Peptidyl α-ketoamides with nucleobases, methylpiperazine, and dimethylaminoalkyl substituents as calpain inhibitors

Article abstract of DOI:10.1021/jm901221v

A series of peptidyl α-ketoamides with the general structure Cbz-l-Leu-d,l-AA-CONH-R were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. Nucleobases, methylpiperazine, and dimethylaminoalkyl groups were incorporated into the primed region of the inhibitors to generate compounds that potentially cross the blood-brain barrier. Two of these compounds (Cbz-Leu-d,l-Abu-CONH-(CH ₂)₃-adenin-9-yl and Cbz-Leu-d,l-Abu-CONH-(CH ₂)₃-(4-methylpiperazin-1-yl) have been shown to have useful concentrations in the brain in animals. The best inhibitor for calpain I was Cbz-Leu-d,l-Abu-CONH-(CH₂)₃-2-methoxyadenin-9-yl (K_i = 23 nM), and the best inhibitor for calpain II was Cbz-Leu-d,l-Phe-CONH-(CH₂)₃-adenin-9-yl (K_i = 68 nM). On the basis of the crystal structure obtained with heterocyclic peptidyl α-ketoamides, we have improved inhibitor potency by introducing a small hydrophobic group on the adenine ring. These inhibitors have good potential to be used in the treatment of neurodegenerative diseases.

Full text of DOI:10.1021/jm901221v

6326 J. Med. Chem. 2010, 53, 6326–6336
DOI: 10.1021/jm901221v
Peptidyl r-Ketoamides with Nucleobases, Methylpiperazine, and Dimethylaminoalkyl
Substituents as Calpain Inhibitors
†
§
†
§,
Asli Ovat,†,‡ Zhao Zhao Li,†,‡ Christina Y. Hampton, Seneshaw A. Asress, Facundo M. Fernandez, Jonathan D. Glass, and
ꢀ
James C. Powers*,†,‡
†School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, ‡Parker H. Petit Institute
for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, Georgia 30332, §Center for Neurodegenerative
Disease, Emory University School of Medicine, 615 Michael Street, Atlanta, Georgia 30322, and Department of Neurology, Emory University
School of Medicine, 615 Michael Street, Atlanta, Georgia 30322
Received August 14, 2009
A series of peptidyl R-ketoamides with the general structure Cbz-L-Leu-D,L-AA-CONH-R were
synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin
B. Nucleobases, methylpiperazine, and dimethylaminoalkyl groups were incorporated into the primed
region of the inhibitors to generate compounds that potentially cross the blood-brain barrier.
Two of these compounds (Cbz-Leu-D,L-Abu-CONH-(CH2)3-adenin-9-yl and Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-(4-methylpiperazin-1-yl) have been shown to have useful concentrations in the brain in
animals. The best inhibitor for calpain I was Cbz-Leu-D,L-Abu-CONH-(CH2)3-2-methoxyadenin-9-yl
(Ki = 23 nM), and the best inhibitor for calpain II was Cbz-Leu-D,L-Phe-CONH-(CH2)3-adenin-9-yl
(Ki = 68 nM). On the basis of the crystal structure obtained with heterocyclic peptidyl R-ketoamides,
we have improved inhibitor potency by introducing a small hydrophobic group on the adenine ring.
These inhibitors have good potential to be used in the treatment of neurodegenerative diseases.
Introduction
acyloxymethyl ketones,47 diazomethyl ketones,48 and chloro-
methyl ketones49 are examples of irreversible peptidyl inhibi-
tors of calpain. Reversible inhibitors of calpain are favored
over the irreversible inhibitors for drug development, since
there are many isoforms of calpains and nonspecific inhibition
of these isoforms can cause severe side effects. Calpain inhibi-
tors have been reviewed.50-52
Synthetic calpain inhibitors protect against neuronal loss and
improve neurological function in animal models of Alzheimer’s
disease,53 traumatic brain injury,54 chronic progressive experi-
mental autoimmune encephalomyelitis,55 cerebral ischemia,56
optic nerve degeneration,57 spinal cord injury,58,59 and paclitaxel-
induced sensory neuropathy.60 In addition, calpain inhibitors are
effective for the treatment of cataracts61 and have antimalarial
activity.62 The neuroprotective effects of calpain inhibitors are well
established, but their use in treatment of human diseases is chal-
lenged by their inability to cross the blood-brain barrier (BBBa).
Calpains are cysteine proteases that require calcium for
activation. They belong to Clan CA of cysteine proteases
together with cruzain, rhodesain, papain, and cathepsins.
There are multiple isoforms of calpain that are both ubiqui-
tous and tissue specific. Calpain I (μ-calpain) and calpain II
(m-calpain) are the two major calpain isoforms that are widely
distributed in mammalian cells. These two isoforms are very
similar and differ in the calcium concentration that they require
to become activated. Calpain I is activated by micromolar
concentrations of Ca2þ, whereas calpain II is activated by
millimolar concentrations of Ca2þ. Calpains are involved in a
variety of calcium-regulated biological processes, such as cell
proliferation and differentiation, apoptosis, membrane fusion,
signal transduction, and platelet activation. Enhanced calpain
activity has been observed in a number of diseases including
ischemic1,2 and traumatic3,4 brain injury, cancer,5-7 muscular
dystrophy,8,9 cataracts,10 strokes,11 and neurological disorders
like Alzheimer’s,12,13 Huntington’s,14 and Parkinson’s15,16 dis-
eases and multiple sclerosis.17,18 Involvement of calpains in a
wide variety of biological processes and diseases makes them
important targets for the development of inhibitors. There are
several reviews on the roles of calpains in diseases.19-26
a Abbreviations: Abu, 4-aminobutyric acid; AMC, 7-amino-4-
methylcoumarin; BBB, blood-brain barrier; Brij, polyoxyethylene
lauryl ether; Bzl, benzyl, CH2Ph; Cbz, benzyloxycarbonyl; CDCl3,
deuterated chloroform; CHAPS, 3-[(3-cholamidopropyl)dimethyl-
ammonio]-1-propanesulfonate; CHT, high affinity choline transporter;
CNS, central nervous system; CNT, concentrative nucleoside transpor-
ters; DCC, dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyri-
dine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
DMSO-d6, deuterated dimethyl sulfoxide; DTT, dithiothreitol; EDC,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EDTA,
ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid;
Et2O, diethyl ether; EtOAc, ethyl acetate; EtOH, ethanol; HOBt, N-
hydroxybenzotriazole; iBCF, isobutyl chloroformate; LC-MS, liquid
chromatography-mass spectrometry; LC-MS/MS, liquid chromatogra-
phy-tandem mass spectrometry; MeOH, methanol; Nva, norvaline;
NMM, N-methylmorpholine; OBzl, benzyloxy; Phe, phenylalanine;
RT, room temperature; TLC, thin layer chromatography; Tris, tris-
(hydroxymethyl)aminomethane; RFU, relative fluorescence unit; VAChT,
vesicular acetylcholine transporter.
Synthetic calpain inhibitors can be divided into two groups:
peptidic inhibitors and nonpeptidic inhibitors. Peptidic inhibi-
tors can be further divided into two groups: reversible inhibitors
and irreversible inhibitors. Peptidyl aldehydes,27-35 R-keto-
acids,36,37 R-ketoesters,36 R-ketoamides,36,38-40 R-diketones,41
and R-keto phosphorus42 are examples of reversible peptidyl inhi-
bitors, whereas peptidyl epoxysuccinates,43-45 vinyl sulfones,46
*To whom correspondence should be addressed. Phone: (404) 894-
4038. Fax: (404) 894-2295. Email: james.powers@chemistry.gatech.edu.
r
pubs.acs.org/jmc
Published on Web 08/06/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6327
The BBB is a structural and physiological barrier that restricts
the passage of various chemical substances into the central
nervous system (CNS).63 The “protective” function of the
BBB is also a major obstacle to the delivery of pharmacologic
agents to the CNS for the treatment of neurological disorders.
Our lead compound, Cbz-Leu-D,L-Abu-CONH-(CH2)3-mor-
pholine (AK295, 1, Figure 1), is a reversible peptidyl R-
ketoamide calpain inhibitor that is neuroprotective in models
of head trauma,54 focal brain ischemia,64 and axonal degen-
eration caused by axotomy or exposure to vincristine65 and
paclitaxel.60 The data document the potential for AK295 to be
a potentially effective compound for the treatment of human
disease, but the development of 1 as a drug may be hampered
by its inability to cross the BBB. In order to design new
analogues of 1 that may cross the BBB, we replaced the
morpholine ring with structural features that could be recog-
nized by the intrinsic BBB transport systems.
