Synthesis of dipeptide-bound epoxides and α,β-unsaturated amides as potential irreversible transglutaminase inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00292-9

Herein we report the synthesis of 24 novel peptides as potential irreversible inactivators of transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gly, known to be a good TGase substrate, and to include either α,β-unsaturated amide groups or the corresponding epoxide groups. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Copyright

Full text of DOI:10.1016/S0968-0896(01)00292-9

Bioorganic & Medicinal Chemistry 10 (2002) 355–360
Synthesis of Dipeptide-Bound Epoxides and ꢀ,ꢁ-Unsaturated
Amides as Potential Irreversible Transglutaminase Inhibitors
Pierre de Macedo, Claudio Marrano and Jeffrey W. Keillor*
´ ´ ´ ´ ´
Departement de chimie, Universite de Montreal, C.P. 6128, Succursale Centre-ville, Montreal, Quebec, Canada H3C 3J7
Received 6 April 2001; accepted 8 August 2001
Abstract—Herein we report the synthesis of 24 novel peptides as potential irreversible inactivators of transglutaminase (TGase).
These peptides were designed to resemble Cbz-l-Gln-Gly, known to be a good TGase substrate, and to include either a,b-unsatu-
rated amide groups or the corresponding epoxide groups. The side chain length of the amino acid residue bearing the inhibitor
group was also varied in order to permit investigation of this effect. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
iodoacetates,30ꢀ32 isocyanates,33 thioureas,34 acivicin
derivatives,35,36 sulfonium methyl ketones,37 thioaceto-
nyl heterocycles,38 and electrophilic glutamine analo-
gues.39 However, their lack of specificity limits their
therapeutic utility. Herein, we report the synthesis of
potential inhibitors designed to have high TGase affinity.
Transglutaminases (TGases, EC 2.3.2.13) constitute a
class of calcium-dependent acyltransferases that
catalyze the formation of an amide bound between the
g-carboxamide groups of peptide-bound glutamine resi-
dues and various primary amines. These enzymes are
widely distributed in mammalian tissues, plasma and
epidermis. Tissue TGases are involved in diverse biolo-
gical functions such as endocytosis,1,2 apoptosis3 and
cell growth regulation.4 Plasma-soluble TGase (Factor
XIIIa) catalyses the formation of fibrin cross-links, sta-
bilizing blood clots during their formation.5ꢀ7 Epi-
dermal TGases are responsible for the formation of the
cornified envelope of epidermal keratinocytes.8ꢀ11
Results and Discussion
The basic structure of the inhibitors shown herein is
based on that of carbobenzyloxy-l-glutaminylglycine
(Cbz-Gln-Gly), a commonly used dipeptide TGase sub-
strate.26,40,41 In the enzymatic mechanism, the acylation
step consists of the attack of the active site cysteine thiol
residue on the carbonyl of the g-carboxamide function
of the substrate glutamine side chain. We therefore
chose to place reactive functional groups at the end of
the peptide side chain. Furthermore, we designed ana-
logues with different chain lengths in order to study the
effect of this variation.
High TGase activities have also been implicated in a
number of disease states, including acne,12,13 catar-
acts,14 immunologic diseases,15 psoriasis,16ꢀ18 and Alz-
heimer’s disease.19ꢀ25 Inhibitors may be useful in the
treatment of TGase dysfunction. The TGase mechanism
involves the transfer of an acyl group from a substrate
to an active site cysteine thiol residue of the enzyme.26
TGases are therefore inactivated by a,b-unsaturated
amides (Michael acceptors) and epoxide derivatives,27
as are many enzymes with an active site thiol group,
through the nucleophilic attack of the cysteine thiol on
the inhibitor resulting in the formation of a covalent
bond that is resistant to subsequent hydrolysis or ami-
nolysis reactions. During the past decades, TGase has
been shown to be irreversibly inactivated by a number
The starting points of our parallel syntheses were the
corresponding side chain primary amine derivatives.
Products 3a and 4a are commercially available, whereas
products 1a–2a were obtained after the rearrangement
of their corresponding amides Na-carbobenzyloxy-l-
asparagine and Na-carbobenzyloxy-l-glutamine respec-
tively (Scheme 1).42 This reaction was carried out in the
presence of bis(trifluoroacetoxy)phenyliodine and the
desired products were obtained after precipitation with
yields around 80%.
28,29
of differentcompounds, including sulfonamides,
Compounds 1b–4b were prepared by allowing acryloyl
chloride to react with compounds 1a–4a (where the
*Corresponding author. Tel.: +1-514-343-6219; fax: +1-514-343-
7586; e-mail: keillorj@chimie.unmontreal.ca
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00292-9

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P. de Macedo et al. / Bioorg. Med. Chem. 10 (2002) 355–360
356
compound number corresponds to the number of
methylene units of the side chain) in the presence of
triethylamine in methanol (Scheme 2). The isolated
yields varied from 73–90% according to the length of
the side chain, since the less polar molecules were more
easily isolated during the work up conditions.
compounds bearing a Michael acceptor function (1b–
4d) into the corresponding epoxides 1e–4g. DMDO was
43
firstdescribed by Adam etal.
and has been employed
as a reagent that is particularly useful for the epoxida-
tion of conjugated double bonds,44 as a preferred alter-
native to m-CPBA, magnesium peroxyphthalate,
hydrogen peroxide in basic conditions and hydrogen
peroxide with tungsten tetroxide.45 Since the epoxida-
tion reactions are quantitative, this method is advanta-
geous because pure product is obtained the after
evaporation of excess DMDO and acetone. Using this
method, compounds 1e–4g were obtained in essentially
quantitative yield, since no further purification was
required after the transformation of starting materials
1b--4d. Like classical epoxidation methods, this pro-
cedure resulted in the racemic formation of the epoxide
group. Due to the presence of an additional stereocenter
on the Cbz-Gln-Gly scaffold, each epoxide dipeptide
was thus obtained as a mixture of two diastereoisomers.
