Design and synthesis of the stabilized analogs of belactosin A with the unnatural cis-cyclopropane structure

Article abstract of DOI:10.1039/c3ob41338a

The belactosin A analog 2a, having the unnatural cis-cyclopropane structure instead of the trans-cyclopropane structure in belactosin A, is a much more potent proteasome inhibitor than belactosin A. However, its cell growth inhibitory effect is rather lower than that expected from its remarkable proteasome inhibitory effect, probably due to its instability under cellular conditions. We hypothesized that the instability of 2a was due to chemical and enzymatic hydrolysis of the strained β-lactone moiety. Thus, to increase the stability of 2a by chemical modification, its analogs with a sterically more hindered β-lactone moiety and/or cyclopropylic strain-based conformational restriction were designed and synthesized, resulting in the identification of a stabilized analog 6a as a proteasome inhibitor with cell growth inhibitory effects. Our findings suggest that the chemical and biological stability of 2a is significantly affected by the steric hindrance around its β-lactone carbonyl moiety and the conformational flexibility of the molecule.

Full text of DOI:10.1039/c3ob41338a

Organic &
Biomolecular Chemistry
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PAPER
Design and synthesis of the stabilized analogs of
belactosin A with the unnatural cis-cyclopropane
structure†
Cite this: Org. Biomol. Chem., 2013, 11,
6615
Shuhei Kawamura,a Yuka Unno,b Akira Asai,b Mitsuhiro Arisawaa and
Satoshi Shuto*a,c
The belactosin A analog 2a, having the unnatural cis-cyclopropane structure instead of the trans-cyclo-
propane structure in belactosin A, is a much more potent proteasome inhibitor than belactosin
A. However, its cell growth inhibitory effect is rather lower than that expected from its remarkable pro-
teasome inhibitory effect, probably due to its instability under cellular conditions. We hypothesized that
the instability of 2a was due to chemical and enzymatic hydrolysis of the strained β-lactone moiety. Thus,
to increase the stability of 2a by chemical modification, its analogs with a sterically more hindered
β-lactone moiety and/or cyclopropylic strain-based conformational restriction were designed and syn-
thesized, resulting in the identification of a stabilized analog 6a as a proteasome inhibitor with cell
growth inhibitory effects. Our findings suggest that the chemical and biological stability of 2a is signifi-
cantly affected by the steric hindrance around its β-lactone carbonyl moiety and the conformational flexi-
bility of the molecule.
Received 29th June 2013,
Accepted 8th August 2013
DOI: 10.1039/c3ob41338a
www.rsc.org/obc
physiologically important cellular processes, such as signal
transduction,4 cell cycle progression,5 and unfolded protein
Introduction
The chemical and biological stability of small molecules response (UPR).6 Because inhibition of the proteasome causes
depends on their chemical structures, and therefore it can be cell cycle arrest to induce apoptosis, the proteasome is an
regulated by structural modifications.1 In the drug discovery attractive target for the development of anti-cancer drugs.7 For
process, compounds with insufficient stability often degrade example, a proteasome inhibitor bortezomib is clinically
rapidly in vivo and sometimes bind covalently to off-target effective for the treatment of multiple myeloma8 and mantle
molecules, resulting in the absence of the desired pharmaco- cell lymphoma.9
logical effect, and even worse, producing an undesired toxic
Belactosin A is a proteasome inhibitor isolated from the
side-effect.2 The chemical and biological instability of com- Streptomyces sp. by Asai,10 which inhibits the proteasome co-
pounds can be improved by changing the steric and/or electro- valently by acylating the active site Thr residue via ring-clea-
static properties of the labile moiety. Furthermore, when the vage of its strained β-lactone moiety.11 Because the binding
compound is unstable in vivo due to enzymatic degradation, it site of belactosin derivatives differs from that of other protea-
can be stabilized by changing structural features such as mole- some inhibitors,11,12 belactosin A is an attractive potential lead
cular size, electrostatic property, hydrophobicity, and confor- for the development of novel proteasome inhibitors (Fig. 1). In
mation to reduce the affinity for the degrading enzyme.
