Effective irreversible alkylating reagents based on the structure of clavulones

Article abstract of DOI:10.1016/j.bmcl.2003.12.026

We describe the design and synthesis of alkylating reagents based on the structure of clavulones. They are composed of cross-conjugated dienone system and irreversibly reacted with two nucleophiles under mildly basic conditions via β-elimination. Hydroxyl derivative 7b showed the highest reactivity toward thiols and showed the strongest cytotoxicity in Hela S3 cells among the three derivatives having a different protecting group at the tert-hydroxyl group.

Full text of DOI:10.1016/j.bmcl.2003.12.026

Bioorganic & Medicinal Chemistry Letters 14 (2004) 837–840
Effective irreversible alkylating reagents based on the
structure of clavulones
Hiroshi Tanaka,a Makoto Kitade,a,y Makoto Iwashima,b,{
Kazuo Iguchib and Takashi Takahashia,*
aDepartment of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama,
Meguro, Tokyo 152-8552, Japan
bSchool of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 31 October 2003; revised 4 December 2003; accepted 5 December 2003
Abstract—We describe the design and synthesis of alkylating reagents based on the structure of clavulones. They are composed of
cross-conjugated dienone system and irreversibly reacted with two nucleophiles under mildly basic conditions via b-elimination.
Hydroxyl derivative 7b showed the highest reactivity toward thiols and showed the strongest cytotoxicity in Hela S3 cells among the
three derivatives having a different protecting group at the tert-hydroxyl group.
# 2003 Elsevier Ltd. All rights reserved.
Chemical genetics is a research approach that uses
biologically active small molecules as probes to study
protein function in cells or organisms.1 Chemical
irreversible modification on specific proteins with the
small molecules should be effective not only to elucidate
function of the binding proteins, but also to identify the
target proteins from living cells or isolated protein
mixtures.2 Therefore, selectively and strongly alkylating
agents are required as chemical probes.
that the metabolic stability of prostaglandin A2 (2) was
dependant on the structure of the side chain.6
Marine prostanoid clavulone I (3) isolated from the
Okinawan soft coral features the same cross-conjugate
dienone system along with a tert-acetoxyl group at the
C12 position and shows strong cytotoxicity.7,8 The
cross-conjugated dienone 3 could undergo sequential
irreversible alkylation to provide the di-coupling
Cross-conjugated dienone prostanoids such as 15-deoxy-
Á
12,14-prostaglandin J2 (15d-PGJ2) (1) and Á7-prostag-
landin A2 (2) display varied biological activities.3 Their
mechanism of action is based on reversible and selective
alkylation with specific proteins at their C11 position to
provide thermodynamically stable adducts 4a.4
Recently, Oliva J. L. and Rojas J. M. et al. have reported
15-deoxy-Á12,14-prostaglandin J2 (1) selectively elicited
H-Ras activation by formation of a covalent bond.5
Additionally, Suzuki and Noyori et al. have elucidated
Keywords: Michael reactions; Clavulones; Prostanoids; Alkylating
reagents; Aldol reactions.
* Corresponding author. Tel.: +81-3-5734-2471; fax: +81-3-5734-
2884; e-mail: thiroshi@apc.titech.ac.jp
y
Present address: Cancer Research Lab., Hanno Research Center,
Taiho Parmaceutical Co., Ltd., 1-27, Misugidai, Hanno-shi,
Saitama, 357-8527, Japan.
Present address: Department of Pharmacognosy, Faculty of
{
Pharmaceutical Sciences, Toyama Medical and Pharmaceutical
University, 2630, Sugitani, Toyama, Toyama 930-0194, Japan.
Scheme 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.026

838
H. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 837–840
would be effective to diversify the two side-chains. At
the first stage of the project, we planned to prepare
hydroxyl and acetoxyl derivatives 7a and 7b bearing two
phenyl side-chains.
