [M + K]⁺), 326 (42, [M + Na]⁺), 321

(100, [M + NH₄]⁺), 304 (79, [M + H]⁺), 248 (90, [M - (CH₃)₂-

CCH₂+ H]⁺), 204 (57, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

362 (43, [M + CH₃COO]^-), 302 (99, [M - H]^-), 228 (100, [M -

tBuOH - H]^-). Anal. (C₁₆H₂₁N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-phenylalanyl-sarcosine-ni-

trile (37). This compound was prepared from Boc-Phe-OH

(0.64 g, 2.40 mmol) and N-methyl-aminoacetonitrile hydro-

chloride (0.28 g, 2.64 mmol) according to the General Proce-

dure. The obtained oily crude material was purified by

fractionation on silica gel using petrol ether/ethyl acetate (2:

1) as eluent to yield a solid residue, which was recrystallized

from n-hexane/ethyl acetate, yield 62%: mp 96-98 °C; [R]²⁰

D

) 0.4 (c ) 1.28, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.38

(w) & 1.40 (i) (s, 9H), 2.68 (s, 3H), 2.93 (dd, J ) 12.9, 8.9 Hz,

1H), 3.00 (dd, J ) 13.0, 5.7 Hz, 1H), 3.98 (d, J ) 17.1 Hz, 1H),

4.44 (d, J ) 17.1 Hz, 1H), 4.75-4.83 (m, 1H), 5.29 (d, J ) 8.7

Hz, 1H), 7.14-7.20 (m, 2H), 7.21-7.26 (m, 1H), 7.26-7.31 (m,

2H); ¹³C NMR (125 MHz, CDCl₃) δ 27.89 (w) & 28.24 (i), 35.17,

35.85, 37.03, 51.85, 78.34, 116.55, 126.55, 128.26, 129.44,

137.44, 155.31, 172.47; MS (ESI) m/z (rel intensity) (pos.) 356

(8, [M + K]⁺), 340 (10, [M + Na]⁺), 335 (29, [M + NH₄]⁺) 318

(59, [M + H]⁺), 262 (100, [M - (CH₃)₂CCH₂+ H]⁺), 218 (64,

[M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 376 (41, [M +

CH₃COO]^-), 316 (79, [M - H]^-), 242 (100, [M - tBuOH - H]^-).

Anal. (C₁₇H₂₃N₃O₃) C, H, N.

N-(tert-Butoxycarbonyl)-1′-naphthylalanyl-glycine-ni-

trile (33). The crude product was recrystallized from petrol

ether/ethyl acetate, yield 42%: mp 167 °C; [R]²⁰_D) -5.5 (c )

1.07, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.35 (br s, 9H),

3.48 (br s, 2H), 3.94 (d, J ) 5.1 Hz, 2H), 4.45-4.52 (m, 1H),

5.16 (br s, 1H), 6.36 (br s, 1H), 7.28 (d, J ) 7.0 Hz, 1H), 7.39

(dd, J ) 8.2, 6.9 Hz, 1H), 7.45-7.54 (m, 2H), 7.76 (d, J ) 8.2

Hz, 1H), 7.85 (d, J ) 8.4 Hz, 1H), 8.06 (d, J ) 8.2 Hz, 1H); ¹³

C

NMR (125 MHz, CDCl₃) δ 27.20, 28.19, 35.65, 55.17, 80.69,

115.31, 123.27, 125.59, 125.94, 126.63, 127.93, 128.10, 128.99,

131.71, 132.18, 133.92, 155.57, 171.75; MS (ESI) m/z (rel

intensity) (pos.) 392 (4, [M + K]⁺), 376 (49, [M + Na]⁺), 371

(100, [M + NH₄]⁺), 354 (36, [M + H]⁺), 298 (44, [M - (CH₃)₂-

CCH₂+ H]⁺), 254 (4, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.)

412 (6, [M + CH₃COO]^-), 352 (100, [M - H]^-), 278 (64, [M -

tBuOH - H]^-). Anal. (C₂₀H₂₃N₃O₃) C, H, N.

N-(2-Naphthylsulfonyl)-glycyl-glycine-nitrile (38). This

compound was prepared according to the General Procedure

for dipeptide nitriles to give a pure crude product, yield 19%:

mp 165 °C (dec); ¹H NMR (500 MHz, DMSO-d₆) δ 3.51 (s, 2H),

4.06 (d, J ) 5.4 Hz, 2H), 7.64-7.72 (m, 2H), 7.82 (dd, J ) 9.3,

1.9 Hz, 1H), 8.01-8.05 (m, 1H), 8.11 (d, J ) 8.8 Hz, 1H), 8.13-

8.16 (m, 1H), 8.20 (br s, 1H), 8.43 (d, J ) 1.6 Hz, 1H), 8.64 (t,

J ) 5.5 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 27.21, 45.15,

117.46, 122.42, 127.61, 127.68, 127.96, 128.87, 129.32, 129.47,

131.81, 134.36, 137.27, 168.72; MS (ESI) m/z (rel intensity)

(pos.) 326 (14, [M + Na]⁺), 321 (100, [M + NH₄]⁺), 304 (42, [M

+ H]⁺) (neg.) 362 (5, [M + CH₃COO]^-), 302 (100, [M - H]^-),

275 (25, [M - HCN - H]^-). Anal. (C₁₄H₁₃N₃O₃S) C, H; N: calcd

13.85, found 12.62.

