Relative and absolute stereochemistries and total synthesis of (+)-macrosphelides A and B, potent, orally bioavailable inhibitors of cell-cell adhesion

Full text of DOI:10.1021/ja971657w

J. Am. Chem. Soc. 1997, 119, 10247-10248
10247
program directed toward the structure elucidation and synthesis
of important bioregulatory natural products, we describe here
the determination of the complete relative and absolute stereo-
chemistries of (+)-macrosphelides A and B (1 and 2) and the
first total synthesis of these materials.
Relative and Absolute Stereochemistries and Total
Synthesis of (+)-Macrosphelides A and B, Potent,
Orally Bioavailable Inhibitors of Cell-Cell
Adhesion
Toshiaki Sunazuka, Tomoyasu Hirose, Yoshihiro Harigaya,
Satoshi Takamatsu, Masahiko Hayashi,
Kanki Komiyama, and Satoshi Ohmura*
Research Center for Biological Function
The Kitasato Institute
School of Pharmaceutical Sciences
Kitasato UniVersity, Minato-ku, Tokyo 108, Japan
Paul A. Sprengeler and Amos B. Smith, III*
Department of Chemistry, Laboratory for Research
on the Structure of Matter
Initially we deduced the connectivity of 1 and 2 via a series
Monell Chemical Senses Center, UniVersity of PennsylVania
of NMR studies, including H-1H and H-13C COSY and
HMBC experiments, in conjunction with FAB MS and IR data
and chemical characterization of the derived di- and monoac-
etates, respectively.12 Single-crystal X-ray diffraction has now
been employed to elucidate the relative stereochemistry of 1
and verify the planar structure (Figure 1).14
1
1
Philadelphia, PennsylVania 19104
ReceiVed May 21, 1997
Critical early events in inflammation,1-3 the allergic re-
sponse,4-6 and tumor metastasis7-9 involve interactions between
leukocytes and endothelial cells. A variety of cytokinins and
related chemical mediators control both leukocyte adhesion and
subsequent intercellular invasion by regulating the expression
of cellular adhesion molecules.10,11 Inhibition of cell-cell
adhesion thus holds promise for the treatment of diverse
pathologies.
Recently we reported the isolation, planar structures, and
preliminary biological evaluation of (+)-macrosphelides A and
B (1 and 2).12 These novel macrolides, produced by Mi-
crosphaeropsis sp. FO-5050, are the first 16-membered-ring
antibiotics embodying three lactone linkages (i.e., macrotri-
olides). The macrosphelides strongly inhibit the adhesion of
human-leukemia HL-60 cells to human-umbilical-vein endo-
thelial cells (HUVEC) in dose-dependent fashion (IC50 3.5 and
36 µM, respectively).12 Preliminary studies suggest that 1 and
2 prevent cell-cell adhesion by inhibiting the binding of sialyl
Lewis x to E-selectin.13 Macrosphelide A also proved to be
orally active against lung metastasis of B16/BL6 melanoma in
mice (50 mg/kg). Importantly, 1 did not inhibit the growth of
various mammalian cell lines (0.2 mg/mL) or microorganisms
(1 mg/mL) in vitro. No acute toxicity was observed upon
intraperitoneal injection into BDF1 mice (200 mg/kg for 5
days).13 The macrosphelides also display significant activity
against the rodent-ear edema reaction induced by arachidonic
acid and, thus, may serve as valuable leads for the development
of lipoxygenase inhibitors.13 In conjunction with our continuing
Figure 1. ORTEP plot for (+)-macrosphelide A (1).
We next sought to determine the absolute configuration via
the Kakisawa-Kashman modification15 of the Mosher NMR
method.16 To this end, the bis(Mosher ester) derivatives (-)-3
and (+)-4 were prepared by treatment of 1 with (S)-(-)- and
(R)-(+)-R-methoxy-R-(trifluoromethyl)phenylacetic acid (MTPA)
in the presence of dicyclohexylcarbodiimide (DCC) and 4-(di-
methylamino)pyridine (DMAP) (THF, room temperature).15 The
1H NMR spectra of 3 and 4 were completely assigned via
selective 1H decoupling. Application of the Kakisawa-Kash-
man test15 to the 1H ∆δ values for 3 and 4 (Figure 2) indicated
that the absolute configurations at C(8) and C(14) are R; thus,
(+)-macrosphelide A (1) contains two (4R,5S)-4,5-dihydroxy-
pentenoic acid moieties and a (3S)-3-hydroxybutanoic acid unit.