The following step consisted of the coupling of tert-
butyl glycine ester with products 1b–4b, previously acti-
vated by 1-(3-dimethylaminopropyl)-3-ethyl-carbodii-
mide (EDC) in the presence of triethylamine in
dichloromethane. Compounds 1c–4c were thus obtained
with yields between 70 and 93%, the less polar deriva-
tives being once again more easily isolated. The tert-
butyl ester groups were hydrolyzed by bubbling hydro-
gen chloride through dichloromethane solutions. In this
way, products 1d–4d were obtained in quantitative
yields. Compounds 1b–4d bear an a,b-unsaturated
amide and are thus potential irreversible (Michael
acceptor) inhibitors of cysteine-activity dependent
enzymes.
Conclusion
These compounds also serve as the synthetic precursors
of the corresponding epoxide analogues, which also
have the potential of forming irreversible adduct pro-
ducts with active site thiol residues. A novel method
using dimethyldioxirane (DMDO),43ꢀ45 which is the
peroxide of acetone, was used to transform the
Efficient synthetic routes have been developed for the
synthesis of 24 new potential irreversible inhibitors of
TGases. All of these compounds are analogues of Cbz-
Gln-Gly, a commonly used substrate for TGases. Based
on this similarity of structure and the known irreversible
inhibition efficacy of Michael acceptor and epoxide
functional groups, we expect to obtain promising enzy-
matic inhibition results. The results of our studies of the
inhibitory activity of these compounds, using a TGase
activity assay recently developed in our laboratories,46
will be reported shortly.47
Scheme 1.
Experimental
Materials and methods
1H and 13C NMR spectra were recorded on a Bruker
400 MHz spectrometer. Solvents are indicated in the
text and the chemical shifts are reported in ppm with
internal reference to TMS. Mass spectra (MS) were
recorded on a Micromass 1212 spectrometer. Infrared
(IR) spectra were recorded in the range 4000–600 cmꢀ1
using a Perkin-Elmer 298 infrared spectrometer. Spectra
of liquids were taken as films of CHCl3 solutions
between NaCl plates and spectra of solids with KBr
disks. Melting points (mp) were determined with a
capillary tube Thomas Hoover melting point apparatus
and are reported as uncorrected values. Flash chroma-
tography was carried out on silica gel (200–430 mesh)
obtained from Silicycle. The starting compounds were
obtained from Sigma-Aldrich.
General amide rearrangement procedure A: synthesis of
compounds 1a–2a. To a solution of 6.45 g of bis(tri-
fluoroacetoxy)phenyliodine (15 mmol) in dimethylfor-
mamide/water (60 mL; 1:1 v/v) were added at room
temperature 2.66 g of Na-carbobenzyloxy-l-asparagine
or Na-carbobenzyloxy-l-glutamine (10 mmol). After
15 min, 1.6 mL of pyridine (20 mmol) was added and
Scheme 2.

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P. de Macedo et al. / Bioorg. Med. Chem. 10 (2002) 355–360
357
stirring was continued for 3 h. The solvents were
removed under reduced pressure and the residue was
dissolved in water (100 mL). The solution was washed
with diethyl ether and the aqueous layer was evaporated
under reduced pressure. Crude compounds 1a or 2a
were dissolved in ethanol and precipitated with ether.
The precipitate was filtered and washed with ether to
give pure product.
A to give the product as a white powder (80% yield, mp
212 ꢁC). 1H NMR (D2O, 400 MHz) d 7.21–7.13 (m, 5H),
4.38 (s, 2H), 3.98 (t, 1H, J=10.0 Hz), 3.55 (d, 2H,
J=10.0 Hz); 13C NMR (D2O, 100 MHz) d 178.59,
158.76, 137.14, 129.53, 129.11, 128.51, 67.71, 59.72,
43.58. IR nmax (cmꢀ1) 3130 (OH); 3050 (NH2); 1751,
1690 (C¼O). MS (FAB+) 239.1 (MH+).
Nꢀ-Carbobenzyloxy-L-2,4-diaminobutyricacid (2a). This
compound was prepared using general procedure A to
give the product as a white powder (81% yield, p
191 ꢁC). 1H NMR (D2O, 400 MHz) d 7.25–7.14 (m, 5H),
4.40 (s, 2H), 3.94 (t, 1H, J=9.9 Hz), 3.59 (t, 2H,
J=5.0 Hz), 2.11 (td, 2h, J=9.8 Hz, J=5.0 Hz); 13C
NMR (D2O, 100 MHz) d 178.52, 158.57, 137.09, 129.51,
129.12, 128.47, 67.77, 54.68, 37.48, 30.50. IR nmax
(cmꢀ1) 3115 (OH); 3010 (NH2); 1748, 1692 (C¼O). MS
(FAB+) 253.1 (MH+)
General acryloyl amidation procedure B: synthesis of
compounds 1b–4b. In 300 mL of methanol 10 mmol of
the corresponding amino derivatives 1a–4a was dis-
solved under nitrogen. The solution was cooled to 0 ꢁC.
After the addition of 14 mL of triethylamine
(100 mmol), 34.8 mL of acryloyl chloride (60 mmol) was
added dropwise. The mixture was allowed to warm to
room temperature overnight with stirring. The reaction
mixture was then evaporated under reduced pressure
and the residue was dissolved in water (100 mL). The
pH was adjusted around to 1.5 with 1 M HCl, and
the product was extracted with ethyl acetate (3ꢂ100 mL).
The organic layer was dried over MgSO4, filt ered and
concentrated under reduced pressure to give pure product.