N-(tert-Butoxycarbonyl)-2′-naphthylalanyl-glycine-ni-

trile (34). The crude product was recrystallized from petrol

ether/ethyl acetate, yield 39%: mp 147 °C; [R]²⁰) 18.7 (c )

D

1

1.27, MeOH); H NMR (500 MHz, CDCl₃) δ 1.35 (s, 9H), 3.21

(d, J ) 6.9 Hz, 2H), 4.00 (dd, J ) 17.3, 5.7 Hz, 1H), 4.06 (dd,

J ) 17.3, 6.0 Hz, 1H), 4.39-4.49 (m, 1H), 5.06 (br s, 1H), 6.67

(br s, 1H), 7.51 (dd, J ) 8.2, 1.6 Hz, 1H), 7.41-7.48 (m, 2H),

7.61 (s, 1H), 7.74-7.81 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) δ

N-(tert-Butoxycarbonyl)-3′-biphenylalanyl-glycine-ni-

trile (39). General Procedure for Biphenyl Derivatives

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7703

39-40. Compound 29 (0.20 g, 0.52 mmol), phenylboronic acid

(0.1 g, 0.78 mmol), and Na₂CO₃(0.12 g, 1.14 mmol) were

dissolved in dimethoxyethane (8 mL) and H₂O (4 mL). After

degassing with Ar, Pd(PPh₃)₄(0.057 g, 0.05 mmol) was added

under an inert atmosphere. The resulting mixture was placed

into a microwave reactor (sealed tube) and irradiated for 30

min at 30 W and 70 °C. The reaction mixture was transferred

into a round-bottom flask and evaporated. The residue was

suspended in H₂O (5 mL) and extracted with ethyl acetate (320

mL). The combined organic layers were washed with sat.

NaHCO₃(215 mL) and brine (15 mL), treated with silica gel,

and evaporated. The obtained brownish residue was dissolved

in a mixture of MeOH (24 mL) and H₂O (6 mL) and subjected

to solid-phase extraction with a C₁₈cartridge (J. T. Baker),

and the cartridge was washed with the same amount of MeOH/

H₂O. The combined eluates were partly evaporated, and the

remaining aqueous suspension was diluted with the required

amount of MeOH (18 mL) to obtain a solution, which was

fractionated by preparative HPLC (isocratic elution, MeOH/

H₂O 60:40). The combined product fractions were concentrated

(10 mL), dried over P₄O₁₀, and recrystallized from petrol ether/

ethyl acetate to obtain 42 (0.15 g, 20%): mp 180-182 °C (lit.⁶²

mp 190-192 °C); [R]²⁰_D) 7.4 (c ) 1.43, MeOH); ¹H NMR (500

MHz, CDCl₃) δ 1.97 (s, 3H), 3.07 (dd, J ) 7.3, 2.5 Hz, 2H),

4.04 (dd, J ) 5.7, 1.9 Hz, 2H), 4.60-4.66 (m, 1H), 6.03 (d, J )

7.6 Hz, 1H), 6.68 (br s, 1H), 7.16-7.20 (m, 2H), 7.23-7.27 (m,

1H), 7.28-7.33 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 23.13,

27.34, 37.63, 54.38, 115.34, 127.37, 128.96, 129.14, 135.90,

170.69, 171.20; MS (ESI) m/z (rel intensity) (pos.) 268 (32, [M

+ Na]⁺), 263 (93, [M + NH₄]⁺), 246 (100, [M + H]⁺), 190 (47,

[M - H₂NCH₂CN + H]⁺) (neg.) 304 (36, [M + CH₃COO]^-), 244

(100, [M - H]^-), 217 (50, [M - HCN - H]^-). Anal. (C₁₃H₁₅N₃O₂)

C, H, N.

N-Benzoyl-phenylalanyl-glycine-nitrile (43). To a solu-

tion of 41 (0.9 g, 3.0 mmol) in H₂O (10 mL), NaHCO₃was

added as solid until saturation (pH 8-9). Benzoyl chloride

(0.31 mL, 2.7 mmol) was added dropwise to this mixture under

ice cooling. After stirring for 30 min, H₂O (30 mL) was added

and the remaining precipitate was filtered off, washed with

H₂O (10 mL), dried over P₄O₁₀, and recrystallized from H₂O/

and lyophilized to obtain 39 (43 mg, 22%): mp 154 °C; [R]²⁰

EtOH to obtain 43 (0.22 g, 27%): mp 199-200 °C; [R]²⁰

)

D

1

-38.5 (c ) 1.10, dioxane); ¹H NMR (500 MHz, DMSO-d₆) δ

3.01 (dd, J ) 13.7, 10.6 Hz, 1H), 3.13 (dd, J ) 13.9, 4.4 Hz,

1H), 4.15 (dd, J ) 5.5, 1.2 Hz, 2H), 4.66-4.71 (m, 1H), 7.13-

7.18 (m, 1H), 7.22-7.27 (m, 2H), 7.30-7.34 (m, 2H), 7.41-

7.46 (m, 2H), 7.48-7.53 (m, 1H), 7.78-7.82 (m, 2H), 8.65 (d,

J ) 8.5 Hz, 1H), 8.75 (t, J ) 5.5 Hz, 1H); ¹³C NMR (125 MHz,

DMSO-d₆) δ 27.33, 36.94, 54.83, 117.63, 126.43, 127.59, 128.24,

128.27, 129.22, 131.47, 133.98, 138.25, 166.47, 172.17; MS

(ESI) m/z (rel intensity) (pos.) 330 (33, [M + Na]⁺), 325 (21,

[M + NH₄]⁺), 308 (100, [M + H]⁺), 252 (23, [M - H₂NCH₂CN

+ H]⁺) (neg.) 366 (6, [M + CH₃COO]^-), 306 (100, [M - H]^-),

279 (29, [M - HCN - H]^-). Anal. (C₁₈H₁₇N₃O₂‚0.25H₂O) C, H;

N: calcd 13.47, found 12.94.

