Aza-peptidyl michael acceptor and epoxide inhibitors - Potent and selective inhibitors of Schistosoma mansoni and Ixodes ricinus legumains (asparaginyl endopeptidases)

Article abstract of DOI:10.1021/jm900849h

Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH=CHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P10 position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.

Full text of DOI:10.1021/jm900849h

7192 J. Med. Chem. 2009, 52, 7192–7210
DOI: 10.1021/jm900849h
Aza-Peptidyl Michael Acceptor and Epoxide Inhibitors;Potent and Selective Inhibitors of
Schistosoma mansoni and Ixodes ricinus Legumains (Asparaginyl Endopeptidases)
†
†
†
†
‡
‡
§
ꢀꢁ
Asli Ovat, Fanuel Muindi, Crystal Fagan, Michelle Brouner, Elizabeth Hansell, Jan Dvorak, Daniel Sojka,
§
‡
‡
,†
ꢁꢀ
Petr Kopacek, James H. McKerrow, Conor R. Caffrey, and James C. Powers*
†School of Chemistry and Biochemistry, and the Petit Institute for Bioscience and Bioengineering, Georgia Institute of Technology, Atlanta,
Georgia 30332-0400, ‡Sandler Center for Basic Research in Parasitic Diseases, California Institute for Quantitative Biosciences, University of
California, San Francisco, California 94158, and Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic,
§
ꢀ
Ceskeꢁ Budeꢀjovice, CZ-370 05, Czech Republic
Received June 10, 2009
Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-
CHdCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of
asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the
hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of
41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted
amides to monosubstituted amides in the P10 position, and potency increased as we increased the
hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased
potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2.
Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for
some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given
the central functions of these enzymes in both parasites, the data presented here may facilitate the
eventual design of selective antiparasitic drugs.
Introduction
The helmintic disease, schistosomiasis, infects over 200
million people in approximately 70 countries, with 779 million
at risk, 85% of whom are in Africa alone.14 Immature and adult
bloodflukes live in the cardiovascular system, ingest red blood
cells, and utilize hemoglobin as a source of amino acids for
growth, development, and reproduction. In the parasite gut,
cysteine and aspartic proteases contribute to the proteolytic
degradation of hemoglobin.15 Among these are a legumain
(SmAE) that, in vitro, degrades hemoglobin16 and processes
and trans-activates the major gut protease cathepsin B.17 As a
central digestive enzyme, therefore, SmAE may represent a
useful target for selective inhibitors that impair the parasite’s
ability to feed.
Hard ticks of the genus Ixodes are vectors of Lyme disease
caused by the spirochetes Borrelia burgdorferi sensu lato. The
multiplication of spirochetes in the tick gut and subsequent
infection of the host requires unimpaired bloodmeal uptake
and digestion.18 As for SmAE, a gut-associated AE in Ixodes
ricinus (IrAE) is thought to contribute to hemoglobin degra-
dation either directly or indirectly by the trans-activation of
other digestive cysteine and aspartic proteases.8,19 Accord-
ingly, IrAE may represent a drug or vaccine target to prevent
or retard transmission of disease.
Asparaginyl endopeptidases (AEs) or legumains (EC.
3.4.22.34) belong to the C13 family of clan CDa cysteine proteases,
which also include caspases, gingipains, clostripain, and separase.1
AEs are acidic lysosomal enzymes that cleave substrates specifi-
cally after an asparagine residue in the P1 position.2 Legumains
were first discovered in the human bloodfluke, Schistosoma
mansoni,3 then in plants,4 mammals,5-7 and most recently in
the ticks, Ixodes ricinus8 and Haemaphysalis longicornis.9 Plant
legumain functions as a processing enzyme of storage proteins
during seed germination.10 Mammalian legumain is involved in
the inhibition of osteoclast formation and bone resorption.11 It
has also been shown that human legumain is highly expressed in
many tumors, including carcinomas of the breast, colon, and
prostate.12 Recently, it was found that mice lacking legumain
develop disorders resembling hemophagocytic syndrome.13
*To whom correspondence should be addressed. Phone: (404)
894-4038. Fax: (404) 894-2295. E-mail: james.powers@chemistry.
gatech.edu.
a Abbreviations: AAsn, aza-asparagine; AMC, 7-amino-4-methyl-
coumarin; Boc, t-butoxycarbonyl; Bzl, benzyl, CH2Ph; C13, a family
of enzymes in the CD clan of cysteine proteases; Cbz, benzyloxycarbo-
nyl; CD, a clan of cysteine proteases; CDCl3, deuterated chloroform;
DCC, dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide; DMSO-d6, dimethylsulfoxide deuterated;
EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
EP, epoxide (C2H2O); EtOAc, ethyl acetate; EtOH, ethanol; HOBt, N-
hydroxybenzotriazole; iBCF, isobutyl chloroformate; IrAE, Ixodes
ricinus asparaginyl endopeptidase; MeOH, methanol; Mu, morpholino-
carbonyl; NMM, N-methylmorpholine; OBzl, benzyloxy; Pip, pipera-
dinocarbonyl; Piz, piperazinocarbonyl; SmAE, Schistosoma mansoni
asparaginyl endopeptidase; TFA, trifluoroacetic acid.
Synthetic AE inhibitors that have been tested against
mammalian AEs20,21 include aza-Asn halomethylketones
(Cbz-Ala-Ala-AAsn-CH2Cl, kobs/[I] = 139 000 M-1 s-1),
Michael acceptors derived from Asn (Cbz-Ala-Ala-NHCH-
(CH2CONH2)CHdCH2CO2CH2CHdCH2, kobs/[I] up to
766 M-1 s-1), and acyloxymethylketones (kobs/[I] from 769
up to 109 000 M-1 s-1). It has also been shown that the
r
pubs.acs.org/jmc
Published on Web 10/22/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7193
Scheme 1. Synthesis of Fumarate Precursorsa
aspartyl peptidyl fluoromethyl ketones designed for caspase
inhibition also moderately inhibit mammalian AE.22 We
recently reported that aza-asparaginyl Michael acceptor inhi-
bitors are potent and selective inhibitors of S. mansoni,
I. ricinus, and Trichomonas vaginalis (a parasitic protozoan)
AEs.23 We designed the aza-peptide epoxides and Michael
acceptor inhibitors to closely resemble an extended peptide
legumain substrate.24,25 Placement of the carbonyl group of
the epoxide moiety or fumarate derivative in the inhibitor
places it in a location identical to that of the carbonyl of the
scissile peptidebondin a good legumain substrate. This design
allows the peptide chain of the inhibitor to exactly match that
of a good substrate from the N-terminus across to the scissile
peptide carbonyl group. The warhead epoxide or Michael
acceptor is then located adjacent to the active site cysteine
residue. Aza-peptide epoxides and Michael acceptors have the
advantage of being easily extended in the P0 direction, allow-
ing interactions with the S0 subsites of AE.
a (i) NMM, iBCF, CH2Cl2, HNR1R2; (ii) NaOH; (iii) HCl, EtOH.
Scheme 2. Synthesis of Epoxysuccinate Derivativesa
In our earlier paper, the inhibitor sequence Cbz-Ala-Ala-
AAsn was maintained, while substituents on the reactive
warhead were changed in order to measure the effects of
modifying the P10 position. SAR studies revealed that the
most potent inhibitors have esters or disubstituted amides
with aromatic groups in the P10 position. Work by other
investigators with the vinyl sulfone inhibitors of cysteine
proteases demonstrated that introduction of the appropriate
N-terminal capping groups could increase compound bio-
availability.26 Accordingly, we expand upon our previous
design of aza-peptide Michael acceptors and epoxides by
introducing groups at the N-terminus in order to both extend
the compounds to P4 and P5 and potentially increase bio-
availability. In this paper, wereport the design and synthesis of
forty one aza-peptide Michael acceptors with the general struc-
ture Boc-AAsn-trans-CHdCHCOR, YCO-Ala-AAsn-trans-
CHdCHCOR and YCO-Ala-Ala-AAsn-trans-CHdCHCOR
where YCO- are potentially bioavailable capping groups. In
addition, eight epoxide inhibitors with the general structure
YCO-Ala-Ala-AAsn-EP-COR, and five aza-peptide substrate
analogues, were synthesized and evaluated. Some of the new
inhibitors have the fastest inhibition rates thus far measured for
these parasitic enzymes.
a (i) NMM, iBCF, CH2Cl2, HNR1R2; (ii) KOH; (iii) HCl, EtOH.
Scheme 3. Synthesis of the Aza-Asparagine Precursora
a (i) BrCH2COOEt, NMM, DMF; (ii) NH3, cat. NaCN, MeOH,
DMF.
monoethyl epoxysuccinate and the corresponding primary or
secondary amines using the standard mixed anhydride cou-
pling procedure with NMM and iBCF, followed by deprotec-
tion of the ethyl ester in ethanol using aqueous KOH
(Scheme 2). The monoethyl ester of cis-oxirane-2,3-dicarbo-
xylic acid monoethyl ester (5) was synthesized following a
previously described procedure.28 Coupling of the dibenzyla-
mine to the monoethyl ester epoxysuccinate to form com-
pound 6t was performed using the standard mixed anhydride
coupling procedure and followed by deprotection as described
above. Thedisubstituted aromatic amines weresynthesizedby
reductive amination starting with an aromatic aldehyde pre-
cursor and an aromatic primary amine.
The preparation of the aza-asparagine precursors (Boc-
NHNHCH2CONH2, Y-CO-Ala-NHNHCH2CONH2, Y-CO-
Ala-Ala-NHNHCH2CONH2, and Cbz-Ala-Val(Ile, Phe)-
NHNHCH2CONH2) are shown in Schemes 3, 4, 5, and 6. The
hydrazide Boc-NHNHCH2CONH2 was synthesized by the
monoalkylation of tert-butyl carbazate (7) with ethyl bro-
moacetate, and the conversion of the ethyl ester to the
amide by ammonolysis was done with catalytic amounts
of NaCN according to the procedure of Hogberg et al.
(Scheme 3).29 For the synthesis of acyl dipeptide aza-
asparagine precursors (12-14), we first prepared alanine
methyl ester isocyanate (10) by reacting the hydrochloride
salt of alanine (9) with phosgene according to the procedure
by Nowick et al.30 Alanine methyl ester isocyanate (10) was
then reacted with piperidine, morpholine, tert-butoxycarbo-
nylpiperazine, and benzyloxycarbonylpiperazine to form
Chemistry
The syntheses of the aza-peptide fumarate and epoxide
analogues are based on the previous syntheses of aza-peptide
Michael acceptor24 and epoxide inhibitors.27 The inhibitors
are obtained by coupling a peptidyl hydrazide to a monoester
or amide of fumaric acid or to a monoester or amide of
epoxysuccinic acid. Monoethyl fumarate (2a) was commer-
cially available, and all of the Michaelacceptor warheads were
synthesized using this compound as a precursor. The mono-
benzyl fumarate (2b) was formed from monoethyl fumarate
and benzyl alcohol using NMM and DCC as the coupling
reagent and followed by deprotection of the ethyl ester in
ethanol using aqueous NaOH. The fumarate precursors
(2c-p) were prepared from monoethyl fumarate and the
corresponding primary or secondary amines by standard
mixed anhydride coupling using NMM and iBCF, followed
by hydrolysis of the ethyl ester with aqueous NaOH
(Scheme 1).
A previously described procedure27 was used for the synth-
esis of (2S,3S)-oxirane-2,3-dicarboxylic acid monoethyl ester
(3). The epoxide warheads (4r, 4s) were synthesized from

7194 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
a
Scheme 4. Synthesis of the Acyl Dipeptide Aza-Asparagine
Scheme 6. Synthesis of Cbz-Ala-AA-NHNHCH2CONH2
Precursorsa
a (i) HCl H-Val-OCH3, HCl H-Ile-OCH3 or HCl H-Phe-OCH3,
3
3
3
HOBt, DCC, DMF (ii) H2NNH2, MeOH, (iii) BrCH2COOEt, NMM,
DMF; (iv) NH3, cat. NaCN, MeOH, DMF.
Aza-peptide Michael acceptors and epoxides with the Boc-
Piz-Ala-Ala-AAsn peptide sequence (29, 36a-r) were treated
with TFA in methylene chloride to form the simple piperazine
derivatives Piz-Ala-Ala-AAsn (37, 38a-r).
a (i) H2NNH2, MeOH; (ii) BrCH2COOEt, NMM, DMF; (iii) NH3,
cat. NaCN, MeOH, DMF.
Results and Discussion
Scheme 5. Synthesis of the Acyl Tripeptide Aza-Asparagine
Synthetic Design. Aza-peptide Michael acceptors and
epoxide inhibitors are selective and potent inhibitors of
SmAE and IrAE.23,25 Previously synthesized inhibitors have
the peptide sequence of Cbz-Ala-Ala-AAsn as a template
since Cbz-Ala-Ala-Asn-NHMec (NHMec = 7-(4-methyl)-
coumarylamide) is an optimal substrate sequence for legu-
main.34 In this study, we used Boc-NHNHCH2CONH2,
YCO-Ala-NHNHCH2CONH2, and YCO-Ala-Ala-NHNH-
CH2CONH2 peptide sequences where the Y groups are
piperidine, morpholine, pipezarine, tert-butoxypiperazine,
and benzyloxycarbonyl piperazine. These groups were cho-
sen to increase the bioavailability of the inhibitors and to
study the interactions of the P4 (YCO-Ala-NHNH-
CH2CONH2) and P5 (YCO-Ala-Ala-NHNHCH2CONH2)
positions of the inhibitor with SmAE and IrAE. For the
inhibitor design, the R carbon of the asparagine (Asn) residue
is replaced by nitrogen. This replacement results in an aza-
peptide containing the aza-asparagine (AAsn) residue. The
presence of an aza-asparagine residue at P1 makes the
synthesis of aza-peptide Michael acceptor and epoxide in-
hibitors easier compared to peptide Michael acceptor and
epoxide inhibitors as the synthesis involves simple coupling
of an acid and an aza-peptide precursor. Aza-peptides are
ideal inhibitors because they are resistant to cleavage by
proteases in vivo35,36 and can incorporate a reactive war-
head. We chose the epoxide and Michael acceptor double
bonds as these warheads can be easily modified in the P0
position to study interactions with the S0 subsites of the
enzymes.
Inhibition of SmAE and IrAE with Aza-Peptidyl Michael
Acceptors. The IC50 values of 41 aza-peptide Michael accep-
tor inhibitors are listed in Table 1. We synthesized four (Boc-
protected aza-asparagine) inhibitors (23a,d,f,h) with differ-
ent Michael acceptor warheads. Only the monoethyl ester
derivative (SmAE IC50 > 2000 nM, IrAE IC50 = 750 nM)
inhibited the enzymes, whereas no inhibition was detected
with the other three compounds.
Due to this result, we synthesized acyl dipeptide aza-
asparagine Michael acceptor inhibitors. Monosubstituted
amides (24k, 25c,e,i,h,k, 26j,k) were either poor or non-
inhibitory. Replacement of the monosubstituted amides with
the disubstitued amides (24k, 25k, 26j-k) yielded increased
potency. Inhibitors 24k, 25k, and 26k have the same iso-
quinoline moiety in their P10 position; however, they have
different acyl groups. Both enzymes seem to favor the
presence of piperidine residue in the P3 position rather than
Precursorsa
a (i) NaOH then HCl; (ii) HCl H-Ala-OCH3, HOBt, DCC, DMF;
(iii) H2NNH2, MeOH; (iv) BrCH2COOEt, NMM, DMF; (v) NH3, cat.
NaCN, MeOH, DMF.
3
Y-CO-Ala-OCH3 (11u-x), respectively.31 Piperidine and
morpholine were commercially available, however; tert-
butoxycarbonylpiperazine32 and benzyloxycarbonylpiper-
azine33 were synthesized according to the literature. The
ester Y-CO-Ala-OCH3 (11u-x) was then reacted with
excess hydrazine (NH2NH2) in methanol to form Y-CO-
Ala-NH-NH2. To introduce the aza-asparagine side chain,
Y-CO-Ala-NH-NH2 was reacted with ethyl bromoacetate
and NMM. The ethyl ester group was then reacted with 7 N
NH3 in methanol in the presence of catalytic NaCN to form
the acyl dipeptide aza-asparagine precursors (Y-CO-Ala-
NHNHCH2CONH2, 12-14) (Scheme 4). For the synthesis
of the acyl tripeptide aza-asparagine precursors (15-18),
we first hydrolyzed the esters 11u-x (Y-CO-Ala-OCH3)
with 1 M NaOH in methanol to form acyl alanine deriva-
tives (Y-CO-Ala-OH). The acyl alanine then was coupled to
alanine methyl ester using HOBt and DCC to form the
dipeptides Y-CO-Ala-Ala-OCH3. The dipeptide esters
Y-CO-Ala-Ala-OCH3 were then reacted with excess hydra-
zine, and the asparagine side chain was introduced as
mentioned previously to form the acyl tripeptide aza-aspar-
agine precursors (Y-CO-Ala-Ala-NHNHCH2CONH2, 15-
18) (Scheme 5).
For the synthesis of Cbz-Ala-Val(Ile, Phe)-NHNHCH2-
CONH2, we reacted Cbz-Ala-OH with valine methyl ester,
isoleucine methyl ester, or phenylalanine methyl ester. The
peptidyl methyl ester was then reacted with hydrazine, and
finally, the asparagine side chain was attached by alkyla-
tion with ethyl bromoacetate followed by ammonolysis
(Scheme 6).
The aza-asparagine precursors (8, 12-14, 15-18, 20-22)
were then coupled to a variety of substituted fumarate analo-
gues (2a-p) and epoxide moieties (4q-s and 6t) using HOBt
and EDC to complete the synthesis of the aza-peptidyl
inhibitors (Scheme 7).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7195
Scheme 7. Coupling of Fumarate Precursors and Epoxide Moieties to Aza-Asparagine Precursors
Table 1. Inhibition of SmAE and IrAE by Aza-Peptide Michael Acceptorsa
inhibitor
Boc-AAsn-CHdCH-COOEt
SmAE IC50 (nM)
IrAE IC50 (nM)
23a
23d
23f
23h
24k
25c
25e
25i
>2000
N.I.
N.I.
N.I.
600
N.I.
N.I.
>2000
>2000
750
750
750
80
750
N.I.
N.I.
N.I.
150
N.I.
N.I.
>2000
N.I.
600
850
800
15
Boc-AAsn-CHdCH-NHCH2-cyclohexyl
Boc-AAsn-CHdCH-CONHBzl
Boc-AAsn-CHdCH-CONHCH2CH2CH2Ph
Pip-Ala-AAsn-CHdCH-CO-tetrahydroisoquinoline
Mu-Ala-AAsn-CHdCH-CONH-cyclopropyl
Mu-Ala-AAsn-CHdCH-CONHCH2-furyl
Mu-Ala-AAsn-CHdCH-CONHCH2Ph-3,4-OCH3
Mu-Ala-AAsn-CHdCH-CONHCH2CH2CH2Ph
Mu-Ala-AAsn-CHdCH-CO-tetrahydroisoquinoline
Cbz-Piz-Ala-AAsn-CHdCH-CO-tetrahydroquinoline
Cbz-Piz-Ala-AAsn-CHdCH-CO-tetrahydroisoquinoline
Pip-Ala-Ala-AAsn-CHdCH-COOEt
25h
25k
26j
26k
27a
27g
27l
Pip-Ala-Ala-AAsn-CHdCH-CONHCH2CH2Ph
Pip-Ala-Ala-AAsn-CHdCH-CON(Bzl)2
750
80
400
17
27o
27p
28a
28h
28k
28p
37a
37b
37l
Pip-Ala-Ala-AAsn-CHdCH-CON(Bzl)-CH2-1-naphthyl
Pip-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
Mu-Ala-Ala-AAsn-CHdCH-COOEt
Mu-Ala-Ala-AAsn-CHdCH-CONHCH2CH2CH2Ph
Mu-Ala-Ala-AAsn-CHdCH-CO-tetrahydroisoquinoline
Mu-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
Piz-Ala-Ala-AAsn-CHdCH-COOEt
100
57
2.4
8
101
1450
103
45
0.23
>2000
65
3.3
8.5
300
250
90
Piz-Ala-Ala-AAsn-CHdCH-COOBzl
Piz-Ala-Ala-AAsn-CHdCH-CON(Bzl)2
13
31
37n
37p
29a
29b
29l
Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
Piz-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
Boc-Piz-Ala-Ala-AAsn-CHdCH-COOEt
40
70
3.8
2.6
80
45
8.5
7.5
Boc-Piz-Ala-Ala-AAsn-CHdCH-COOBzl
Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(Bzl)2
Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
Cbz-Piz-Ala-Ala-AAsn-CHdCH-COOEt
Cbz-Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
Cbz-Piz-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
Cbz-Ala-Val-AAsn-CHdCH-COOEt
Cbz-Ala-Val-AAsn-CHdCH-CONHCH2-1-naphthyl
Cbz-Ala-Val-AAsn-CHdCH-CO-tetrahydroquinoline
Cbz-Ala-Ile-AAsn-CHdCH-COOEt
Cbz-Ala-Phe-AAsn-CHdCH-CONHCH2-furyl
Cbz-Ala-Phe-AAsn-CHdCH-CO-tetrahydroquinoline
Cbz-Ala-Phe-AAsn-CHdCH-CO-tetrahydroisoquinoline
45
58
30
20
29n
29p
30a
30n
30p
31a
31m
31j
68
70
5
0.14
0.35
0.37
65
70
50
100
1050
138
280
>2000
>2000
>2000
350
13
32a
33e
33j
0.52
1800
500
500
33k
a AE zymogens from Ixodes ricinus and Schistosoma mansoni were expressed in Pichia pastoris and activated as described previously.8,40 Inhibition
assays were performed in 0.1 M citrate-phosphate, 4 mM DTT, pH 6.8, <1% DMSO for SmAE, or 0.1 M citrate-phosphate, 4 mM DTT, 0.1 M NaCl,
pH 5.5 for IrAE, <1% DMSO, with a final concentration of 10 μM Cbz-Ala-Ala-AAsn-AMC (Cbz = PhCH2-OCO-, AMC = 7-amino-4-methyl-
coumarin) as substrate.
morpholine or benzyloxypiperazine. Probably, the oxygen of
the morpholine is disturbing the H-bond network around the
active site of the AEs, whereas the benzyloxypiperazine is not
accommodated in the P3 position due to the bulkiness of the

