Synthesis of 2,3- and 3,4-methanoamino acid equivalents with stereochemical diversity and their conversion into the tripeptide proteasome inhibitor belactosin a and its highly potent cis-cyclopropane stereoisomer

Article abstract of DOI:10.1021/ol8013304

(Chemical Equation Presented) A series of chiral 2,3- and 3,4-methanoamino acid equivalents of stereochemical diversity were designed and synthesized from our chiral cyclopropane units, using a diastereoselective Grignard addition with (R)- or (S)-t-butanesulfinyl imines as the key step. These equivalents were converted into the proteasome inhibitor belactosin A and its cis-cyclopropane stereoisomer. The unnatural cis-isomer was shown to be more than twice as potent as belactosin A as a proteasome inhibitor.

Full text of DOI:10.1021/ol8013304

ORGANIC
LETTERS
2008
Vol. 10, No. 16
3571-3574
Synthesis of 2,3- and 3,4-Methanoamino
Acid Equivalents with Stereochemical
Diversity and Their Conversion into the
Tripeptide Proteasome Inhibitor
Belactosin A and Its Highly Potent
Cis-Cyclopropane Stereoisomer
Keisuke Yoshida,† Kazuya Yamaguchi,† Takayuki Sone,† Yuka Unno,‡
Akira Asai,‡ Hideyoshi Yokosawa,† Akira Matsuda,† Mitsuhiro Arisawa,† and
Satoshi Shuto*,†
Faculty of Pharmaceutical Sciences, Hokkaido UniVersity, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan, and Graduate School of Pharmaceutical Sciences,
UniVersity of Shizuoka, Yata, Shizuoka 422-8526, Japan
shu@pharm.hokudai.ac.jp
Received June 13, 2008
ABSTRACT
A series of chiral 2,3- and 3,4-methanoamino acid equivalents of stereochemical diversity were designed and synthesized from our chiral
cyclopropane units, using a diastereoselective Grignard addition with (R)- or (S)-t-butanesulfinyl imines as the key step. These equivalents
were converted into the proteasome inhibitor belactosin A and its cis-cyclopropane stereoisomer. The unnatural cis-isomer was shown to be
more than twice as potent as belactosin A as a proteasome inhibitor.
The development of useful peptide mimetics may be
achieved by replacing a key amino acid of biologically active
peptides with a conformationally restricted amino acid
analogue having the side chain in the bioactive conforma-
tion.1 Because of its small, rigid structural features, a
cyclopropane restricts the conformation of molecules, which
can improve the biological activity of the lead molecules
due to an entropic advantage.2,3 Thus, the cyclopropane-
containing amino acids have been synthesized and used
(2) Excellent reviews of cyclopropane-containing amino acids were
recently published: Brackmann, F.; de Meijere, A Chem. ReV. 2007, 107,
4493–4537, 4538-4583
.
(3) (a) Kazuta, Y.; Matsuda, A.; Shuto, S. J. Org. Chem. 2002, 67, 1669–
1677. (b) Kazuta, Y.; Hirano, K.; Natsume, K.; Yamada, S.; Kimura, R.;
Matsumoto, S.; Furuichi, K.; Matsuda, A.; Shuto, S. J. Med. Chem. 2003,
46, 1980–1988. (c) Watanabe, M.; Kazuta, Y.; Hayashi, H.; Yamada, S.;
Matsuda, A.; Shuto, S J. Med. Chem. 2006, 49, 5587–5596, and references
† Hokkaido University.
‡ University of Shizuoka.
(1) (a) Hanessian, S.; McNaughton-Smith, G.; Lombart, J.-G.; Lubell,
W. D. Tetrahedron 1997, 53, 12789–12854. (b) Souers, A. J.; Ellman, J. A.
Tetrahedron 2001, 57, 7431–7488.
therein
.
10.1021/ol8013304 CCC: $40.75
Published on Web 07/19/2008
2008 American Chemical Society

