Synthesis and evaluation of peptidic irreversible inhibitors of tissue transglutaminase

Article abstract of DOI:10.1016/j.bmc.2006.09.011

Herein we report the synthesis and the evaluation of eight novel compounds as irreversible inhibitors of transglutaminase (TGase). These compounds are based on a minimal peptidic scaffold shown previously [Chem. Biol. 2005, 12, 469-475] to confer affinity for the TGase active site and bear electrophilic groups such as α,β-unsaturated amide, chloroacetamide or maleimide; their general structure being Cbz-Phe-spacer-electrophile. The affinity conferred by the Cbz-Phe scaffold was determined by comparison to N-propylacrylamide and the length of the spacer was also varied to evaluate its importance. The inhibitory efficiencies (k_inact/K_I) of these compounds vary up to 10⁵ M^-1 min^-1, among the highest reported for derivatives based on this simple Cbz-Phe peptidic scaffold.

Full text of DOI:10.1016/j.bmc.2006.09.011

Bioorganic & Medicinal Chemistry 14 (2006) 8379–8385
Synthesis and evaluation of peptidic irreversible inhibitors of tissue
transglutaminase
Christophe Pardin, Steve M. F. G. Gillet and Jeffrey W. Keillor*
De´partement de chimie, Universite´ de Montre´al, C.P. 6128, Succursale Centre-ville, Montre´al, Que., Canada H3C 3J7
Received 8 June 2006; revised 29 August 2006; accepted 7 September 2006
Available online 27 September 2006
Abstract—Herein we report the synthesis and the evaluation of eight novel compounds as irreversible inhibitors of transglutaminase
(TGase). These compounds are based on a minimal peptidic scaffold shown previously [Chem. Biol. 2005, 12, 469–475] to confer
affinity for the TGase active site and bear electrophilic groups such as a,b-unsaturated amide, chloroacetamide or maleimide; their
general structure being Cbz-Phe-spacer-electrophile. The affinity conferred by the Cbz-Phe scaffold was determined by comparison
to N-propylacrylamide and the length of the spacer was also varied to evaluate its importance. The inhibitory efficiencies (kinact/KI)
of these compounds vary up to 105 Mꢀ1 minꢀ1, among the highest reported for derivatives based on this simple Cbz-Phe peptidic
scaffold.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In order to regulate excess TGase activity, a number of
potential TGase inactivators have been developed,
including dihydroisoxazole derivatives,1 gluten peptide
analogs,9 and dipeptide-bound a,b-unsaturated amides,
epoxides, and 1,2,4-thiadiazoles.10–12 Herein we report
the synthesis and the evaluation of eight novel com-
pounds as irreversible TGase inhibitors. The peptidic
scaffold (Cbz-Phe–) of these compounds was similar to
those of recently published TGase irreversible inhibi-
tors1 but bearing functional groups such as a,b-unsatu-
rated amide, chloroacetamide or maleimide, allowing
comparison of the relative efficiency of the different elec-
trophilic groups. The chain length of the spacer between
the phenylalanine and the electrophilic group was also
varied in order to permit investigation of its importance.
Finally, N-propylacrylamide was also synthesized and
evaluated to determine the significance of the Cbz-Phe
scaffold on TGase affinity.
Transglutaminases (EC 2.3.2.13) are a family of Ca2+
-
dependent enzymes that catalyze acyl transfer reactions.
Catalytic activity is exhibited toward the c-carboxamide
moiety of a protein- or peptide-bound glutamine residue
which serves as acyl donor to either protein- or peptide-
bound lysine residues or free amines that serve as acyl
acceptors, resulting in new intermolecular amidic cross-
links.2–4
Tissue transglutaminase has been identified as a con-
tributor to the formation of cataracts and to Celiac dis-
ease, and a growing body of evidence suggests that it
may be involved in atherosclerosis, inflammation,
fibrosis, diabetes, cancer metastases, autoimmune dis-
eases, lamellar ichthyosis, and psoriasis (for a review,
see Ref. 5). TGase is also suspected to play a role in
neurodegenerative diseases associated with an increase
in polyglut- amine-containing peptides in the brain
such as Huntington’s disease, Alzheimer disease,
Parkinson disease, and supranuclear palsy.6–8
2. Results and discussion
The starting point of our parallel syntheses is the com-
mercially available Cbz-Phe (1). The corresponding p-
nitrophenyl ester (2) was obtained in 68% isolated yield
according to a previously established procedure13 using
p-nitrophenyl chloroformate, triethylamine as a base,
and catalytic dimethylaminopyridine in acetonitrile
(Scheme 1). This activated Cbz-Phe ester (2) was then
coupled with different diamino alkanes in acetonitrile
Abbreviations: Cbz, carbobenzyloxy; DMAP, 4-dimethylaminopyri-
dine; DMF, dimethyl formamide; GABA, c-aminobutyric acid;
TGase, transglutaminase.
Keywords: Transglutaminase; Enzyme inhibition; Peptide.
