Studies of the synthesis of all stereoisomers of MG-132 proteasome inhibitors in the tumor targeting approach

Article abstract of DOI:10.1021/jm901619n

MG-132 is a tripeptide aldehyde (Z-L-leu-L-leu-L-leu-H, 2) proteasome inhibitor that exerts antitumor activity and enhances cytostatic/cytotoxic effects of chemo- and radiotherapy. Because of a troublesome synthesis of tripeptides with a non-natural configuration and modified side chains of amino acids, only two stereoisomers of MG-132 have been reported. Here, we propose a new approach to the synthesis of tripeptide aldehydes based on the Ugi reaction. Chiral, enantiomerically stable 2-isocyano-4-methylpentyl acetates were used as substrates for Ugi reaction resulting in a formation of tripeptide skeletons. Further functionalization of the obtained products led to a synthesis of tripeptide aldehydes. All stereoisomers of MG-132 were synthesized and studied as potential inhibitors of chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities of proteasome. These studies demonstrated the influence of absolute configuration of chiral aldehydes on the cytostatic/cytotoxic effects of the synthesized compounds and revealed that only (S,R,S)-(-)-2 stereoisomer is a more potent. proteasome inhibitor than MG-132.

Full text of DOI:10.1021/jm901619n

J. Med. Chem. 2010, 53, 1509–1518 1509
DOI: 10.1021/jm901619n
Studies of the Synthesis of All Stereoisomers of MG-132 Proteasome Inhibitors in the
Tumor Targeting Approach
Michaz Mroczkiewicz,† Katarzyna Winkler,†,‡ Dominika Nowis,§ Grzegorz Placha, Jakub Golab,§ and Ryszard Ostaszewski*,‡
†Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland, ‡Institute of Organic Chemistry,
Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland, §Department of Immunology, Center of Biostructure Research,
Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Poland, and Department of Internal Diseases, Hypertension, and Vascular Disease,
Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Poland
Received July 27, 2009
MG-132 is a tripeptide aldehyde (Z-L-leu-L-leu-L-leu-H, 2) proteasome inhibitor that exerts antitumor
activity and enhances cytostatic/cytotoxic effects of chemo- and radiotherapy. Because of a troublesome
synthesis of tripeptides with a non-natural configuration and modified side chains of amino acids, only
two stereoisomers of MG-132 have been reported. Here, we propose a new approach to the synthesis of
tripeptide aldehydes based on the Ugi reaction. Chiral, enantiomerically stable 2-isocyano-4-methyl-
pentyl acetates were used as substrates for Ugi reaction resulting in a formation of tripeptide skeletons.
Further functionalization of the obtained products led to a synthesis of tripeptide aldehydes. All
stereoisomers of MG-132 were synthesized and studied as potential inhibitors of chymotrypsin-like,
trypsin-like, and peptidylglutamyl peptide hydrolyzing activities of proteasome. These studies demon-
strated the influence of absolute configuration of chiral aldehydes on the cytostatic/cytotoxic effects of
the synthesized compounds and revealed that only (S,R,S)-(-)-2 stereoisomer is a more potent
proteasome inhibitor than MG-132.
1. Introduction
lymphoma was bortezomib (1, Figure 1). It is a dipeptidyl
boronic acid, which decreases proliferation, induces apoptosis,
enhances the activity of chemotherapy and radiation, and
reverses chemoresistance in a variety of hematologic and solid
malignancy models both in vitro and in vivo.18
Recent progress in biomedical research is focused, among
others, on small peptides that reveal wide spectrum of biolo-
gical activities.1-3 Peptides with a C-terminal aldehyde group
have been mostly reported as proteolytic enzyme inhibitors.
Thus, tripeptide aldehydes (TPAsa) are known as inhibitors
of IκB degradation,4 human rhinovirus protease,5 human
calpain,6 and proteasome.7,8
A ubiquitin-proteasome system (UPS) is responsible for
the nonlysosomal ATP-dependent turnover and degradation
of the majority of eukaryotic intracellular proteins. It partici-
pates in the degradation of normal and abnormal proteins
involved in cell cycle control, apoptosis, and tumor growth.9,10
Dysregulation of the system results in the development of
multiple diseases such as malignancy,11 neurodegenerative,12
cardiovascular,13 inflammatory, and autoimmune diseases.10
Proteasome inhibitors represent a novel class of anticancer
drugs that induce apoptosis and cell cycle arrest in tumor cells.
The majority of proteasome inhibitors have a structure of small
cyclic14 and linear15-17 peptides. The first inhibitor of protea-
some that has been approved for clinical use in the treatment of
drug-resistant multiple myeloma and recently also for mantle
A special attention in proteasome inhibition studies is paid to
tripeptide aldehydes with the best characterized representative
being Z-L-leu-L-leu-L-leu-H known as MG-132 (2). MG-132
exerts both direct antiproliferative and cytotoxic effects toward
tumor cells and increases apoptosis induced by other agents. It
also potentiates antitumor effects of chemo- and radiotherapy.
Tripeptide 2 has been reported a: (a) an effective inducer of
apoptosis in human osteosarcoma which is the most common
malignant bone tumor, mainly occurring in children and ado-
lescents,19 (b) a potentiator of the cancer cells death induced by
the histone deacetylase inhibitors,20 and (c) an agent sensitizing
prostate cancer cells to ionizing radiation.21 MG-132 also exerts
activities not associated with anticancer effects such as inter-
ference with proteasome-dependent proteolysis in skeletal mus-
cle and increase of whole-body protein turnover.22 Recent
studies have revealed a new application of MG-132, which
effectively prevents the development of morphine tolerance in
rats.23 Tripeptide aldehydes similar to MG-132 also reveal wide
spectrum of activities. They quite often possess the activity of
proteasome inhibitors, and sometimes they are more potent
than aldehyde 2. It has been confirmed that the presence of
N-terminal benzyloxycarbonyl group and C-terminal leucine
residue in TPAs is crucial for the achievement of high protea-
some inhibition activity. The most active inhibitor of 20S (cata-
lytic particle) proteasome having structure of TPA is Z-(2-naph-
thyl)alanyl-(1-naphthyl)alanylleucinal (3) with Ki = 0.015 nM
against 4.0 nM for MG-132 and 0.62 nM for bortezomib.
*To whom correspondence should be addressed. Telephone: þ48 22
343 2120. Fax: þ48 22 632 6681. E-mail: rysza@icho.edu.pl.
aAbbreviations: TPA, tripeptide aldehyde; UPS, ubiquitin-protea-
some system; MG-132, Z-L-leu-L-leu-L-leu-H; DMB-NH2, 2,4-dimethox-
ybenzylamine; HPLC, high performance liquid chromatography, DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran;
WGL, wheat germ lipase; ChTL, chymotrypsin-like; TL, trypsin-like;
PGPH peptidylglutamyl peptide hydrolysis; DCM, methylene chloride;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDTA,
ethylenediaminetetraacetic acid.
r
2010 American Chemical Society
Published on Web 01/29/2010
pubs.acs.org/jmc

1510 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Mroczkiewicz et al.
2. Results and Discussion
Here, we present extension and application of the pre-
viously described general methodology for the synthesis of
tripeptide aldehydes35 together with the evaluation of their
activityof proteasomeinhibition. Wehaveobtained a series of
all eight diastereoisomers of tripeptide Z-leu-leu-leu-H (MG-
132) by the application of chiral, enantiomerically pure
carboxylic acids and isocyanides as substrates for the Ugi
reaction. A general approach is based on (a) formation of a
tripeptide scaffold by Ugi reaction, (b) separation of epimers,
(c) deprotection of the amide function, and (d) formation of
the C-terminal aldehyde group (Scheme 1).
2.1. Synthesis of Isocyanides. At first, a new chiral non-
racemizable isocyanide derived from leucine should be de-
signed and synthesized. Application of this isocyanide for the
Ugi reaction should avoid the formation of mixtures com-
posed of more than two diastereoisomeric products. Because
of the possibility of racemization, we rejected the use of
isocyanides derived from amino esters as (S)- and (R)-2-
isocyano-4-methylpentanoates,36 the analogue of the best
known group representative (S)-2-isocyano-3-phenylpro-
pionate.32,33 We decided to use, deriving from β-amino
alcohol, both enantiomers of 2-isocyano-4-methylpentyl
acetate ((S)-(þ)-8 and (R)-(-)-8). A replacement of electron
withdrawing carboxylic ester group with a distant electron
donating acetoxy moiety should avoid commonly observed
racemization. The isocyanides were obtained by four-step
procedure from (S)-(-)- and (R)-(þ)-leucine (4) (Scheme 2).
Reduction of the carboxylic group with borane generated in
situ from sodium borohydride with iodine gave correspond-
ing leucinols (5). (S)-(þ)- and (R)-(-)-leucinols (5) have been
N-formylated with ethyl formate in quantitative yields to
(S)-(-)-6 and (R)-(þ)-6 and then directly O-acetylated with
acetic anhydride catalyzed by pyridine. (S)-(-)- and (R)-(þ)-
N-formylo-O-acetyloleucinol (7) have been obtained as
white crystals in good 73% and 74% yields, respectively.
We have found in aging tests that these precursors of
isocyanides are almost completely enantiomerically stable
under storage conditions (4 °C). Optical rotations for the
fresh obtained compounds were -47.3 and þ46.8 (c 1.0,
CHCl3), respectively, and after 4 months of storage of these
samples the values have almost not decreased, -46.2 and
þ44.9, respectively. This indicates that progress of racemiza-
tion is about 0.5-1.0% per month. Next, the dehydratation
of formamide to isocyanide group was investigated. In most
cases chiral isocyanides are synthesized from amino esters by
the application of diphosgene or triphosgene as dehydrating
agent in the presence of N-methylmorpholine as a base. Such
conditions together with the lower temperature (-78 °C)
enables isocyanide synthesis without racemization.32,36 The
standard procedure of formamide deprotection with phos-
phoryl oxychloride and triethylamine results in total race-
mization of isocyanides derived from amino esters.32 In our
methodology we decided to try both deprotection proce-
dures. At first, reaction of formyl derivatives (S)-(-)-7 and
(R)-(þ)-7 with phosphorus oxychloride and triethylamine at
-60 to -30 °C resulted in isocyanides (S)-(þ)-8 and (R)-(-)-
8 formation in good to excellent yields, 100% and 75%, res-
pectively. The [R]D for both enantiomers amounted to þ2.66
and -2.75, respectively. After 4 months of storage at -20 °C
the [R]D amounted to þ2.60 and -2.70, respectively. Thus,
it confirms that the isocyanides are almost nonracemiza-
ble under storage conditions (∼0.5% per month). Next,
Figure 1. Examples of proteasome inhibitors.
