Macrocyclic ketone analogues of halichondrin B

Article abstract of DOI:10.1016/j.bmcl.2004.08.069

Synthesis and SAR studies of structurally simplified analogues of marine natural product halichondrin B resulted in the discovery of E7389, a new potential antitumor agent currently undergoing Phase I clinical trials. Structurally simplified macrocyclic ketone analogues of halichondrin B were prepared by total synthesis and found to retain the potent cell growth inhibitory activity in vitro, stability in mouse serum, and in vivo efficacy of the natural product.

Full text of DOI:10.1016/j.bmcl.2004.08.069

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5551–5554
Macrocyclic ketone analogues of halichondrin B
Wanjun Zheng,a Boris M. Seletsky,a Monica H. Palme,a Paul J. Lydon,a Lori A. Singer,a
Charles E. Chase,a Charles A. Lemelin,a Yongchun Shen,a Heather Davis,a
Lynda Tremblay,a Murray J. Towle,b Kathleen A. Salvato,b Bruce F. Wels,b
Kimberley K. Aalfs,b Yoshito Kishi,c Bruce A. Littlefieldb and Melvin J. Yua,*
aDepartment of Medicinal Chemistry, Eisai Research Institute, 4 Corporate Drive, Andover, MA 01810, USA
bDepartment of Anticancer Research, Eisai Research Institute, 4 Corporate Drive, Andover, MA 01810, USA
cAdvisory Board, Eisai Research Institute, 4 Corporate Drive, Andover, MA 01810, USA
Received 30 July 2004; accepted 31 August 2004
Available online 21 September 2004
This paper is dedicated to memory of Bruce F. Wels, our friend and colleague
Abstract—Structurally simplified macrocyclic ketone analogues of halichondrin B were prepared by total synthesis and found to
retain the potent cell growth inhibitory activity in vitro, stability in mouse serum, and in vivo efficacy of the natural product.
Ó 2004 Elsevier Ltd. All rights reserved.
Halichondrin B (HB) is a structurally complex marine
natural product.1,2 Despite extensive synthetic studies,
the only successful total synthesis of HB and norhalich-
ondrin B was reported by Kishi and co-workers in
1992.3 The discovery that the right half (RH) of HB
and analogue 1 exhibited potent cell growth inhibitory
activity in vitro4,5 and antitumor efficacy in vivo,6
respectively, provided a compelling starting point for a
drug discovery program. In the preceding paper7 we
reported our SAR investigation and identification of a
series of macrolactone analogues (e.g., 2 and 3) that
retained the remarkable in vitro biological profile of
HB. However, these compounds failed to demonstrate
salutary effects in vivo. Herein, we report our efforts to
optimize the in vivo properties of these structurally sim-
plified analogues, culminating in the discovery of E7389
(4), a fully synthetic analogue of HB that is currently
undergoing Phase I clinical evaluation.
mouse serum in the 3–4day growth inhibition assay,
whereas activity of the tetrahydropyran or tetrahydrofu-
ran series of analogues exemplified by 2 and 3, respec-
tively, were completely abrogated by mouse serum at
concentrations as low as 0.01%. Since the seco acid of
Instability of the macrolactone acyl moiety was regarded
as the most likely explanation for the observed lack of
in vivo efficacy associated with the structurally simpli-
fied macrolactone series. Consistent with this hypothe-
sis, in vitro activity of octahydropyrano[3,2-b]pyran
analogue 1 was not affected by inclusion of up to 1%
*
Corresponding author. Tel.: +1 978 661 7202; fax: +1 978 657
7715; e-mail: melvin_yu@eri.eisai.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.069