7196 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
Table 2. Inhibition of SmAE and IrAE by Aza-Peptide Epoxidesa
inhibitor
SmAE IC50 (nM)
IrAE IC50 (nM)
34q
34r
34s
35s
35r
35t
38r
36r
Pip-Ala-Ala-AAsn-EP(S,S)-COOEt
220
72
30
12
Pip-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
Pip-Ala-Ala-AAsn-EP(S,S)-CONHCH2-1-naphthyl
Mu-Ala-Ala-AAsn-EP(S,S)-CONHCH2-1-naphthyl
Mu-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
Mu-Ala-Ala-AAsn-EP(cis)-CON(Bzl)2
Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
Boc-Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
160
60
8.5
6
90
1.7
N.I.
11
N.I.
150
80
270
a AE zymogens from Ixodes ricinus and Schistosoma mansoni were expressed in Pichia pastoris and activated as described previously.8,40 Inhibition
assays were performed in 0.1 M citrate-phosphate, 4 mM DTT, pH 6.8, <1% DMSO for SmAE, or 0.1 M citrate-phosphate, 4 mM DTT, 0.1 M NaCl,
pH 5.5 for IrAE, <1% DMSO, with a final concentration of 10 μM Cbz-Ala-Ala-AAsn-AMC (Cbz = PhCH2-OCO-, AMC = 7-amino-4-methyl-
coumarin) as substrate.
group. Although the disubstituted amides were better inhi-
bitors than the monosubstituted ones, they were still weak.
In view of these results, acyl tripeptide aza-asparagine
Michael acceptors were synthesized. Among the inhibitors
with Pip-Ala-Ala-AAsn sequence, the ethyl ester derivative
Interestingly, all of the compounds with Boc-Piz-Ala-Ala-
AAsn and Cbz-Ala-Ala-AAsn peptide sequences were po-
tent inhibitors of both SmAE and IrAE. We speculate that
there is a hydrophobic pocket that is interacting with hydro-
phobic groups (Boc and Cbz) at P4 or P5. In particular, the
Cbz-Ala-Ala-AAsn peptide sequence (30a,n,p) produced the
best inhibitors of IrAE (IC50=0.14, 0.35, and 0.37 nM, res-
pectively) herein reported. As the Boc-Ala-Ala-AAsn com-
pounds were not as potent as those containing Cbz-Ala-Ala-
AAsn, we conclude that the increased π-stacking of the
aromatic residues in either the S4 or S5 pocket improves
inhibitor binding.
(27a) was as effective (SmAE IC50 = 80 nM, IrAE IC50
=
15 nM) as the dibenzyl amide analogue (27l) (SmAE IC50=
80 nM, IrAE IC50=17 nM). Again, the disubstituted amides
(27l,o,p) were more potent than the monosubstituted amide
analogue (27g). The dinaphthyl derivative (27p) was the most
potent of the acyl tripeptide inhibitors against SmAE
(IC50=57 nM), whereas the N-benzyl-N-naphthyl derivative
was the best against IrAE (IC50=2.4 nM).
To study the effect of P2 residue on inhibitor potency, we
synthesized seven compounds with the Cbz-Ala-AA-AAsn
(AA=Val, Ile, and Phe) peptide sequence. Compounds with
valine (31a,m,j) or isoleucine (32a) residue at P2 were good to
poor inhibitors, whereas those containing phenylalanine at
P2 (33e,j,k) were extremely poor against SmAE and IrAE,
confirming that both enzymes do not prefer large hydro-
phobic groups in the S2 position. With respect to the ethyl
ester compounds 31a and 32a that differ only at P2, SmAE
reacted with both equally, whereas inhibition of IrAE was
100-fold better with 32a. This is a second example of
divergent SAR between these enzymes and suggests that
IrAE can accept larger aliphatic hydrophobic residues at
S2. Previous studies with positional scanning substrate
combinatorial libraries tested against SmAE34 and IrAE8
demonstrated that the preferred amino acids at P2 are Ala>
Val>Ile>Phe and Ile>Ala>Val>Phe, respectively. Thus,
our results are in good agreement with these data.
Among the inhibitors with the Mu-Ala-Ala-AAsn peptide
sequence, the ethyl ester analogue (28a) was as effective
(IC50=101 nM) as the disubstituted amide analogue (28k)
(IC50 = 103 nM) against SmAE. However, in the case of
IrAE, replacement of the ethyl ester (IC50=0.23 nM) with
the disubstituted amide analogue, tetrahydroisoquinoline
(IC50=65 nM), results in 300-fold decrease in potency. This
is one of very few examples where the parasite AEs diverged
markedly in their SAR. We conclude that, even though IrAE
does prefer disubstituted amides, for example, the di-
naphthyl analogue (28p) (IC50=3.3 nM), it does not favor
constrained amides in the P10 position. The monoamide
analogue (28h) (SmAE IC50=1450 nM; IrAE IC50 >2000
nM) was a poor inhibitor of both enzymes. There can be two
reasons for this; either the alkyl spacer puts the phenyl group
in an unfavorable position or the presence of a hydrogen
bond donor group is not favored in the S10 position.
Next, we synthesized five inhibitors with the Piz-Ala-Ala-
AAsn peptide sequence. The two ester derivatives (ethyl
ester, 37a, and benzyl ester, 37b) were moderate inhibitors
of both SmAE (IC50 =300 and 250 nM, respectively) and
IrAE (IC50 = 8.5 and 13 nM, respectively). However, the
disubstituted amide derivatives (37l,n,p) were better against
SmAE (IC50=90,40, and 70 nM). Because these compounds
contain aromatic groups in their warheads, we conclude that
the presence of such groups at P10 improves binding of the
inhibitors due to π-stacking in either S10 or S20 of SmAE.
Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
Inhibition of SmAE and IrAE with Aza-Peptidyl Epoxides.
The IC50 values of eight aza-peptide epoxide inhibitors are
listed in Table 2. Comparison of the three aza-peptidyl
epoxide inhibitors with Pip-Ala-Ala-AAsn sequence showed
that the ester analogue (34q) and the monosubstituted
amide analogue (34s) were moderate inhibitors against
SmAE with the IC50 values of 220 and 160 nM, respec-
tively; however, the disubstituted amide derivative (34r) was
best with an IC50 value of 72 nM. For IrAE, all compounds
were moderate inhibitors, but unlike SmAE, IrAE favored
the monosubstituted amide analogue (IC50=8.5 nM) to the
ethyl ester analogue (IC50 =30 nM) and the disubstituted
amide analogue (IC50 = 12 nM). These results showed
that the two enzymes differ in their S10 or S20 positions
slightly. Replacement of the piperidine residue at P4
(37n) was the most potent inhibitor against SmAE (IC50
=
40 nM) among all the compounds synthesized. Probably,
the methyl group interacts with a small hydrophobic
pocket in the S10 subsite, whereas the naphthyl group
reaches to a larger hydrophobic pocket in the S20 subsite.
Next, we synthesized eight compounds with protecting
groups on the piperazine; five of these (29a,b,l,n,p) contain
a tert-butoxycarbonyl (Boc) group, whereas the other
three (30a,n,p) contain a benzyloxycarbonyl (Cbz) group.
of compound 34s (SmAE IC50 = 160 nM, IrAE IC50
=
8.5 nM) with a morpholine residue in compound 35s
(IC50 = 60 nM) resulted in an almost 3-fold increase in
potency against SmAE, whereas it had almost no effect
against IrAE (IC50=6 nM).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7197
Table 3. Second-Order Rate Constants for Inhibition of Parasite AEs by Aza-Peptide Michael Acceptors and Epoxidesa
SmAE IC50
(nM)
SmAE kinact/Kiapp
or kass (M-1 s-1) ꢀ10-6
IrAE IC50
(nM)
IrAE kinact/Kiapp
or kass (M-1 s-1) ꢀ10-6
inhibitor
Cbz-Ala-Ala-AAsn-CHdCH-COOEt24
37b Piz-Ala-Ala-AAsn-CHdCH-COOBzl
29b Boc-Piz-Ala-Ala-AAsn-CHdCH-COOBzl
38r Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
34r Pip-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
31
250
45
0.16
0.046
0.023
0.03
0.037
0.01
0.04
0.4
4.5
13
0.64
0.17
0.67
0.10
0.45
0.39
0.35
1.98
0.77
1.79
0.22
7.5
11
150
72
12
36r Boc-Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2
29l Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(Bzl)2
28p Mu-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
27p Pip-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2
30n Cbz-Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
37n Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl
80
45
270
30
45
57
3.3
8
0.27
0.084
0.16
70
40
0.35
3.8
a AE zymogens from Ixodes ricinus and Schistosoma mansoni were expressed in Pichia pastoris and activated as described previously.8,40 Inhibition
assays were performed in 0.1 M citrate-phosphate, 4 mM DTT, pH 6.8, <1% DMSO for SmAE, or 0.1 M citrate-phosphate, 4 mM DTT, 0.1 M NaCl,
pH 5.5 for IrAE, <1% DMSO, with a final concentration of 10 μM Cbz-Ala-Ala-AAsn-AMC (Cbz = PhCH2-OCO-, AMC = 7-amino-4-methyl-
coumarin) as substrate. Second-order rate constants were determined by linear or nonlinear regression analysis using GRAPHPAD PRISM 3.0a, as
described in the text.
To study the effect of stereochemistry on inhibitor po-
tency, we synthesized two dibenzyl amide epoxide warheads
with S,S and cis configurations and coupled them to the
Mu-Ala-Ala-AAsn peptidyl precursor to form compounds
35r and 35t, respectively. Compound 35t (cis) was inactive
against both enzymes, whereas the S,S compound retained
potency. Accordingly, we suggest that the cis stereochemis-
try of the epoxide unfavorably changes the orientation of the
prime side substituents to cause a loss of inhibition. Piz-Ala-
Ala-AAsn-EP(S,S)-CON(Bzl)2 (38r) was a moderate inhibi-
tor against both SmAE and IrAE with IC50 values of 150 and
11 nM, respectively. Replacement of the piperazine with tert-
butoxycarbonyl piperazine (36r) increased the potency
2-fold against SmAE. On the other hand, the same replace-
ment resulted in 24-fold decrease in potency against IrAE.
Extension of the aza-peptides to the P5 position by introduc-
Figure 1. Mechanism of inhibition of cysteine proteases by Michael
tion of Boc group resulted in increased potency in all
compounds except this one example.
acceptor and epoxide inhibitors.
nucleophilic attack by the catalytic cysteine residue on the
Michael acceptor double bond at C2 or epoxide at C3
forming a covalent bond and irreversibly inhibiting the
enzyme (Figure 1).37 The mechanism is supported by
NMR studies,23 X-ray crystal structures of aza-peptide
Michael acceptor inhibitors bound to caspase-3 and cas-
pase-8,38 and crystal structures of aza-peptide epoxide
inhibitors bound to caspase-3.39 In view of these data, we
suggest that, for AEs, the site of attack is the same in both
warheads, that is, the carbon closest to the aza-peptide
nitrogen atom (Figure 1).
Finally, we synthesized five aza-peptidyl substrate analo-
gue inhibitors such as Mu-Ala-AAsn-CH2CH2Ph; however,
none of these compounds inhibited SmAE or IrAE (data not
shown). The structures of these compounds can be found in
the Supporting Information.
Second-Order Inhibition Rate Constants. Second-order in-
hibition rate constants for a selection of the better aza-peptide
Michael acceptors and epoxides are presented in Table 3. We
compared the rates of the new compounds with the gold
standard inhibitor Cbz-Ala-Ala-AAsn-CHdCH-COOEt24
(kinact/Kiapp = 160 000 M-1 s-1 for SmAE and 640 000
M-1 s-1 for IrAE). In the case of SmAE, two compounds
(28p, 400 000 M-1 s-1, and 27p, 270 000 M-1 s-1) have
higher rates than Cbz-Ala-Ala-AAsn-CHdCH-COOEt,
and one compound (37n, 160 000 M-1 s-1) has the same
rate. All three compounds have Michael acceptors as a
warhead and disubstituted amides with aromatic residues.
In general, aza-peptide Michael acceptors inhibit SmAE
more rapidly than epoxide analogues. For IrAE, three com-
pounds (29b, 670 000 M-1 s-1, 30n, 1 790 000 M-1 s-1, and
28p, 1 980 000 M-1 s-1) have higher rates than Cbz-Ala-Ala-
AAsn-CHdCH-COOEt. Again, all compounds were aza-
peptide Michael acceptors with aromatic residues in the P10
position.
Conclusion. We demonstrate that aza-peptide Michael ac-
ceptors and epoxides with the general structure Boc-AAsn-
trans-CHdCHCOR, YCO-Ala-AAsn-trans-CHdCHCOR,
YCO-Ala-Ala-AAsn-trans-CHdCHCOR, and YCO-Ala-
Ala-AAsn-EP-COR are inhibitors of SmAE and IrAE. We
have shown that tripeptides (YCO-Ala-Ala-AAsn-trans-
CHdCHCOR) are favored over dipeptides (YCO-Ala-
AAsn-trans-CHdCHCOR) that, in turn, are favored over
simple AAsn derivatives (Boc-AAsn-trans-CHdCHCOR).
Extension of the inhibitor structure to P5 resulted in the most
potent compounds against IrAE with IC50 values as low as
0.14 nM and inhibition rates as high as 1 980 000 M-1 s-1
.
Aza-peptide Michael acceptor inhibitors are more potent than
aza-peptide epoxide inhibitors. We have also determined that
the stereochemistry of the epoxide warhead is important; S,S
is favored over cis. In addition, both enzymes prefer small
Mechanism of Inhibition. Aza-peptide Michael acceptors
and epoxides are irreversible inhibitors of clan CD cys-
teine proteases.24 The mechanism of inhibition involves

7198 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
hydrophobic amino acids such as Val and Ile in the P2
position, whereas large hydrophobic amino acids such as
Phe are not tolerated. The results and SAR generated here
may facilitate the design of potent and selective AE inhibitors
with antiparasite bioactivity.
stirred vigorously for 16 h at room temperature. The solvent was
then removed under vacuum.
To synthesize tert-butoxycarbonyl piperazine (Boc-Piz), a
solution of di-tert-butyl dicarbonate (1 equiv) in dichloro-
methane was added slowly to a solution of piperazine (2 equiv)
in dichloromethane at 0 °C. The reaction mixture was stirred at
room temperature overnight. The dichloromethane solution was
washed with saturated NaHCO3 and water. The organic phase
was dried with anhydrous Na2SO4 and evaporated. Purification
by column chromatography using 10% MeOH/CH2Cl2 as the
eluent gave the product in 57% yield: 1H NMR (CDCl3) δ 1.42
(s, 9H, Boc), 2.00 (br s, 1H), 2.78 (m, 4H, piperazine), 3.36 (m, 4H,
piperazine); MS (ESI) m/z 187.1 [(M þ 1)þ].
Experimental Section
Material and Methods. Materials were obtained from Acros,
Bachem Bioscience Inc., or Sigma Aldrich and used without
further purification. The purity of each compound was con-
1
firmed by TLC, H NMR, MS, and elemental analysis. Ele-
mental analysis was carried out by Atlantic Microlab Inc.,
Norcross, GA. Elemental analysis revealed that the purity of
all target compounds after purification was higher than 95%.
Chemical shifts are reported in parts per million relative to an
internal standard (trimethylsilane). TLC was performed on
Sorbent Technologies (250 μm) silica gel plates. The 1H NMR
spectra were obtained on a Varian Mercury 400 MHz spectro-
meter. Electrospray ionization (ESI), fast atom bombardment
(FAB), and high-resolution mass spectrometry were obtained
using Micromass Quattro LC and VG Analytical 70-SE instru-
ments. Elemental analysis was carried out by Atlantic Microlab
Inc., Norcross, GA.
To synthesize benzyloxycarbonyl piperazine (Cbz-Piz),
monoprotection of piperazine was done with benzyl chloro-
formate (1 equiv) and Et3N (1 equiv) in dichloromethane. The
solvent was evaporated, and the crude product was purified by
column chromatography using 10% MeOH/CH2Cl2 as the
1
eluent to obtain the product in 55% yield: H NMR (DMSO-
d6) δ 2.33 (s, 1H, NH), 2.62 (m, 4H, piperazine), 3.30-3.36 (m,
4H, piperazine), 5.00 (s, 2H, Cbz), 7.15-7.22 (m, 5H, Ph); MS
(ESI) m/z 221.0 [(M þ 1)þ].
The piperidine derivative Pip-Ala-OCH3 was prepared by
adding piperidine to alanine isocyanate and purified by column
chromatography on silica gel using 2:18:5 MeOH/CH2Cl2.
EtOAc as the eluent: white solid, yield 83%; 1H NMR
(CDCl3-d) δ 1.32 (d, 3H, Ala-CH3), 1.48 (m, 6H, piperidine),
3.27 (m, 4H, piperidine), 3.67 (s, 3H, OCH3), 4.41 (m, 1H, R-H),
5.05 (d, 1H, NH); MS (ESI) m/z 215.0 [(M þ 1)þ].
Determination of IC50 Values and Second-Order Inhibition
Rates for Asparaginyl Endopeptidases. IC50 values were deter-
mined exactly as previously described.23 For second-order rate
inhibitions, the substrate Z-Ala-Ala-Asn-AMC and inhibitors
were prepared as 10 and 20 mM stock solutions, respectively, in
DMSO. Assays were performed at 25 °C and incorporated
either activated recombinant I. ricinus legumain8 or activated
recombinant S. mansoni legumain.40 An aliquot of (100 μL)
S. mansoni legumain in assay buffer (0.1 M citrate-phosphate
buffer at pH 6.8 containing 4 mM DTT) was added to 100 μL of
inhibitor in assay buffer with 20 μM substrate and inhibitor
present at 2, 1, 0.8, 0.6, 0.4, 0.2, and 0.1 μM (or DMSO alone)
added as a 1 μL aliquot. The progress of inhibition was
followed for 2-5 min, while the uninhibited activity was linear.
An aliquot of (100 μL) I. ricinus legumain in assay buffer (0.1 M
citrate-phosphate buffer at pH 5.5 containing 4 mM DTT with
0.1 M NaCl) was added to inhibitor in 100 μL assay buffer with
inhibitor present at 1, 0.8, 0.5, 0.25, 0.125, 0.0625, 0.03125 μM
and DMSO alone, as above. Release of free AMC was mea-
sured at emission and excitation wavelengths of 355 and 460
nm, respectively, in a Molecular Devices Flex-Station fluoro-
meter. Measurements were taken every 1.52 s. The rate of
inhibition (kobs) was determined at each inhibitor concentra-
tion according to [P]=vo/kobs ꢀ [1 - exp(-kobs ꢀ t)], where [P]
is the concentration of product formed over time t and vo is the
initial rate, using nonlinear regression analysis (GRAPHPAD
PRISM 3.0a). If the kobs versus inhibitor concentration
could reliably fit a two-step irreversible mechanism (r2>0.9),
the inhibition constant Ki and the inactivation constant kinact
were determined by nonlinear regression analysis according to
kobs= kinact ꢀ [I]o/([I]o þ KI* ꢀ (1 þ [S]o/Km)), where [I]o and
[S]o are the concentrations of inhibitor and substrate, respec-
tively, and K*I =Ki ꢀ (1=[S]o/Km). For those inhibitors with
which kobs was linear with increasing concentrations of inhi-
bitor, linear regression analysis was used to obtain the associa-
tion constant kass using kobs= kass ꢀ [I]o/(1 þ [S]o/Km).
Multiple determinations at each inhibitor concentration were
performed.
The morpholine derivative Mu-Ala-OCH3 was prepared by
adding morpholine to alanine isocyanate and purified by col-
umn chromatography on silica gel using 10% MeOH/CH2Cl2 as
1
the eluent: yellowish solid, yield 93%; H NMR (CDCl3-d) δ
1.29 (d, 3H, Ala-CH3), 3.28 (m, 4H, morpholine), 3.58 (m, 4H,
morpholine), 3.64 (s, 3H, OCH3), 4.38 (m, 1H, R-H), 5.25 (s, 1H,
NH); MS (ESI) m/z 217.0 [(M þ 1)þ].
The tert-butoxycarbonyl piperazine derivative Boc-Piz-Ala-
OCH3 was prepared by adding tert-butoxycarbonyl piperazine to
alanine isocyanate and purified by column chromatography on
silica gel using 10% MeOH/CH2Cl2 as the eluent: white solid, yield
80%; 1H NMR (CDCl3-d) δ 1.37 (d, 3H, Ala-CH3), 1.44 (s, 9H,
Boc), 3.34-3.44 (m, 8H, piperazine), 3.72 (s, 3H, OCH3), 4.47
(m, 1H, R-H), 5.08 (s, 1H, NH); MS (ESI) m/z 316.2 [(M þ 1)þ].
The benzyloxycarbonyl piperazine derivative Cbz-Piz-Ala-
OCH3 was prepared by adding benzyloxycarbonyl piperazine to
alanine isocyanate and was purified by column chromatography
on silica gel using 2:18:5 MeOH/CH2Cl2/EtOAc as the eluent:
white solid, yield 72%; 1H NMR (CDCl3-d) δ 1.31 (d, 3H,
Ala-CH3), 3.34-3.36 (m, 4H, piperazine), 3.44-3.47 (m, 4H,
piperazine), 3.65 (s, 3H, OCH3), 4.39 (m, 1H, R-H), 5.07
(s, 2H, Cbz), 5.41 (d, 1H, NH), 7.23-7.29 (m, 5H, Ph); MS
(ESI) m/z 350.2 [(M þ 1)þ].
General Procedure for the Synthesis of Capped Dipeptide
Methyl Ester (YCO-Ala-Ala-OCH3). The methyl ester group
of Y-Ala-OCH3 was hydrolyzed in MeOH using 1 M aqueous
NaOH (1.1 equiv) under standard deblocking conditions. The
Y-Ala-OH derivative was then coupled to alanine methyl
ester hydrochloride (HCl H-Ala-OCH3) using the DCC/HOBt
3
coupling method. To a stirred solution of the Y-Ala-OH
(1 equiv) in DMF at -15 °C was added HOBt (1.5 equiv). The
hydrochloride salt of the alanine methyl ester was pretreated
with NMM (1.5 equiv) at -15 °C in DMF prior to the addition.
The reagent DCC (1.5 equiv) was added to the solution, and
the reaction mixture was allowed to react for 16 h at room
temperature. The DMF was evaporated, and the residue was
redissolved in EtOAc. The organic layer was washed with 2%
citric acid, saturated NaHCO3, and saturated NaCl, dried over
MgSO4, and concentrated. Purification on a silica gel column
with an appropriate eluent gave the product with yields of
60-89%.
General Procedure for the Synthesis of Capped Alanine Methyl
Ester (YCO-Ala-OCH3). Alanine methyl ester isocyanate,
which was synthesized from alanine hydrochloride salt accord-
ing to the procedure by Nowick et al.31 was dissolved in CH2Cl2
and cooled to 0 °C. Y-H groups, which are commercially
available except for benzyloxycarbonyl piperazine and tert-
butoxycarbonyl piperazine, were added to the stirred solution
of alanine methyl ester isocyanate. The reaction mixture was