The larger ∆δ shifts observed for the protons â to the secondary
(1) Arm, J. P.; Lee, T. H. AdV. Immunol. 1992, 51, 323-382.
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(14) Compound (+)-1, C16H22O8, crystallizes in the monoclinic space
group P21 with a ) 10.387(4), b ) 5.656(5), and c ) 16.392(4) Å, â )
106.49(2)°, V ) 923.4(9) Å3, Z ) 2 and dcalcd ) 1.231 g/cm3. The cell
constants were determined from a least-squares fit of the setting angles for
15 accurately centered reflections. X-ray intensity data were collected on a
Rigaku AFC5S diffractometer employing Cu KR radiation (λ ) 1.541 78
Å) and the ω-2θ scan technique. A total of 1904 reflections were measured
with 2θmax ) 140.3°. The intensity data were corrected for Lorentz and
polarization effects but not for absorption. The structure was solved by
direct methods. For refinement, 1522 unique reflections with F2 > 3σ(F2)
were used. Full-matrix least-squares refinement based on F, minimizing
(3) Pang, G.; Couch, L.; Batey, R.; Clancy, R.; Cripps, A. Clin. Exp.
Immunol. 1994, 96, 437-443.
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1805-1809.
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Gosset, P. Clin. Exp. Immunol. 1994, 98, 344-349.
(7) Lauri, D.; Needham, L.; Martin-Padura, I.; Dejana, E. J. Natl. Cancer
Inst. 1991, 83, 1321-1324.
the quantity ∑w(|Fo| - |Fc|)2 with w ) 4Fo /σ2(Fo ), converged to R )
2
2
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Immunol. Immunopathol. 1994, 72, 129-136.
0.082 and Rw ) 0.092.
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55, 610-616.
(15) (a) Kusumi, T.; Ohtani, I.; Inouye, Y.; Kakisawa, H. Tetrahedron
Lett. 1988, 29, 4731-4734. (b) Ohtani, I.; Kusumi, T.; Ishitsuka, M. O.;
Kakisawa, H. Tetrahedron Lett. 1989, 30, 3147-3150. (c) Kusumi, T.;
Fujita, Y.; Ohtani, I.; Kakisawa, H. Tetrahedron Lett. 1991, 32, 2923-
2926. (d) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Org. Chem.
1991, 56, 1296-1298. (e) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa,
H. J. Am. Chem. Soc. 1991, 113, 4092-4096.
(10) Springer, T. A. Nature 1990, 346, 425-434.
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(12) (a) Hayashi, M.; Kim, Y.-P.; Hiraoka, H.; Natori, M.; Takamatsu,
S.; Kawakubo, T.; Masuma, R.; Komiyama, K.; Ohmura, S. J. Antibiot. 1995,
48, 1435-1439. (b) Takamatsu, S.; Kim, Y.-P.; Hayashi, M.; Hiraoka, H.;
Natori, M.; Komiyama, K.; Ohmura, S. J. Antibiot. 1996, 49, 95-98.
(13) Hayashi, M.; Hiraoka, H.; Kim, Y.-P.; Takamatsu, S.; Komiyama,
K.; Ohmura, S. Submitted for publication.
(16) (a) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34,
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S0002-7863(97)01657-0 CCC: $14.00 © 1997 American Chemical Society

10248 J. Am. Chem. Soc., Vol. 119, No. 42, 1997
Communications to the Editor
Scheme 1
Figure 2. Absolute stereochemistry determination: ∆δ values for the
bis(Mosher ester) derivatives 3 and 4 (ppm, 500 MHz; ∆δ ) δS - δR
) δ3 - δ4).
hydroxyls [i.e., the C(6) and C(13) vinyl and C(9) and C(15)
methyl protons] are consistent with the proposed solution
conformations, wherein the MTPA aromatic rings lie closer to
the â hydrogens than to the R hydrogens.
To secure the relative and absolute stereochemistries of 2,
we subjected (+)-macrosphelide A to pyridinium dichromate
(PDC) oxidation in CH2Cl2 (room temperature, 3 h). Preparative
TLC gave a mixture of the 14- and 8-monoketones 217 and 517
as well as pure 8,14-diketone 617 (16% yield) and recovered 1
(45%). HPLC separation then afforded 2 and 5 in 21 and 18%
yields. Synthetic 2 proved to be indistinguishable from the
natural product (1H and 13C NMR, IR, high-resolution mass
spectrometry, and optical rotation). Accordingly, the configura-
tions of (+)-macrosphelides A (1) and B (2) are (3S, 8R, 9S,
14R, 15S) and (3S, 8R, 9S, 15S), respectively. These assign-
ments were confirmed by total synthesis.