recent years, we have investigated the three-dimensional struc-
The ubiquitin–proteasome system is the major degradation ture–activity relationship (SAR) study of belactosin A and
pathway of intracellular proteins,3 which are involved in many identified the unnatural cis-cyclopropane isomer 1 as a more
potent proteasome inhibitor than belactosin A having the
trans-cyclopropane structure.13 Furthermore, we investigated
the SAR of 1 resulting in identification of the optimized inhi-
aFaculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan
bGraduate School of Pharmaceutical Sciences, University of Shizuoka, Yada,
bitor 2a, which appeared to be as potent as the clinical drug
bortezomib (Fig. 2).14 Despite its remarkable proteasome
inhibitory effect, however, its inhibitory effect on cell growth is
not so strong, compared with other potent inhibitors such as
bortezomib15 or carfilzomib,16 as summarized in Table 1. In
our previous study, we investigated the stability of 2b, instead
Shizuoka 422-8526, Japan
cCenter for Research and Education on Drug Discovery, Hokkaido University, Kita-12,
Nishi-6, Kita-ku, Sapporo 060-0812, Japan. E-mail: shu@pharm.hokudai.ac.jp;
Fax: +81-11-706-3769; Tel: +81-11-706-3769
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob41338a
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Table 1 Inhibitory effect of 2a, bortezomib and carfilzomib on proteasome
chymotrypsin-like (CT-L) activity and HCT116 cell growth
IC50 [nM]
Proteasome
(CT-L activity)
Cell growth
(HCT116)
IC50 ratio (cell
growth/CT-L activity)
Compound
2a
5.7
4.5
6.3
1820
5.0
8.5
319
1.1
1.3
Bortezomib
Carfilzomib
Fig. 3 Stability of 2b in 0.1 M TEAA buffer (pH 7.4) and human AB serum at
37 °C.
Furthermore, it was found that 2b is significantly unstable
under biological conditions (t1/2 = 2.3 min in serum), which
might be correlated with the relatively weak cell growth inhi-
bitory effect of 2a, because 2a can be as unstable as 2b (Fig. 3).
Thus, we planned to develop stabilized derivatives of 2a. Here
we describe the design, synthesis, biological activities, and
chemical and biological stability of the newly designed
compounds.
Fig. 1 Known proteasome inhibitors.
Results and discussion
Design of compounds
The reactivity of the carbonyl group with nucleophiles is
affected by the steric hindrance around its carbon atom,18 and
we therefore designed compounds 3a–5a with various substitu-
ents at the α-carbon of the β-lactone carbonyl group of 2a
(Fig. 4a) to change the bulkiness at the position. The order of
the steric hindrance around the β-lactone carbonyl group is
thought to be 3a < 2a < 4a < 5a, as depicted in Fig. 4a.
On the other hand, because enzyme recognition can be
influenced by the three-dimensional structure of the substrate,
conformational restriction of 2a and its analogs might result
in lowering the affinity for the degrading enzyme, and we
therefore designed 6a as a conformationally restricted analog.
The cis-oriented adjacent substituents on the cyclopropane
ring are fixed in the eclipsed orientation, and accordingly, they
exert significant mutual steric repulsion, which we previously
termed “cyclopropylic strain”.19 Due to this characteristic
Fig. 2 Previous SAR studies of belactosin A performed by our laboratory.
of 2a due to its poor solubility in aqueous medium, and structural feature, conformation of the substituents (Fig. 5a)
demonstrated that 2b is gradually degraded in aqueous on a cyclopropane ring can be restricted, and therefore, in
medium, while its half-life (t1/2 = 10 h in pH 7.4 buffer)14b compound 5a, conformers A (anti, the cyclopropane ring
is longer than that of other β-lactone-type proteasome inhi- “down”/the side chain “up”) and B (syn, the cyclopropane ring
bitors (omuralide, 13 min; salinosporamide A, 56 min).17 “down”/the side chain “down”) would be preferable (Fig. 5b).