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W. Zheng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5551–5554
RH was not active, the lack of in vivo efficacy for struc-
turally simplified macrolactone analogues in the mouse
xenograft model was most likely attributed to sensitivity
of the macrolactone ring to nonspecific esterases present
in serum.
to the corresponding dimesylates. The desired isomer
14 was separated by column chromatography and
subjected to cyclization in the presence of Triton B.
Methylmagnesium bromide mediated desulfonylation13
followed by methylation of the corresponding alcohol
furnished 15. Adjusting protecting groups on the C.32
side chain, selective cleavage of the benzyl ether under
Raney-Nickel conditions14 and Swern oxidation15 af-
forded aldehyde 5.
To further test this hypothesis, nonhydrolyzable ester
bioisosteres were prepared to identify derivatives that
would retain activity in the presence of mouse serum.
Of the surrogate groups examined, only the ketone bio-
isostere provided analogues with sufficient in vitro po-
tency to warrant further investigation.
Nozaki–Hiyama–Kishi coupling18 of aldehyde 5 with
vinyl iodide 63a and subsequent KHMDS-promoted
cyclization afforded a 3:1 mixture of two C.27 diastereo-
mers favoring the desired product (Scheme 3). The
MPM-ethers were cleaved using DDQ19 and two C.27
isomers were separated by flash chromatography. The
major product 16 was converted to sulfone 17 in four
steps. Coupling of sulfone 17 with aldehyde 8 followed
by oxidation resulted in 18. SmI2 mediated desulfonyla-
tion20 followed by Nozaki–Hiyama–Kishi macrocycliza-
tion18 and subsequent allylic alcohol oxidation gave
enone 19. Treatment with TBAF and PPTS afforded
the desired macrocyclic ketone 9.
The macrocyclic ketone analogues were prepared in a
convergent manner from three major fragments 5,
6,3a,8 and 89 in a way similar to that developed by Kishi
and co-workers for the total synthesis of HB (Scheme 1).
Aldehyde 5 was prepared as outlined in Scheme 2. Reg-
ioselective opening of epoxide 1110 with the lithium
anion of 1011 in the presence of BF3ÆOEt2 furnished a
3:1 mixture of structural isomers favoring desired prod-
uct 12.12 Hydrogenation using Lindlarꢀs catalyst fol-
lowed by acetylation furnished cis-olefin 13. Alkene
dihydroxylation with osmium tetroxide afforded an 8:1
mixture of diastereomers, which was directly converted
The vicinal diol on the C.32 side chain provided a con-
venient handle for subsequent derivatization. Analogues
Scheme 1. Final assembly from key fragments.
Scheme 3. Synthesis of macrocyclic ketone 9. Reagents and condi-
tions: (a) 0.5% NiCl2/CrCl2, 6, THF/DMF (4:1), rt; (b) KHMDS,
THF, 0°C, 75%; (c) DDQ, CH2Cl2/pH7 buffer, rt, 59%; (d) MsCl,
Et3N, CH2Cl2, 0°C, 98%; (e) PhSH, i-Pr2NEt, DMF, rt, 96%; (f)
TPAP, NMO, CH3CN, rt, 81%; (g) DIBAL, CH2Cl2, À78°C, 97%; (h)
n-BuLi, 8, DME ,À40 to À26°C; (i) Dess–Martin,16 CH2Cl2, rt, 82%;
(j) SmI2, THF/MeOH, À78°C, 82%; (k) 1% NiCl2/CrCl2, THF/DMF
(4:1), rt; (l) Dess–Martin,16 CH2Cl2, rt, 73%; (m) TBAF/imidaz-
oleÆHCl, THF, rt; (n) PPTS, CH2Cl2, rt, 90%.17
Scheme 2. Synthesis of aldehyde 5. Reagents and conditions: (a) n-
BuLi, 11, BF3ÆOEt, toluene, À50°C, 69%; (b) H2, 5% Pd/CaCO3,
quinoline, hexanes/CH2Cl2, rt, 99%; (c) Ac2O, pyridine, DMAP,
CH2Cl2, rt, 99%; (d) OsO4, NMO, t-BuOH/H2O, rt; (e) MsCl,
pyridine, rt, 58%; (f) Triton B, MeOH, rt; (g) MeMgBr, THF, 45°C;
(h) t-BuOK, MeI, THF, 0°C, 84%; (i) HCl, THF/MeOH/H2O, rt; (j)
TBSCl, imidazole, DMF, rt; (k) H2, Raney Ni, EtOH, 84%; (l)
(COCl)2, DMSO, Et3N, CH2Cl2, À60 to 0°C, 73%.