) 11.3 (c ) 1.02, MeOH); H NMR (500 MHz, CDCl₃) δ 1.38

(s, 9H), 3.10 (dd, J ) 13.6, 7.6 Hz, 1H), 3.16 (dd, J ) 13.9, 6.6

Hz, 1H), 4.05 (dd, J ) 17.4, 4.1 Hz, 1H), 4.12 (dd, J ) 17.5,

6.1 Hz, 1H), 4.35-4.43 (m, 1H), 4.96 (br s, 1H), 6.61 (br s, 1H),

7.15 (d, J ) 7.6 Hz, 1H), 7.31-7.35 (m, 1H), 7.37 (s, 1H), 7.38-

7.44 (m, 3H), 7.46-7.49 (m, 1H), 7.53-7.57 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃) δ 27.33, 28.20, 37.83, 55.70, 80.99, 115.46,

126.05, 127.12, 127.50, 127.97, 128.11, 128.80, 129.33, 136.58,

140.63, 141.79, 155.70, 171.65; MS (ESI) m/z (rel intensity)

(pos.) 418 (3, [M + K]⁺), 402 (35, [M + Na]⁺), 397 (100, [M +

NH₄]⁺), 380 (38, [M + H]⁺), 324 (39, [M - (CH₃)₂CCH₂+ H]⁺),

280 (6, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 378 (100, [M -

H]^-), 304 (62, [M - tBuOH - H]^-). Anal. (C₂₂H₂₅N₃O₃) C, N;

H: calcd 6.64, found 7.19.

N-(4-Phenylbenzoyl)-phenylalanyl-glycine-nitrile (44).

This compound was prepared according to the General Pro-

cedure for dipeptide nitriles using biphenyl-4-carbonic acid (0.5

g, 2.50 mmol) instead of the protected amino acid and 41 (0.84

g, 2.76 mmol) instead of aminoacetonitrile sulfate. The crude

N-(tert-Butoxycarbonyl)-2′-biphenylalanyl-glycine-ni-

trile (40): yield 21%; mp 79-83 °C; [R]²⁰) 4.2 (c ) 1.04,

D

MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.32 (s, 9H), 2.82-2.98

(m, 1H), 3.22 (dd, J ) 14.2, 5.1 Hz, 1H), 3.97 (dd, J ) 17.7,

5.0 Hz, 1H), 4.02 (dd, J ) 17.8, 6.1 Hz, 1H), 4.03-4.12 (m,

1H), 4.64 (d, J ) 7.9 Hz, 1H), 6.40 (br s, 1H), 7.21-7.24 (m,

1H), 7.26-7.33 (m, 5H), 7.35-7.39 (m, 1H), 7.41-7.46 (m, 2H);

¹³C NMR (125 MHz, CDCl₃) δ 27.25, 28.14, 34.65, 55.42, 80.62,

115.52, 127.09, 127.38, 127.88, 128.61, 129.34, 129.98, 130.35,

133.87, 141.13, 142.45, 155.64, 171.76; MS (ESI) m/z (rel

intensity) (pos.) 418 (48, [M + K]⁺), 402 (35, [M + Na]⁺), 397

(100, [M + NH₄]⁺), 380 (85, [M + H]⁺), 324 (52, [M - (CH₃)₂-

CCH₂+ H]⁺), 280 (13, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.)

438 (7, [M + CH₃COO]^-), 378 (100, [M - H]^-), 304 (76, [M -

tBuOH - H]^-). Anal. (C₂₂H₂₅N₃O₃‚0.5H₂O) C, H, N.

product was recrystallized from petrol ether/ethyl acetate, yield

1

17%: mp 214-215 °C; [R]²⁰) -44.2 (c ) 1.24, dioxane); H

D

NMR (500 MHz, DMSO-d₆) δ 3.03 (dd, J ) 13.9, 10.8 Hz, 1H),

3.15 (dd, J ) 13.9, 4.4 Hz, 1H), 4.14 (dd, J ) 5.7, 1.0 Hz, 2H),

4.67-4.75 (m, 1H), 7.14-7.18 (m, 1H), 7.23-7.27 (m, 2H),

7.32-7.35 (m, 2H), 7.37-7.42 (m, 1H), 7.46-7.50 (m, 2H),

7.70-7.76 (m, 4H), 7.87-7.92 (m, 2H), 8.72 (d, J ) 8.5 Hz,

1H), 8.77 (t, J ) 5.5 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆)

δ 27.35, 36.95, 54.85, 117.64, 126.44, 126.50, 126.99, 128.19,

128.26, 128.29, 129.14, 129.23, 132.77, 138.28, 139.28, 143.04,

166.11, 172.18; MS (ESI) m/z (rel intensity) (pos.) 406 (24, [M

+ Na]⁺), 401 (11, [M + NH₄]⁺), 384 (100, [M + H]⁺), 328 (25,

[M - H₂NCH₂CN + H]⁺) (neg.) 442 (5, [M + CH₃COO]^-), 400

(6, [M + H₂O - H]^-), 382 (100, [M - H]^-), 355 (18, [M - HCN

- H]^-). Anal. (C₂₄H₂₁N₃O₂‚0.25H₂O) C, H, N.

Phenylalanyl-glycine-nitrile Hydroperchlorate (41).