extensively for peptide mimetic studies.2 However, the
bioactive conformation of biologically active peptides, such
as peptide ligands targeting a G protein-coupled receptor
(GPCR), is usually unclear. In these cases, a series of
cyclopropane-containing amino acids with stereochemical
diversity, where the side chains are located in a variety of
spatial arrangements owing to the conformational restriction,
can be used to identify the bioactive conformation and to
develop peptide mimetics with high potency.
Figure 1. Structures of belactosin A (3) and its cis-cyclopropane
stereoisomer (4).
We describe here an efficient synthetic method for a series
of chiral 2,3- and 3,4-methanoamino acid equivalents III
(Scheme 1) with cis/trans, D/L, and syn/anti stereochemical
equivalents of L- or D-2,3- and -3,4-methanoamino acids IV,
since a vinyl group is stable under various reaction conditions
and can be converted easily into a carboxyl group.7 Com-
pounds III would be derived from compounds II, the
symmetric CH2dCHC*H(NH)- moiety of which would be
constructed via the diastereoselective Grignard addition to
the (R)- and (S)-t-butanesulfinyl imines I.8 Grignard additions
to the t-butanesulfinyl imines are known to occur highly
stereoselectively, where the stereochemical outcome is
dependent on the configuration at the sulfinyl moiety.8
Scheme 1
Table 1. Grignard Reaction of (S)-t-Butanesulfinyl Imine 6
diversity. Using these equivalents, we synthesized belactosin
A (3),4,5 a tripeptidic proteasome inhibitor containing a
3-(trans-2-aminocyclopropyl)-L-alanine (trans-3,4-methano-
L-ornithine), and its cis-cyclopropane stereoisomer 4 (Figure
1). The biological evaluation showed that the unnatural cis-
isomer 4 is a more potent proteasome inhibitor than the
natural belactosin A having the trans-cyclopropane structure.
The stereoselective synthesis of cyclopropane derivatives
with a desired stereochemistry is often troublesome.2,3,6 To
solve this problem, we recently developed chiral units
composed of four stereoisomeric cyclopropanes, 1 and 2,
and their enantiomers ent-1 and ent-2 (Scheme 1), for
preparing various cyclopropane derivatives bearing adjacent
carbon substituents in a cis or a trans relationship.3a These
units were employed as synthons in this study.
d
entrya,b
temp (°C)
conc (M)c
yield (%)
D/L
1
2
3
4
5
6
7
0
rt
rt
rt
rt
110
110
0.1
0.1
0.1
0.1
0.01
0.1
0.01
quant
quant
quant
97
98
94
1:4.6
1:6.7
1:5.1
1:7.4
1:10
1:8.1
1:16
quant
a Solvent: entries 1 and 2, CH2Cl2; entry 3, THF; entries 4-7, toluene.
b Equivalent of Grignard reagent: entries 1-5, 3 equiv; entry 6, 1.5 equiv;
entry 7, 1.1 equiv. c Concentration of the substrate. D or L indicates the
d
stereochemistry of the cyclopropane amino acid by replacing the vinyl group
of the Grignard reaction product with a carboxyl, and the ratio was
determined by HPLC.
We designed the cyclopropane derivatives III with a
CH2dCHC*H(NHPg)- side chain (Scheme 1) as versatile
We investigated the Grignard reaction using a series of (R)-
and (S)-t-butanesulfinyl imines 6-13 with a cis- or trans-
cyclopropane structure (Tables 1 and 2).9 The trans-cyclopro-
pane t-butanesulfinyl imines 6-9 were prepared from the chiral
trans-cyclopropane unit 1 (Scheme 2). Similarly, the cis-
(4) (a) Asai, A.; Hasegawa, A.; Ochiai, K.; Yamashita, Y.; Mizukami,
T. J. Antibiot. 2000, 53, 81–83. (b) Asai, A.; Tsujita, T.; Sharma, S. V.;
Yamashita, Y.; Akinaga, S.; Funakoshi, M.; Kobayashi, H.; Mizukami, T.
Biochem. Pharmacol. 2004, 67, 227–234
.
(5) Total synthesis of belactosin A: (a) Armstrong, A.; Scutt, J, N. Chem.
Commun. 2004, 510–511. (b) Larionov, O. V.; de Meijere, A. Org. Lett.
(8) (a) Cogan, D. A.; Liu, G.; Kim, K.; Backes, B. J.; Ellman, J. A.
J. Am. Chem. Soc. 1998, 120, 8011–8019. (b) Cogan, D. A.; Liu, G.; Ellman,
J. A. Tetrahedron 1999, 55, 8883–8904. (c) Davis, F. A.; McCoull, W. J.
Org. Chem. 1999, 64, 3396–3397. (d) Senanayake, C. H.; Krishnamurthy,
D.; Lu, A.-H.; Han, Z.; Gallou, I. Aldrichimica Acta 2005, 38, 93–104.
(9) Stereochemistries of the Grignard reaction products were determined
by the modified Mosher method.
2004, 6, 2153–2156
.
(6) For examples see: (a) Stammer, C. H. Tetrahedron 1990, 46, 2231–
2254. (b) Kodama, H.; Shimohigashi, Y. J. Synth. Org. Chem. Jpn. 1994,
52, 180–191. (c) Shimamoto, K.; Ohfune, Y. J. Med. Chem. 1996, 39, 407–
¨
423. (d) Demir, A. S.; Sesenoglu, O HelV. Chim. Acta 2004, 87, 106–119,
and references therein
(7) Cooper, T. S.; Laurent, P.; Moody, C. J.; Takle, A. K. Org. Biol.
.
(10) (a) Adamas, J. Nat. ReV. Cancer 2004, 4, 349–360. (b) Ciechanover,
A. Angew. Chem., Int. Ed. 2005, 44, 5944–5996.
Chem. 2004, 2, 265–267
.
3572
Org. Lett., Vol. 10, No. 16, 2008

