Structurally simplified macrolactone analogues of halichondrin B

Article abstract of DOI:10.1016/j.bmcl.2004.08.068

Structurally simplified macrolactone analogues of halichondrin B (e.g., 47, IC₅₀ = 0.67 nM) were identified that retain the potent cell growth inhibitory activity of the natural product in vitro. A structurally simplified macrolactone analogue of halichondrin B was identified that retains the potent cell growth inhibitory activity of the natural product in vitro.

Full text of DOI:10.1016/j.bmcl.2004.08.068

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5547–5550
Structurally simplified macrolactone analogues of halichondrin B
Boris M. Seletsky,a Yuan Wang,a Lynn D. Hawkins,a Monica H. Palme,a
Gregory J. Habgood,a Lucian V. DiPietro,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
Abstract—A structurally simplified macrolactone analogue of halichondrin B was identified that retains the potent cell growth inhib-
itory activity of the natural product in vitro.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Halichondrin B (HB) is a structurally complex marine
natural product that exhibits extraordinary cytotoxic
activity in vitro1,2 and antitumor efficacy in vivo.3 Pre-
clinical development as a new anticancer chemothera-
peutic, however, was hampered by extremely limited
supply of this agent isolated from natural sources. De-
spite synthetic studies from a number of laboratories,
the only successful total synthesis of HB and norhalich-
ondrin B was reported by Kishi and co-workers in
1992.4
The discovery that macrolactone right half (RH) syn-
thetic intermediates 1 and 2 exhibited potent cell growth
inhibitory activity in vitro5,6 provided an exciting and
compelling starting point for a drug discovery effort
around this important class of antitumor agents. Our
initial structure–activity relationship study explored
modifications to the C.29–C.36 octahydropyrano[3,2-
b]pyran ring system.7 Herein, we report our efforts to
further simplify the structure of these macrolactones
while maintaining potent cell growth inhibitory activity.
2. Synthesis
Macrolactone analogues represented generically by 6
were assembled from three key fragments 3, 4,4a,8 and
54a,9 in a manner identical to that described by Kishi
and co-workers in their total synthesis of HB (Scheme
1). Preparation of aldehyde 3 generically represented
below is outlined in Schemes 2–4.
L-Glucose acetate 7 was converted to aldehyde 14 as
summarized in Scheme 2. Stereoselective C-glycosida-
tion10 and adjustment of protecting groups furnished
diol 8. Tosylation at C.32 and opening of the intermediate
epoxide with methyl Grignard afforded intermediate 9.
*
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.2004.08.068