Compound 16 (8.4 g, 27.7 mmol) was dissolved in formic acid

(50 mL) and stirred for 6 h at room temperature. The solvent

was thoroughly removed under reduced pressure and the

residue was dissolved in iPrOH (30 mL). The resulting solution

was treated with 70% HClO₄(2.4 mL, 27.7 mmol) under ice

cooling. Since precipitation did not occur, the solution was

evaporated (CAUTION: Perchloric acid is explosive!) and

the remaining residue dissolved in H₂O (70 mL). The resulting

mixture was extracted with Et₂O (2 × 20 mL). The aqueous

phase was lyophilized to obtain 41 as a semisolid, yellow

residue (6.64 g, 79%), which is pure enough for further steps:

¹H NMR (500 MHz, DMSO-d₆) δ 2.98 (dd, J ) 13.9 Hz, J )

7.3 Hz, 1H), 3.05 (dd, J ) 13.9 Hz, J ) 6.6 Hz, 1H), 4.00 (br

s, 1H), 4.20 (d, J ) 5.7 Hz, 2H), 7.19-7.23 (m, 2H), 7.25-7.30

tert-Butoxycarbonylbenzylidenehydrazine (45). Benz-

aldehyde (0.77 mL, 7.6 mmol) was added dropwise to a solution

of tert-butyl carbazate (1 g, 7.6 mmol) in THF (10 mL). After

stirring for 4 h at room temperature, the solvent was removed

under reduced pressure to obtain 45 (1.47 g, 88%) as white

solid, which was used without further purification in the next

step: mp 203 °C (lit.⁶³mp 186 °C); ¹H NMR (500 MHz, CDCl₃)

δ 1.52 (s, 9H), 7.30-7.36 (m, 3H), 7.62-7.68 (m, 2H), 7.82 (br

s, 1H), 8.03 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 28.26,

81.46, 127.16, 128.54, 129.80, 133.89, 143.68, 152.46.

1-tert-Butoxycarbonyl-2-benzylhydrazine × BH₂CN

(46). To a solution of 45 (1.3 g, 5.9 mmol) in THF (15 mL) and

AcOH (9 mL) NaBH₃CN (0.93 g, 14.8 mmol) was added as solid

under ice cooling. The resulting mixture was stirred for 18 h

at room temperature. Subsequently, ethyl acetate (50 mL) and

sat. NaHCO₃(50 mL) were added and the two phases were

separated. The aqueous phase was alkalized with solid

NaHCO₃and extracted with ethyl acetate (2 × 50 mL). The

combined organic phases were washed with sat. NaHCO₃(1

× 25 mL) and brine (25 mL), dried over Na₂SO₄, and

3

(m, 1H), 7.31-7.36 (m, 2H), 8.21 (s, 3H), 9.06 (t, J ) 5.5 Hz,

1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 27.26, 36.97, 53.58,

117.00, 127.47, 128.79, 129.57, 134.62, 168.87.

N-Acetyl-phenylalanyl-glycine-nitrile (42). To a solution

of 41 (0.9 g, 3.0 mmol) in H₂O (10 mL), NaHCO₃was added

as solid until saturation (pH 8-9). Acetic anhydride (0.42 mL,

4.5 mmol) was added dropwise to this mixture under ice

cooling. After stirring for 30 min, H₂O (30 mL) was added and

the remaining precipitate was filtered off, washed with H₂O

7704 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Lo¨ser et al.

evaporated under reduced pressure to obtain 46 (1.54 g, 100%)

as a white residue: mp 143-144 °C (dec) (lit.³⁶mp 152 °C)

(petrol ether/ethyl acetate); ¹H NMR (500 MHz, CDCl₃) δ 1.39

(s, 9H) 4.11 (dd, J ) 12.8, 5.8 Hz, 1H), 4.30 (dd, J ) 12.8, 4.9

Hz, 1H), 6.40 (br s, 1H), 6.86 (br s, 1H); ¹³C NMR (125 MHz,

CDCl₃) δ 27.91, 62.70, 84.89, 128.92, 129.75, 130.40, 130.81,

154.41; MS (ESI) m/z (rel intensity) (pos.) 279 (74, [C₁₂H₁₈N₂O₂

acid is explosive!). The precipitate was collected by filtration

to obtain 50 (0.68 g, 72%): mp 175-178 °C; ¹H NMR (500

MHz, DMSO-d₆) δ 3.16 (s, 3H), 3.71 (s, 3H), 3.88 (s, 2H), 7.98

(s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 32.04, 39.32, 61.60,

166.99.

N-(tert-Butoxycarbonyl)-phenylalanyl-glycine-N′-meth-

oxy-N′-methylamide (51). Boc-Phe-OH (0.8 g, 3.0 mmol) was

dissolved in THF (15 mL) and cooled to -25 °C. Subsequently,

N-methylmorpholine (0.33 mL, 3.0 mmol) and isobutyl chlo-

roformate (0.39 mL, 3.0 mmol) were added under vigorous

stirring. Immediately, a solution of 50 (0.72 g, 3.3 mmol) in

H₂O (0.5 mL) and 1 N NaOH (3.3 mL) was added. The

resulting mixture was allowed to warm to room temperature.