Our approach to the construction of 1 and 2 entailed the
enantioselective preparation of two differentially protected
derivatives of trans-(4R,5S)-4,5-dihydroxy-2-hexenoic acid. The
third building block, (3S)-3-hydroxybutyric acid, is com-
mercially available. As our point of departure, we selected the
asymmetric dihydroxylation18 of (E,E)-hexa-2,4-dienoic acid
tert-butyl ester (7),19 which afforded the (4S,5S)-diol (-)-817
in 62% yield (Scheme 1). Selective monosilylation of (-)-8
[tert-butyldimethylsilyl chloride (TBSCl), DMAP, CH2Cl2]
provided the desired ether (+)-917 (56% yield; 78% based on
recovered 8) plus the 4-silyloxy isomer (11%, not shown).
Mitsunobu inversion [PPh3, diethyl azodicarboxylate (DEAD),
HCO2H; dilute NH3/MeOH] at C(4) of 9 furnished (+)-1017
(83% yield); the enantiomeric purities of both 9 and 10 were
85% ee as determined by Mosher analysis.16 After protection
of (+)-10 as the (methoxyethoxy)methyl (MEM) ether (-)-1117
(87% yield), saponification (0.2 N NaOH, MeOH/THF/H2O)
gave (-)-1217 in 94% yield, whereas desilylation generated the
second building block (-)-1317 (Bu4NF, THF, 100%).
Condensation of carboxylic acid (-)-12 and alcohol (-)-13
via the Keck protocol20 [DCC, DMAP, camphorsulfonic acid
(CSA), CH2Cl2, 77% yield] and desilylation of the resultant ester
(-)-1417 (3:1:1 AcOH/THF/H2O) produced (-)-1517 in 83%
yield. The third fragment, TBS ether (+)-16,21 was prepared
from (3S)-3-hydroxybutyric acid and coupled with (-)-15 in
96% yield (DCC, DMAP, CSA, CH2Cl2). Removal of the silyl
and tert-butyl moieties in (-)-1717 [5:1:5 trifluoroacetic acid
(TFA)/CH2Cl2/thioanisole]22 provided seco acid (-)-1817 (64%
yield), which smoothly underwent Yamaguchi macrolactoniza-
tion23 (DMAP, 2,4,6-trichlorobenzoyl chloride, 91%). Finally,
deprotection of (-)-1917 (1:1 TFA/CH2Cl2) gave synthetic 1 in
90% yield, identical in all respects (400 MHz 1H and 100 MHz
13C NMR, IR, high-resolution FAB MS, optical rotation, melting
point and mixed melting point, TLC and HPLC in four solvent
systems) with a sample of the natural product.
The first total synthesis of (+)-macrosphelide A (1) has thus
been achieved via a highly convergent, efficient strategy (11
steps, 10.6% overall yield). In conjunction with the conversion
of 1 to 2, the successful route also constitutes a formal
construction of (+)-macrosphelide B, confirming the assigned
structures of both congeners. Further refinements of the
synthetic scheme and the preparation and biological evaluation
of macrosphelide analogs will be reported in due course.
Acknowledgment. Financial support was provided by the Ministry
of Education, Science and Culture (Japan), the Japan Keirin Associ-
tation, a Kitasato University Research Grant for Young Researchers,
and by the National Institutes of Health (Institute of General Medical
Sciences) through grant GM-29028. The authors are also indebted to
Dr. George T. Furst for assistance in interpreting the NMR data.
(17) All synthetic compounds were purified by flash chromatography
on silica gel. The structure assigned to each new compound is in accord
with its IR, 400 or 270 MHz 1H NMR, and 100 or 67.5 MHz 13C NMR
spectra, as well as appropriate parent ion identification by high-resolution
mass spectrometry. In addition, compounds 8-19 gave satisfactory combus-
tion analyses.
Supporting Information Available: Preparative procedures and
characterization data for 1-6, 8-19; tables of X-ray data for 1 (18
pages). See any current masthead page for ordering and Internet access
instructions.
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114, 7570-7571.
JA971657W
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