Nꢀ-Carbobenzyloxy-L-2-amino-3-acryloylaminopropionic
acid (1b). This compound was prepared using general
procedure B to give the product as a light-yellow oil
(73% yield). 1H NMR (CDCl3, 400 MHz) d 9.93 (s, 1H),
7.30–7.26 (m, 5H), 7.03 (s, 1H), 6.21 (t, 1H, J=8.1 Hz),
6.07 (d, 2H, J=8.2 Hz), 5.51 (s, 1H), 5.03 (s, 2H), 4.27
(t, 1H, J=5.3 Hz), 3.27 (d, 2H, J=5.3 Hz); 13C NMR
(CDCl3, 100 MHz) d 172.80, 167.65, 156.58, 136.07,
130.10, 128.49, 128.15, 127.98, 127.59, 67.09, 54.61,
41.32. IR nmax (cmꢀ1) 3310 (OH); 1746, 1690, 1663
(C¼O). MS (FAB+) 293.1 (MH+).
General peptide coupling procedure C: synthesis of com-
pounds 1c–4c. Compounds 1b–4b (6 mmol) were acti-
vated by reaction with 1.15 g of EDC hydrochloride
(6 mmol) and 0.92 mL of triethylamine (6.6 mmol) in
dichloromethane (50 mL). After 15 min, 1.00 g of glycine
tert-butyl ester hydrochloride (6 mmol) was added and
the mixture was stirred overnight at room temperature.
The mixture was then washed with water (3ꢂ100 mL)
and the organic layer was dried over MgSO4, filtered,
and concentrated under reduced pressure. The crude
productwas purified by column chromatography (ethyl
acetate eluant) to give pure product.
Nꢀ-Carbobenzyloxy-L-2-amino-4-acryloylaminobutyric
acid (2b). This compound was prepared using general
procedure B to give the product as a light orange oil
(80% yield). 1H NMR (CDCl3, 400 MHz) d 9.94 (s, 1H),
7.27–7.23 (m, 5H), 7.02 (s, 1H), 6.19 (t, 1H, J=7.9 Hz),
6.05 (d, 2H, J=7.9 Hz), 5.48 (s, 1H), 5.05 (s, 2H), 4.27
(t, 1H, J=4.8 Hz), 3.23 (t, 2H, J=4.7 Hz), 2.11 (td, 2H,
J=4.8 Hz, J=4.8 Hz); 13C NMR (CDCl3, 100 MHz)
d 174.18, 167.01, 156.70, 136.10, 130.43, 128.44, 128.08,
127.84, 126.97, 66.97, 51.80, 36.07, 31.75. IR nmax
(cmꢀ1) 3314 (OH); 1743, 1694, 1669 (C¼O). MS
(FAB+) 307.1 (MH+).
General tert-butyl ester deprotection procedure D: syn-
thesis of compounds 1d–4d. Compounds 1c–4c (3 mmol)
were dissolved in dichloromethane (50 mL). Gaseous
hydrochloric acid was bubbled through the solution for
4 h at room temperature. The solvent was removed
under reduced pressure to give pure product.
General epoxidation procedure E: synthesis of com-
pounds 1e–4e, 1f–4f, 1g–4g. DMDO was prepared by
distillation under reduced pressure from a mixture of
58 g of NaHCO3 (690 mmol), 120 g of OXONE1
(potassium peroxymonosulfate, 178 mmol) in water
(250 mL) and acetone (192 mL) using a dry ice trap. A
large excess of a freshly prepared 2 M solution of
DMDO in acetone was then added to compounds 1b–
4b, 1c–4c, and 1d–4d (1 mmol) in order to complete the
epoxidation. The mixture was stirred for 5 h at 0 ꢁC and
then allowed to warm to room temperature overnight
with stirring. The solvent and any excess DMDO were
subsequently removed under reduced pressure to give
pure product.
Nꢀ-Carbobenzyloxy-L-2-amino-5-acryloylaminopentanoic
acid (3b). This compound was prepared using general
procedure B to give the product as a white powder
(83% yield, mp 60 ꢁC). H NMR (CDCl3, 400 MHz) d
1
9.95 (s, 1H), 7.30–7.26 (m, 5H), 7.01 (s, 1H), 6.17 (t, 1H,
J=8.1 Hz), 6.01 (d, 2H, J=7.9 Hz), 5.50 (s, 1H), 5.02 (s,
2H), 4.25 (t, 1H, J=4.8 Hz), 3.20 (t, 2H, J=4.7 Hz),
1.75–1.35 (m, 4H); 13C NMR (CDCl3, 100 MHz)
d 172.08, 164.22, 153.94, 133.66, 128.05, 125.93, 125.57,
125.37, 124.25, 64.37, 51.03, 36.59, 26.99, 22.66. IR nmax
(cmꢀ1) 3310 (OH); 1744, 1691, 1668 (C¼O). MS
(FAB+) 321.1 (MH+).
Nꢀ-Carbobenzyloxy-L-2-amino-6-acryloylaminohexanoic
acid (4b). This compound was prepared using general
procedure B to give the product as an orange powder
Spectral data
(90% yield, mp 65 ꢁC). H NMR (CDCl3, 400 MHz) d
1
Nꢀ-Carbobenzyloxy-L-2,3-diaminopropioniciadc (1a).
This compound was prepared using general procedure
9.93 (s, 1H), 7.30–7.27 (m, 5H), 7.04 (s, 1H), 6.17 (t, 1H,
J=7.9 Hz), 6.02 (d, 2H, J=7.9 Hz), 5.50 (s, 1H), 5.04 (s,

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P. de Macedo et al. / Bioorg. Med. Chem. 10 (2002) 355–360
358
2H), 4.27 (t, 1H, J=4.6 Hz), 3.21 (t, 2H, J=4.5 Hz),
1.81–1.26 (m, 6H); 13C NMR (CDCl3, 100 MHz)
d 174.68, 166.51, 156.34, 136.08, 130.51, 128.34, 127.97,
127.77, 126.56, 66.77, 53.57, 39.11, 31.58, 28.44, 22.35.