Here we describe new calpain inhibitors that contain nu-
cleobases, methylpiperazine, and dimethylaminoalkyl moi-
eties in the primed region of the inhibitor. We hypothesized
that these compounds could be recognized by BBB transport
systems in the brain and thus would penetrate into the brain
and spinal cord to inhibit calpain activation during the
progression of neurological diseases.
Leu-D,L-Abu-CO2Et and Cbz-Leu-D,L-Phe-CO2Et were pre-
pared by a two-step Dakin-West reaction from the cor-
responding dipeptide acids Cbz-Leu-Abu-OH and Cbz-Leu-
Phe-OH. The dipeptide acids were reacted with ethyloxalyl
chloride in the presence of pyridine and 4-dimethylamino-
pyridine (DMAP) to form peptidyl R-enol esters. The peptidyl
R-enol esters were then converted to peptidyl R-ketoesters by
reacting with triethylamine. The peptidyl R-ketoacids were
obtained by the hydrolysis of the peptidyl R-ketoesters with 1
M NaOH under standard deblocking conditions to give 2 and
3 (Figure 2).
Some of the P0 amines such as N,N-dimethylpropane-1,3-
diamine and N,N-dimethylethane-1,2-diamine were commer-
cially available. Other amines such as 9-(3-aminopropyl)-
adenine66 and 9-(3-aminopropyl)-2-methoxyadenine were
synthesized in three steps using the procedure described by
Woollins and co-workers.66 Reaction of 2-chloroadenine (6)
with sodium methoxide gave2-methoxyadenine (7).67 Adenine
and 2-methoxyadenine (7) were reacted with 1-bromo-3-
chloropropane toadd the linker by a single alkylation reaction.
The chloro group on the linker was then reacted with sodium
azide to obtain the corresponding azide derivatives. Cata-
lytic reduction of the azide in the presence of palladium acti-
vated on carbon and hydrogen gas gave the precursor amines
9-(3-aminopropyl)adenine and 9-(3-aminopropyl)-2-methoxy-
adenine (8). For the synthesis of 1-(3-aminopropyl)cytosine,68
N-acetylcytosine was reacted with 1-bromo-3-chloropropane
and then with sodium azide to form 1-(3-azidopropyl)-
N-acetylcytosine. The acetyl group was deblocked with
the ammonia, and then catalytic reduction of azide to the
amine was completed in the presence of palladium activa-
ted on carbon and hydrogen gas. For the synthesis of 1-(3-
aminopropyl)-4-methylpiperazine,69 N-methylpiperazine
was reacted with N-(3-bromopropyl)phthalimide and the
corresponding amine was obtained after reacting N-(3-(4-
methylpiperazin-1-yl)propyl)phthalimide with hydrazine
monohydrate.
Chemistry
We previously reported synthetic methods for the pre-
paration of peptidyl R-ketoamides.36,38 The R-ketoesters Cbz-
Figure 1. Structure of Cbz-Leu-D,L-Abu-CONH-(CH2)3-morpho-
linyl (1).
Figure 2. Synthesis of the peptidyl R-ketoamides.

6328 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ovat et al.
The target peptidyl R-ketoamides were obtained in low
yields by coupling the appropriate R-ketoacid (2, 3) and the
appropriate amine (R2NH2) using N-hydroxybenzotriazole
(HOBt) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC) (Figure 2).
Results and Discussion
Synthetic Design. We designed a series of new peptidyl R-
ketoamides extending to the primed region as calpain in-
hibitors. Peptidyl R-ketoamides inactivate the cysteine pro-
teases by forming a reversible hemithioketal adduct with the
active site cysteine residue that resembles the transition state
for peptide bond hydrolysis. This intermediate is quite
stable, and thus, low Ki values have been observed with
peptidyl R- ketoamide transition-state inhibitors. Peptidyl R-
ketoamides have been extensively studied by our group36,38
and other investigators.70-73 Our previous work has shown
that extension of the inhibitors to the primed region in-
creased the potency of the inhibitors and N-monosubstituted
peptidyl R-ketoamides were more potent than the corre-
sponding N,N-disubstituted peptidyl R-ketoamides.38 It
has also been observed that R-aminobutyric acid (Abu),
phenylalanine (Phe), or norvaline (Nva) in the P1 position
and leucine in the P2 position were preferred.74 To generate
compounds capable of crossing the BBB, we designed pepti-
dyl R-ketoamides with nucleobases, methylpiperazine, and
dimethylaminoalkyl structures in the primed region.
Charged and polar compounds such as nucleotides and
choline, which are essential for the brain, do not cross the
BBB, and there are multiple transport systems to facilitate
the delivery of these compounds to the brain.75 Choline, a
positively charged molecule, has a critical role in the CNS as
a precursor to the neurotransmitter acetylcholine but does
not cross the BBB, and its uptake into the brain is dependent
upon carrier-mediated transport. Several BBB choline trans-
porters such as the high affinity choline transporter (CHT)
and the vesicular acetylcholine transporter (VAChT) are
responsible for the transport of choline across the BBB.76
The choline transporters also deliver choline analogues such
as N-n-octylcholine, N-n-decylnicotinium iodide, bis-pyridinium
cyclophanes,77 and nicotine to the brain.78 Several cationic drugs
such as verapamil, diphenhydramine, and donepezil are trans-
ported by or are competitive inhibitors of the choline trans-
porters.79,80 We synthesized several peptidyl R-ketoamides
Cbz-Leu-D,L-Abu-CONH-(CH2)3-(4-methylpiperazin-1-yl)
(4d), Cbz-Leu-D,L-Phe-CONH-(CH2)3-(4-methylpiperazin-
1-yl) (5d), Cbz-Leu-D,L-Phe-CONH-(CH2)3-N-(CH3)2 (5e),
and Cbz-Leu-D,L-Phe-CONH-(CH2)2-N-(CH3)2 (5f) that
contain N-methylpiperazine or dimethylaminoalkyl groups
in the primed region. The methylpiperazine derivatives are
similar to our lead structure 1 where there is a P0 morpholine
ring but not a methylated tertiary amine. The methylpiper-
azine ring has some features in common with donepezil
(a nitrogen heterocycle), verapamil (a methyl tertiary amine),
and nicotine (both). It was hypothesized that these struc-
tural features would provide sufficient recognition for some
of the choline transporters to enable these compounds to
penetrate the brain. The dimethylaminoalkyl moiety in keto-
amides 5e and 5f is found in diphenhydramine and could
also be recognized by the choline transporters. We planned
to methylate these compounds to increase their resemblance
to choline but abandoned that strategy when the dimethyl-
aminoalkyl compounds proved to be difficult to purify and
Figure 3. Proposed binding interactions of the peptidyl R-ketoa-
mides 4a and 4b with the active site of calpain I.
when the nucleobase derivatives proved to be more potent
inhibitors.
Nucleosides, nucleotides, and heterocyclic bases, the
building blocks of RNA and DNA, are hydrophilic com-
pounds and do not readily penetrate cell membranes by
passive diffusion. Instead they are transported by several
concentrative nucleoside transporters (CNT1, CNT2, and
CNT3)81 that are specific for the transport of different hetero-
cyclic bases and nucleotides. Several of the compounds (Cbz-
Leu-D,L-Abu-CONH-(CH2)3-adenin-9-yl (4a), Cbz-Leu-D,
L-Abu-CONH-(CH2)3-cytosin-3-yl (4c), Cbz-Leu-D,L-Phe-
CONH-(CH2)3-adenin-9-yl (5a), and Cbz-Leu-D,L-Phe-
CONH-(CH2)3-cytosin-3-yl (5c)) that we synthesized have
nucleobases such as adenine and cytosine in the primed
region to facilitate their recognition by BBB nucleoside trans-
porter systems. Several drugs, such as nucleoside reverse
transcriptase inhibitors that are used in the treatment of HIV
infection, are transported into the CNS by these nucleoside
transporters, while many protease inhibitors are not effec-
tively transported.82-86 Hence, attachment of structural
features for recognition by brain transporter systems to
calpain inhibitors appears to be a promising strategy for
facilitating incorporation of these molecules into the brain.
The effectiveness of this strategy for other tissue types has
been demonstrated by Meier and co-workers who attached
ketoamide calpain inhibitors to various muscle cell target-
ing capping groups to assist with accumulation of calpain
inhibitors in muscle cells for the treatment of Duchenne
muscular dystrophy and observed improved uptake of calpain
inhibitors into muscle cells.87
Mechanism of Inhibition and Binding Mode. The mechan-
ism of inhibition of calpain by R-ketoamides involves the
formation of a reversible enzyme-inhibitor complex prior to
attack of the active site cysteine residue (Cys115) on the keto
carbonyl group of the R-ketoamides. This leads to the
formation of a stable but reversible tetrahedral hemithioke-
tal adduct (Figure 3) containing a hydrogen bond between
the newly formed hydroxyl group of the tetrahedral adduct
and the imidazole ring of His272.