After 2 h, THF was removed under reduced pressure and the

aqueous residue was diluted with a small volume of H₂O,

adjusted to pH 1-2 (10% NaHSO₄), and extracted with ethyl

acetate (3 × 20 mL). The combined organic layers were washed

with H₂O (10 mL), sat. NaHCO₃(2 × 10 mL), and brine (10

mL), dried over Na₂SO₄, and evaporated to dryness to obtain

+

¹¹BH₂CN + NH₄]⁺), 278 (20, [C₁₂H₁₈N₂O₂

NH₄]⁺), 262 (97, [C₁₂H₁₈N₂O₂¹¹BH₂CN + H]⁺), 261 (27,

[C₁₂H₁₈N₂O₂+

¹⁰BH₂CN + H]⁺), 206 (100, [C₁₂H₁₈N₂O₂

+

¹⁰BH₂CN +

+

¹¹BH₂CN - (CH₃)₂CCH₂+ H]⁺), 205 (30, [C₁₂H₁₈N₂O₂+ ¹⁰BH₂-

CN - (CH₃)₂CCH₂+ H]⁺) (neg.) 260 (98, [C₁₂H₁₈N₂O₂+ ¹¹BH₂-

CN - H]^-), 259 (29, [C₁₂H₁₈N₂O₂+ ¹⁰BH₂CN - H]^-), 233 (100,

[C₁₂H₁₈N₂O₂+ ¹¹BH₂CN - HCN - H]^-), 232 (31, [C₁₂H₁₈N₂O₂

+

¹¹BH₂CN - HCN - H]^-.

1-tert-Butoxycarbonyl-2-benzylhydrazine (47). Com-

pound 46 (1.51 g, 5.8 mmol) was dissolved in MeOH (10 mL)

and treated with 1 N NaOH (10 mL) for 2 h at room

temperature. After removal of the organic solvent, the aqueous

residue was extracted with ethyl acetate (3 × 20 mL). The

combined organic phases were washed with brine (20 mL),

dried over Na₂SO₄, and concentrated to obtain 47 as a colorless

oil (1.24 g, 96%), which was pure enough for further conver-

sion: ¹H NMR (500 MHz, CDCl₃) δ 1.44 (s, 9H), 3.96 (s, 2H),

4.15 (br s, 1H), 6.01 (br s, 1H), 7.25-7.35 (m, 5H); ¹³C NMR

(125 MHz, CDCl₃) δ 28.32, 55.87, 80.72, 127.58, 128.55, 128.94,

137.41, 156.79.

1

51 (1.07 g, 98%): mp 65 °C; H NMR (500 MHz, DMSO-d₆) δ

1.20 (w) & 1.28 (i) (s, 9H), 2.72 (dd, J ) 13.9, 10.7 Hz, 1H),

3.03 (dd, J ) 13.9, 3.8 Hz, 1H), 3.11 (s, 3H), 3.70 (s, 3H), 3.99

(dd, J ) 17.5, 4.8 Hz, 1H), 4.07 (dd, J ) 17.6, 5.4 Hz, 1H),

4.19-4.26 (m, 1H), 6.90 (d, J ) 8.8 Hz, 1H), 7.13-7.21 (m,

1H), 7.22-7.32 (m, 4H), 8.00 (t, J ) 5.2 Hz, 1H); ¹³C NMR

(125 MHz, DMSO-d₆) δ 28.26, 32.23, 37.67, 39.83, 55.47, 61.30,

78.13, 126.23, 128.09, 129.30, 138.44, 155.34, 169.63, 172.19.

N-(tert-Butoxycarbonyl)-phenylalanyl-glycine-alde-

hyde (52). A solution of 51 (0.56 g, 1.53 mmol) in dry Et₂O

(20 mL) was added all at once to a suspension of LiAlH₄(0.32

g, 8.42 mmol) in dry Et₂O (80 mL) under Ar at -35 °C. The

resulting mixture was allowed to warm to room temperature

over 2 h. Subsequently, it was cooled to -35 °C again and

treated with 25% KHSO₄(5 mL). The mixture was filtered over

Celite, and the residue was washed with Et₂O (2 × 20 mL).

The combined filtrates were washed with 10% NaHSO₄(25

mL), sat. NaHCO₃(2 × 25 mL), and brine (25 mL), dried over

Na₂SO₄, and evaporated to dryness to obtain 52 as white foam

N-(tert-Butoxycarbonyl)-azaphenylalanyl-glycine-ni-

trile (48). To a solution of N,N′-carbonyldiimidazole (0.36 g,

2.25 mmol) in DMF (10 mL) solid aminoacetonitrile sulfate

(0.24 g, 2.25 mmol) and diisopropylethylamine (0.38 mL, 2.25

mmol) were added under ice cooling. After 1 h, a solution of

47 (0.5 g, 2.25 mmol) in DMF (7 mL) was added. After stirring

for 13 h at room temperature, the solvent was removed under

reduced pressure. The residue was suspended in H₂O (5 mL),

acidified to pH 1 (10% NaHSO₄), and extracted with ethyl

acetate (3 × 15 mL). The combined organic layers were washed

with H₂O (10 mL), sat. NaHCO₃(2 × 10 mL) and brine (10

mL), dried over Na₂SO₄, and evaporated to obtain an oily

residue, which was fractionated on silica gel (petrol ether/ethyl

acetate 1:1). The fractions containing the main product were

combined and delivered from solvent to obtain 48 (0.35 g,

51%): mp 131-133 °C; ¹H NMR (500 MHz, DMSO-d₆, 70 C) δ

1.35 (s, 9H), 4.04 (d, J ) 6.0 Hz, 2H), 4.55 (br s, 2H), 7.16-

7.22 (m, 1H), 7.23-7.33 (m, 5H), 8.68 (br s, 1H); ¹³C NMR (125

MHz, DMSO-d₆, 70 C) δ 27.78, 28.77, 51.58, 79.77, 117.93,

126.87, 127.89, 128.38, 136.98, 154.01, 157.40; MS (ESI) m/z

(rel intensity) (pos.) 327 (22, [M + Na]⁺), 322 (100, [M +

NH₄]⁺), 305 (51, [M + H]⁺), 249 (74, [M - (CH₃)₂CCH₂+ H]⁺),

205 (16, [M - (CH₃)₂CCH₂- CO₂+ H]⁺) (neg.) 363 (36, [M +

CH₃COO]^-), 303 (99, [M - H]^-), 229 (100, [M - tBuOH - H]^-).

