Structure-activity relationship of boronic acid derivatives of tyropeptin: Proteasome inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.07.122

The structure-activity relationship of the boronic acid derivatives of tyropeptin, a proteasome inhibitor, was studied. Based on the structure of a previously reported boronate analog of tyropeptin (2), 41 derivatives, which have varying substructure at the N-terminal acyl moiety and P2 position, were synthesized. Among them, 3-phenoxyphenylacetamide 6 and 3-fluoro picolinamide 22 displayed the most potent inhibitory activity toward chymotryptic activity of proteasome and cytotoxicity, respectively. The replacement of the isopropyl group in the P2 side chain to H or Me had negligible effects on the biological activities examined in this study.

Full text of DOI:10.1016/j.bmcl.2010.07.122

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5839–5842
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of boronic acid derivatives of tyropeptin:
Proteasome inhibitors
a
b
a
b
Takumi Watanabe a, , Hikaru Abe , Isao Momose , Yoshikazu Takahashi , Daishiro Ikeda ,
*
Yuzuru Akamatsu a
a Institute of Microbial Chemistry, Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
b Institute of Microbial Chemistry, Numazu, 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:
The structure–activity relationship of the boronic acid derivatives of tyropeptin, a proteasome inhibitor,
was studied. Based on the structure of a previously reported boronate analog of tyropeptin (2), 41 deriv-
atives, which have varying substructure at the N-terminal acyl moiety and P2 position, were synthesized.
Among them, 3-phenoxyphenylacetamide 6 and 3-fluoro picolinamide 22 displayed the most potent
inhibitory activity toward chymotryptic activity of proteasome and cytotoxicity, respectively. The
replacement of the isopropyl group in the P2 side chain to H or Me had negligible effects on the biological
activities examined in this study.
Received 20 July 2010
Revised 26 July 2010
Accepted 27 July 2010
Available online 1 August 2010
Keywords:
Proteasome inhibitor
Boronic acid
Ó 2010 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Cytotoxicity
Multiple myeloma
Tyropeptin
Proteasome, a multicatalytic threonine protease, is responsible
for ubiquitin-dependent nonlysosomal proteolysis.1 This enzyme
has three distinct active sites that are individually responsible for
the chymotrypsin-like, caspase-like, and trypsin-like proteolytic
activities.2 Among these, the chymotrypsin-like activity is of great-
est interest, and much research in medicinal chemistry has been
focused on it.3,4
Elevated levels of the proteasome have been implicated in many
diseases including cancer. In fact, it has been reported that the
anti-cancer activity of proteasome inhibitors is due to inhibition
trypsin-like activity of human proteasome when compared to tyro-
peptin A (Fig. 1).
Encouraged by these results, we conducted further SAR studies
of tyropeptin-boronic acid derivatives. In the present study, the ef-
fect of acyl moiety located at the N-terminus on the proteasome-
inhibitory activity and cytotoxicity against RPMI8226 cells derived
from multiple myeloma was investigated. Proteasome-inhibitory
activities were determined using purified human erythrocyte-de-
rived 20S proteasome (Enzo Life Sciences. Plymouth Meeting, PA)
as previously described.13
Scheme 1 summarizes the procedure for synthesizing the tyro-
peptin-boronic acid derivatives used in this study. According to the
method reported previously,15 41 analogs of 2 that have a variety
of acyl groups at the N-terminus were prepared using WSCꢀHCl
as a coupling reagent with corresponding carboxylic acids and
the peptide boronate 4.16
Table 1 shows the inhibitory activity toward proteasome and
the cytotoxicity of tyropeptin-boronic acid derivatives synthesized
for this study. The almost identical biological activities were ob-
served for the previously reported 1-naphtylacetyl derivative 2
and its regioisomer 5. The most potent inhibitor of chymotryp-
sin-like activity was analog 6, which has a 3-phenoxyphenylacetyl
group at the N-terminal acyl moiety; almost ninefold more potent
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 Indeed, proteasome inhibitors have been recognized as
promising candidates for anti-cancer agent,10,11 since the US Food
and Drug Administration approved the first clinical use of a com-
pound from this class, bortezomib 3 (also referred to as PS-341,
VelcadeÒ), for the treatment of multiple myeloma.