IR nmax (cmꢀ1): 3305 (OH); 1740, 1691, 1665 (C¼O).
MS (FAB+) 335.1 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-3-acryloylamino)propio-
nylglycine (1d). This compound was prepared using
general procedure D to give the product as a soft white
powder (100% yield, mp 115 ꢁC). H NMR (CD3OD,
1
400 MHz) d 7.35–7.28 (m, 5H), 6.17 (d, 2H, J=8.0 Hz),
5.55 (t, 1H, J=8.1 Hz), 5.08 (s, 2H), 4.21 (t, 1H,
J=6.5 Hz), 3.72 (s, 2H), 3.21 (d, 2H, J=6.6 Hz); 13C
NMR (CD3OD, 100 MHz) d 173.10, 171.87, 168.90,
158.28, 137.98, 131.82, 129.64, 129.19, 128.99, 127.68,
67.96, 56.50, 42.13, 41.26. IR nmax (cmꢀ1) 3318 (OH);
1745, 1689, 1658, 1649 (C¼O). MS (FAB+) 350.1
(MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-3-acryloylamino)propio-
nylglycine tert-butyl ester (1c). This compound was pre-
pared using general procedure C to give the product as a
softwhiet powder (70% yield, mp 70 ꢁC). 1H NMR
(CDCl3, 400 MHz) d 7.30–7.22 (m, 5H), 6.82 (s, 1H),
6.21 (t, 1H, J=8.2 Hz), 6.09 (d, 2H, J=8.1 Hz), 5.49 (s,
1H), 5.47 (s, 1H), 5.01 (s, 2H), 4.19 (t, 1H, J=5.7 Hz),
3.91 (s, 2H), 3.33 (d, 2H, J=5.9 Hz), 1.47 (s, 9H); 13C
NMR (CDCl3, 100 MHz) d 170.96, 168.80, 166.76,
156.35, 136.21, 130.75, 128.23, 128.06, 127.67, 126.18,
(Nꢀ-Carbobenzyloxy-L-2-amino-4-acryloylamino)butyryl-
glycine (2d). This compound was prepared using gen-
eral procedure D to give the product as a soft-white
powder (100% yield, mp 124 ꢁC). H NMR (CD3OD,
1
81.71, 66.57, 55.36, 41.26, 41.29, 27.77. IR nmax (cmꢀ1
)
400 MHz) d 7.34–7.29 (m, 5H), 6.19 (d, 2H, J=8.3 Hz),
5.60 (t, 1H, J=8.3 Hz), 5.04 (s, 2H), 4.18 (t, 1H,
J=6.7 Hz), 3.72 (s, 2H), 3.20 (t, 2H, J=6.5 Hz), 2.18
(td, 2H, J=6.4 Hz, J=6.6 Hz); 13C NMR (CD3OD,
100 MHz) d 175.00, 172.88, 168.60, 158.37, 137.99,
131.64, 129.62, 129.17, 128.96, 127.67, 67.93, 54.40,
42.03, 37.50, 32.86. IR nmax (cmꢀ1) 3311 (OH); 1749,
1692, 1660, 1651 (C¼O). MS (FAB+) 364.2 (MH+).
1753, 1739, 1657, 1648 (C¼O). MS (FAB+) 406.2
(MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-4-acryloylamino)butyryl-
glycine tert-butyl ester (2c). This compound was pre-
pared using general procedure C to give the product as a
white powder (80% yield, mp 120 ꢁC). 1H NMR
(CDCl3, 400 MHz) d 7.28–7.21 (m, 5H), 6.80 (s, 1H),
6.18 (t, 1H, J=8.2 Hz), 6.09 (d, 2H, J=8.0 Hz), 5.48
(s, 1H), 5.45 (s, 1H), 5.00 (s, 2H), 4.18 (t, 1H,
J=5.6 Hz), 3.89 (s, 2H), 3.30 (t, 2H, J=6.1 Hz), 1.93
(td, 2H, J=5.8 Hz, J=5.7 Hz), 1.45 (s, 9H); 13C
NMR (CDCl3, 100 MHz) d 172.14, 168.74, 166.47,
156.29, 136.23, 130.86, 128.38, 127.97, 126.19, 81.85,
(Nꢀ-Carbobenzyloxy-L-2-amino-5-acryloylamino)pentan-
oylglycine (3d). This compound was prepared using
general procedure D to give the product as a soft white
powder (100% yield, mp 144 ꢁC). H NMR (CD3OD,
1
400 MHz) d 7.36–7.30 (m, 5H), 6.23 (d, 2H, J=8.5 Hz),
5.69 (t, 1H, J=8.4 Hz), 5.07 (s, 2H), 4.14 (t, 1H,
J=6.3 Hz), 3.74 (s, 2H), 3.19 (t, 2H, J=6.5 Hz), 1.98–
1.42 (m, 4H). 13C NMR (CD3OD, 100 MHz) d 175.32,
171.67, 168.72, 158.30, 138.13, 131.18, 129.70, 129.23,
129.00, 128.45, 67.84, 56.10, 42.10, 40.55, 30.83, 26.55.
IR nmax (cmꢀ1) 3313 (OH); 1747, 1694, 1662, 1654
(C¼O). MS (FAB+) 378.1 (MH+).