*
Corresponding author. Tel.: +514 343 6219; fax: +514 343 7586;
e-mail: jw.keillor@umontreal.ca
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.011

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C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
O
O
O
H
N
a
b
O
O
NH2
( )n
O
N
H
O
N
H
O
N
H
OH
O
O
1
2
3a n=2
3b n=4
3c n=6
3d n=8
NO2
Scheme 1. Synthesis of the common amine scaffolds. Reagents and conditions: (a) p-nitrophenyl chloroformate, NEt3, DMAP, CH3CN, 50 min, rt.
(b) Diaminoalkane, CH3CN, 1 h, rt.
to give Cbz-Phe–NH–(CH2)n–NH2 (3a–3d, n = 2,4,6,8)
in 27–82% isolated yield.
it shows 80% homology with human tissue TGase,15
thereby validating its use as a model for the evaluation
of inhibitors of potential therapeutic utility.
From this common amine scaffold, compounds 4a–4d
were prepared by allowing acryloyl chloride to react
with compounds 3a–3d in the presence of triethylamine
in acetonitrile and obtained after purification in 29–46%
isolated yield. Alternatively, compounds 5a–5c and 9
were prepared by allowing chloroacetic anhydride to
react with compounds 3a–3c and 8 in the presence of
triethylamine in acetonitrile and obtained after purifica-
tion in 28–56% isolated yield. Furthermore, a maleimide
derivative was prepared by allowing maleic anhydride to
react with compound 3b in the presence of triethylamine
in dichloromethane to give the open form of maleimide
6b, which was then cyclized in the presence of acetic
anhydride and sodium acetate to give compound 7b in
37% isolated yield over the two reaction steps. Although
none of the reported yields were optimized, all final
compounds were easily obtained in sufficient yield to al-
low thorough kinetic evaluation as TGase inhibitors (see
Schemes 2–4).
Mono-exponential time-dependent inhibition was ob-
served for all compounds showing inhibitory activity,
consistent with consumption of the enzyme upon irre-
versible inactivation. Irreversibility was confirmed by
the lack of activity even after removal of excess inhibitor
by dilution. Kinetic evaluation of inhibitors was carried
out using direct continuous colorimetric15 and/or fluoro-
metric16 assays, giving comparable results. Apparent
inactivation rate constants were used to determine the
kinetic parameters kinact and KI (Scheme 5) as described
in Section 4.
Shown in Table 1 are the kinetic parameters determined
for a,b-unsaturated amides 4a–4d using a fluorogenic
substrate. The KI values were found to vary only slightly
across the series, from 3.5 to 13 lM. Khosla has previ-
ously reported the synthesis and evaluation of similar
inhibitors composed of the same Cbz-Phe scaffold but
bearing a dihydroisoxazole electrophile.1 The affinity
constants of these dihydroisoxazole derivatives are
ꢁ50-fold higher than compounds 4a–4d, suggesting the
larger cyclic electrophilic group may be less easily
accommodated by the sterically constrained active site
of TGase.17 We previously reported the synthesis11
and evaluation10 of a series of analogous inhibitors
where the same electrophilic group was introduced on
the side chain of a Cbz-Xaa-Gly peptidic framework
(Fig. 1). The affinity constants of those inhibitors are
roughly an order of magnitude lower than those of com-
pounds 4a–4d.
Recombinant guinea pig liver TGase was expressed in
Escherichia coli and subsequently purified according to
a procedure developed in our laboratories.14 In addition
to being easy to obtain in excellent yield and solubility,
guinea pig liver TGase was chosen for this study because
O
3a n=2
3b n=4
3c n=6
3d n=8
4a n=2
4b n=4
4c n=6
4d n=8
H
N
H
a
N
( )n
O
N
H
O
O
Furthermore, the inhibitory efficiencies (kinact/KI) of
compounds 4a–4d show the same trends, being 3–20
times lower than the inhibitors developed earlier in our
laboratories, and 4–50 times higher than Khosla’s corre-
sponding dihydroisoxazole inhibitors. The efficiency of
compounds 4a–4d also appears to increase slightly with
the length of the spacer. This tendency was observed for
three other series of irreversible inhibitors10,12 and may
be explained in terms of the structure of the acyl-donor
substrate binding site. TGase is designed to exclude
asparagine residues as acyl-donor substrates, reacting
only with glutamine residues. Molecular modeling of tis-
sue TGase17 suggests this specificity involves the binding
of peptide backbone of potential acyl-donor substrates
at a site sufficiently distant from the site of acylation
H
NH2
N
8
9
O
Scheme 2. Synthesis of a,b-unsaturated amide derivatives. Reagents
and condition: (a) acryloyl chloride, NEt3, CH3CN, overnight, rt.
O
H
H
3a n=2
3b n=4
3c n=6
5a n=2
5b n=4
5c n=6
a
N
N
( )n
O
N
Cl
H
O
O
Scheme 3. Synthesis of chloroacetamide derivatives. Reagents and
condition: (a) chloroacetic anhydride, NEt3, CH3CN, overnight, rt.

C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
8381
O
O
O
H
N
H
N
H
N
a
b
N
( )n
( )n
3b n=4
O
N
O
N
H
H
O
O
O
CO2H
O
6b n=4
7b n=4
Scheme 4. Synthesis of maleimide derivative. Reagents and conditions: (a) maleic anhydride, CH2Cl2, 1 h, rt. (b) Ac2O, NaOAc, 2 h, rt.