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Fig. 4 Structure of newly designed compounds 3–6 and their parent compound 2. (a) Relative steric hindrance around the β-lactone carbonyl group is also shown.
(b) The structure of conformationally restricted analog 6.
in its anti-form (Fig. 5b). Notably, this cyclopropylic strain-
based conformational restriction can be achieved by the
minimal structural change, i.e., only the introduction of a
methyl group, allowing us to more rigorously investigate the
relationship between conformation and stability.
Compounds 3a–6a were thought to be poorly soluble in
aqueous medium; therefore we also planned to synthesize
compounds 3b–6b, which are analogs of 3a–6a without the
N-terminal Cbz group, to evaluate their stability under
aqueous conditions instead of 3a–6a.
Synthesis
The target compounds 3a–6a would be obtained by conden-
sation between the unit A or B and the unit C, D, or E.
Although the synthesis of units A and B has been described in
our previous report,14b,20 we needed to prepare the β-lactone
units C–E (Scheme 1). In particular, in the synthesis of D and
E, construction of the chiral all-carbon quaternary center adja-
cent to the β-lactone carbonyl group would be a key step.
The β-lactone unit C was prepared as shown in Scheme 2,
Fig. 5 The cyclopropylic strain-based conformational restriction. (a) General
using a procedure similar to that for the preparation of the
representation of the cyclopropylic strain. (b) Presumed stable conformation of 5
β-lactone unit in the total synthesis of belactosin A by
(syn/anti) and 6 (syn).
Armstrong et al.21 4-Methylpentanoic acid 7 was condensed with
(4R)-4-benzyl-2-oxazolidinone by the mixed anhydride method
using LiCl as an additive22 to give 8, which was treated with
Previously, we demonstrated that the bioactive conformation
of the cis-cyclopropane belactosin derivatives seems to be
syn.20 Therefore, we designed conformationally restricted
analog 6a (Fig. 4b), whose conformation is restricted in the
syn-form due to the significant steric repulsion between the
introduced 1′R-methyl group and the cis-oriented amide group
BrCH2CO2t-Bu/NaHMDS at −78 °C in THF to afford 9 stereose-
lectively.23,24 The oxazolidinone moiety of 9 was removed by
hydrolysis with LiOH/H2O2 in aqueous THF to give 10.25 The
α-position of the t-butyl ester in 10 was diastereoselectively
chlorinated with CCl4/LiHMDS in THF at −78 °C,26 which
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Scheme 1 Synthetic plan of 3a–6a.
Scheme 2 Synthesis of β-lactone units C and D.
seemed to proceed through the Li-chelated seven-membered
dianion transition state,23b followed by the ring-closing reac-
tion under alkaline two-phase conditions to afford the
β-lactone 11 (unit C, Pg = t-Bu).27
The synthesis of unit D is also shown in Scheme 2. Starting
from propionic acid 12, the β-lactone 16 was prepared accord-
ing to the same procedure used for the synthesis of 11.
Methanolysis of 16 yielded the ring-opened product 17, the
substrate for the key reaction forming the asymmetric quatern-
ary carbon center. Treatment of 17 with LiHMDS/3-bromo-2-
methylpropene in THF at −78 °C to 0 °C afforded the desired
alkylated product 18 as a single isomer.28 The reaction seemed
to proceed through the Li-chelated six-membered transition
state, in which the bulky t-butyl ester group prevents access of
the electrophile from the upper side as shown in Scheme 2.29
Hydrogenation of 18 afforded 19, and subsequently its
methyl ester moiety was selectively hydrolyzed with LiOH in
aqueous THF, followed by ring-closing reaction with PyBOP30
to afford the β-lactone 20 (unit D, Pg = t-Bu). The relative
stereochemistry of 20 was determined by NOE experiments
(Fig. 6a).31
Fig. 6 NOE experiments of 20 (a) and 32 (b).