The preparation of the di-phenyl derivatives 7a and 7b
is shown in Scheme 3. Treatment of 4-hydroxy-cyclo-
pentenone (11) with lithiated 1-trimethylsilylpropyne at
ꢀ78 ꢁC provided diol 12 in 58% yield.10 Selective
oxidation of the secondary alcohol in diol 12 with
MnO2 afforded enone 13 in 71% yield. Introduction of
the aromatic ring at the o-chain was achieved by one-
pot silyl deprotection and Sonogashira coupling
reaction11 of 13 with phenyl iodide in the presence of
tetrabutyl ammonium fluoride and a catalytic amount
of Pd(PPh3)4 and CuI to give phenyl acetylene 14 in
99% yield. Subsequent protection of the tert-alcohol
with 3,4-dihydro-2H-pyran (DHP) in the presence of
pyridinium p-toluenesufonate (PPTS) provided enone
15 in 89% yield. Aldol condensation of enone 15 with
benzaldehyde to form the cross-conjugated dienone was
investigated. Exposure of 15 to several bases to generate
the enolate resulted in b-elimination of the protected
tert-hydroxyl group to give dienone 16 as the major
product. The dianion enolate from b-hydroxyl ketone
14 was readily decomposed at ꢀ78 ꢁC. Further exami-
nation of the aldol condensation reaction revealed that
treatment of ketone 15 with an equivalent of KHMDS
in THF at ꢀ78 ꢁC for 15 min, followed by addition of
benzaldehyde at the same temperature afforded the
desired cross-conjugated dieone 17 in 8% yield along
with dienone 16 (32%) and the starting material 15
(38%). Removal of the tetrahydropyranyl ether of 17
under acidic conditions provided alcohol 7b12 in 99%
yield. X-ray crystallographic analysis of alcohol 7b
showed that the newly formed double bond had the
(E)-configuration.13 Acetylation of alcohol 7b with
Ac2O in the presence of pyridine provided acetate 7a in
99% yield.14
Scheme 2. Strategy for the synthesis of 7.
product 5b, followed by b-elimination of the C12
acetoxyl group to provide enone 6. The irreversible
alkylation would be more effective to inhibit protein
functions than the reversible one.9 Therefore, clavulone
derivatives having appropriate side-chains could
become useful chemical probes. In this report, we
describe the synthesis of simple clavulone derivatives
with two aromatic side-chains, and the relationship
between reactivity of the double Michael acceptors and
their cytotoxicity (Scheme 1).
Cross-conjugated dienones 7 bearing two aromatic side-
chains were designed as irreversible alkylating reagents
(Scheme 2).9 Substituents on the aromatic side-chains
would be effective not only to vary their electrostatic
and steric parameters for the selective alkylation to
specific proteins, but also to tune the reactivity of the
second alkylation reaction. Electron withdrawing
protecting groups at the C12 tert-hydroxyl group should
enhance the subsequent b-elimination. However, its
bulkiness could reduce the reactivity of Michael
acceptor at C11 position.
Our strategy for the synthesis of 7 involves the coupling
of terminal acetylene 8 and aryl halide 10 with palla-
dium catalyst, followed by aldol condensation of 9, and
Scheme 3. Reactions and conditions: (a) 1-trimethylsilyl-1-propyne, BuLi, ꢀ78 ꢁC, 58%; (b) MnO2, Et2O, 71%; (c) Pd(PPh3)4, CuI, phenyl iodide,
Bu4NF, 99%; (d) DHP, PPTS, 89%; (e) KHMDS, ꢀ78 ꢁC, then benzaldehyde, 32% for 11, 8% for 12, and 38% for recovery of 10; (f) 5%TFA/
CH2Cl2, 99%; (g) Ac2O, pyridine, CH2Cl2, 99%.

H. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 837–840
Table 1. Michael addition of cross-conjugate dienones 7a, 7b and 17
839
Entry
Substrate
Recovered starting
material (%)
Yield of
18 (%)
Yield of
20 (%)
1
2
3
7a
7b
17
14
014
85
078
65
06
Next, alkylation reaction of thiols with the cross-
conjugated dinenones 7a, 7b and 17 was investigated
(Scheme 4 and Table 1). Treatment of the cross-con-
jugated dienone 7a with mercaptoethanol (2.0equiv) in
the presence of triethylamine (2.0equiv) at room temp-
erature for 24 h provided bis-thioethers 20 in 78% yield
as two diastereomers (1:1), instead of the expected
cyclopentenone 19, along with the recovered starting
material 7a in 14% yield. The highly conjugated phenyl
acetylene moiety at the o-chain could promote the
unexpected b-elimination to form the exocyclic double
bond, or isomerization of 19 to 20 would occur because
of sever steric repulsion among substituents on the
conjugated enone. Structure determination of bis-thio-
ether 20 was achieved by analysis of 1D and 2D-NMR
spectra of each of the separated isomers 20a15 and
20b.16 1H NOE experiment shows the exo-double bond
had the (E)-configuration. The relative stereochemistry
of the two isomers 20a and 20b were not determined.