Article
Pip-Ala-OH was obtained by hydrolysis of Pip-Ala-OCH3
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7199
(d, 1H, NH), 6.54 (d, 1H, NH), 7.36-7.32 (m, 5H, Ph); MS (ESI)
m/z 337.1 [(M þ 1)þ].
in MeOH using 1 M aqueous NaOH (1.1 equiv): white solid,
yield 85%; 1H NMR (DMSO-d6) δ 1.23 (d, 3H, J=7.4 Hz, Ala-
CH3), 1.33-1.44 (m, 4H, piperidine), 1.49 (d, 2H, J=5.0 Hz,
piperidine), 3.24 (s, 4H, piperidine), 4.03 (q, 1H, J = 7.2 Hz,
R-H), 6.50 (d, 1H, J=7.3 Hz, NH).
After coupling Cbz-Ala-OH with HCl H-Ile-OCH3, Cbz-Ala-
3
Ile-OCH3 was purified by column chromatography on silica gel
using 1:1 EtOAc/hexane as the eluent: white solid, yield 89%; 1H
NMR (CDCl3-d) δ 0.85-0.89 (m, 6H, 2 ꢀ Ile-CH3), 1.08-
1.19 (m, 2H, CH2), 1.29-1.43 (m, 3H, Ala-CH3), 1.86 (m, 1H,
CH), 3.70 (s, 3H, OCH3), 4.34 (q, 1H, R-H), 4.55 (q, 1H, R-H),
5.09 (m, 2H, Cbz), 5.63 (d, 1H, NH), 6.82 (d, 1H, NH), 7.26-7.36
(m, 5H, Ph).
After coupling Pip-Ala-OH with HCl H-Ala-OCH3, Pip-
3
Ala-Ala-OCH3 was purified by column chromatography on silica
gel using 10% MeOH/CH2Cl2 as the eluent: white solid, yield
78%; 1H NMR (CDCl3-d) δ 1.37-1.40 (m, 6H, 2 ꢀ Ala-CH3),
1.54-1.59 (m, 6H, piperidine), 3.31-3.34 (m, 4H, piperidine),
3.73 (s, 3H, OCH3), 4.45-4.53 (m, 2H, 2 ꢀ R-H), 5.21 (s, 1H,
NH), 7.12 (s, 1H, NH); MS (FAB) m/z 286.0 [(M þ 1)þ].
Mu-Ala-OH was obtained by hydrolysis of Mu-Ala-OCH3 in
MeOH using 1 M aqueous NaOH (1.1 equiv): white solid, yield
89%; 1H NMR (DMSO-d6) δ 1.24 (d, 3H, J=7.4 Hz, Ala-CH3),
3.26 (t, 4H, morpholine), 3.50-3.52 (m, 4H, morpholine),
4.01-4.08 (m, 1H, R-H), 6.65 (d, 1H, J=7.4 Hz, NH), 12.21
(br s, 1H, COOH); MS (FAB) m/z 202.9 [(M þ 1)þ].
After coupling Cbz-Ala-OH with HCl H-Phe-OCH3, Cbz-
3
Ala-Phe-OCH3 was purified by column chromatography on
silica gel using 2:1 EtOAc/hexane as the eluent: white solid, yield
86%; 1H NMR (CDCl3-d) δ 1.32 (d, 3H, Ala-CH3), 3.03-3.16
(m, 2H, CH2Ph), 3.70 (s, 3H, OCH3), 4.25 (q, 1H, R-H), 4.85
(q, 1H, R-H), 5.08 (m, 2H, Cbz), 5.38 (d, 1H, NH), 7.08 (d,
1H, NH), 7.21-7.34 (m, 10 H, 2 ꢀ Ph); MS (ESI) m/z 385.0
[(M þ 1)þ].
Peptidyl Hydrazides. Anhydrous hydrazine (10 equiv) was
added to a stirred solution of a peptidyl methyl ester (1 equiv) in
MeOH, and the reaction mixture was stirred vigorously for 16 h
at room temperature. Excess hydrazine and solvent were re-
moved under vacuum, and the residue was washed with ether
several times to give the peptidyl hydrazide as a white solid.
Compounds were purified further on silica gel column using the
eluent 10% MeOH/CH2Cl2 when needed.
After coupling Mu-Ala-OH with HCl H-Ala-OCH3, Mu-
3
Ala-Ala-OCH3 was purified by column chromatography on
silica gel using 10% MeOH/CH2Cl2 as the eluent: white solid,
yield 60%; 1H NMR (CDCl3-d) δ 1.37 (d, 3H, J=7.2 Hz, Ala-
CH3), 1.39 (d, 3H, Ala-CH3), 3.34-3.36 (m, 4H, morpholine),
3.65-3.67 (m, 4H, morpholine), 3.73 (s, 3H, OCH3), 4.45-4.53
(m, 2H, 2 ꢀ R-H), 5.32 (s, 1H, NH), 7.02 (d, 1H, J=6.7 Hz, NH);
MS (FAB) m/z 288.2 [(M þ 1)þ].
Pip-Ala-NH-NH2: white solid, yield 97%; 1H NMR (DMSO-
d6) δ 1.15 (d, 3H, Ala-CH3), 1.36-1.41 (m, 4H, piperidine),
1.46-1.49 (m, 2H, piperidine), 3.22-3.25 (m, 4H, piperidine),
4.06-4.12 (m, 3H, R-H and NH2), 6.28 (d, 1H, NH), 8.96 (s, 1H,
NH); MS (ESI) m/z 215.0 [(M þ 1)þ].
Boc-Piz-Ala-OH was obtained by hydrolysis of Boc-Piz-Ala-
OCH3 in MeOH using 1 M aqueous NaOH (1.1 equiv): white
solid, yield 94%; 1H NMR (DMSO-d6) δ 1.24 (d, 3H, J =
7.4 Hz, Ala-CH3), 1.38 (s, 9H, Boc), 3.26 (s, 8H, piperazine),
3.97-4.10 (m, 1H, R-H), 6.70 (d, 1H, J=7.3 Hz, NH).
Mu-Ala-NH-NH2: white solid, yield 97%; 1H NMR (DMSO-
d6) δ 1.16 (d, 3H, Ala-CH3), 3.23-3.26 (m, 4H, morpholine),
3.49-3.52 (m, 4H, morpholine), 4.07-4.14 (m, 3H, R-H and
NH2), 6.45 (d, 1H, NH), 8.97 (s, 1H, NH); MS (ESI) m/z 217.0
[(M þ 1)þ].
After coupling Boc-Piz-Ala-OH with HCl H-Ala-OCH3,
3
Boc-Piz-Ala-Ala-OCH3 was purified by column chromato-
graphy on silica gel using 10% MeOH/CH2Cl2 as the eluent:
white solid, yield 85%; 1H NMR (CDCl3-d) δ 1.36-1.39 (m, 6H,
2 ꢀ Ala-CH3), 1.44 (s, 9H, Boc), 3.34-3.39 (m, 8H, piperazine),
3.72 (s, 3H, OCH3), 4.44-4.54 (m, 2H, 2 ꢀ R-H), 5.38 (d, 1H, J=
7.3 Hz, NH), 7.09 (d, 1H, J=7.2 Hz, NH).
Cbz-Piz-Ala-NH-NH2: white solid, yield 98%; 1H NMR
(DMSO-d6) δ 1.16 (d, 3H, AlaCH3), 3.30-3.34 (m, 8H,
piperazine), 4.11 (m, 3H, R-H and NH2), 5.07 (s, 2H, Cbz),
6.51 (d, 1H, NH), 7.34 (m, 5H, Ph), 8.95 (s, 1H, NH).
Pip-Ala-Ala-NH-NH2: white solid, yield 91%; 1H NMR
(DMSO-d6) δ 1.15-1.18 (m, 6H, 2 ꢀ Ala-CH3), 1.38-1.40
(m, 4H, piperidine), 1.48-1.52 (m, 2H, piperidine), 3.25-3.27
(m, 4H, piperidine), 4.06 (m, 1H, R-H), 4.17-4.21 (m, 3H, R-H
and NH2), 6.40 (d, 1H, NH), 7.71 (d, 1H, NH), 9.01 (s, 1H, NH);
MS (ESI) m/z 286.0 [(M þ 1)þ].
Cbz-Piz-Ala-OH was obtained by hydrolysis of Cbz-Piz-Ala-
OCH3 in MeOH using 1 M aqueous NaOH (1.1 equiv): white
solid, yield 87%; 1H NMR (DMSO-d6) δ 1.25 (d, 3H, Ala-CH3),
3.32-3.34 (m, 8H, piperazine), 4.06 (m, 1H, R-H), 5.07 (s, 2H,
Cbz), 6.72 (d, 1H, NH), 7.28-7.37 (m, 5H, Ph).
After coupling Cbz-Piz-Ala-OH with HCl H-Ala-OCH3, Cbz-
3
Piz-Ala-Ala-OCH3 was purified by column chromatography on
silica gel using 10% MeOH/CH2Cl2 as the eluent: white solid, yield
89%; 1H NMR (CDCl3-d) δ 1.38 (m, 6H, Ala-CH3), 3.38 (m, 4H,
piperazine), 3.50 (m, 4H, piperazine), 3.72 (s, 3H, OCH3), 4.46
(m, 1H, R-H), 5.12 (s, 2H, Cbz), 7.34 (m, 5H, Ph).
Mu-Ala-Ala-NH-NH2: white solid, yield 92%; 1H NMR
(DMSO-d6) δ 1.15-1.18 (m, 6H, 2 ꢀ Ala-CH3), 3.25-3.28 (m,
4H, morpholine), 3.51-3.53 (m, 4H, morpholine), 4.09 (q, 1H,
J=7.2 Hz, R-H), 4.16-4.23 (m, 3H, R-H and NH2), 6.53 (d, 1H,
J=7.1 Hz, NH), 7.77 (d, 1H, J=7.7 Hz, NH), 8.99 (s, 1H, NH).
Boc-Piz-Ala-Ala-NH-NH2: white solid, yield 66%; 1H NMR
(DMSO-d6) δ 1.15-1.19 (m, 6H, 2 ꢀ Ala-CH3), 1.38 (s, 9H,
Boc), 3.27 (s, 8H, piperazine), 4.05-4.12 (m, 1H, R-H),
4.18-4.19 (m, 3H, R-H and NH2), 6.56 (d, 1H, J = 7.0 Hz,
NH), 7.76 (d, 1H, J=7.7 Hz, NH), 8.99 (s, 1H, NH).
Cbz-Piz-Ala-Ala-NH-NH2: white solid, yield 98%; 1H NMR
(DMSO-d6) δ 1.15-1.18 (m, 6H, 2 ꢀ Ala-CH3), 3.45 (s, 8H,
piperazine), 4.03-4.08 (m, 1H, R-H), 4.15-4.19 (m, 3H, R-H
and NH2), 5.06 (s, 2H, Cbz), 6.58 (d, 1H, NH), 7.29-7.35 (m,
5H, Ph), 7.78 (d, 1H, NH), 9.00 (s, 1H, NH).
General Procedure for the Synthesis of Cbz-Capped Dipeptide
Methyl Esters (Cbz-Ala-Val(Ile, Phe)-OCH3). The Cbz-Ala-OH
derivative was coupled to valine methyl ester (or isoleucine
methyl ester or phenylalanine methyl ester) using the DCC
and HOBt coupling method. To a stirred solution of the
Cbz-Ala-OH (1 equiv) in DMF at -15 °C was added HOBt
(1.5 equiv). The hydrochloride salt of the valine methyl ester was
pretreated with NMM (1.5 equiv) at -15 °C in DMF prior to the
addition. The reagent DCC (1.5 equiv) was added to the
solution, and the reaction mixture was allowed to react for
16 h at room temperature. The DMF was evaporated, and the
residue was redissolved in EtOAc. The organic layer was washed
with 2% citric acid, saturated NaHCO3, and saturated NaCl,
dried over MgSO4, and concentrated.
Cbz-Ala-Val-NH-NH2: white solid, yield 98%; 1H NMR
(DMSO-d6) δ 0.78-0.81 (m, 6H, 2 ꢀ Val-CH3), 1.15 (d, 3H,
Ala-CH3), 1.82 (m, 1H, Val-CH), 4.02-4.11 (m, 2H, 2 ꢀ R-H),
4.23 (s, 2H, NH2), 4.99 (s, 2H, Cbz), 7.29-7.34 (m, 5H, Ph), 7.46
(d, 1H, NH), 7.68 (d, 1H, NH), 9.15 (s, 1H, NH).
After coupling Cbz-Ala-OH with HCl H-Val-OCH3, Cbz-
3
Ala-Val-OCH3 was purified by column chromatography on
silica gel using 10% MeOH/CH2Cl2 as the eluent: white solid,
yield 85%; 1H NMR (CDCl3-d) δ 0.88 (m, 6H, 2 ꢀ Val-CH3),
1.39 (d, 3H, Ala-CH3), 2.16 (m, 1H, CH), 3.74 (s, 3H, OCH3),
4,27 (m, 1H, R-H), 4.52 (m, 1H, R-H), 5.12 (s, 2H, Cbz), 5.36
Cbz-Ala-Ile-NH-NH2: white solid, yield 98%; 1H NMR
(DMSO-d6) δ 0.71-0.84 (m, 6H, 2 ꢀ Ala-CH3), 0.97-1.05 (m,
1H, CH), 1.15 (d, 3H, CH3), 1.38-1.44 (m, 1H, CH), 1.61-1.63
(m, 1H, CH), 4.05-4.13 (m, 2H, 2 ꢀ R-H), 4.23 (s, 2H, NH2),