Crystal structures of 4a (Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-adenin-9-yl) and 4d (Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-(4-methylpiperazin-1-yl) bound to the rat calpain I
protease core (μI-II) have previously been reported by Camp-
bell et al.88 Figure 3 shows a schematic drawing of the
interaction of 4a with the active site of calpain. Important
features in this structure are stacking of the adenine moiety of
4a against a tryptophan (Trp298) in the catalytic site of
calpain I, formation of a hydrogen bond between the amino
group of adenine and the side chain of Glu300, formation
of two hydrogen bonds between the carbonyl oxygen of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6329
Table 1. Inhibition of Calpain I, Calpain II, and Cathepsin B by Peptidyl R-Ketoamidesa
Ki (μM)
compd
Cal I
Cal II
0.041
Cat B
6.9
Cal I/Cat B
1
Cbz-Leu-D,L-Abu-CONH-(CH2)3-morpholine38
Cbz-Leu-D,L-Abu-CONH-(CH2)3-adenin-9-yl
Cbz-Leu-D,L-Phe-CONH-(CH2)3-adenin-9-yl
0.150 ( 0.029
0.053 ( 0.001
0.055 ( 0.009
0.023 ( 0.006
0.041 ( 0.012
0.165 ( 0.020
0.48 ( 0.06
0.640 ( 0.137
1.37 ( 0.126
0.226 ( 0.036
0.711 ( 0.159
46
4a
5a
4b
5b
4c
5c
4d
5d
5e
5f
0.070 ( 0.010
0.068 ( 0.006
0.077 ( 0.025
0.209 ( 0.029
1.14 ( 0.06
0.438 ( 0.079
0.286 ( 0.074
6.36 ( 1.06
0.80 ( 0.15
1.75 ( 0.18
0.88 ( 0.02
2.34 ( 0
0.75 ( 0.06
0.44 ( 0.03
1.42 ( 0.34
111 ( 17
15.1
31.8
38.3
57.1
4.54
0.92
2.22
81.0
334
Cbz-Leu-D,L-Abu-CONH-(CH2)3-2-methoxyadenin-9-yl
Cbz-Leu-D,L-Phe-CONH-(CH2)3-2-methoxyadenin-9-yl
Cbz-Leu-D,L-Abu-CONH-(CH2)3-cytosin-3-yl
Cbz-Leu-D,L-Phe-CONH-(CH2)3-cytosin-3-yl
Cbz-Leu-D,L-Abu-CONH-(CH2)3-(4-methylpiperazin-1-yl)
Cbz-Leu-D,L-Phe-CONH-(CH2)3-(4-methylpiperazin-1-yl)
Cbz-Leu-D,L-Phe-CONH-(CH2)3-N-(CH3)2
0.844 ( 0.317
3.52 ( 0.54
75.5 ( 6.8
475 ( 66
Cbz-Leu-D,L-Phe-CONH-(CH2)2-N-(CH3)2
668
a Calpain assays were performed in 50 mM Tris HCl, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% CHAPS, pH 7.5, 10 mM DTT, 5 mM CaCl2,
and <5% DMSO. Calpain I from porcine erythrocytes and calpain II from porcine kidney were used in the assays. The human liver cathepsin B assays
were performed in 0.1 M NaHPO4, 1.25 mM EDTA, 0.01% Brij, pH 6.0 buffer, and <5% DMSO.
carboxamide with Gln109 and Cys115, and formation of
hydrogen bonds between the inhibitor backbone and Gly208,
Gly271 (Figure 3). Neither the stacking interaction with Trp298
nor hydrogen bond formation with Glu300 was observed
between the piperazinyl ring of compound 4d and the primed
side region of the enzyme.
Interestingly, a hydrophobic pocket formed by Ala262,
Ile263, and Val269 was observed in the crystal structure near
the C2 carbon of the adenine moiety (Figure 3). To facilitate
interactions with this hydrophobic pocket, we have syn-
thesized several peptidyl R-ketoamides with a 2-methoxy-
adenine moiety in the primed region (Cbz-Leu-D,L-Abu-
CONH-(CH2)3-2-methoxyadenin-9-yl (4b) and Cbz-Leu-D,
L-Phe-CONH-(CH2)3-2-methoxyadenin-9-yl (5b)).
The inhibitory potency of the new inhibitors toward
calpain I, calpain II, and cathepsin B is shown in Table 1.
Calpain I Inhibition. As expected from the crystal struc-
ture, inhibitors with adenine (4a, 5a) and 2-methoxyadenine
(4b, 5b) in the primed region were 3- to 7-fold more potent
than our lead compound 1 (Ki = 150 nM). The best calpain I
inhibitors were Cbz-Leu-D,L-Abu-CONH-(CH2)3-2-meth-
oxyadenin-9-yl (4b) and Cbz-Leu-D,L-Phe-CONH-(CH2)3-
2-methoxyadenin-9-yl (5b) with Ki values of 23 and 41 nM,
respectively, while compounds Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-adenine-9-yl (4a) and Cbz-Leu-D,L-Phe-CON-
H-(CH2)3-adenine-9-yl (5a) were slightly less potent than
the 2-methoxyadenine derivatives but still have Ki values of
53 and 55 nM, respectively. The increased potency of the
2-methoxyadenine derivatives confirmed that the 2-meth-
oxy group in these compounds is probably interacting with
the hydrophobic pocket (Ala262, Ile263, and Val269) in the
primed region (Figure 3). Compounds 4c (Ki = 165 nM)
and 5c (Ki = 480 nM) with cytosine in the primed side (Cbz-
Leu-D,L-Abu-CONH-(CH2)3-cytosin-3-yl, Ki = 165 nM;
Cbz-Leu-D,L-Phe-CONH-(CH2)3-cytosin-3-yl, Ki=480 nM)
were 3- to 8-fold less potent than the adenine derivatives.
However, the cytosine compounds are still very potent in-
hibitors, since they can also form a hydrogen bond with
Glu300 and can stack on Trp298, although not as well as
the adenine or 2-methoxyadenine derivatives. Among the
compounds with nucleobases in the primed region, Abu in
the P1 position is slightly favored over Phe.
primed side but are still reasonable inhibitors of calpain I.
The decreased potency is probably due to the lack of the
stacking interactions with Trp298 and the hydrogen bond
with Glu300. Changing the amino acid in the P1 position
from an Abu to a Phe resulted in a 100-fold increase in
potency in compound 5e, while decreasing the alkyl spacer
by one methylene group in compound 5f resulted in a 3-fold
decrease in potency.
Calpain II Inhibition. In general, the inhibitors were more
inhibitory toward calpain I but the order of reactivity of
calpain II is similar to that of calpain I. Compounds with
nucleobases (4a, 4b, 5a, and 5b) in the primed region were
more potent than those with dimethylaminoalkyl groups
(5e,f). Compounds Cbz-Leu-D,L-Abu-CONH-(CH2)3-ade-
nin-9-yl (4a) and Cbz-Leu-D,L-Phe-CONH-(CH2)3-adenin-
9-yl (5a) were the most potent inhibitors of calpain II with
Ki values of 70 and 68 nM, respectively. Introduction of
the methoxy group to the C2 carbon of adenine did not
significantly change the potency for Cbz-Leu-D,L-Abu-
CONH-(CH2)3-2-methoxyadenin-9-yl (4b) (Ki = 77 nM)
but resulted in a 3-fold decrease in potency for Cbz-Leu-D,
L-Phe-CONH-(CH2)3-2-methoxyadenin-9-yl (5b (Ki = 209
nM). The cytosine derivatives Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-cytosin3-yl (4c) (Ki = 1.14 μM) and Cbz-Leu-D,
L-Phe-CONH-(CH2)3-cytosin-3-yl (5c) (Ki = 438 nM)
were less potent than the adenine and 2-methoxyadenine
derivatives.