Under the reaction conditions, mono-adduct inter-
mediate 18c was not observed. Exposure of alcohol
7b to the same reaction conditions resulted in the
disappearance of starting material 7b and yielded the
bis-thioether 20 in 65% yield along with mono-adduct
18b17 in 14% yield as a single stereoisomer. On the
other hand, reaction of the THP derivative 17 with thiol
under the same conditions provided only bis-thioether
20 in 6% yield along with dienone 17 (85%). These
results indicate that sequential Michael reactions were
initiated by conjugate addition of thiol to the endo-
enone and steric hindrance of the protecting group at 12
position could block both two Micheal reactions.
Scheme 4. Reactions and conditions: HOCH2CH2SH (2.0equiv),
NEt3 (2.0equiv) CH 2Cl2, rt.
Table 2. Cytotoxicity of cross-conjugated dienone derivatives 7a, 7b,
17, 18b, 20a, and 20b in HeLa S3 cellsa
Entry
Compd
IC50 (nmol/L)
1
2
3
4
5
6
7a
7b
17
18b
20a
20b
24
15
1.1ꢂ103
21
>1.0ꢂ106
>1.0ꢂ106
a Cell proliferation assay was carried out using Cell Counting Kit
(Wako Pure Chemical Industries Ltd., Osaka, Japan). In brief, cells
were plated in triplicate in 96-well plates at a density of 10ꢂ103 cells/
well in EMEM medium. Following overnight culture, compounds 7a,
7b, 17, 18b, 20a, and 20b were added and the cells were incubated for
72 h. After 72 h, WST-8 solution was added and incubated for 1 h.
The plates were read at a wavelength of 450nm using Microplate
Reader Model 3550(Bio-Rad, Richmond, CA). Results are presented
as the mean ꢃS.D. of three wells.
aromatic side-chains. Dieones 7a, 7b, and 17 reacted
with two equivalents of thiol at room temperature.
Alcohol 7b had the highest reactivity in the Michael
reaction and showed the strongest biological activity in
comparison with acetate 7a and THP ether 17. The
protecting group of the tert-hydroxyl affected the
reactivity of the Michael acceptor with thiol and their
cytotoxicity. The synthesis of a combinatorial library
of cross-conjugated derivatives varying at the two
aromatic rings is in progress.
Random alkylation reaction to biomolecules in the cells
would result in antiproliferative effects. To test the
applicability of the sequential Michael reaction to bio-
molecules in the cell, the cytotoxicity of the derivatives
7a, 7b, 17, 18b, 20a, and 20b in HeLa S3 cells (uter-
ocervical carcinoma) was tested18 (Table 2). Alcohol 7b
showed the strongest activity (IC50=15.0nM) among
the three dienones 7a, 7b and 17. Cytotoxicity of mono-
adduct 18b was almost the same as dienone 7b. On the
other hand, bis-thioethers 20a and 20b did not show
cytotoxicity. These results suggested that retro-Michael
reaction of 18b proceeded to generate the cross-con-
jugate dienone 7b in situ. Furthermore, the highly reac-
tivity of the Michael acceptor would be essential for the
strong biological activity. These results indicate that
their biological activity should be tunable by the steric
hindrance of the protecting group.
Acknowledgements
This work was supported by a Grand-in Aid for
Scientific Research on Priority Area (S) from the
Ministry of Education, Culture, Sports, Science, and
Technology (Grant-in-aid No.14103013). We thank
Hiroaki Tsujimoto (Taiho Pharmaceutical Co. Ltd)
for the cytotoxic assay and Kanji Endoh (Taiho
Pharmaceutical Co. Ltd) for X-ray crystallographic
analysis of 7b.
In conclusion, we have reported the synthesis of
cross-conjugated dienones 7a, 7b, and 17 bearing two

840
H. Tanaka et al. / Bioorg. Med. Chem. Lett. 14 (2004) 837–840
NMR (100 MHz, CDCl3): d 195.2, 160.8, 136.1, 135.3,
References and notes
134.4, 133.4, 132.1, 131.6, 130.0, 128.8, 128.3, 128.2,
122.8, 84.1, 84.0, 78.1, 27.4; IR (neat) 3418, 3074, 2910,
1681, 1589 cmꢀ1; MS(ESI-TOF) 301 [M+H]+.