7200 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
4.99 (m, 2H, Cbz), 7.29-7.36 (m, 5H, Ph), 7.45 (d, 1H, NH), 7.70
(d, 1H, NH), 9.16 (s, 1H, NH).
Cbz-Piz-Ala-Ala-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 57%; 1H NMR (DMSO-d6) δ 1.12-1.18
(m, 9H, 2 ꢀ Ala-CH3 and NHCH2COCH2CH3), 3.32-3.34 (m,
8H, piperazine), 3.44 (d, 2H, NHCH2COCH2CH3), 4.03-4.10
(m,4H, 2ꢀ R-H and NHCH2COCH2CH3), 5.04(s, 2H, Cbz), 6.59
(d, 1H, NH), 7.29-7.35 (m, 5H, Ph), 7.81 (d, 1H, NH), 9.29
(d, 1H, NH).
Cbz-Ala-Val-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 64%; 1H NMR (DMSO-d6) δ
0.78-0.84 (m, 6H, 2 ꢀ CH3), 1.14-1.18 (m, 6H, 2 ꢀ CH3),
1.84 (m, 1H, CH), 3.48 (d, 2H, NHCH2COCH2CH3), 4.02-4.08
(m, 4H, 2 ꢀ R-H and NHCH2COCH2CH3), 4.99 (m, 2H, Cbz),
7.28-7.34 (m, 5H, Ph), 7.46 (d, 1H, NH), 7.70 (d, 1H, NH).
Cbz-Ala-Ile-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as
the eluent: white solid, yield 55%; 1H NMR (DMSO-d6) δ
0.72-0.82 (m, 6H, 2 ꢀ CH3), 0.97 (m, 1H, CH), 1.14 (m, 6H,
2 ꢀ CH3), 1.35 (m, 1H, CH), 1.60 (m, 1H, CH), 3.38 (s, 2H,
NHCH2CO), 4.03-4.19 (m, 4H, COCH2CH3 and 2ꢀR-H), 4.99
(m, 2H, Cbz), 7.29-7.34 (m, 5H, Ph), 7.46 (d, 1H, NH), 7.66 (d,
1H, NH), 9.44 (s, 1H, NH).
Cbz-Ala-Phe-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 10% MeOH/CH2Cl2 as
the eluent: white solid, yield 53%; 1H NMR (DMSO-d6) δ 1.09
(d, 3H, Ala-CH3), 1.15-1.19 (t, 3H, NHCH2COCH2CH3),
2.71 (s, 1H, NH), 2.76-2.92 (m, 2H, CH2Ph), 3.33-3.39
(m, 2H, NHCH2COCH2CH3), 3.98-4.08 (m, 3H, R-H and
NHCH2COCH2CH3), 4.21 (s, 1H, NH), 4.43 (q, 1H, R-H),
4.99 (m, 2H, Cbz), 7.15-7.41 (m, 10H, 2 ꢀ Ph), 7.93 (d, 1H,
NH), 9.13 (s, 1H, NH).
General Procedure for the Synthesis of Peptidyl-NH-NH-
CH2CONH2. The peptidyl-NH-NHCH2COOEt (1 equiv) was
dissolved in a 7 N solution (100 equiv) of NH3 in methanol and a
small amount of DMF and allowed to stir in an ice bath. To this
solution was added a catalytic amount of NaCN (0.1 equiv). The
flask was closed with a rubber septum and allowed to stir at 4 °C
for 5 days. The solvent was evaporated, and the product was
purified by column chromatography on a silica gel using the
proper eluent.
Cbz-Ala-Phe-NH-NH2: white solid, yield 97%; 1H NMR
(DMSO-d6) δ 1.08 (d, 3H, Ala-CH3), 2.78-2.97 (m, 2H, CH2Ph),
3.99 (q, 1H, R-H), 4.20 (br s, 2H, NH2), 4.42 (q, 1H, R-H), 4.99
(m, 2H, Cbz), 7.14-7.39 (m, 11 H, 2 ꢀ Ph and NH), 7.92 (d, 1H,
NH), 9.14 (s, 1H, NH); MS (ESI) m/z 385.2 [(M þ 1)þ].
General Procedure for the Synthesis of Peptidyl-NH-NH-
CH2COOEt. The reagent tert-butyl bromoacetate (1.1 equiv)
was added dropwise at -15 °C to a stirred solution of a peptidyl
hydrazide (1 equiv) in DMF and NMM (1.1 equiv), and the
mixture was allowed to react for 36 h at room temperature. The
solvent was removed under vacuum, and the residue was
purified on a silica gel column with the proper eluent.
Boc-NH-NH-CH2COOEt was purified by column chromatog-
raphy on silica gel using 15% MeOH/CH2Cl2 as the eluent: white
1
solid, yield 90%; H NMR (DMSO-d6) δ 1.26 (t, 3H, NHCH2-
COCH2CH3), 1.45 (m, 9H, Boc), 4.11 (s, 2H, NCH2CO), 4.19
(q, 2H, NHCH2COCH2CH3); MS (ESI) m/z 219.2 [(M þ 1)þ].
Pip-Ala-NH-NH-CH2COOEt was purified by column chro-
matography on silica gel using 15% MeOH/CH2Cl2 as the eluent:
yellow oil, yield 26%; 1H NMR (DMSO-d6) δ 0.78-0.84 (m, 3H,
NHCH2COCH2CH3), 1.13-1.19 (m, 3H, Ala-CH3), 1.38-1.41
(m, 4H, piperidine), 1.47-1.50 (m, 2H, piperidine), 3.23-3.25
(m, 4H, piperidine), 3.45 (d, 2H, J = 4.4 Hz, NHCH2CO-
CH2CH3), 4.03-4.09 (m, 3H, R-H and NHCH2COCH2CH3),
5.09 (d, 1H, J=5.2 Hz, NH), 6.32 (d, 1H, J=7.5 Hz, NH), 9.16
(d, 1H, J=5.4 Hz, NH); MS (ESI) m/z 301.0 [(M þ 1)þ].
Mu-Ala-NH-NH-CH2COOEt was purified by column chro-
matography on silica gel using 15% MeOH/CH2Cl2 as the eluent:
yellow oil, yield 41%; 1H NMR (DMSO-d6) δ 0.78-0.84 (m, 3H,
NHCH2COCH2CH3), 1.14-1.18 (m, 3H, Ala-CH3), 3.20-3.29
(m, 4H, morpholine), 3.45 (d, 2H, NHCH2COCH2CH3),
3.49-3.55 (m, 4H, CH2OCH2 morpholine), 4.03-4.10 (m, 3H,
R-H and NHCH2COCH2CH3), 5.10 (d, 1H, NH), 6.48 (d, 1H,
NH), 9.23 (d, 1H, NH); MS (ESI) m/z 303.0 [(M þ 1)þ].
Cbz-Piz-Ala-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 44%; 1H NMR (DMSO-d6) δ 1.14-1.18
(m, 6H, Ala-CH3 and NHCH2COCH2CH3), 3.30-3.33 (m, 8H,
piperazine) 3.45 (d, 2H, NHCH2COCH2CH3), 4.03-4.10
(m, 3H, R-H and NHCH2COCH2CH3), 5.07 (s, 2H, Cbz), 6.55
(d, 1H, NH), 7.29-7.36 (m, 5H, Ph), 9.23 (d, 1H, NH); MS (ESI)
m/z 436.3 [(M þ 1)þ].
Boc-NH-NH-CH2CONH2 was purified by column chromato-
graphy on silica gel using 15% MeOH/CH2Cl2 as the eluent:
white solid, yield 80%; 1H NMR (DMSO-d6) δ 1.36 (s, 9H,
Boc), 3.16 (s, 2H, NCH2CO), 4.96 (d, 1H, NH), 7.16 (s, 1H,
NH), 7.38 (s, 1H, NH), 8.24 (s, 1H, NH); MS (ESI) m/z 189.9
[(M þ 1)þ].
Pip-Ala-Ala-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 20%; 1H NMR (DMSO-d6) δ 1.13-1.18
(m, 9H, 2 ꢀ Ala-CH3 and NHCH2COCH2CH3), 1.37-1.40
(m, 4H, piperidine), 1.48-1.52 (m, 2H, piperidine), 3.23-3.25
(m, 4H, piperidine), 3.44 (d, 2H, NHCH2COCH2CH3),
4.04-4.09 (m, 3H, R-H and NHCH2COCH2CH3), 4.15 (m, 1H,
R-H), 5.15 (m, 1H, NH), 6.40 (d, 1H, NH), 7.74 (d, 1H, NH), 9.32
(d, 1H, NH).
Mu-Ala-Ala-NH-NH-CH2COOEt was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 30%; 1H NMR (DMSO-d6) δ 0.78-0.84
(m, 6H, 2 ꢀ CH3), 1.14-1.18 (m, 6H, 2 ꢀ CH3), 3.25-3.32 (m,
4H, morpholine), 3.44 (d, 2H, J=4.9 Hz, NHCH2COCH2CH3),
3.50-3.53 (m, 4H, morpholine), 4.04-4.09 (m, 3H, R-H and
NHCH2COCH2CH3), 4.18 (q, 1H, R-H), 5.15 (q, 1H, J=5.0 Hz,
NH), 6.53 (d, 1H, J=7.2 Hz, NH), 7.81 (d, 1H, J=7.5 Hz, NH),
9.29 (d, 1H, J=5.9 Hz, NH).
Boc-Piz-Ala-Ala-NH-NH-CH2COOEt was purified by col-
umn chromatography on silica gel using 15% MeOH/CH2Cl2
as the eluent: white solid, yield 50%; 1H NMR (DMSO-d6) δ 1.26
(t, 3H, NHCH2COCH2CH3), 1.34-1.37 (m, 6H, 2 ꢀ Ala-CH3),
1.45 (s, 9H, Boc), 3.37-3.42 (m, 10H, piperazine and NH-
CH2COCH2CH3), 4.15-4.22 (m, 2H, NHCH2COCH2CH3),
4.39 (m, 1H, R-H), 4.46 (m, 1H, R-H and NHCH2COCH2CH3),
5.54 (d, 1H, NH), 7.35 (d, 1H, NH), 8.89 (s, 1H, NH).
Pip-Ala-NH-NH-CH2CONH2 was purified by column chro-
matography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 86%; 1H NMR (DMSO-d6) δ 1.15
(d, 3H, Ala-CH3), 1.38-1.41 (m, 4H, piperidine), 1.47-1.50
(m, 2H, piperidine), 3.17 (d, 2H, NCH2CO), 3.23-3.26 (m, 4H,
piperidine), 4.04 (q, 1H, R-H), 5.13 (d, 1H, NH), 5.36 (d, 1H,
NH), 7.10 (s, 1H, NH), 7.46 (s, 1H, NH), 9.18 (d, 1H, NH); MS
(ESI) m/z 272.0 [(M þ 1)þ].
Mu-Ala-NH-NH-CH2CONH2 was purified by column chro-
matography on silica gel using 15% MeOH/CH2Cl2 as the eluent:
white solid, yield 53%; 1H NMR (DMSO-d6) δ 1.16 (d, 3H, J=
7.1 Hz, Ala-CH3), 3.18 (d, 2H, J=3.5 Hz, NCH2CO), 3.25-3.26
(m, 4H, morpholine), 3.50-3.52 (m, 4H, morpholine), 4.02-4.09
(m, 1H, R-H), 5.14 (d, 1H, J=3.9 Hz, NH), 6.52 (d, 1H, J=
7.4 Hz, NH), 7.12 (s, 1H, NH), 7.46 (s, 1H, NH), 9.23 (d, 1H, J=
3.8 Hz, NH); MS (ESI) m/z 274.0 [(M þ 1)þ].
Cbz-Piz-Ala-NH-NH-CH2CONH2 was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 62%; 1H NMR (DMSO-d6) δ 1.16
(d, 3H, Ala-CH3), 3.18 (d, 2H, NCH2CO), 3.31-3.33 (m, 8H,
piperazine), 4.05 (m, 1H, R-H), 5.07 (s, 2H, Cbz), 5.15 (d, 1H,
NH), 6.58 (d, 1H, NH), 7.11 (s, 1H, NH), 7.30-7.38 (m, 5H, Ph),
7.45 (s, 1H, NH), 7.85 (d, 1H, NH), 9.23 (d, 1H, NH).

Article
Pip-Ala-Ala-NH-NH-CH2CONH2 was purified by column
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7201
and the residue was redissolved in ethyl acetate and washed with
2% citric acid, saturated NaHCO3, and saturated NaCl, dried
over MgSO4, and concentrated. The product was purified by
column chromatography as needed.
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 58%; 1H NMR (DMSO-d6) δ
1.15-1.18 (m, 6H, 2 ꢀ Ala-CH3), 1.40 (s, 4H, piperidine),
1.48-1.51 (m, 2H, piperidine), 3.17 (d, 2H, J = 3.6 Hz,
NCH2CO), 3.21-3.27 (m, 4H, piperidine), 4.05 (q, 1H, J=6.9
Hz, R-H), 4.14 (q, 1H, J = 7.0 Hz, R-H), 5.19 (d, 1H, J =
4.1 Hz, NH), 6.40 (d, 1H, J=6.9 Hz, NH), 7.11 (s, 1H, NH), 7.42
(s, 1H, NH), 7.79 (d, 1H, J=7.5 Hz, NH), 9.29 (d, 1H, J=3.8 Hz,
NH); MS (ESI) m/z 343.1 [(M þ 1)þ].
trans-3-Benzyloxycarbonylpropenoic Acid or Monobenzyl
Fumarate (2b, HOOCCHdCHCOOBzl). Equimolar amounts
of fumaric acid and benzyl alcohol were dissolved in anhydrous
DMF. The reagent NMM (1 equiv) was added at 0 °C followed
by EDC after 15 min. The reaction mixture was stirred overnight
at room temperature. The DMF was evaporated, and the crude
residue was redissolved in EtOAc. The product was extracted
with saturated aqueous NaHCO3. The aqueous layer was then
acidified with 1 N HCl to pH 2. The product was extracted with
EtOAc, and the organic layer was washed with water and dried
with MgSO4. The solvent was evaporated, and purification by
column chromatography using 5% MeOH/CH2Cl2 as the eluent
gave a white powder (51% yield): 1H NMR (DMSO-d6) δ 5.21
(s, 2H, CHdCH-COOCH2Ph), 6.73 (m, 2H, CHdCH-COOC-
H2Ph), 7.29-7.43 (m, 5H, Ph); MS (ESI) m/z 207 [(M þ 1)þ].
trans-3-Cyclopropylcarbamoylpropenoic Acid Ethyl Ester
(EtOOCCHdCHCONH-Cyclopropyl) was obtained by mixed
anhydride coupling of equimolar amounts of monoethyl fuma-
rate and cyclopropylamine. Purification by column chromato-
graphy using 2:1 hexane/EtOAc as the eluent gave a white
Mu-Ala-Ala-NH-NH-CH2CONH2 was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 48%; 1H NMR (DMSO-d6) δ
1.16-1.19 (m, 6H, 2 ꢀ Ala-CH3), 3.17 (d, 2H, J = 4.1 Hz,
NCH2CO), 3.26-3.27 (m, 4H, morpholine), 3.50-3.53 (m,
4H, morpholine), 4.04-4.18 (m, 2H, 2 ꢀ R-H), 5.19 (d, 1H, J=
4.2 Hz, NH), 6.54 (d, 1H, J=7.0 Hz, NH), 7.12 (s, 1H, NH), 7.42
(s, 1H, NH), 7.86 (d, 1H, J=7.5 Hz, NH), 9.27 (d, 1H, J=4.3
Hz, NH).
Boc-Piz-Ala-Ala-NH-NH-CH2CONH2 was purified by col-
umn chromatography on silica gel using 15% MeOH/CH2Cl2 as
the eluent: white solid, yield 67%; 1H NMR (DMSO-d6) δ
1.16-1.19 (m, 6H, 2 ꢀ Ala-CH3), 1.39 (s, 9H, Boc), 3.17 (d,
2H, J = 2.9 Hz, NCH2CO), 3.27-3.33 (m, 8H, piperazine),
4.03-4.14 (m, 2H, 2 ꢀ R-H), 5.19 (d, 1H, NH), 6.57 (d, 1H, J=
6.9 Hz, NH), 7.11 (s, 1H, NH), 7.41 (s, 1H, NH), 7.85 (d, 1H,
J=7.5 Hz, NH), 9.26 (d, 1H, J=3.2 Hz, NH).
Cbz-Piz-Ala-Ala-NH-NH-CH2CONH2 was purified by col-
umn chromatography on silica gel using 15% MeOH/CH2Cl2 as
the eluent: white solid, yield 34%; 1H NMR (DMSO-d6) δ
1.15-1.19 (m, 6H, 2 ꢀ Ala-CH3), 3.17 (d, 2H, J = 3.7 Hz,
NCH2CO), 3.32-3.35 (m, 8H, piperazine), 4.06-4.14 (m, 2H,
2 ꢀ R-H), 5.07 (s, 2H, Cbz), 5.20 (d, 1H, NH), 6.60 (d, 1H, J=6.9
Hz, NH), 7.12 (s, 1H, NH), 7.23-7.35 (m, 5H, Ph), 7.41 (s, 1H,
NH), 7.87 (d, 1H, J=7.5 Hz, NH), 9.27 (d, 1H, J=4.0 Hz, NH).
Cbz-Ala-Val-NH-NH-CH2CONH2 was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 41%; 1H NMR (DMSO-d6) δ
0.78-0.81 (m, 6H, 2 ꢀ Val-CH3), 1.16 (d, 3H, Ala-CH3), 1.85
(m, 1H, CH), 3.40 (d, 2H, NHCH2CO), 4.09-4.13 (m, 2H, 2 ꢀ
R-H), 4.99 (m, 2H, Cbz), 5.27 (d, 1H, NH), 7.13 (s, 1H, NH),
7.28-7.32 (m, 5H, Ph), 7.45 (s, 1H, NH), 7.73 (d, 1H, NH), 9.46
(d, 1H, NH).
1
powder (84% yield): H NMR (CDCl3) δ 0.56-0.59 (m, 2H,
CH2), 0.80-0.82 (m, 2H, CH2), 1.28 (t, 3H, CH3), 2.81-2.84 (m,
1H, CH), 4.20 (q, 2H, CH2CH3), 6.77-6.81 (d, 2H, CHd
CHCON and NH), 6.92 (d, 1H, J=15.5 Hz, CHdCHCON).
trans-3-Cyclopropylcarbamoylpropenoic Acid (2c, HOOC-
CHdCHCONH-Cyclopropyl).
EtOOCCHdCHCONH-
cyclopropyl was hydrolyzed in EtOH using NaOH (1 M aqu-
eous, 1.1 equiv) under standard deblocking conditions to give a
white solid (91% yield): 1H NMR (DMSO-d6) δ 0.41-0.45 (m,
2H, CH2), 0.64-0.69 (m, 2H, CH2), 2.69-2.76 (m, 1H, CH),
6.47 (d, 1H, J=15.5 Hz, CHdCHCON), 6.80 (d, 1H, J=15.5
Hz, CHdCHCON), 8.53 (d, 1H, NH).
trans-3-Cyclohexylcarbamoylpropenoic Acid Ethyl Ester (EtO-
OCCHdCHCONH-cyclohexyl) was obtained by mixed an-
hydride coupling of equimolar amounts of monoethyl fumarate
and cyclohexylamine. Purification by column chromatography
using 2:1 EtOAc/hexane as the eluent gave a white powder (63%
yield).
trans-3-Cyclohexylcarbamoylpropenoic Acid (2d, HOOC-
Cbz-Ala-Ile-NH-NH-CH2CONH2 was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 25%; 1H NMR (DMSO-d6) δ
0.76-0.79 (m, 6H, 2 ꢀ Ile-CH3), 1.02 (m, 1H, CH), 1.14 (d,
3H, Ala-CH3), 1.40 (m, 1H, CH), 1.63 (m, 1H, CH), 3.18 (d, 2H,
NHCH2CO), 4.00-4.10 (m, 2H, 2 ꢀ R-H), 4.99 (m, 2H, Cbz),
5.29 (d, 1H, NH), 7.15-7.46 (m, 7H, Ph and 2 ꢀ NH), 7.77
(d, 1H, NH), 9.47 (d, 1H, NH).
CHdCHCONH-cyclohexyl).
EtOOCCHdCHCONH-
cyclohexyl was hydrolyzed in EtOH using NaOH (1 M aqu-
eous, 1.1 equiv) under standard deblocking conditions to give
a white solid (81% yield): 1H NMR (DMSO-d6) δ 0.82-0.90
(m, 2H, CH2), 1.07-1.39 (m, 4H, 2 ꢀ CH2), 1.59-1.65 (m, 5H,
2 ꢀ CH2 and CH), 2.98 (t, 2H, J=6.1 Hz, NHCH2), 6.47 (d,
1H, J = 15.6, CHdCHCON), 6.93 (d, 1H, J = 15.6,
CHdCHCON), 8.43 (t, 1H, J=5.5 Hz, NH).
Cbz-Ala-Phe-NH-NH-CH2CONH2 was purified by column
chromatography on silica gel using 15% MeOH/CH2Cl2 as the
eluent: white solid, yield 64%; 1H NMR (DMSO-d6) δ 1.10
(d, 3H, Ala-CH3), 2.80-2.89 (m, 2H, CH2Ph), 3.13 (d, 2H,
NHCH2CO), 4.00 (q, 1H, R-H), 4.38 (q, 1H, R-H), 4.99 (m, 2H,
Cbz), 5.21 (d, 1H, NH), 7.12-7.40 (m, 12H, 2 ꢀ Ph and 2 ꢀ
NH), 7.93 (s, 1H, NH), 7.97 (s, 1H, NH).
General Procedure for the Synthesis of Fumaric Acid Mono-
amides by the Mixed Anhydride Coupling Method. Coupling of
the amine precursors to monoethyl fumarate was accomplished
using the mixed anhydride coupling method. To a solution of
the monoethyl fumarate (1 equiv) in CH2Cl2 at -20 °C was
added N-methylmorpholine (NMM, 1 equiv) followed by iso-
butyl chloroformate (iBCF, 1 equiv). After the reaction mixture
was allowed to stir for 30 min, the amine (1 equiv) was added to
the mixture. Hydrochloride salts of the amine were pretreated
with NMM (1 equiv) at -20 °C in CH2Cl2 prior to addition.
After 30 min, the reaction mixture was allowed to stir overnight
at room temperature. The methylene chloride was evaporated,
trans-3-(2-Furyl)carbamoylpropenoic Acid Ethyl Ester (EtOO-
CCHdCHCONHCH2-2-furyl) was obtained by mixed anhy-
dride coupling of equimolar amounts of monoethyl fumarate
and 2-furylamine. Purification by column chromatography
using 5% MeOH/CH2Cl2 as the eluent gave a yellow oil (84%
1
yield): H NMR (CDCl3) δ 1.26 (t, 3H, CH3CH2), 4.14-4.19
(m, 2H, CH3CH2), 4.50 (d, 2H, NHCH2), 6.22-6.23 (d, 1H,
furyl), 6.28-6.29 (d, 1H, furyl), 6.81 (d, 1H, J = 15.5 Hz,
CHdCHCON), 6.92 (t, 1H, NH), 7.00 (d, 1H, J = 15.5 Hz,
CHdCHCON), 7.32 (s, 1H, furyl).
trans-3-(2-Furyl)carbamoylpropenoic Acid (2e, HOOCCHd
CHCONHCH2-2-furyl). EtOOCCHdCHCONHCH2-2-furyl
was hydrolyzed in EtOH using NaOH (1 M aqueous, 1.1 equiv)
under standard deblocking conditions to give a white solid (79%
yield): 1H NMR (DMSO-d6) δ 4.38 (d, 2H, NHCH2), 6.29-6.30
(d, 1H, J = 7.8 Hz, furyl), 6.40-6.41 (d, 1H, furyl), 6.53-
6.57 (d, 1H, J=15.5 Hz, CHdCHCON), 6.95 (d, 1H, J=15.6
Hz, CHdCHCON), 7.60 (s, 1H, furyl), 8.96 (t, 1H, J = 5.5
Hz, NH).