Previously, we reported the isolation and structural determina-
tion of the novel proteasome inhibitors tyropeptins A (1) produced
by Kitasatospora sp. MK993-dF2,12,13 and structure–activity rela-
tionship (SAR) studies of tyropeptin derivatives.14,15 In these stud-
ies, tyropeptin-boronic acid derivatives (2 as a representative)
were found to exhibit enhanced inhibitory activity against chymo-
than bortezomib 3 (IC50: 0.0041 for 6 and 0.039 lM for 3). Unfor-
tunately these compounds showed weak antitumor activity,17
which prompted us to use different acyl groups. Instead, we chose
* Corresponding author. Tel.: +81 3 3441 4173; fax: +81 3 3441 7589.
E-mail address: twatanabe@bikaken.or.jp (T. Watanabe).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.122

5840
T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5839–5842
Figure 1. Structure of tyropeptin A (1), a tyropeptin-boronic acid derivative (2), and bortezomib (3).
Scheme 1. Reagents and conditions: (a) carboxylic acid, WSCꢀHCl, HOBt, iPr2NEt, CH2Cl2; (b) (i) H-Gly-OBn or H-Ala-OBn, WSCꢀHCl, HOBt, iPr2NEt, CH2Cl2; (ii) H2, Pd/C,
MeOH; (c) 47, WSCꢀHCl, HOBt, iPr2NEt, CH2Cl2; (d) (i) TFA, CHCl3; (ii) iBuB(OH)2, 1 M HCl, hexane; (e) 2-picolinic acid, WSCꢀHCl, HOBt, iPr2NEt, CH2Cl2.
various N-heteroaromatic rings because bortezomib 3 has a pyra-
zine carboxamide moiety.
First, amide derivatives of commercially available carboxylic
acids having quinoline, isoquinoline, pyrazine, and pyridine nuclei

T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5839–5842
5841
Table 1
displayed one of the most potent cytotoxicities against RPMI8226
among the tyropeptin-related compounds synthesized in our labo-
Biological activities of tyropeptin-boronic acid derivatives, and bortezomib (IC50: lM)
ratory (IC50: 0.0049 lM). Here again, the potency of the inhibitory
activity toward proteasome and cytotoxicity did not coincide with
Compounds Chymotrypsin-
like activity
Caspase-
Trypsin-
Cytotoxicity
like activity like activity (RPMI8226)
each other. In fact, 22 showed only a moderate activity toward pro-
teasome (IC50: 0.14 lM).
Other than the deleterious effect of an OH group on the inhibi-
tion of chymotryptic activity, no obvious relationship was ob-
served between the biological activities examined in this study
and the structure of the pyridyl moiety.
5
6
7
8
9
0.022
0.0041
0.041
0.059
0.38
39
29
19
11
>40
16
32
24
16
33
23
>40
>40
>40
30
30
30
25
30
27
20
21
16
23
26
20
17
24
27
25
28
29
>40
26
20
21
35
34
21
34
>40
39
0.75
12
1.1
10
9
0.17
0.19
0.034
0.093
0.26
0.073
0.054
0.049
0.056
0.017
0.013
0.87
>40
5.4
10
8.6
18
19
40
>40
>40
20
20
14
17
24
14
20
20
15
15
10
13
17
14
14
14
20
19
>40
11
31
16
14
21
19
15
>40
>40
>40
>40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
52
53
2
0.10
0.056
0.049
0.093
0.24
0.23
0.50
2.3
0.085
0.14
0.12
0.088
0.14
0.081
0.11
0.083
0.088
0.083
0.10
0.053
0.061
0.095
0.093
0.092
0.15
0.11
0.39
0.24
0.13
0.087
0.094
0.059
0.19
0.11
0.26
In addition, a preliminary study to evaluate the effect of the P2
side chain was conducted. Based on the structure of 15, two ana-
logs, in which the P2 valine was replaced with either glycine (52)
or alanine (53), were prepared using a procedure that was analo-
gous to the synthesis of the above-mentioned tyropeptin deriva-
tives. As a result, removal of all or part of the P2 side chain of 15
did not influence the biological activity tested in this study.16
In summary, boronic acid derivatives of tyropeptin were syn-
thesized and tested for proteasome-inhibitory activity and cyto-
toxicity against RPMI8226 in this study. The most potent
compounds found were 3-phenoxyphenylaceamide 6 (for protea-
some-inhibitory activity) and 3-fluoropicolinamide 22 (for cyto-
toxicity). The structural change in P2 did not affect the in vitro
activities tested in this study. In order to clarify whether the struc-
tural change of P2 side chain can alter the physicochemical proper-
ties of analogs without affecting the biological activities, a SAR
study on this moiety is currently under way. Moreover, full details
of the antitumor activities of these compounds will be also re-
ported in due course.