This indicates that aldehyde 3 is about 270 and 40 times more
potent than MG-132 and bortezomib, respectively.15,24 It was
found that more hydrophobic and hindered groups as side
chains at P2 and P3 positions (counting from the C end) of
tripeptides result in enhanced of proteasome inhibitory acti-
vity.15 A great number of studied TPAs as well as 3 contain side
chains of noncoded amino acids,25 but they very rarely have
the opposite to natural absolute configuration at any of
R-carbon.4,26,27 It is caused by the troublesome synthesis
and very narrow access to commercially available precursors
of peptides with non-natural configuration.
Generally, N-benzyloxycarbonyl tripeptide aldehydes, in-
cluding MG-132, are synthesized by the classical coupling
of N-protected amino acid with a dipeptide containing a
C-terminal Weinreb amide group. In most cases, an aldehyde
group is introduced to the structure of tripeptides by the
reduction of the Weinreb amide group with lithium aluminum
hydride.4,24,25,28 However, this methodology is strongly lim-
ited by the availability of amino acids with non-natural side
chains or/and configuration at R-carbon as precursors for the
peptide synthesis. This results in very narrow access to other
stereoisomers of small peptides, as MG-132 and restricted
biological activity data of these compounds.24
Recently, multicomponent reactions as Ugi and Passerini
reactions create a new tool for the small peptides synthesis.
However, these reactions have only been used for the synthesis
of simple peptides. Application of the Passerini reaction fol-
lowed by the enzymatic resolution of enantiomers and amino
acid coupling led to tripeptides in a six-step procedure,29
whereas use of Ugi reaction resulted in tripeptides directly.
However, the application of Ugi reaction in the previous reports
was limited to the synthesis of simple tripeptides containing
glycine residues.30,31 According to the mechanism of Ugi reac-
tion when chiral, nonracemic substrates are used, products form
as a mixture of two epimers that differ in configuration at the
R-carbon of P2. However, synthesis of enantiomerically pure
R-isocyanoacetates is problematic because of their tendency
to racemization during the step of formamide group dehydra-
tionp; this is why special conditions have to be used.32 More-
over, although no significant racemization of chiral isocyanides
is observed during Passerini reaction,33 the same substrates are
enantiomerically unstable under conditions of Ugi condensa-
tion.34 In these cases Ugi reaction gives a mixture of more than
two diastereoisomers. Therefore, studies on the preparation of
new chiral nonracemizable isocyanides are also required to
develop new strategies for the synthesis of tripeptide aldehydes.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1511
Scheme 1. General Methodology for the Synthesis of Tripeptide Aldehydesa
a R1, R3 = H, alkyl; X = N-amide bond protection; Y = aldehyde group equivalent.
Scheme 2. Synthesis of Isocyanide (S)-(-)-8a
Scheme 3. Synthesis of Tripeptide Aldehydes 2a
a Reagents and conditions: (a) NaBH4, I2, THF, reflux, 16 h, 73%; (b)
HCOOEt, reflux, 4 h, 100%; (c) Ac2O, pyridine, DMAP, DCM, room
temp, 2 h, 73%; (d) POCl3, Et3N, DCM, -70 to -40 °C, 2.5 h; 100%; (e)
ClCOOCCl3, NMM, DCM, -40 °C, 1 h, 84%. Enantiomer (R)-(þ)-8
was obtained by the same methodology.
milder conditions like diphosgene as dehydrating agent and
N-methylmorpholine have been applied. Herein, the (S)-(þ)-
2-isocyano-4-methylpentyl acetate has been obtained in 84%
and the [R]D amounted to þ2.87. The difference between the
results of the methods is negligible, which indicates that both
dehydration conditions do not induce the racemization
effect. The next experiments also indicate that the isocya-
nides were obtained as enantiomerically pure compounds.
2.2. Synthesis of Tripeptide Aldehydes. For Ugi reactions
we have applied the isocyanides obtained by the phosphorus
oxychloride dehydration process, according to the previous
reported methodology, as the other components N-benzy-
loxycarbonylleucines (S)-(-)-9 and (R)-(þ)-9, 2,4-dimethox-
ybenzylamine (DMB-NH2) (10) and isovaleraldehyde (11)
have been used.35 Cbz-leucines (9) enable the insertion of
leucine residue at P3 position without the risk of racemiza-
tion under the Ugi reaction conditions. Application of
isovaleraldehyde as a carbonyl compound gives an oppor-
tunity to insert the isobutyl group as leucine residue at the P2
position of tripeptide. According to the mechanism of the
Ugi reaction, products are formed as a mixture of two
epimers differing in the absolute configuration at the R-
carbon of the P2 position. Application of 2,4-dimethoxy-
benzylamine enables further easy cleavage of amide bond
protection formed during the Ugi reaction.35 At first,
(S)-(þ)-2-isocyano-4-methylpentyl acetate ((S)-(þ)-8) was
used together with N-Cbz-leucines (S)-(-)-9 and (R)-(þ)-9
in two reactions. As a result, two pairs of epimers (S,S,
S)-(þ)-12/(S,R,S)-(-)-12 and (R,S,S)-12/(R,R,S)-12 were
a (a) MeOH, room temp, 48 h; (b) TFA, DCM, 50 °C, 1 h; c) NaOHaq.
MeOH, room temp, 30 min; (d) Dess-Martin reagent, DCM, room
temp, 1.5 h. Enantiomers of presented aldehydes were obtained by the
same methodology.
,
obtained (Scheme 3). HPLC analyses confirmed no racemiza-
tion appearance during the multicomponent condensation
and revealed only two signals of main products in each of
reaction mixtures. Products have been obtained as almost
equimolar mixtures, both in 51:49 ratio and in good 82% and
76% yields, respectively. Moreover, each signal was derived
from a different diastereoisomer, which indicates that none of
them was the enantiomer of the other one. The corresponding
Ugi reactions of (R)-(-)-8 with (S)-(-)-9 and (R)-(þ)-9, 10,
and 11 resulted in the formation of the rest of the epimer pairs

1512 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Mroczkiewicz et al.
Table 1. Optical Activities of Tripeptide Aldehydes 2
in 80% and 76% yields, respectively. The results demonstrate
that the 2-isocyano-4-methylpentyl acetates (S)-(þ)-8 and
(R)-(-)-8 are nonracemizable both under Ugi reaction and
under storage conditions. To our knowledge, this is the first
example of isocyanides derived from amino acids that are not
susceptible to racemization during the Ugi reaction.
tripeptide aldehyde 2
enantiomer
[R]2D5
(S,S,S)-(þ)
(S,R,S)-(-)
(S,I,R)-(-)
(S,II,R)-(þ)
(R,I,S)-(þ)
(R,II,S)-(-)
(R,R,R)-(-)
(R,S,R)-(þ)
A
B
þ37.1
-57.3
-47.1
þ29.0
þ45.9
-27.1
-36.5
þ55.5
C
D
C0
D0
A0
B0
Next, we focused on a methodology for the separation of
the epimers mixtures. First, because of the large experience of
our group in the enzymatic resolution of stereoisomeric
mixtures of compounds with the acetoxyl group,29,37,38 we
have examined an enzymatic approach with the application
of lipases to a diastereoselective hydrolysis of the mixture of
epimers (S,S,S)-(þ)-12 and (S,R,S)-(-)-12. We have exam-
ined 18 commercially available lipases and 12 enzyme pre-
parations as acetone powders from animal tissues (livers and
kidneys). The products were observed in the reactions cata-
lyzed by acetone powders from pig, rabbit, and turkey livers
and lipases from wheat germ, C. cylindracea, P. roqueforti,
and recombinant R. miehei. However, in standard screening
procedure (phosphate buffer (pH 7.0)/acetone, 9:1) the best
results have been obtained for the application of wheat germ
lipase (WGL); therefore, for the next experiments we have
used WGL and we performed a screening of nonaqueous
cosolvents as additives to the reaction medium in different
ratios. Thus, such solvents as acetone, i-Pr2O, Et2O, tert-
butyl methyl ether, cyclohexane, DMF, DMSO, THF, to-
luene, methanol, and ethanol were examined. Herein, pro-
ducts were observed in the mixtures with acetone, DMSO,
and methanol additives. We repeated these reactions in
preparative scale and products ratios were examined by
HPLC. The best diastereoisomeric excess was obtained with
the acetone and then with DMSO and methanol: 90%, 76%,
and 60%, respectively. However, the yields were inversely
proportional to the values of 17%, 52%, and 83%, respec-
tively. So the results were not satisfactory because of low
yield for the best diastereoisomerically enriched product.
Because the application of enzymes did not give the
expected results, we decided to apply the physical methods
for the separation of epimers. The TLC’s analysis revealed
that epimers (S,S,S)-(þ)-12 and (S,R,S)-(-)-12 are charac-
terized by the significant difference in polarities.
Thus, we tried to separate the epimers by column chro-
matography on silica gel. Triple repetition of the process
allowed us to obtain pure diastereoisomers of (S,S,S)-(þ)-12
and (S,R,S)-(-)-12 in 35% and 33% yields, respectively. The
purities of the products were higher than 95% (HPLC).
However, a similar separation of epimers (R,S,S)-12 and
(R,R,S)-12 was not possible because of small difference
between polarities of these compounds, but the problem of
separation was resolved in the next step of the studies.