Table 2. Kinetic parameters determined for chloroacetamide inhibi-
tors 5a–5c
S
I
E + P
E•S
E-I
E
Inhibitor
n
kinact (minꢀ1
)
KI (lM) kinact/KI (lMꢀ1 minꢀ1
No inhibition observed
73 17 0.010 0.001
0.053 0.017
)
KI
5a
5b
5c
2
4
6
0.76 0.11
0.32 0.04
kinact
E•I
6
2
Scheme 5. Kinetic scheme used to evaluate irreversible inhibitors.
of Michael acceptor 4a. As such, its electrophilic group
may not be positioned to react efficiently with the active
site cysteine thiol group.
Table 1. Kinetic parameters determined for a,b-unsaturated amide
inhibitors 4a–4d
Inhibitor
n
kinact (minꢀ1
)
KI (lM) kinact/KI (lMꢀ1 minꢀ1
0.047 0.009
4.5 1.0 0.080 0.020
13 0.069 0.017
)
Compound 7b was evaluated using a chromogenic sub-
strate15 and time-dependent inhibition was observed.
However, its low solubility and relatively high inhibition
constant (KI > 160 lM) did not allow saturating condi-
tions to be achieved, nor its kinetic parameters to be
measured precisely. As was discussed above, the volume
of the maleimide moiety may lead to steric hindrance in
the narrow tunnel of the acyl-donor substrate binding
site.17
4a
4b
4c
4d
2
4
6
8
0.42 0.03
0.36 0.03
0.90 0.12
0.38 0.02
9
2
3
3.5 1.0 0.109 0.036
CO2H
O
NH
O
H
N
O
N
H
Subsequently, N-propylacrylamide 9 was prepared and
evaluated to see if the Cbz-Phe moiety was really re-
quired for recognition by TGase. Compound 9 was ob-
served to inactivate TGase and the rate of inactivation
showed linear dependence on its concentration up to
2 mM, consistent with the second-order reaction of 9
with TGase in solution. Hence it would appear the
Cbz-Phe moiety is essential for TGase affinity and spe-
cific inhibition, leading to the rapid pre-formation of a
discrete Michaelis complex and the saturation kinetics
observed for compounds 4, 5, and 7. As suggested pre-
viously,18 the aromatic groups of these and other TGase
inhibitors1 may direct binding to the hydrophobic
groove present on the surface of the enzyme, allowing
insertion of the pendant electrophilic group into the glu-
tamine side-chain binding tunnel leading to the active
site.17
n
O
n = 1 - 4
kinact = 0.46 - 1.34 min-1
KI = 0.15 - 2.75 μM
kinact/KI = 0.49 - 3.00 μM-1min-1
Figure 1. Analogous a,b-unsaturated amides studied previously as
irreversible TGase inhibitors.10,11
such that asparagine b-carboxamide groups are unable
to acylate the active site thiol residue. However, it can
be supposed that TGase cannot exclude longer acyl-do-
nor substrates (or irreversible inhibitors) that possess the
conformational flexibility required to properly position
their pendant reactive groups near the active site thiol.
On a practical level, spacers longer than n = 8 were
not pursued due to difficulties with their preparation
(strong emulsion formation) and their kinetic evaluation
(low solubility).
Finally, compounds 4c and 5c were tested, as represen-
tative TGase inhibitors, for their reactivity toward
glutathione, as a measure of their resistance to non-spe-
cific decomposition upon reaction with other nucleo-
philes present in living cells. The formation of
glutathione adduct upon incubation with a physiological
concentration of glutathione at 37 ꢁC (pH 7) was fol-
lowed quantitatively by mass spectrometry (see Section
4). After 24 h, only ꢁ10% of either inhibitor was con-
sumed by glutathione, in sharp contrast to their rapid
reaction with TGase. The specificity of their reactivity
with TGase bodes well for the in vivo application of
these inhibitors.
The chloroacetamide derivatives 5a–5c were evaluated
using a direct continuous colorimetric method15 and
their kinetic parameters are shown in Table 2. Cursory
inspection shows that the values determined for these
parameters are similar to those measured for the a,b-un-
saturated amides 4a–4c. It is noteworthy that compound
5a does not inhibit the activity of TGase. This may be
due to the fact that 5a is the shortest inhibitor studied,
the electrophilic carbon of its chloroacetamide moiety
being even nearer to the Cbz-Phe group than in the case

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C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
3. Conclusions
each reaction mixture was determined at time zero and
after 1, 2, 6, and 24 h by mass spectrometry, providing
the relative proportion of unreacted inhibitor to that
of glutathione adduct. Mass measurements were per-
formed by direct injection of 0.2 lL aliquots on a LC-
MSD-Tof instrument in positive electrospray mode.
The protonated molecular ions (M+H)+ of targeted spe-
cies and internal standard were integrated and used for
quantitation.