The 4-methylpiperazine derivatives Cbz-Leu-D,L-Abu-
CONH-(CH2)3-(4-methylpiperazin-1-yl) (4d) (Ki = 286 nM)
and Cbz-Leu-D,L-Phe-CONH-(CH2)3-(4-methylpiperazin-1-
yl) (5d) (Ki = 6.36 μM) were less potent than the adenine
derivatives 4a and 5a. The dimethylaminoalkyl analogues 5e
and 5f have Ki values of 25.9 μM, 844 nM, and 3.52 μM,
respectively. Again, replacing Abu with Phe in the P1 position
resulted in a 30-fold increase in potency for compound 5e and
decreasing the alkyl spacer length by one methylene group
decreased the potency 4-fold for Cbz-Leu-D,L-Phe-CON-
H-(CH2)2-N-(CH3)2 (5f).
Cathepsin B Inhibition and Selectivity. In order to deter-
mine the selectivity of the new inhibitors, we measured
inhibitory potency with cathepsin B. In general, the peptidyl
R-ketoamides displayed lower affinity toward cathepsin B.
The most selective calpain I inhibitors among the ones with
nucleobases in the primed region were the 2-methoxyade-
nine derivatives. The inhibitor 4b (Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-2-methoxyadenin-9-yl) with a Ki value of 23 nM
Compounds containing 4-methylpiperazine (4d, Ki =
640 nM; 5d, Ki = 1.37 μM) or dimethylamino alkyl groups
(5e, Ki = 226 nM; 5f, Ki = 711 nM) in the primed side were
less potent than 1 and compounds with nucleobases in the

6330 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ovat et al.
for calpain I is 3- and 38-fold poorer with calpain II and
cathepsin B, respectively. The Ki of 5b (Cbz-Leu-D,L-Phe-
CONH-(CH2)3-2-methoxyadenin-9-yl) is 41 nM, which
makes it 5- and 57-fold more potent on calpain I than on
calpain II and cathepsin B. The small hydrophobic pocket
observed in the active site of calpain I is not present in
cathepsin B; thus, both selectivity and increased potency
toward calpain I have been obtained by the introduction of a
small hydrophobic group on to the adenine ring to interact
with the pocket.
Except for 4d (Ki = 1.42 μM), most of the inhibitors with
4-methylpiperazine and dimethylaminoalkyl substituents
in the primed region were poor inhibitors of cathepsin B
(Cbz-Leu-D,L-Phe-CONH-(CH2)3-(4-methylpiperazin-1-yl)
(5d, Ki = 111 μM), Cbz-Leu-D,L-Phe-CONH-(CH2)3-N-
(CH3)2 (5e, Ki = 75.5 μM), Cbz-Leu-D,L-Phe-CONH-
(CH2)2-N-(CH3)2 (5f, Ki = 475 μM) except Cbz-Leu-D,L-
Abu-CONH-(CH2)3-(4-methylpiperazin-1-yl) with a Ki value
of 1.42 μM). The most selective calpain I inhibitors among
those with N-methylpiperazine and dimethylaminoalkyl sub-
stituents in the primed region were Cbz-Leu-D,L-Phe-CON-
H-(CH2)3-N-(CH3)2 (5e) and Cbz-Leu-D,L-Phe-CONH-
(CH2)2-N-(CH3)2 (5f). Inhibitor 5e (Ki = 0.226 μM toward
calpain I) is 3- and 334-fold less potent on calpain II and
cathepsin B, respectively, while inhibitor 5f (Ki = 0.711 μM
toward calpain I) is 5- and 668-fold less potent on calpain II
and cathepsin B, respectively. The calpain inhibitors 5e and
5f, although less potent than several other inhibitors, have
increased specificity for calpain and may be more useful in
biological experiments.
Table 2. Concentrations of 1 in Mouse Serum and Tissue Samplesa
concn of 1, μg/(g of tissue)
subcutaneous
(N = 2; 1 h)
intravenous
(N = 3; 1 h)
oral
(N = 2; 4 h)
tissue
brain
heart
kidney
ND
ND
ND
NQ
NQ
NQ
ND
NQ
NQ
NQ
0.541 ( 0.008
0.936 ( 0.068
1.550 ( 0.068
ND
0.561 ( 0.036
1.816 ( 0.161
0.943 ( 0.052
ND
liver
spinal cord
serum
0.895 ( 0.196
NQ
NQ
0.333 ( 0.009b
1.316 ( 0.067
peripheral nerve
spleen
1.380 ( 0.174
a Concentration of 1 detected in the brain, heart, kidney, liver, spinal
cord, serum, peripheral nerve, or spleen obtained from mice dosed with
the inhibitor 1 at 24 mg/kg body weight. The mice received the drug by
subcutaneous (N = 2, mice sacrificed after 1 h), intravenous (N = 3,
mice sacrificed after 1 h), or oral (N = 2, mice sacrificed after 4 h)
administration. A calibration curve was measure at varying doses of 1 in
mouse plasma. The quantitation limit was 0.004 mg/mL plasma, and the
detection limit was 0.001 mg/mL (1 ng/mL) plasma. No calibration
curves were determined with individual tissues, and the quantitation
limit is likely higher in tissue. The errors in the measurements were
determined using multiple HPLC sample injections from the individual
animals. ND = not detected. NQ = not quantifiable. b The value listed
for the sciatic nerve with 1 was from three injections of a sample from one
mouse dosed intravenously. The other two mice were NQ.
Table 3. Concentration of Calpain Inhibitors in the Mouse Brain after
Subcutaneous Administrationa
μg/(g of tissue)
compd
N = 3; 1 h
N = 3; 2 h
N = 3; 4 h
N = 3; 8 h
1.17 ( 0.01b
ND
4a
4d
ND
NQc
ND
1.14 ( 0.02b
ND
ND
Overall, cathepsin B was inhibited by the new peptidyl
R-ketoamides, but good selectivity was obtained for calpain I
and calpain II in R-ketoamides 5d, 5e, and 5f. The cytosine
derivative 5c was equally potent with calpain I, calpain II,
and cathepsin B.
a Concentration of 4a and 4d detected in the brain obtained from mice
dosed with the inhibitors at 24 mg/kg body weight. The mice received the
drug by subcutaneous administration and were sacrificed after 1, 2, 4,
and 4 h. A calibration curve was measured at varying doses of 4a and 4d
in mouse plasma. The quantitation limit was 0.16 μg/mL plasma for 4a
and was 0.23 μg/mL plasma for 4d. No calibration curves were deter-
mined for brain tissue. ND = not detected. NQ = not quantifiable.
b The value listed is three injections of a sample from one mouse, and this
group is N = 1. The two other mice were NQ. c The inhibitor was
detected in all three mice.
Brain Permeability. In our initial studies demonstrating
axonal protection in the animal model of peripheral neuro-
pathy,60 1 was delivered continuously from a subcutane-
ous minidiffusion pump at a dose of 1 at 24 (mg/kg)/day.
These studies showed that 1 (Figure 1) is an effective calpain
inhibitor in vivo, and we therefore choose this dose to use in
our animal studies. Liquid chromatography tandem mass
spectrometric (LC-MS/MS) experiments were performed in
order to determine the concentration of 1 in the brain, heart,
kidney, liver, spinal cord, serum, peripheral (sciatic) nerve,
and spleen of mice dosed with 24 mg of 1/kg body weight
via subcutaneous (sample cohort N = 2 mice), intravenous
(N = 3 mice), or oral (N = 2 mice) administration and sacri-
ficed after 1 (sc), 1 (iv), or 4 (oral) h, respectively (Table 2).
The inhibitor 1 could not be detected in the brain but
was present in the liver, heart, kidney, and spleen at levels
of >0.5 μg/g of tissue after a single subcutaneous or intra-
venous dose, indicating that the bioavailability of 1 via
subcutaneous or oral administration was good, although
the inhibitor passed through the excretory system without
penetrating the BBB.
tissue. The N-methylpiperazine derivative 4d could be de-
tected in the three mice at the 1 h time point but could not be
quantitated with sufficient accuracy. After 2 h, 4d could be
detected and quantitated in one mouse at 1.14 ( 0.02 μg/g
tissue. The concentrations observed for 4a and 4d are app-
roximately 2 μM, which is several-fold higher than the Ki
values for inhibition of calpain I and II, and thus, this is likely
a therapeutically useful concentration.
The detection of the two compounds in the brains of
several animals, but not all the animals, is certainly encoura-
ging. However, this result only shows that the compounds
are incorporated into the brain. This could result from either
passive diffusion or active transport by one of the BBB
barrier transporters. Future experiments should involve in
vitro studies with various BBB transporters to determine if
the compounds are indeed interacting with the transporters
or are simply entering the brain by passive diffusion. It will
also be necessary to develop a more sensitive and routine
analytical method for 4a and 4d in tissue samples in order to
test our hypothesis further and to validate our design strat-
egy. The current analytical method was optimized for 1 and
did not allow us to detect and quantitate 4a and 4d in the
majority of animals dosed.