7, D/L ) 1:16) under 0.01 M substrate concentration
conditions in toluene at 110 °C, in which the reaction was
complete with only 1.1 equiv of the Grignard reagent.
By employing the low substrate concentration (0.01M)/
high temperature (110 °C) reaction conditions, we carried
out the Grignard reactions with a variety of the (S)-t-
butanesulfinyl imines and the (R)-t-butanesulfinyl imines as
the substrates (Table 2). In all of these cases, the addition
products equivalent to an L-amino acid were selectively
formed from the (S)-t-butanesulfinyl imines (entries 1, 4, and
5), and the products equivalent to a D-amino acid were
selectively formed from the (R)-t-butanesulfinyl imines
(entries 2, 3, 6, and 7), respectively, regardless of the
stereochemistry of the cyclopropane backbone or the side-
chain length of the substrates. The stereochemical outcome
is in accord with the chelated transition state model reported
by Ellman.8a,b
Table 2. Grignard Reaction of the (S)- and (R)-t-Butanesulfinyl
Imines 7-13 under the Low Substrate Concentration/High
Temperature Conditionsa,b
Thus, we have successfully developed a general procedure
for the preparation of a series of 2,3- and 3,4-methanoamino
acid equivalents with cis/trans, D/L, and syn/anti stereochem-
ical diversity. This is, to our knowledge, the first synthetic
method for systematically providing chiral 2,3- and 3,4-
methanoamino acids with stereochemical diversity.2,6
Using the 3,4-methanoamino acid equivalents obtained by
the Grignard reaction as the key units, we next planned to
synthesize belactosin A (3) and its cis-cyclopropane stereo-
isomer 4.
a R ) CH2OTBDPS. b D or L indicates the stereochemistry by replacing
the vinyl group with a carboxyl, and the ratio was determined by 1H NMR
(entries 1-4) or HPLC (entries 5-7).
Scheme 2
Belactosin A, a tripeptide consisting of L-Ala, 3-((1R,2S)-
2-aminocyclopropyl)-L-Ala, and a chiral carboxy-ꢀ-lactone,
was isolated by Asai. Belactosin A may be an effective
anticancer drug lead since it prevents tumor cell cycle
progression due to proteasome inhibition.4,10 The cyclopro-
pane part of 3 would restrict the orientation of the L-Ala
and the ꢀ-lactone moieties to determine the three-dimensional
structure of the molecule, which might be critical for its
interaction with the proteasome. Thus, our interest derives
from the biological activity of the cis-cyclopropane isomer
4, in which the L-Ala and the ꢀ-lactone moieties can be
restricted in a spatial arrangement different from that in 3
with the trans-cyclopropane structure.
These two target compounds would be obtained by
condensation between L-Ala (A), trans- or cis-3,4-metha-
noamino acid (B), and the chiral carboxy-ꢀ-lactone (C)
(Figure 2). The chiral ꢀ-lactone can be prepared from L-Ile,
according to the method reported by Armstrong,5a and the
Grignard reaction products 15 and 16 (Figure 2) would be
equivalents of the trans- and cis-3,4-methanoamino acids (B).
Compound 15, including some of the minor diastereomer,
was treated with HCl to remove the N- and O-protecting
groups, and the resulting free amino group was protected
with a Fmoc group to give the diastereomerically pure 17
after silica gel column chromatography. Oxidation of the
hydroxymethyl moiety of 17, followed by treatment with
diphenyl phosphoryl azide (DPPA)/Et3N, gave the corre-
sponding acid azide, which was heated in t-BuOH to afford
the Curtius rearrangement product 18. After removal of the
Fmoc group of 18, the product was condensed with the mixed
anhydride Cbz-L-Ala-OPiv to form the dipeptide 19 in 72%
cyclopropane t-butanesulfinyl imines 10-13 were prepared
from the chiral cis-cyclopropane unit 2.
The Grignard reaction was first investigated with the trans-
cyclopropane (S)-t-butanesulfinyl imine 6 as the substrate.
As shown in Table 1, the reaction gave a mixture of the
diastereomers 14a with the D-amino acid stereochemistry and
14b with the L-amino acid stereochemistry in excellent yield
under all of the reaction conditions examined, where the
diastereomer 14b was always the major product (entries
1-7). The reaction was carried out with 0.1 M substrate
concentration in CH2Cl2 at 0 °C and gave the addition
products in a D/L ratio of 1:4.6, while 3.0 equiv of the reagent
was needed to complete the reaction (entry 1). The stereo-
selectivity increased in a similar reaction at room temperature
(entry 2, D/L ) 1:6.7). Although the stereoselectivity
decreased with THF as solvent (entry 3, D/L ) 1:5.1), it
increased somewhat with toluene as solvent (entry 4, D/L )
1:7.4). Although the Grignard reactions with the t-butane-
sulfinyl imines usually occur highly stereoselectively under
low-temperature conditions,8 we found that the stereoselec-
tivity clearly improved when the reaction was performed with
a lower substrate concentration of 0.01 M (entry 5, D/L )
1:10) or at a higher temperature (entry 6, D/L ) 1:8.1). Thus,
the diastereomer 14b was obtained highly selectively (entry
Org. Lett., Vol. 10, No. 16, 2008
3573