Eight novel TGase inhibitors were prepared by straight-
forward synthetic routes, diverging from common amine
intermediates based on the aromatic peptidic scaffold
Cbz-Phe. The acrylamide (4a–4d), chloroacetamide
(5a–5c), and maleimide (7b) derivatives thus prepared
showed varying degrees of affinity and reactivity, similar
to the analogous dihydroisoxazole derivatives reported
previously by Khosla.1 The length of the spacer between
the Cbz-Phe– group and the electrophilic group seems to
require a certain minimal length in order to allow inhi-
bition, probably due to the inherent capacity of TGase
to discriminate glutamine from asparagines based in
turn on the length of the glutamine side-chain binding
tunnel.17 Further kinetic and structural characterization
experiments are underway to verify these hypotheses
and to establish guidelines for the design of future
TGase inhibitors.
4.4. Synthesis
The various diamino alkanes used herein, as well as N-
Cbz-L-phenylalanine (Cbz-Phe–OH), p-nitrophenylchlo-
roformate, acryloyl chloride, chloroacetic anhydride,
1-propylamine, maleic anhydride, and DMAP, were ob-
tained from Aldrich. Triethylamine and DMF were ob-
tained from ACP. Sodium acetate and acetic anhydride
were obtained from Anachemia. Acetonitrile and
dichloromethane were obtained from EMD. N-Cbz-
Glu(c-p-nitrophenyl ester)Gly15 and Cbz-Phe-GABA
umbelliferyl ester16 were synthesized in our laboratory
according to published procedures.
4. Experimental
4.1. Enzyme preparation
1H and 13C NMR spectra were recorded on a Bruker
300 MHz spectrometer in the solvent indicated in the
text. Chemical shifts are reported in ppm with internal
reference to TMS. High-resolution mass spectra
(HRMS) were recorded on a LC-MSD-Tof instrument
from Agilent technologies in positive electrospray mode.
Either protonated molecular ions (M+H)+ or sodium
adducts (M+Na)+ were used for empirical formula
confirmation.
Recombinant guinea pig liver TGase was over-expressed
in E. coli and subsequently purified according to a pro-
tocol developed in our laboratory.14
4.2. Kinetics
Mono-exponential time-dependent inactivation was ob-
served for all of the inhibitors studied herein. When the
inhibition reaction mixture was diluted 100-fold to re-
move excess inhibitor, no enzyme activity was observed
or recovered, relative to the same concentration of unin-
hibited enzyme used as a positive control, consistent
with irreversible inhibition. For kinetic runs, either the
chromogenic substrate Cbz-Glu(c-p-nitrophenyl es-
ter)Gly15 or the fluorogenic substrate Cbz-Phe-GABA
umbelliferyl ester16 was used, at concentrations corre-
sponding to 2· KM in the presence of 1.25–160 lM of
each inhibitor. Stock solutions of all inhibitors were pre-
pared in DMF such that the final concentration of this
co-solvent was constant at 5% v/v. Kinetic evaluation
was carried out by the method of Stone and Hofste-
enge,19 as described previously.10–12 Observed first-order
rate constants of inactivation were determined from
non-linear regression, to a mono-exponential model.
These rate constants were in turn fit to Eq. 1 by non-lin-
ear regression, providing the kinetic parameters kinact
and KI as defined in Scheme 5.
4.4.1. Procedure A: Spacer coupling. Cbz-Phe p-nitroph-
enylester (2) (0.5 mmol) was dissolved in 50 mL of
CH3CN. Diaminoalkane (5 mmol) was added in one ali-
quot and the reaction mixture was stirred for 1 h. The
solvent was evaporated under reduced pressure and
40 mL of EtOAc was added. The organic phase was
washed with a solution of 1 N NaOH until the aqueous
phase was colorless, then dried over MgSO4, filtered and
evaporated under reduced pressure. The residue was
precipitated with CH2Cl2/hexane to give the desired
amide product as a solid.
4.4.2. Procedure B: Acryloyl chloride reaction. Cbz-Phe–
NH–(CH2)n–NH2 (0.15 mmol) was dissolved in 3 mL of
CH3CN and NEt3 (104 lL, 0.75 mmol). The reaction
mixture was cooled to 0 ꢁC, acryloyl chloride (30 lL,
0.375 mmol) was added dropwise, and the mixture was
then stirred overnight at room temperature. The final
product was isolated by filtration in the cases where it
precipitated from solution. Otherwise, the pure product
was obtained by extraction and flash chromatography.
kinact½Iꢂ
ꢀ
ꢁ
kobs
¼
:
ð1Þ
½Sꢂ
½Iꢂ þ KI 1 þ K
M
4.4.3. Procedure C: Chloroacetic anhydride reaction.
Cbz-Phe–NH–(CH2)n–NH2 (0.15 mmol) was dissolved
in 3 mL of CH3CN and NEt3 (104 lL, 0.75 mmol).
The reaction mixture was cooled to 0 ꢁC, a chloroacetic
anhydride (64 mg,0.375 mmol) was added portionwise,
and the mixture was then stirred overnight at room tem-
perature. The final product was isolated by filtration in
4.3. Glutathione reactivity
Compounds 4c and 5c (0.2 mM) were each incubated
with 1.6 mM of glutathione and 3.4 mM glutamine as
an internal standard in 10 mM ammonium bicarbonate
buffer (10% DMSO, pH 7) at 37 ꢁC. The composition of

C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
8383
the cases where it precipitated from solution. Otherwise,
the pure product was obtained by extraction and flash
chromatography.
