Synthesis of boronic acid derivatives of tyropeptin: Proteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.117

Boronic acid derivatives of tyropeptin were synthesized with TP-110 as the lead compound. Due to the lability of the aminoboronic acid moiety, careful design of the deprotection and coupling sequence was required. Liquid-liquid partition chromatography wa

Full text of DOI:10.1016/j.bmcl.2009.02.117

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2343–2345
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of boronic acid derivatives of tyropeptin: Proteasome inhibitors
b,
a
a
a
a
Takumi Watanabe a, , Isao Momose , Masatoshi Abe , Hikaru Abe , Ryuichi Sawa , Yoji Umezawa ,
*
*
Daishiro Ikeda b, Yoshikazu Takahashi a, Yuzuru Akamatsu a
a Molecular Structure Research Group, Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
b Numazu Bio-Medical Research Institute, Microbial Chemistry Research Center, 18-24 Miyamoto, Numazu 410-0301, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Boronic acid derivatives of tyropeptin were synthesized with TP-110 as the lead compound. Due to the
lability of the aminoboronic acid moiety, careful design of the deprotection and coupling sequence
was required. Liquid–liquid partition chromatography was found to be a powerful tool for purification
of compounds of this class. The obtained derivatives showed potent inhibitory activities against the
human 20S proteasome in vitro.
Received 19 November 2008
Revised 9 February 2009
Accepted 11 February 2009
Available online 4 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Proteasome
Inhibitor
Boronic acid
Centrifugal liquid–liquid partition
chromatography
The proteasome, a multicatalytic threonine protease, is respon-
sible for ubiquitin-dependent nonlysosomal proteolysis.1 This en-
zyme has three distinct active sites that are individually
responsible for the chymotryptic, peptidyl-glutamyl peptide-
hydrolyzing (PGPH), and tryptic proteolytic activities.2 Among
these, the chymotrypsin-like activity is of greatest interest, and
much research in medicinal chemistry has focused on it.3,4
against human prostate cancer PC-3 cells. Encouraged by these re-
sults, another SAR study was performed in which TP-110 was used
as the lead compound (Fig. 1).
In 2003, FDA approved the first clinical use of the proteasome
inhibitor bortezomib 4 (also referred to as PS-341, VelcadeÒ) for
the treatment of multiple myeloma. The most characteristic struc-
tural feature of this compound is the presence of the boronic acid
moiety, a functional group found in various bioactive agents.15,16 A
report by Adams et al.17 showed that in comparison with the
antagonistic activity of the aldehyde congener, bortezomib is a
>2500-fold more potent antagonist of the chymotrypsin-like activ-
Elevated levels of the proteasome have been implicated in many
diseases including cancer. In fact, it has been reported that the
anticancer activity of proteasome inhibitors is due to inhibition
of the transcriptional factor NF-j
B;5,6 stabilization of p21, p27,
and p53;7,8 and suppression of the unfolded protein response
(UPR).9 Thus, proteasome inhibitors are recognized as promising
anticancer agents.10,11
Previously, we reported the isolation and structural determina-
tion of the novel proteasome inhibitors tyropeptins A and B (1a
and 1b) produced by Kitasatospora sp. MK993-dF2.12,13 Tyropeptins
have a tripeptide structure with a formyl functionality at the P1
position, which can form a hemiacetal with the nucleophilic hydro-
xyl group in the catalytic site of the proteasome. An SAR study of
tyropeptins was carried out later in which more potent compounds
that were selective inhibitors of chymotryptic activity were de-
signed and synthesized.14 It is noteworthy that TP-110, one of
these derivatives, exhibited outstanding growth inhibitory activity
* Corresponding authors. Tel.: +81 3 3441 4173; fax: +81 3 3441 7589 (T.W.), tel.:
+81 55 924 0601; fax: +81 55 922 6888 (I.M.).
E-mail addresses: twatanabe@bikaken.or.jp (T. Watanabe), imomose@bikaken.
or.jp (I. Momose).