66.74, 53.45, 41.94, 35.86, 27.91. IR nmax (cmꢀ1
)
1759, 1746, 1664, 1651 (C¼O). MS (FAB+) 420.2
(MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-5-acryloylamino)penta-
noylglycine tert-butyl ester (3c). This compound was
prepared using general procedure C to give the product
as a yellow powder (85% yield, mp 84 ꢁC). H NMR
(Nꢀ-Carbobenzyloxy-L-2-amino-6-acryloylamino)hexano-
ylglycine (4d). This compound was prepared using gen-
eral procedure D to give the product as a light green
1
(CDCl3, 400 MHz) d 7.29–7.18 (m, 5H), 6.77 (s, 1H),
6.17 (t, 1H, J=8.0 Hz), 6.06 (d, 2H, J=8.1 Hz), 5.47 (s,
1H), 5.44 (s, 1H), 5.02 (s, 2H), 4.18 (t, 1H, J=5.0 Hz),
3.80 (s, 2H), 3.24 (t, 2H, J=6.0 Hz), 1.95–1.27 (m, 13H).
13C NMR (CDCl3, 100 MHz) d 170.20, 166.23, 163.59,
153.84, 133.73, 128.55, 125.76, 125.33, 125.21, 123.19,
79.12, 64.03, 51.63, 39.27, 35.94, 27.41, 25.29, 22.81. IR
nmax (cmꢀ1) 1755, 1746, 1662, 1654 (C¼O). MS
(FAB+) 434.1 (MH+).
powder (100% yield, mp 116 ꢁC). H NMR (CD3OD,
1
400 MHz) d 7.36–7.30 (m, 5H), 6.22 (d, 2H, J=8.6 Hz),
5.64 (t, 1H, J=8.4 Hz), 5.09 (s, 2H), 4.15 (t, 1H,
J=6.5 Hz), 3.72 (s, 2H), 3.26 (t, 2H, J=6.5 Hz), 1.90–
1.40 (m, 6H); 13C NMR (CD3OD, 100 MHz) d 175.59,
171.67, 168.68, 158.33, 138.11, 131.20, 129.68, 129.22,
128.96, 128.33, 67.81, 56.51, 42.06, 40.74, 32.96, 29.71,
24.17. IR nmax (cmꢀ1) 3310 (OH); 1745, 1690, 1660,
1650 (C¼O). MS (FAB+) 392.4 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-6-acryloylamino)hexan-
oylglycine tert-butyl ester (4c). This compound was pre-
pared using general procedure C to give the product as a
white powder (93% yield, mp 116 ꢁC). 1H NMR
(CDCl3, 400 MHz) d 7.30–7.19 (m, 5H), 6.78 (s, 1H),
6.17 (t, 1H, J=8.1 Hz), 6.08 (d, 2H, J=8.0 Hz), 5.49 (s,
1H), 5.45 (s, 1H), 5.02 (s, 2H), 4.20 (t, 1H, J=5.3 Hz),
3.80 (s, 2H), 3.26 (t, 2H, J=6.2 Hz), 1.96–1.20 (m, 15H).
13C NMR (CDCl3, 100 MHz) d 172.55, 168.67, 165.91,
156.34, 136.10, 130.93, 128.27, 127.88, 127.73, 125.78,
81.76, 66.62, 54.65, 41.76, 38.68, 31.82, 28.57, 27.80,
22.38. IR nmax (cmꢀ1) 1752, 1745, 1660, 1652 (C¼O).
MS (FAB+) 448.2 (MH+).
Nꢀ-Carbobenzyloxy-L-2-amino-3-(oxiranecarbonylamino)-
propionicacid (1e). This compound was prepared using
general procedure E to give the product as a light-yel-
low oil (100% yield). 1H NMR (CD3OD, 400 MHz)
d 7.33–7.29 (m, 5H), 5.12 (s, 2H), 4.21 (t, 1H,
J=6.9 Hz), 3.33 (t, 1H, J=4.7 Hz), 3.25 (d, 2H,
J=7.1 Hz), 2.80 (d, 2H, J=4.6 Hz); 13C NMR
(CD3OD, 100 MHz) d 173.43, 172.08, 158.45, 138.04,
129.57, 129.12, 128.94, 67.83, 55.15, 49.99, 47.64, 41.25.
IR nmax (cmꢀ1) 3340 (OH); 1750, 1700, 1686 (C¼O).
MS (FAB+) 309.1 (MH+).

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P. de Macedo et al. / Bioorg. Med. Chem. 10 (2002) 355–360
359
Nꢀ-Carbobenzyloxy-L-2-amino-4-(oxiranecarbonylamino)-
butyricaicd (2e). This compound was prepared using
general procedure E to give the product as a light
41.93, 35.45, 32.97, 27.93. IR nmax (cmꢀ1) 1752, 1747,
1684, 1675 (C¼O). MS (FAB+) 436.2 (MH+).
1
orange oil (100% yield). H NMR (CD3OD, 400 MHz)
(Nꢀ-Carbobenzyloxy-L-2-amino-5-(oxiranecarbonylami-
no))pentanoylglycine tert-butyl ester (3f). This com-
pound was prepared using general procedure E to give
the product as a dark yellow oil (100% yield). 1H NMR
(CDCl3, 400 MHz) d 7.35–7.30 (m, 5H), 6.79 (s, 1H),
6.73 (s, 1H), 5.98 (s, 1H), 5.07 (s, 2H), 4.11 (t, 1H,
J=7.1 Hz), 3.84 (s, 2H), 3.46 (t, 1H, J=4.5 Hz), 3.10 (t,
2H, J=7.0 Hz), 2.79 (d, 2H, J=4.5 Hz), 1.90–1.41 (m,
13H); 13C NMR (CDCl3, 100 MHz) d 173.06, 169.36,
170.30, 156.35, 136.10, 128.55, 128.10, 127.94, 82.86,
67.76, 55.95, 49.99, 47.50, 42.83, 39.49, 30.63, 28.41,
26.73. IR nmax (cmꢀ1) 1755, 1745, 1680, 1672 (C¼O).
MS (FAB+) 450.3 (MH+).
d 7.35–7.32 (m, 5H), 5.10 (s, 2H), 4.20 (t, 1H,
J=7.0 Hz), 3.34 (t, 1H, J=4.6 Hz), 3.28 (t, 2H,
J=7.2 Hz), 2.85 (d, 2H, J=4.6 Hz), 2.09 (td, 2H,
J=7.0 Hz, J=7.1 Hz); 13C NMR (CD3OD, 100 MHz) d
175.52, 171.66, 171.58, 158.61, 138.08, 129.63, 129.16,
128.93, 67.86, 53.13, 50.14, 47.74, 36.94, 32.16. IR nmax
(cmꢀ1) 3330 (OH); 1743, 1690, 1681 (C¼O). MS
(FAB+) 323.1 (MH+).