The synthesis of unit E is shown in Scheme 3. L-Isoleucine
(21) was deaminated32 to afford 22, which was converted to the
alcohol 25 according to the same procedure used for the syn-
thesis of 17 described above. Next, we tried to construct the
asymmetric quaternary carbon center by stereoselective
methylation of 25 as in the synthesis of unit D. Although the
reaction was investigated under various conditions, it did not
proceed at all. Because the bulky (S)-sec-butyl side chain of 25
seems to lower the reactivity, we next examined the
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Scheme 3 Synthesis of β-lactone unit E.
methylation reaction with the β-lactone33 27 as a substrate, product was successively treated with TFAA and with benzyl
which was prepared by removal of the t-butyl group of 24 with alcohol, which gave the desired benzyl ester 31 exclusively.35
TFA. Thus, when 27 was treated with LDA/MeI in THF at Finally, 31 was treated with PyBOP30 to yield the β-lactone 32
−78 °C to −40 °C, the desired methylated product 28 was (unit E, Pg = Bn). The relative stereochemistry of 32 was deter-
obtained as a diastereomeric mixture (dr 3 : 1), while the yield mined by NOE experiments (Fig. 6b).36
was low. The stereoselectivity of the reaction might be caused
The synthesized β-lactone units 11, 20 and 32 were depro-
by steric repulsion due to the carboxy group as depicted in tected and finally condensed with unit A or B to yield 3a–6a.
Scheme 3. The carboxy group of 28 was re-protected with a Compounds 3b–6b were also synthesized by hydrogenolysis of
t-butyl group and subsequent methanolysis gave 26, which was 3a–6a (Scheme 4).
obtained as a single isomer after silica gel column chromato-
graphy purification. The secondary alcohol moiety of 26 was
Chemical and biological stability of 2b–6b
oxidized with Dess–Martin periodinane and subsequent
reduction of the resulting carbonyl group with (R)-2-methyl-
CBS-1,3,2-oxazaborolidine34 resulted in complete inversion of
its stereochemistry to give the corresponding epimer 29.
Although we attempted to selectively hydrolyze the methyl
ester moiety of 29, the desired mono-ester 30 was not obtained
at all, even under SN2 reaction conditions. Thus, we hydrolyzed
both the methyl and t-butyl ester moieties of 29, and then the
We evaluated the chemical and biological stability of 2b–6b.
The compounds were incubated in 0.1 M TEAA buffer (pH 7.4)
or human AB serum at 37 °C, and the time courses were ana-
lyzed by HPLC to obtain the half-life (t1/2), the results of which
are shown in Fig. 7. In 0.1 M TEAA buffer, the order of their
stability was 3b < 2b < 4b, 5b, 6b, which clearly corresponds to
the order of the steric hindrance around their β-lactone carbo-
nyl group (Fig. 4a), as we expected. Notably, 4b, 5b, and 6b
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Scheme 4 Synthesis of 3a–6a and 3b–6ba. a Reagents and conditions: (a) TFA/CH2Cl2, −5 °C; (b) Pd/C, H2, THF, quant.; (c) TFA/CH2Cl2; (d) PivCl, Et3N, CH2Cl2, 0 °C
to rt, 62% (3a, 2 steps from A), 100% (4a, 2 steps from A); (e) EDC·HCl, HOAt, Et3N, CH2Cl2, 0 °C, quant. (5a, 2 steps from A), 91% (6a, 2 steps from B); (f ) Pd/C, H2,
TFA–THF, 0 °C, quant. (3b–6b).
Fig. 7 Chemical and biological stability of 2b–6b. (a) Time courses of 2b–6b in 0.1 M TEAA buffer (pH 7.4) at 37 °C analyzed by HPLC. (b) Time courses of 2b–6b
in human AB serum at 37 °C analyzed by HPLC. (c) Calculated half-life of 2b–6b under each condition. ND: not degraded.