Two of the newly synthesized compounds (4a and 4d),
which were designed to cross the BBB, were also analyzed
in the brain of mice. For these studies, inhibitors (24 mg/
kg body weight) were subcutaneously administered to mice
sacrificed after 1 (N = 3), 2 (N = 3), 4 (N = 3), or 8 (N = 3) h
(Table 3). The adenine compound 4a, was only detected in
quantifiable levels in 1 mouse out of 12. This mouse at the 8 h
time point had a concentration of 4a of 1.17 ( 0.01 μg/g

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6331
Conclusions
mice. Each experiment was done with cohorts of two to three
animals per time point and dosed with 24 mg inhibitor/kg body
weight, administered subcutaneously (N = 2 for 1, N = 3 for 4a
and 4d), intravenously (N = 3 for 1), or orally via oral gavage
(N = 2 for 1). For compound 1, mice dosed subcutaneously,
intravenously, or orally were sacrificed after 1, 1, or 4 h,
respectively. For compounds 4a and 4d, mice (N = 3) were
sacrificed 1, 2, 4, or 8 h after the dose was administered. At the
designated time, each animal was perfused with buffered saline
at 37 °C to clear all blood vessels and was sacrificed. Serum and
extracted tissue samples were frozen in liquid nitrogen and
stored at -80 °C until analyzed by LC-MS/MS.
LC-MS/MS Assays. For the measurement of plasma phar-
macokinetics, tissue distribution, and permeability into the
nervous system of 1, 4a, and 4d, a LC-MS/MS assay was
developed using another of our calpain inhibitor compounds,
Cbz-Leu-Nva-CONH-(CH2)3-morpholine (ZLAK74), as an in-
ternal standard. The internal standard was added to serum or
tissues early in the sample preparation procedure to compensate
for incomplete analyte extraction. All drug standards and
tissues were stored at -80 °C until needed. Control tissues from
undosed mice were used to prepare calibration standards with
concentrations of 0.10, 0.30, 0.50, and 1.00 μM for inhibitor 1.
The calibration standards for 4a and 4d where prepared at
identical concentration in mouse plasma. Sample preparation
for the spiked control tissues (or plasma) and the sample tissues
was identical and based on a protocol reported by Guo and co-
workers.89 First, the tissues were homogenized with water in a
5% (weight/volume) ratio (i.e., 5 g tissue/mL water) and 100 μL
aliquots of each homogenate were pipetted into centrifuge tubes
and spiked with 1.00 μM Cbz-Leu-Nva-CONH-(CH2)3-mor-
pholine as an internal standard. The mixture was sonicated
for 10 min to ensure homogeneity in the sample, and an amount
of 300 μL of 99.9:0.1 v/v acetonitrile/formic acid was added
to precipitate proteins. The sample was briefly vortexed and
sonicated for 15 min followed by centrifugation at 13000g for
30 min. The supernatant was removed and evaporated at 45 °C
for 3 h, and the residue was reconstituted with 100 μL of 30:69.9:0.1
v/v acetonitrile/water/formic acid. The final solution was filte-
red using a 0.45 μm syringe filter (Acrodisc, Pall) and analyzed
by LC-MS/MS.
Wehaveshown thatpeptidylR-ketoamideswiththe general
structure of Cbz-L-Leu-D,L-AA-CONH-R, where R is a het-
erocyclic base, are effective inhibitors of the cysteine proteases
calpain I, and II. It has been observed that Abu, which is a
small hydrophobic residue, is slightly favored over the large
hydrophobic residue Phe in the P1 position. It was observed
that nucleobases were favored over dimethylaminoalkyl or
methylpiperazine substituents in the primed region because of
stacking interactions of the nucleobases with a Trp residue
near the active site. Our hypothesis that introduction of a
hydrophobic group on the adenine ring would facilitate inter-
actions with the hydrophobic pocket observed in the crystal
structure resulting in increased potency was verified experi-
mentally for the new calpain inhibitors 4b and 5b with a
2-methoxyadenine group. In an effort to further extend per-
meability across the BBB to other structures, peptidyl R-
ketoamides containing cytosine in the primed side were also
synthesized but were less effective than the adenine derivatives.
Although our lead compound 1 was found in the peripheral
nerve and other tissue, it was not detected in the brain.
However, two compounds, 4a and 4d, have been detected at
therapeutically useful concentrations in the brain of some
mice after subcutaneous administration. Increased levels of
calpain activity have been observed in a number of neurode-
generative diseases with brain involvement including Alzheimer’s,
Huntington’s, and Parkinson’s diseasesand multiple sclerosis.
Development of selective calpain inhibitors that can cross
the BBB is required for the treatment of these diseases. Here,
we have shown that peptidyl R-ketoamide calpain inhibitors
potentially can be designed to cross the BBB and thus may
be useful in the treatment of a variety of neurodegenerative
diseases.
Experimental Section
Material and Methods. Materials were obtained from Acros,
Bachem Bioscience Inc., or Sigma Aldrich and used without
further purification. The structures and purity of each target
compound were confirmed by TLC, 1H NMR, MS, HPLC
analysis, and/or elemental analysis. 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. Chemical shifts are reported in ppm relative to an internal
standard (trimethylsilane). Electrospray ionization (ESI), fast-
atom-bombardment (FAB), and high-resolution mass spectro-
metry (HRMS) were obtained using Micromass Quattro LC
and VG Analytical 70-SE instruments. The purity of com-
pounds 5a, 4b, 5b, 4c, and 5c after purification was determined
by elemental analysis and was higher than 95%. The elemental
composition for each of these compounds is given in the
Experimental Section for that compound. Elemental analyses
were carried out by Atlantic Microlab Inc., Norcross, GA. The
purity of 5d, 5e, and 5f was determined by HPLC. The analysis
was run on a Beckman Coulter HPLC running 32Karat, version
4.0, software. The Alltech/Applied Science C18 column used
was 250 mm ꢀ 4.6 mm and packed with 5 μm Sperisorb ODS 2.
The column was eluted with an isocratic mixture of 60% 0.1%
TFA in acetonitrile and 40% 0.1% TFA in water. Detection was
at 220 and 254 nm, and the area percent was measured using the
32Karat software in duplicate HPLC runs. Compound 5d was
95-99% pure, 5e was 89-91% pure, and 5f was 96-99% pure.
The synthesis of compounds 1, 4a, and 4d has previously been
reported.36,38,88
All measurements were carried out in an Agilent HPLC 1100
system coupled to a ThermoFinnigan LCQ Deca XPþ ion trap
mass spectrometer using an ESI source operating in positive ion
mode. For experiments with compound 1, an analytical Zorbax
˚
Extend C18 column (1.0 mm ꢀ 150 mm, 5 μm particles, 80 A
pore size, Agilent) was used with the following binary gradient
program: 0.0 min, 30% B; 0.5 min, 30% B; 0.7 min, 85% B; 2.5
min, 85% B; 2.70 min, 30% B; 5.5 min, 30% B. An analytical
Symmetry Shield reverse-phase C18 column (1.0 mm ꢀ 150 mm,
˚
3.5 μm particles, 100 A pore size; Waters) preceded by a Zorbax
RX-C18 guard column (4.6 mm ꢀ 12.5 mm, 5.0 μm particles, 2
μm pore size; Agilent) was used for experiments with com-
pounds 4a and 4d. For separations using this column, the binary
solvent gradient program used was as follows: 0.0 min, 30% B;
0.5 min, 30% B; 0.7 min, 100% B; 4.5 min, 100% B; 5.0 min
-30% B; 6.0 min, 30% B. The mobile phases used for all
separations were as follows: A=0.1% formic acid in water and
B = 0.1% formic acid in acetonitrile. The flow rate was 80 μLmin-1
,
with an injection volume of 15 μL.
The mass spectrometer was operated in multiple reaction
monitoring mode using the following precursor f fragment
transitions, 1: m/z 505.2 f 443.2. Cbz-Leu-Nva-CONH-
(CH2)3-morpholine (internal standard): m/z 519.2 f 457.2. 4a:
m/z 553.2 f 509.2. 4d: m/z 518.3 f 410.3. The following settings
were used: ESI needle voltage þ4.0 kV; sheath gas 15 arbitrary
units (∼0.6 L min-1); capillary temperature 275 °C; capillary
voltage 34 V (1), 28 V (4a), 18 V (4d); capillary skimmer voltage
25 V (1), 55 V (4a), 45 V (4d). For all experiments, the mass
analyzer was set with the automatic gain control on 1 ꢀ 107 with
Animal Studies. All experiments involving animals were
approved by the Emory University Institutional Animal Care
and Use Committee. Animals were 7-week-old female C57BL/6

6332 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ovat et al.