Figure 1. Structure of tyropeptins (1a, 1b) and TP-110 (2).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.117

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T. Watanabe et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2343–2345
diastereoselectivity. The absolute configuration of the newly intro-
duced stereocenter of the major isomer was determined to be S by
converting 8 into the reported compound 9 (1-(4-methoxyphenyl)-
2-butanol) and comparing their signs of optical rotation.20,21 The
chlorine atom of 8 was then substituted with a nitrogen function-
ality in an SN2 manner by treatment with LiHMDS. Acidic desilyla-
tion of 10 resulted in the desired Tyr(Me)-boronic acid 11a in the
form of a TFA salt. The Phe- and Leu-boronic acids (11b and 11c,
respectively) were prepared according to previously described
procedures.18,19
The subsequent coupling reaction of 11a–c and 1-naphtylace-
tyl-Tyr(Me)-Val-OH 12 proceeded without any difficulty (Scheme
2). The stage was then set for final deprotection of the resultant
tripeptides 13a–c, which led to the target compounds 3a–c. Previ-
ous procedures for the syntheses of peptide boronates generally
employed one of two methods: transesterifications with an addi-
tive boronic acid such as i-BuB(OH)2 (condition A) or hydrolysis
of the esters in aqueous media and concomitant oxidative cleavage
of the liberated pinanediol to direct equilibrium to the accumula-
tion of free boronic acids (condition B). However, these methods
failed to give the final products 3a–c, presumably because of the
limited solubility of the substrates in the media. Modification of
the reaction conditions by using various combinations of organic
solvents and reagents were also unsuccessful. These discouraging
results forced us to examine deprotection at an earlier step. In fact,
removal of the pinanediols of 11a–c under the transesterification
conditions resulted in successful synthesis of aminoboronic acid
hydrochlorides 14a–c (Scheme 2). The efficacy of the subsequent
peptide bond formation depended on the coupling reagent: the
use of WSCÁHCl, TBTU, DPPA, or BOP led to boron-proton exchange
to give 15a–c. Only diethyl cyanophosphate resulted in an accept-
able amount of the desired product; however, it could not be sep-
arated from the byproducts derived from the coupling reagent.
After careful optimization of the reaction sequence and purifica-
tion method, a synthetic route was finally established; this is
Figure 2. Boronic acid derivatives of tyropeptin (3a–c) and bortezomib (4).
ity of the proteasome. The boronate moiety can also form covalent
bonds with the nucleophilic hydroxyl group in the catalytic site of
the proteasome. The remarkable enhancement of inhibition is due
to the slow dissociation of oxygen from boron. Inspired by the re-
sults of this study, we synthesized tyropeptin boronic acid deriva-
tives, as shown in Figure 2 (3a–c). Apart from 3a, which was
designed on the basis of TP-110, other structurally related analogs
were prepared that had Leu- or Phe-boronic acid as the P1 residue
(3b and 3c, respectively). By comparing the proteasome-inhibitory
activities of all these compounds, we expected to clarify the mode
of interaction between the S1 cavity of the proteasome and P1 side
chains of the inhibitors.
Scheme 1 illustrates the synthesis of Tyr(Me)-boronic acid 11a
from 4-methoxybenzyl chloride 5.18,19 The Grignard reagent gener-
ated from 5 was treated with triethylborate, and subsequent
hydrolysis with H2SO4 resulted in 4-methoxybenzylboronic acid
6. Subsequent esterification with (+)-pinanediol afforded 7. A ster-
eoselective one-carbon homologation with LiCHCl2 employing the
pinane part as the chiral auxiliary resulted in chloride 8 with >98:2
Scheme 1. Reagents and conditions: (a) (i) Mg, THF, then B(OEt)3; (ii) H2SO4, H2O, 41%; (b) (+)-pinanediol, Et2O, 63%; (c) LiCHCl2, ZnCl2, THF–Et2O, 98% (>96% de); (d) LiHMDS,
THF, 84%; (e) TFA, hexane, 95%.