Anal. (C₁₅H₂₀N₄O₃) C, H; N: calcd 18.41, found 17.47.

N-(tert-Butoxycarbonyl)-glycine-N′-methoxy-N′-meth-

ylamide (49). To a solution of N,O-dimethylhydroxylamine

hydrochloride (3.59 g, 36.8 mmol) in H₂O (5 mL), 1 N NaOH

(36.8 mL) was added under ice cooling. The resulting solution

was added to a suspension of N-(tert-butoxycarbonyl)-glycine

N′-hydroxysuccinimide ester (2.50 g, 9.2 mmol) in dioxane (20

mL) and stirred for 6 h at room temperature. After evaporation

of the solvent, the remaining aqueous residue was adjusted

to pH 1-2 with 10% NaHSO₄and extracted with ethyl acetate

(3 × 30 mL). The combined organic layers were washed with

sat. NaHCO₃, H₂O, and brine (each 20 mL), dried over Na₂SO₄,

and evaporated to obtain 49 (1.24 g, 62%): mp 100 °C; ¹H NMR

(500 MHz, CDCl₃) δ 1.42 (s, 9H), 3.17 (s, 3H), 3.68 (s, 3H),

4.05 (d, J ) 3.8 Hz, 2H), 5.24 (br s, 1H); ¹³C NMR (125 MHz,

CDCl₃) δ 28.33, 32.36, 41.70, 61.43, 79.62, 155.87, 170.21.

(0.33 g, 70%): mp 88-90 °C; [R]²⁰) 6.9 (c ) 1.06, MeOH);

D

¹H NMR (500 MHz, CDCl₃) δ 1.39 (s, 9H), 3.03-3.09 (m, 2H),

4.05 (dd, J ) 20.4, 4.6 Hz, 1H), 4.12 (dd, J ) 20.2, 5.1 Hz,

1H), 4.36-4.45 (m, 1H), 4.99 (br s, 1H), 6.60 (br s, 1H), 7.09-

7.34 (m, 5H), 9.54 (s, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 28.23,

38.32, 50.03, 55.70, 80.46, 127.06, 128.72, 129.23, 136.41,

155.43, 171.74, 195.94; MS (ESI) m/z (rel intensity) (pos.) 339

(22, [M + CH₃OH + H]⁺), 324 (7, [M + NH₄]⁺), 307 (100, [M

+ H]⁺), 251 (36, [M - (CH₃)₂CCH₂+ H]⁺), 207 (5, [M - (CH₃)₂-

CCH₂- CO₂+ H]⁺), (neg.) 397 (33, [M + CH₃OH + CH₃COO]^-),

383 (51, [M + H₂O + CH₃COO]^-), 365 (5, [M + CH₃COO]^-),

305 (100, [M - H]^-), 231 (93, [M - tBuOH - H]^-). Anal.

(C₁₆H₂₂N₂O₄) H, N; C: calcd 62.73, found 61.05.

N-(tert-Butoxycarbonyl)-phenylalanyl-glycine-meth-

ylketone (53). A small portion of a solution of CH₃I (0.95 mL,

15.3 mmol) in dry Et₂O (10 mL) was added dropwise to a

suspension of Mg sparks (0.37 g, 15.3 mmol) in dry Et₂O (5

mL). A drop of Br₂was added to initiate the reaction. The

residual amount of CH₃I was added dropwise over a period of

30 min. After stirring for an additional 1 h almost all of the

metal dissolved. Subsequently, a solution of 51 (0.56 g, 1.53

mmol) in dry Et₂O (10 mL) was added dropwise under ice

cooling. After stirring the mixture for an additional 1 h at room

temperature, sat. NH₄Cl (20 mL) was added and the aqueous

phase was extracted with Et₂O (2 × 20 mL). The combined

organic phases were washed with brine (10 mL), dried over

Na₂SO₄, and evaporated. The obtained oily-crystalline residue

was fractionated on silica gel (petrol ether/ethyl acetate 1:1)

to obtain 53 (245 mg, 55%): mp 110-111 °C; [R]²⁰_D) -6.0 (c

) 1.14, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.38 (s, 9H),

2.14 (s, 3H), 2.98-3.06 (m, 1H), 3.08 (dd, J ) 13.6, 6.6 Hz,

1H), 4.01 (dd, J ) 20.0, 4.1 Hz, 1H), 4.10 (dd, J ) 19.7, 4.9

Hz, 1H), 4.34-4.44 (m, 1H), 4.97 (br s, 1H), 6.51-6.62 (m, 1H),

Glycine N′-methoxy-N′-methylamide Hydroperchlor-

ate (50). Compound 49 (1.19 g, 5.45 mmol) was dissolved in 4

N HCl/dioxane (10 mL) and stirred for 2 h at room tempera-

ture. After removal of the solvent, the residual oil was

dissolved in iPrOH (15 mL) and treated with 70% HClO₄(0.46

mL, 5.45 mmol) under ice cooling (CAUTION: Perchloric

7.15-7.18 (m, 2H), 7.19-7.23 (m, 1H), 7.25-7.29 (m, 2H); ¹³

C

NMR (125 MHz, CDCl₃) δ 27.23, 28.22, 38.35, 49.65, 55.65,

80.27, 126.97, 128.66, 129.21, 136.49, 155.35, 171.28, 202.18;

Interaction of Cysteine Proteases with Nitriles

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7705

MS (ESI) m/z (rel intensity) (pos.) 359 (3, [M + K]⁺), 343 (6,

[M + Na]⁺), 338 (38, [M + NH₄]⁺) 321 (100, [M + H]⁺), 265

(56, [M - (CH₃)₂CCH₂+ H]⁺), 221 [M - (CH₃)₂CCH₂- CO₂+

H]⁺), (neg.) 379 (11, [M + CH₃COO]^-); 319 (28, [M - H]^-),

245 (100, [M - tBuOH - H]^-). Anal. (C₁₇H₂₄N₂O₄) C, H, N.