2 microscans, 400 μs max injection time, 1.2 Da mass selection
window, and 34% normalized collision energy. After acquisi-
tion, peak areas for the chromatographic peaks present in the
extracted ion chromatograms for the internal standard and the
compound of interest were determined using the mass spectro-
meter software Xcalibur 2.0 (Thermo) with the Genesis peak
detection algorithm (15 smoothing points, signal-to-noise ratio
threshold of 3.0). The areas for the compound of interest and the
internal standard were exported to Excel, and the ratio of the
peak area of the compound of interest to peak area of Cbz-Leu-
Nva-CONH-(CH2)3-morpholine (internal standard) was calcu-
lated. The amount of compound present in each sample was
determined by correlating the area ratio to a concentration using
the calibration curves obtained during each experiment.
Calpain I and Calpain II Assays. The fluorogenic substrate
Suc-Leu-Tyr-AMC was obtained from Bachem. Calpain I from
porcine erythrocytes and calpain II from porcine kidney were
purchased from Calbiochem. The fluorescence was monitored
using a Tecan Spectrafluor microplate reader. AMC was used as
the calibration standard, and the calibration curve was plotted
against RFU for different concentrations of AMC within the
range 5-0.08 μM. Inhibitor stock solutions were prepared in
DMSO and kept at 4 °C prior to use. Calpain assays were
performed in 50 mM Tris HCl, 50 mM NaCl, 1 mM EDTA, 1
mM EGTA, 0.1% CHAPS, pH 7.5, 10 mM DTT, 5 mM CaCl2,
and three different substrate (Suc-Leu-Tyr-AMC) concentra-
tions (0.8, 0.4, 0.2 μM). A 10 μL aliquot of DMSO (control) or
inhibitor solution in DMSO (DMSO content of <5%) was
added to 200 μL of buffer. The reaction was initiated by adding a
2 μL aliquot of enzyme (with a final concentration of 10 nM) to
the well. The reaction was monitored by the release of 7-amino-
4-methylcoumarin (λex = 360 nm, λem = 465 nm). The total
volume in the reaction well was 212 μL, and controls were run
every hour. Velocities were determined at room temperature
(RT) at five or more concentrations of inhibitor and at three
fixed concentrations of substrate. A plot of 1/v versus [I] gave
intersecting lines with a correlation coefficient of g0.95. Ki
values were determined by Dixon plots.90
Cathepsin B Assay. The fluorogenic substrate Cbz-Arg-Arg-
AMC was obtained from Bachem. Cathepsin B from human
liver was purchased from Calbiochem. The fluorescence was
monitored and calibrated using the method reported for calpain
I and II above. Inhibitor stock solutions were prepared in
DMSO and kept at 4 °C prior to use. The cathepsin B assay
was performed in 0.1 M NaHPO4, 1.25 mM EDTA, 0.01% Brij,
pH 6.0 buffer, and three different substrate (Cbz-Arg-Arg-
AMC) concentrations (0.5, 0.2, 0.1 μM). A 10 μL aliquot of
DMSO (control) or inhibitor solution in DMSO (DMSO con-
tent <5%) was added to 200 μL of buffer. The reaction was
initiated by adding 5 μL of activated enzyme (with a final
concentration of 0.4 μM) to the well. The enzyme was activated
by the addition of cathepsin B kinetic buffer (267 μL) and 0.1 M
DTT (3 μL) to the enzyme stock solution (30 μL). The reaction
was monitored by the release of 7-amino-4-methylcoumarin
(λex = 360 nm, λem = 465 nm). The total volume in the reaction
well was 215 μL, and controls were run every hour. Veloci-
ties and Ki values were determined using the aforementioned
method for calpain I and II.
Statistical Analysis. The data in Table 1 for calpain I, calpain
II, and cathepsin B were analyzed using the VassarStats Web site
for statistical computation. This Web site was developed by
Professor Richard Lowry. Specifically a one-way ANOVA with
a post hoc Tukey HSD (honestly significant differences) test was
performed. Further details and the comparisons are given in the
Supporting Information. In general, inhibition constants for
calpain I, calpain II, and and cathepsin B that differed by less
than a factor of approximately 2 were considered to be non-
significant in this analysis. In general, differences in potency
between calpain I and calpain II for each compound were
nonsignificant with three exceptions (4b, 5b, and 4c), while the
differences between calpain I and cathepsin B and between
calpain II and cathepsin II were always significant with one
exception (5c).
General Procedure for the Synthesis of Dipeptide r-Ketoesters.
The synthesis of the dipeptide acids Cbz-Leu-Abu-OH and Cbz-
Leu-Phe-OH are given in the Supporting Information. The
dipeptide acid (1 equiv) was dissolved in dry THF, and 4-di-
methylaminopyridine (0.05 equiv), pyridine (3 equiv), and ethyl
oxalyl chloride (2.1 equiv) were added sequentially. The result-
ing mixture was stirred at reflux temperature for 4 h. After
removal of the heat source, 1 M HCl (50 mL) was added to the
brown solution. The mixture was extracted with ethyl acetate
(2 ꢀ 100 mL). The combined extract was washed with 100 mL of
saturated NaCl, dried over MgSO4 overnight, and filtered.
Ethyl acetate was removed from the filtrate to give a mixture
of products containing the dipeptide enol ester. The mixture of
products was dissolved in 20 mL of absolute ethanol and stirred
in an ice bath. Triethylamine (1 equiv) was added, and the
mixture was stirred for 1 h at RT. Solvent was removed from the
final mixture using a rotary evaporator. The crude oil was
subjected to column chromatography to give the dipeptidyl R-
ketoester. These compounds have previously been reported
using a synthetic scheme with more steps.36,38
Cbz-Leu-D,L-Abu-COOEt: light-yellow oil, 76% yield. 1H
NMR (CDCl3): 0.86-0.93 (m, 9H, 2 ꢀ Leu-CH3 and Abu-CH3),
1.26-1.37 (m, 4H, CH3 and Leu-CH), 1.49-1.98 (m, 4H, Abu-
CH2, Leu-CH2 and CH3), 4.23-4.38 (m, 2H, 2ꢀR-H), 4.42-4.46
(m, 2H, CH2), 5.03-5.13 (m, 2H, Cbz), 5.67-5.73 (m, 1H, NH),
7.21-7.32 (m, 6H, Ph and NH). HRMS (FAB) calcd for C21H31-
N2O6: 407.2182. Observed m/z 407.2178 ([M þ H]þ).
Cbz-Leu-D,L-Phe-COOEt: light-yellow oil, 68% yield. 1H
NMR (CDCl3): 0.79-0.90 (m, 6H, 2 ꢀ Leu-CH3), 1.22-1.61
(m, 6H, Leu-CH2, CH3 and Leu-CH), 2.93-3.07 (m, 1H, CH),
3.19-3.28 (m, 1H, CH), 4.14-4.33 (m, 4H, CH2 and 2 ꢀ R-H),
5.08 (d, 2H, Cbz), 5.23-5.33 (m, 1H, NH), 6.77-6.84 (m, 1H,
NH), 7.12-7.29 (m, 5H, Ph), 7.33(s, 5H, Ph). HRMS (FAB) calcd
for C26H33N2O6: 469.2339. Observed m/z 469.2337 ([M þ H]þ).
General Procedure for the Synthesis of Dipeptidyl r-Ketoacids.
Dipeptidyl R-ketoesters (1 equiv) were dissolved in ethanol, and
1 M NaOH solution (1.1 equiv) was added in portions while
stirring in an ice bath. The resulting mixture was stirred at RT
for an hour and extracted with anhydrous ether (4 ꢀ 30 mL).
The aqueous layer was acidified to pH 4 with 2 M HCl in an ice
bath and extracted with diethyl ether (Et2O, 2 ꢀ 50 mL). The
combined ether extract was washed with saturated NaCl, dried
over MgSO4 overnight, and filtered. Ether was removed from
the filtrate by evaporation, and the product was dried under
reduced pressure. These compounds have previously been re-
ported using a synthetic scheme with more steps.36,38
Cbz-Leu-D,L-Abu-COOH (2): pale-yellow hygroscopic flakes,
96% yield. 1H NMR (CDCl3): 0.91 (d, 9H, Abu-CH3 and 2 ꢀ
Leu-CH3), 1.47-1.75 (m, 5H, Leu-CH2, Abu-CH2, Leu-CH),
4.13-4.35 (m, 2H, 2 ꢀ R-H), 5.04-5.13 (m, 3H, Cbz and NH),
7.32 (s, 5H, Ph), 8.35-8.41 (d, 1H, NH). HRMS (FAB) calcd for
C19H27N2O6: 379.1869. Observed m/z 379.1870 ([M þ H]þ).