HRMS (FAB) calcd for C23H32N3O3 ([M+H]+):
398.2438, found 398.2448.
4.4.3.5. N-Carbobenzyloxy-L-phenylalanine 8-ami-
nooctylamide (3d). This compound was prepared using
general procedure A from 1,8-diaminooctane to give the
desired product as a pale yellow solid in 27% isolated
yield. 1H NMR (300 MHz, DMSO) d 7.94 (t, 1H,
J = 5.2 Hz), 7.49 (d, 1H, J = 8.5 Hz), 7.23 (m, 10H),
4.92 (s, 2H), 4.16 (m, 1H), 2.97 (m, 4H), 2.72 (m, 2H),
2.48 (s, 2H), 1.20 (m, 12H). 13C NMR (75 MHz, DMSO)
d 160.6, 158.6, 146.5, 130.3, 129.3, 122.1, 121.2, 121.0,
120.6, 120.4, 119.3, 63.1, 54.9, 41.4, 37.9, 33.7, 30.0,
29.8, 29.6, 27.4, 27.3, 23.9. HRMS (FAB) calcd for
C25H36N3O3 ([M+H]+): 426.2751, found 426.2748.
4.4.3.1. N-Carbobenzyloxy-L-phenylalanine p-nitroph-
enylester (2). N-Cbz-L-phenylalanine (1) (1 mmol) was
dissolved in 10 mL of CH3CN with NEt3 (139 lL,
1 mmol) as base and placed in an ice bath, then p-
nitrophenyl chloroformate (222 mg, 1.1 mmol) was
added. The reaction mixture was stirred for 5 min at
4 ꢁC. Then DMAP (12.2 mg, 0.1 mmol) was added
and the reaction mixture was stirred another 45 min
at 4 ꢁC. The solvent was removed by rotary evapora-
tion and 30 mL of EtOAc was added. The organic
phase was washed with 3· 10 mL of 1 N HCl, dried
over MgSO4, and filtered and evaporated under re-
duced pressure. The mixture was triturated with diethyl
ether and filtered to obtain a white solid in 68% isolat-
4.4.3.6. N-Carbobenzyloxy-L-phenylalanine 2-(acryl-
amido)ethylamide (4a). This compound was prepared
using general procedure B from 3a. The reaction mix-
ture was evaporated and 20 mL of EtOAc was added.
The organic phase was washed with 3· 5 mL of 1 N
HCl and 3· 5 mL of 1 N NaOH, dried with MgSO4,
and filtered and evaporated. The product was obtained
by precipitation in acetone/diethyl ether to give a white
solid in 42% isolated yield. 1H NMR (300 MHz, CDCl3)
d 7.26 (m, 10H), 6.45 (s, 1H), 6.25 (dd, 1H, J = 1.5,
17.0 Hz), 6.22 (s, 1H), 6.03 (dd, 1H, J = 10.2, 17.0 Hz),
5.62 (dd, 1H, J = 1.5, 10.2 Hz), 5.39 (d, 1H,
J = 7.2 Hz), 5.07 (s, 2H), 4.36 (q, 1H, J = 7.3 Hz), 3.32
(s, 4H), 3.05 (d, 2H, J = 7.2 Hz). 13C NMR (75 MHz,
DMSO) d 161.1, 154.8, 146.6, 130.3, 129.3, 124.4,
122.0, 121.2, 121.0, 120.7, 120.5, 119.3, 118.3, 63.1,
54.9, 38.3, 37.7. HRMS (FAB) calcd for C22H26N3O4
([M+H]+): 396.1917, found 396.1924.
1
ed yield. H NMR (300 MHz, CDCl3) d 8.22 (d, 2H,
J = 8.9 Hz), 7.33 (m, 10H), 7.10 (d, 2H, J = 8.9 Hz),
5.34 (d, 1H, J = 7.8 Hz), 5.12 (s, 2H), 4.84 (m, 1H),
3.24 (m, 2H). 13C NMR (75 MHz, CDCl3) d 162.3,
149.5, 148.6, 140.1, 131.3, 130.5, 125.2, 124.9, 124.5,
124.3, 124.2, 123.6, 121.4, 118.7, 68.1, 56.9, 41.2.
HRMS (FAB) calcd for C23H21N2O6 ([M+H]+):
421.1394, found 421.1396.
4.4.3.2. N-Carbobenzyloxy-L-phenylalanine 2-amino-
ethylamide (3a). This compound was prepared using
general procedure A from 1,2-diaminoethane to give
the desired product as a white solid in 56% isolated
1
yield. H NMR (300 MHz, DMSO-d6) d 7.97 (t, 1H,
J = 5.5 Hz), 7.49 (d, 1H, J = 8.5 Hz), 7.23 (m, 10H),
4.92 (s, 2H), 4.17 (m, 1H), 3.03 (m, 2H), 2.92 (m, 2H),
2.74 (t, 2H, J = 10.1 Hz), 2.49 (s, 2H). 13C NMR
(75 MHz, DMSO) d 163.7, 149.8, 132.0, 131.6, 125.3,
124.7, 124.6, 124.3, 124.1, 123.2, 67.9, 58.3, 44.9, 43.9,
42.1. HRMS (FAB) calcd for C19H24N3O3 ([M+H]+):
342.1812, found 342.1815.