Scheme 3
Figure 2. Representation of the synthetic targets 3 and 4.
yield. After removal of the Boc group of 19, the resulting
amine 20 was condensed with the mixed anhydride 22,
prepared from the known ꢀ-lactone 21,5a in the presence of
NaHCO3 in DMF/CH2Cl2 to form the tripeptide 23 in
excellent yield. Oxidation of the vinyl group to a carboxyl
group with a NaIO4/KMnO4/NaHCO3 combination in aque-
ous acetone, followed by removal of the Cbz group, finally
produced belactosin A (3) in 77% yield (Scheme 3). By the
same procedure, the cis-cyclopropane isomer 4 was synthe-
sized from the cis-3,4-methanoamino acid equivalent 16.
In measuring the inhibitory effect of belactosin A (3) and
its cis-cyclopropane stereoisomer 4 on the chymotrypsin-
like activity of the purified human 20S proteasome, it was
noted that, while the synthesized belactosin A showed
significant inhibitory effect (IC50 ) 304 ( 153 nM), as
reported previously,4a the isomer 4 (IC50 ) 150 ( 47 nM)
was about twice as potent as belactosin A.
with stereochemical diversity can be used effectively as the
key units to identify biologically active peptidic compounds.
Acknowledgment. We are grateful to Dr. Y. Kanda
1
(Kyowa Hakko Kogyo) for providing an H NMR chart of
In summary, we designed and synthesized a series of chiral
2,3- and 3,4-methanoamino acid equivalents of stereochem-
ical diversity. Two of these equivalents were effectively used
for the synthesis of belactosin A (3) and its cis-cyclopropane
isomer 4. The isomer 4 was identified as a more potent
proteasome inhibitor than belactosin A itself. These results
suggest that the 2,3- and 3,4-methanoamino acid equivalents
natural belactosin A.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data of all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL8013304
3574
Org. Lett., Vol. 10, No. 16, 2008