Nꢀ-Carbobenzyloxy-L-2-amino-5-(oxiranecarbonylamino)-
pentanoicacid (3e). This compound was prepared using
general procedure E to give the product as a soft-white
powder (100% yield, mp 102 ꢁC). H NMR (CD3OD,
1
400 MHz) d 7.37–7.29 (m, 5H), 5.09 (s, 2H), 4.21 (t, 1H,
J=7.1 Hz), 3.36 (t, 1H, J=4.8 Hz), 3.26 (t, 2H,
J=7.1 Hz), 2.86 (d, 2H, J=4.7 Hz), 1.85–1.42 (m, 4H);
13C NMR (CD3OD, 100 MHz) d 176.18, 171.62, 158.70,
137.98, 129.72, 129.28, 128.98, 67.90, 55.19, 50.17,
47.85, 39.70, 30.03, 26.79. IR nmax (cmꢀ1) 3332 (OH);
1741, 1698, 1683 (C¼O). MS (FAB+) 337.1 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-6-(oxiranecarbonylami-
no))hexanoylglycine tert-butyl ester (4f). This com-
pound was prepared using general procedure E to give
the product as a light orange oil (100% yield). 1H NMR
(CDCl3, 400 MHz) d 7.33–7.29 (m, 5H), 6.81 (s, 1H),
6.75 (s, 1H), 5.86 (s, 1H), 5.09 (s, 2H), 4.15 (t, 1H,
J=7.0 Hz), 3.83 (s, 2H), 3.42 (t, 1H, J=4.6 Hz), 3.09 (t,
2H, J=7.0 Hz), 2.82 (d, 2H, J=4.6 Hz), 1.87–1.35 (m,
15H); 13C NMR (CDCl3, 100 MHz) d 172.38, 168.79,
168.64, 156.24, 136.09, 128.25, 127.87, 127.72, 81.84,
66.66, 54.43, 49.21, 47.05, 41.75, 38.21, 31.80, 28.50,
27.76, 22.26. IR nmax (cmꢀ1) 1760, 1749, 1682, 1667
(C¼O). MS (FAB+) 464.4 (MH+).
Nꢀ-Carbobenzyloxy-L-2-amino-6-(oxiranecarbonylamino)-
hexanoicacid (4e). This compound was prepared using
general procedure E to give the product as a white
powder (100% yield, mp 95 ꢁC). 1H NMR (CD3OD,
400 MHz) d 7.37–7.27 (m, 5H), 5.10 (s, 2H), 4.22 (t, 1H,
J=7.0 Hz), 3.33 (t, 1H, J=4.9 Hz), 3.25 (t, 2H,
J=6.9 Hz), 2.82 (d, 2H, J=4.9 Hz), 1.85–1.38 (m, 6H);
13C NMR (CD3OD, 100 MHz) d 175.36, 171.70, 158.34,
138.13, 129.58, 129.13, 128.94, 67.78, 56.32, 50.03,
47.51, 39.82, 32.96, 29.91, 23.97. IR nmax (cmꢀ1) 3312
(OH); 1744, 1701, 1689 (C¼O). MS (FAB+) 351.2
(MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-3-(oxiranecarbonylami-
no))propionylglycine (1g). This compound was prepared
using general procedure E to give the product as a light-
1
yellow oil (100% yield). H NMR (CD3OD, 400 MHz)
d 7.39–7.33 (m, 5H), 5.13 (s, 2H), 4.11 (t, 1H,
J=6.6 Hz), 3.81 (s, 2H), 3.35 (t, 1H, J=4.6 Hz), 3.20 (d,
2H, J=6.6 Hz), 2.79 (d, 2H, J=4.6 Hz); 13C NMR
(CD3OD, 100 MHz) d 172.86, 172.10, 171.79, 158.24,
137.99, 129.62, 129.20, 129.06, 67.97, 52.95, 50.11.
47.74, 42.07, 41.18. IR nmax (cmꢀ1) 3320 (OH); 1751,
1718, 1682, 1660 (C¼O). MS (FAB+) 366.1 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-3-(oxiranecarbonylami-
no))propionylglycine tert-butyl ester (1f). This com-
pound was prepared using general procedure E to give
the product as a white powder (100% yield, mp 80 ꢁC).