4.4.3.7. N-Carbobenzyloxy-L-phenylalanine 4-(acryl-
amido)butylamide (4b). This compound was prepared
using general procedure B from 3b. The reaction mix-
ture was evaporated and 20 mL of EtOAc was added.
The organic phase was washed with 3· 5 mL of 1 N
HCl and 3· 5 mL of 1 N NaOH, dried with MgSO4,
and filtered and evaporated. The desired product was
obtained by precipitation from acetone/diethyl ether to
give a white solid in 29% isolated yield. 1H NMR
(300 MHz, CDCl3) d 7.24 (m, 10H), 6.27 (dd, 1H,
J = 1.5, 16.9 Hz), 6.07 (dd, 1H, J = 10.2, 17.0 Hz), 5.96
(s, 1H), 5.86 (s, 1H), 5.62 (dd, 1H, J = 1.5, 10.2 Hz),
5.38 (d, 1H, J = 5.4 Hz), 5.06 (s, 2H), 4.34 (q, 1H,
J = 7.4 Hz), 3.24 (m, 6H), 1.38 (m, 4H). 13C NMR
(75 MHz, DMSO) d 160.7, 154.5, 146.5, 130.9, 129.3,
124.5, 122.1, 121.2, 121.0, 120.7, 120.5, 119.3, 118.0,
63.1, 54.9, 38.7, 38.3, 27.5. HRMS (FAB) calcd for
C24H30N3O4 ([M+H]+): 424.2230, found 424.2232.
4.4.3.3. N-Carbobenzyloxy-L-phenylalanine 4-amino-
butylamide (3b). This compound was prepared using
general procedure A from 1,4-diaminobutane to give
the desired product as a white solid in 82% isolated
yield. 1H NMR (300 MHz, DMSO) d 7.98 (t, 1H,
J = 5.5 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.23 (m, 10H),
4.91 (s, 2H), 4.16 (m, 1H), 2.97 (m, 4H), 2.72 (t, 2H,
J = 10.2 Hz), 1.77 (s, 2H), 1.39 (m, 4H). 13C NMR
(75 MHz, DMSO) d 160.6, 146.4, 130.2, 129.2, 122.0,
121.1, 120.9, 120.5, 120.3, 119.2, 63.0, 54.9, 40.9, 38.9,
37.8, 30.8, 27.4. HRMS (FAB) calcd for C21H28N3O3
([M+H]+): 370.2125, found 370.2123.
4.4.3.4. N-Carbobenzyloxy-L-phenylalanine 6-amino-
hexylamide (3c). This compound was prepared using
general procedure A from 1,6-diaminohexane to give
4.4.3.8. N-Carbobenzyloxy-L-phenylalanine 6-(acryl-
amido)hexylamide (4c). This compound was prepared
using general procedure B from 3c. The reaction mixture
was filtered and the solid was purified by flash chroma-
tography (EtOAc) to give pure product as a white solid
1
the product as a white solid in 52% isolated yield. H
NMR (300 MHz, DMSO) d 7.95 (t, 1H, J = 4.9 Hz),
7.49 (d, 1H, J = 8.6 Hz), 7.23 (m, 10H), 4.92 (s, 1H),
4.16 (m, 1H), 2.97 (m, 4H), 2.72 (m, 2H), 2.48 (s, 2H),
1.23 (m, 8H). 13C NMR (75 MHz, DMSO) d 160.7,
146.5, 130.3, 129.3, 122.1, 121.2, 121.0, 120.6, 120.4,
119.3, 63.1, 54.9, 41.2, 38.6, 37.9, 33.2, 29.8, 27.3, 27.2.
1
in 46% isolated yield. H NMR (300 MHz, CDCl3) d
7.24 (m, 10H), 6.26 (dd, 1H, J = 1.6, 17.0 Hz), 6.07
(dd, 1H, J = 10.2, 17.0 Hz), 6.01 (s, 1H), 5.91 (s, 1H),
5.61 (dd, 1H, J = 1.6, 10.2 Hz), 5.42 (d, 1H,

8384
C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
J = 6.3 Hz), 5.05 (s, 2H), 4.35 (q, 1H, J = 7.5 Hz), 3.27
(m, 2H), 3.12 (m, 4H), 1.46 (m, 2H), 1.23 (m, 6H). 13C
NMR (75 MHz, DMSO) d 160.6, 154.5, 146.5, 130.2,
129.3, 124.5, 122.1, 121.2, 121.0, 120.7, 120.5, 119.3,
118.0, 63.1, 54.9, 38.5, 37.9, 29.9, 29.8, 27.2, 27.1.