7202 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
trans-3-Benzylcarbamoylpropenoic Acid Ethyl Ester (EtO-
OCCHdCHCONHBzl) was obtained by mixed anhydride cou-
pling of equimolar amounts of monoethyl fumarate and ben-
zylamine. Purification by column chromatography using 5%
MeOH/CH2Cl2 gave a white powder (81% yield): 1H NMR
(DMSO-d6) δ 1.20-1.24 (t, 3H, J = 14.4 Hz, CH2CH3),
4.14-4.19 (q, 2H, J=7.1 Hz, CH2CH3), 4.37 (d, 2H, J=5.9
Hz, NCH2Ph), 6.59 (d, 1H, J=15.2 Hz, CHdCHCON), 7.05
(d, 1H, J=15.2 Hz, CHdCHCON), 7.22-7.34 (m, 5H, Ph), 9.04
(s, 1H, NH).
monoethyl fumarate and 1,2,3,4-tetrahydroquinoline to give a
brown syrup (83% yield): 1H NMR (CDCl3) δ 1.29 (t, 3H,
CH3CH2OC), 1.99-2.02 (m, 2H, N-CH2-CH2-CH2), 2.73-2.76
(t, 1H, N-CH2-CH2-CH2), 3.86-3.98 (t, 1H, N-CH2-CH2-
CH2), 4.24-4.30 (q, 2H, CH3CH2OOC), 6.80 (d, 1H, J=14.8
Hz, CHdCHCON), 7.18-7.22 (m, 4H, quinoline), 7.46 (d, 1H,
J=14.8 Hz, CHdCHCON).
trans-3-(3,4-Dihydro-2H-quinolin-1-ylcarbonyl)propenoic Acid
(2j, HOOCCHdCHCO-tetrahydroquinoline). EtOOCCHd
CHCO-tetrahydroquinoline was hydrolyzed in EtOH using
NaOH (1 M aqueous, 1.1 equiv) under standard deblocking
conditions to give a clear syrup, which was recrystallized using
cold EtOAc to give a yellow powder (78% yield): 1H NMR
(DMSO-d6) δ 1.84-1.91 (m, 2H, N-CH2-CH2-CH2), 2.68-2.71
(t, 1H, N-CH2-CH2-CH2), 3.71-3.75 (t, 1H, N-CH2-CH2-
CH2), 6.58 (d, 1H, J = 15.6 Hz, CHdCHCON), 7.06-7.24
(m, 5H, quinoline and CHdCHCON).
trans-3-(3,4-Dihydro-2H-quinolin-1-ylcarbonyl)propenoic Acid
Ethyl Ester (EtOOCCHdCHCO-tetrahydroisoquinoline) was
obtained by mixed anhydride coupling of equimolar amounts
of monoethyl fumarate and 1,2,3,4-tetrahydroisoquinoline to
give a brown syrup (83% yield): 1H NMR (CDCl3) δ 1.29 (t, 3H,
CH3CH2OC), 1.99-2.02 (m, 2H, N-CH2-CH2-CH2), 2.73-2.76
(t, 1H, N-CH2-CH2-CH2), 3.86-3.98 (t, 1H, N-CH2-CH2-
CH2), 4.24-4.30 (q, 2H, CH3CH2OOC), 6.80 (d, 1H, J=14.8
Hz, CHdCHCON), 7.20 (m, 4H, quinoline), 7.46 (d, 1H, J=
14.8 Hz, CHdCHCON).
trans-3-Benzylcarbamoylpropenoic Acid (2f, HOOCCHd
CHCONHBzl). EtOOCCHdCHCONHBzl was hydrolyzed in
EtOH using NaOH (1 M aqueous, 1.1 equiv) under standard
deblocking conditions to give a white solid (73% yield): 1H
NMR (DMSO-d6) δ 4.37 (d, 2H, J = 5.9 Hz, NCH2Ph),
6.52-6.56 (d, 1H, J = 15.2 Hz, CHdCHCON), 6.95-6.99
(d, 1H, J=15.2 Hz, CHdCHCON), 7.23-7.33 (m, 5H, Ph),
8.98 (t, 1H, J=5.6 Hz, NH); MS (ESI) m/z 206 [(M þ 1)þ].
trans-3-Phenylethylcarbamoylpropenoic Acid Ethyl Ester
(EtOOCCHdCHCONHCH2CH2Ph) was obtained by a mixed
anhydride coupling of equimolar amounts of monoethyl
fumarate and phenethylamine to give a clear colorless syrup
1
(78% yield): H NMR (DMSO-d6) δ 2.74 (t, 2H, J=7.3 Hz,
NCH2CH2Ph), 3.33-3.43 (m, 2H, NCH2CH2Ph), 6.56 (d, 1H,
J = 15.6 Hz, CHdCHCON), 6.97 (d, 1H, J = 15.6 Hz,
CHdCHCON), 7.15-7.28 (m, 5H, Ph), 8.63 (t, 1H, J = 5.4
Hz, NH).
trans-3-Phenylethylcarbamoylpropenoic Acid (2g, HOOC-
CHdCHCONHCH2CH2Ph). EtOOCCHdCHCONHCH2-
CH2Ph was hydrolyzed in EtOH using NaOH (1 M aqueous,
1.1 equiv) under standard deblocking conditions to give a clear,
colorless syrup (81% yield): 1H NMR (DMSO-d6) δ 3.54 (t, 2H,
N-CH2-CH2-Ph), 3.61 (t, 2H, N-CH2-CH2-Ph), 6.45z (d, 1H,
J=15.2 Hz, CHdCHCON), 7.00 (d, 1H, J=15.6 Hz, CHd
CHCON), 7.14-7.31 (m, 5H, Ph).
trans-3-Phenylpropylcarbamoylpropenoic Acid Ethyl Ester
(EtOOCCHdCHCONHCH2CH2CH2Ph) was obtained by
mixed anhydride coupling of equimolar amounts of monoethyl
fumarate and 3-phenyl-1-propylamine. Purification by column
chromatography using 1:1 EtOAc/hexane gave a white powder
(80% yield).
trans-3-Phenylpropylcarbamoylpropenoic Acid (2h, HOOC-
CHdCHCONHCH2CH2CH2Ph). EtOOCCHdCHCONH-
CH2CH2CH2Ph was hydrolyzed in EtOH using NaOH
(1 M aqueous, 1.1 equiv) under standard deblocking condi-
tions to give a white powder (81% yield): 1H NMR (DMSO-
d6) δ 1.68-1.76 (m, 2H, NH-CH2-CH2-CH2-Ph), 2.49-2.59
(m, 2H, NH-CH2-CH2-CH2-Ph), 3.12-3.17 (m, 2H, NH-
CH2-CH2-CH2-Ph), 6.49 (d, 1H, J=15.6 Hz, CHdCHCON),
6.92 (d, 1H, J=15.6 Hz, CHdCHCON), 7.15-7.28 (m, 5H,
Ph), 8.51 (t, 1H, J=5.5 Hz, NH).
trans-3-(3,4-Dihydro-2H-quinolin-1-ylcarbonyl)propenoic Acid
(2k, HOOCCHdCHCO-tetrahydroisoquinoline). EtOOCCHd
CHCO-tetrahydroquinoline was hydrolyzed in EtOH using
NaOH (1 M aqueous, 1.1 equiv) under standard deblocking
conditions to give a clear syrup, which was recrystallized using
cold EtOAc to give a yellow powder (68% yield): 1H NMR
(DMSO-d6) δ 1.2.78-2.87 (m, 2H, CH2NCH2CH2), 3.71-
3.78 (m, 2H, CH2NCH2CH2), 4.66-4.76 (d, 2H, CH2-
NCH2CH2CH2), 6.52 (d, 1H, J = 15.6 Hz, CHdCHCON),
7.16-7.21 (m, 4H, quinoline), 7.43-7.49 (d, 1H, J=15.6 Hz,
CHdCHCON).
trans-3-Dibenzylcarbamoylpropenoic Acid Ethyl Ester (EtO-
OCCHdCHCON(Bzl)2) was obtained by mixed anhydride
coupling of equimolar amounts of monoethyl fumarate and
dibenzylamine to give a clear, pink syrup (87% yield).
trans-3-Dibenzylcarbamoylpropenoic Acid (2l, HOOCCHd
CHCON(Bzl)2). EtOOCCHdCHCON(Bzl)2 was hydrolyzed
in EtOH using NaOH (1 M aqueous, 1.1 equiv) under standard
1
deblocking conditions to give a white powder (91% yield): H
NMR (DMSO-d6) δ 4.57 (s, 2H, NCH2Ph), 4.65 (s, 2H,
NCH2Ph), 6.61-6.65 (d, 1H, J = 15.2 Hz, CHdCHCON),
7.15-7.17 (d, 1H, J = 15.2 Hz, CHdCHCON), 7.25-7.50
(m, 10H, 2 ꢀ Ph).
trans-3-(1-Naphthylmethylcarbamoyl)propenoic Acid Ethyl
Ester (EtOOCCHdCH-CONHCH2-1-Napth) was obtained
by mixed anhydride coupling of equimolar amounts of mono-
ethyl fumarate and 1-naphthylmethylamine. Purification by
column chromatography using 2:1 hexane/EtOAc to give white
trans-3-(3,4-Dimethoxybenzylcarbamoyl)propenoic Acid Ethyl
Ester (EtOOCCHdCH-CONHCH2Ph-3,4-OCH3) was ob-
tained by mixed anhydride coupling of equimolar amounts of
monoethyl fumarate and 3,4-dimethoxyphenylmethylamine.
Purification by column chromatography using 5% MeOH/
1
powder (86% yield): H NMR (CDCl3) δ 1.20 (t, 3H, CH3),
1
3.99 (q, 2H, CH2), 4.93 (d, 2H, CH2-naphth), 6.84 (d, 1H,
CHdCHCON), 6.90 (d, 1H, CHdCHCON), 7.38-7.44
(m, 2H, naphthyl), 7.47-7.54 (m, 2H, naphthyl), 7.79 (d, 1H,
naphthyl), 7.84 (d, 1H, naphthyl), 7.95 (d, 1H, naphthyl).
trans-3-(1-Naphthylmethylcarbamoyl)propenoic Acid (2m,
HOOCCHdCH-CONHCH2-1-Napth). EtOOCCHdCHCO-
NHCH2-1-Napth was hydrolyzed in EtOH using NaOH (1 M
aqueous, 1.1 equiv) under standard deblocking conditions to
give a white solid (76% yield): 1H NMR (DMSO-d6) δ 4.82
(d, 2H, J=5.5 Hz, CH2-naphthyl), 6.58 (d, 1H, J=15.6 Hz,
CHdCHCON), 6.93 (d, 1H, J = 15.6 Hz, CHdCHCON),
7.40-7.57 (m, 4H, naphthyl), 7.84-7.86 (m, 1H, naphthyl),
7.94 (d, 1H, J = 7.7 Hz, naphthyl), 8.03 (d, 1H, J = 8.2 Hz,
naphthyl), 9.00 (t, 1H, J=5.4 Hz, NH).
CH2Cl2 gave a white powder (69% yield): H NMR (DMSO-
d6) δ 1.22 (t, 3H, CH3), 3.70-3.72 (d, 6H, 2 ꢀ OCH3), 4.16
(q, 2H, CH2CH3), 4.29 (d, 2H, NHCH2), 6.57-6.61 (d, 1H, J=
15.6 Hz, CHdCHCON), 6.76-6.79 (d, 1H, Ph), 6.87-6.89
(d, 2H, Ph), 7.04 (d, 1H, J = 15.6 Hz, CHdCHCON), 8.92
(t, 1H, NH).
trans-3-(3,4-Dimethoxybenzylcarbamoyl)propenoic Acid (2i,
HOOCCHdCH-CONHCH2Ph-3,4-OCH3). EtOOCCHd
CH-CONHCH2Ph-3,4-OCH3 was hydrolyzed in EtOH
using NaOH (1 M aqueous, 1.1 equiv) under standard
deblocking conditions to give a white powder (72% yield).
trans-3-(3,4-Dihydro-2H-quinolin-1-ylcarbonyl)propenoic Acid
Ethyl Ester (EtOOCCHdCHCO-tetrahydroquinoline) was ob-
tained by a mixed anhydride coupling of equimolar amounts of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7203
trans-3-(N-Methyl-1-naphthylmethylcarbamoyl)propenoic Acid
Ethyl Ester (EtOOCCHdCHCON(CH3)CH2-1-Naphthyl) was
obtained by mixed anhydride coupling of equimolar amounts of
monoethyl fumarate and N-methyl-1-naphthylmethylamine hy-
drochloride. Purification by column chromatography using 1:1
EtOAc/hexane gave a white powder (62% yield): 1H NMR
(CDCl3) δ 1.29 (t, 3H, CH3), 3.01 (s, 3H, CH3), 5.11 (s, 2H,
CH2), 6.89 (d, 1H, J = 15.2 Hz, CHdCHCON), 7.40-7.52
(m, 5H, CHdCHCON and naphthyl), 7.78-7.89 (m, 3H,
naphthyl).
1H NMR (CDCl3) δ 1.27-1.36 (m, 3H, OCH2CH3), 3.75-3.81
(m, 2H, epoxy CH), 4.25-4.37 (m, 2H, OCH2CH3).
General Procedure for Coupling of the Mono Ethyl Ester
Epoxysuccinates to Amines. The method used was the mixed
anhydride coupling method. The monoethyl epoxysuccinate
(1 equiv) was dissolved in CH2Cl2 and cooled to -20 °C. To
the reaction mixture were added NMM (3 equiv) and then iBCF
(3 equiv). The reaction mixture was stirred at -20 °C for 15-20
min, and then the amine (3 equiv) was added. The reaction
mixture was then stirred at -20 °C for 1 h and then at room
temperature overnight. The solvent was removed, and the crude
product was dissolved in EtOAc. The organic layer was then
washed with 2% citric acid, saturated NaHCO3, and brine. The
product was purified with column chromatography as needed.
Hydrolysis of the ethyl ester with 1 M NaOH (1.5 equiv) in
EtOH gave the desired amides.
trans-3-(N-Methyl-1-naphthylmethylcarbamoyl)propenoic Acid
(2n, HOOCCHdCHCON(CH3)CH2-1-Naphthyl). EtOOCCHd
CHCON(CH3)CH2-1-Naphthyl was hydrolyzed in EtOH using
NaOH (1 M aqueous, 1.1 equiv) under standard deblocking
1
conditions to give a white solid (13% yield): H NMR (DMSO-
d6) δ 3.01 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.63 (d, 1H, J =
15.2 Hz, CHdCHCON), 7.19 (d, 1H, J = 15.2 Hz, CHd
CHCON), 7.37-7.60 (m, 4H, naphthyl), 7.85-8.01 (m, 3H,
naphthyl).
(2S,3S)-2-Carboxylic acid-3-(dibenzylcarbamoyl)oxirane (4r,
HOOC-EP-CON(Bzl)2) was obtained by mixed anhydride cou-
pling of the monoethyl epoxysuccinate and dibenzylamine to
give a clear oil and which was then hydrolyzed under basic
trans-3-(N-Benzyl-1-naphthylmethylcarbamoyl)propenoic Acid
Ethyl Ester (EtOOCCHdCHCON(Bzl)-CH2-1-Naphthyl) was
obtained by mixed anhydride coupling of equimolar amounts of
monoethyl fumarate and N-benzyl-1-naphthylmethylamine.
Purification by column chromatography using 1:1 EtOAc/
hexane gave a yellow oil (89% yield): 1H NMR (CDCl3) δ 1.31
(t, 3H, CH3CH2), 4.23 (q, 2H, CH3CH2), 4.55-4.69 (d, 2H,
N-CH2), 4.71-4.81 (d, 2H, N-CH2), 6.93 (d, 1H, J=14.4 Hz,
CHdCHCON), 7.17 (d, 1H, J = 14.4 Hz, CHdCHCON),
7.23-7.57 (m, 10H, naphthyl and Ph), 7.85 (m, 2H, naphthyl).
trans-3-(N-Benzyl-1-naphthylmethylcarbamoyl)propenoic Acid
(2o, HOOCCHdCHCON(Bzl)-CH2-1-Naphthyl). EtOOCCHd
CHCON(Bzl)-1-CH2-Napth was hydrolyzed in EtOH using
NaOH (1 M aqueous, 1.1 equiv) under standard deblocking
conditions to give a white solid after recrystallization from
1
conditions to the corresponding acid: H NMR (DMSO-d6) δ
3.51 (d, 1H, epoxy CH), 4.03 (d, 1H, epoxy CH), 4.50 (dd, 2H,
NCH2Ph), 4.70 (dd, 2H, NCH2Ph), 7.18-7.36 (m, 10H, 2 ꢀ Ph).
(2S,3S)-2-Carboxylic acid-3-(1-naphthylmethylcarbamoyl)-
oxirane (4s, HOOC-EP-CONH-CH2-1-Naphthyl) was obtained
by a mixed anhydride coupling of the monoethyl epoxysuccinate
and 1-naphthylmethylamine to give a yellow powder, which was
then hydrolyzed under basic conditions to the corresponding
acid: 1H NMR (acetone-d6) δ 3.59 (d, 1H, epoxy CH), 3.82
(d, 1H, epoxy CH), 5.21-5.36 (m, 4H, N(1-CH2-Napth)2),
7.37-7.56 (m, 8H, N(1-CH2-naphthyl)2), 7.84-7.96 (m, 5H,
N(1-CH2-naphthyl)2), 8.15-8.18 (m, 1H, N(1-CH2-naphthyl)2).
cis-2-Carboxylic acid-3-(dibenzylcarbamoyl)oxirane (6t, HO-
OC-EP-CON(Bzl)2) was obtained by a mixed anhydride cou-
pling of the monoethyl epoxysuccinate and dibenzylamine to
give a clear oil, which was then hydrolyzed under basic condi-
1
cold EtOAc (77% yield): H NMR (DMSO-d6) δ 4.82 (d, 2H,
N-CH2), 5.01 (d, 2H, N-CH2), 6.63 (d, 1H, J = 15.2 Hz,
CHdCHCON), 7.19 (d, 1H, J = 15.2 Hz, CHdCHCON),
7.37-7.60 (m, 9H, naphthyl and Ph), 7.85-8.01 (m, 3H,
naphthyl).
1
tions to the corresponding acid: H NMR (DMSO-d6) δ 3.79
(d, 1H, epoxy CH), 4.13 (d, 1H, epoxy CH), 4.23-4.63 (dd, 4H,
NCH2Ph), 7.16 (d, 2H, Ph), 7.24-7.37 (m, 8H, Ph).
trans-3-(Di-1-naphthylmethylcarbamoyl)propenoic Acid Ethyl
Ester (EtOOCCHdCHCON(CH2-1-Naphthyl)2) was obtained
by a mixed anhydride coupling of equimolar amounts of mono-
ethyl fumarate and N,N-di(1-naphthylmethyl)amine. Purifica-
tion by column chromatography using 2:1 hexane/EtOAC gave
white solid (40% yield): 1H NMR (DMSO-d6) δ 1.10 (t, 3H, J=
7.2 Hz, CH3), 4.04 (q, 2H, J=7.1 Hz, CH2), 5.22 (s, 4H, N-1-
CH2-Napth), 6.75 (d, 1H, J = 15.2 Hz, CHdCHCON), 7.19
(d, 1H, J = 15.2 Hz, CHdCHCON), 7.17-8.11 (m, 14H,
N(1-CH2-Napth)2).
trans-3-(Di-1-naphthylmethylcarbamoyl)propenoic Acid (2p,
HOOCCHdCHCON(CH2-1-Naphthyl)2). EtOOCCHdCHC-
ON(CH2-1-Napth)2) was hydrolyzed in EtOH using NaOH
(1 M aqueous, 1.1 equiv) under standard deblocking conditions
to give a white solid (69% yield): 1H NMR (DMSO-d6) δ 5.21
(s, 4H, N-1-CH2-Napth), 6.61 (d, 1H, J = 15.2 Hz, CHd
CHCON), 6.70 (d, 1H, J = 15.2 Hz, CHdCHCON), 7.19-
8.10 (m, 14H, N(1-CH2-Napth)2).
(2S,3S)-Oxirane-2,3-dicarboxylic Acid Monoethyl Esters (4q,
Monoethyl Epoxysuccinates, HOOC-EP-COOEt). The diethyl
trans-epoxysuccinates were synthesized stereoselectively by a
procedure adapted from one described previously by Mori and
Iwasawa.41 One ethyl ester functional group was hydrolyzed
using 1 M KOH (1 equiv) in EtOH at 0 °C: 1H NMR (DMSO-d6)
δ 1.19 (t, 3H, OCH2CH3), 3.55 (d, 1H, epoxy CH), 3.64 (d, 1H,
epoxy CH), 4.15 (q, 2H, OCH2CH3).
cis-Oxirane-2,3-dicarboxylic Acid Monoethyl Ester (Mono-
ethyl Epoxysuccinate, HOOC-EP-COOEt). The diethyl ester
cis-epoxysuccinate was synthesized using the procedure de-
scribed by Meth-Cohn.42 One ethyl ester functional group was
selectively hydrolyzed as described by Rich and Schaschke:43,44
General Procedure for the Synthesis of Aza-Peptide Michael
Acceptors and Aza-Peptide Epoxides by the HOBt/EDC Coupl-
ing Method. To a stirred solution of the fumaric acid or epoxide
precursor (1.5 equiv) in DMF at -10 °C were added HOBt (1.5
equiv), the peptidyl hydrazide precursor (1 equiv), and EDC (1.5
equiv). The mixture was allowed to react for 16 h at room
temperature. The DMF was evaporated, and the residue was
redissolved in EtOAc. The organic layer was washed with 2%
citric acid, saturated NaHCO3, and saturated NaCl, dried over
MgSO4, and concentrated. Column chromatography on silica
gel afforded the aza-peptide Michael acceptor and aza-peptide
epoxide derivatives.
N2-tert-Butoxycarbonyl-N1-carbamoylmethyl-N1-trans-(3-eth-
oxycarbonylpropenoyl)hydrazine (23a, Boc-AAsn-CHdCH-
COOEt). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a white powder (47% yield): 1H NMR
(DMSO-d6) δ 1.19 (t, 3H, CH3), 1.39 (s, 9H, Boc), 3.59 (d, 1H,
CH), 4.17 (q, 2H, CH2), 4.43 (d, 1H, CH), 6.59 (d, 1H,
CHdCHCON), 7.19-7.25 (m, 3H, CHdCHCON and NH),
7.43 (s, 1H, NH), 9.93 (s, 1H, NH); MS (FAB) m/z 316.1 [(M þ
1)þ]. Anal. (C13H21N3O6 0.15CH2Cl2 0.2H2O) C, H, N.
3
3
N2-tert-Butoxycarbonyl-N1-carbamoylmethyl-N1-trans-(3-cyclo-
hexylcarbamoylpropenoyl)hydrazine (23d, Boc-AAsn-CHd
CH-NHCH2-cyclohexyl). This compound was obtained using
the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder
(24% yield): 1H NMR (DMSO-d6) δ 0.84-0.89 (m, 2H, cyclo-
hexyl), 1.12-1.19 (m, 4H, cyclohexyl), 1.39 (s, 9H, Boc),