1. (a) Schreiber, S. L. Bioorg. Med. Chem. 1988, 6, 1127. (b)
Stocwell, B. R.; Hardwick, J. S.; Tong, J. K.; Schreiber,
S. L. J. Am. Chem. Soc. 1999, 121, 10662.
2. Campbell, D. A.; Szardenings, A. K. Curr. Opin. Chem.
Biol. 2003, 7, 296.
3. (a) Needleman, P.; Turk, J.; Kakschik, B. A.; Morrison,
A. R.; Lefkowith, J. B. Ann. Rev. Biochem. 1986, 55, 69.
(b) Rozera, C.; Carattoli, A.; De Marco, A.; Amici, C. J.;
Santoro, M. G. J. Clin. Invest. 1996, 97, 1795. (c) Santoro,
M. G. Trends Microbiol 1997, 5, 276. (d) Ross, A.; Kapa-
chi, P.; Natori, T.; Takahashi, T.; Chen, Y.; Karin, N.
Nature 2000, 403, 103.
4. Suzuki, M.; Mori, M.; Niwa, T.; Hirata, R.; Furuta, K.;
Ishikawa, T.; Noyori, R. J. Am. Chem. Soc. 1997, 119,
2376.
5. Oliva, J. L.; Perez-Sala, D.; Castrillo, A.; Martınez, N.;
Canada, F. J.; Bosca, L.; Rojas, J. M. P. Natl. Acad.
U.S.A 2003, 100, 4772.
6. Suzuki, M.; Mori, M.; Niwa, T.; Hirata, R.; Furuta, K.;
Ishikawa, T.; Noyori, R. J. Am. Chem. Soc. 1997, 119,
2376.
7. (a) For isolation of clavulone I, see: Kikuchi, H.; Tsuki-
tani, Y.; Iguchi, K.; Yamada, Y. Tetrahedron Lett. 1982,
23, 5171. (b) Kobayashi, M.; Yasuzawa, T.; Yoshihara,
M.; Akutsu, H.; Kyogoku, Y.; Kitagawa, I. Tetrahedron
Lett. 1982, 23, 5331.
8. (a) For synthesis of clavulones and related compounds,
see: Corey, E. J.; Mehrotra, M. J. Am. Chem. Soc. 1984,
106, 3384. (b) Nagaoka, H.; Miyakoshi, T.; Yamada, Y.
Tetrahedron Lett. 1984, 25, 3621. (c) Hashimoto, S.; Arai,
Y.; Hamanaka, N. Tetrahedron Lett. 1985, 26, 2679. (d)
Shibasaki, M.; Ogawa, Y. Tetrahedron Lett. 1985, 26,
3841. (e) Kuhn, C.; Skaltsuounis, L.; Monneret, C.; Flor-
ent, J.-C. Eur. J. Org. Chem. 2003, 2585 and references
therein.
13. Crystallographic data (excuding structure factors) for the
structure of 7b have been deposited with the Cambridge
Crystallographic Data Center asꢁsupplementary publica-
tion number CCDC 197097; 146 C.
1
14. Spectra 7a: H NMR (400 MHz, CDCl3): d 2.71 (s, 3H),
3.04 (d, 1H, J=16.9 Hz), 3.42 (d, 1H, J=16.9 Hz), 6.64
(d, 1H, J=6.30Hz), 7.27–7.30(m, 5H), 7.33–7.39 (m,
3H), 7.53 (s, 1H), 7.68 (d, 1H, J=6.3 Hz), 7.98 (brd, 2H,
J=8.2 Hz); 13C NMR (100MHz, CDCl3): d 193.7, 168.8,
157.0, 135.5, 134.2, 133.8, 133.3, 131.5, 131.1, 129.9, 128.8,
128.2, 128.1, 122.8, 84.2, 83.6, 83.0, 27.2, 21.3; IR (neat):
3074, 1751, 1705, 1632 cmꢀ1; MS(ESI-TOF) 343 [M+H]+.