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B. M. Seletsky et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5547–5550
Scheme 1. Final assembly from key fragments.
Scheme 4. Synthesis of aldehydes 25a/b and 26a/b. Reagents and
conditions: (a) (i) allylTMS, BF3ÆEt2O, hexane, 0°C;10 (ii) K2CO3,
MeOH; (iii) separate isomers (10:1), 87% for 20, 9% for 21; (b) (i)
TBSCl, imidazole, CH2Cl2, rt; (ii) separate isomers (4:1); (iii) MeI,
NaH, THF–DMF, 0°C, 54%; (iv) TBAF, THF, rt; (v) PvCl, pyridine,
CH2Cl2, 0°C to rt, 88%; (c) (i) MPMOTCI, BF3ÆEt2O, CH2Cl2, 0°C,
85%; (ii) OsO4, NMO, acetone–H2O, rt (89%); (iii) separate isomers
(1:1); (d) (i) TBSOTf, Et3N, CH2Cl2, 0°C; (ii) LAH, Et2O, 0°C, 91%;
(iii) (COCl)2, DMSO, Et3N, CH2Cl2, À78°C to 0°C; (iv)
(MePPh3)+BrÀ, n-BuLi, THF–DMSO, 0°C to rt, 76%; (e) (i)
BH3ÆTHF, THF, 0°C; (ii) NaBO3, H2O, rt, 63%; (iii) (COCl)2,
DMSO, Et3N, CH2Cl2, À78°C to 0°C, 100%.
Scheme 2. Synthesis of aldehyde 14. Reagents and conditions: (a) (i)
allylTMS, BF3ÆEt2O, MeCN, 80°C, 67%;10 (ii) NaOMe, MeOH, rt,
84%; (iii) p-MeOC6H4CH(OMe)2, p-TsOH, DMF, rt, 90%; (b) (i)
TsCl, Pyr, rt, 71%; (ii) NaOMe, MeOH, rt, 79%; (iii) MeMgCl, Et2O,
reflux, 75%; (c) (i) (COCl)2, DMSO, Et3N, CH2Cl2, À78°C to 0°C; (ii)
Et3N, DMF, rt, 74%; (d) (i) LAH, Et2O, 0°C, 88%; (ii) TBSOTf,
DIEA, CH2Cl2, 0°C; (e) (i) O3, CH2Cl2, À78°C; (ii) NaBH4, EtOH, rt,
80%; (iii) TBDPSCl, imidazole, DMAP, DMF, rt, 73%; (f) (i) DIBAL-
H, CH2Cl2, À78°C, 59%; (ii) (COCl)2, DMSO, Et3N, CH2Cl2, À78°C
to 0°C; (iii) (MePPh3)+BrÀ, n-BuLi, THF–DMSO, 96%; (g) (i)
BH3ÆDMS, THF, À10°C to rt; (ii) NaBO3, H2O, rt, 46%; (iii) Dess–
Martin oxidation,11 65%.
aldehyde 14 was subsequently obtained following a
series of standard transformations.
Our earlier SAR studies with RH demonstrated a degree
of steric tolerance at the C.31 center.7 If the methyl
group could be replaced with a methoxyl substituent,
then the corresponding aldehyde fragment could be pre-
pared in a more straightforward manner. Toward that
end, diol 8 was selectively methylated12 and converted
to aldehyde 18 (Scheme 3).
Aldehydes 25a/b and 26a/b were prepared from L-arabi-
nose derivative 1913 as outlined in Scheme 4. The two
C.34-diastereomeric diols 23a/b were chromatographi-
cally separated and carried forward separately to final
products 25a and 25b. C.32a-isomer 21 was converted
to C.34-epimers 26a and 26b.
Aldehydes 14, 18, 25a/b, and 26a/b were coupled with
fragments 4 and 5 (Scheme 1) and carried forward to
final compounds 27, 28, 41, 42, 46, and 47, respectively.
The other macrolactone analogues were prepared in a
similar manner.
Scheme 3. Synthesis of aldehyde 18. Reagents and conditions: (a) (i)
Bu2SnO, toluene, reflux;12 (ii) KF, MeI, DMF; (iii) separate 15, 70%;
(b) (i) TBSCl, imidazole, DMF, rt; (ii) DIBAL-H, CH2Cl2–toluene,
À40°C to À5°C; (iii) PvCl, Pyr, CH2Cl2, 0°C to rt, 80%; (iv) OsO4,
NMO, acetone–H2O, rt; (v) NaIO4, MeOH–H2O, rt; (vi) NaBH4,
MeOH–Et2O, 0°C; (vii) TBDPSCl, imidazole, DMF, rt, 96%; (c) (i)
LAH, Et2O, 0°C; (ii) (COCl)2, DMSO, Et3N, CH2Cl2, À78°C to 0°C;
(iii) (MePPh3)+BrÀ, n-BuLi. THF–DMSO, 0°C to rt, 66%; (iv)
BH3ÆTHF, THF, 0°C; (v) NaBO3, H2O, rt, 60%; (vi) (COCl)2, DMSO,
Et3N, CH2Cl2, À78°C to 0°C, 99%.
3. Results
The compounds were evaluated for cell growth inhibi-
tory activity against DLD-1 human colon cancer cells
under continuous exposure conditions (intrinsic po-
tency), and for the ability to maintain a complete mitotic
Inversion of this center was accomplished by an oxida-
tion, epimerization, and reduction sequence. The desired