According to our previously reported procedures,35 sepa-
rated epimers of (S,S,S)-(þ)-12 and (S,R,S)-(-)-12 were
subjected to deprotection of the amide bond by cleavage of
the DMB group with TFA, resulting in (S,S,S)-(þ)-13 and
(S,R,S)-(-)-13 in excellent 100% and 91% yields, respec-
tively. Next, basic hydrolysis of the acetoxy group gave (S,S,
S)-(þ)-15 and (S,R,S)-(-)-15 in 100% and 93% yields, res-
pectively. Finally, selective oxidation of alcohol groups with
Dess-Martin reagent gave aldehydes (S,S,S)-(þ)-2 and
(S,R,S)-(-)-2 in 79% and 63% yields, respectively (Scheme 3,
route A). Because no racemization was observed during the
whole process, the products were obtained as single diaster-
eoisomers of tripeptide aldehydes. We assigned the absolute
configurations of tripeptide aldehydes and their precursors
by a comparison of commercially available MG-132 proper-
ties with obtained final products. The enantiomers (R,R,R)-
(-)-2 and (R,S,R)-(þ)-2 were obtained in an analogous
manner in route A (Scheme 3) with similar results.
Because the separation of epimers (R,S,S)-12 and (R,R,S)-12
was not possible, we subjected them to a deprotection process
directly, but we have realized that it did not change the
difference between the polarities of compounds. The separation
was also not possible after the hydrolysis stage. Thus, we tried to
change the order of the reactions and to perform hydrolysis
of the ester group (Scheme 3, route B). Herein, we obtained
N-DMB protected alcohols (R,I,S)-(þ)-14 and (R,II,S)-(-)-14,
which were characterized by considerable difference in polarity,
which allowed us to separate the epimers by triple column
chromatography. The products have been obtained in good
purities (>95%, HPLC) and 36% and 25% yields, respectively,
but at this time we are not able to determine the absolute
configurations at R-carbon of P2 residues; therefore, we have
described the less polar compounds as (R,I,S) and more polar
compounds as (R,II,S). Next, we subjected the alcohols to a
deprotection stage with TFA. We have obtained deprotected
tripeptide alcohols (R,I,S)-(þ)-15 and (R,II,S)-(-)-15 in 49%
and 61% yields, respectively. The lower yields are the result of
byproducts formation which were not observed during the
deprotection of N-DNB protected esters. And finally the alco-
hol was oxidized with Dess-Martin reagent to aldehydes (R, I,
S)-(þ)-2 and (R,II,S)-(-)- 2 in 45% and 56% yields, respec-
tively. For the synthesis of enantiomers (S,I, R )-(-)- 2 and
(S,II,R)-(þ)-2 the same methodology (route B) was applied and
similar results were obtained. Thus, the presented methodology
allowed synthesis of all eight diastereoisomers of tripeptide
aldehyde Z-leu-leu-leu-H (Table 1) which were then analyzed
as inhibitors of chymotrypsin-like (ChTL) proteasome activity.
2.3. Inhibition of Proteasome Activity. In a preliminary
experiment MG-132 ((S,S,S)-(þ)-2) and its stereoisomers
were investigated for inhibition of proteasome activity in
lysates of J558L multiple myeloma cells or EMT6 breast
cancer cells. Only MG-132 and (S,R,S)-(-)- 2 were capable
of inhibiting proteasome activity at 100 nM in lysates ob-
tained from both J558L and EMT6 cells. At 1000 nM a partial
proteasome inhibition was also observed with (S, I,R)-(-)-2,
(R,R,R)-(-)- 2, and (R,II, S)-(-)-2 (Tables 2 and 3).
Although proteasomes are responsible for most intracel-
lular proteolysis, other cytosolic (tripeptidyl peptidases) and
lysosomal (cathepsins) enzymes are present in tumor cell
lysates and might influence the processing of tripeptide alde-
hydes used as fluorogenic substrates.39 Therefore, MG-132
((S,S,S)-(þ)-2) and its stereoisomers were investigated for
inhibition of ChTL, trypsin-like (TL) and peptidylglutamyl
peptide hydrolyzing (PGPH) activities of purified 20S pro-
teasomes isolated from human erythrocytes. These experi-
ments revealed that only two stereoisomers ((S,R,S)-(-)-2
and (S,I,R)-(-)-2) as well as MG-132 itself inhibit ChTL

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1513
Table 2. Influence of MG-132 and Its Stereoisomers on the Cleavage of Suc-leu-leu-Val-Tyr-AMC in J558L Cells
proteasome activity at 100 nMa
30 min
proteasome activity at 1000 nMa
compd
DMSO
15 min
60 min
15 min
30 min
60 min
100 ( 5
115 ( 3
104 ( 2
100 ( 5
92 ( 3
87 ( 2
84 ( 3
71 ( 2
25 ( 2
32 ( 3
100 ( 3
118 ( 2
105 ( 3
110 ( 2
93 ( 1
89 ( 2
91 ( 3
77 ( 2
36 ( 2
44 ( 4
100 ( 2
123 ( 2
107 ( 5
112 ( 1
94 ( 2
89 ( 1
95 ( 5
82 ( 2
50 ( 2
60 ( 5
100 ( 5
114 ( 3
107 ( 3
102 ( 2
55 ( 3
58 ( 2
71 ( 4
30 ( 1
11 ( 0
15 ( 0
100 ( 3
117 ( 2
106 ( 3
96 ( 8
64 ( 3
66 ( 2
72 ( 2
40 ( 2
21 ( 1
25 ( 1
100 ( 2
121 ( 3
106 ( 3
99 ( 3
77 ( 4
71 ( 3
74 ( 3
53 ( 4
38 ( 2
44 ( 3
(R,I,S)-(þ)-2
(S,II,R)-(þ)-2
(R,S,R)-(þ)-2
(S,I,R)-(-)-2
(R,R,R)-(-)-2
(S,S,S)-(þ)-2
(R,II,S)-(-)-2
(S,R,S)-(-)-2
MG-132
a Data refer to % of control activity ( SD measured after 15, 30, or 60 min.
Table 3. Influence of MG-132 and Its Stereoisomers on the Cleavage of Suc-leu-leu-Val-Tyr-AMC in EMT6 cells
proteasome activity at 100 nMa
30 min
proteasome activity at 1000 nMa
30 min
compd
DMSO
15 min
60 min
15 min
60 min
100 ( 5
97 ( 1
110 ( 2
95 ( 1
90 ( 4
71 ( 3
87 ( 4
17 ( 4
20 ( 3
100 ( 4
95 ( 1
103 ( 1
91 ( 2
90 ( 3
77 ( 4
85 ( 5
22 ( 4
25 ( 3
100 ( 4
95 ( 1
99 ( 2
91 ( 2
91 ( 2
86 ( 5
84 ( 7
31 ( 4
35 ( 2
100 ( 5
104 ( 6
118 ( 10
44 ( 4
62 ( 3
29 ( 3
61 ( 3
5 ( 0
100 ( 4
100 ( 5
111 ( 8
46 ( 4
70 ( 4
37 ( 2
61 ( 3
7 ( 0
100 ( 4
99 ( 3
106 ( 7
53 ( 4
78 ( 6
50 ( 2
67 ( 3
13 ( 1
19 ( 1
(R,I,S)-(þ)-2
(S,II,R)-(þ)-2
(S,I,R)-(-)-2
(R,R,R)-(-)-2
(S,S,S)-(þ)-2
(R,II,S)-(-)-2
(S,R,S)-(-)-2
MG-132
12 ( 1
14 ( 1
a Data refer to % of control activity ( SD measured after 15, 30, or 60 min.
Table 4. Influence of MG-132 and Its Stereoisomers on the ChTL, TL,
and PGPH Activities of Purified Human Erythrocyte 20S Proteasomes
obtained as enantiomerically stable under storage and Ugi
reaction conditions. This is the first attempt to synthesize
the complete group of eight possible diastereoisomers of
such compounds. This is also the first attempt at full analytical
and biological characterization of MG-132 and its stereoi-
somers; however, assignment of the absolute configuration
at all R-carbons of all compounds based on magnetic reso-
nance spectra was not possible because of the affinity of three
equal isobutyl side chains and their vicinity on the tripeptide
structure. All stereoisomers of 2 were studied as inhibitors of
ChTL, TL, and PGPH activities of purified human 20S
proteasomes, and intriguingly stereoisomers (S,R,S)-(-)-2
and (S,I,R)-(-)-2, having non-natural absolute configuration,
were capable of inhibiting the activity of purified 20S protea-
somes with (S,R,S)-(-)-2being 5-fold more effective than MG-
132 in terms of inhibiting ChTL activity. Cytostatic/cytotoxic
effects exerted by the stereoisomers against tumor cells corre-
lated with the ability to inhibit ChTL activity of the protea-
some.
IC50 (μM)
compd
ChTL
TL
PGPH
(R,I,S)-(þ)-2
(S,II,R)-(þ)-2
(R,S,R)-(þ)-2
(S,I,R)-(-)-2
(R,R,R)-(-)-2
(S,S,S)-(þ)-2
(R,II,S)-(-)-2
(S,R,S)-(-)-2
MG-132
>100
>100
>100
0.97
>100
>100
>100
91.33
>100
>100
>100
34.4
>100
>100
>100
22.82
11.29
>100
17.25
2.95
9.81
5.46
1.79
0.22
0.89
104.43
5.70
proteasome activity with an IC50 of less than 1 μM (Table 4).
TL and PGPH activities were inhibited at markedly higher
concentrations, but again (S,R,S)-(-)-2 was more effective
than MG-132. Significant cytostatic/cytotoxic effects against
J588L and EMT6 tumor cells were observed only with the
compounds that blocked ChTL activity of 20S proteasomes
with IC50 below 1 μM (Figures 1 and 2 of Supporting Infor-
mation). However, (S,R,S)-(-)-2, despite being the most
effective inhibitor, having a 5-fold lower IC50 than MG-132
(0.22 μM versus 0.89 μM) did not exert stronger cytostatic/
cytotoxic effects against tumor cells. Interestingly, (R,II,
S)-(-)-2 did not exert any cytostatic/cytotoxic effects against
tumor cells despite being able to inhibit the ChTL activity of
purified 20S proteasomes with an IC50 of 1.79 μM.