Scheme 2. Reagents and conditions: (a) WSCÁHCl, HOBt, i-Pr2EtN, CH2Cl2; (b) condition A: NaIO4, H2O, organic co-solvent (acetone, THF, or DMF); condition B: i-BuB(OH)2,
0.1 M HCl–hexane; (c) i-BuB(OH)2, 0.1 M HCl–hexane; (d) 12, diethyl cyanophosphate, DMF; (e) condition A: 12, WSCÁHCl, HOBt, i-Pr2EtN, CH2Cl2; condition B: 12, TBTU,
HOBt, i-Pr2EtN, THF; condition C: 12, DPPA, TEA, DMF; condition D: 12, BOP reagent, TEA, CH2Cl2.

T. Watanabe et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2343–2345
2345
Scheme 3. Reagents and conditions: (a) 11a–c, WSCÁHCl, HOBt, i-Pr2EtN, CH2Cl2, 69% for 17a, 42% for 17b, 48% for 17c; (b) (i) TFA, CHCl3; (ii) i-BuB(OH)2, 0.1 M HCl–hexane,
88% for 18a, 62% for 18b, 80% for 18c; (c) 1-naphthylacetic anhydride, DMAP, CH2Cl2, 72% for 3a, 72% for 3b, quant. for 3c.
Table 1
In summary, boronic acid derivatives of tyropeptin were syn-
Proteasome-inhibitory activities9 of tyropeptin boronic acid derivatives (3a–c and
thesized in this study, and careful design of the reaction sequence
18a–c), TP-110 (2), and bortezomib (4)
and CPC purification resulted in the desired compounds without
Compounds
Chymotrypsin-like
activity
PGPH
activity
Trypsin-like
activity
loss of the boronate moiety. The tyropeptin derivatives 3a–c are
good potential inhibitors of the chymotryptic activity of protea-
some. In particular, the Leu-boronate derivative 3c displayed an
IC50 value that was even lower than that of bortezomib. Further
investigations on the biological activities of these compounds,
including their cytotoxicity and anti-tumor activity, are currently
underway and will be reported in due course.
3a
3b
3c
18a
0.0063
0.0044
0.0026
0.13
>40
7.4
0.94
16
7.7
1.4
5.6
4.6
2.8
3.7
4.0
1.9
>40
>40
18b
18c
TP-110 (2)
Bortezomib (4)
0.10
0.021
0.083
0.024
>40
0.73
Supplementary data
The IC50 values are in lM.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.117.
shown in Scheme 3. Boc-Tyr(Me)-Val-OH (16), which was already
synthesized in the previous study,14 was attached to 11a–c using
WSCÁHCl to provide the tripeptide intermediates 17a–c. The Boc
group and chiral auxiliary were removed in the usual manner to
obtain the tripeptide boronates as HCl salts (18a–c). Centrifugal li-
quid–liquid partition chromatography (CPC) using a solvent sys-
tem of CHCl3/MeOH/H2O (5:6:4) in the ascending mode was
found to be effective for the purification of the relatively unstable
intermediates. Finally, acylation of the amino groups of 18a–c with
1-naphthylacetyl anhydride provided the desired tyropeptin-boro-
nate analogs 3a–c, which were also purified by CPC (CHCl3/MeOH/
H2O (5:6:4), in the descending mode).
References and notes
1. Ciechanover, A. Angew. Chem., Int. Ed. 2005, 44, 5944.
2. Orlowski, M.; Cardozo, C.; Michaud, C. Biochemistry 1993, 32, 1563.
3. Lopes, U. G.; Erhardt, P.; Yao, R.; Cooper, G. M. J. Biol. Chem. 1997, 272, 12893.
4. An, B.; Goldfarb, R. H.; Siman, R.; Dou, Q. P. Cell Death Differ. 1998, 5, 1062.
5. Frankel, A.; Man, S.; Elliot, P.; Adams, J.; Kerbel, R. S. Clin. Cancer Res. 2000, 6,
33719.
6. Sunwoo, J. B.; Chen, Z.; Dong, G.; Yeh, N.; Bancroft, C. C.; Sausville, E.; Adams, J.;
Elliot, P.; Van Waes, C. Clin. Cancer Res. 2001, 7, 1419.