HRMS (FAB) calcd for C26H34N3O4 ([M+H]+):
452.2543, found 452.2532.
overnight, the solvent was removed and 40 mL of
EtOAc was added. The organic phase was washed with
3· 6 mL of 1 N HCl and 3· 6 mL of 1 N NaOH, then
dried over MgSO4, filtered and evaporated. The pure
product was obtained by trituration with EtOAc to give
a white solid in 43% isolated yield. 1H NMR (300 MHz,
CDCl3) d 7.24 (m, 10H), 6.59 (s, 1H), 5.67 (s, 1H), 5.42
(d, 1H, J = 7.5 Hz), 5.06 (s, 2H), 4.32 (q, 1H,
J = 6.8 Hz), 4.01 (s, 2H), 3.25 (q, 2H, J = 6.4 Hz), 3.12
(m, 4H), 1.48 (m, 2H), 1.23 (m, 6H). 13C NMR
(75 MHz, DMSO) d 160.6, 155.7, 146.5, 130.3, 129.3,
122.1, 121.2, 121.0, 120.7, 120.5, 119.3, 63.1, 54.9,
42.4, 38.5, 37.9, 29.9, 29.8, 27.2, 27.1. HRMS (FAB)
calcd for C25H33N3O4Cl ([M+H]+): 474.2154, found
474.2157.
4.4.3.9. N-Carbobenzyloxy-L-phenylalanine 8-(acryl-
amido)octylamide (4d). This compound was prepared
using general procedure B from 3d. The reaction mix-
ture was evaporated and 20 mL of EtOAc was added.
The organic phase was washed with 3· 5 mL of 1 N
HCl, 3· 5 mL of 1 N NaOH and 5 mL of brine, dried
with MgSO4, and filtered and evaporated. The product
was purified by flash chromatography (EtOAc) to give
a
white solid in 46% isolated yield. 1H NMR
4.4.3.13.
N-Carbobenzyloxy-L-phenylalanine
4-
(300 MHz, CDCl3) d 7.24 (m, 10H), 6.25 (dd, 1H,
J = 1.6, 17.0 Hz), 6.06 (dd, 1H, J = 10.2, 16.9 Hz), 5.66
(s, 1H), 5.65 (s, 1H), 5.60 (dd, 1H, J = 1.6, 10.2 Hz),
5.42 (d, 1H, J = 7.3 Hz), 5.05 (s, 2H), 4.31 (q, 1H,
J = 7.0 Hz), 3.28 (m, 2H), 3.09 (m, 4H), 1.49 (m, 2H),
1.24 (m, 10H). 13C NMR (75 MHz, DMSO) d 160.6,
154.5, 146.5, 130.3, 129.3, 124.5, 122.1, 121.2, 121.0,
120.7, 120.4, 119.3, 118.0, 63.1, 54.9, 38.6, 37.9, 29.9,
29.8, 29.5, 27.5, 27.3. HRMS (FAB) calcd for
C28H38N3O4 ([M+H]+): 480.2856, found 480.2869.
(maleamido)butylamide (6b). The compound 3b
(0.15 mmol) was dissolved in 3 mL of CH2Cl2 and male-
ic anhydride (0.165 mmol) was added. The mixture was
stirred for 1 h at room temperature under nitrogen. The
precipitated was filtered and washed with CH2Cl2 to
give the pure product as a white solid in 87% isolated
1
yield. H NMR (300 MHz, DMSO) d 9.1 (s, 1H), 7.99
(t, 1H, J = 5.3 Hz), 7.48 (d, 1H, J = 8.5 Hz), 7.24 (m,
10H), 6.39 (d, 1H, J = 12.6 Hz), 6.23 (d, 1H,
J = 12.5 Hz), 4.92 (s, 2H), 4.16 (m, 1H), 3.10 (m, 4H),
2.76 (m, 2H), 1.38 (s, 4H). 13C NMR (75 MHz, DMSO)
d 160.8, 155.4, 146.5, 130.2, 129.3, 125.6, 124.4, 122.1,
121.2, 121.0, 120.7, 120.5, 119.3, 63.1, 54.9, 38.2, 37.9,
27.5, 26.8. HRMS (FAB) calcd for C25H30N3O6
([M+H]+): 468.2129, found 468.2143.
4.4.3.10. N-Carbobenzyloxy-L-phenylalanine 2-(chlo-
roacetylamido)ethylamide (5a). This compound was pre-
pared using general procedure C from 3a. After stirring
overnight, the reaction mixture was filtered and the solid
obtained was washed with diethyl ether to give the pure
product as a white solid in 56% isolated yield. 1H NMR
(300 MHz, CDCl3) d 7.24 (m, 10H), 7.14 (s, 1H), 6.95 (s,
1H), 5.27 (d, 1H, J = 6.7 Hz), 5.06 (s, 2H), 4.32 (q, 1H,
J = 7.2 Hz), 3.95 (s, 2H), 3.29 (s, 4H), 3.02 (m, 2H). 13C
NMR (75 MHz, DMSO) d 161.2, 156.0, 146.6, 130.3,
129.3, 122.1, 121.2, 121.0, 120.7, 120.5, 119.3, 63.2,
55.0, 42.4, 38.7, 38.2, 37.7. HRMS (FAB) calcd for
C21H25N3O4Cl ([M+H]+): 418.1528, found 418.1538.
4.4.3.14.