0.87
0.014
0.014
0.014
0.014
0.0049
0.019
0.015
0.0097
0.039
0.039
0.029
0.014
0.013
0.047
0.046
0.044
0.053
0.047
0.052
0.34
0.041
0.013
0.013
0.051
0.048
0.044
0.024
0.057
0.028
0.0088
Acknowledgments
The authors thank Dr. Ryuich Sawa and Ms. Yumiko Kubota at
Institute of Microbial Chemistry, Tokyo, for collecting analytical
data. The authors are also grateful to Ms. Shoko Kakuda at Institute
of Microbial Chemistry, Numazu, for evaluation of biological
activity.
0.11
0.019
0.039
References and notes
3
1. Ciechanover, A. Angew. Chem., Int. Ed. 2005, 44, 5944.
2. Kisselev, A. F.; Goldberg, A. L. Methods Enzymol. 2005, 398, 364.
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.
9. Lee, A.-H.; Iwakoshi, N. N.; Anderson, K. C.; Glimcher, L. H. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 9946.
10. Sterz, J.; von Metzler, I.; Hahne, J.-C.; Lamottke, B.; Rademacher, J.; Heider, U.
Expert Opin. Invest. Drugs 2008, 17, 879.
11. Yang, H.; Zonder, J. A.; Dou, P. Expert Opin. Invest. Drugs 2009, 18, 957.
12. Momose, I.; Sekizawa, R.; Hashizume, H.; Kinoshita, N.; Homma, Y.; Hamada,
M.; Iinuma, H.; Takeuchi, T. J. Antibiot. 2001, 54, 997.
without any substituents were prepared (7–17). Except for com-
pound 10, the quinoline and isoquinoline derivatives inhibited
the chymotrypsin-like activity of proteasome more effectively than
the pyrazine and pyridine congeners. It is noteworthy that inhibi-
tion of proteasome did not necessarily correlate with the cytotox-
icity. Indeed, the most potent cytotoxicity, comparable to that of
bortezomib, was observed for picolinic acid amide 15, albeit a
modest inhibitory activity against proteasome. Because the analog
15 displayed an antitumor activity in a preliminary experiment,17
further SAR studies were performed starting with this analog to
clarify the effects of substituents on the pyridine ring. To this
end, various picolinic groups installed with one (or two in the case
of 3,6-dichloroderivative 29) functional group were introduced at
the N-terminus (18–43): Me, F, Cl, Br, CF3, OH, OMe, NO2, or
NMe2 derivatives.
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.
In most cases, when tested against the chymotrypsin-like activ-
ity of proteasome, analogs with additional substituents showed
15. Watanabe, T.; Momose, I.; Abe, M.; Abe, H.; Sawa, R.; Umezawa, Y.; Ikeda, D.;
Takahashi, Y.; Akamatsu, Y. Bioorg. Med. Chem. Lett. 2009, 19, 2343.
16. All the new compounds showed satisfactory analytical data. The
IC50 values lower than that of 15 (0.23
derivatives 36 and 37 (IC50: 0.39 and 0.24
ticular, the 3,6-Cl2 (29), 3-Br (30), and 6-Me (41) derivatives
lM) except for hydroxylated
representatives are listed below: 6: a white powder: mp 102–105 °C; ½a D20
ꢁ
lM, respectively). In par-
ꢂ36.2 (c 0.150, CHCl3); IR (KBr) mmax 3294, 1643, 1512, 1250, 1034 cmꢂ1
;
1H
NMR (CD3OD, 600 MHz); d 7.32 (2H, m), 7.20 (1H, t, J = 7.9 Hz), 7.12 (2H, d,
J = 8.6 Hz), 7.08 (1H, m), 7.06 (2H, d, J = 8.6 Hz), 6.94 (2H, d, J = 7.9 Hz), 6.87–
6.80 (5H, m), 6.75 (2H, d, J = 8.6 Hz), 4.62 (1H, m), 4.31 (1H, d, J = 7.6 Hz), 3.73
(3H, s), 3.71 (3H, s), 3.40 (2H, d, J = 14.4 Hz), 3.02 (1H, dd, J = 14.1, 5.1 Hz),
2.82–2.74 (2H, m), 2.51 (1H, dd, J = 14.1, 10.0 Hz), 2.07 (1H, m), 0.95–0.92 (6H,
m); 13C NMR (CD3OD, 150 MHz); d 177.8, 173.8, 173.5, 160.0, 159.7, 158.8,
showed comparable potency (IC50: 0.053, 0.061, and 0.059 lM,
respectively) to that of bortezomib 3.