Cbz-Leu-D,L-Phe-COOH (3): pale-yellow hygroscopic flakes,
89% yield. 1H NMR (CDCl3): 0.77-0.86 (m, 6H, 2 ꢀ Leu-CH3),
1.09-1.56 (m, 3H, Leu-CH2 and Leu-CH), 2.49-2.51 (m, 1H,
CH), 2.75-2.91 (m, 1H, CH), 4.01-4.08 (m, 2H, 2 ꢀ R-H),
4.89-5.06 (m, 3H, Cbz and NH), 7.18-7.40 (m, 10H, 2 ꢀ Ph),
8.49 (t, 1H, NH). HRMS (FAB) calcd for C24H29N2O6: 441.2026.
Observed m/z 441.2025 ([M þ H]þ).
Synthesis of 9-(3-Aminopropyl)adenine.66 The synthesis has
been previously reported and experimental details are given in
Supporting Information.
Synthesis of 9-(3-Aminopropyl)-2-methoxyadenine. A mixture
of 2-chloroadenine (1 equiv) and sodium methoxide (7.5 equiv)
in anhydrous methanol (50 mL) was sealed in pressure vessel.
The reaction mixture was held at an internal temperature of
100 °C for 24 h before cooling to RT. Once cooled, the pressure

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6333
vessel was opened and the suspension was diluted with water
(50 mL). The resulting solution was evaporated under reduced
pressure to give a final volume of 70 mL; water (30 mL) was
added to this solution to give a final volume of 100 mL. The
solution was transferred to a three-neck flask equipped with a
stirrer, thermometer, and pH meter. The solution was heated to
60 °C (internal temperature), and 50% aqueous HCl was added to
adjust the pH to 9.5. The resulting suspension was stirred at 60 °C
for 1 h, cooled slowly to RT, and stirred for 16 h. The suspension
was filtered, and the filter cake was washed with water (10 mL)
and methanol (2 ꢀ 10 mL). The solid was dried under vacuum to
give 2-methoxyadenine in 70% yield. 1H NMR (DMSO-d6): 3.76
(s, 3H, OCH3), 7.12 (s, 2H, NH2), 7.86 (s, 1H, CH).
8.08-8.14 (m, 2H, 2 ꢀ adenine-CH), 8.33-8.39 (d, 1H, NH),
8.83-8.89 (t, 1H, NH). HRMS (FAB) calcd for C32H39N8O5:
615.3043. Observed m/z 615.3094 ([M þ H]þ). Anal. (C32H38-
N8O5 0.75H2O) C, H, N.
3
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(6-amino-2-meth-
oxy-9H-purin-9-yl)propyl)-2-oxopentanamide (4b, Cbz-Leu-D,L-
Abu-CONH-(CH2)3-2-methoxyadenin-9-yl). The ketoamide pro-
duct Cbz-Leu-D,L-Abu-CONH-(CH2)3-2-methoxyadenin-9-yl
was obtained from9-(3-aminopropyl)-2-methoxyadenine andthe
ketoacid Cbz-Leu-D,L-Abu-COOH using the EDC/HOBt cou-
pling method, purified by column chromatography on silica gel
with 85:15 CH2Cl2/MeOH as the eluent, then recrystallized from
1
EtOAc/hexane to give a yellowish white solid (26% yield). H
NMR (DMSO-d6): 0.72-0.93 (m, 9H, 2 ꢀ CH3 of Leu and CH3
of Abu), 1.37-1.60 (m, 4H, CH2 of Leu and CH2 of Abu), 1.76
(m, 1H, CH of Leu), 1.93-1.97 (m, 2H, CH2), 3.09 (m, 2H, CH2),
3.78 (s, 3H, OCH3), 3.98-4.14 (m, 3H, CH2 and R-H), 4.84 (m,
1H, R-H), 4.99 (s, 2H, Cbz), 7.20 (s, 2H, NH2), 7.28-7.40 (m,
6H, Ph and NH), 7.93 (s, 1H, CH of adenine), 8.24-8.31 (m,
1H, NH), 8.77 (m, 1H, NH). HRMS (FAB) calcd for C28H41-
N6O6: 583.2993. Observed m/z 583.2900 ([M þ H]þ). Anal.
A mixture of 2-methoxyadenine (1 equiv), 1-bromo-3-chlor-
opropane (4.3 equiv), and potassium carbonate (2.35) in DMF
(200 mL) was stirred at RT under argon for 4 days, filtered, and
evaporated to dryness. The crude product was purified by
column chromatography and gave 9-(3-chloropropyl)-2-meth-
oxyadenine in 66% yield. MS (ESI) m/z 241.9 ([M þ H]þ).
A mixture of 9-(3-chloropropyl)-2-methoxyadenine (1 equiv)
and sodium azide (3 equiv) in DMF was stirred at 80 °C for 24 h,
cooled to RT, and filtered. The crude product was purified by
column chromatography to give 9-(3-azidopropyl)-2-methox-
(C28H39N8O6 0.55H2O) C, H, N.
3
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(6-amino-2-methoxy-
9H-purin-9-yl)propyl)-2-oxophenylbutanamide (5b, Cbz-Leu-
D,L-Phe-CONH-(CH2)3-2-methoxyadenin-9-yl). The ketoamide
product Cbz-Leu-D,L-Phe-CONH-(CH2)3-2-methoxyadenin-9-yl
was obtained from 9-(3-aminopropyl)-2-methoxyadenine and the
ketoacid Cbz-Leu-D,L-Phe-COOH using the EDC/HOBt coup-
ling method, purified by column chromatography on silica gel
with 85:15 CH2Cl2/MeOH as the eluent, then recrystallized from
EtOAc/hexane to give a yellow solid (24% yield). 1H NMR
(DMSO-d6): 0.73-0.75 (d, 6H, 2 ꢀ CH3 of Leu), 1.11-1.35 (m,
4H, CH2 Leu and CH2 of Phe), 1.54 (m, 1H CH of Leu),
1.95-1.98 (m, 2H, CH2), 3.09-3.12 (m, 2H, CH2), 3.77 (s, 3H,
OCH3), 4.03 (m, 3H, CH2 and R-H), 4.97 (s, 2H, Cbz), 5.17 (m,
1H, R-H), 7.20-7.33 (m, 12H, 2 ꢀ Ph and NH2), 7.93 (d, 1H, CH
of adenine), 8.38 (d, 1H, NH), 8.84 (m, 1H, NH). HRMS (FAB)
calcd for C33H41N6O6: 645.3149. Observed m/z 645.3067 ([M þ
1
yadenine as a white crystalline solid in 74% yield. H NMR
(DMSO-d6): 1.99-2.06 (m, 2H, CH2), 3.33-3.37 (m, 2H, CH2),
3.80 (s, 3H, OCH3), 4.10 (t, 2H, CH2), 7.21 (s, 2H, NH2), 7.92 (s,
1H, CH). MS (ESI) m/z 249.0 ([M þ H]þ).
A mixture of 9-(3-azidopropyl)-2-methoxyadenine and 5%
palladium on carbon in MeOH was reacted with hydrogen gas at
RT for 20 h. The catalyst was removed by filtration and the
solvent removed to give 9-(3-aminopropyl)-2-methoxyadenine
as a white solid in 75% yield. 1H NMR (DMSO-d6): 1.82-1.85
(m, 2H, CH2), 2.46-2.49 (m, 2H, CH2), 3.03 (s, 2H, NH2), 3.79
(s, 3H, OCH3), 4.09 (t, 2H, CH2), 7.20 (s, 2H, NH2), 7.92 (s, 1H,
CH). MS (ESI) m/z 223.2 ([M þ H]þ).
Synthesis of 1-(3-Aminopropyl)cytosine.68 The synthesis has
been previously reported, and experimental details are given in
Supporting Information.
Synthesis of 1-(3-Aminopropyl)-4-methylpiperazine.69 The
synthesis has been previously reported, and experimental details
are given in Supporting Information.
H]þ). Anal. (C33H40N8O6 0.25H2O) C, H, N.