B. M. Seletsky et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5547–5550
5549
block (CMB) 10h after drug washout using flow cyto-
metric analysis of U937 human histiocytic lymphoma
cells (reversibility assay).14 In addition, susceptibility
to P-glycoprotein (PgP) mediated drug efflux using mur-
b]pyran analogue 2. As expected, replacement of the
C.31-methyl group with a methoxyl substituent (e.g.,
28) was well tolerated. Replacement with an ethoxyl
group, however, led to a 30-fold loss in intrinsic potency
(35 vs 36 or 37). Inverting the C.32 stereocenter as in 31
and 33 resulted in a three- to five-fold improvement over
28 and 32, respectively, in ability to maintain a CMB.
Shortening the C.33-side chain had no effect (32 vs
28), but extending the side chain to a propanediol
(e.g., 36 and 37) led to improved activity in the
ine P388/VMDRC.04 cells,
a multidrug resistant
(MDR) subline of murine P388/S leukemia cells,15 was
determined (Table 1).
Tetrahydropyran derivative 27 exhibited biological pro-
file similar to that of C.29–C.36 octahydropyrano[3,2-
Table 1. In vitro activity profile for compounds of generic structure 6
Compound
R1
R2
R3
R4
DLD-1a IC50, nM (n)
U937b IC99, nM (n)
FRc
HB
2
0.74 0.03 (6)
3.4 0.04 (4)
25 (4)
220 (6)
2.8
12
Tetrahydropyran series
27
28
29
30
31
32
33
34
35
36
37
38
Me
OH
OH
OH
OMe
H
H
CH2CH2OH
CH2CH2OH
CH2CH2OMe
CH2CH2OH
CH2CH2OH
CH2OH
CH2OH
H
2.5 0.20 (3)
1.8 0.00 (3)
1.8 0.10 (3)
2.1 0.06 (3)
1.8 0.30 (3)
2.6 0.40 (3)
2.0 0.30 (3)
>1000 (2)
170 (3)
300 (3)
>1000 (2)
>1000 (2)
100 (2)
300 (2)
65 (2)
5.6
10
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
H
H
1.6
2.6
12
H
OH
H
OH
H
8.5
13
OH
OH
OH
OH
OH
H
ND
ND
ND
ND
23
H
X
490 90.0 (3)
19.0 1.70 (4)
12.0 1.60 (3)
11.0 0.90 (3)
OMe
OMe
H
X
X (C.34-epi)
53 (3)
65 (2)
>1000
H
20
1.7
Tetrahydrofuran series
39
40
41
42
43
44
45
46
47
48
49
H
OMe
OMe
OMe
OMe
H
CH2OH
CH2CH2OH
H
1.0 0.20 (3)
0.97 0.07 (3)
1.4 0.10 (3)
6.4 0.80 (3)
220 18.0 (3)
1.9 0.10 (3)
0.67 0.09 (3)
0.67 0.07 (3)
0.67 0.09 (3)
>1000 (2)
100
100
2.8
2.8
8.8
8.2
ND
15
H
H
H
X (C.34-R)
X (C.34-S)
X (C.34-R)
H
30 (2)
100 (1)
ND
H
H
OMe
H
H
OMe
OMe
OMe
OMe
Y
Z
H
30 (2)
30 (2)
20 (2)
15 (4)
ND
H
H
4.5
9.3
6.2
ND
ND
H
H
H
X (C.34-R)
X (C.34-S)
H
>1000 (2)
ND
a Cell growth inhibition under continuous exposure conditions for 3–4days, IC50 SEM (n = number of experiments).
b Mitotic block reversibility assay; ND—not determined.
c Fold resistance calculated as the IC50 ratio between retrovirally transformed P388/VMDRC.04 and parental P388 cells; ND—not determined.