15. Spectra 20a: 1H NMR (400 MHz, CDCl3): d 2.40–2.60 (m,
3H), 2.98 (dd, 1H, J=6.8, 19.3 Hz), 3.13 (t, 2H, J=6.3
Hz), 3.53 (t, 2H, J=6.3 Hz) 3.84 (t, 2H, J=6.3 Hz), 3.92
(s, 1H), 3.99 (s, 1H), 4.04 (brd, 1H, J=6.8 Hz), 6.59 (s,
1H), 7.15 (m, 2H), 7.27–7.31 (m, 8H); 13C NMR
(100 MHz, CDCl3): d 202.5, 162.4, 152.6, 142.6, 141.0,
134.5, 129.1, 129.0, 127.7, 127.3, 127.0, 126.8, 113.3, 61.4,
60.2, 53.4, 45.0, 44.0, 42.0, 34.0, 32.3; IR (neat): 3360,
2920, 1668, 1615, 1533 cmꢀ1
[M+H]+.
; MS (ESI-TOF) 439
16. Spectra 20b: 1H NMR (400 MHz, CDCl3): d 2.53 (dd, 2H,
J=1.9, 18.8 Hz), 2.76 (t, 2H, J=6.3 Hz), 2.94 (dd, 1H,
J=6.8, 18.8 Hz), 3.05–3.20 (m, 2H), 3.81 (t, 2H, J=6.3
Hz), 3.83 (t, 2H, J=5.7 Hz), 3.89 (s, 1H), 4.01(s, 1H), 4.10
(brd, 1H, J=6.8 Hz), 6.48 (s, 1H), 7.20–7.33 (m, 10H);
13C NMR (100 MHz, CDCl3): d 202.5, 163.3, 152.1, 142.1,
141.0, 134.3, 129.1, 129.0, 127.7, 127.4, 127.1, 127.0,
114.1, 61.3, 60.2, 53.6, 44.9, 44.6, 42.6, 34.0, 32.8; IR
(neat): 3384, 2855,1687, 1618, 1527 cmꢀ1; MS (ESI-TOF)
439 [M+H]+.
17. Spectra 18b: 1H NMR (400 MHz, CDCl3): d 2.71 (dd, 1H,
J=6.8, 18.8 Hz), 2.89 (m, 1H), 2.96 (m, 1H), 3.04 (dd, 1H,
J=8.2, 18.8 Hz), 3.07 (m, 2H), 3.82 (brs, 3H), 3.91 (dd,
1H, J=6.8, 8.2 Hz), 7.26–7.32 (m, 5H), 7.39–7.43 (m,
3H), 7.72 (s, 1H), 7.92 (m, 2H); 13C NMR (100 MHz,
CDCl3): d 201.1, 140.8, 137.2, 133.3, 132.2, 131.5, 131.4,
130.3, 128.3, 128.2, 122.8, 84.8, 83.8, 79.0, 61.4, 50.3, 43.9,
9. Recently, the design and synthesis of a mono-alkylating
reagent based on the Michael reaction followed by b-
elimination have been reported: Sakakura, A.; Takaya-
nagi, Y.; Kigoshi, H. Tetrahedron Lett. 2002, 43, 6055.
10. Corey, E. J.; Kirst, H. A. Tetrahedron Lett. 1968, 9, 5041.
11. Sonogashira, K.; Tohda, Y. Tetrahedron Lett. 1975, 16,
4467.
12. Spectra 7b : 1H NMR (400 MHz, CDCl3): d 2.81 (brs,
1H), 2.90(d, 1H, J=17.0Hz), 3.28 (d, 1H, J=17.0Hz),
6.51 (d, 1H, J=6.8 Hz), 7.27–7.30(m 3H), 7.33–7.39 (m
2H), 7.33–7.39 (m 2H), 7.41–7.45 (m 3H), 7.52 (s 1H),
7.66 (d, 1H, J=6.8 Hz), 7.98 (brd, 2H, J=8.7 Hz); 13C
36.0, 28.6; IR (neat): 3412, 2924, 1713, 1610 cmꢀ1
MS(ESI-TOF) 379 [M+H]+.
;
18. (a) Ishiyama, M.; Miyamoto, Y.; Sasamoto, K.; Ohkura,
Y.; Ueno, K. Talanta 1997, 44, 1299. (b) Tominaga, H.;
Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.;
Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47.