4. Experimental Section
4.1. General. NMR spectra were measured with Varian 200
GEMINI, Varian 400 GEMINI, and Bruker AM 500 spectro-
meters, with TMS used as an internal standard. TLCs were
performed with silica gel 60 (230-400 mesh, Merck) and silica
gel 60 PF254 (Merck). CHN analysis was performed on a
Perkin-Elmer 240 elemental analyzer. MS spectra were recorded
on an API-365 (SCIEX) apparatus. IR spectra were recorded on
a Perkin-Elmer FT-IR Spectrum 2000 spectrometer. Optical
rotations were measured with a JASCO P-2000 polarimeter.
HPLC experiments were carried out on a KROMASIL 100 C-18
column, λ = 230 nm: method 1 (eluent methanol/water 85:15 (v/
v), flow of 1 mL/min); method 2 (eluent methanol/water 80:20
(v/v), flow of 1.2 mL/min); method 3 (eluent methanol/water
83:17 (v/v), flow of 1.0 mL/min). All key compounds were
proven by these methods to show >95% purity.
3. Conclusions
Application of Ugi reaction gives the opportunity for a quick
and easy synthesis of small peptides with non-natural config-
uration at R-carbon. The presented methodology allowed the
synthesis of all stereoisomers of tripeptide aldehyde Z-leu-
leu-leu-H (MG-132, 2) with the application of isocyanides (8)

1514 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Mroczkiewicz et al.
4.2. Synthesis of Isocyanides. 4.2.1. Synthesis of (S)-(þ)-2-
Isocyano-4-methylpentyl Acetate (S)-(þ)-8. 4.2.1.a. (S)-(þ)-2-
Amino-4-methylpentan-1-ol (S)-(þ)-Leucinol (S)-(þ)-5. To a
suspension of sodium borohydride (5.77 g, 152.4 mmol) in
THF (100 mL), L-leucine (10.0 g, 76.2 mmol) was added under
nitrogen atmosphere. The reaction mixture was cooled to 0 °C,
and a solution of iodine (19.3 g, 76.2 mmol) in THF (50 mL) was
added for 1 h. Then the reaction mixture was stirred until the gas
bubbles stopped evolving and was refluxed for 16 h and cooled
to room temperature, and methanol was added until the mixture
became clear. After 30 min, the volatiles were evaporated. The
residue was dissolved in aqueous solution of potassium hydro-
xide (100 mL, 20%), stirred for 3 h, and extracted with DCM
(4 ꢀ 75 mL). The combined organic layers were dried (MgSO4),
and the solvent was evaporated. The residual dense oil was
distilled under reduced pressure (57 °C, 1.0 mmHg). Yield: 73%,
6.49 g (55.3 mmol) of a colorless oil. Rf = 0.42 (CHCl3/MeOH/
NH4OH, 10:2:0.25, v/v/v). 1H NMR (200 MHz, CDCl3) δ 0.89
(t, J = 6.4 Hz, 6H), 1.17 (t, J = 7.0 Hz, 2H), 1.64-1.70 (m, 1H),
2.10 (bs, 3H), 2.82-2.96 (m, 1H), 3.21 (dd, J = 10.6 Hz, J = 7.8
Hz, 1H), 3.55 (dd, J = 10.6 Hz, J = 3.8 Hz, 1H). 13C NMR (50
MHz, CDCl3) δ 22.5, 23.7, 25.0, 44.0, 50.9, 67.4. [R]2D5 þ4.1 (c
4.0, EtOH) (lit.40 þ4.2 (c 0.9, EtOH)).
(CH2-O) cm-1. Anal. Calcd for C9H15NO2: C, 63.86; H, 8.94;
N, 8.28. Found: C, 63.91; H, 8.82; N, 8.24. [R]2D5 2.66 (c 1.0,
CHCl3).
4.2.1.d.2. Method 2. To a solution of (S)-(-)-7 (1.0 g, 5.34
mmol) in DCM (15 mL) cooled to -40 °C N-methylmorpholine
(1.76 mL, 16.0 mmol) was added. Diphosgene (0.322 mL, 2.67
mmol) in DCM (3 mL) was added dropwise for 5 min. The
reaction mixture was stirred for 2.5 h, when the temperature was
allowed to increase to -30 °C. Then the reaction mixture was
poured to an ice cooled water (50 mL, 1 M). Layers were sepa-
rated, and the aqueous layer was extracted with DCM (3 ꢀ
30 mL). Organic layers were combined, dried (MgSO4), and
concentrated. The residual brownish oil was purified by column
chromatography (silica gel, 70-230 mesh, 19 g, DCM). Yield:
84%, 0.76 g (4.50 mmol) of colorless oil. 1H NMR (200 MHz,
CDCl3) δ 0.90 (dd, J = 8.7 Hz, J = 6.6 Hz, 6H), 1.12-1.32 (m,
1H), 1.48-1.70 (m, 1H), 1.71-1.92 (s, 1H), 2.08 (s, 3H), 3.70-
3.88 (m, 1H), 3.94-4.18 (m, 2H). 13C NMR (50 MHz, CDCl3) δ
20.9, 21.5, 23.2, 24.9, 40.2, 52.3, 65.5, 170.7. IR (film) νmax: 2962
(CH3), 2142 (NtC), 1744 (CdO), 1236 (CH2-O) cm-1. Anal.
Calcd for C9H15NO2: C, 63.86; H, 8.94; N, 8.28. Found: C,
63.94; H, 8.80; N, 8.30. [R]2D5 þ2.87 (c 1.0, CHCl3).
4.2.2. Synthesis of (R)-(-)-2-Isocyano-4-methylpentyl Acetate
(R)-(-)-8. Product has been obtained by method 1 for the
synthesis of (S)-(þ)-8. Yield: 75%, 1.35 g (7.95 mmol) of yellow
oil. 1H NMR (200 MHz, CDCl3) δ 0.90 (dd, J = 8.8 Hz, J = 6.7
Hz, 6H), 1.12-1.34 (m, 1H), 1.48-1.70 (m, 1H), 1.70-1.92 (s,
1H), 2.08 (s, 3H), 3.70-3.92 (m, 1H), 3.92-4.20 (m, 2H). 13C
NMR (50 MHz, CDCl3) δ 20.8, 21.4, 23.3, 25.0, 40.3, 52.3, 65.7,
170.6. IR (film) νmax: 2960 (CH3), 2140 (NtC), 1745 (CdO),
1236 (CH2-O) cm-1. Anal. Calcd for C9H15NO2: C, 63.86; H,
8.94; N, 8.28. Found: C, 63.98; H, 8.75; N, 8.15. [R]2D5 -2.75 (c
1.0, CHCl3).
4.3. General Procedures for Tripeptides Synthesis. 4.3.1. Gen-
eral Procedure 1 for the Ugi Reaction. A solution of an amine (1
equiv) and an aldehyde (1 equiv) in methanol (2 mL/1 mmol)
was stirred for 15 min at room temperature. Then an acid (1
equiv) in methanol (2 mL/1 mmol) was added. After an addi-
tional 15 min an isocyanide (1 equiv) was added. The reaction
mixture was stirred at room temperature for 2 days. The solvent
was evaporated, and the products were purified by gradient
flash column chromatography (silica gel, 230-400 mesh, hex-
ane/EtOAc).
4.3.2. General Procedure 2 for the Deprotection of Amide
Bond. To a solution of Ugi reaction product (1.0 mmol) in
DCM (5 mL), trifluoroacetic acid (0.5 mL) was added. The
reaction mixture was stirred at 50 °C for 1 h and then cooled in
an ice-water bath, diluted with DCM (5 mL), and neutralized
with an aqueous saturated solution of sodium bicarbonate until
the violet color disappeared. Layers were separated, and the
aqueous layer was extracted with DCM (3 ꢀ 15 mL). The
combined organic layers were washed with water (20 mL) and
brine (20 mL), dried (MgSO4) and evaporated. Product(s) was-
(were) purified by gradient flash column chromatography (silica
gel, 230-400 mesh, hexane/EtOAc).
4.3.3. General Procedure 3 for the Hydrolysis of Acetic Ester
Group. To a solution of an ester (1 equiv) in MeOH (5 mL/1
mmol), an aqueous solution of sodium hydroxide (2.5 equiv, 4
M) was added. The reaction mixture was stirred at room
temperature for 30 min, and then the solvent was evaporated.
The residue was diluted with EtOAc (15 mL) and washed with
an aqueous solution of hydrochloric acid (15 mL, 1 M). Layers
were separated, and the aqueous layer was extracted with
EtOAc (2 ꢀ 15 mL). The combined organic layers were dried
(MgSO4), and solvent was evaporated. Product were purified by
gradient flash column chromatography (silica gel, 70-230
mesh, hexane/EtOAc).
4.2.1.b. (S)-(-)-N-[-1-(Hydroxymethyl)-3-methylbutyl]for-
mamide (S)-(-)-6. A solution of (S)-(þ)-5 (6.3 g, 53.8 mmol)
in ethyl formate (60 mL) was refluxed for 3 h. Then the reaction
mixture was cooled, and volatiles were evaporated to give 7.68 g
(53.7 mmol) of crude product as slightly yellow oil. Yield 100%.
Product was used without further purification. Rf = 0.69
(CHCl3/MeOH/NH4OH, 10:2:0.25, v/v/v). 1H NMR (200
MHz, CDCl3) δ 0.93 (d, J = 6.6 Hz, 6H), 1.20-1.48 (m, 2H),
1.50-1.66 (m, 1 H), 3.40-3.78 (m, 3H), 4.12 (bs, 1H), 6.11 (bs,
1H), 8.18 (s, 1H) . 13C NMR (50 MHz, CDCl3) δ 22.4, 23.4, 25.2,
40.4, 49.2, 65.8, 162.3. IR (film) νmax: 3280 (O-H), 2958 (CH3),
1660 (CdO) cm-1. [R]D25 -32.5 (c 4.0, CHCl3).