7204 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
1.42-1.64 (m, 5H, cyclohexyl), 2.98 (t, 2H, CH2), 3.57 (s, 1H,
CH), 4.38 (s, 1H, CH), 6.89 (d, 1H, CHdCHCON), 7.08 (d, 1H,
J=15.2 Hz, CHdCHCON), 7.17 (s, 1H, NH), 7.41 (s, 1H, NH),
8.43 (t, 1H, J=5.5 Hz, NH), 9.88 (s, 1H, NH); MS (FAB) m/z
(m, 4H, morpholine), 4.11-4.18 (m, 1H, R-H), 4.34 (d, 2H,
NHCH2), 6.24-6.25 (d, 1H, furyl), 6.37-6.38 (d, 1H, furyl), 6.71
(d, 1H, NH), 6.84-6.88 (d, 1H, CHdCHCON), 7.09-7.13 (d, 1H,
CHdCHCON), 7.18 (s, 1H, NH), 7.54-7.56 (d, 2H, NH and furyl),
8.86 (t, 1H, NH), 10.59 (s, 1H, NH); HRMS (FAB) calcd for
383.2 [(M þ 1)þ]. Anal. (C18H30N4O5 0.15H2O) C, H, N.
3
N2-tert-Butoxycarbonyl-N1-carbamoylmethyl-N1-trans-(3-
benzylcarbamoylpropenoyl)hydrazine (23f, Boc-AAsn-CHdCH-
CONHBzl). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a white powder (44% yield): 1H NMR
(DMSO-d6) δ 0.84-0.89 (m, 2H, cyclohexyl), 1.12-1.19 (m,
4H, cyclohexyl), 1.39 (s, 9H, Boc), 3.59 (s, 1H, CH), 4.35-4.41
(m, 3H, CH and CH2), 6.92-6.96 (d, 1H, J = 15.2 Hz,
CHdCHCON), 7.14-7.33 (m, 7H, Ph and CHdCHCON and
NH), 7.41 (s, 1H, NH), 8.98 (t, 1H, J=5.4 Hz, NH), 9.89 (s, 1H,
NH); MS (FAB) m/z 377.2 [(M þ 1)þ]. Anal. (C18H24N4O5)
C, H, N.
C19H27N6O7 451.1936, observed m/z 451.2037. Anal. (C19H26N6O7
1.3H2O) C, H, N.
3
N2-(N-Morpholinocarbonylalanyl)-N1-carbamoylmethyl-N1-
trans-(3-(3,4-dimethoxybenzyl)carbamoylpropenoyl)hydrazine
(25i, Mu-Ala-AAsn-CHdCH-CONHCH2Ph-3,4-OCH3). This
compound was obtained using the HOBt/EDC coupling method
and purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane gave
a white powder (14% yield): 1H NMR (DMSO-d6) δ 1.24 (d, 3H,
CH3), 3.25-3.26 (m, 4H, morpholine), 3.32 (s, 2H, NCH2CO),
3.49-3.51 (m, 4H, morpholine), 3.70-3.71 (d, 6H, 2 ꢀ OCH3),
4.11-4.18 (m, 1H, R-H), 4.28 (d, 2H, NHCH2), 6.70-6.72 (d, 1H,
NH), 6.74-6.76 (d, 1H, CHdCHCON), 6.86-6.91 (m, 3H, Ph
and CHdCHCON), 7.10-7.18 (m, 2H, Ph and NH), 7.53 (s, 1H,
NH), 8.82 (t, 1H, NH), 10.59 (s, 1H, NH); HRMS (FAB) calcd
for C23H33N6O8 521.2354, observed m/z 521.2431. Anal.
(C23H32N6O8 0.1EtOAc 1H2O) C, H, N.
N2-tert-Butoxycarbonyl-N1-carbamoylmethyl-N1-trans-(3-phe-
nylpropylcarbamoylpropenoyl)hydrazine (23h, Boc-AAsn-CHd
CH-CONHCH2CH2CH2Ph). This compound was obtained
using the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent. Re-
crystallization with EtOAc/hexane gave a white powder (53%
yield): 1H NMR (DMSO-d6) δ 1.39 (s, 9H, Boc), 1.67-1.74 (m,
2H, NHCH2CH2CH2Ph), 2.55-2.58 (t, 2H, NHCH2CH2-
CH2Ph), 3.11-3.16 (m, 2H, NHCH2CH2CH2Ph), 3.59 (s, 1H,
CH), 4.41 (s, 1H, NH), 6.87-6.91 (d, 1H, J = 15.2 Hz,
CHdCHCON), 7.08-7.28 (m, 7H, Ph and CHdCHCON and
NH), 7.41 (s, 1H, NH), 8.51 (t, 1H, J=5.5 Hz, NH), 9.89 (s, 1H,
3
3
N2-(N-Morpholinocarbonylalanyl)-N1-carbamoylmethyl-N1-
trans-(3-phenylpropylcarbamoylpropenoyl)hydrazine (25h, Mu-
Ala-AAsn-CHdCH-CONHCH2CH2CH2Ph). This compound
was obtained using the HOBt/EDC coupling method and puri-
fied by column chromatography using 10% MeOH/CH2Cl2 as
the eluent. Recrystallization with EtOAc/hexane gave a white
powder (41% yield): 1H NMR (DMSO-d6) δ 1.24 (d, 3H, CH3),
1.67-1.75 (m, 2H, NHCH2CH2CH2Ph), 2.55-2.59 (t, 2H,
NHCH2CH2CH2Ph), 3.09-3.18 (m, 2H, NHCH2CH2CH2Ph),
3.26-3.28 (m, 4H, morpholine), 3.33 (s, 2H, NCH2CO),
3.50-3.52 (m, 4H, morpholine), 4.11-4.18 (m, 1H, R-H), 6.71
(d, 1H, NH), 6.84-6.88 (d, 1H, CHdCHCON), 7.09-7.14
(d, 1H, CHdCHCON), 7.15-7.28 (m, 6H, Ph and NH), 7.53
(s, 1H, NH), 8.45 (t, 1H, NH), 10.58 (s, 1H, NH); HRMS (FAB)
calcd for C23H33N6O6 489.2462, observed m/z 489.2486. Anal.
NH); MS (FAB) m/z 405.3 [(M þ 1)þ]. Anal. (C20H28N4O5
3
1.2hexane) C, H, N.
N2-(N-Piperidinylcarbonylalanyl)-N1-carbamoylmethyl-N1-trans-
(3-(3,4-dihydro-1H-isoquinolin-2-ylcarbonyl)propenoyl)hydrazine
(24k, Pip-Ala-AAsn-CHdCHCO-tetrahydroisoquinoline). This
compound was obtained using the HOBt/EDC coupling method
and purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane gave
a white powder (41% yield): 1H NMR (DMSO-d6) δ 1.23 (d, 3H,
CH3), 1.37 (s, 4H, piperidine), 1.48 (2H, piperidine), 1.74-1.77
(m, 2H, N-CH2-CH2-CH2), 2.79-2.87 (m, 2H, N-CH2-CH2-
CH2), 3.23 (m, 4H, piperidine), 3.60 (s, 2H, NCH2CO),
3.72-3.75 (m, 2H, N-CH2-CH2), 4.12 (m, 1H, R-H), 6.55
(d, 1H, NH), 7.06-7.37 (m, 7H, isoquinoline and CHdCHCON
and NH and CHdCHCON), 7.57 (s, 1H, NH), 10.53 (s, 1H,
NH); HRMS (FAB) calcd for C22H30N5O8 485.2507, observed
(C23H32N6O6 0.5H2O) C, H, N.
3
N2-(N-Morpholinocarbonylalanyl)-N1-carbamoylmethyl-N1-
trans-(3-(3,4-dihydro-1H-isoquinolin-2-ylcarbonyl)propenoyl)-
hydrazine (25k, Mu-Ala-AAsn-CHdCHCO-tetrahydroisoquino-
line). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a white powder (27% yield): 1H NMR
(DMSO-d6) δ 1.23 (d, 3H, J = 7.1 Hz, Ala-CH3), 2.78-2.86
(m, 2H, N-CH2-CH2-CH2), 3.22-3.34 (m, 6H, morpholine
and NCH2CO), 3.47-3.58 (m, 6H, N-CH2-CH2-CH2 and
morpholine), 3.71-3.73 (m, 2H, N-CH2-CH2), 4.11-4.15 (m,
1H, R-H), 6.69 (d, 1H, J = 6.6 Hz, NH), 7.05-7.39 (m, 7H,
CHdCHCON and CHdCHCON and isoquinoline and NH),
7.55 (s, 1H, NH), 10.58 (s, 1H, NH); HRMS (FAB) calcd for
C23H31N6O6 485.2507, observed m/z 485.2579. Anal. (C23H30-
m/z 485.2579. Anal. (C24H32N6O5 1.3H2O) C, H, N.
3
N2-(N-Morpholinylcarbonylalanyl)-N1-carbamoylmethyl-N1-
trans-(3-cyclopropylcarbamoylpropenoyl)hydrazine (25c, Mu-
Ala-AAsn-CHdCH-CONH-cyclopropyl). This compound
was obtained using the HOBt/EDC coupling method and
purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane
gave a white powder (67% yield): 1H NMR (DMSO-d6) δ
0.40-0.43 (m, 2H, CH2), 0.63-0.68 (m, 2H, CH2), 1.24
(d, 3H, CH3), 2.69-2.73 (m, 1H, CH), 3.28-3.29 (m, 4H,
morpholine), 3.33 (s, 2H, NCH2CO), 3.51-3.53 (m, 4H,
morpholine), 4.11-4.18 (m, 1H, R-H), 6.71-6.77 (m, 2H, NH
and CHdCHCON), 7.03-7.07 (d, 1H, CHdCHCON), 7.17
(s, 1H, NH), 7.53(s, 1H, NH), 8.47 (d, 1H, NH), 10.58 (s, 1H,
NH); HRMS (FAB) calcd for C17H27N6O6 411.1987, observed
N6O6 1.4H2O) C, H, N.
3
N2-(4-(Benzyloxycarbonyl)piperazin-1-ylcarbonylalanyl)-N1-car-
bamoylmethyl-N1-trans-(3-(3,4-dihydro-2H-quinolin-1-ylcarbonyl)-
propenoyl)hydrazine (26j, Cbz-Piz-Ala-AAsn-CHdCHCO-tetra-
hydroquinoline). This compound was obtained using the HOBt/
EDC coupling method and purified by column chromato-
graphy using 10% MeOH/CH2Cl2 as the eluent. Recrystallization
with EtOAc/hexane gave a yellow powder (29% yield): 1H NMR
(DMSO-d6) δ 1.27 (d, 3H, J = 7.0 Hz, Ala-CH3), 1.84-1.89
(m, 2H, N-CH2-CH2-CH2), 2.67-2.71 (m, 2H, N-CH2-CH2-
CH2), 3.30-3.33 (m, 10H, piperazine and NCH2CO), 3.70-3.76
(m, 2H, N-CH2-CH2), 4.13-4.20 (m, 1H, R-H), 5.07 (s, 2H,
Cbz), 6.79 (d, 1H, J = 6.6 Hz, NH), 7.05-7.37 (m, 12H, Ph,
quinoline, NH, CHdCHCON and CHdCHCON), 7.53 (s, 1H,
NH), 10.60 (s, 1H, NH); HRMS (FAB) calcd for C31H38-
m/z 411.1981. Anal. (C17H26N6O6 1H2O) C, H, N.
3
N2-(N-Morpholinocarbonylalanyl)-N1-carbamoylmethyl-N1-trans-
(3-(2-furylmethyl)carbamoylpropenoyl)hydrazine (25e, Mu-Ala-
AAsn-CHdCH-CONHCH2-2-furyl). This compound was ob-
tained using the HOBt/EDC coupling method and purified
by column chromatography using 10% MeOH/CH2Cl2 as the
eluent. Recrystallization with EtOAc/hexane gave a yellow powder
1
(48% yield): H NMR (DMSO-d6) δ 1.24 (d, 3H, CH3), 3.27-
N7O7 620.2827, observed m/z 620.2891. Anal. (C31H37N7O7
0.9H2O) C, H, N.
3
3.28 (m, 4H, morpholine), 3.34 (s, 2H, NCH2CO), 3.50-3.52

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7205
N2-(4-(Benzyloxycarbonyl)piperazin-1-ylcarbonylalanyl)-N1-car-
1.27 (d, 3H, Ala-CH3), 1.37 (s, 4H, piperidine), 1.48 (m, 2H,
piperidine), 3.24 (s, 4H, piperidine), 3.33 (s, 2H, NCH2CO),
4.13 (m, 1H, R-H), 4.28 (m, 1H, R-H), 4.65-4.67 (m, 2H,
CH2Ph), 5.06-5.17 (m, 2H, CH2Naph) 6.39 (t, 1H, NH),
7.12-7.56 (m, 13H, Ph and Naph and CHdCHCON and
CHdCHCON and 2 ꢀ NH) 7.84-8.05 (m, 4H, Naph and
NH), 10.72 (s, 1H, NH); HRMS (FAB) calcd for C36H44N7O6
bamoylmethyl-N1-trans-(3-(3,4-dihydro-1H-isoquinolin-2-ylcar-
bonyl)propenoyl)hydrazine (26k, Cbz-Piz-Ala-AAsn-CHdCHCO-
tetrahydroisoquinoline). This compound was obtained using the
HOBt/EDC coupling method and purified by column chromato-
graphy using 10% MeOH/CH2Cl2 as the eluent. Recrystallization
1
with EtOAc/hexane gave a white powder (46% yield): H NMR
(DMSO-d6) δ 1.23 (d, 3H, J=7.0 Hz, CH3), 2.75-2.87 (m, 2H, N-
CH2-CH2-CH2), 3.30-3.33 (m, 10H, piperazine and NCH2CO),
3.69-3.77 (m, 2H, N-CH2-CH2), 4.10-4.15 (m, 1H, R-H),
4.64-4.74 (m, 2H, NCH2C), 5.07 (s, 2H, Cbz), 6.76 (d, 1H, J=
6.5 Hz, NH), 7.05-7.39 (m, 12H, Ph, isoquinoline, NH,
CHdCHCON and CHdCHCON), 7.55 (s, 1H, NH), 10.57 (s,
1H, NH); HRMS (FAB) calcd for C31H38N7O7 620.2827, observed
m/z 620.2879. Anal. (C31H37N7O7 1H2O 0.5EtOAc) C, H, N.
670.3348, observed m/z 670.3374. Anal. (C36H43N7O6
1.2H2O) C, H, N.
3
N2-(N-Piperidinocarbonylalanylalanyl)-N1-carboxymethyl-N1-
trans-(3-(di-1-naphthylmethylcarbamoyl)propenoyl)hydrazine (27p,
Pip-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2). This com-
pound was obtained using the HOBt/EDC coupling method and
purified by column chromatography using 10% MeOH/CH2Cl2 as
the eluent. Recrystallization with EtOAc/hexane gave a white
powder (63% yield): 1H NMR (DMSO-d6) δ 1.20 (d, 3H, J=6.8
Hz, Ala-CH3), 1.28 (d, 3H, J = 6.9 Hz, Ala-CH3), 1.35 (s, 4H,
piperidine), 1.42-1.46 (m, 2H, piperidine), 3.23 (s, 4H, piperidine),
3.33(s, 2H, NCH2CO), 4.06-4.20 (m, 1H, R-H), 4.22-4.34 (m, 1H,
R-H), 5.21 (s, 4H, 2 ꢀ N-CH2-naphthyl), 6.38 (d, 1H, J=7.6 Hz,
NH), 7.13-7.56 (m, 12H, naphthyl and CHdCHCON and
CHdCHCON and 2 ꢀ NH), 7.82-8.10 (m, 7H, naphthyl and
NH), 10.72 (s, 1H, NH); HRMS (FAB) calcd for C40H46N7O6
3
3
N2-(N-Piperidinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-ethoxycarbonylpropenoyl)hydrazine (27a, Pip-Ala-
Ala-AAsn-CHdCH-COOEt). This compound was obtained
using the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder
1
(35% yield): H NMR (DMSO-d6) δ 1.17-1.25 (m, 9H, 2 ꢀ
Ala-CH3 and COCH2CH3), 1.39 (s, 4H, piperidine), 1.48-1.52
(m, 2H, piperidine), 3.25 (s, 4H, piperidine), 3.32 (s, 2H,
NCH2CO), 4.09-4.19 (m, 3H, R-H and COCH2CH3),
4.22-4.26 (m, 1H, R-H), 6.35 (d, 1H, J = 6.7 Hz, NH), 6.59
(d, 1H, J = 15.5 Hz, CHdCHCON), 7.18-7.20 (m, 2H,
CHdCHCON and NH), 7.51 (s, 1H, NH), 8.04 (br s, 1H,
NH), 10.75 (s, 1H, NH); HRMS (FAB) calcd for C20H33N6O7
720.3568, observed m/z 720.350409. Anal. (C40H45N7O6 1.3H2O)
3
C, H, N.
N2-(N-Morpholinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-ethoxycarbonylpropenoyl)hydrazine (28a, Mu-Ala-
Ala-AAsn-CHdCH-COOEt). This compound was obtained
using the HOBt/EDC coupling method and purified by column
chromatography using 15% MeOH/CH2Cl2 as the eluent. Re-
crystallization with EtOAc/hexane gave a white powder (36%
yield): 1H NMR (DMSO-d6) δ 1.17-1.25 (m, 9H, 2 ꢀ Ala-CH3
and COCH2CH3), 3.24-3.26 (m, 4H, morpholine), 3.32 (s, 2H,
NCH2CO), 3.50-3.52 (m, 4H, morpholine), 4.09-4.18 (m, 3H,
R-H and COCH2CH3), 4.20-4.25 (m, 1H, R-H), 6.50 (d, 1H, J=
7.3 Hz, NH), 6.59 (d, 1H, J=15.5 Hz, CHdCHCON), 7.17-7.20
(m, 2H, CHdCHCON and NH), 7.51 (s, 1H, NH), 8.08 (s, 1H,
NH), 10.73 (s, 1H, NH); HRMS (FAB) calcd for C19H31N6O8
469.2405, observed m/z 469.2426. Anal. (C20H32N6O7 0.9H2O)
3
C, H, N.
N2-(N-Piperidinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-phenylethylcarbamoylpropenoyl)hydrazine (27g, Pip-Ala-
Ala-AAsn-CHdCH-CONHCH2CH2Ph). This compound was ob-
tained using the HOBt/EDC coupling method and purified by
column chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder (48%
yield): 1H NMR (DMSO-d6) δ 1.19-1.28 (m, 6H, 2 ꢀ CH3), 1.39
(s, 4H, piperidine), 1.50 (m, 2H, piperidine), 2.75 (t, 2H, CH2),
3.25-3.28 (m, 4H, piperidine), 3.34 (s, 2H, NCH2CO), 3.35-3.40
(m, 2H, CH2), 4.13 (m, 1H, R-H), 4.27 (m, 1H, R-H), 6.41 (d, 1H,
NH), 6.83-6.87 (d, 1H, CHdCHCON), 7.03-7.07 (d, 1H,
CHdCHCON), 7.18-7.31 (m, 6H, Ph and NH), 7.52 (s, 1H,
NH), 8.07 (s, 1H, NH), 8.56 (t, 1H, NH), 10.69 (br s, 1H, NH);
HRMS (FAB) calcd for C26H38N7O6 544.2878, observed m/z
471.2227, observed m/z 471.219789. Anal. (C19H30N6O8 1H2O)
3
C, H, N.
N2-(N-Morpholinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-phenylpropylcarbamoylpropenoyl)hydrazine (28h,
Mu-Ala-Ala-AAsn-CHdCH-CONHCH2CH2CH2Ph). This
compound was obtained using the HOBt/EDC coupling
method and purified by column chromatography using 10%
MeOH/CH2Cl2 as the eluent. Recrystallization with EtOAc/
hexane gave a white powder (32% yield): 1H NMR (DMSO-d6)
δ 1.18 (d, 3H, J=7.2 Hz, Ala-CH3), 1.25 (d, 3H, J=7.1 Hz, Ala-
CH3), 1.68-1.75 (m, 2H, NHCH2CH2CH2Ph), 2.55-2.59
(t, 2H, NHCH2CH2CH2Ph), 3.11-3.16 (m, 2H, NHCH2-
CH2CH2Ph), 3.25-3.29 (m, 4H, morpholine), 3.35 (s, 2H,
NCH2CO), 3.49-3.51 (m, 4H, morpholine), 4.10-4.17 (m,
1H, R-H), 4.22-4.28 (m, 1H, R-H), 6.53 (d, 1H, J=7.3 Hz,
NH), 6.87 (d, 1H, J=15.2 Hz, CHdCHCON), 7.05 (d, 1H, J=
15.2 Hz, CHdCHCON), 7.14-7.28 (m, 6H, Ph and NH), 7.50
(s, 1H, NH), 8.10 (s, 1H, NH), 8.49 (t, 1H, J=5.5 Hz, NH),
544.3001. Anal. (C26H37N7O6 0.1H2O) C, H, N.
3
N2-(N-Piperidinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-dibenzylcarbamoylpropenoyl)hydrazine (27l, Pip-
Ala-Ala-AAsn-CHdCH-CON(Bzl)2). This compound was
obtained using the HOBt/EDC coupling method and purified
by column chromatography using 10% MeOH/CH2Cl2 as
the eluent. Recrystallization with EtOAc/hexane gave a white
powder (52% yield): 1H NMR (DMSO-d6) δ 1.18-1.27 (m, 6H,
2 ꢀ Ala-CH3), 1.37 (s, 4H, piperidine), 1.46-1.49 (m, 2H,
piperidine), 3.23-3.25 (m, 4H, piperidine), 3.33 (s, 2H,
NCH2CO), 4.11-4.16 (m, 1H, R-H), 4.24-4.30 (m, 1H, R-H),
4.56-4.63 (d, 4H, J=27.4 Hz, 2 ꢀ CH2Ph), 6.38 (d, 1H, J=7.2
Hz, NH), 7.14-7.37 (m, 13H, 2 ꢀ Ph and CHdCHCON and
CHdCHCON and NH), 7.49 (s, 1H, NH), 8.05 (s, 1H, NH),
10.72 (s, 1H, NH); HRMS (FAB) calcd for C32H42N7O6
10.67 (s, 1H, NH); HRMS (FAB) calcd for C26H38-
N7O7 560.2834, observed m/z 560.2817. Anal. (C26H37N7O7
0.4H2O) C, H, N.
3
N2-(N-Morpholinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-(3,4-dihydro-1H-isoquinolin-2-ylcarbonyl)propenoyl)-
hydrazine (28k, Mu-Ala-Ala-AAsn-CHdCHCO-tetrahydroiso-
quinoline). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10%MeOH/CH2Cl2 as the eluent. RecrystallizationwithEtOAc/
hexane gave a white powder (29% yield): 1H NMR (DMSO-d6) δ
1.16-1.23 (m, 6H, 2 ꢀ Ala-CH3), 2.79-2.85 (m, 2H, N-CH2-
CH2-CH2), 3.25-3.35 (m, 8H, morpholine and NCH2CO and
N-CH2-CH2-CH2), 3.50 (m, 4H, morpholine), 3.72-3.75 (m, 2H,
N-CH2-CH2), 4.12 (m, 1H, R-H), 4.25 (m, 1H, R-H), 4.65-4.75
620.3191, observed m/z 620.3143. Anal. (C32H41N7O6 1.1-
3
H2O) C, H, N.
N2-(N-Piperidinocarbonylalanylalanyl)-N1-trans-(3-benzyl-
1-naphthylmethylcarbamoylpropenoyl)-N1-carbamoylmethyl-
hydrazine (27o, Pip-Ala-Ala-AAsn-CHdCH-CON(Bzl)-
CH2-1-naphthyl). This compound was obtained using the
HOBt/EDC coupling method and purified by column chro-
matography using 10% MeOH/CH2Cl2 as the eluent. Re-
crystallization with EtOAc/hexane gave a white powder
1
(27% yield): H NMR (DMSO-d6) δ 1.20 (d, 3H, Ala-CH3),