1H NMR (CDCl3, 400 MHz) d 7.35–7.30 (m, 5H), 6.80
(s, 1H), 6.74 (s, 1H), 5.91 (s, 1H), 5.10 (s, 2H), 4.10 (t,
1H, J=7.0 Hz), 3.84 (s, 2H), 3.46 (t, 1H, J=4.5 Hz),
3.02 (d, 2H, J=7.1 Hz), 2.79 (d, 2H, J=4.6 Hz), 1.48 (s,
9H); 13C NMR (CDCl3, 100 MHz) d 170.96, 168.96,
168.80, 156.46, 136.19, 128.41, 128.02, 127.86, 82.03,
66.88, 49.22, 46.96, 41.98, 41.87, 27.91, 27.68. IR nmax
(cmꢀ1) 1761, 1748, 1680, 1663 (C¼O). MS (FAB+)
422.2 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-4-(oxiranecarbonylami-
no))butyrylglycine (2g). This compound was prepared
using general procedure E to give the product as a light-
yellow powder (100% yield, mp 111 ꢁC). 1H NMR
(CD3OD, 400 MHz) d 7.38–7.32 (m, 5H), 5.12 (s, 2H),
4.13 (t, 1H, J=6.7 Hz), 3.79 (s, 2H), 3.28 (t, 1H,
J=4.5 Hz), 3.23 (t, 2H, J=6.6 Hz), 2.81 (d, 2H,
J=4.6 Hz), 2.12 (td, 2H, J=6.5 Hz, J=6.5 Hz); 13C
NMR (CD3OD, 100 MHz) d 174.82, 171.76, 171.56,
158.34, 138.03, 129.57, 129.13, 128.97, 67.90, 54.12,
50.06, 47.59, 41.96, 36.80, 32.95. IR nmax (cmꢀ1) 3333
(OH); 1750, 1717, 1686, 1674 (C¼O). MS (FAB+)
380.2 (MH+).
(Nꢀ-Carbobenzyloxy-L-2-amino-4-(oxiranecarbonylami-
no))butyrylglycine tert-butyl ester (2f). This compound
was prepared using general procedure E to give the
productas a white powder (100% yield, mp 118 ꢁC). 1H
NMR (CDCl3, 400 MHz) d 7.34–7.29 (m, 5H), 6.79 (s,
1H), 6.72 (s, 1H), 5.90 (s, 1H), 5.09 (s, 2H), 4.10 (t, 1H,
J=7.0 Hz), 3.84 (s, 2H), 3.45 (t, 1H, J=4.6 Hz), 3.08 (t,
2H, J=7.1 Hz), 2.77 (d, 2H, J=4.6 Hz), 1.90 (td, 2H,
J=7.0 Hz, J=7.1 Hz), 1.46 (s, 9H); 13C NMR (CDCl3,
100 MHz) d 171.96, 169.84, 168.71, 156.34, 136.21,
128.41, 128.02, 127.83, 81.98, 66.84, 52.21, 49.34, 47.15,
(Nꢀ-Carbobenzyloxy-L-2-amino-5-(oxiranecarbonylami-
no))pentanoylglycine (3g). This compound was prepared
using general procedure E to give the product as a dark-
yellow oil (100% yield). 1H NMR (CD3OD, 400 MHz) d
7.35–7.30 (m, 5H), 5.08 (s, 2H), 4.15 (t, 1H, J=6.5 Hz),
3.82 (s, 2H), 3.39 (t, 1H, J=4.2 Hz), 3.19 (t, 2H,

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P. de Macedo et al. / Bioorg. Med. Chem. 10 (2002) 355–360
360
J=6.6 Hz), 2.88 (d, 2H, J=4.3 Hz), 1.89–1.35 (m, 4H);
13C NMR(CD3OD, 100 MHz) d 175.26, 171.78, 171.44,
158.35, 138.07, 129.58, 129.14, 128.95, 67.81, 55.97,
17. Schroeder, W. T.; Thatcher, S. M.; Stewart-Galetka, S.;
Annarella, M.; Chema, D.; Siciliano, M. J.; Davies, P. J. A.;
Tang, H. Y.; Sowa, B. A.; Duvic, M. J. Invest. Dermatol. 1992,
99, 27.
18. Wolf, R.; Schiavo, A. L. Int. J. Dermatol. 1997, 36, 10.
19. Selkoe, D. J.; Ihara, Y.; Salazar, F. Science 1982, 215,
1243.
50.05, 47.58, 42.04, 41.94, 30.63, 26.67. IR nmax (cmꢀ1
)
3335 (OH); 1745, 1725, 1677, 1669 (C¼O). MS (FAB+)
394.2 (MH+).
20. Selkoe, D. J.; Abraham, C.; Ihara, Y. Proc. Natl. Acad.
Sci. U.S.A. 1982, 79, 6070.
21. Schlaepfer, W. In Biological Aspects of Alzheimer’s Dis-
ease, Katzman, R., Ed.; Cold Spring Harbor Laboratory:
Cold Spring Harbor, 1983; p 107.
(Nꢀ-Carbobenzyloxy-L-2-amino-6-(oxiranecarbonylami-
no))hexanoylglycine (4g). This compound was prepared
using general procedure E to give the product as a light-
1
yellow oil (100% yield). H NMR (CD3OD, 400 MHz)
22. Farmer, P. M.; Peck, A.; Terry, R. D. J. Neuropathol.
Exp. Neurol. 1976, 35, 367.
23. Amendola, A.; Lombardi, G.; Oliverio, S.; Colizzi, V.;
Piacentini, M. FEBS Lett. 1994, 339, 258.
24. Benzinger, T. L. S.; Gregory, D. M.; Burkoth, T. S.;
Miller-Auer, H.; Lynn, D. G.; Botto, R. E.; Meredith, S. C.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13407.
25. Norlund, M. A.; Lee, J. M.; Zainelli, G. M.; Muma, N. A.
Brain Res. 1999, 851, 154.
d 7.36–7.33 (m, 5H), 5.09 (s, 2H), 4.15 (t, 1H,
J=6.8 Hz), 3.80 (s, 2H), 3.32 (t, 1H, J=4.3 Hz), 3.20 (t,
2H, J=6.5 Hz), 2.78 (d, 2H, J=4.5 Hz), 1.92–1.37 (m,
6H); 13C NMR (CD3OD, 100 MHz) d 175.36, 171.70,
171.18, 158.34, 138.13, 129.58, 129.13, 128.94, 67.78,
56.32, 50.03, 47.51, 41.93, 39.82, 32.96, 29.91, 23.97. IR
nmax (cmꢀ1) 3340 (OH); 1750, 1720, 1680, 1670 (C¼O).