5550
B. M. Seletsky et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5547–5550
reversibility assay, albeit with increased susceptibility to
PgP-mediated drug efflux. This inverse relationship was
also observed when the hydroxyl groups were converted
to methyl ethers 29 and 30 or to an acetonide 38, result-
ing in diminished activity in the reversibility assay, and
decreased susceptibility to PgP-mediated drug efflux.
Removal of the C.33-side chain (i.e., 34) led to complete
loss of activity.
References and notes
1. Uemura, D.; Takahashi, K.; Yamamoto, T.; Katayama,
C.; Tanaka, J.; Okumura, Y.; Hirata, Y. J. Am. Chem.
Soc. 1985, 107, 4796–4798.
2. Hirata, Y.; Uemura, D. Pure Appl. Chem. 1986, 58, 701–
710.
3. (a) Fodstad, Ø.; Breistøl, K.; Pettit, G. R.; Shoemaker, R.
H.; Boyd, M. R. J. Exp. Therap. Oncol. 1996, 1, 119–125;
(b) Towle, M. J.; Aalfs, K. K.; Budrow, J. A.; Kishi, Y.;
Littlefield, B. A. Annual Meeting of the American
Association for Cancer Research, Toronto, Ontario,
March 18–22, 1995, 2342.
4. (a) Aicher, T. D.; Buszek, K. R.; Fang, F. G.; Forsyth, C.
J.; Jung, S. H.; Kishi, Y.; Matelich, M. C.; Scola, P. M.;
Spero, D. M.; Yoon, S. K. J. Am. Chem. Soc. 1992, 114,
3162–3164; (b) Aicher, T. D.; Buszek, K. R.; Fang, F. G.;
Forsyth, C. J.; Jung, S. H.; Kishi, Y.; Scola, P. M.
Tetrahedron Lett. 1992, 33, 1549–1552; (c) Buszek, K. R.;
Fang, F. G.; Forsyth, C. J.; Jung, S. H.; Kishi, Y.; Scola,
P. M.; Yoon, S. K. Tetrahedron Lett. 1992, 33, 1553–1556;
(d) Fang, F. G.; Kishi, Y.; Matelich, M. C.; Scola, P. M.
Tetrahedron Lett. 1992, 33, 1557–1560.
Since the C.31 substituent appeared to play a critical
role, we hypothesized that alternative ring systems that
preserve the spatial relationship of this group relative
to the macrolactone ring may stabilize the bioactive con-
formation of the molecule and lead to analogues with
improved biological activity. In the X-ray crystal struc-
ture of norhalichondrin A p-bromophenacyl ester,1 the
C.29–C.33 tetrahydropyran ring adopted a twist boat
conformation that placed the C.31 methyl group in a
pseudoequatorial position. Since a tetrahydrofuran ring
could similarly orient the C.31 methyl group in a pseu-
doequatorial position, we prepared a series of tetra-
hydrofuran derivatives to determine if the biological
activity limitations observed with the tetrahydropyran
analogues could be circumvented.
5. (a) Towle, M. J.; Kishi, Y.; Littlefield, B. A., unpublished
observations, 1992; (b) Kishi, Y.; Fang, F. G.; Forsyth, C.
J.; Scola, P. M.; Yoon, S. K. U.S. Patent 5,436,238.
6. Stamos, D. P.; Sean, S. C.; Kishi, Y. J. Org. Chem. 1997,
62, 7552–7553.
Gratifyingly, tetrahydrofuran 41 exhibited a superior
potency and reversibility profile to that found for the
tetrahydropyran series (cf. 41 and 27–38). Inversion of
stereochemistry at C.31 led to a 150-fold loss of potency
(43 vs 41), confirming the importance of this group in
both the tetrahydropyran and tetrahydrofuran series.
As expected based on conformational arguments, activ-
ity was also critically dependent on the configuration at
C.29 and C.30 of the macrolactone ring (e.g., 48 and 49,
respectively). The trifluoromethyl ketone analogue 45
exhibited an in vitro biological profile similar to 41 sug-
gesting the bioisosteric equivalence of a hydrated tri-
fluoromethyl ketone acting as a formal geminal diol
with a vicinal diol moiety.16 The influence of stereo-
chemistry at C.32 was further investigated. C.34-epi-
meric diols 46 and 47 showed superior activity in the
reversibility assay compared with the corresponding
C.32 diastereomers 41 and 42. From these studies, tetra-
hydrofuran 47 emerged as the most promising analogue
based on intrinsic potency, ability to maintain a CMB,
and susceptibility to PgP-mediated drug efflux.
7. Wang, Y.; Habgood, G. J.; Christ, W. J.; Kishi, Y.;
Littlefield, B. A.; Yu, M. J. Bioorg. Med. Chem. Lett.
2000, 10, 1029–1032.
8. (a) Xie, C.; Nowak, P.; Kishi, Y. Org. Lett. 2002, 4, 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. Stamos, D. P.; Kishi, Y. Tetrahedron Lett. 1996, 37, 8643–
8646.
10. Lewis, M. D.; Cha, J. K.; Kishi, Y. J. Am. Chem. Soc.
1982, 104, 4976–4978.
11. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155–
4156.
12. Nashed, M. A.; Anderson, L. Tetrahedron Lett. 1976,
3503–3506.
13. L-Arabinose derivative 19 was prepared in three steps
from L-arabinose dithioacetal: (a) TBDPSCl, Et3N; (b) I2,
NaHCO3; (c) Ac2O, pyridine.
14. Towle, M. J.; Salvato, K. A.; Budrow, J.; Wels, B. F.;
Aalfs, K. K.; Zheng, W.; Seletsky, B. M.; Palme, M. H.;
Habgood, G. J.; Singer, L. A.; DiPietro, L. V.; Wang, Y.;
Chen, J. J.; Lydon, P. J.; Quincy, D. A.; Kishi, Y.;
Yoshimatsu, K.; Yu, M. J.; Littlefield, B. A. Annual
Meeting of the American Association for Cancer
Research, New Orleans, LA, March 24–28, 2001, 1976.
15. Yang, J.-M.; Goldenberg, S.; Gottesman, M. M.; Hait, W.
N. Cancer Res. 1994, 54, 730–737.
In summary, structurally simplified macrolactone
analogues of HB were identified that retain the
potent cell growth inhibitory activity of the natural
product in vitro, and may represent the minimum phar-
macophore for the halichondrin class of antimitotic
agents.
16. Patel, D. V.; Rielly-Gauvin, K.; Ryono, D. E.; Free, C. A.;
Smith, S. A.; Petrillo, E. W. J. Med. Chem. 1993, 36, 2431–
2447.