N-Carbobenzyloxy-L-phenylalanine
4-
(maleimido)butylamide (7b). In a flask contained 6b
(0.15 mmol), sodium acetate (0.06 mmol) and 1 mL of
acetic anhydride were added. The reaction mixture was
stirred for 2 h at 100 ꢁC then 10 mL of 1 N NaOH was
added and the precipitate obtained was filtered. The sol-
id was purified by flash chromatography using EtOAc as
1
eluant to give a white solid in 37% isolated yield. H
NMR (300 MHz, DMSO) d 7.24 (m, 10H), 6.66 (s,
2H), 5.66 (s, 1H), 5.33 (d, 1H, J = 4.1 Hz), 5.06 (s,
2H), 4.30 (q, 1H, J = 6.7 Hz), 3.42 (t, 2H, J = 6.9 Hz),
3.13 (m, 4H), 1.38 (m, 4H). 13C NMR (75 MHz,
DMSO) d 160.7, 146.5, 130.2, 129.3, 126.9, 122.1,
121.2, 121.0, 120.7, 120.5, 119.3, 63.1, 54.9, 38.1, 37.9,
37.0, 27.3, 26.5. HRMS (FAB) calcd for C25H28N3O5
([M+H]+): 450.2023, found 450.2026.
4.4.3.11. N-Carbobenzyloxy-L-phenylalanine 4-(chlo-
roacetylamido)butylamide (5b). This compound was pre-
pared using general procedure C from 3b. After stirring
overnight, the solvent was removed and 20 mL of
EtOAc was added. The organic phase was washed with
3· 5 mL of 1 N HCl and 3· 5 mL of 1 N NaOH, then
dried with MgSO4, filtered and evaporated. The desired
product was then purified by flash chromatography
1
(EtOAc) to give a white solid in 28% isolated yield. H
4.4.3.15. N-Propylacrylamide (9). This compound was
prepared using general procedure B from 8. The reac-
tion mixture was evaporated and 20 mL of EtOAc was
added. The organic phase was washed with 3· 5 mL of
1 N HCl, 3· 5 mL of 1 N NaOH and then 5 mL of
brine, dried with MgSO4, and filtered and evaporated.
The product was purified by flash chromatography
(EtOAc) to give a colorless oil in 65% isolated yield.
1H NMR (300 MHz, CDCl3) d 6.25 (dd, 1H, J = 1.5,
16.9 Hz), 6.06 (dd, 1H, J = 10.2 Hz, 16.9 Hz), 5.60 (dd,
1H, J = 1.5, 10.2 Hz), 3.28 (q, 2H, J = 6.4 Hz), 1.54
(m, 2H), 0.92 (t, 3H, J = 7.4 Hz). 13C NMR (75 MHz,
CDCl3) d 161.8, 126.6, 126.0, 48.9, 27.0, 16.7. HRMS
NMR (300 MHz, CDCl3) d 7.24 (m, 10H), 6.60 (s,
1H), 5.78 (s, 1H), 5.33 (d, 1H, J = 6.7 Hz), 5.06 (s,
2H), 4.32 (q, 1H, J = 6.6 Hz), 4.01 (s, 2H), 3.12 (m,
6H), 1.36 (m, 4H). 13C NMR (75 MHz, DMSO) d
160.7, 155.7, 146.5, 130.3, 129.3, 122.1, 121.2, 121.0,
120.7, 120.5, 119.3, 63.1, 54.9, 42.4, 38.3, 37.9, 27.4,
27.3. HRMS (FAB) calcd for C23H29N3O4Cl
([M+H]+): 446.1841, found 446.1842.
4.4.3.12. N-Carbobenzyloxy-L-phenylalanine 6-(chlo-
roacetylamido)hexylamide (5c). This compound was pre-
pared using general procedure C from 3c. After stirring

C. Pardin et al. / Bioorg. Med. Chem. 14 (2006) 8379–8385
8385
(FAB) calcd for C6H12NO ([M+H]+): 114.0913, found
114.0912.
6. Cooper, A. J. L.; Jeitner, T. M.; Gentile, V.; Blass, J. P.
Neurochem. Int. 2002, 40, 53–67.
7. Singer, S. M.; Zainelli, G. M.; Norlund, M. A.; Lee, J. M.;
Muma, N. A. Neurochem. Int. 2002, 40, 17–30.
8. Karpuj, M. V.; Becker, M. W.; Steinman, L. Neurochem.
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Acknowledgments
9. Hausch, F.; Halttunen, T.; Ma¨ki, M.; Khosla, C. Chem.
Biol. 2003, 10, 225–231.
The authors thank Professor Joelle Pelletier of this
department for helpful discussion and Dr. Alexandra
Furtos of the departmental mass spectrometry service
for her timely assistance.
´
10. de Macedo, P.; Marrano, C.; Keillor, J. W. Bioorg. Med.
Chem. 2002, 10, 355–360.
´
11. Marrano, C.; de Macedo, P.; Keillor, J. W. Bioorg. Med.
Chem. 2001, 9, 1923–1928.
´
12. Marrano, C.; de Macedo, P.; Gagnon, P.; Lapierre, D.;
Gravel, C.; Keillor, J. W. Bioorg. Med. Chem. 2001, 9,
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