7206 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
(m, 2H, N-CH2-C), 6.51 (d, 1H, J=7.2 Hz, NH), 7.04-7.42 (m,
7H, isoquinoline, CHdCHCON, CHdCHCON and NH), 7.53
(s, 1H, NH), 8.08 (s, 1H, NH), 10.68 (s, 1H, NH); HRMS (FAB)
calcd for C26H36N7O7 558.2676, observed m/z 558.2660. Anal.
C31H41N8O6 621.3179, observed m/z 621.314358. Anal. (C31-
H40N8O6 1.7TFA 1.1H2O) C, H, N.
3
3
N2-(N-Piperazinocarbonylalanylalanyl)-N1-carbamoylmethyl-N1-
trans-(3-(methyl-1-naphthylmethylcarbamoyl)propenoyl)hydrazine
(37n, Piz-Ala-Ala-AAsn-CHdCH-CON(CH3)CH2-1-naphthyl).
This compound was obtained by the removal of the tert-butyl
protecting group of Boc-Piz-Ala-Ala-AAsn-CHdCH-CON-
(CH3)CH2-1-naphthyl with trifluoroacetic acid/methylene chlor-
ide (1:1) for 3 h at room temperature. The volatiles were
evaporated, and the TFA salt was washed several times with
diethyl ether to give a white solid (99% yield): 1H NMR (DMSO-
d6) δ 1.19-1.28 (m, 6H, 2 ꢀ Ala-CH3), 2.98-3.03 (m, 7H,
piperazine and N-CH3), 3.50-3.51 (m, 6H, piperazine and
NCH2CO), 4.13-4.29 (m, 2H, 2 ꢀ R-H), 5.04-5.09 (m, 2H,
N-CH2-naphthyl), 5.21 (s, 1H, NH), 6.76 (d, 1H, J = 6.9 Hz,
NH), 7.07-7.61 (m, 8H, naphthyl and CHdCHCON and CHd
CHCON and 2 ꢀ NH), 7.86-8.13 (m, 3H, naphthyl), 8.83
(s, 1H, NH), 10.71 (s, 1H, NH); HRMS (FAB) calcd for C29H39-
(C26H35N7O7 1.8H2O) C, H, N.
3
N2-(N-Morpholinocarbonylalanylalanyl)-N1-carboxymethyl-
N1-trans-(3-(di-1-naphthylmethylcarbamoyl)propenoyl)hydra-
zine (28p, Mu-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naph-
thyl)2). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization
with EtOAc/hexane gave a white solid (40% yield): 1H NMR
(DMSO-d6) δ 1.21 (d, 3H, J=7.1 Hz, Ala-CH3), 1.29 (d, 3H,
J=7.1 Hz, Ala-CH3), 3.21-3.26 (m, 4H, morpholine), 3.29
(s, 2H, NCH2CO), 3.47-3.49 (m, 4H, morpholine), 4.13-4.20
(m, 1H, R-H), 4.25-4.32 (m, 1H, R-H), 5.20 (s, 4H, 2 x
N-CH2-naphthyl), 6.52 (d, 1H, J=7.4 Hz, NH), 7.12-7.55
(m, 12H, naphthyl and CHdCHCON and CHdCHCON and
NH2), 7.82-8.10 (m, 7H, naphthyl and NH), 10.71 (s, 1H,
N8O6 595.2987, observed m/z 595.2947. Anal. (C29H38N8O6
3
NH); HRMS (FAB) calcd for
C
39H44N7O7 722.3340,
1.4TFA 2H2O) C, H, N.
3
observed m/z 722.329674. Anal. (C39H43N7O7 1.6H2O)
N2-(N-Piperazinocarbonylalanylalanyl)-N1-carboxymethyl-N1-
trans-(3-(di-1-naphthylmethylcarbamoyl)propenoyl)hydrazine (37p,
Piz-Ala-Ala-AAsn-CHdCH-CON(CH2-1-naphthyl)2). This
compound was obtained by the removal of the tert-butyl
protecting group of Boc-Piz-Ala-Ala-AAsn-CHdCH-CON-
(CH2-1-naphthyl)2 with trifluoroacetic acid/methylene chloride
(1:1) for 3 h at room temperature. The volatiles were evaporated,
and the TFA salt was washed several times with diethyl ether to
give a white solid (93% yield): 1H NMR (DMSO-d6) δ 1.21
(d, 3H, Ala-CH3), 1.29 (d, 3H, Ala-CH3), 1.37 (s, 9H, Boc),
3.03 (s, 4H, piperazine), 3.50-3.73 (m, 6H, piperazine and
NCH2CO), 4.14-4.19 (m, 1H, R-H), 4.25-4.31 (m, 1H, R-H),
5.21 (s, 4H, 2 ꢀ N-CH2-naphthyl), 6.77 (d, 1H, J=7.3 Hz, NH),
7.13-7.56 (m, 12H, naphthyl and CHdCHCON and CHd
CHCON and 2 ꢀ NH), 7.82-8.14 (m, 7H, naphthyl and NH),
8.82 (s, 1H, NH), 10.73 (s, 1H, NH); HRMS (FAB) calcd
for C39H45N8O6 721.3384, observed m/z 721.3379. Anal.
(C39H44N8O6 1.6TFA 1H2O) C, H, N.
3
C, H, N.
N2-(N-Piperazinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-ethoxycarbonylpropenoyl)hydrazine (37a, Piz-Ala-
Ala-AAsn-CHdCH-COOEt). This compound was obtained
by the removal of the tert-butyl protecting group of Boc-Piz-
Ala-Ala-AAsn-CHdCH-COOEt with trifluoroacetic acid/
methylene chloride (1:1) for 3 h at room temperature. The
volatiles were evaporated, and the TFA salt was washed several
1
times with diethyl ether to give a white solid (98% yield): H
NMR (DMSO-d6) δ 1.18-1.25 (m, 9H, 2 ꢀ Ala-CH3 and
COCH2CH3), 3.04 (s, 4H, piperazine), 3.36-3.50 (m, 6H,
piperazine and NCH2CO), 4.13-4.26 (m, 4H, 2 ꢀ R-H and
COCH2CH3), 6.59 (d, 1H, J = 15.4 Hz, CHdCHCON), 6.75
(d, 1H, J=7.0 Hz, NH), 7.17-7.21 (m, 2H, CHdCHCON and
NH), 7.53 (s, 1H, NH), 8.12 (s, 1H, NH), 8.78 (s, 1H, NH), 10.75
(s, 1H, NH); HRMS (FAB) calcd for C19H32N7O7 470.2358,
observed m/z 470.2386. Anal. (C19H31N7O7 1.2TFA 1.5H2O)
3
3
3
3
N2-(4-(tert-Butoxycarbonyl)piperazin-1-yl carbonylalanylala-
nyl)-N1-carbamoylmethyl-N1-trans-(3-ethoxycarbonylpropenoyl)-
hydrazine (29a, Boc-Piz-Ala-Ala-AAsn-CHdCH-COOEt). This
compound was obtained using the HOBt/EDC coupling method
and purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane gave
a white powder (34% yield): 1H NMR (DMSO-d6) δ 1.17-1.25
(m, 9H, 2 x Ala-CH3 and COCH2CH3), 1.38 (s, 9H, Boc), 3.26 (s,
8H, piperazine), 3.32 (s, 2H, NCH2CO), 4.08-4.26 (m, 4H, 2 ꢀ
R-H and COCH2CH3), 6.54 (d, 1H, J=7.1 Hz, NH), 6.59 (d, 1H,
J=15.7 Hz, CHdCHCON), 7.17-7.20 (m, 2H, CHdCHCON
and NH), 7.51 (s, 1H, NH), 8.08 (s, 1H, NH), 10.72 (s, 1H, NH);
HRMS (FAB) calcd for C24H40N7O9 570.2882, observed m/z
C, H, N.
N2-(N-Piperazinocarbonylalanylalanyl)-N1-trans-(3-benzyl-
oxycarbonylpropenoyl)-N1-carbamoylmethylhydrazine (37b,
Piz-Ala-Ala-AAsn-CHdCH-COOBzl). This compound was
obtained by the removal of the tert-butyl protecting group
of Boc-Piz-Ala-Ala-AAsn-CHdCH-COOBzl with trifluoro-
acetic acid/methylene chloride (1:1) for 3 h at room temperature.
The volatiles were evaporated, and the TFA salt was washed
several times with diethyl ether to give a white solid (98% yield):
1H NMR (DMSO-d6) δ 1.18 (s, 6H, 2 ꢀ Ala-CH3), 3.03 (s, 4H,
piperazine), 3.37-3.49 (m, 6H, piperazine and NCH2CO),
4.08-4.23 (m, 1H, R-H), 4.37-4.52 (m, 1H, R-H), 5.19 (s, 2H,
OCH2Ph), 6.64-6.74 (m, 2H, CHdCHCON and NH),
7.20-7.37 (m, 7H, Ph and CHdCHCON and NH), 7.52
(s, 1H, NH), 8.11 (s, 1H, NH), 8.85 (s, 1H, NH), 10.76 (s, 1H,
NH); HRMS (FAB) calcd for C24H34N7O7 532.2520, observed
m/z 532.2505. Anal. (C24H33N7O7 1.05TFA 1.5H2O) C, H, N.
570.2906. Anal. (C24H39N7O9 0.9H2O) C, H, N.
3
N2-(4-(tert-Butoxycarbonyl)piperazin-1-yl carbonylalanylala-
nyl)-N1-trans-(3-benzyloxycarbonylpropenoyl)-N1-carbamoylme-
thylhydrazine (29b, Boc-Piz-Ala-Ala-AAsn-CHdCH-CO-
OBzl). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization
with EtOAc/hexane gave a white powder (25% yield): 1H
NMR (DMSO-d6) δ 1.17 (d, 6H, J=7.1 Hz, 2 ꢀ CH3), 1.38
(s, 9H, Boc), 3.25 (s, 8H, piperazine), 3.33 (s, 2H, NCH2CO),
4.07-4.13 (m, 1H, R-H), 4.19-4.24 (m, 1H, R-H), 5.19 (s, 2H,
O-CH2-Ph), 6.54 (d, 1H, J=7.1 Hz, NH), 6.65 (d, 1H, J=15.3
Hz, CHdCHCON), 7.22-7.39 (m, 7H, Ph and CHdCHCON
and NH), 7.52 (s, 1H, NH), 8.09 (s, 1H, NH), 10.45 (s, 1H, NH);
HRMS (FAB) calcd for C29H42N7O9 632.6771, observed m/z
3
3
N2-(N-Piperazinocarbonylalanylalanyl)-N1-carbamoylmethyl-
N1-trans-(3-dibenzylcarbamoylpropenoyl)hydrazine (37l, Piz-
Ala-Ala-AAsn-CHdCH-CON(Bzl)2). This compound was
obtained by the removal of the tert-butyl protecting group of
Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(Bzl)2 with trifluoro-
acetic acid/methylene chloride (1:1) for 3 h at room temperature.
The volatiles were evaporated, and the TFA salt was washed
several times with diethyl ether to give a white solid (99% yield):
1H NMR (DMSO-d6) δ 1.19-1.27 (m, 6H, 2 ꢀ Ala-CH3), 3.03
(s, 4H, piperazine), 3.49-3.61 (m, 6H, piperazine and NCH2-
CO), 4.11-4.27 (m, 1H, R-H), 4.23-4.30 (m, 1H, R-H), 4.56
(s, 2H, NCH2Ph), 4.64 (s, 2H, NCH2Ph), 6.76 (d, 1H, J=7.4 Hz,
NH), 7.14-7.35 (m, 13H, 2 ꢀ Ph and CHdCHCON and
CHdCHCON and NH), 7.50 (s, 1H, NH), 8.13 (s, 1H, NH),
8.81 (s, 1H, NH), 10.71 (s, 1H, NH); HRMS (FAB) calcd for
632.6753. Anal. (C29H41N7O9 0.9H2O) C, H, N.
3
N2-(4-(tert-Butoxycarbonyl)piperazin-1-yl carbonylalanylala-
nyl)-N1-carbamoylmethyl-N1-trans-(3-dibenzylcarbamoylpropenoyl)-
hydrazine (29l, Boc-Piz-Ala-Ala-AAsn-CHdCH-CON(Bzl)2). This

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7207
compound was obtained using the HOBt/EDC coupling method
and purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane gave a
white powder (50% yield): 1H NMR (DMSO-d6) δ 1.19 (d, 3H, J=
7.2 Hz, Ala-CH3), 1.26 (d, 3H, J=7.1 Hz, Ala-CH3), 1.38 (s, 9H,
Boc), 3.25 (s, 8H, piperazine), 3.32 (s, 2H, NCH2CO), 4.11-4.18 (m,
1H, R-H), 4.23-4.30 (m, 1H, R-H), 4.56 (s, 2H, N-CH2-Ph), 4.63
(s, 2H, N-CH2-Ph), 6.55 (d, 1H, J=7.4 Hz, NH), 7.14-7.37 (m,
13H, 2 ꢀ Ph and CHdCHCON and CHdCHCON and NH),
7.82-8.10 (m, 7H, naphthyl and NH), 7.48 (s, 1H, NH), 8.09 (s, 1H,
NH), 10.69 (s, 1H, NH); HRMS (FAB) calcd for C36H49N8O8
6.60 (t, 1H, J=7.7 Hz, NH), 7.07-7.61 (m, 12H, naphthyl and
CHdCHCON and CHdCHCON and NH2), 7.85-8.10 (m, 4H,
naphthyl), 10.43 (s, 1H, NH); HRMS (FAB) calcd for C37H45-
N8O8 729.3355, observed m/z 729.3376. Anal. (C37H44N8O8
1.3H2O) C, H, N.
3
N2-(4-(Benzyloxycarbonyl)piperazin-1-ylcarbonylalanylalanyl)-
N1-carboxymethyl-N1-trans-(3-(di-1-naphthylmethylcarbamoyl)-
propenoyl)hydrazine (30p, Cbz-Piz-Ala-Ala-AAsn-CHdCH-
CON(CH2-1-naphthyl)2). This compound was obtained using the
HOBt/EDC coupling method and purified by column chromatog-
raphy using 10% MeOH/CH2Cl2 as the eluent. Recrystallization
1
721.3668, observed m/z 721.3660. Anal. (C36H48N8O8 1.5H2O)
3
with EtOAc/hexane gave a white powder (39% yield): H NMR
C, H, N.
(DMSO-d6) δ 1.20 (d, 3H, Ala-CH3), 1.28 (d, 3H, Ala-CH3), 3.31
(m, 8H, piperazine), 3.34 (s, 2H, NCH2CO), 4.16 (m, 1H, R-H), 4.27
(m, 1H, R-H), 5.06(s, 2H, Cbz), 5.20(s, 4H, N-CH2-Ph and N-CH2-
naphthyl), 6.59 (d, 1H, NH), 7.12-7.54 (m, 17H, naphthyl and Ph
and CHdCHCON and CHdCHCON and NH2), 7.82-8.13 (m,
5H, naphthyl and NH), 10.44 (s, 1H, NH); HRMS (FAB) calcd for
C47H51N8O8 855.3838, observed m/z 855.382438. Anal. (C47H50-
N2-(4-(tert-Butoxycarbonyl)piperazin-1-ylcarbonylalanylalanyl)-
N1-carbamoylmethyl-N1-trans-(3-(methyl-1-naphthylmethylcarba-
moyl)propenoyl)hydrazine (29n, Boc-Piz-Ala-Ala-AAsn-CHd
CH-CON(CH3)CH2-1-naphthyl). This compound was obtained
using the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent. Re-
crystallization with EtOAc/hexane gave a white powder (29%
yield): 1H NMR (DMSO-d6) δ 1.18-1.26 (m, 6H, 2 ꢀ CH3), 1.37
(s, 9H, Boc), 2.99 (d, 3H, J = 10.5 Hz, N-CH3), 3.26 (s, 8H,
piperazine), 3.34 (s, 2H, NCH2CO), 4.10-4.16 (m, 1H, R-H),
4.23-4.29 (m, 1H, R-H), 4.99-5.10 (m, 2H, N-CH2-naphthyl),
5.21 (s, 1H, NH), 6.58 (d, 1H, J=7.2 Hz, NH), 7.07-7.61 (m, 8H,
naphthyl and CHdCHCON and CHdCHCON and NH2),
7.85-8.10 (m, 3H, naphthyl), 8.12 (s, 1H, NH), 10.42 (s, 1H,
NH), 10.72 (s, 1H, NH); HRMS (FAB) calcd for C34H47N8O8
N8O8 1.2H2O) C, H, N.
3
N2-(N-Benzyloxycarbonylalanylvalyl)-N1-carbamoylmethyl-
N1-trans-(3-ethoxycarbonylpropenoyl)hydrazine (31a, Cbz-
Ala-Val-AAsn-CHdCH-COOEt). This compound was ob-
tained using the HOBt/EDC coupling method and purified by
column chromatography using 10% MeOH/CH2Cl2 as the
eluent. Recrystallization with EtOAc/hexane gave a white
powder (25% yield): 1H NMR (DMSO-d6) δ 0.84-0.85
(d, 6H, J=6.2 Hz, 2ꢀVal-CH3), 1.15-1.21 (m, 6H, Ala-CH3
and COCH2CH3), 1.97 (s, 1H, CH), 3.32 (s, 2H, NHCH2CO),
4.08-4.17 (m, 4H, 2 ꢀ R-H and COCH2CH3), 4.95-5.02 (m,
2H, Cbz), 6.59 (d, 1H, J=15.6 Hz, CHdCHCON), 7.18-7.44
(m, 7H, Ph and CHdCHCON and NH), 7.50 (s, 1H, NH), 7.92
(s, 1H, NH), 10.90 (s, 1H, NH); HRMS (FAB) calcd for
695.3511, observed m/z 695.3559. Anal. (C34H46N8O8 1.4H2O)
3
C, H, N.
N2-(4-(tert-Butoxycarbonyl)piperazin-1-ylcarbonylalanylalanyl)-
N1-carboxymethyl-N1-trans-(3-di-1-CH2-naphthylcarbamoylacry-
loyl)hydrazine (29p, Boc-Piz-Ala-Ala-AAsn-CHdCH-CON-
(CH2-1-naphthyl)2). This compound was obtained using the
HOBt/EDC coupling method and purified by column chromato-
graphy using 10% MeOH/CH2Cl2 as the eluent. Recrystalliza-
C
24H34N5O8 520.2456, observed m/z 520.24019. Anal.
(C24H33N5O8 1.9CH2Cl2) C, H, N.
3
N2-(Benzyloxycarbonylalanylvalyl)-N1-carbamoylmethyl-N1-
trans-(3-(1-naphthylmethylcarbamoyl)propenoyl)hydrazine (31m,
Cbz-Ala-Val-AAsn-CHdCH-CONHCH2-1-naphthyl). This com-
pound was obtained using the HOBt/EDC coupling method and
purified by column chromatography using 10% MeOH/CH2Cl2
as the eluent. Recrystallization with EtOAc/hexane gave a white
powder (11% yield): 1H NMR (DMSO-d6) δ 0.85 (s, 6H, 2 ꢀ Val-
CH3), 1.17 (d, 3H, J=6.8 Hz, Ala-CH3), 2.00 (s, 1H, CH), 3.32
(s, 2H, NHCH2CO), 4.11-4.22 (m, 2H, 2 ꢀ R-H), 4.81 (d, 2H,
J=4.7 Hz, NHCH2-Naph), 4.99 (s, 2H, Cbz), 6.93 (d, 1H, J=15.2
Hz, CHdCHCON), 7.13-7.54 (m, 12H, Ph and Naph and
CHdCHCON and NH), 7.73-8.02 (m, 3H, Naph and NH), 8.96
(s, 1H, NH), 10.85 (s, 1H, NH); HRMS (FAB) calcd for C33H39-
1
tion with EtOAc/hexane gave a white powder (25% yield): H
NMR (DMSO-d6) δ 1.20 (d, 3H, CH3), 1.28 (d, 3H, CH3), 1.37
(s, 9H, Boc), 3.25 (s, 8H, piperazine), 3.33 (s, 2H, NCH2CO), 4.15
(m, 1H, R-H), 4.25 (m, 1H, R-H), 5.20 (s, 4H, 2 ꢀ N-CH2-
naphthyl), 6.57 (d, 1H, NH), 7.11-7.56 (m, 12H, naphthyl and
CHdCHCON and CHdCHCON and NH2), 7.82-8.10 (m, 7H,
naphthyl and NH), 10.43 (s, 1H, NH); HRMS (FAB) calcd
for C44H53N8O8 821.4025, observed m/z 821.3981. Anal.
(C44H52N8O8 1.1H2O) C, H, N.
3
N2-(4-(Benzyloxycarbonyl)piperazin-1-ylcarbonylalanylalanyl)-
N1-carbamoylmethyl-N1-trans-(3-ethoxycarbonylpropenoyl)hydra-
zine (30a, Cbz-Piz-Ala-Ala-AAsn-CHdCH-COOEt). This com-
pound was obtained using the HOBt/EDC coupling method and
purified by column chromatography using 15% MeOH/CH2Cl2
as the eluent. Recrystallization with EtOAc/hexane gave a white
powder (41% yield): 1H NMR (DMSO-d6) δ 1.16-1.28 (m, 9H,
2 ꢀ Ala-CH3 and COCH2CH3), 3.31-3.34 (m, 10H, piperazine
and NCH2CO), 4.09-4.18 (m, 3H, R-H and COCH2CH3),
4.20-4.29 (m, 1H, R-H), 5.07 (s, 2H, Cbz), 6.55-6.61 (m, 2H,
NH and CHdCHCON), 7.18-7.38 (m, 7H, Ph and NH and
CHdCHCON), 7.50 (s, 1H, NH), 8.09 (d, 1H, J=5.4 Hz, NH),
10.72 (s, 1H, NH); HRMS (FAB) calcd for C27H38N7O9 604.2726,
N6O7 631.2875, observed m/z 631.2885. Anal. (C33H38N6O7
1.4H2O) C, H, N.
3
N2-(N-Benzyloxycarbonylalanylvalyl)-N1-carbamoylmethyl-N1-
trans-(3-(3,4-dihydro-2H-quinolin-1-ylcarbonyl)propenoyl)hydra-
zine (31j, Cbz-Ala-Val-AAsn-CHdCH-CO-tetrahydroquino-
line). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a yellowish solid (36% yield): 1H NMR
(DMSO-d6) δ 0.86-0.88 (d, 6H, J=6.4 Hz, 2 ꢀ CH3), 1.79 (d,
3H, J=7.0 Hz, Ala-CH3), 1.83-1.89 (m, 2H, N-CH2-CH2-CH2),
2.00 (s, 1H, CH), 2.67-2.70 (t, 2H, J=6.4 Hz, N-CH2-CH2-
CH2), 3.32 (s, 2H, NHCH2CO), 3.70-3.73 (m, 2H, N-CH2-
CH2-CH2), 4.07-4.16 (m, 1H, R-H), 4.21-4.25 (m, 1H, R-H),
4.96-5.03 (m, 2H, Cbz), 7.07-7.44 (m, 13H, Ph, quinoline,
CHdCHCON, CHdCHCON and NH), 7.49 (s, 1H, NH),
7.93 (s, 1H, NH), 10.88 (s, 1H, NH); HRMS (FAB) calcd for C31-
observed m/z 604.2777. Anal. (C27H37N7O9 0.9H2O) C, H, N.
3
N2-(4-(Benzyloxycarbonyl)piperazin-1-ylcarbonylalanylalanyl)-
N1-carbamoylmethyl-N1-trans-(3-(methyl-1-naphthylmethylcarba-
moyl)propenoyl)hydrazine (30n, Cbz-Piz-Ala-Ala-AAsn-CHdCH-
CON(CH3)CH2-1-naphthyl). This compound was obtained using
the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent. Re-
crystallization with EtOAc/hexane gave a white powder (38%
yield): 1H NMR (DMSO-d6) δ 1.15-1.28 (m, 6H, 2 ꢀ CH3), 2.99
(d, 3H, J=9.0 Hz, N-CH3), 3.32 (s, 8H, piperazine), 3.34 (s, 2H,
NCH2CO), 4.07-4.20 (m, 1H, R-H), 4.21-4.32 (m, 1H, R-H),
4.97-5.12 (m, 4H, Cbz and N-CH2-naphthyl), 5.20 (s, 1H, NH),
H39N6O7 607.2875, observed m/z 607.2910. Anal. (C31H38N6O7
3
0.3H2O) C, H, N.
N2-(N-Benzyloxycarbonylalanylisoleucyl)-N1-carbamoylmethyl-
N1-trans-(3-ethoxycarbonylpropenoyl)hydrazine (32a, Cbz-Ala-Ile-
AAsn-CHdCH-COOEt). This compound was obtained using