N-(tert-Butoxycarbonyl)-phenylalanyl-propargyl-

amide (54). General Procedure for Unsaturated Amides

54 and 55. Boc-Phe-OH (1 g, 3.8 mmol) was dissolved in THF

(20 mL) and cooled to -25 °C. Subsequently, N-methylmor-

pholine (0.41 mL, 3.8 mmol) and isobutyl chloroformate (0.51

mL, 3.8 mmol) were added under vigorous stirring. Im-

mediately, propargylamine (0.29 mL, 4.2 mmol) was added.

The suspension was allowed to warm to room temperature.

After 2 h, THF was removed under reduced pressure and the

solid residue was dissolved in H₂O (10 mL), adjusted to pH

1-2 (10% NaHSO₄), and extracted with ethyl acetate (3 × 20

mL). The combined organic layers were washed with H₂O (10

mL), sat. NaHCO₃(2 × 10 mL), and brine (10 mL), dried over

Na₂SO₄, and evaporated to dryness to obtain a solid residue,

which was recrystallized from cyclohexane to obtain 54 (0.75

to the experimental data, where v is the rate, v₀is the rate in

the absence of inhibitor, [I] is the inhibitor concentration and

K_i′ is the apparent inhibition constant. The true inhibition

constants K_iwere calculated by correction of K_i′ according to

K_i) K_i′/(1 + [S]/K_m)

(2)

where [S] is the substrate concentration and K_mis the

Michaelis constant.

To determine the K_mvalue, the total hydrolysis of Z-Phe-

Arg-NHNp (480 µM) was followed in the presence of a papain

concentration which was 20-fold higher than in the inhibition

assays; other conditions were as noted above. Hydrolysis went

to completion in 30 min. Progress curves of six independent

experiments were analyzed by fitting the following set of

differential equations to the experimental data using the

program EASY-FIT,

([S₀] + [P₀] - [P])

d[P]

dt

) k_cat[E_t]

(3)

(4)

g, 65%): mp 102-104 °C (lit.⁶⁴mp 98-100 °C); [R]²⁰) +3.6

K_m+ ([S₀] + [P₀] - [P])

D

1

(c ) 1.32, MeOH); H NMR (500 MHz, CDCl₃) δ 1.22 (w) &

1.28 (i) (s, 9H), 2.71 (dd, J ) 12.2, 10.8 Hz, 1H), 2.92 (dd, J )

13.7, 4.5 Hz, 1H), 3.09 (t, J ) 2.5, 1H), 3.83-3.88 (m, 2H),

4.10-4.17 (m, 1H), 6.85 (d, J ) 8.4 Hz, 1H), 7.15-7.20 (m,

1H), 7.21-7.30 (m, 4H), 8.33 (t, J ) 5.1 Hz, 1H); ¹³C NMR

(125 MHz, CDCl₃) δ 28.15, 28.27, 37.65, 55.69, 73.17, 78.11,

81.13, 126.29, 128.11, 129.32, 138.1, 155.31, 171.53; MS (ESI)

m/z (rel intensity) (pos.) 341 (3, [M + K]⁺), 325 (6, [M + Na]⁺),

303 (54, [M + H]⁺), 247 (72, [M - (CH₃)₂CCH₂+ H]⁺), 203

(100, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 361 (47, [M +

CH₃COO]^-), 301 (66, [M - H]^-), 227 (100, [M - tBuOH - H]^-).

Anal. (C₁₇H₂₂N₂O₃) C, H, N.

d[E_t]

K_m

) -k_inac[E_t]

dt

K_m+ ([S₀] + [P₀] - [P])

where [P] is the product concentration (dependent experimen-

tal variable) and t is the time (independent experimental

variable). For the calculation of the pNA concentrations, an

extinction coefficient ꢀ₄₀₅) 9.96 cm²/µmol was used. The

following parameters were obtained by nonlinear regression:

[E_t] (total concentration of active enzyme), K_m(Michaelis

constant), [S₀] (initial substrate concentration), [P₀] (initial

product concentration), and k_inac(rate constant for enzyme

inactivation). As the determination of k_cat(catalytic constant)

was beyond the scope of this investigation, a reasonable value

was taken from the literature (k_cat) 1980 min^-1).⁶⁶The

following results were obtained: K_m) 965 ( 244 µM, [S₀] )

437 ( 2 µM, [P₀] ) 40 ( 0.8 µM.

Cathepsins. Enzyme activities were calculated from kinetic

measurements performed by fluorimetric detection of the

product AMC at 37 °C in a stirred cell. The wavelengths for

excitation and emission were 360 and 440 nm, respectively.