4.2.1.c. (S)-(-)-2-Formamido-4-methylpentyl Acetate (S)-(-)-7.
To a solution of (S)-(-)-6 (7.66 g, 52.8 mmol) in DCM (100 mL),
pyridine (8.5 mL, 105.6 mmol), acetic anhydride (15.0 mL, 158.4
mmol), and N,N-dimethylaminopyridine (20 mg) were added
under nitrogen atmosphere. The reaction mixture was stirred for
2 h at room temperature and washed with an aqueous solution
of copper sulfate (3 ꢀ 25 mL, 5%), a saturated aqueous solution
of sodium bicarbonate (2 ꢀ 25 mL), and water (2 ꢀ 25 mL). The
organic layer was dried (MgSO4) and the solvent was evapo-
rated to leave a crude product that was crystallized from diethyl
eter. Yield: 73%, 7.25 g (38.8 mmol) of white crystals. Mp 45-
48 °C. Rf = 0.50 (hexane/EtOAc, 5:5, v/v). 1H NMR (200 MHz,
CDCl3) δ 0.80-1.00 (m, 6H), 1.28-1.48 (m, 2H), 1.50-1.75 (m,
1H), 2.08 (s, 3H), 3.90-4.20 (m, 2H), 4.30-4.50 (m, 1H), 5.72
(bs, 1H), 8.19 (s, 1H). IR (film) νmax: 2960 (CH3), 1740 (NCdO),
1660 (OCdO), 1242 (CH2-O) cm-1. [R]D25 -47.3 (c 1.5, CHCl3).
4.2.1.d. (S)-(þ)-2-Isocyano-4-methylpentyl Acetate (S)-(þ)-
8. 4.2.1.d.1. Method 1. To a solution of (S)-(-)-7 (2.0 g, 10.7
mmol) in DCM (30 mL) cooled to -40 °C triethylamine (7.44
mL, 53.4 mmol) was added. The mixture was cooled to -70 °C,
and a solution of phosphorus oxychloride (1.08 mL, 11.7 mmol)
in DCM (5 mL) was added dropwise for 5 min. The reaction
mixture was stirred for 2.5 h, when the temperature was allowed
to increase to -30 °C. Then the reaction mixture was poured to
an ice cooled solution of sodium bicarbonate (100 mL, 1 M).
Layers were separated, and the aqueous layer was extracted with
DCM (3 ꢀ 50 mL). Organic layers were combined, dried
(MgSO4), and concentrated. The residual dark oil was purified
by column chromatography (silica gel, 70-230 mesh, 37 g,
DCM). Yield: 100%, 1.80 g (10.6 mmol) of yellow oil. 1H
NMR (200 MHz, CDCl3) δ 0.89 (dd, J = 8.8 Hz, J = 6.6 Hz,
6H), 1.14-1.35 (m, 1H), 1.50-1.70 (m, 1H), 1.71-1.90 (s, 1H),
2.06 (s, 3H), 3.70-3.90 (m, 1H), 3.94-4.20 (m, 2H). 13C NMR
(50 MHz, CDCl3) δ 20.9, 21.4, 23.2, 24.9, 40.3, 52.3, 65.6, 170.7.
IR (film) νmax: 2960 (CH3), 2142 (NtC), 1745 (CdO), 1235
4.3.4. General Procedure 4 for the Oxidation of Alcohol Group.
To a solution of alcohol (1 equiv) in DCM (2 mL), Dess-Martin
reagent (2 equiv) was added. The reaction mixture was stirred at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1515
room temperature for 1.5 h. An aqueous solution of sodium
hydroxide (2 mL, 5%) was added, and the mixture was stirred
for an additional 10 min. Then water (10 mL) and DCM (10 mL)
were added, layers were separated, and the aqueous layer was
extracted with DCM (3 ꢀ 10 mL). The combined organic layers
were dried (MgSO4), and solvent was evaporated. Product was
purified by column chromatography (silica gel, 70-230 mesh,
hexane/EtOAc).
4.4. Synthesis of Aldehydes (S,S,S)-(þ)-2 (Cbz-L-leu-L-leu-L-
leu-CHO) and (S,R,S)-(-)-2 (Cbz-L-leu-D-leu-L-leu-CHO). 4.4.1.
Synthesis of Compounds (S,S,S)-(þ)-12 (Cbz-L-leu-n(DMB)-L-
leu-L-leu-CH2OAc) and (S,R,S)-(-)-12 (Cbz-L-leu-n(DMB)-D-
leu-L-leu-CH2OAc). General Procedure 1. Products (S,S,S)-(þ)-
12 and (S,R,S)-(-)-12 were separated by triple flash column
chromatography.
13C NMR (100 MHz, CDCl3) δ 20.8, 22.0, 22.2, 22.6, 22.9, 23.0,
24.6, 24.8, 40.4, 40.6, 41.3, 46.4, 51.9, 53.6, 66.2, 67.1, 128.0,
128.3, 128.5, 136.0, 156.2, 171.0, 171.2, 172.2. HRMS calcd for
C28H45N3O6Na [M þ Na]þ: 542.3201. Found: 542.3175. [R]2D5
-63.1 (c 1.0, CHCl3). Retention time (method 3) tR = 5.99 min.
4.4.4. Synthesis of Compound (S,S,S)-(þ)-15 (Cbz-L-leu-L-leu-
L-leu-CH2OH). General Procedure 3. Yield: 95%, 204 mg (0.43
mmol) of white crystals. Mp 111-113 °C. Rf = 0.20 (CHCl3/
MeOH, 95:5, v/v). 1H NMR (400 MHz, CDCl3) δ 0.86-0.96 (m,
18H), 1.18-1.30 (m, 1H), 1.32-1.43 (m, 1H), 1.44-1.57 (m,
2H), 1.57-1.68 (m, 4H), 1.68-1.80 (m, 1H), 3.26 (br s, 1H), 3.38
(dd, J = 6.4 Hz, J = 11.5 Hz, 1H), 3.61 (dd, J = 3.4 Hz, J =
11.4 Hz, 1H), 3.96-4.09 (m, 1H), 4.10-4.18 (m, 1H), 4.42-4.52
(m, 1H), 5.02 (d, J = 12.3 Hz, 1H), 5.11 (d, J = 12.2 Hz, 1H),
5.68 (d, J = 7.0 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 7.25 (br s, 1H),
7.29-7.36 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 21.9, 22.1,
22.8, 22.9, 23.1, 24.7, 24.8, 24.8, 39.9, 40.1, 41.0, 49.9, 52.0, 54.0,
65.2, 67.2, 128.0, 128.2, 128.5, 135.9, 156.6, 172.4, 173.1. HRMS
calcd for C26H43N3O5Na [M þ Na]þ: 500.3095. Found:
500.3093. [R]2D5 10.3 (c 1.0, CHCl3). Retention time (method 2)
Compound (S,S,S)-(þ)-12. Yield: 35%, 459 mg (0.69 mmol)
of colorless oil. Rf = 0.38 (hexane/AcOEt, 7:3, v/v). 1H NMR
(400 MHz, CDCl3) δ 0.69 (d, J = 6.3 Hz, 3H), 0.80-0.96 (m,
15H), 1.20-1.40 (m, 3H), 1.45-1.68 (m, 6H), 2.04 (s, 3H), 3.78
(s, 3H), 3.79 (s, 3H), 3.88 (dd, J = 6.0 Hz, J = 11.1 Hz, 1H), 3.96
(dd, J = 4.4 Hz, J = 11.1 Hz, 1H), 4.16 (octet, J = 4.5 Hz, 1H),
4.34 (d, J = 17.2 Hz, 1H), 4.62 (t, J = 7.7 Hz, 1H), 4.71 (d, J =
17.0 Hz, 1H), 5.08 (q, J = 11.9 Hz, 2H), 5.37 (d, J = 7.6 Hz, 1H),
6.43 (d, J = 3.9 Hz, 1H), 6.44 (d, J = 4.1 Hz, 1H), 6.76 (d, J =
t
R = 4.73 min.
4.4.5. Synthesis of Compound (S,R,S)-(-)-15 (Cbz-L-leu-D-
leu-L-leu-CH2OH). General Procedure 3. Yield: 84%, 148 mg
(0.31 mmol) of white crystals. Mp 161-163 °C. Rf = 0.19
(CHCl3/MeOH, 95:5, v/v). 1H NMR (400 MHz, CDCl3) δ
0.85-0.96 (m, 18H), 1.27-1.36 (m, 1H), 1.36-1.46 (m, 1H),
1.46-1.56 (m, 2H), 1.56-1.67 (m, 4H), 1.67-1.78 (m, 1H), 2.76
(br s, 1H), 3.52 (dd, J = 5.7 Hz, J = 11.3 Hz, 1H), 3.65 (dd, J =
3.5 Hz, J = 11.3 Hz, 1H), 3.95-4.07 (m, 1H), 4.12-4.22 (m,
1H), 4.35-4.44 (m, 1H), 5.10 (s, 2H), 5.47 (d, J = 5.9 Hz, 1H),
6.72 (d, J = 6.3 Hz, 1H), 6.78 (d, J = 6.5 Hz, 1H), 7.30-7.38 (m,
5H). 13C NMR (100 MHz, CDCl3) δ 22.0, 22.1, 22.9, 23.0, 24.7,
24.8, 24.9, 39.8, 40.4, 41.1, 50.2, 52.4, 54.0, 65.6, 67.3, 128.1,
128.3, 128.6, 135.9, 156.5, 172.1, 172.7. HRMS calcd for
C26H43N3O5Na [M þ Na]þ: 500.3095. Found: 500.3087. [R]2D5
-63.6 (c 1.0, CHCl3). Retention time (method 2) tR = 4.47 min.