MS (FAB+) 410.2 (MH+).
26. Folk, J. E.; Finlayson, J. S. Adv. Protein Chem. 1977, 31,
1.
27. Rich, D. H. In Proteinase Inhibitors; Barret, A. J., Salve-
sen, G., Eds.; Elsevier: New York, 1986, p 153.
28. Lorand, L.; Rule, N. G.; Ong, H. H.; Furlanetto, R.;
Jacobsen, A.; Downey, J.; Oner, N.; Bruner-Lorand, J. Bio-
chemistry 1968, 7, 1214.
Acknowledgements
The authors gratefully acknowledge the financial sup-
port of Apotex Research, Inc. and the Natural Sciences
and Engineering Research Council of Canada
(NSERC).
29. Stenberg, P.; Nilsson, L.; Erickson, O.; Lunden, R. Acta
Pharm. Suec. 1971, 8, 415.
30. Holbrook, J. J.; Cooke, R. D.; Kingston, I. B. Biochem. J.
1973, 135, 901.
31. Chung, S. I.; Lewis, M. S.; Folk, J. E. J. Biol. Chem. 1974,
249, 940.
References and Notes
32. Curtis, C. G.; Brown, K. L.; Credo, R. B.; Domanik,
R. A.; Gray, A.; Stenberg, P.; Lorand, L. Biochemistry 1974,
13, 3774.
1. Levitzki, A.; Willingham, M.; Pastan, I. H. Proc. Natl.
Acad. Sci. U.S.A. 1980, 77, 2706.
2. Davies, P. J.; Davies, D. R.; Levitzki, A.; Maxfield, F. R.;
Milhaud, P.; Willingham, M. C.; Pastan, I. H. Nature (Lon-
don) 1980, 283, 162.
3. Fesus, L.; Thomazy, V.; Falus, A. FEBS Lett. 1987, 224,
104.
4. Birckbichler, P. J.; Orr, G. R.; Patterson, M. K.; Conway,
E.; Carter, H. A.; Maxwell, M. D. Biochim. Biophys. Acta
1983, 763, 27.
5. Matacic, S.; Loewy, A. G. Biochim Biophys Res. Commun.
1968, 30, 356.
6. Lorand, L. Ann N. Y. Acad. Sci. 1972, 202, 6.
7. Pisano, J. J.; Finlayson, J. S.; Peyton, M. P. Science 1968,
160, 892.
8. Rice, R. H.; Green, H. Cell 1979, 18, 681.
9. Kim, S. Y.; Chung, S. I.; Steinert, P. J. Biol. Chem. 1995,
270, 18026.
10. Nemes, Z.; Demeny, M.; Marekov, L. N.; Fesus, L.; Stei-
ner, P. M. J. Biol. Chem. 2000, 275, 2636.
11. Candi, E.; Tarcsa, E.; Digiovanna, J. J.; Compton, J. G.;
Elias, P. M.; Marekov, L. N.; Steinert, P. M. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 2067.
33. Gross, M.; Whetzel, N. K.; Folk, J. E. J. Biol. Chem.
1975, 250, 7693.
34. Lee, K. N.; Fesus, L.; Yancey, S. T.; Girard, J. E.; Chung,
S. I. J. Biol. Chem. 1985, 260, 14689.
35. Castelhano, A. L.; Billedeau, R.; Pliura, D. H.; Bona-
ventura, B. J.; Krantz, A. Bioorg. Chem. 1988, 16, 335.
36. Auger, M.; McDermott, A. E.; Robinson, V.; Casthel-
hano, A. L.; Billedeau, R. J.; Pliura, D. H.; Krantz, A.; Grif-
fin, R. G. Biochemistry 1993, 32, 3930.
37. Pliura, D. H.; Bonaventura, B. J.; Pauls, H. W.; Killackey,
J. F.; Krantz, A. J. Enzyme Inhib. 1992, 6, 181.
38. Freund, K. F.; Doshi, K. P.; Gaul, S. L.; Claremon, D. A.;
Remy, D. C.; Baldwin, J. J.; Pitzenberger, S. M.; Stern, A. M.
Biochemistry 1994, 33, 10109.
39. Doyle, P. M.; Harris, C. J.; Carter, K. R.; Simpkin,
D. S. A.; Bailey-Smith, P.; Stone, D.M; Russel, L.; Blackwell,
G. J. Biochem. Soc. Trans. 1990, 18, 1318.
40. Folk, J. E. Adv. Enzymol. Relat. Areas Mol. Biol. 1983, 54,
1.
41. Folk, J. E.; Chung, S. I. Methods Enzymol. 1985, 113, 358.
42. Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266.
43. Adam, W.; Bialas, J.; Hadjiarapoglou, L. Chem. Ber.
1991, 124, 2377.
12. De Young, L.; Ballaron, S.; Epstein, W. J. Invest. Derma-
tol. 1984, 82, 275.
13. Dalziel, K.; Dykes, P. J.; Marks, R. Br. J. Exp. Pathol.
1984, 65, 107.
44. Adam, W.; Hadjiarapoglou, L.; Smerz, A. Chem. Ber.
1991, 124, 227.
14. Azari, P.; Rahim, J.; Clarkson, D. P. Curr. Eye Res. 1981,
463.
15. Fesus, L. Surv. Immunol. Res. 1982, 1, 297.
16. Bernard, B. A.; Reano, A.; Darmon, Y. M.; Thivolet, J.
Brit. J. Dermatol. 1986, 114, 279.
45. de Macedo, P.; Perie, J. J. Synth. Commun. 1998, 28, 2679.
46. de Macedo, P.; Marrano, C.; Keillor, J. W. Anal. Biochem.
2000, 285, 16.
47. Marrano, C.; de Macedo, P.; Keillor, J. W. Bioorg. Med.
Chem. 2001, 9, 1923.