The reaction volume of the assay was 2 mL. To assay cathepsin

L, Z-Phe-Arg-NHMec was used as substrate at a concentration

of 10 µM in 100 mM sodium phosphate pH 6.0, 100 mM NaCl,

5 mM EDTA, 0.01% Brij 35, 25 µM DTT, and 1% DMSO. For

cathepsin S, Z-Val-Val-Arg-NHMec was chosen as substrate

at a concentration of 40 µM. The assay medium consisted of

50 mM potassium phosphate pH 6.5, 50 mM NaCl, 2 mM

EDTA, 0.01% Triton X-100, 25 µM DTT, and 1% DMSO. In

the cathepsin K assay, Z-Leu-Arg-NHMec was used as sub-

strate at a concentration of 20 µM in 100 mM sodium citrate

pH 5.0, 100 mM NaCl, 1 mM EDTA, 0.01% CHAPS, 25 µM

DTT, and 1% DMSO. Stock solutions of the substrates were

prepared in DMSO in a 1000-fold higher concentration than

the final concentrations. Dilutions were done in the corre-

sponding assay medium without DTT and DMSO. Stock

solutions of the inhibitors were prepared in DMSO. The

enzyme dilutions were daily prepared from a stock with the

corresponding assay medium without DMSO, containing 5 mM

DTT (the complete amount of DTT required for the final

concentration noted above), and kept on ice.

N-(tert-Butoxycarbonyl)-phenylalanyl-allylamide (55).

The compound was obtained as pure crude product, yield

100%: mp 105-107 °C (lit.⁶⁵mp 88-90 °C); [R]²⁰) +1.0 (c

D

) 2.11, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 1.22 (w) & 1.29

(i) (s, 9H), 2.74 (dd, J ) 13.7, 10.0 Hz, 1H), 2.93 (dd, J ) 13.8,

4.9 Hz, 1H), 3.63-3.74 (m, 2H), 4.12-4.19 (m, 1H), 5.02 (dd,

J ) 10.3, 1.3 Hz, 1H), 5.09 (dd, J ) 17.2, 1.7 Hz, 1H), 5.69-

5.80 (m, 1H), 6.85 (d, J ) 8.7 Hz, 1H), 7.15-7.20 (m, 1H),

7.21-7.30 (m, 4H), 7.99 (t, J ) 5.3 Hz); ¹³C NMR (125 MHz,

CDCl₃) δ 28.27, 37.80, 40.9, 55.93, 78.08, 115.06, 126.27,

128.11, 129.31, 135.27, 138.31, 155.32, 171.53; MS (ESI) m/z

(rel intensity) (pos.) 343 (2, [M + K]⁺), 327 (6, [M + Na]⁺),

305 (45, [M + H]⁺), 249 (69, [M - (CH₃)₂CCH₂+ H]⁺), 205

(100, [M - (CH₃)₂CCH₂- CO₂+ H]⁺), (neg.) 363 (60, [M +

CH₃COO]^-), 303 (31, [M - H]^-), 229 (100, [M - tBuOH - H]^-).

Anal. (C₁₇H₂₄N₂O₃) C, H, N.

Inhibition Experiments. Papain. Enzyme activities were

calculated from kinetic measurements performed by spectro-

photometric detection of the product p-nitroaniline (pNA) at

25 °C in a multicell holder (parallel measurements for each

single inhibitor determination; final volume 1 mL) at a

wavelength of 405 nm. A 4 mM stock solution of the chro-

mogenic substrate Z-Phe-Arg-NHNp was prepared in DMSO;

the final concentration was 200 µM. The assay medium was

0.1 M sodium phosphate pH 6.5, 2.5 mM EDTA (ethylenedi-

aminetetraacetic acid), 300 µM DTT, and 12% DMSO. Stock

solutions of the inhibitors were prepared in DMSO. In the

absence of inhibitor, 70 µL of DMSO was added to the cuvette.

A papain stock solution was prepared in 1 mM HCl. For daily

activation, the papain stock solution was diluted 1:100 in 15

mM DTT, 0.1 M sodium phosphate pH 6.5, 2.5 mM EDTA and

incubated at 25 °C for 1 h. The activated enzyme was kept on

ice. After thermal equilibration, the reaction was initiated by

addition of the enzyme (20 µL); its final concentration cata-

lyzed the conversion of the substrate with a rate of 1-2 µM/

min. Progress curves were monitored over 10 min. Rates were

determined for 7 different inhibitor concentrations in duplicate.

The apparent inhibition constants K_i′ were determined by

fitting

After thermal equilibration, 10 µL of the enzyme solution

was added and product formation was monitored over 5 min.

The inhibition constants were computed from the initial

velocities determined at 10 to 12 different inhibitor concentra-

tions and duplicate or triplicate measurements in the absence

of the inhibitor. The K_i′ values were obtained from nonlinear

regressions according to eq 1, and K_iwas calculated from eq

2. Michaelis constants were determined at 10 to 12 different

substrate concentrations by the standard method to obtain the

following values: cathepsin L, K_m) 5.3 ( 0.3 µM; cathepsin

S, K_m) 19.2 ( 0.8 µM; cathepsin K, K_m) 6.2 ( 1.2 µM.

Progress curves of the reactions of cathepsins L, S, and K in

v ) v₀/(1 + [I]/K_i′)

(1)

7706 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

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the presence of inhibitor 53 were biphasic and were analyzed

by nonlinear regression using eq 5,

[P] ) v_st + {(v₀- v_s)(1 - exp(-k_obst))}/k_obs+ d (5)

where v_sis the steady state rate, v₀is the initial rate, k_obsis

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k_obs) k_on[I] + k_off

(6)

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(7)

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Acknowledgment. The authors are grateful to Prof.

B. Wiederanders, Jena, and Prof. D. Bro¨mme, Vancou-

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Supporting Information Available: Elemental analyti-

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JM050686B

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Interaction of papain-like cysteine proteases with dipeptide-derived nitriles

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