W. Zheng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5551–5554
5553
20–40 were prepared from diol 9 using standard trans-
formations summarized in Scheme 4.
under continuous exposure conditions, and the ability
to maintain a complete mitotic block (CMB) 10h after
drug washout using flow cytometric analysis of U937
human histiocytic lymphoma cells (reversibility assay).21
In addition, susceptibility to P-glycoprotein (PgP)
The compounds were evaluated for cell growth inhibi-
tory activity against DLD-1 human colon cancer cells
Scheme 4. C.32 Side chain modifications. Reagents and conditions: (a) i. DEAD, Ph3P, p-NO2C6H4CO2H, Et2O, rt; ii. K2CO3, MeOH, rt, 60%; (b) i.
NaIO4, MeOH–H2O (4:1), rt, ii. NaBH4, MeOH–CH2Cl2 (4:1), À78°C to rt, 89%; (c) i. TBDPSCl, imidazole, DMF, rt; ii. Dess–Martin reagent,
CH2Cl2, rt; iii. TBAF, imidazoleÆHCl, THF, rt, 77%; (d) ArNCO, Et3N, CH2Cl2, rt, 83–92%; (e) TsCl, pyridine, CH2Cl2, rt, 72%; or MsCl, collidine,
CH2Cl2, 0°C, 90%; (f) KCN, DMSO, 60°C, 67% from 26; (g) n-Bu4NN3, DMF, 80°C, 85% from 26; (h) i. NaI, acetone, 60°C, ii. ArSH, DMF, 59–
85% from 26, rt; (i) mCPBA, CH2Cl2, 0°C, 61–74%; (j) R1R2NH, MeOH, rt, 90–100% from 27; (k) RCO2H, EDCI, CH2Cl2, rt, 31–70%.
Table 1. In vitro activity profile for macrocyclic ketone analogues of HB
Compound
DLD-1a IC50, nMa (n)
U937b IC99, 0h, nM
U937b IC99, 10h, nM (n)
Reversibility ratio
FRc
HB
1
0.74 0.03 (6)
3.4 0.40 (4)
1.8 0.00 (3)
0.67 0.09 (3)
13.0 0.20 (10)
1.0 0.10 (18)
1.9 0.10 (4)
0.66 0.11 (4)
1.1 0.20 (5)
1.0 0.20 (4)
1.3 0.06 (3)
0.86 0.07 (3)
2.0 0.20 (3)
0.80 0.01 (3)
0.49 0.07 (4)
0.53 0.01 (4)
1.2 0.10 (3)
0.71 0.12 (3)
0.66 0.06 (4)
0.74 0.03 (3)
3.3 0.10 (3)
1.2 0.20 (3)
0.47 0.08 (3)
0.43 0.03 (3)
0.69 0.03 (4)
0.72 0.06 (4)
0.87 0.03 (4)
8
9
25 (4)
220 (6)
300 (3)
15 (4)
10 (4)
38 (2)
100 (1)
100 (1)
30 (2)
65 (2)
65 (2)
30 (3)
30 (2)
30 (1)
30 (2)
30 (2)
200 (2)
30 (2)
30 (2)
30 (2)
10 (2)
10 (2)
10 (2)
20 (2)
20 (2)
10 (2)
10 (2)
3
24
30
8
2.8
12
2
10
2
10
3
4 (E7389)
9 (ER-076349)
6.2
15
10
3
1
13
33
33
10
22
22
10
4
9.7
13
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
3
3
8.5
3.7
5.7
8.6
5.8
2.4
3.0
7.0
1.9
2.0
1.7
3.2
3.4
7.0
22
3
3
3
3
7
3
10
15
10
20
10
10
10
1
2
3
10
3
3
3
10
1
10
10
7
1
17
3
4.7
5.9
20
3
7
2
5
3
3
3.6
a Cell growth inhibition under continuous exposure conditions for 3–4days, IC50 SE M (n = number of experiments).
b Mitotic block reversibility assay.
c Fold resistance calculated as the IC50 ratio between retrovirally transformed P388/VMDRC.04 and parental P388 cells.

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W. Zheng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5551–5554
K. A.; Wels, B. F.; Aalfs, K. K.; Kishi, Y.; Littlefield, B.
A.; Yu, M. J. Bioorg. Med. Chem. Lett. 2004, 14,
preceding paper. doi:10.1016/j.bmcl.2004.08.068.
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4427–4429; (b) Choi, H.; Nakajima, K.; Demeke, D.;
Kang, F.-A.; Jun, H.-S.; Wan, Z.-K.; Kishi, Y. Org. Lett.
2002, 4, 4435–4438.
9. Aldehyde 8 was prepared by DIBAL reduction of methyl
ester 7 described earlier: Ref. 3a and Stamos, D. P.; Kishi,
Y. Tetrahedron Lett. 1996, 37, 8643–8646.
10. Compound 11 was prepared from the corresponding
allylic epoxy alcohol (Hirai, Y.; Chintani, M.; Yamazaki,
T.; Momose, T. Chem. Lett. 1989, 1449–1452) by treat-
ment with MPMCl and t-BuOK in 93% yield.
11. Smith, A. B.; Chen, S. S.-Y.; Nelson, F. C.; Reichert, J.
M.; Salvatore, B. A. J. Am. Chem. Soc. 1995, 117, 12013–
12014.
12. Yamaguchi, M.; Hirao, I. Tetrahedron Lett. 1983, 24, 391–
394.
13. Cossy, J.; Ranaivosata, J.-L.; Bellosta, V.; Wietzke, R.
Synth. Commun. 1995, 25, 3109–3112.
14. (a) Oikawa, Y.; Tanaka, T.; Horita, K.; Yonemitsu, O.
Tetrahedron Lett. 1984, 25, 5397–5400; (b) Horita, K.;
Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu, O.
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mediated drug efflux was determined using murine P388/
VMDRC.04 cells22 (Table 1).
Macrocyclic ketone 9 (ER-076349, NSC 707390) exhib-
ited an in vitro profile very similar to the corresponding
macrolactone analogue 3, thus confirming bioisosteric
equivalence of the ketone and ester functional groups.
Compound 9 remained active in vitro even after inclu-
sion of 100% mouse serum, and exhibited antitumor
efficacy in a variety of human tumor xenograft models.23
Modifications to the C.32 side chain had little impact on
in vitro biological activity (Table 1). Interestingly, amine
4 (E7389, previously ER-086526, also NSC-707389)
exhibited a reversibility ratio of one in U937 cells. Sev-
eral other amine (34–37) and amide (38–40) derivatives
were subsequently prepared and also found to exhibit
a low reversibility ratio (e.g., 34). E7389 demonstrated
remarkable efficacy (0.05–1mg/kg) in a variety of human
tumor xenograft models.23 This compound was selected
for development as a new potential anticancer chemo-
therapeutic agent, and is currently in collaborative
Phase I clinical trials with the United States NCI.
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