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which have a quaternary carbon adjacent to their β-lactone car- cyclopropylic strain resulted in significant stabilization in
bonyl group were quite stable and no degradation was human serum probably due to the decreased affinity for meta-
observed under the conditions.
bolic enzymes. The correlation between conformation and
Similarly, in human AB serum, the order of their stability metabolic stability has not been well studied, and this study
was 3b < 2b < 4b < 5b < 6b, where the relative stability of 2b–5b presents an interesting example of their clear correlation.
also depended on the steric hindrance around their β-lactone
carbonyl group, while their half-life was remarkably short com-
pared with those in 0.1 M TEAA buffer, suggesting that 2b–6b
were degraded enzymatically in human AB serum. Further-
Abbreviations
more, 6b, the conformationally restricted analog of 5b, was sig- Bn
nificantly more stable than 5b, where the t1/2 was longer than Boc
1 h. Therefore, the conformational restriction might result in CBS
lowering the affinity for the degradation enzyme, as we hypoth- Cbz
esized. This finding is an interesting example of the corre- CT-L
lation between conformational flexibility and biological DMP
instability. Thus, we successfully identified 6a as a chemically EDC
Benzyl
t-Butoxycarbonyl
Corey–Bakshi–Shibata
Benzyloxycarbonyl
Chymotrypsin-like
Dess–Martin periodinane
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Ethyl
and biologically stable analog of 2a.
Et
HOAt
HPLC
LDA
1-Hydroxy-7-azabenzotriazole
High-pressure liquid chromatography
Lithium diisopropylamide
Pharmacological effects of 6a
We investigated the inhibitory effect of the highly stable 6a on
the CT-L activity of the proteasome and HCT116 cell growth
(Table 2). Notably, 6a (IC50 = 4.0 μM) showed a cell growth
inhibitory effect comparable to that of 2a (IC50 = 1.8 μM),
despite its significantly lowered proteasome inhibitory activity
(IC50 = 1.3 μM) compared with that of 2a (IC50 = 0.0057 μM).
The IC50 ratio (cell growth/CT-L activity) of 6a was 3.1, which is
remarkably improved over that of 2a (319), and it was almost
the same as those of bortezomib and carfilzomib (Table 1).
These findings suggest that the lower cell growth inhibitory
effect of 2a arises from its instability as we expected, and that
structural optimization of 6a might lead to the development of
highly potent cell growth inhibitors.
LiHMDS Lithium hexamethyldisilazide
Me
Piv
PyBOP
Methyl
Pivaloyl
Benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate
NaHMDS Sodium hexamethyldisilazide
NOE
SAR
TEAA
TFA
TFAA
THF
Thr
Nuclear Overhauser effect
Structure–activity relationship
Triethylammonium acetate
Trifluoroacetic acid
Trifluoroacetic anhydride
Tetrahydrofuran
Threonine
TMS
UPR
Trimethylsilyl
Unfolded protein response
In summary, by chemical modification of 2a, we success-
fully developed a chemically and biologically stabilized analog
6a, in which the steric hindrance around the unstable
β-lactone moiety and the cyclopropylic strain-based confor-
mational restriction would work together to stabilize the mole-
cule. The cell growth inhibitory activity of 6a is comparable to
its proteasome inhibitory activity, so that the structural optim-
ization of 6a might result in highly potent cell growth inhibi-
tors. The chemical and biological stability of 2a derivatives is
well correlated to the steric hindrance around their β-lactone
carbonyl group due to the bulkiness of their α-carbon substitu-
ents. Furthermore, conformational restriction by the
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reported previously by Armstrong et al. using (R)-oxazolidi-
none instead of (S)-oxazolidinone (ref. 21). The [α]D and
NMR data of 24 are different from those of the reported
diastereomer. Thus, absolute configuration of 32 was deter-
mined as shown in Scheme 3.
6622 | Org. Biomol. Chem., 2013, 11, 6615–6622
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