4.4.6. Synthesis of (S,S,S)-(þ)-2 (Cbz-L-leu-L-leu-L-leu-
CHO). General Procedure 4. Yield: 79%, 55 mg (0.12 mmol)
of colorless oil. Rf = 0.22 (CHCl3/MeOH, 95:5, v/v). 1H NMR
(400 MHz, CDCl3) δ 0.88-1.00 (m, 18H, CH(CH3)2), 1.38-
1.48 (m, 1H, CH(CH3)2), 1.49-1.58 (m, 1H, CH(CH3)2), 1.60-
1.74 (m, 6H, CH2CH(CH3)2), 1.74-1.86 (m, 1H, CH(CH3)2),
4.04-4.15 (m, 1H, CHCHO), 4.40-4.49 (m, 1H, NHCHCO),
4.50-4.58 (m, 1H, NHCHCO), 5.00-5.14 (m, 2H, OCH2Ph),
5.29 (d, J = 6.4 Hz, 1H, NHC(O)O), 6.59 (d, J = 8.3 Hz, 1H,
NHCHCHO), 7.06 (d, J = 7.1 Hz, 1H, NH), 7.29-7.38 (m, 5H,
ArH), 9.49 (s, 1H, CHO). 13C NMR (100 MHz, CDCl3) δ 21.6,
21.7, 22.1, 22.7, 23.0, 23.1, 24.7, 24.7, 24.9, 37.4, 40.1, 40.6, 51.6,
54.1, 57.2, 67.3, 128.0, 128.3, 128.6, 135.8, 156.5, 172.2, 172.6,
199.8. HRMS calcd for C26H41N3O5Na [M þ Na]þ: 498.2938.
Found: 498.2943. [R]2D5 37.1 (c 0.5, CHCl3).
8.8 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 7.30-7.40 (m, 6H). 13
C
NMR (100 MHz, CDCl3) δ 20.7, 20.8, 22.0, 22.3, 22.6, 23.1,
23.5, 24.4, 24.7, 25.3, 36.9, 40.5, 42.3, 46.1, 50.2, 55.3, 55.4, 66.3,
66.9, 98.6, 103.8, 116.9, 128.0, 128.2, 128.5, 129.1, 136.2, 156.1,
158.0, 160.8, 170.2, 171.0, 175.3. HRMS calcd for C37H55N3O8-
Na [M þ Na]þ: 692.3885. Found: 692.3862. [R] þ6.1 (c 1.0,
25
D
CHCl3). Retention time (method 1) tR = 17.42 min.
Compound (S,R,S)-(-)- 12. Yield: 33%, 438 mg (0.65 mmol)
of colorless oil. Rf = 0.30 (hexane/AcOEt, 7:3, v/v). 1H NMR
(400 MHz, CDCl3 ) δ 0.80-0.96 (m, 18H), 1.12-1.40 (m, 3H),
1.44-1.63 (m, 4H), 1.65 (m, 2H), 1.99 (s, 3H), 3.77 (d, J = 4.3
Hz, 1H), 3.80 (s. 3H), 3.81 (s, 3H), 4.06-4.14 (m, 1H), 4.14-4.24
(m, 1H), 4.34 (d, J = 16.0 Hz, 1H), 4.62 (d, J = 16.2 Hz, 1H),
4.88 (t, J = 8.5 Hz, 1H), 5.04-5.16 (m, 2H), 5.53 (d, J = 8.6 Hz,
1H), 6.03 (d, J = 8.8 Hz, 1H), 6.43-6.49 (m, 2H), 7.11 (d, J =
8.8 Hz, 1H), 7.30-7.38 (m, 6H). 13C NMR (100 MHz, CDCl3) δ
20.7, 21.3, 22.3, 22.6, 22.7, 23.3, 23.5, 23.6, 24.3, 24.6, 25.3, 29.7,
37.0, 40.5, 43.1, 45.9, 50.4, 55.3, 55.4, 66.3, 66.7, 98.7, 104.4,
116.2, 127.9, 128.0, 128.1, 128.3, 128.5, 130.0, 136.4, 155.9,
158.2, 161.1, 170.3, 170.9, 173.4. HRMS calcd for C37H55N
3O8Na [M þ Na]þ: 692.3881. Found: 692.3885. [R]2D5 -54.2 (c
1.0, CHCl3). Retention time (method 1) tR = 15.39 min.
4.4.2. Synthesis of Compound (S,S,S)-(þ)-13 (Cbz-L-leu-L-leu-
L-leu-CH2OAc). General Procedure 2. Yield: 91%, 265 mg (0.51
mmol) of white crystals. Mp 128-131 °C. Rf = 0.25 (hexane/
AcOEt, 7:3, v/v). 1H NMR (400 MHz, CDCl3) δ 0.87-0.97 (m,
18H), 1.26-1.33 (m, 1H), 1.37-1.45 (m, 1H), 1.45-1.55 (m,
2H), 1.56-1.71 (m, 4H), 1.74-1.85 (m, 1H), 2.04 (s, 3H), 4.00
(dd, J = 5.7 Hz, J = 11.1 Hz, 1H), 4.04-4.16 (m, 2H),
4.18-4.28 (m, 1H), 4.38-4.49 (m, 1H), 5.05 (d, J = 12.3 Hz,
1H), 5.14 (d, J = 12.1 Hz, 1H), 5.30 (d, J = 6.9 Hz, 1H), 6.37 (d,
J = 6.7 Hz, 1H), 6.55 (br s, 1H), 7.30-7.39 (m, 5H). 13C NMR
(100 MHz, CDCl3) δ 20.9, 21.7, 22.0, 22.1, 22.8, 23.0, 24.7, 24.7,
24.9, 40.1, 40.3, 40.7, 46.8, 51.7, 54.1, 66.3, 67.2, 128.0, 128.3,
128.6, 135.9, 156.4, 171.2, 171.4, 172.2. HRMS calcd for
C28H45N3O6Na [M þ Na]þ: 542.3201. Found: 542.3193. [R]2D5
þ0.27 (c 1.0, CHCl3). Retention time (method 3) tR = 6.31 min.
4.4.3. Synthesis of Compound (S,R,S)-(-)-13 (Cbz-L-leu-D-
leu-L-leu-CH2OAc). General Procedure 2. Yield: 82%, 245 mg
(0.47 mmol) of white crystals. Mp 144-146 °C. Rf = 0.24
(hexane/AcOEt, 7:3, v/v). 1H NMR (400 MHz, CDCl3) δ
0.85-0.97 (m, 18H), 1.22-1.31 (m, 1H), 1.32-1.42 (m, 1H),
1.44-1.54 (m, 3H), 1.54-1.66 (m, 3H), 1.66-1.77 (m, 1H), 2.05
(s, 3H), 3.98-4.10 (m, 2H), 4.16-4.29 (m, 2H), 4.38 (q, J = 7.6
Hz, 1H), 5.04-5.18 (m, 2H), 5.39 (d, J = 7.6 Hz, 1H), 6.35 (d,
J = 8.4 Hz, 1H), 6.61 (d, J = 7.8 Hz, 1H), 7.29-7.38 (m, 5H).
4.4.7. Synthesis of Compound (R,R,R)-(-)-2 (Cbz-D-leu-D-leu-
D-leu-CHO). General Procedure 4. Yield: 76%, 35 mg (0.073
mmol) of colorless oil. Rf = 0.23 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (400 MHz, CDCl3) δ 0.88-0.96 (m, 18H, CH(CH3)2),
1.35-1.47 (m, 1H, CH(CH3)2), 1.47-1.57 (m, 1H, CH(CH3)2),
1.58-1.74 (m, 6H, CH2CH(CH3)2), 1.74-1.86 (m, 1H, CH-
(CH3)2), 4.05-4.15 (m, 1H, CHCHO), 4.38-4.49 (m, 1H,
NHCHCO), 4.49-4.57 (m, 1H, NHCHCO), 4.98-5.15 (m,
2H, OCH2Ph), 5.29 (d, J = 6.5 Hz, 1H, NHC(O)O), 6.56 (d,
J = 8.8 Hz, 1H, NHCHCHO), 7.07 (d, J = 6.3 Hz, 1H, NH),
7.29-7.38 (m, 5H, ArH), 9.49 (s, 1H, CHO). 13C NMR (100
MHz, CDCl3) δ 21.6, 21.7, 22.1, 22.7, 23.0, 23.1, 24.7, 24.7, 24.9,
26.9, 37.4, 40.1, 40.7, 41.5, 51.6, 54.1, 57.2, 67.2, 128.0, 128.3,
128.6, 135.9, 156.5, 172.2, 172.6, 199.7. HRMS calcd for
C26H41N3O5Na [M þ Na]þ: 498.2938. Found: 498.2928. [R]2D5
-36.5 (c 0.5, CHCl3).
4.4.8. Synthesis of Compound (S,R,S)-(-)-2 (Cbz-L-leu-D-leu-
L-leu-CHO). General Procedure 4. Yield: 63%, 39 mg (0.082
mmol) of colorless oil. Rf = 0.22 (CHCl3/MeOH, 95:5, v/v).

1516 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Mroczkiewicz et al.
1H NMR (400 MHz, CDCl3) δ 0.88-0.98 (m, 18H, CH(CH3)2),
1.40-1.58 (m, 3H, CH(CH3)2), 1.59-1.79 (m, 6H, CH2CH-
(CH3)2), 4.12-4.22 (m, 1H, CHCHO), 4.40-4.54 (m, 2H,
NHCHCO), 5.10 (s, 2H, OCH2Ph), 5.24 (d, J = 7.5 Hz, 1H,
NHC(O)O), 6.50 (d, J = 7.9 Hz, 1H, NHCHCHO), 6.77 (d, J =
7.0 Hz, 1H, NH), 7.32-7.39 (m, 5H, ArH), 9.53 (s, 1H, CHO).
13C NMR (100 MHz, CDCl3) δ 21.7, 21.9, 22.0, 22.8, 22.9, 23.1,
24.6, 24.7, 24.8, 37.6, 40.3, 41.1, 51.7, 53.7, 57.2, 67.3, 128.0,
128.3, 128.6, 135.9, 156.4, 171.9, 172.3, 199.6. HRMS calcd for
C26H41N3O5Na [M þ Na]þ: 498.2938. Found: 498.2951. [R]2D5
-57.3 (c 0.5, CHCl3).