3
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-amino-2-oxo-
pyrimidin-1(2H)-yl)propyl))-2-oxopentanamide (4c, Cbz-Leu-
D,L-Abu-CONH-(CH2)3-cytosin-3-yl). The ketoamide product
Cbz-Leu-D,L-Abu-CONH-(CH2)3-cytosin-3-yl was obtained
from 1-(3-aminopropyl)cytosine and the ketoacid Cbz-Leu-D,L-
Abu-COOH using the EDC/HOBt coupling method, purified by
column chromatography on silica gel with 85:15 CH2Cl2/MeOH
as the eluent, then recrystallized from EtOAc/hexane to give
a yellowish white solid (11% yield). 1H NMR (DMSO-d6):
0.72-0.95 (m, 9H, 2 ꢀ CH3 of Leu and CH3 of Abu), 1.41-
1.73 (m, 5H, CH2 of Leu, CH2 of Abu and CH of Leu), 2.13-2.19
(m, 2H, CH2), 3.07 (m, 2H, CH2), 3.56 (m, 1H, R-H), 3.93-4.10
(m, 3H, R-H and CH2), 4.99 (s, 2H, Cbz), 5.60 (d, 1H, CH
of cytosine), 6.97 (d, 2H, NH2), 7.32-7.44 (m, 6H, Ph and
NH), 7.68 (d, 1H, CH of cytosine), 8.22-8.28 (m, 1H, NH),
8.73 (m, 1H, NH). HRMS (FAB) calcd for C26H37N6O6: 529.2775.
General Procedure for the Synthesis of Target Peptide
r-Ketoamides by the HOBt and EDC Coupling Method. HOBt
(1.5 equiv) was added to a stirred solution of the dipeptidyl
R-ketoacid (1.5 equiv) in DMF at -10 °C, followed by addition
of the heterocyclic amine (1 equiv) and EDC (1.5 equiv). The
mixture was allowed to react for 16 h at RT. DMF was
evaporated, and the residue was redissolved in EtOAc. The
organic layer was washed with 2% citric acid, saturated NaH-
CO3, and saturated NaCl before being dried over MgSO4 and
concentrated. Column chromatography on silica gel was used to
purify the peptidyl R-ketoamides.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(6-amino-9H-pur-
in-9-yl)propyl)-2-oxopentanamide (4a, Cbz-Leu-D,L-Abu-CON-
H-(CH2)3-adenin-9-yl). This ketoamide has previously been
reported,88 and characterization data are shown in the Support-
ing Information.
Observed m/z529.2781 ([M þ H]þ). Anal. (C26H36N6O6 1EtOAc)
3
C, H, N.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(6-amino-9H-pur-
in-9-yl)propyl)-2-oxophenylbutanamide (5a, Cbz-Leu-D,L-Phe-
CONH-(CH2)3-adenin-9-yl). The ketoamide product Cbz-Leu-
D,L-Phe-CONH-(CH2)3-adenin-9-yl was obtained from 9-(3-
aminopropyl)adenine and the ketoacid Cbz-Leu-D,L-Phe-
COOH using the EDC/HOBt coupling method, purified by
column chromatography on silica gel with 85:15 CH2Cl2/MeOH
as the eluent, then recrystallized from EtOAc/hexane to give a
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-amino-2-oxo-
pyrimidin-1(2H)-yl)propyl))-2-oxophenylbutanamide (5c, Cbz-
Leu-D,L-Phe-CONH-(CH2)3-cytosin-3-yl). The ketoamide pro-
duct Cbz-Leu-D,L-Phe-CONH-(CH2)3-cytosin-3-yl was ob-
tained from 1-(3-aminopropyl)cytosine and the ketoacid
Cbz-Leu-D,L-Phe-COOH using the EDC/HOBt coupling
method, purified by column chromatography on silica gel with
85:15 CH2Cl2/MeOH as the eluent, then recrystallized from
1
1
white solid (21% yield). H NMR (DMSO-d6): 0.69-0.86 (m,
EtOAc/hexane to give a yellow solid (12% yield). H NMR
9H, 2 ꢀ Leu-CH3 and Abu-CH3), 1.15-1.36 (m, 5H, 2 ꢀ CH2
and CH), 1.92-2.00 (m, 2H, CH2), 3.04-3.15 (m, 4H, 2 ꢀ CH2),
4.07-4.14 (m, 2H, CH2 and 2 ꢀ R-H), 4.95-5.01 (m, 2H, Cbz),
5.18 (s, 1H, NH), 7.12-7.40 (m, 12H, 2 ꢀ Ph and NH2),
(DMSO-d6): 0.74-0.76 (d, 6H, 2 ꢀ CH3 of Leu), 1.11-1.37 (m,
4H, CH2 Leu and CH2 of Phe), 1.59 (m, 1H CH of Leu),
2.11-2.15 (m, 2H, CH2), 3.01-3.10 (m, 2H, CH2), 3.82-4.03
(m, 3H, R-H and CH2), 4.99 (s, 2H, Cbz), 5.18 (m, 1H, NH),

6334 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ovat et al.
5.61 (d, 1H, CH of cytosine), 6.97 (d, 2H, NH2), 7.13-7.57
(m, 11H, 2 ꢀ Ph and CH of cytosine), 8.36 (d, 1H, NH), 8.80
(m, 1H, NH). HRMS (FAB) calcd for C31H39N6O6: 591.2886.
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Observed m/z 591.2852 ([M þ H]þ). Anal. (C31H38N6O6
3
1EtOAc) C, H, N.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-methylpipera-
zin-1-yl)propyl)-2-oxopentanamide (4d, Cbz-Leu-D,L-Abu-
CONH-(CH2)3-(4-methylpiperazin-1-yl). This ketoamide has
previously been reported,88 and characterization data are shown
in the Supporting Information.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-methylpipera-
zin-1-yl)propyl)-2-oxophenylbutanamide (5d, Cbz-Leu-D,L-Phe-
CONH-(CH2)3-(4-methylpiperazin-1-yl)). The dipeptide ketoamide
product Cbz-Leu-D,L-Phe-CONH-(CH2)3-(4-methylpiperazin-
1-yl was obtained from Cbz-Leu-D,L-Phe-COOH and 1-methyl-
4-(3-aminopropyl)piperazine using the EDC/HOBt coupling
method and purified twice by column chromatography on silica
gel with 85:15 CH2Cl2/MeOH as the eluent to give a yellow
semisolid in 10% yield. 1H NMR (CDCl3): 0.81 (m, 6H, CH3 of
Leu), 1.40-1.60 (m, 5H, CH2 and CH), 2.28 (s, 6H, CH3), 3.00
(m, 2H, CH2), 3.20 (m, 2H, CH2), 4.05 (m, 2H, CH2), 4.50 (b, 1H,
R-H), 5.02 (m, 3H, Cbz and R-H), 6.70 (b, 1H, NH), 7.05-7.30
(m, 7H, Ph and NH). The purity was 95-99% by HPLC using
either 220 or 254 nm detection.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(dimethylamino)-
propyl)-2-oxophenylbutanamide (5e, Cbz-Leu-D,L-Phe-CON-
H-(CH2)3-N(CH3)2). The dipeptide ketoamide product Cbz-Leu-
D,L-Phe-CONH-(CH2)3-N(CH3)2 was obtained from Cbz-Leu-D,
L-Phe-COOH and (CH3)2N(CH2)3NH2 using the EDC/HOBt
coupling method. Purification by column chromatography
twice on silica gel with 80:20 CH2Cl2/MeOH as the eluent
provided a yellow semisolid in 10% yield. 1H NMR (CDCl3):
0.84 (m, 6H, CH3 of Leu), 1.50-1.80 (m, 5H, CH2 and CH),
2.12 and 2.19 (d, 6H, CH3), 3.00 (m, 2H, CH2), 3.20 (m, 2H,
CH2), 4.15 (m, 2H, CH2), 4.50 (b, 1H, R-H), 5.10 (m, 3H, Cbz
and R-H), 6.82 (b, 1H, NH), 7.05-7.30 (m, 6H, Ph and NH),
7.40 (b, 1H, NH). The purity was 89-91% by HPLC using
either 220 or 254 nm detection. The impurity is likely the
dipeptide Cbz-Leu-Phe-NH-(CH2)3-N(CH3)2. HRMS (FAB)
for C29H41N4O5: m/z 525.3077 ([M þ H]þ).
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Acknowledgment. This publication was made possible by
Grant R21 NS053801 from the National Center for Research
Resources (NCRR), a component of the National Institutes
of Health (NIH). Its contents are solely the responsibility of
the authors and do not necessarily represent the official views
of NCRR or NIH.
Supporting Information Available: Synthesis of precursor
dipeptides and amines; characterization of previously reported
peptidyl ketoamides; statistical analysis of the results in Table 1
using a one-way ANOVA with a post hoc Tukey HSD (honestly
significant differences) test. This material is available free of
charge via the Internet at http://pubs.acs.org.

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