Substantial loss of cytotoxicity toward RPMI8226 was not ob-
served for the compounds of this class. Notably, 3-F derivative 22

5842
T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5839–5842
158.7, 138.6, 133.8, 131.3, 130.91, 130.88, 130.0, 125.1, 124.4, 120.7, 119.9,
Compound 22:
a
white powder: mp 126–128 °C;
½
a 2D0
ꢁ
ꢂ39.8 (c 0.215,
1H NMR
118.2, 115.0, 114.9, 56.5, 55.8, 55.70, 55.65, 43.4, 37.8, 37.3, 31.9, 19.4, 18.8;
CHCl3); IR (KBr) mmax 3305, 1658, 1512, 1246, 1176, 1034 cmꢂ1
;
HRMS (ESI-Orbitrap) m/z calcd for
C
38H44BN3NaO8 [(M+Na)+] 704.3114,
(CD3OD, 600 MHz); d 8.60 (1H, m), 7.74–7.70 (1H, m), 7.64–7.61 (1H, m), 7.17
(2H, d, J = 8.6 Hz), 7.09 (2H, d, J = 8.6 Hz), 6.83 (2H, d, J = 8.6 Hz), 6.71 (2H, d,
J = 8.6 Hz), 4.36 (1H, d, J = 7.2 Hz), 3.74 (3H, s), 3.72 (3H, s), 3.10–3.03 (2H, m),
2.84 (1H, dd, J = 8.9, 6.2 Hz), 2.76 (1H, dd, J = 14.2, 6.2 Hz), 2.54 (1H, dd, J = 14.2,
8.9 Hz), 2.07 (1H, m), 2.13 (1H, m), 0.84 (3H, d, J = 6.5 Hz), 0.81 (3H, d,
J = 6.5 Hz); 13C NMR (CD3OD, 150 MHz); d 178.0, 173.7, 164.3 (d, J = 5.7 Hz),
160.5 (d, J = 268.6 Hz), 160.3, 159.6, 145.9 (d, J = 5.7 Hz), 138.6 (d, J = 5.7 Hz),
133.9, 131.5, 130.9, 130.1 (d, J = 5.7 Hz), 129.6, 127.5 (d, J = 20.1 Hz), 115.1,
114.9, 56.9, 56.2, 55.71, 55.69, 38.7, 37.0, 31.1, 19.4, 18.6; HRMS (ESI-Orbitrap)
þ
found: 704.3096. Compound 15: a white powder: mp 123–125 °C; ½a D20
ꢁ
ꢂ48.0
(c 0.260, CHCl3); IR (KBr) mmax 3305, 1658, 1512, 1246, 1176, 1034 cmꢂ1
;
1H
NMR (CD3OD, 600 MHz); d 8.60 (1H, m), 8.00 (1H, m), 7.92 (1H, dt, J = 7.7,
1.8 Hz), 7.53 (1H, m), 7.16–7.13 (4H, m), 6.85 (2H, d, J = 8.7 Hz), 6.78 (2H, d,
J = 8.7 Hz), 4.36 (1H, d, J = 8.0 Hz), 3.74 (3H, s), 3.71 (3H, s), 3.16 (1H, dd,
J = 13.9, 5.2 Hz), 2.98 (1H, dd, J = 13.9, 8.5 Hz), 2.90 (1H, m), 2.84 (1H, dd,
J = 14.2, 5.5 Hz), 2.53 (1H, dd, J = 14.2, 10.0 Hz), 2.13 (1H, m), 1.00 (3H, d,
J = 6.2 Hz), 0.99 (3H, d, J = 6.2 Hz); 13C NMR (CD3OD, 150 MHz); d 177.6, 173.6,
166.1, 160.1, 159.7, 150.4, 149.8, 138.8, 133.8, 131.4, 130.8, 129.7, 128.0, 123.1,
m/z calcd for C30H36BFN4NaO7 [(M+Na)+] 617.2553, found: 617.2568.
þ
115.0, 114.9, 56.6, 55.9, 55.7, 55.6, 38.4, 37.2, 31.9, 19.4, 18.9; HRMS (ESI-TOF)
17. Manuscript in preparation.
[(M+Na)+] 599.2648, found: 599.2639.
þ
m/z calcd for
C30H37BN4NaO7