4.4.9. Synthesis of Compound (R,S,R)-(þ)-2 (Cbz-D-leu-L-leu-
D-leu-CHO). General Procedure 4. Yield: 25%, 15 mg (0.032
mmol) of colorless oil. Rf = 0.22 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (500 MHz, CDCl3) δ 0.88-0.98 (m, 18H, CH(CH3)2),
1.40-1.57 (m, 3H, CH(CH3)2), 1.58-1.79 (m, 6H, CH2CH-
(CH3)2), 4.12-4.22 (m, 1H, CHCHO), 4.40-4.52 (m, 2H,
NHCHCO), 5.10 (s, 2H, OCH2Ph), 5.28 (d, J = 7.6 Hz, 1H,
NHC(O)O), 6.53 (d, J = 6.2 Hz, 1H, NHCHCHO), 6.77 (d, J =
7.0 Hz, 1H, NH), 7.30-7.38 (m, 5H, ArH), 9.52 (s, 1H, CHO).
13C NMR (125 MHz, CDCl3) δ 21.8, 21.9, 22.0, 22.8, 22.9, 23.1,
24.7, 24.7, 24.8, 37.6, 40.4, 41.1, 51.8, 53.8, 57.3, 67.2, 128.0,
128.1, 128.3, 128.6, 128.6, 136.0, 156.4, 172.0, 172.4, 199.3.
HRMS calcd for C27H45N3O6Na [M þ CH3OH þ Na]þ:
530.3201. Found: 530.3177. [R]2D5 55.5 (c 0.5, CHCl3).
2.42 (br s, 1H), 3.43 (dd, J = 6.1 Hz, J = 11.3 Hz, 1H), 3.59 (dd,
J = 3.1 Hz, J = 11.4 Hz, 1H), 3.79 (s, 3H), 3.80 (s, 3H), 3.82-
3.89 (m, 1H), 4.39 (d, J = 16.5 Hz, 1H), 4.62-4.72 (m, 1H), 4.74
(d, J = 16.5 Hz, 1H), 5.04-5.16 (m, 2H), 5.40 (d, J = 7.7 Hz,
1H), 6.44 (s, 2H), 6.64 (d, J = 6.6 Hz, 1H), 7.11 (d, J = 8.6 Hz,
1H), 7.35 (s, 6H). 13C NMR (100 MHz, CDCl3 ) δ 20.9, 22.2,
22.2, 22.7, 23.0, 23.5, 24.5, 24.8, 25.3, 37.0, 39.7, 42.0, 46.0, 50.2,
50.7, 55.3, 55.4, 58.3, 65.9, 67.0, 98.7, 103.9, 116.5, 128.0, 128.1,
128.5, 129.7, 136.2, 156.3, 158.2, 161.0, 171.5, 175.1. Anal. Calcd
for C35H53N3O7: C, 66.96; H, 8.51; N, 6.69. Found: C, 66.63; H,
8.44; N, 6.45. [R]2D5 -29.7 (c 1.0, CHCl3). Retention time
(method 2) tR = 11.42 min.
4.5.3. Synthesis of Compound (R,I,S)-(þ)-15 (Cbz-D-leu-I-leu-
L-leu-CH2OH). General Procedure 2. Yield: 49%, 73 mg (0.152
mmol) of white crystals. Mp 124-127 °C. Rf = 0.21 (CHCl3/
MeOH, 95:5, v/v). 1H NMR (500 MHz, CDCl3) δ 0.88-0.96 (m,
18H), 1.24-1.34 (m, 1H), 1.38-1.46 (m, 1H), 1.46-1.56 (m,
2H), 1.56-1.69 (m, 4H), 1.70-1.79 (m, 1H), 2.77 (br s, 1H),
3.40-3.47 (m, 1H), 3.58-3.66 (m, 1H), 3.97-4.06 (m, 1H),
4.11-4.19 (m, 1H), 7.38-4.46 (m, 1H), 5.05 (d, J = 12.1 Hz,
1H), 5.12 (d, J = 12.0 Hz, 1H), 5.40 (d, J = 5.2 Hz, 1H), 6.57 (d,
J = 6.9 Hz, 1H), 6.73 (d, J = 7.2 Hz, 1H), 7.30-7.37 (m, 5H).
13C NMR (125 MHz, CDCl3) δ 21.8, 22.1, 22.8, 22.9, 23.0, 24.8,
24.9, 39.9, 40.3, 41.0, 50.1, 52.1, 54.2, 65.5, 67.4, 128.1, 128.3,
128.6, 135.9, 156.5, 172.2, 172.8. HRMS calcd for C26H43N3O5-
Na [M þ Na]þ: 500.3095. Found: 500.3079. [R]2D5 38.4 (c 1.0,
CHCl3). Retention time (method 2) tR = 4.73 min.
4.5.4. Synthesis of Compound (R,II,S)-(-)-15 (Cbz-D-leu-II-
leu-L-leu-CH2OH). General Procedure 2. Yield: 61%, 88 mg
(0.184 mmol) of white crystals. Mp 107-109 °C. Rf = 0.19
(CHCl3/MeOH, 95:5, v/v). 1H NMR (500 MHz, CDCl3) δ
0.87-0.97 (m, 18H), 1.29-1.36 (m, 1H), 1.38-1.47 (m, 1H),
1.49-1.56 (m, 2H), 1.57-1.68 (m, 4H), 1.75-1.85 (m, 1H), 2.47
(br s, 1H), 3.44-3.51 (m, 1H), 3.62 (dd, J = 3.5 Hz, J = 11.3 Hz,
1H), 3.95-4.03 (m, 1H), 4.03-4.10 (m, 1H), 4.35-4.42 (m, 1H),
5.05 (d, J = 12.1 Hz, 1H), 5.14 (d, J = 12.0 Hz, 1H), 5.34 (br s,
1H), 6.43 (br s, 1H), 6.66 (br s, 1H), 7.30-7.38 (m, 5H). 13C
NMR (125 MHz, CDCl3) δ 21.6, 22.1, 22.2, 22.7, 23.0, 24.7,
24.8, 25.0, 29.7, 39.8, 40.4, 40.8, 50.2, 52.6, 54.3, 65.5, 67.4,
128.0, 128.3, 128.6, 135.8, 156.6, 172.0, 172.7. HRMS calcd for
C26H43N3O5Na [M þ Na]þ: 500.3095. Found: 500.3090. [R]2D5
-24.0 (c 1.0, CHCl3). Retention time (method 2) tR = 4.87 min.
4.5.5. Synthesis of Compound (R,I,S)-(þ)-2 (Cbz-D-leu-I-leu-
L-leu-CHO). General Procedure 4. Yield: 45%, 33 mg (0.069
mmol) of colorless oil. Rf = 0.23 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (500 MHz, CDCl3) δ 0.87-0.98 (m, 18H), 1.40-1.57 (m,
2H), 1.57-1.69 (m, 6H), 1.69-1.83 (m, 1H), 4.11-4.21 (m, 1H),
4.40-4.55 (m, 2H), 5.09 (s, 2H), 5.24 (d, J = 5.9 Hz, 1H), 6.49
(d, J = 8.2 Hz, 1H), 6.86 (d, J = 6.5 Hz, 1H), 7.30-7.38 (m, 5H),
9.49 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 21.7, 21.8, 22.9,
22.9, 23.1, 24.7, 24.8, 24.9, 29.7, 37.4, 40.3, 41.0, 51.6, 54.2, 57.3,
67.4, 128.0, 128.0, 128.3, 128.6, 135.9, 156.6, 172.1, 172.3, 199.5.
HRMS calcd for C27H45N3O6Na [M þ CH3OH þ Na]þ:
530.3201. Found: 530.3212. [R]2D5 45.9 (c 0.5, CHCl3).
4.5.6. Synthesis of Compound (S,I,R)-(-)-2 (Cbz-L-leu-I-leu-
D-leu-CHO). General Procedure 4. Yield: 40%, 19 mg (0.041
mmol) of colorless oil. Rf = 0.23 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (500 MHz, CDCl3) δ 0.80-1.02 (m, 18H), 1.42-1.57 (m,
2H), 1.58-1.69 (m, 6H), 1.69-1.82 (m, 1H), 4.13-4.20 (m, 1H),
4.40-4.56 (m, 2H), 5.09 (s, 2H), 5.30 (d, J = 5.9 Hz, 1H), 6.57
(d, J = 8.1 Hz, 1H), 6.91 (d, J = 6.8 Hz, 1H), 7.30-7.38 (m, 5H),
9.48 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 21.7, 21.8, 22.8,
22.9, 23.1, 24.7 24.8, 24.9, 29.7, 37.4, 40.3, 41.0, 51.6, 54.2, 57.3,
67.4, 128.0, 128.1, 128.4, 128.6, 135.9, 156.6, 172.1, 172.4, 199.6.
HRMS calcd for C26H41N3O5Na [M þ Na]þ: 498.2938. Found:
498.2947. [R]2D5 -47.1 (c 0.5, CHCl3).
4.5. Synthesis of Aldehydes (R,I,S)-2 (Cbz-D-leu-I-leu-L-leu-
CHO) and (R,II,S)-2 (Cbz-D-leu-II-leu-L-leu-CHO). 4.5.1.