7. Hideshima, T.; Richardson, P.; Chauhau, D.; Palombella, V. J.; Elliot, P. J.; Adams,
J.; Anderson, K. C. Cancer Res. 2001, 61, 3071.
8. Ling, Y.-H.; Liebes, L.; Jiang, J.-D.; Holland, J. F.; Elliot, P. J.; Adams, J.; Muggia, F.
M.; Perez-Solar, R. Clin. Cancer Res. 2003, 9, 1145.
The synthesized tyropeptin boronic acid derivatives22 were sub-
jected to in vitro assays for measuring the proteasome-inhibitory
activities (Table 1, the human 20S proteasome was purchased from
Biomol, Plymouth Meeting, PA).12 To evaluate the influence of the
acyl group at the N-terminus on the inhibitory effect, the tripeptide
HCl salts 18a–c were also tested. Substitution of the formyl group
of TP-110 (2) with boronic acid (3a) resulted in moderate improve-
ment in the inhibition of the chymotryptic proteasome activity. It
is noteworthy that compound 3c, which has an aliphatic side chain
at P1, is an approximately ninefold more potent inhibitor of the
chymotryptic activity of proteasome than bortezomib. The lower
in vitro activity of 3a and 3b in comparison to that of 3c implies
that the S1 pocket of proteasome favors aliphatic side chains over
benzylic ones. Comparison of the results of 3a and 3b suggests that
the existence of the methoxy group on the phenyl group in the P1
side chain has only a small impact on the affinity to the catalytic
site of the proteasome. Removal of the 1-naphthylacetyl group
(18a–c) resulted in substantial loss of activity. Moreover, all syn-
thesized tyropeptin derivatives showed selectivity toward the chy-
motrypsin-like activity.
9. Lee, A.-H.; Iwakoshi, N. N.; Anderson, K. C.; Glimcher, L. H. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 9946.
10. Mitsiades, C. S.; Mitsiades, N.; Hideshima, T.; Richardson, P. G.; Anderson, K. C.
Curr. Drug Targets 2006, 7, 1341.
11. Rajkumar, S. V.; Richardson, P. G.; Hideshima, T.; Anderson, K. C. J. Clin. Oncol.
2005, 23, 630.
12. Momose, I.; Sekizawa, R.; Hashizume, H.; Kinoshita, N.; Homma, Y.; Hamada,
M.; Iinuma, H.; Takeuchi, T. J. Antibiot. 2001, 54, 997.
13. Momose, I.; Sekizawa, R.; Hirosawa, S.; Ikeda, D.; Naganawa, H.; Iinuma, H.;
Takeuchi, T. J. Antibiot. 2001, 54, 1004.
14. Momose, I.; Umezawa, Y.; Hirosawa, S.; Iinuma, H.; Ikeda, D. Bioorg. Med. Chem.
Lett. 2005, 15, 1867.
15. Yang, W.; Gao, X.; Wang, B. Med. Res. Rev. 2003, 23, 346.
16. Groziak, M. P. Am. J. Ther. 2001, 8, 321. See also references cited therein.
17. Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.; Greiner, L.;
Klunder, J. M.; Ma, Y.-T.; Plamondon, L.; Stein, R. L. Bioorg. Med. Chem. Lett.
1998, 8, 333.
18. Matteson, D. S.; Sadhu, K. M.; Lienhard, G. E. J. Am. Chem. Soc. 1981, 103, 5241.
19. Kettner, C. A.; Shenvi, A. S. J. Biol. Chem. 1984, 259, 15106.
20. Ferraboschi, P.; Grisenti, P.; Manzocchi, A.; Santaniello, E. J. Chem. Soc., Perkin
Trans. 1 1990, 2469.
21. Acetti, D.; Brenna, E.; Fuganti, C. Tetrahedron: Asymmetry 2007, 18, 488.
22. All compounds were characterized by 1H NMR, 13C NMR, and HRMS. Details of
the synthetic procedure and physicochemical data of the unreported
compounds are provided as Supplementary data.