7208 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Ovat et al.
the HOBt/EDC coupling method and purified by column chro-
matography using 10% MeOH/CH2Cl2 as the eluent. Recrystalli-
zation with EtOAc/hexane gave a white solid (23% yield): H
NMR (DMSO-d6) δ 0.79-0.82 (m, 6H, 2 ꢀ Ile-CH3), 1.15-1.24
(m, 7H, CH and Ala-CH3 and COCH2CH3), 1.39-1.46 (m, 1H,
CH), 1.70-1.76 (m, 1H, CH), 3.32 (s, 2H, NHCH2CO), 4.06-4.24
(m, 4H, 2 x R-H and COCH2CH3), 4.99 (s, 2H, Cbz), 6.60 (d, 1H,
J = 15.5 Hz, CH = CHCON), 7.19-7.36 (m, 7H, Ph and
CHdCHCON and NH), 7.49 (s, 1H, NH), 7.94 (s, 1H, NH),
10.90 (s, 1H, NH); HRMS (FAB) calcd for C25H36N5O8 534.2558,
(s, 1H, NH), 7.51 (s, 1H, NH), 8.06 (s, 1H, NH), 10.72 (s, 1H, NH);
HRMS (FAB) calcd for C20H33N6O8 485.2354, observed m/z
1
485.2363. Anal. (C20H32N6O8 1.2H2O) C, H, N.
3
(2S,3S)-2-(Dibenzylcarbamoyl)-3-(N2-(N-piperidinocarbonyl-
alanylalanyl)-N1-carbamoylmethylhydrazinocarbonyl)oxirane (34r,
Pip-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2). This compound was ob-
tained using the HOBt/EDC coupling method and purified by
column chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder (29%
yield): 1H NMR (DMSO-d6) δ 1.17-1.19 (m, 6H, 2 ꢀ Ala-CH3),
1.34-1.43 (m, 4H, piperidine), 1.47-1.51 (m, 2H, piperidine),
3.22-3.29 (m, 4H, piperidine), 3.32 (s, 2H, NCH2CO), 3.80 (s, 1H,
epoxy CH), 4.05-4.12 (m, 1H, R-H), 4.23-4.74 (m, 6H, R-H and
epoxy CH and 2 ꢀ N-CH2-Ph), 6.41 (d, 1H, J=6.0 Hz, NH),
7.21-7.39 (m, 11H, 2 ꢀ Ph and NH) 7.47 (s, 1H, NH) 8.09 (s, 1H,
NH), 10.68 (s, 1H, NH); HRMS (FAB) calcd for C32H42N7O7
observed m/z 534.2600. Anal. (C25H35N5O8 0.8H2O) C, H, N.
3
N2-(N-Benzyloxycarbonylalanylphenylalanyl)-N1-carbamoyl-
methyl-N1-trans-(3-(2-furyl)carbamoylpropenoyl)hydrazine (33e,
Cbz-Ala-Phe-AAsn-CHdCH-CONHCH2-2-furyl). This com-
pound was obtained using the HOBt/EDC coupling method
and purified by column chromatography using 10% MeOH/
CH2Cl2 as the eluent. Recrystallization with EtOAc/hexane gave
a yellowish solid (7% yield): 1H NMR (DMSO-d6) δ 1.11 (d, 3H,
Ala-CH3), 2.81-2.98 (m, 2H, CH2Ph), 3.32 (d, 2H, NHCH2CO),
4.01 (m, 1H, R-H), 4.34 (d, 2H, CH2-furyl), 4.53 (m, 1H, R-H),
4.96-4.99 (m, 2H, Cbz), 6.24 (d, 1H, furyl CH), 6.36 (t, 1H, furyl
CH), 6.87-6.91 (d, 1H, CHdCHCON), 7.09-7.13 (d, 1H,
CHdCHCON), 7.18-7.34 (m, 11H, 2 ꢀ Ph and NH), 7.43
(s, 1H, NH), 7.55 (d, 1H, furyl CH), 8.19 (s, 1H, NH), 8.91
(t, 1H, NH), 10.89 (s, 1H, NH); HRMS (FAB) calcd for C31H35-
636.3140, observed m/z 636.3129. Anal. (C32H41N7O7 1.1H2O)
3
C, H, N.
(2S,3S)-2-(1-Naphthylmethylcarbamoyl)-3-(N2-(N-piperidino-
carbonylalanylalanyl)-N1-carbamoylmethylhydrazinocarbonyl)-
oxirane (34s, Pip-Ala-Ala-AAsn-EP(S,S)-CONHCH2-1-naph-
thyl). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization with
1
EtOAc/hexane gave a yellowish solid (47% yield): H NMR
N6O8 619.2511, observed m/z 619.2575. Anal. (C31H34N6O8
0.75H2O) C, H, N.
(DMSO-d6) δ 1.14-1.20 (m, 6H, 2 ꢀ Ala-CH3), 1.33-1.42
(m, 4H, piperidine), 1.46-1.50 (m, 2H, piperidine), 3.22-3.26
(m, 4H, piperidine), 3.32 (s, 2H, NCH2CO), 3.52 (s, 1H, epoxy
CH), 4.10-4.20 (m, 3H, 2 x R-H and epoxy CH), 4.73-4.78
(m, 2H, N-CH2-naphthyl), 6.36 (d, 1H, J=7.2 Hz, NH), 7.21
(s, 1H, NH), 7.43-7.58 (m, 4H, naphthyl), 7.45-7.56 (m, 4H,
naphthyl), 7.84-8.03 (m, 4H, naphthyl and NH), 9.00 (s, 1H,
NH), 10.77 (s, 1H, NH); HRMS (FAB) calcd for C29H38N7O7
3
N2-(N-Benzyloxycarbonylalanylphenylalanyl)-N1-carbamoyl-
methyl-N1-trans-(3-(3,4-dihydro-2H-quinolin-1-ylcarbonyl)prope-
noyl)hydrazine (33j, Cbz-Ala-Phe-AAsn-CHdCH-CO-tetra-
hydroquinoline). This compound was obtained using the HOBt/
EDC coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a yellowish solid (27% yield): 1H NMR
(DMSO-d6) δ 1.12 (d, 3H, J = 6.8 Hz, Ala-CH3), 1.84-1.89
(m, 2H, N-CH2-CH2-CH2), 2.67-2.70 (t, 2H, J=6.5 Hz, N-CH2-
CH2-CH2), 2.87-3.01 (m, 2H, CH2Ph), 3.32 (s, 2H, NH-
CH2CO), 3.70-3.71 (m, 2H, N-CH2-CH2-CH2), 3.99-4.06
(m, 1H, R-H), 4.55-4.60 (m, 1H, R-H), 4.94-5.02 (m, 2H, Cbz),
7.05-7.34 (m, 17H, 2 ꢀ Ph, quinoline, CHdCHCON, CHd
CHCON and NH), 7.48 (s, 1H, NH), 8.18 (s, 1H, NH), 10.92
(s, 1H, NH); HRMS (FAB) calcd for C35H39N6O7 655.2875,
596.2827, observed m/z 596.2847. Anal. (C29H37N7O7 1.4H2O)
3
C, H, N.
(2S,3S)-2-(1-Naphthylmethylcarbamoyl)-3-(N2-(N-morpholino-
carbonylalanylalanyl)-N1-carbamoylmethylhydrazinocarbonyl)-
oxirane (35s, Mu-Ala-Ala-AAsn-EP(S,S)-CONHCH2-1-naph-
thyl). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a white powder (22% yield): 1H NMR
(DMSO-d6) δ 1.15-1.21 (m, 6H, 2 ꢀ Ala-CH3), 3.23-3.29 (m,
4H, morpholine), 3.34 (s, 2H, NCH2CO), 3.44-3.55 (m, 4H,
morpholine), 4.12-4.20 (m, 4H, 2 ꢀ R-H and 2 ꢀ epoxy CH),
4.70-4.82 (m, 2H, N-CH2-naphthyl), 6.52 (d, 1H, J=6.9 Hz,
NH), 7.21 (s, 1H, NH), 7.43-7.58 (m, 4H, naphthyl), 7.84-8.10
(m, 4H, naphthyl and NH), 9.00 (s, 1H, NH), 10.77 (s, 1H, NH);
HRMS (FAB) calcd for C28H36N7O8 598.2620, observed m/z
observed m/z 655.2923. Anal. (C35H38N6O7 0.4H2O) C, H, N.
3
N2-(N-Benzyloxycarbonylalanylphenylalanyl)-N1-carbamoyl-
methyl-N1-trans-(3-(3,4-dihydro-2H-isoquinolin-1-ylcarbonyl)prope-
noyl)hydrazine (33k, Cbz-Ala-Phe-AAsn-CHdCH-CO-tetra-
hydroisoquinoline). This compound was obtained using the HOBt/
EDC coupling method and purified by column chromatography
using 10% MeOH/CH2Cl2 as the eluent. Recrystallization with
EtOAc/hexane gave a white powder (11% yield): 1H NMR
(DMSO-d6) δ 1.10 (d, 3H, J = 6.0 Hz, Ala-CH3), 2.76-2.98
(m, 4H, CH2Ph and N-CH2-CH2-CH2), 3.35 (s, 2H, NHCH2CO),
3.66-3.76 (m, 2H, N-CH2-CH2), 3.98-4.04 (m, 1H, R-H),
4.50-4.58 (m, 1H, R-H), 4.65-4.73 (m, 2H, N-CH2-C), 4.94-
5.02 (m, 2H, Cbz), 7.00-7.33 (m, 12H, Ph, isoquinoline,
CHdCHCON, CHdCHCON and NH), 7.51 (s, 1H, NH), 8.18
(s, 1H, NH), 10.89 (s, 1H, NH); HRMS (FAB) calcd for C35H39-
598.2656. Anal. (C28H35N7O8 1.7H2O) C, H, N.
3
(2S,3S)-2-(Dibenzylcarbamoyl)-3-(N2-(N-morpholinocarbonylal-
anylalanyl)-N1-carbamoylmethylhydrazinocarbonyl)oxirane (35r,
Mu-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2). This compound was
obtained using the HOBt/EDC coupling method and purified
by column chromatography using 10% MeOH/CH2Cl2 as the
eluent. Recrystallization with EtOAc/hexane gave a white pow-
der (19% yield): 1H NMR (DMSO-d6) δ 1.13-1.25 (m, 6H, 2 ꢀ
Ala-CH3), 3.21-3.27 (m, 4H, morpholine), 3.34 (s, 2H,
NCH2CO), 3.42-3.57 (m, 4H, morpholine), 3.80-4.25 (m, 4H,
2 ꢀ R-H, 2 ꢀ epoxy CH), 4.55-4.78(m, 4H, 2 ꢀ N-CH2-Ph), 6.55
(d, 1H, NH), 7.22-7.47 (m, 12H, 2 ꢀ Ph and 2 ꢀ NH), 8.15
(s, 1H, NH), 10.66 (s, 1H, NH); HRMS (FAB) calcd for C31H40-
N6O7 655.2875, observed m/z 655.2893. Anal. (C35H38N6O7
0.75EtOAc) C, H, N.
3
(2S,3S)-3-(N2-(N-Piperidinocarbonylalanylalanyl)-N1-carbamoyl-
methylhydrazinocarbonyl)oxirane-2-carboxylic Acid Ethyl Ester
(34q, Pip-Ala-Ala-AAsn-EP(S,S)-COOEt). This compound was
obtained using the HOBt/EDC coupling method and purified by
column chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder (3%
yield): 1H NMR (DMSO-d6) δ1.12-1.20 (m, 9H, 2 ꢀ Ala-CH3 and
COCH2CH3), 1.33-1.44 (m, 4H, piperidine), 1.46-1.50 (m, 2H,
piperidine), 3.19-3.29 (m, 4H, piperidine), 3.32 (s, 2H, NCH2CO),
3.49 (s, 1H, epoxy CH), 3.67 (s, 1H, epoxy CH), 4.05-4.28 (m, 4H,
2 ꢀ R-H and COCH2CH3), 6.41 (d, 1H, J= 6.2 Hz, NH), 7.23
N7O8 638.2938, observed m/z 638.2937. Anal. (C31H39N7O8
1.4H2O) C, H, N.
3
cis-2-(Dibenzylcarbamoyl)-3-(N2-(N-morpholinocarbonylalanyl-
alanyl)-N1-carbamoylmethylhydrazinocarbonyl)oxirane (35t, Mu-
Ala-Ala-AAsn-EP(cis)-CON(Bzl)2). This compound was obtained
using the HOBt/EDC coupling method and purified by column
chromatography using 10% MeOH/CH2Cl2 as the eluent.
Recrystallization with EtOAc/hexane gave a white powder

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7209
(13% yield): 1H NMR (DMSO-d6) δ 1.16-1.25 (m, 6H, 2 ꢀ Ala-
CH3), 3.20-3.28 (m, 4H, morpholine), 3.33 (s, 2H, NCH2CO),
3.46-3.53 (m, 4H, morpholine), 4.04-4.25 (m, 4H, 2 ꢀ R-H, 2 ꢀ
epoxy CH), 4.37-4.73 (m, 4H, 2 ꢀ N-CH2-Ph), 6.53 (d, 1H, J=7.3
Hz, NH), 7.15-7.41 (m, 12H, 2 ꢀ Ph and 2 ꢀ NH), 8.16 (d, 1H, J=
5.9 Hz, NH), 10.87 (s, 1H, NH); HRMS (FAB) calcd for C31H40-
Endopeptidase with Restricted Specificity. Biochem. J. 1999, 339
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N7O8 638.2938, observed m/z 638.2943. Anal. (C31H39N7O8
1.5H2O) C, H, N.
3
(2S,3S)-2-(Dibenzylcarbamoyl)-3-(N2-(N-piperazinocarbonylala-
nylalanyl)-N1-carbamoylmethylhydrazinocarbonyl)oxirane (38r,
Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2). This compound was
obtained by the removal of the tert-butyl protecting group of
Boc-Piz-Ala-Ala-AAsn-EP(S,S)-CON(Bzl)2 with trifluoroacetic
acid/methylene chloride (1:1) for 3 h at room temperature. The
volatiles were evaporated, and the TFA salt was washed several
times with diethyl ether to give a white solid (91% yield):
1H NMR (DMSO-d6) δ 1.18-1.21 (m, 6H, 2 ꢀ Ala-CH3), 3.03
(s, 4H, piperazine), 3.37-3.51 (m, 6H, piperazine and NCH2CO),
3.83 (s, 1H, epoxide CH), 4.10-4.33 (m, 3H, 2 ꢀ R-Handepoxide
CH), 4.53-4.57 (m, 4H, 2 ꢀ NCH2Ph), 6.76 (d, 1H, J=7.0 Hz,
NH), 7.21-7.39 (m, 11H, 2 ꢀ Ph and NH), 7.47 (s, 1H, NH), 8.18
(s, 1H, NH), 8.71 (s, 1H, NH), 10.62 (s, 1H, NH); HRMS (FAB)
calcd for C31H41N8O7 637.3093, observed m/z 637.3032. Anal.
(C31H40N8O7 1.1TFA 2H2O) C, H, N.
3
3
(2S,3S)-2-(Dibenzylcarbamoyl)-3-(N2-(4-(tert-butoxycarbonyl)-
piperazin-1-yl carbonylalanylalanyl)-N1-carbamoylmethylhydrazi-
nocarbonyl)oxirane (36r, Boc-Piz-Ala-Ala-AAsn-EP(S,S)-CON-
(Bzl)2). This compound was obtained using the HOBt/EDC
coupling method and purified by column chromatography using
10% MeOH/CH2Cl2 as the eluent. Recrystallization with EtOAc/
hexane gave a white powder (22% yield): 1H NMR (DMSO-d6) δ
1.18 (s, 6H, 2 ꢀ Ala-CH3), 1.38 (s, 9H, Boc), 3.25-3.31 (m, 10H,
piperazine and NCH2CO), 3.81 (s, 1H, epoxide CH), 4.08-4.30
(m, 3H, 2 ꢀ R-H and epoxide CH), 4.57 (s, 2H, NCH2Ph), 4.70
(s, 2H, NCH2Ph), 6.57 (d, 1H, J=7.6 Hz, NH), 7.17-7.36 (m,
11H, 2 ꢀ Ph and NH), 7.46 (s, 1H, NH), 8.14 (s, 1H, NH), 10.67
(s, 1H, NH); HRMS (FAB) calcd for C36H49N8O9 737.3617,
observed m/z 737.3630. Anal. (C36H48N8O9 1.4H2O) C, H, N.
3
(18) Fikrig, E.; Narasimhan, S. Borrelia Burgdorferi;Traveling
Acknowledgment. The work was supported by the Sandler
Foundation. D.S. and P.K. were supported by the Grant 206/
06/0865 from the Grant Agencyof the CzechRepublic and the
Research Center No. LC06009.
Incognito? Microbes Infect. 2006, 8, 1390–1399.
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Gut of the Tick Ixodes ricinus Reveals an Evolutionarily Conserved
Network of Aspartic and Cysteine Peptidases. Parasit. Vectors
2008, 1, 7.
Supporting Information Available: Synthesis of aza-peptide
substrate analogues (figure), inhibition data (table), and syn-
thetic details. This material is available free of charge via the
Internet at http://pubs.acs.org.
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E.; Watts, C. Novel Cell-Permeable Acyloxymethylketone
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Gerhartz, B.; Demuth, H. U. Inhibition of Mammalian Legumain
by Michael Acceptors and Azaasn-Halomethylketones. Biol.
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