Synthesis of Compounds (R,I,S)-12 (Cbz-D-leu-n(DMB)-I-leu-L-
leu-CH2OAc) and (R,II,S)-12 (Cbz-D-leu-n(DMB)-II-leu-L-leu-
CH2OAc). General Procedure 1. Products (R,I,S)-12 and (R,II,
S)-12 were obtained as a mixture in 51:49 ratio. Yield: 82%,
1.098 g (1.64 mmol) of colorless oil. Rf = 0.65 (hexane/AcOEt,
1
7:3, v/v). H NMR (200 MHz, CDCl3) δ 0.70 (d, J = 6.4 Hz,
2H), 0.74-1.06 (m, 16H), 1.06-1.40 (m, 3H), 1.40-1.70 (m,
6H), 2.03 (s, 3H), 3.77 (s, 3H), 3.79 (s, 3H), 3.85-4.45 (m, 3H),
4.50-5.0 (m, 3H), 5.02-5.15 (m, 2H), 5.34 (d, J = 6.9 Hz,
0.5H), 5.53 (d, J = 6.8 Hz, 0.5H), 6.02 (d, J = 7.2 Hz, 0.5H),
6.35-6.50 (m, 2H), 6.71 (d, J = 7.0 Hz, 0.5H), 7.04 (d, J = 7.4
Hz, 0.5H), 7.12 (d, J = 7.5 Hz, 0.5H), 7.35 (s, 6H). 13C NMR (50
MHz, CDCl3) δ 21.2, 22.2, 22.5, 23.0, 23.4, 23.9, 25.0, 25.6, 37.3,
40.6, 42.7, 43.3, 45.8, 46.7, 50.5, 55.7, 58.6, 66.6, 67.1, 67.3, 99.0,
104.1, 117.2, 128.3, 128.8, 129.6, 156.4, 170.6, 171.2, 175.4.
Anal. Calcd for C37H55N3O8: C, 66.34; H, 8.28; N, 6.27. Found:
C, 66.18; H, 8.34; N, 6.12. Retention time of epimers (method 1):
t
R(R,I,S)-12 = 19.48 min; tR(R,II,S)-12 = 16.17 min.
4.5.2. Synthesis of Compounds (R,I,S)-(þ)-14 (Cbz-D-leu-n-
(DMB)-I-leu-L-leu-CH2OH) and (R,II,S)-(-)-14 (Cbz-D-leu-n-
(DMB)-II-leu-L-leu-CH2OH). General Procedure 3. Products
(R,I,S)-(þ)-14 and (R,II,S)-(-)-14 were separated by flash
column chromatography.
Compound (R, I,S)-(þ)-14. Yield: 36%, 328 mg (0,52 mmol)
of white crystals. Mp 80-83 °C. Rf = 0.46 (hexane/AcOEt, 7:3,
v/v). 1H NMR (400 MHz, CDCl3) δ 0.76 (d, J = 6.2 Hz, 6H),
0.82-1.04 (m, 15H), 1.04-1.15 (m, 1H), 1.38-1.58 (m, 2H),
1.60-1.70 (m, 1H), 1.70-1.90 (m, 2H), 2.73 (br s, 1H), 3.13 (dd,
J = 5.8, J = 11.5, 1H), 3.52 (dd, J = 2.6 Hz, J = 11.5 Hz, 1H),
3.81 (s, 6H), 3.86-3.95 (m, 1H), 4.07 (d, J = 14.9 Hz, 1H), 4.84
(d, J = 14.7 Hz, 1H), 5.02-5.11 (m, 1H), 5.12 (s, 2H), 5.41 (d,
J = 8.6 Hz, 1H), 5.54 (d, J = 8.8 Hz, 1H), 6.50 (s, 2H), 7.24 (d,
J = 7.9 Hz, 1H), 7.28-7.41 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 20.9, 21.0, 21.3, 21.9, 22.0, 22.3, 23.0, 23.3, 23.5, 24.4,
24.8, 25.3, 37.4, 39.9, 41.9, 48.3, 49.3, 50.7, 55.4, 55.5, 58.4, 65.4,
66.9, 99.2, 104.6, 115.1, 128.0, 128.4, 131.8, 136.4, 156.7, 158.8,
161.7, 170.3, 172.9. Anal. Calcd for C35H53N3O7: C, 66.96; H,
8.51; N, 6.69. Found: C, 66.84; H, 8.44; N, 6.48. [R]2D5 29.4 (c 1.0,
CHCl3). Retention time (method 2) tR = 14.18 min.
Compound (R,II,S)-(-)-14. Yield: 25%, 225 mg (0.36 mmol)
of white crystals. Mp 55-58 °C. Rf = 0.34 (hexane/AcOEt, 7:3,
v/v). 1H NMR (400 MHz, CDCl3) δ 0.73 (d, J = 6.4 Hz, 3H),
0.81-0.94 (m, 15H), 1.20-1.44 (m, 3H), 1.46-1.68 (m, 6H),
4.5.7. Synthesis of Compound (R,II,S)-(-)-2 (Cbz-D-leu-II-
leu-L-leu-CHO). General Procedure 4. Yield: 56%, 30 mg (0.063
mmol) of colorless oil. Rf = 0.21 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (400 MHz, CDCl3) δ 0.86-0.99 (m, 18H), 1.39-1.57 (m,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1517
2H), 1.58-1.73 (m, 6H), 1.75-1.85 (m, 1H), 4.04-4.15 (m, 1H),
4.38-4.46 (m, 1H), 4.46-4.58 (m, 1H), 4.97-5.06 (m, 1H),
5.06-5.13 (m, 1H), 5.25 (d, J = 5.9 Hz, 1H), 6.41 (d, J = 6.5 Hz,
1H), 7.07 (br s, 1H), 7.30-7.36 (m, 5H), 9.45 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 21.6, 21.7, 22.1, 22.8, 23.0, 23.1, 23.1, 24.7,
24.7, 24.9, 40.3, 40.9, 51.8, 54.2, 57.2, 67.3, 128.0, 128.3, 128.6,
135.9, 156.5, 172.1, 172.5, 199.5. HRMS calcd for C26H41N3O5-
Na [M þ Na]þ: 498.2938. Found: 498.2937. [R]2D5 -27.1 (c 0.5,
CHCl3).
4.5.8. Synthesis of Compound (R,II,S)-(þ)-2 (Cbz-L-leu-II-leu-
D-leu-CHO). General Procedure 4. Yield: 32%, 15 mg (0.031
mmol) of colorless oil. Rf = 0.22 (CHCl3/MeOH, 95:5, v/v). 1H
NMR (500 MHz, CDCl3) δ 0.89-0.97 (m, 18H), 1.40-1.58 (m,
2H), 1.60-1.72 (m, 6H), 1.74-1.85 (m, 1H), 4.06-4.14 (m, 1H),
4.39-4.45 (m, 1H), 4.46-4.54 (m, 1H), 4.98-5.05 (m, 1H),
5.07-5.12 (m, 1H), 5.28 (d, J=6.5 Hz, 1H), 6.46 (d, J=7.3 Hz,
1H), 7.00 (br s, 1H), 7.29-7.38 (m, 5H), 9.49 (s, 1H). 13C NMR
(125 MHz, CDCl3) δ 21.7, 21.7, 22.1, 22.7, 23.0, 23.1, 24.7, 24.7,
24.9, 37.4, 40.4, 41.0, 51.9, 54.2, 57.2, 67.3, 128.0, 128.3, 128.6,
135.9, 156.5, 172.1, 172.5, 199.6. HRMS calcd for C26H41N3O5Na
[M þ Na]þ: 498.2938. Found: 498.2945. [R]D25 29.0 (c 0.5, CHCl3).
4.6. Cell Lines and Reagents. Murine multiple myeloma
(J588L) and murine breast cancer (EMT6) cell lines were
purchased from ATCC (Manassas, VA). Cells were cultured
in RPMI-1640 (J588L) or Dulbecco’s modified Eagle’s medium
(EMT6) supplemented with 10% heat-inactivated fetal calf
serum, antibiotics, 2-mercaptoethanol (50 mM), and L-gluta-
mine (2 mM) (all from Invitrogen, Carlsbad, CA). MG132 was
purchased from Calbiochem/EMD (San Diego, CA) and was
dissolved in DMSO to 10 mM stock concentration.
AMC=7-amido-4-methylcoumarin) (Bachem, Weil am Rhein,
Germany). Purified 20S proteasomes isolated from human
erythrocytes were purchased from Enzo Life Sciences (www.
enzolifesciences.com). Purified 20S proteasomes (300 ng) were
incubated with 20 μM fluorogenic peptide substrates for
chymotrypsin-like, PGPH, and trypsin-like proteasomal acti-
vities (Suc-leu-leu-Val-Tyr-AMC, Z-leu-leu-Glu-βNA and
Ac-Arg-leu-Arg-AMC, respectively, all purchased from Bachem,
Weil am Rhein, Germany) in 100 μL assay buffer (20 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM NaN3, 1 mM DTT) in
the presence of tested compounds for 30 min at 37 °C, followed
by measurement of hydrolysis of the fluorogenic substrates
using a Victor2 D fluorometer (Perkin-Elmer, Waltham, MA)
equipped with 355 nm excitation and 460 nm emission filters.
Corresponding solvent (DMSO) concentrations were used for
the controls.
Acknowledgment. This work was financially supported by
Polish State Committee for Scientific Research, Grants N405
007 31/0544 and N N401 3240 33. J.G. is a recipient of the
Mistrz Award from the Foundation for Polish Science.
Supporting Information Available: Cytostatic/cytotoxic ef-
fects exerted by MG-132 and its stereoisomers against J558L
and EMT6 cells. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
4.7. Cytotoxic/Cytostatic Assays. The cytostatic/cytotoxic
effects in EMT6 cells were measured using crystal violet staining.
Briefly, tumor cells were dispensed into 96-well plates (Sarstedt,
Numbrecht, Germany) at 1.5 ꢀ 103 cells per well and allowed to
attach overnight. The following day the investigated agents were
added at indicated concentrations. After 24 h of incubation the
cells were rinsed with PBS and stained with 0.5% crystal violet in
2% ethanol for 10 min at room temperature. Next, plates were
washed four times with tap water and cells were lysed with 1%
SDS solution. Absorbance was measured at 595 nm using an
ELISA reader (Bio-Rad, Hercules, CA).
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